HFE gene mutations in patients with primary iron overload: is there a significant improvement in molecular diagnosis yield with HFE sequencing?

PubMed

Santos, Paulo C J L; Pereira, Alexandre C; Cançado, Rodolfo D; Schettert, Isolmar T; Sobreira, Tiago J P; Oliveira, Paulo S L; Hirata, Rosario D C; Hirata, Mario H; Figueiredo, Maria Stella; Chiattone, Carlos S; Krieger, Jose E; Guerra-Shinohara, Elvira M

2010-12-15

Rare HFE variants have been shown to be associated with hereditary hemochromatosis (HH), an iron overload disease. The low frequency of the HFE p.C282Y mutation in HH-affected Brazilian patients may suggest that other HFE-related mutations may also be implicated in the pathogenesis of HH in this population. The main aim was to screen for new HFE mutations in Brazilian individuals with primary iron overload and to investigate their relationship with HH. Fifty Brazilian patients with primary iron overload (transferrin saturation>50% in females and 60% in males) were selected. Subsequent bidirectional sequencing for each HFE exon was performed. The effect of HFE mutations on protein structure were analyzed by molecular dynamics simulation and free binding energy calculations. p.C282Y in homozygosis or in heterozygosis with p.H63D were the most frequent genotypic combinations associated with HH in our sample population (present in 17 individuals, 34%). Thirty-six (72.0%) out of the 50 individuals presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n=11, 22.0%). One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior indicated that the p.V256I mutation does not reduce the binding affinity between HFE and β2-microglobulin (β2M) in the same way the p.C282Y mutation does compared with the native HFE protein. In conclusion, screening of HFE through direct sequencing, as compared to p.C282Y/p.H63D genotyping, was not able to increase the molecular diagnosis yield of HH. The novel p.V256I mutation could not be implicated in the molecular basis of the HH phenotype, although its role cannot be completely excluded in HH-phenotype development. Our molecular modeling analysis can help in the analysis of novel, previously undescribed, HFE mutations. Copyright © 2010 Elsevier Inc. All rights reserved.

Molecular epidemiology of HFE gene polymorphic variants (C282Y, H63D and S65C) in the population of Espírito Santo, Brazil.

PubMed

Alves, L N R; Santos, E V W; Stur, E; Silva Conforti, A M A; Louro, I D

2016-04-27

Hereditary hemochromatosis (HH) is an autosomal recessive disorder that leads to progressive iron accumulation and may cause cirrhosis, hepatocellular carcinoma, diabetes, and heart failure. Most cases of HH have been linked to mutations in genes associated with iron homeostasis. There have been three major variants in the high Fe (HFE) gene associated with the disease: C282Y, H63D and S65C. In this context, we aimed to evaluate the prevalence of the polymorphic variants (C282Y, H63D and S65C) of the HFE gene in the population of the Espírito Santo State (ES), Brazil by analyzing three different groups: general population (N = 120), Pomeranian descendants (N = 59), and patients with HH (N = 20). Using genomic DNA extracted from peripheral blood, polymorphic variant identification was performed by polymerase chain reaction-restriction fragment length polymorphism. Statistically significant differences were observed for genotype distribution of C282Y (P < 0.001) and H63D (P = 0.013) between the general population and the patients diagnosed with HH. This is the first study to analyze HFE gene allele frequencies for the general population, Pomeranian subpopulation, and patients with HH of ES, Brazil.

Association of HFE gene mutations with nonalcoholic fatty liver disease in the Iranian population.

PubMed

Saremi, L; Lotfipanah, S; Mohammadi, M; Hosseinzadeh, H; Sayad, A; Saltanatpour, Z

2016-10-31

To determine whether the HFE gene variants H63D and C282Y are associated with NAFLD in persons with type 2 diabetes, we conducted a case-control study including 145 case of NAFLD patients with a history of type 2 diabetes and 145 matching control. The genomic DNA was extracted from the peripheral venous blood and the genotyping of HFE gene mutations was analyzed using the PCR-RFLP technique. Statistical analysis was performed using SPSS 12.0 software by χ2 test, t test and ANOVA (P<0.05). Data showed no increased frequency of HFE mutations in persons with type 2 diabetes and no association between H63D mutation and NAFLD in the study population. Also, we analyzed index of physiological variables including FBS, lipid profile (TC, TG, LDL-C, and HDL-C), BMI, HbA1c, and micro albuminuria and Cr levels). Data showed there are no relationship between these indexes and HFE gene mutations and either NAFLD as a complication of diabetes. But our results showed a relationship between C282Y mutation and NAFLD in persons with type 2 diabetes. C282Y mutation might be a genetic marker of NAFLD in Iranian population.

HFE Genotyping in Patients with Elevated Serum Iron Indices and Liver Diseases

PubMed Central

Evangelista, Andreia Silva; de Araújo, Thiago Ferreira; Abrantes-Lemos, Clarice Pires; Deguti, Marta Mitiko; Cançado, Eduardo Luiz Rachid

2015-01-01

Iron abnormalities in chronic liver disease may be the result of genetic diseases or secondary factors. The present study aimed to identify subjects with HFE-HH in order to describe the frequency of clinical manifestations, identify risk factors for iron elevation, and compare the iron profile of HFE-HH to other genotypes in liver disease patients. A total of 108 individuals with hepatic disease, transferrin saturation (TS) > 45%, and serum ferritin (SF) > 350 ng/mL were tested for HFE mutations. Two groups were characterized: C282Y/C282Y or C282Y/H63D genotypes (n = 16) were the HFE hereditary hemochromatosis (HFE-HH) group; and C282Y and H63D single heterozygotes, the H63D/H63D genotype, and wild-type were considered group 2 (n = 92). Nonalcoholic liver disease, alcoholism, and chronic hepatitis C were detected more frequently in group 2, whereas arthropathy, hepatocarcinoma, diabetes, and osteoporosis rates were significantly higher in the HFE-HH group. TS > 82%, SF > 2685 ng/mL, and serum iron > 178 μg/dL were the cutoffs for diagnosis of HFE-HH in patients with liver disease. Thus, in non-Caucasian populations with chronic liver disease, HFE-HH diagnosis is more predictable in those with iron levels higher than those proposed in current guidelines for the general population. PMID:25654085

Effect of HFE gene polymorphism on sustained virological response in patients with chronic hepatitis C and elevated serum ferritin.

PubMed

Coelho-Borges, Silvia; Cheinquer, Hugo; Wolff, Fernando Herz; Cheinquer, Nelson; Krug, Luciano; Ashton-Prolla, Patricia

2012-01-01

Abnormal serum ferritin levels are found in approximately 20%-30% of the patients with chronic hepatitis C and are associated with a lower response rate to interferon therapy. To determine if the presence of HFE gene mutations had any effect on the sustained virological response rate to interferon based therapy in chronic hepatitis C patients with elevated serum ferritin. A total of 44 treatment naÏve patients with histologically demonstrated chronic hepatitis C, all infected with hepatitis C virus genotype non-1 (38 genotype 3; 6 genotype 2) and serum ferritin above 500 ng/mL were treated with interferon (3 MU, 3 times a week) and ribavirin (1.000 mg, daily) for 24 weeks. Sustained virological response was defined as negative qualitative HCV-RNA more than 24 weeks after the end of treatment. Serum HCV-RNA was measured by qualitative in house polymerase chain reaction with a limit of detection of 200 IU/mL. HFE gene mutation was detected using restriction-enzyme digestion with RsaI (C282Y mutation analysis) and BclI (H63D mutation analysis) in 16 (37%) patients, all heterozygous (11 H63D, 2 C282Y and 3 both). Sustained virological response was achieved in 0 of 16 patients with HFE gene mutations and 11 (41%) of 27 patients without HFE gene mutations (P = 0.002; exact Fisher test). Heterozigozity for H63D and/or C282Y HFE gene mutation predicts absence of sustained virological response to combination treatment with interferon and ribavirin in patients with chronic hepatitis C, non-1 genotype and serum ferritin levels above 500 ng/mL.

The prevalence of primary hereditary hemochromatosis in central Anatolia.

PubMed

Karaca, Halit; Güven, Kadri; Önal, Müge; Gürsoy, Şebnem; Başkol, Mevlüt; Özkul, Yusuf

2013-01-01

Hereditary hemochromatosis is an autosomal recessive disorder associated with the HFE genes. Early identification and diagnosis is important as end stage organ damage may occur if treatment is delayed.. This study aimed to identify the prevalence of hereditary hemochromatosis in Kayseri and surroundings known as Central Anatolia. 2304 participants (1220 males, 1084 females) who were older then the age of 17 were included in the study conducted between December 2005 and December 2006 in Kayseri, Turkey. Transferin saturation was measured from overnight fasting blood samples. Serum iron, total iron binding capacity, and transferin saturation were measured. Serum ferritin levels and hereditary hemochromatosis genetic analysis were also performed after an overnight fasting blood samples from participants whose transferin saturation results were more than 50% in man and more than 45% in women. The homozygote C282Y mutation and heterozygote C282Y mutation prevalences were found as 0.08% (1/1220) and 0.08% (1/1220) in male participants, respectively. The heterozygote H63D mutation prevalence was found in 0.09% (1/1084) of female participants. Calculated prevalences in general population are as follows; The homozygote C282Y mutation prevalence is 0.043% (1/2304), the heterozygote C282Y mutation prevalence is 0.043% (1/2304) and the heterozygote H63D mutation prevalence is 0.043% (1/2304). The prevalence of hereditary hemochromatosis in Central Anatolia is 0.043% (1/2304). Because of the relatively low frequency, population screening studies are not cost-effective.

Iron overload in HFE C282Y heterozygotes at first genetic testing: a strategy for identifying rare HFE variants.

PubMed

Aguilar-Martinez, Patricia; Grandchamp, Bernard; Cunat, Séverine; Cadet, Estelle; Blanc, François; Nourrit, Marlène; Lassoued, Kaiss; Schved, Jean-François; Rochette, Jacques

2011-04-01

Heterozygotes for the p.Cys282Tyr (C282Y) mutation of the HFE gene do not usually express a hemochromatosis phenotype. Apart from the compound heterozygous state for C282Y and the widespread p.His63Asp (H63D) variant allele, other rare HFE mutations can be found in trans on chromosome 6. We performed molecular investigation of the genes implicated in hereditary hemochromatosis in six patients who presented with iron overload but were simple heterozygotes for the HFE C282Y mutation at first genetic testing. Functional impairment of new variants was deduced from computational methods including molecular modeling studies. We identified four rare HFE mutant alleles, three of which have not been previously described. One mutation is a 13-nucleotide deletion in exon 6 (c.1022_1034del13, p.His341_Ala345 > LeufsX119), which is predicted to lead to an elongated and unstable protein. The second one is a substitution of the last nucleotide of exon 2 (c.340G > A, p.Glu114Lys) which modifies the relative solvent accessibility in a loop interface. The third mutation, p.Arg67Cys, also lies in exon 2 and introduces a destabilization of the secondary structure within a loop of the α1 domain. We also found the previously reported c.548T > C (p.Leu183Pro) missense mutation in exon 3. No other known iron genes were mutated. We present an algorithm at the clinical and genetic levels for identifying patients deserving further investigation. Conclusions Our results suggest that additional mutations in HFE may have a clinical impact in C282Y carriers. In conjunction with results from previously described cases we conclude that an elevated transferrin saturation level and elevated hepatic iron index should indicate the utility of searching for further HFE mutations in C282Y heterozygotes prior to other iron gene studies.

[Haemochromatosis screening in 120 patients complaining with persistant fatigue].

PubMed

Vital Durand, D; François, S; Nové-Josserand, R; Durupt, S; Durieu, I; Morel, Y; Rousset, H

2004-09-01

Chronic fatigue is the more frequent symptom identified in the course of hereditary haemochromatosis. A screening for this disorder was carried out in 120 primary care patients consulting for unexplained chronic fatigue. Transferrin saturation and serum ferritin were determined in all patients. If transferrin saturation was >or= 45% and serum ferritin >or= 300 microg/l, HFE1 genotyping for mutations C282Y and H63D was completed. One hundred and twenty patients were recruited, 19-86 years old, including 62 males and 58 females. 45 patients (38%) presented with serum ferritin >or= 300 microg/l. Thirty two patients (27%) presented with transferrin saturation >or= 45%. Twenty two patients (18%) presented with these two pathological values. Four C282Y/H63D compound heterozygous, one H63D/H63D homozygous, and eight simplex heterozygous (6 H63D and 2 C282Y) genotypes were found. Patients with serum ferritin >or= 300 microg/l were predominantly male (89%), older (57 year) and plethoric (BMI: 26.4) corresponding mainly to dysmetabolic hyperferritinemia. None of these 120 patients consulting for unexplained chronic fatigue was found with hereditary haemochromatosis. Therefore observed prevalence is 0, with upper limit of 95% confidence interval at 2.5%. But the high prevalence (38%) of serum ferritin >or= 300 microg/l must be emphasized, corresponding usually to dysmetabolic hyperferritinemia.

Haemochromatosis HFE gene polymorphisms as potential modifiers of hereditary nonpolyposis colorectal cancer risk and onset age.

PubMed

Shi, Zumin; Johnstone, Daniel; Talseth-Palmer, Bente A; Evans, Tiffany-Jane; Spigelman, Allan D; Groombridge, Claire; Milward, Elizabeth A; Olynyk, John K; Suchy, Janina; Kurzawski, Grzegorz; Lubinski, Jan; Scott, Rodney J

2009-07-01

Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by germline mutations in DNA mismatch repair genes; however, variation in disease expression suggests that there are potential modifying factors. Polymorphisms of the HFE gene, which cause the iron overload disorder hereditary haemochromatosis, have been proposed as potential risk factors for the development of colorectal cancer (CRC). To understand the relationship between HNPCC disease phenotype and polymorphisms of the HFE gene, a total of 362 individuals from Australia and Poland with confirmed causative MMR gene mutations were genotyped for the HFE C282Y and H63D polymorphisms. A significantly increased risk of developing CRC was observed for H63D homozygotes when compared with combined wild-type homozygotes and heterozygotes (hazard ratio = 2.93, p = 0.007). Evidence for earlier CRC onset was also observed in H63D homozygotes with a median age of onset 6 years earlier than wild type or heterozygous participants (44 vs. 50 years of age). This effect was significant by all tests used (log-rank test p = 0.026, Wilcoxon p = 0.044, Tarone-Ware p = 0.035). No association was identified for heterozygosity of either polymorphism and limitations on power-prevented investigation of C282Y homozygosity or compound C282Y/H63D heterozygosity. In the Australian sample only, women had a significantly reduced risk of developing CRC when compared with men (hazard ratio = 0.58, p = 0.012) independent of HFE genotype for either single nucleotide polymorphisms. In conclusion, homozygosity for the HFE H63D polymorphism seems to be a genetic modifier of disease expression in HNPCC. Understanding the mechanisms by which HFE interrelates with colorectal malignancies could lead to reduction of disease risk in HNPCC.

Association between the HFE C282Y, H63D Polymorphisms and the Risks of Non-Alcoholic Fatty Liver Disease, Liver Cirrhosis and Hepatocellular Carcinoma: An Updated Systematic Review and Meta-Analysis of 5,758 Cases and 14,741 Controls

PubMed Central

Yin, Wei-Li; Wang, Feng-Mei; Han, Tao

2016-01-01

Background Conflicting results have been obtained for the association between two common polymorphisms (C282Y, H63D) of human HFE (hereditary hemochromatosis) gene and the risks of the liver diseases, including non-alcoholic fatty liver disease (NAFLD), liver cirrhosis and hepatocellular carcinoma (HCC). Methods An updated systematic review and meta-analysis was conducted to evaluate the potential role of HFE polymorphisms in the susceptibility to NAFLD, liver cirrhosis and HCC. After retrieving articles from online databases, eligible studies were enrolled according to the selection criteria. Stata/SE 12.0 software was utilized to perform the statistical analysis. Results In total, 43 articles with 5,758 cases and 14,741 controls were selected. Compared with the control group, a significantly increased risk of NAFLD was observed for the C282Y polymorphism in the Caucasian population under all genetic models and for the H63D polymorphism under the allele, heterozygote and dominant models (all OR>1, Passociation<0.05). However, no significant difference between liver cirrhosis cases and the control group was observed for HFE C282Y and H63D (all Passociation>0.05). In addition, we found that HFE C282Y was statistically associated with increased HCC susceptibility in the overall population, while H63D increased the odds of developing non-cirrhotic HCC in the African population (all OR>1, Passociation<0.05). Moreover, a positive association between compound heterozygosity for C282Y/H63D and the risk of NAFLD and HCC, but not liver cirrhosis, was observed. Conclusion Our meta-analysis provides evidence that the HFE C282Y and H63D polymorphisms confer increased genetic susceptibility to NAFLD and HCC but not liver cirrhosis. Additional well-powered studies are required to confirm our conclusion. PMID:27657935

Iron overload and HFE gene mutations in Czech patients with chronic liver diseases.

PubMed

Dostalikova-Cimburova, Marketa; Kratka, Karolina; Stransky, Jaroslav; Putova, Ivana; Cieslarova, Blanka; Horak, Jiri

2012-01-01

The aim of the study was to identify the prevalence of HFE gene mutations in Czech patients with chronic liver diseases and the influence of the mutations on iron status. The presence of HFE gene mutations (C282Y, H63D, and S65C) analyzed by the PCR-RFLP method, presence of cirrhosis, and serum iron indices were compared among 454 patients with different chronic liver diseases (51 with chronic hepatitis B, 122 with chronic hepatitis C, 218 with alcoholic liver disease, and 63 patients with hemochromatosis). Chronic liver diseases patients other than hemochromatics did not have an increased frequency of HFE gene mutations compared to controls. Although 33.3% of patients with hepatitis B, 43% of patients with hepatitis C, and 73.2% of patients with alcoholic liver disease had elevated transferrin saturation or serum ferritin levels, the presence of HFE gene mutations was not significantly associated with iron overload in these patients. Additionally, patients with cirrhosis did not have frequencies of HFE mutations different from those without cirrhosis. This study emphasizes the importance, not only of C282Y, but also of the H63D homozygous genetic constellation in Czech hemochromatosis patients. Our findings show that increased iron indices are common in chronic liver diseases but {\\it HFE} mutations do not play an important role in the pathogenesis of chronic hepatitis B, chronic hepatitis C, and alcoholic liver disease.

The association between the C282Y and H63D polymorphisms of HFE gene and the risk of Parkinson's disease: A meta-analysis.

PubMed

Xia, Jianjian; Xu, Huamin; Jiang, Hong; Xie, Junxia

2015-05-19

Impaired brain iron homeostasis has been considered as an important mechanism in Parkinson's diseases (PD). There are indications that C282Y and H63D polymorphisms of HFE genes involved in iron metabolism might contribute to the pathogenesis of PD in some cases. However, the investigation of the relationship between PD and the two polymorphisms had produced contradictory results. We performed a meta-analysis to assess the C282Y and H63D polymorphisms of HFE in PD susceptibility. PubMed, EMBASE and Web of Science were systematically searched to identify relevant researches. The strict selection criteria and exclusion standard were applied. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations. A fixed-effect or random-effect model was selected, depending on the results of the heterogeneity test. Fifteen studies were included in the meta-analysis (eight studies with 1631 cases and 4548 controls for C282Y; seven studies with 1192 cases and 4065 controls for H63D). For the C282Y polymorphism, significant associations were observed in the Recessive model (YY vs CY+CC: OR=0.22, 95% CI=0.09-0.57, P=0.002). This indicated that the C282Y polymorphism in HFE might be a potential protective factor for PD. However, no significant associations were found for any genetic model for the H63D polymorphism, suggesting that the H63D polymorphism might not be associated with PD. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Reverse cascade screening of newborns for hereditary haemochromatosis: a model for other late onset diseases?

PubMed

Cadet, E; Capron, D; Gallet, M; Omanga-Léké, M-L; Boutignon, H; Julier, C; Robson, K J H; Rochette, J

2005-05-01

Genetic testing can determine those at risk for hereditary haemochromatosis (HH) caused by HFE mutations before the onset of symptoms. However, there is no optimum screening strategy, mainly owing to the variable penetrance in those who are homozygous for the HFE Cys282Tyr (C282Y) mutation. The objective of this study was to identify the majority of individuals at serious risk of developing HFE haemochromatosis before they developed life threatening complications. We first estimated the therapeutic penetrance of the C282Y mutation in people living in la Somme, France, using genetic, demographic, biochemical, and follow up data. We examined the benefits of neonatal screening on the basis of increased risk to relatives of newborns carrying one or two copies of the C282Y mutation. Between 1999 and 2002, we screened 7038 newborns from two maternity hospitals in the north of France for the C282Y and His63Asp (H63D) mutations in the HFE gene, using bloodspots collected on Guthrie cards. Family studies and genetic counselling were undertaken, based on the results of the baby's genotype. In la Somme, we found that 24% of the adults homozygous for the C282Y mutation required at least 5 g iron to be removed to restore normal iron parameters (that is, the therapeutic penetrance). In the reverse cascade screening study, we identified 19 C282Y homozygotes (1/370), 491 heterozygotes (1/14) and 166 compound heterozygotes (1/42) in 7038 newborns tested. The reverse cascade screening strategy resulted in 80 adults being screened for both mutations. We identified 10 previously unknown C282Y homozygotes of whom six (four men and two women) required venesection. Acceptance of neonatal screening was high; parents understood the risks of having HH and the benefits of early detection, but a number of parents were reluctant to take the test themselves. Neonatal screening for HH is straightforward. Reverse cascade screening increased the efficiency of detecting affected adults with undiagnosed haemochromatosis. This strategy allows almost complete coverage for HH and could be a model for efficient screening for other late onset genetic diseases.

Metabolic alterations, HFE gene mutations and atherogenic lipoprotein modifications in patients with primary iron overload.

PubMed

Meroño, Tomás; Brites, Fernando; Dauteuille, Carolane; Lhomme, Marie; Menafra, Martín; Arteaga, Alejandra; Castro, Marcelo; Saez, María Soledad; Ballerga, Esteban González; Sorroche, Patricia; Rey, Jorge; Lesnik, Philippe; Sordá, Juan Andrés; Chapman, M John; Kontush, Anatol; Daruich, Jorge

2015-05-01

Iron overload (IO) has been associated with glucose metabolism alterations and increased risk of cardiovascular disease (CVD). Primary IO is associated with mutations in the HFE gene. To which extent HFE gene mutations and metabolic alterations contribute to the presence of atherogenic lipoprotein modifications in primary IO remains undetermined. The present study aimed to assess small, dense low-density lipoprotein (LDL) levels, chemical composition of LDL and high-density lipoprotein (HDL) particles, and HDL functionality in IO patients. Eighteen male patients with primary IO and 16 sex- and age-matched controls were recruited. HFE mutations (C282Y, H63D and S65C), measures of insulin sensitivity and secretion (calculated from the oral glucose tolerance test), chemical composition and distribution profile of LDL and HDL subfractions (isolated by gradient density ultracentrifugation) and HDL functionality (as cholesterol efflux and antioxidative activity) were studied. IO patients compared with controls exhibited insulin resistance (HOMA-IR (homoeostasis model assessment-estimated insulin resistance): +93%, P< 0.001). Metabolic profiles differed across HFE genotypes. C282Y homozygotes (n=7) presented a reduced β-cell function and insulin secretion compared with non-C282Y patients (n=11) (-58% and -73%, respectively, P< 0.05). In addition, C282Y homozygotes featured a predominance of large, buoyant LDL particles (C282Y: 43±5; non-C282Y: 25±8; controls: 32±7%; P< 0.001), whereas non-C282Y patients presented higher amounts of small, dense LDL (C282Y: 23±5; non-C282Y: 39±10; controls: 26±4%; P< 0.01). HDL particles were altered in C282Y homozygotes. However, HDL functionality was conserved. In conclusion, metabolic alterations and HFE gene mutations are involved in the presence of atherogenic lipoprotein modifications in primary IO. To what extent such alterations could account for an increase in CVD risk remains to be determined.

C282Y polymorphism in the HFE gene is associated with risk of breast cancer.

PubMed

Liu, Xiaoyan; Lv, Chunlei; Luan, Xiaorong; Lv, Ming

2013-10-01

The C282Y and H63D polymorphisms in the HFE gene have been implicated in susceptibility of breast cancer, but a number of studies have reported inconclusive results. The aim of this study is to investigate the association between the C282Y and H63D polymorphisms in the HFE gene and breast cancer risk by meta-analysis. We searched PubMed and Embase databases, covering all related studies until March 2, 2013. Statistical analysis was performed using STATA 10.0. A total of 7 studies including 1,720 cases and 18,296 controls for HFE C282Y polymorphism and 5 studies including 942 cases and 1,571 controls for HFE H63D polymorphism were included in the meta-analysis. The results showed that HFE C282Y polymorphism was significantly associated with increased risk of breast cancer under homozygotes vs. wild-type model (OR = 2.06, 95%CI = 1.19-3.58) and recessive model (OR = 1.98, 95%CI = 1.14-3.44) but not under heterozygotes vs. wild-type model (OR = 0.97, 95%CI = 0.70-1.35), dominant model (OR = 1.00, 95%CI = 0.72-1.40) and multiplicative model (OR = 1.04, 95%CI = 0.76-1.42). However, we did not find any association between HFE H63D polymorphism and breast cancer risk under all genetic models. This current meta-analysis suggested that C282Y polymorphism rather than H63D might be associated with increased risk of breast cancer.

The hepcidin gene promoter nc.-1010C > T; -582A > G haplotype modulates serum ferritin in individuals carrying the common H63D mutation in HFE gene.

PubMed

Silva, Bruno; Pita, Lina; Gomes, Susana; Gonçalves, João; Faustino, Paula

2014-12-01

Hereditary hemochromatosis is an autosomal recessive disorder characterized by severe iron overload. It is usually associated with homozygosity for the HFE gene mutation c.845G > A; p.C282Y. However, in some cases, another HFE mutation (c.187C > G; p.H63D) seems to be associated with the disease. Its penetrance is very low, suggesting the possibility of other iron genetic modulators being involved. In this work, we have screened for HAMP promoter polymorphisms in 409 individuals presenting normal or increased serum ferritin levels together with normal or H63D-mutated HFE genotypes. Our results show that the hepcidin gene promoter TG haplotype, originated by linkage of the nc.-1010C > T and nc.-582A > G polymorphisms, is more frequent in the HFE_H63D individuals presenting serum ferritin levels higher than 300 μg/L than in those presenting the HFE_H63D mutation but with normal serum ferritin levels or in the normal control group.Moreover, it was observed that the TG haplotype was associated to increased serum ferritin levels in the overall pool of HFE_H63D individuals. Thus, our data suggest that screening for these polymorphisms could be of interest in order to explain the phenotype. However, this genetic condition seems to have no clinical significance.

Non-HFE iron overload as a surrogate marker of disease severity in patients of liver cirrhosis.

PubMed

Noor, Mohd Talha; Tiwari, Manish; Kumar, Ravindra

2016-01-01

Decompensated liver cirrhosis is an important cause of mortality worldwide. Various modifiable and non-modifiable factors are involved in the pathogenesis of cirrhosis and its complications. This study was aimed to evaluate the association of iron overload and disease severity in patients of liver cirrhosis and its association with HFE gene mutation. Forty-nine patients with decompensated liver cirrhosis were recruited. Clinical and laboratory parameters were compared in patients with and without iron overload. C282Y and H63D gene mutation analysis was performed in all patients with iron overload. Iron overload was found in 20 (40.82%) patients. A significant positive correlation of transferrin saturation with Child-Turcotte-Pugh (CTP) score (r = 0.705, p < 0.001) and model for end-stage liver disease (MELD) score (r = 0.668, p < 0.001) was found. Transferrin saturation was also independently associated with high CTP and MELD score on multivariate analysis. Mortality over 3 months was significantly more common in iron-overloaded patients (p = 0.028). C282Y homozygosity or C282Y/H63D compound heterozygosity was not found in any of the patients with iron overload. Iron overload was significantly associated with disease severity and reduced survival in patients of decompensated liver cirrhosis.

HFE C282Y/H63D compound heterozygotes are at low risk of hemochromatosis-related morbidity.

PubMed

Gurrin, Lyle C; Bertalli, Nadine A; Dalton, Gregory W; Osborne, Nicholas J; Constantine, Clare C; McLaren, Christine E; English, Dallas R; Gertig, Dorota M; Delatycki, Martin B; Nicoll, Amanda J; Southey, Melissa C; Hopper, John L; Giles, Graham G; Anderson, Gregory J; Olynyk, John K; Powell, Lawrie W; Allen, Katrina J

2009-07-01

The risk of hemochromatosis-related morbidity is unknown among HFE compound heterozygotes (C282Y/H63D). We used a prospective population-based cohort study to estimate the prevalence of elevated iron indices and hemochromatosis-related morbidity for compound heterozygotes. In all, 31,192 subjects of northern European descent were genotyped for HFE C282Y and H63D. An HFE-genotype stratified random sample of 1,438 subjects, followed for an average of 12 years to a mean age of 65 years, completed questionnaires and gave blood. Clinical examinations were blinded to HFE genotype. A total of 180 (84 males) clinically examined C282Y/H63D participants were compared with 330 (149 males) controls with neither HFE mutation; 132 (65 males) and 270 (122 males), respectively, had serum iron measures at both timepoints. Mean serum ferritin (SF) and transferrin saturation (TS) were significantly greater for male and female compound heterozygotes than for wild-types at baseline and follow-up (all P < 0.02) except for females who were premenopausal at baseline, where SF was similar in both genotype groups. For subjects with serum measures from both baseline and follow-up, mean SF and TS levels did not change significantly for men or for postmenopausal women, but for premenopausal women SF levels increased from 43 to 109 microg/L for compound heterozygotes and from 35 to 64 microg/L for wild-types (both P < 0.001). Male and female compound heterozygotes had a similar prevalence of hemochromatosis-related morbidity to wild-types. One of 82 males and zero of 95 females had documented iron overload-related disease. For male compound heterozygotes, mean iron indices do not change during middle age but for female compound heterozygotes menopause results in increased mean SF. Although compound heterozygotes might maintain elevated iron indices during middle age, documented iron overload-related disease is rare.

Analysis of single nucleotide variants of HFE gene and association to survival in The Cancer Genome Atlas GBM data.

PubMed

Lee, Sang Y; Zhu, Junjia; Salzberg, Anna C; Zhang, Bo; Liu, Dajiang J; Muscat, Joshua E; Langan, Sara T; Connor, James R

2017-01-01

Human hemochromatosis protein (HFE) is involved in iron metabolism. Two major HFE polymorphisms, H63D and C282Y, have been associated with an increased risk of cancers. Previously, we reported decreased gender effects in overall survival based on H63D or C282Y HFE polymorphisms patients with glioblastoma multiforme (GBM). However, the effect of other single nucleotide variation (SNV) in the HFE gene on the cancer development and progression has not been systematically studied. To expand our finding in a larger sample, and to identify other HFE SNV, we analyzed the frequency of somatic SNV in HFE gene and its relationship to survival in GBM patients using The Cancer Genome Atlas (TCGA) GBM (Caucasian only) database. We found 9 SNVs with increased frequency in blood normal of TCGA GBM patients compared to the 1000Genome. Among 9 SNVs, 7 SNVs were located in the intron and 2 SNVs (i.e., H63D, C282Y) in the exon of HFE gene. The statistical analysis demonstrated that blood normal samples of TCGA GBM have more H63D (p = 0.0002, 95% Confidence interval (CI): 0.2119-0.3223) or C282Y (p = 0.0129, 95% CI: 0.0474-0.1159) HFE polymorphisms than 1000Genome. The Kaplan-Meier survival curve for the 264 GBM samples revealed no difference between wild type (WT) HFE and H63D, and WT HFE and C282Y GBM patients. In addition, there was no difference in the survival of male/female GBM patients based on HFE genotype. There was no correlation between HFE expression and survival. In conclusion, the current results suggest that somatic HFE polymorphisms do not impact GBM patients' survival in the TCGA data set of GBM.

Precipitating factors of porphyria cutanea tarda in Brazil with emphasis on hemochromatosis gene (HFE) mutations. Study of 60 patients.

PubMed

Vieira, Fatima Mendonça Jorge; Nakhle, Maria Cristina; Abrantes-Lemos, Clarice Pires; Cançado, Eduardo Luiz Rachid; Reis, Vitor Manoel Silva dos

2013-01-01

Porphyria cutanea tarda is the most common form of porphyria, characterized by the decreased activity of the uroporphyrinogen decarboxylase enzyme. Several reports associated HFE gene mutations of hereditary hemochromatosis with porphyria cutanea tarda worldwide, although up to date only one study has been conducted in Brazil. Investigation of porphyria cutanea tarda association with C282Y and H63D mutations in the HFE gene. Identification of precipitating factors (hepatitis C, HIV, alcoholism and estrogen) and their link with HFE mutations. An ambispective study of 60 patients with PCT was conducted during the period from 2003 to 2012. Serological tests for hepatitis C and HIV were performed and histories of alcohol abuse and estrogen intake were investigated. HFE mutations were identified with real-time PCR. Porphyria cutanea tarda predominated in males and alcohol abuse was the main precipitating factor. Estrogen intake was the sole precipitating factor present in 25% of female patients. Hepatitis C was present in 41.7%. All HIV-positive patients (15.3%) had a history of alcohol abuse. Allele frequency for HFE mutations, i.e., C282Y (p = 0.0001) and H63D (p = 0.0004), were significantly higher in porphyria cutanea tarda patients, compared to control group. HFE mutations had no association with the other precipitating factors. Alcohol abuse, hepatitis C and estrogen intake are prevalent precipitating factors in our porphyria cutanea tarda population; however, hemochromatosis in itself can also contribute to the outbreak of porphyria cutanea tarda, which makes the research for HFE mutations necessary in these patients.

HFE gene variants affect iron in the brain.

PubMed

Nandar, Wint; Connor, James R

2011-04-01

Iron accumulation in the brain and increased oxidative stress are consistent observations in many neurodegenerative diseases. Thus, we have begun examination into gene mutations or allelic variants that could be associated with loss of iron homeostasis. One of the mechanisms leading to iron overload is a mutation in the HFE gene, which is involved in iron metabolism. The 2 most common HFE gene variants are C282Y (1.9%) and H63D (8.9%). The C282Y HFE variant is more commonly associated with hereditary hemochromatosis, which is an autosomal recessive disorder, characterized by iron overload in a number of systemic organs. The H63D HFE variant appears less frequently associated with hemochromatosis, but its role in the neurodegenerative diseases has received more attention. At the cellular level, the HFE mutant protein resulting from the H63D HFE gene variant is associated with iron dyshomeostasis, increased oxidative stress, glutamate release, tau phosphorylation, and alteration in inflammatory response, each of which is under investigation as a contributing factor to neurodegenerative diseases. Therefore, the HFE gene variants are proposed to be genetic modifiers or a risk factor for neurodegenerative diseases by establishing an enabling milieu for pathogenic agents. This review will discuss the current knowledge of the association of the HFE gene variants with neurodegenerative diseases: amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and ischemic stroke. Importantly, the data herein also begin to dispel the long-held view that the brain is protected from iron accumulation associated with the HFE mutations.

Serum ferritin is a biomarker for liver mortality in the Hemochromatosis and Iron Overload Screening Study.

PubMed

Adams, Paul C; Barton, James C; Guo, Helen; Alter, David; Speechley, Mark

2015-01-01

We identified no reports of long-term follow-up of participants in hemochromatosis screening programs. We evaluated causes of death and survival in non-C282Y homozygous Canadian participants in the primary care-based hemochromatosis and iron overload screening (HEIRS) study. Initial screening (IS) included transferrin saturation (TS), serum ferritin (SF), HFE genotyping (C282Y, H63D), and health questionnaire responses. By definition, participants without C282Y or H63D had HFE wt/wt. We linked 20,306 Canadian participants to the Ontario Death Registry for dates and causes of death 9 y after IS. We computed Cox proportional hazards to identify factors with increased death risks and Kaplan-Meier curves to estimate survival of non-C282Y homozygous participants with SF ≤ 1,000 μg/L and > 1,000 μg/dL. There were 19,052 evaluable participants (IS mean age 49 y; 60% women; 93 C282Y homozygotes). There were 988 deaths. Significantly increased hazard ratios for all-cause mortality were positively associated with TS, SF, men, and C282Y homozygosity, and liver disease, diabetes, and heart failure reports. Non-C282Y homozygous participants with SF > 1,000 μg/L had lower survival than those with SF ≤ 1,000 μg/L (p < 0.0001). Nine years after initial screening, non-C282Y homozygous participants and SF > 1,000 μg/L was associated with decreased survival.

Precipitating factors of porphyria cutanea tarda in Brazil with emphasis on hemochromatosis gene (HFE) mutations. Study of 60 patients*

PubMed Central

Vieira, Fatima Mendonça Jorge; Nakhle, Maria Cristina; Abrantes-Lemos, Clarice Pires; Cançado, Eduardo Luiz Rachid; dos Reis, Vitor Manoel Silva

2013-01-01

BACKGROUND Porphyria cutanea tarda is the most common form of porphyria, characterized by the decreased activity of the uroporphyrinogen decarboxylase enzyme. Several reports associated HFE gene mutations of hereditary hemochromatosis with porphyria cutanea tarda worldwide, although up to date only one study has been conducted in Brazil. OBJECTIVES Investigation of porphyria cutanea tarda association with C282Y and H63D mutations in the HFE gene. Identification of precipitating factors (hepatitis C, HIV, alcoholism and estrogen) and their link with HFE mutations. METHODS An ambispective study of 60 patients with PCT was conducted during the period from 2003 to 2012. Serological tests for hepatitis C and HIV were performed and histories of alcohol abuse and estrogen intake were investigated. HFE mutations were identified with real-time PCR. RESULTS Porphyria cutanea tarda predominated in males and alcohol abuse was the main precipitating factor. Estrogen intake was the sole precipitating factor present in 25% of female patients. Hepatitis C was present in 41.7%. All HIV-positive patients (15.3%) had a history of alcohol abuse. Allele frequency for HFE mutations, i.e., C282Y (p = 0.0001) and H63D (p = 0.0004), were significantly higher in porphyria cutanea tarda patients, compared to control group. HFE mutations had no association with the other precipitating factors. CONCLUSIONS Alcohol abuse, hepatitis C and estrogen intake are prevalent precipitating factors in our porphyria cutanea tarda population; however, hemochromatosis in itself can also contribute to the outbreak of porphyria cutanea tarda, which makes the research for HFE mutations necessary in these patients PMID:24068123

Analysis of single nucleotide variants of HFE gene and association to survival in The Cancer Genome Atlas GBM data

PubMed Central

Zhang, Bo; Liu, Dajiang J.; Muscat, Joshua E.; Langan, Sara T.; Connor, James R.

2017-01-01

Human hemochromatosis protein (HFE) is involved in iron metabolism. Two major HFE polymorphisms, H63D and C282Y, have been associated with an increased risk of cancers. Previously, we reported decreased gender effects in overall survival based on H63D or C282Y HFE polymorphisms patients with glioblastoma multiforme (GBM). However, the effect of other single nucleotide variation (SNV) in the HFE gene on the cancer development and progression has not been systematically studied. To expand our finding in a larger sample, and to identify other HFE SNV, we analyzed the frequency of somatic SNV in HFE gene and its relationship to survival in GBM patients using The Cancer Genome Atlas (TCGA) GBM (Caucasian only) database. We found 9 SNVs with increased frequency in blood normal of TCGA GBM patients compared to the 1000Genome. Among 9 SNVs, 7 SNVs were located in the intron and 2 SNVs (i.e., H63D, C282Y) in the exon of HFE gene. The statistical analysis demonstrated that blood normal samples of TCGA GBM have more H63D (p = 0.0002, 95% Confidence interval (CI): 0.2119–0.3223) or C282Y (p = 0.0129, 95% CI: 0.0474–0.1159) HFE polymorphisms than 1000Genome. The Kaplan-Meier survival curve for the 264 GBM samples revealed no difference between wild type (WT) HFE and H63D, and WT HFE and C282Y GBM patients. In addition, there was no difference in the survival of male/female GBM patients based on HFE genotype. There was no correlation between HFE expression and survival. In conclusion, the current results suggest that somatic HFE polymorphisms do not impact GBM patients’ survival in the TCGA data set of GBM. PMID:28358914

Highly sensitivity adhesion molecules detection in hereditary haemochromatosis patients reveals altered expression.

PubMed

Norris, S; White, M; Mankan, A K; Lawless, M W

2010-04-01

Several abnormalities in the immune status of patients with hereditary haemochromatosis (HH) have been reported, suggesting an imbalance in their immune function. This may include persistent production of, or exposure to, altered immune signalling contributing to the pathogenesis of this disorder. Adhesion molecules L-, E- and P-Selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) are some of the major regulators of the immune processes and altered levels of these proteins have been found in pathological states including cardiovascular diseases, arthritis and liver cancer. The aim of this study was to assess L-, E- and P-Selectin, ICAM-1 and VCAM-1 expression in patients with HH and correlate these results with HFE mutation status and iron indexes. A total of 139 subjects were diagnosed with HH (C282Y homozygotes = 87, C282Y/H63D = 26 heterozygotes, H63D homozygotes = 26), 27 healthy control subjects with no HFE mutation (N/N), 18 normal subjects heterozygous for the H63D mutation served as age-sex-matched controls. We observed a significant decrease in L-selectin (P = 0.0002) and increased E-selectin and ICAM-1 (P = 0.0006 and P = 0.0059) expression in HH patients compared with healthy controls. This study observes for the first time that an altered adhesion molecules profile occurs in patients with HH that is associated with specific HFE genetic component for iron overload, suggesting that differential expression of adhesion molecules may play a role in the pathogenesis of HH.

Hemochromatosis (HFE) gene mutations and response to chloroquine in porphyria cutanea tarda.

PubMed

Stölzel, Ulrich; Köstler, Erich; Schuppan, Detlef; Richter, Matthias; Wollina, Uwe; Doss, Manfred O; Wittekind, Christian; Tannapfel, Andrea

2003-03-01

To examine the role of hemochromatosis (HFE) gene mutations, which are associated with porphyria cutanea tarda (PCT), in the therapeutic response to chloroquine. We retrospectively analyzed a database (Excel version 2001 [Microsoft Excel, Redmond, Wash]; date range of search, 1985-1999) of chloroquine-treated patients with PCT on whether HFE mutations (C282Y and H63D) might have influenced the clinical response, urinary porphyrin excretion, liver enzyme activities, and serum iron markers. Serum samples and corresponding complete sets of data before and after therapy were available in 62 of 207 patients with PCT who were treated exclusively with chloroquine. Academic teaching hospital. For treatment, low-dose chloroquine diphosphate, 125 to 250 mg twice weekly, was used during a median time of 16 months (range, 12-26 months). Of the 62 German patients with PCT, 37 (60%) carries HFE mutations. Chloroquine therapy was accompanied by clinical remission and reduced urinary porphyrin excretion (P<.001) in the 24 patients (39%) with HFE wild type as well as in 35 HFE heterozygous patients with PCT (56%). Decreases of serum iron markers following chloroquine therapy were limited to patients with PCT and HFE wild type. All patients homozygous for the C282Y mutation (3 [5%] of 62) had high serum iron, ferritin, and transferrin saturation and failed to respond to chloroquine treatment. The therapeutic response to chloroquine was not compromised by C282Y heterozygosity and compound heterozygosity of HFE mutations. Because HFE C282Y homozygotes (+/+) did not respond to chloroquine and a decrease in serum iron concentration was limited to patients with PCT and HFE wild type, phlebotomy should be first-line therapy in patients with PCT and HFE mutations.

Influence of diet, menstruation and genetic factors on iron status: a cross-sectional study in Spanish women of childbearing age.

PubMed

Blanco-Rojo, Ruth; Toxqui, Laura; López-Parra, Ana M; Baeza-Richer, Carlos; Pérez-Granados, Ana M; Arroyo-Pardo, Eduardo; Vaquero, M Pilar

2014-03-06

The aim of this study was to investigate the combined influence of diet, menstruation and genetic factors on iron status in Spanish menstruating women (n = 142). Dietary intake was assessed by a 72-h detailed dietary report and menstrual blood loss by a questionnaire, to determine a Menstrual Blood Loss Coefficient (MBLC). Five selected SNPs were genotyped: rs3811647, rs1799852 (Tf gene); rs1375515 (CACNA2D3 gene); and rs1800562 and rs1799945 (HFE gene, mutations C282Y and H63D, respectively). Iron biomarkers were determined and cluster analysis was performed. Differences among clusters in dietary intake, menstrual blood loss parameters and genotype frequencies distribution were studied. A categorical regression was performed to identify factors associated with cluster belonging. Three clusters were identified: women with poor iron status close to developing iron deficiency anemia (Cluster 1, n = 26); women with mild iron deficiency (Cluster 2, n = 59) and women with normal iron status (Cluster 3, n = 57). Three independent factors, red meat consumption, MBLC and mutation C282Y, were included in the model that better explained cluster belonging (R2 = 0.142, p < 0.001). In conclusion, the combination of high red meat consumption, low menstrual blood loss and the HFE C282Y mutation may protect from iron deficiency in women of childbearing age. These findings could be useful to implement adequate strategies to prevent iron deficiency anemia.

A European allele map of the C282Y mutation of hemochromatosis: Celtic versus Viking origin of the mutation?

PubMed

Lucotte, Gérard; Dieterlen, Florent

2003-01-01

The aim of this new meta-analysis (to the end of 2002) is to compile the Y allele frequencies of the C282Y mutation of hereditary hemochromatosis (HFE gene) for 63 European populations, representing a total of 10,708 unrelated people concerning control samples. A new allele map of C282Y frequencies in Europe was constructed. The highest European frequencies are observed in the Celtic populations in Ireland, in the United Kingdom, and in France, but elevated frequencies are also observed in Scandinavia.

The Effect of the Hemochromatosis (HFE) Genotype on Lead Load and Iron Metabolism among Lead Smelter Workers

PubMed Central

Fan, Guangqin; Du, Guihua; Li, Huijun; Lin, Fen; Sun, Ziyong; Yang, Wei; Feng, Chang; Zhu, Gaochun; Li, Yanshu; Chen, Ying; Jiao, Huan; Zhou, Fankun

2014-01-01

Background Both an excess of toxic lead (Pb) and an essential iron disorder have been implicated in many diseases and public health problems. Iron metabolism genes, such as the hemochromatosis (HFE) gene, have been reported to be modifiers for lead absorption and storage. However, the HFE gene studies among the Asian population with occupationally high lead exposure are lacking. Objectives To explore the modifying effects of the HFE genotype (wild-type, H63D variant and C282Y variant) on the Pb load and iron metabolism among Asian Pb-workers with high occupational exposure. Methods Seven hundred and seventy-one employees from a lead smelter manufacturing company were tested to determine their Pb intoxication parameters, iron metabolic indexes and identify the HFE genotype. Descriptive and multivariate analyses were conducted. Results Forty-five H63D variant carriers and no C282Y variant carrier were found among the 771 subjects. Compared with subjects with the wild-type genotype, H63D variant carriers had higher blood lead levels, even after controlling for factors such as age, sex, marriage, education, smoking and lead exposure levels. Multivariate analyses also showed that the H63D genotype modifies the associations between the blood lead levels and the body iron burden/transferrin. Conclusions No C282Y variant was found in this Asian population. The H63D genotype modified the association between the lead and iron metabolism such that increased blood lead is associated with a higher body iron content or a lower transferrin in the H63D variant. It is indicated that H63D variant carriers may be a potentially highly vulnerable sub-population if they are exposed to high lead levels occupationally. PMID:24988074

The effect of the hemochromatosis (HFE) genotype on lead load and iron metabolism among lead smelter workers.

PubMed

Fan, Guangqin; Du, Guihua; Li, Huijun; Lin, Fen; Sun, Ziyong; Yang, Wei; Feng, Chang; Zhu, Gaochun; Li, Yanshu; Chen, Ying; Jiao, Huan; Zhou, Fankun

2014-01-01

Both an excess of toxic lead (Pb) and an essential iron disorder have been implicated in many diseases and public health problems. Iron metabolism genes, such as the hemochromatosis (HFE) gene, have been reported to be modifiers for lead absorption and storage. However, the HFE gene studies among the Asian population with occupationally high lead exposure are lacking. To explore the modifying effects of the HFE genotype (wild-type, H63D variant and C282Y variant) on the Pb load and iron metabolism among Asian Pb-workers with high occupational exposure. Seven hundred and seventy-one employees from a lead smelter manufacturing company were tested to determine their Pb intoxication parameters, iron metabolic indexes and identify the HFE genotype. Descriptive and multivariate analyses were conducted. Forty-five H63D variant carriers and no C282Y variant carrier were found among the 771 subjects. Compared with subjects with the wild-type genotype, H63D variant carriers had higher blood lead levels, even after controlling for factors such as age, sex, marriage, education, smoking and lead exposure levels. Multivariate analyses also showed that the H63D genotype modifies the associations between the blood lead levels and the body iron burden/transferrin. No C282Y variant was found in this Asian population. The H63D genotype modified the association between the lead and iron metabolism such that increased blood lead is associated with a higher body iron content or a lower transferrin in the H63D variant. It is indicated that H63D variant carriers may be a potentially highly vulnerable sub-population if they are exposed to high lead levels occupationally.

The Study of HFE Genotypes and Its Expression Effect on Iron Status of Iranian Haemochromatosis, Iron Deficiency Anemia Patients, Iron-Taker and Non Iron-Taker Controls.

PubMed

Beiranvand, Elham; Abediankenari, Saeid; Rostamian, Mosayeb; Beiranvand, Behnoush; Naazeri, Saeed

2015-01-01

The role of HFE gene mutations or its expression in regulation of iron metabolism of hereditary haemochromatosis (HH) patients is remained controversial. Therefore here the correlation between two common HFE genotype (p.C282Y, p.H63D) and HFE gene expression with iron status in HH, iron deficiency anemia (IDA) and healthy Iranian participants was studied. For this purpose genotype determination was done by polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP). Real-Time PCR was applied for evaluation of HFE gene expression. Biochemical parameters and iron consumption were also assessed. Homozygote p.H63D mutation was seen in all HH patients and p.C282Y was not observed in any member of the population. A significant correlation was observed between serum ferritin (SF) level and gender or age of HH patients. p.H63D homozygote was seen to be able to significantly increase SF and transferrin saturation (TS) level without affecting on liver function. Our results also showed that iron consumption affects on TS level increasing. HFE gene expression level of IDA patients was significantly higher than other groups. Also the HFE gene expression was negatively correlated with TS. Finally, the main result of our study showed that loss of HFE function in HH is not derived from its gene expression inhibition and much higher HFE gene expression might lead to IDA. However we propose repeating of the study for more approval of our finding.

Differences in hepatic phenotype between hemochromatosis patients with HFE C282Y homozygosity and other HFE genotypes.

PubMed

Cheng, Raymond; Barton, James C; Morrison, Elizabeth D; Phatak, Pradyumna D; Krawitt, Edward L; Gordon, Stuart C; Kowdley, Kris V

2009-07-01

There are limited data comparing hepatic phenotype among hemochromatosis patients with different HFE genotypes. The goal of this study was to compare hepatic histopathologic features and hepatic iron concentration (HIC) among patients with phenotypic hemochromatosis and different HFE genotypes. We studied 182 US patients with phenotypic hemochromatosis. Degree of hepatic fibrosis, pattern of iron deposition, presence of steatosis or necroinflammation, and HIC were compared among different HFE genotypes. C282Y/H63D compound heterozygotes and patients with HFE genotypes other than C282Y/C282Y were more likely to have stainable Kupffer cell iron (31.1% vs. 9.5%; P=0.02), portal or lobular inflammation (28.9% vs. 15.6%; P=0.03), and steatosis (33.3% vs. 10.2%; P<0.01) on liver biopsy than C282Y homozygotes. Mean log10 HIC (P<0.05) and log10 ferritin (P<0.05) were higher among C282Y homozygotes than in patients with other HFE genotypes. In a logistic regression analysis using age, sex, HFE genotype, log10 ferritin, and log10 HIC as independent variables, log10 serum ferritin (P=0.0008), male sex (P=0.0086), and log10 HIC (P=0.047), but not HFE genotype (P=0.0554) were independently associated with presence or absence of advanced hepatic fibrosis. C282Y/H63D compound heterozygotes and other non-C282Y homozygotes which express the hepatic hemochromatosis phenotype frequently have evidence of steatosis or chronic hepatitis and lower body iron stores than C282Y homozygotes. These data suggest that presence of concomitant liver disease may explain expression of the hemochromatosis phenotype among non-C282Y homozygotes. Increased age, HIC, and ferritin are associated with advanced hepatic fibrosis, regardless of HFE genotype.

Comparison of oxidative stress and the frequency of polymorphisms in the HFE gene between hemoglobin S trait blood donors and sickle cell disease patients.

PubMed

Viana-Baracioli, L M S; Tukamoto Junior, N C; Ricci Junior, O; Mattos, L C; Ângulo, I L; Bonini-Domingos, C R

2011-12-08

It is well documented that Hb S and iron affect blood cells, and trigger oxidative processes and generation of free radicals with potential for lipid peroxidation. We evaluated the frequency of polymorphisms in the HFE gene in Hb AS blood donors and how these polymorphisms influenced lipid peroxidation and antioxidant capacity. Blood samples were collected from 211 Hb AS blood donors, 119 Hb AA blood donors as a control group, and 28 sickle cell disease patients (Hb SS). The H63D allele was found at a frequency of 10.5% in the Hb AS samples, and the C282Y allele frequency was 0.7%. In the control group, the frequencies of the H63D and C282Y alleles were 13.4 and 2.1%, respectively. In the sickle-cell disease patients, the H63D and the C282Y allele frequencies were 10.7 and 3.5%, respectively. The frequencies of the C282Y and H63D polymorphisms in Hb AS blood donors are similar to those reported for the Brazilian population. Serum malondialdehyde values, indicative of lipid peroxidation, were highest in sickle cell patients, independent of the polymorphisms in the HFE gene, with significant differences, showing the influence of Hb S allele in the levels of lipid peroxidation. However, the trolox equivalent antioxidant capacity average levels, indicative of the antioxidant capacity, were reduced with significant differences, indicating that in spite of a lipid peroxidation raise, this is not followed by the increased of the antioxidant capacity, leading to oxidative stress.

Clinical manifestations of hemochromatosis in HFE C282Y homozygotes identified by screening.

PubMed

McLaren, Gordon D; McLaren, Christine E; Adams, Paul C; Barton, James C; Reboussin, David M; Gordeuk, Victor R; Acton, Ronald T; Harris, Emily L; Speechley, Mark R; Sholinsky, Phyliss; Dawkins, Fitzroy W; Snively, Beverly M; Vogt, Thomas M; Eckfeldt, John H

2008-11-01

Patients with hemochromatosis may suffer organ damage from iron overload, often with serious clinical consequences. To assess prevalences of self-reported symptoms and clinical signs and conditions in persons homozygous for the hemochromatosis gene (HFE) mutation (C282Y) identified by screening. Participants were adults 25 years of age or older enrolled in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. C282Y homozygotes (n=282) were compared with control participants without the HFE C282Y or H63D alleles (ie, wild type/wild type; n=364). Previously diagnosed C282Y homozygotes and newly diagnosed homozygotes with elevated serum ferritin levels had higher prevalences of certain symptoms such as chronic fatigue (OR 2.8; 95% CI 1.34 to 5.95, and OR 2.0; 95% CI 1.07 to 3.75, respectively), and had more hyperpigmentation on physical examination (OR 4.7; 95% CI 1.50 to 15.06, and OR 3.7; 95% CI 1.10 to 12.16, respectively) and swelling or tenderness of the second and third metacarpophalangeal joints (OR 4.2; 95% CI 1.37 to 13.03, and OR 3.3; 95% CI 1.17 to 9.49, respectively) than control subjects. Joint stiffness was also more common among newly diagnosed C282Y homozygotes with elevated serum ferritin than among control subjects (OR 2.7; 95% CI 1.38 to 5.30). However, the sex- and age-adjusted prevalences of self-reported symptoms and signs of liver disease, heart disease, diabetes and most other major clinical manifestations of hemochromatosis were similar in C282Y homozygotes and control subjects. Some symptoms and conditions associated with hemochromatosis were more prevalent among C282Y homozygotes identified by screening than among control subjects, but prevalences of most outcomes were similar in C282Y homozygotes and controls in this primary care-based study.

Clinical manifestations of hemochromatosis in HFE C282Y homozygotes identified by screening

PubMed Central

McLaren, Gordon D; McLaren, Christine E; Adams, Paul C; Barton, James C; Reboussin, David M; Gordeuk, Victor R; Acton, Ronald T; Harris, Emily L; Speechley, Mark R; Sholinsky, Phyliss; Dawkins, Fitzroy W; Snively, Beverly M; Vogt, Thomas M; Eckfeldt, John H

2008-01-01

BACKGROUND: Patients with hemochromatosis may suffer organ damage from iron overload, often with serious clinical consequences. OBJECTIVE: To assess prevalences of self-reported symptoms and clinical signs and conditions in persons homozygous for the hemochromatosis gene (HFE) mutation (C282Y) identified by screening. METHODS: Participants were adults 25 years of age or older enrolled in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. C282Y homozygotes (n=282) were compared with control participants without the HFE C282Y or H63D alleles (ie, wild type/wild type; n=364). RESULTS: Previously diagnosed C282Y homozygotes and newly diagnosed homozygotes with elevated serum ferritin levels had higher prevalences of certain symptoms such as chronic fatigue (OR 2.8; 95% CI 1.34 to 5.95, and OR 2.0; 95% CI 1.07 to 3.75, respectively), and had more hyperpigmentation on physical examination (OR 4.7; 95% CI 1.50 to 15.06, and OR 3.7; 95% CI 1.10 to 12.16, respectively) and swelling or tenderness of the second and third metacarpophalangeal joints (OR 4.2; 95% CI 1.37 to 13.03, and OR 3.3; 95% CI 1.17 to 9.49, respectively) than control subjects. Joint stiffness was also more common among newly diagnosed C282Y homozygotes with elevated serum ferritin than among control subjects (OR 2.7; 95% CI 1.38 to 5.30). However, the sex- and age-adjusted prevalences of self-reported symptoms and signs of liver disease, heart disease, diabetes and most other major clinical manifestations of hemochromatosis were similar in C282Y homozygotes and control subjects. CONCLUSIONS: Some symptoms and conditions associated with hemochromatosis were more prevalent among C282Y homozygotes identified by screening than among control subjects, but prevalences of most outcomes were similar in C282Y homozygotes and controls in this primary care-based study. PMID:19018338

Iron-related gene variants and brain iron in multiple sclerosis and healthy individuals.

PubMed

Hagemeier, Jesper; Ramanathan, Murali; Schweser, Ferdinand; Dwyer, Michael G; Lin, Fuchun; Bergsland, Niels; Weinstock-Guttman, Bianca; Zivadinov, Robert

2018-01-01

Brain iron homeostasis is known to be disturbed in multiple sclerosis (MS), yet little is known about the association of common gene variants linked to iron regulation and pathological tissue changes in the brain. In this study, we investigated the association of genetic determinants linked to iron regulation with deep gray matter (GM) magnetic susceptibility in both healthy controls (HC) and MS patients. Four hundred (400) patients with MS and 150 age- and sex-matched HCs were enrolled and obtained 3 T MRI examination. Three (3) single nucleotide polymorphisms (SNPs) associated with iron regulation were genotyped: two SNPs in the human hereditary hemochromatosis protein gene HFE : rs1800562 (C282Y mutation) and rs1799945 (H63D mutation), as well as the rs1049296 SNP in the transferrin gene (C2 mutation). The effects of disease and genetic status were studied using quantitative susceptibility mapping (QSM) voxel-based analysis (VBA) and region-of-interest (ROI) analysis of the deep GM. The general linear model framework was used to compare groups. Analyses were corrected for age and sex, and adjusted for false discovery rate. We found moderate increases in susceptibility in the right putamen of participants with the C282Y (+ 6.1 ppb) and H63D (+ 6.9 ppb) gene variants vs. non-carriers, as well as a decrease in thalamic susceptibility of progressive MS patients with the C282Y mutation (left: - 5.3 ppb, right: - 6.7 ppb, p < 0.05). Female MS patients had lower susceptibility in the caudate (- 6.0 ppb) and putamen (left: - 3.9 ppb, right: - 4.6 ppb) than men, but only when they had a wild-type allele (p < 0.05). Iron-gene linked increases in putamen susceptibility (in HC and relapsing remitting MS) and decreases in thalamus susceptibility (in progressive MS), coupled with apparent sex interactions, indicate that brain iron in healthy and disease states may be influenced by genetic factors.

Characteristics of participants with self-reported hemochromatosis or iron overload at HEIRS Study initial screening

PubMed Central

Barton, James C.; Acton, Ronald T.; Leiendecker-Foster, Catherine; Lovato, Laura; Adams, Paul C.; Eckfeldt, John H.; McLaren, Christine E.; Reiss, Jacob A.; McLaren, Gordon D.; Reboussin, David M.; Gordeuk, Victor R.; Speechley, Mark R.; Press, Richard D.; Dawkins, Fitzroy W.

2013-01-01

There are few descriptions of young adults with self-reported hemochromatosis or iron overload (H/IO). We analyzed initial screening data in 7,343 HEmochromatosis and IRon Overload Screening (HEIRS) Study participants ages 25–29 years, including race/ethnicity and health information; transferrin saturation (TS) and ferritin (SF) measurements; and HFE C282Y and H63D genotypes. We used denaturing high-pressure liquid chromatography and sequencing to detect mutations in HJV, TFR2, HAMP, SLC40A1, and FTL. Fifty-one participants reported previous H/IO; 23 (45%) reported medical conditions associated with H/IO. Prevalences of reports of arthritis, diabetes, liver disease or liver cancer, heart failure, fertility problems or impotence, and blood relatives with H/IO were significantly greater in participants with previous H/IO reports than in those without. Only 7.8% of the 51 participants with previous H/IO reports had elevated TS; 13.7% had elevated SF. Only one participant had C282Y homozygosity. Three participants aged 25–29 years were heterozygous for potentially deleterious mutations in HFE2, TFR2, and HAMP promoter, respectively. Prevalences of self-reported conditions, screening iron phenotypes, and C282Y homozygosity were similar in 1,165 participants aged 30 years or greater who reported previous H/IO. We conclude that persons who report previous H/IO diagnoses in screening programs are unlikely to have H/IO phenotypes or genotypes. Previous H/IO reports in some participants could be explained by treatment that induced iron depletion before initial screening, misdiagnosis, or participant misunderstanding of their physician or the initial screening questionnaire. PMID:17726683

The role of genetic factors in patients with hepatocellular carcinoma and iron overload - a prospective series of 234 patients.

PubMed

Funakoshi, Natalie; Chaze, Iphigénie; Alary, Anne-Sophie; Tachon, Gaëlle; Cunat, Séverine; Giansily-Blaizot, Muriel; Bismuth, Michael; Larrey, Dominique; Pageaux, Georges-Philippe; Schved, Jean-François; Donnadieu-Rigole, Hélène; Blanc, Pierre; Aguilar-Martinez, Patricia

2016-05-01

Iron overload (IO) in HFE-related hereditary haemochromatosis is associated with increased risk of liver cancer. This study aimed to investigate the role of other genes involved in hereditary IO among patients with hepatocellular carcinoma (HCC). Patients with HCC diagnosed in our institution were included in this prospective study. Those with ferritin levels ≥300 μg/L (males) or ≥200 μg/L (females) and/or transferrin saturation ≥50% (males) or ≥45% (females) had liver iron concentration (LIC) evaluated by MRI. HFE C282Y and H63D mutations were screened. Genetic analyses of genes involved in hereditary IO (HFE, HJV/HFE2, HAMP, TFR2, SLC40A1, GNPAT) were performed in patients with increased LIC. A total of 234 patients were included; 215 (92%) had common acquired risk factors of HCC (mainly alcoholism or chronic viral hepatitis). 119 patients had abnormal iron parameters. Twelve (5.1%) were C282Y homozygotes, three were compound C282Y/H63D heterozygotes. LIC was measured by MRI in 100 patients. Thirteen patients with a LIC>70 μmol/g were enrolled in further genetic analyses: two unrelated patients bore the HAMP:c.-153C>T mutation at the heterozygous state, which is associated with increased risk of IO and severe haemochromatosis. Specific haplotypes of SLC40A1 were also studied. Additional genetic risk factors of IO were found in 18 patients (7.7%) among a large series of 234 HCC patients. Screening for IO and the associated at-risk genotypes in patients who have developed HCC, is useful for both determining etiologic diagnosis and enabling family screening and possibly primary prevention in relatives. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Risk factors for development of dementia in a unique six-year cohort study. I. An exploratory, pilot study of involvement of the E4 allele of apolipoprotein E, mutations of the hemochromatosis-HFE gene, type 2 diabetes, and stroke.

PubMed

Percy, Maire; Somerville, Martin J; Hicks, Mark; Garcia, Angeles; Colelli, Teresa; Wright, Emily; Kitaygorodsky, Julia; Jiang, Amy; Ho, Valerie; Parpia, Alyssa; Wong, Michael K

2014-01-01

Risk factors for dementia development are not well-defined. We evaluated several factors alone and in combination in a unique cohort of Caucasian volunteers over an approximately 6-year observation window using a nested case/control design. Factors included: apolipoprotein E (ApoE) gene variants (the E4 allele is the strongest confirmed genetic predisposing factor for Alzheimer's disease), the hemochromatosis-HFE gene mutations (H63D and C282Y), diabetes, and stroke. At study entry, subjects were ≥65 years of age (M ± SD = 73.0 ± 4.9), had an MMSE score ≥24, and no evidence of cerebrovascular disease or current depression. Genotyping was completed on 163 available DNA samples from three different groups at the study end: those who still had normal cognitive function; those who had developed dementia; and those with Mild Cognitive Impairment (MCI). Analyses were interpreted at the 95% confidence level without Bonferroni corrections. In the subgroup with dementia, all cases of diabetes were type 2 and present at study entry, whereas all strokes occurred during the study. The results highlight apparently synergistic interactions between genetic and medical risk factors for dementia development, gender differences in risk factors, and involvement of HFE mutations. Having E4 (i.e., either of E3/4 or E4/4), C282Y, H63D, diabetes, or stroke alone did not attain significance. Significant predisposing factors with post-hoc power ≥80% were: E4 homozygosity (E4/4)males+females, odds ratio (OR) = 56.0); E4+diabetes (males+females, OR = 13.7; E4+H63D+diabetes (females, OR = 52.0); E4+stroke (males, OR = 46.5). The importance of preventing diabetes and stroke to ward off dementia and the possible role of iron dysmetabolism in dementia are discussed.

Mutations in HAMP and HJV genes and their impact on expression of clinical hemochromatosis in a cohort of 100 Spanish patients homozygous for the C282Y mutation of HFE gene.

PubMed

Altès, Albert; Bach, Vanessa; Ruiz, Angels; Esteve, Anna; Felez, Jordi; Remacha, Angel F; Sardà, M Pilar; Baiget, Montserrat

2009-10-01

Most hereditary hemochromatosis (HH) patients are homozygous for the C282Y mutation of the HFE gene. Nevertheless, penetrance of the disease is very variable. In some patients, penetrance can be mediated by concomitant mutations in other iron master genes. We evaluated the clinical impact of hepcidin (HAMP) and hemojuvelin mutations in a cohort of 100 Spanish patients homozygous for the C282Y mutation of the HFE gene. HAMP and hemojuvelin mutations were evaluated in all patients by bidirectional direct cycle sequencing. Phenotype-genotype interactions were evaluated. A heterozygous mutation of the HAMP gene (G71D) was found in only one out of 100 cases. Following, we performed a study of several members of that family, and we observed several members had a digenic inheritance of the C282Y mutation of the HFE gene and the G71D mutation of the HAMP gene. This mutation in the HAMP gene did not modify the phenotype of the individuals who were homozygous for the C282Y mutation. One other patient presented a new polymorphism in the hemojuvelin gene, without consequences in iron load or clinical course of the disease. In conclusion, HAMP and hemojuvelin mutations are rare among Spanish HH patients, and their impact in this population is not significant.

HFE p.C282Y gene variant is associated with varicose veins in Russian population.

PubMed

Sokolova, Ekaterina A; Shadrina, Alexandra S; Sevost'ianova, Kseniya S; Shevela, Andrey I; Soldatsky, Evgenii Yu; Seliverstov, Evgenii I; Demekhova, Marina Yu; Shonov, Oleg A; Ilyukhin, Evgenii A; Smetanina, Mariya A; Voronina, Elena N; Zolotukhin, Igor A; Filipenko, Maxim L

2016-08-01

Recently, the association of polymorphism rs1800562 (p.C282Y) in the hemochromatosis (HFE) gene with the increased risk of venous ulceration was shown. We hypothesized that HFE gene polymorphism might be involved not only in ulceration process, but also in susceptibility to primary varicose veins. We genotyped HFE p.C282Y (rs1800562) and p.H63D (rs1799945) variants in patients with primary varicose veins (n = 463) and in the control group (n = 754). In our study, p.282Y variant (rs1800562 A allele) was significantly associated with the risk of varicose veins (OR 1.79, 95 % CI = 1.11-2.89, P = 0.02). A borderline significant reverse association of p.63D variant (rs1799945 G allele) with venous leg ulcer development was revealed in Russians (OR 0.25, 95 % CI = 0.06-1.00, P = 0.05), but not in the meta-analysis (P = 0.56). We conclude that the HFE gene polymorphism can affect the risk of developing primary varicose veins.

The relationship between selected VDR, HFE and ALAD gene polymorphisms and several basic toxicological parameters among persons occupationally exposed to lead.

PubMed

Szymańska-Chabowska, Anna; Łaczmański, Łukasz; Jędrychowska, Iwona; Chabowski, Mariusz; Gać, Paweł; Janus, Agnieszka; Gosławska, Katarzyna; Smyk, Beata; Solska, Urszula; Mazur, Grzegorz; Poręba, Rafał

2015-08-06

The aim of this study was to find a relationship between polymorphisms of ALAD rs1805313, rs222808, rs1139488, VDR FokI and HFE C282Y and H63D and basic toxicological parameters (lead and ZnPP blood concentration) in people occupationally exposed to lead. We collected data of 101 workers (age 25-63 years) directly exposed to lead. The toxicological lab tests included blood lead, cadmium and ZnPP concentration measurement and arsenic urine concentration measurement. Workers were genotyped for ALAD (rs1805313, rs222808, rs1139488), HFE (C282Y, H63D) and VDR (FokI). Individuals with the lead exposure and coexisting F allel in the locus Fok-I of VDR gene are suspected of higher zinc protoporphyrins concentrations. Workers exposed to the lead with the Y allel in the locus C282Y of the HFE gene are predisposed to lower ZnPP levels and individuals with coexisting H allel in the locus H63D HFE gene are predisposed to lower Pb-B levels. The T allel in the locus rs1805313 of the ALAD gene determines lower Pb-B and ZnPP levels in lead-exposed individuals. The heterozigosity of the locus rs2228083 of the ALAD gene has a strong predilection to higher Pb-B levels. The carriage of the C allel in the locus rs1139488 of the ALAD gene might determine higher Pb-B levels and the heterozigosity of the locus rs1139488 of the ALAD gene might result in higher ZnPP levels. The study revealed relationship between VDR, HFE and ALAD genes polymorphism and basic toxicological parameters in occupationally exposed workers. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Study of the HFE gene common polymorphisms in French patients with sporadic amyotrophic lateral sclerosis.

PubMed

Praline, Julien; Blasco, Hélène; Vourc'h, Patrick; Rat, Valérian; Gendrot, Chantal; Camu, William; Andres, Christian R

2012-06-15

Our objective was to investigate whether the C282Y (p.Cys 282 Tyr) and H63D (p. His 63 Asp) HFE polymorphisms were associated with sporadic amyotrophic lateral sclerosis (SALS) in the French population. We searched for a relation of HFE polymorphisms with the clinical characteristics of the disease. The HFE polymorphisms were studied in 824 patients with SALS and 583 controls. We compared the frequency of the polymorphisms between SALS and controls groups by univariate and multivariate statistics, taking into account gender, site, age-at-onset and survival. We did not observe significant difference in the frequency of H63D polymorphism between SALS and control group. We observed a significant difference for C282Y between patients and controls with a low frequency of the Y allele in patients (3.2%) compared to our control group (5.9%). Disease duration, distribution of gender, site-of-onset, age-at-onset did not differ between groups taking into account genotypes of each polymorphism. Our results in this large cohort of ALS patients indicate that H63D polymorphism is not associated with SALS in the French population. This conclusion does not exclude a weak effect of the HFE gene polymorphisms in certain ALS populations, or an effect of other rare HFE gene variants. Copyright © 2012 Elsevier B.V. All rights reserved.

Heritability of Serum Iron Measures in the Hemochromatosis and Iron Overload Screening (HEIRS) Family Study

PubMed Central

McLaren, Christine E.; Barton, James C.; Eckfeldt, John H.; McLaren, Gordon D.; Acton, Ronald T.; Adams, Paul C.; Henkin, Leora F.; Gordeuk, Victor R.; Vulpe, Chris D.; Harris, Emily L.; Harrison, Barbara W.; Reiss, Jacob A.; Snively, Beverly M.

2013-01-01

Heritability is the proportion of observed variation in a trait among individuals in a population that is attributable to hereditary factors. The HEIRS Family Study estimated heritability of serum iron measures. Probands were HFE C282Y homozygotes or non-C282Y homozygotes with elevated transferrin saturation (TS > 50%, men; TS > 45%, women) and serum ferritin concentration (SF > 300 μg/L, men; SF > 200 μg/L, women). Heritability (h2) was estimated by variance component analysis of TS, natural logarithm (ln) of SF, and unsaturated iron-binding capacity (UIBC). Participants (N=942) were 77% Caucasians, 10% Asians, 8% Hispanics, and 5% other race/ethnicities. Average age (SD) was 49 (16) y; 57% were female. For HFE C282Y homozygote probands and their family members, excluding variation due to HFE C282Y and H63D genotype and measured demographic and environmental factors, the residual h2 (SE) was 0.21 (0.07) for TS, 0.37 (0.08) for ln SF, and 0.34 (0.08) for UIBC (all P < 0.0004 for comparisons with zero). For the non-C282Y homozygote proband group, residual h2 was significant with a value of 0.64 (0.26) for ln SF (p=0.0096). In conclusion, serum iron measures have significant heritability components, after excluding known genetic and non-genetic sources of variation. PMID:20095037

Sample-to-SNP kit: a reliable, easy and fast tool for the detection of HFE p.H63D and p.C282Y variations associated to hereditary hemochromatosis.

PubMed

Nielsen, Peter B; Petersen, Maja S; Ystaas, Viviana; Andersen, Rolf V; Hansen, Karin M; Blaabjerg, Vibeke; Refstrup, Mette

2012-10-01

Classical hereditary hemochromatosis involves the HFE-gene and diagnostic analysis of the DNA variants HFE p.C282Y (c.845G>A; rs1800562) and HFE p.H63D (c.187C>G; rs1799945). The affected protein alters the iron homeostasis resulting in iron overload in various tissues. The aim of this study was to validate the TaqMan-based Sample-to-SNP protocol for the analysis of the HFE-p.C282Y and p.H63D variants with regard to accuracy, usefulness and reproducibility compared to an existing SNP protocol. The Sample-to-SNP protocol uses an approach where the DNA template is made accessible from a cell lysate followed by TaqMan analysis. Besides the HFE-SNPs other eight SNPs were used as well. These SNPs were: Coagulation factor II-gene F2 c.20210G>A, Coagulation factor V-gene F5 p.R506Q (c.1517G>A; rs121917732), Mitochondria SNP: mt7028 G>A, Mitochondria SNP: mt12308 A>G, Proprotein convertase subtilisin/kexin type 9-gene PCSK9 p.R46L (c.137G>T), Plutathione S-transferase pi 1-gene GSTP1 p.I105V (c313A>G; rs1695), LXR g.-171 A>G, ZNF202 g.-118 G>T. In conclusion the Sample-to-SNP kit proved to be an accurate, reliable, robust, easy to use and rapid TaqMan-based SNP detection protocol, which could be quickly implemented in a routine diagnostic or research facility. Copyright © 2012. Published by Elsevier B.V.

Frequency of the S65C mutation in the hemochromatosis gene in Brazil.

PubMed

Oliveira, V C; Caxito, F A; Gomes, K B; Castro, A M; Pardini, V C; Ferreira, A C S

2009-07-14

Development of hereditary hemochromatosis is associated with the C282Y, H63D or S65C mutations in the hemochromatosis gene. Though there is extensive knowledge about the former two, there is little information on the mechanism of action and the allelic frequency of the S65C mutation. We examined the prevalence of the S65C mutation of the hemochromatosis gene in Brazilians with clinical suspicion of hereditary hemochromatosis. Genotyping for this mutation was carried out in 633 individuals with clinical suspicion of hereditary hemochromatosis, using the polymerase chain reaction, followed by enzymatic digestion. The sample comprised 77.1% men and 22.9% women, giving a ratio of approximately 3:1; the mean age was 48.8 +/- 13.8 years. More than half (57.3%) of the individuals in the sample were 41 to 60 years old. The frequency of heterozygotes for this mutation was 0.016; no homozygous mutant patients were found. This is the first analysis of the S65C mutation in individuals suspected of having hereditary hemochromatosis in Brazil.

Thyroid-stimulating hormone and free thyroxine levels in persons with HFE C282Y homozygosity, a common hemochromatosis genotype: the HEIRS study.

PubMed

Barton, James C; Leiendecker-Foster, Catherine; Reboussin, David M; Adams, Paul C; Acton, Ronald T; Eckfeldt, John H

2008-08-01

Relationships of thyroid and iron measures in large cohorts are unreported. We evaluated thyroid-stimulating hormone (TSH) and free thyroxine (T4) in white participants of the primary care-based Hemochromatosis and Iron Overload Screening (HEIRS) Study. We measured serum TSH and free T4 in 176 HFE C282Y homozygotes without previous hemochromatosis diagnoses and in 312 controls without HFE C282Y or H63D who had normal serum iron measures and were matched to C282Y homozygotes for Field Center, age group, and initial screening date. We defined hypothyroidism as having TSH >5.00 mIU/L and free T4 <0.70 ng/dL, and hyperthyroidism as having TSH <0.400 mIU/L and free T4 >1.85 ng/dL. Multivariate analyses were performed using age, sex, Field Center, log(10) serum ferritin (SF), HFE genotype, log(10) TSH, and log(10) free T4. Prevalences of hypothyroidism in C282Y homozygotes and controls were 1.7% and 1.3%, respectively, and of hyperthyroidism 0% and 1.0%, respectively. Corresponding prevalences did not differ significantly. Correlations of log(10) SF with log(10) free T4 were positive (p = 0.2368, C282Y homozygotes; p = 0.0492, controls). Independent predictors of log(10) free T4 were log(10) TSH (negative association) and age (positive association); positive predictors of log(10) SF were age, male sex, and C282Y homozygosity. Proportions of C282Y homozygotes and controls who took medications to supplement or suppress thyroid function did not differ significantly. Prevalences of hypothyroidism and hyperthyroidism are similar in C282Y homozygotes without previous hemochromatosis diagnoses and controls. In controls, there is a significant positive association of SF with free T4. We conclude that there is no rationale for routine measurement of TSH or free T4 levels in hemochromatosis or iron overload screening programs.

Mutation Spectrum and Phenotypic Features in Noonan Syndrome with PTPN11 Mutations: Definition of Two Novel Mutations.

PubMed

Atik, Tahir; Aykut, Ayca; Hazan, Filiz; Onay, Huseyin; Goksen, Damla; Darcan, Sukran; Tukun, Ajlan; Ozkinay, Ferda

2016-06-01

To evaluate the spectrum of PTPN11 gene mutations in Noonan syndrome patients and to study the genotype-phenotype associations. In this study, twenty Noonan syndrome patients with PTPN11 mutations were included. The patients underwent a detailed clinical and physical evaluation. To identify inherited cases, parents of all mutation positive patients were analyzed. Thirteen different PTPN11 mutations, two of them being novel, were detected in the study group. These mutations included eleven missense mutations: p.G60A, p.D61N, p.Y62D, p.Y63C, p.E69Q, p.Q79R, p.Y279C,p.N308D, p.N308S, p.M504V, p.Q510R and two novel missense mutations: p.I56V and p.I282M. The frequency of cardiac abnormalities and short stature were found to be 80 % and 80 %, respectively. Mental retardation was not observed in patients having exon 8 mutations. No significant correlations were detected between other phenotypic features and genotypes. By identifying genotype-phenotype correlations, this study provides information on phenotypes observed in NS patients with different PTPN11 mutations.

Variation in the HFE gene is associated with the development of bleomycin-induced pulmonary toxicity in testicular cancer patients.

PubMed

van der Schoot, Gabriela G F; Westerink, Nico-Derk L; Lubberts, Sjoukje; Nuver, Janine; Zwart, Nynke; Walenkamp, Annemiek M E; Wempe, Johan B; Meijer, Coby; Gietema, Jourik A

2016-05-01

Bleomycin and cisplatin are of key importance in testicular cancer treatment. Known potential serious adverse effects are bleomycin-induced pulmonary toxicity (BIP) and cisplatin-induced renal toxicity. Iron handling may play a role in development of this toxicity. Carriage of allelic variants of the HFE gene induces altered iron metabolism and may contribute to toxicity. We investigated the association between two common allelic variants of the HFE gene, H63D and C282Y, with development of pulmonary and renal toxicity during and after treatment with bleomycin- and cisplatin-containing chemotherapy. In 369 testicular cancer patients treated with bleomycin and cisplatin at the University Medical Center Groningen between 1978 and 2006, H63D and/or C282Y genotypes were determined with an allelic discrimination assay. Data were collected on development of BIP, pulmonary function parameters, renal function, and survival. BIP developed more frequently in patients who were heterozygote (16 in 75, 21%) and homozygote (2 in 4, 50%) for the H63D variant, compared with those who had the HFE wild-type gene (31 in 278, 11%) (p = 0.012). Overall survival, testicular cancer-related survival, and change in renal function were not associated with the H63D variant. We observed an association between presence of one or both H63D alleles and development of BIP in testicular cancer patients treated with bleomycin combination chemotherapy. In patients heterozygote and homozygote for the H63D variant, BIP occurred more frequently compared with wild-type patients. When validated and confirmed, HFE H63D genotyping may be used to identify patients with increased risk for pulmonary bleomycin toxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Using iron studies to predict HFE mutations in New Zealand: implications for laboratory testing.

PubMed

O'Toole, Rebecca; Romeril, Kenneth; Bromhead, Collette

2017-04-01

The diagnosis of hereditary haemochromatosis (HH) is not straightforward because symptoms are often absent or non-specific. Biochemical markers of iron-overloading may be affected by other conditions. To measure the correlation between iron studies and HFE genotype to inform evidence-based recommendations for laboratory testing in New Zealand. Results from 2388 patients genotyped for C282Y, H63D and S65C in Wellington, New Zealand from 2007 to 2013 were compared with their biochemical phenotype as quantified by serum ferritin (SF), transferrin saturation (TS), serum iron (SI) and serum transferrin (ST). The predictive power of these markers was evaluated by receiver operator characteristic (ROC) curve analysis, and if a statistically significant association for a variable was seen, sensitivity, specificity and predictive values were calculated. Test ordering patterns showed that 62% of HFE genotyping tests were ordered because of an elevated SF alone and only 11% of these had a C-reactive protein test to rule out an acute phase reaction. The association between SF and significant HFE genotypes SF was low. However, TS values ≥45% predicted HH mutations with the highest sensitivity and specificity. A SF of >1000 µg/L was found in one at-risk patient (C282Y homozygote) who had a TS <45%. Our analysis highlights the need for clear guidelines for investigation of hyperferritinaemia and HH in New Zealand. Using our findings, we developed an evidence-based laboratory testing algorithm based on a TS ≥45%, a SF ≥1000 µg/L and/or a family history of HH which identified all C282Y homozygotes in this study. © 2016 Royal Australasian College of Physicians.

Hereditary hemochromatosis of tongue.

PubMed

Al Wayli, Hessa; Rastogi, Sanjay; Verma, Neha

2011-01-01

Hereditary hemochromatosis (HH) refers to several inherited disorders of iron metabolism leading to tissue iron overload. Classical HH is associated with mutations in HFE (C282Y homozygotes or C282Y/H63D compound heterozygotes) and is almost exclusively found in populations of northern European descent. Non-HFE-associated HH is caused by mutations in other recently identified genes involved in iron metabolism. Hepcidin is an iron regulatory hormone that inhibits ferroportin-mediated iron export from enterocytes and macrophages. Defective hepcidin gene expression or function may underlie most forms of HH. Target organs and tissues affected by HH include the liver, heart, pancreas, joints, and skin, with cirrhosis and diabetes mellitus representing late signs of disease in patients with markedly elevated liver iron concentration. Recently, we have encountered the rare representation of this disease of the oral cavity associated with generalized burning sensation of the tongue. The diagnosis was established accidently, from the lab investigations, otherwise the patient was healthy and free from classical signs and symptoms of the disease. The patient was adequately treated by phlebotomy. To conclude, all patients with a chief complaint of burning sensation of the oral cavity and tongue should be adequately screened for hereditary hemochromatosis to prevent the associated mortality and morbidity with the hemochromatosis. Copyright © 2011 Mosby, Inc. All rights reserved.

Effect of C282Y genotype on self-reported musculoskeletal complications in hereditary hemochromatosis.

PubMed

Camacho, António; Funck-Brentano, Thomas; Simão, Márcio; Cancela, Leonor; Ottaviani, Sébastien; Cohen-Solal, Martine; Richette, Pascal

2015-01-01

Arthropathy that mimics osteoarthritis (OA) and osteoporosis (OP) is considered a complication of hereditary hemochromatosis (HH). We have limited data comparing OA and OP prevalence among HH patients with different hemochromatosis type 1 (HFE) genotypes. We investigated the prevalence of OA and OP in patients with HH by C282Y homozygosity and compound heterozygosity (C282Y/H63D) genotype. A total of 306 patients with HH completed a questionnaire. Clinical and demographic characteristics and presence of OA, OP and related complications were compared by genotype, adjusting for age, sex, body mass index (BMI), current smoking and menopausal status. In total, 266 of the 306 patients (87%) were homozygous for C282Y, and 40 (13%) were compound heterozygous. The 2 groups did not differ by median age [60 (interquartile range [IQR] 53 to 68) vs. 61 (55 to 67) years, P=0.8], sex (female: 48.8% vs. 37.5%, P=0.18) or current smoking habits (12.4% vs. 10%, P=0.3). As compared with compound heterozygous patients, C282Y homozygous patients had higher median serum ferritin concentration at diagnosis [1090 (IQR 610 to 2210) vs. 603 (362 to 950) µg/L, P<0.001], higher median transferrin saturation [80% (IQR 66 to 91%) vs. 63% (55 to 72%), P<0.001]) and lower median BMI [24.8 (22.1 to 26.9) vs. 26.2 (23.5 to 30.3) kg/m2, P<0.003]. The overall prevalence of self-reported OA was significantly higher with C282Y homozygosity than compound heterozygosity (53.4% vs. 32.5%; adjusted odds ratio [aOR] 2.4 [95% confidence interval 1.2-5.0]), as was self-reported OP (25.6% vs. 7.5%; aOR 3.5 [1.1-12.1]). Patients with C282Y homozygosity may be at increased risk of musculoskeletal complications of HH.

Effect of C282Y Genotype on Self-Reported Musculoskeletal Complications in Hereditary Hemochromatosis

PubMed Central

Simão, Márcio; Cancela, Leonor; Ottaviani, Sébastien; Cohen-Solal, Martine; Richette, Pascal

2015-01-01

Objective Arthropathy that mimics osteoarthritis (OA) and osteoporosis (OP) is considered a complication of hereditary hemochromatosis (HH). We have limited data comparing OA and OP prevalence among HH patients with different hemochromatosis type 1 (HFE) genotypes. We investigated the prevalence of OA and OP in patients with HH by C282Y homozygosity and compound heterozygosity (C282Y/H63D) genotype. Methods A total of 306 patients with HH completed a questionnaire. Clinical and demographic characteristics and presence of OA, OP and related complications were compared by genotype, adjusting for age, sex, body mass index (BMI), current smoking and menopausal status. Results In total, 266 of the 306 patients (87%) were homozygous for C282Y, and 40 (13%) were compound heterozygous. The 2 groups did not differ by median age [60 (interquartile range [IQR] 53 to 68) vs. 61 (55 to 67) years, P=0.8], sex (female: 48.8% vs. 37.5%, P=0.18) or current smoking habits (12.4% vs. 10%, P=0.3). As compared with compound heterozygous patients, C282Y homozygous patients had higher median serum ferritin concentration at diagnosis [1090 (IQR 610 to 2210) vs. 603 (362 to 950) µg/L, P<0.001], higher median transferrin saturation [80% (IQR 66 to 91%) vs. 63% (55 to 72%), P<0.001]) and lower median BMI [24.8 (22.1 to 26.9) vs. 26.2 (23.5 to 30.3) kg/m2, P<0.003]. The overall prevalence of self-reported OA was significantly higher with C282Y homozygosity than compound heterozygosity (53.4% vs. 32.5%; adjusted odds ratio [aOR] 2.4 [95% confidence interval 1.2–5.0]), as was self-reported OP (25.6% vs. 7.5%; aOR 3.5 [1.1–12.1]). Conclusion Patients with C282Y homozygosity may be at increased risk of musculoskeletal complications of HH. PMID:25822977

HFE gene variants modify the association between maternal lead burden and infant birthweight: a prospective birth cohort study in Mexico City, Mexico.

PubMed

Cantonwine, David; Hu, Howard; Téllez-Rojo, Martha Maria; Sánchez, Brisa N; Lamadrid-Figueroa, Héctor; Ettinger, Adrienne S; Mercado-García, Adriana; Hernández-Avila, Mauricio; Wright, Robert O

2010-07-26

Neonatal growth is a complex process involving genetic and environmental factors. Polymorphisms in the hemochromatosis (HFE) iron regulatory genes have been shown to modify transport and toxicity of lead which is known to affect birth weight. We investigated the role of HFE C282Y, HFE H63 D, and transferrin (TF) P570 S gene variants in modifying the association of lead and infant birthweight in a cohort of Mexican mother-infant pairs. Subjects were initially recruited between 1994-1995 from three maternity hospitals in Mexico City and 411 infants/565 mothers had archived blood available for genotyping. Multiple linear regression models, stratified by either maternal/infant HFE or TF genotype and then combined with interaction terms, were constructed examining the association of lead and birthweight after controlling for covariates. 3.1%, 16.8% and 17.5% of infants (N=390) and 1.9%, 14.5% and 18.9% of mothers (N=533) carried the HFE C282Y, HFE H63D, and TF P570 S variants, respectively. The presence of infant HFE H63 D variants predicted 110.3 g (95% CI -216.1, -4.6) decreases in birthweight while maternal HFE H63 D variants predicted reductions of 52.0 g (95% CI -147.3 to 43.2). Interaction models suggest that both maternal and infant HFE H63 D genotype may modify tibia lead's effect on infant birthweight in opposing ways. In our interaction models, maternal HFE H63 D variant carriers had a negative association between tibia lead and birthweight. These results suggest that the HFE H63 D genotype modifies lead's effects on infant birthweight in a complex fashion that may reflect maternal-fetal interactions with respect to the metabolism and transport of metals.

White blood cells and subtypes in HFE p.C282Y and wild-type homozygotes in the Hemochromatosis and Iron Overload Screening Study.

PubMed

Barton, James C; Barton, J Clayborn; Acton, Ronald T

2017-03-01

The major histocompatibility complex is linked to white blood cell (WBC) and lymphocyte counts in subjects unselected for HFE genotypes. We compared age, sex, body mass index, total WBC and subtypes (neutrophils, lymphocytes, monocytes, eosinophils, basophils) (Beckman Coulter® Gen-S), transferrin saturation, and serum ferritin of HFE p.C282Y and wild-type (p.C282Y, p.H63D negative) homozygotes without acquired conditions that influence WBC counts. We performed regressions on WBC and subtypes. There were 161 p.C282Y homozygotes (45.3% men) and 221 wild-type homozygotes (40.3% men). Mean WBC of men and women and between HFE genotypes were similar. Mean lymphocytes were higher in male p.C282Y homozygotes: 1.6×10 9 /L [95% confidence interval: 1.5,1.7] vs. 1.4 [1.3,1.5], p=0.0002. Mean lymphocytes and basophils were higher in female p.C282Y homozygotes: 1.6 [1.5,1.7] vs. 1.4 [1.3,1.5], p=0.0002; and 0.065 [0.059,0.071] vs. 0.052 [0.051,0.054], p=0.0001, respectively. Transferrin saturation was associated with neutrophils (negative; p=0.0163). Age was associated with lymphocytes (negative; p=0.0003) and monocytes (positive; p<0.0001). Regressions on lymphocytes and basophils revealed positive associations with p.C282Y homozygosity (p=0.0043 and 0.0003, respectively). There were significant positive associations of neutrophils, lymphocytes, monocytes, and eosinophils. We conclude that HFE p.C282Y homozygosity is significantly associated with lymphocyte and basophil counts. Copyright © 2016 Elsevier Inc. All rights reserved.

Iranian hereditary hemochromatosis patients: baseline characteristics, laboratory data and gene mutations.

PubMed

Zamani, Farhad; Bagheri, Zohreh; Bayat, Maryam; Fereshtehnejad, Seyed-Mohammad; Basi, Ali; Najmabadi, Hossein; Ajdarkosh, Hossein

2012-10-01

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder in white people, characterized by highly abnormal uptake of iron from the gastrointestinal tracts. Recently, mutation studies have focused to detect the genes responsible for HH. In this cross-sectional study, 12 HH patients were recruited, who were referred to Firoozgar Hospital, Tehran, Iran. In addition to the clinical assessments, a complete laboratory evaluation, imaging modalities, histopathologic assessment, atomic absorption spectrophotometry and gene mutation study were performed. The genetic study for HFE gene mutation was examined for all of the patients since 2006, while non-HFE mutation was conducted since December 2010 (only for 1 of them). Twelve patients were evaluated consisting of 11 men and 1 woman, with the mean age of 39.58±12.68 yr. The average of atomic iron loads was 13.25±4.83-fold higher than normal standards. Four patients had heterozygotic mutation of H63D (33.3%). There was no significant difference in either the iron load of liver (P=0.927) and heart (P=0.164) or serum concentration of ferritin (P=0.907) and TIBC (P=0.937) between the HFE-mutant and without HFE mutation HH cases. In contrast to other studies, C282Y mutation was not detected in any of our Iranian HH patients. Heterozygotic mutations of H63D (HFE) and TFR2 (non-HFE) genes were found to be more common in these patients. Similar to previous reports, these mutations were not found to be significantly associated with severity of presentation in HH patients.

Best practice guidelines for the molecular genetic diagnosis of Type 1 (HFE-related) hereditary haemochromatosis

PubMed Central

King, Caitriona; Barton, David E

2006-01-01

Background Hereditary haemochromatosis (HH) is a recessively-inherited disorder of iron over-absorption prevalent in Caucasian populations. Affected individuals for Type 1 HH are usually either homozygous for a cysteine to tyrosine amino acid substitution at position 282 (C282Y) of the HFE gene, or compound heterozygotes for C282Y and for a histidine to aspartic acid change at position 63 (H63D). Molecular genetic testing for these two mutations has become widespread in recent years. With diverse testing methods and reporting practices in use, there was a clear need for agreed guidelines for haemochromatosis genetic testing. The UK Clinical Molecular Genetics Society has elaborated a consensus process for the development of disease-specific best practice guidelines for genetic testing. Methods A survey of current practice in the molecular diagnosis of haemochromatosis was conducted. Based on the results of this survey, draft guidelines were prepared using the template developed by UK Clinical Molecular Genetics Society. A workshop was held to develop the draft into a consensus document. The consensus document was then posted on the Clinical Molecular Genetics Society website for broader consultation and amendment. Results Consensus or near-consensus was achieved on all points in the draft guidelines. The consensus and consultation processes worked well, and outstanding issues were documented in an appendix to the guidelines. Conclusion An agreed set of best practice guidelines were developed for diagnostic, predictive and carrier testing for hereditary haemochromatosis and for reporting the results of such testing. PMID:17134494

Hemochromatosis (HFE) gene mutations and risk of gastric cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

PubMed

Agudo, Antonio; Bonet, Catalina; Sala, Núria; Muñoz, Xavier; Aranda, Núria; Fonseca-Nunes, Ana; Clavel-Chapelon, Françoise; Boutron-Ruault, Marie Christine; Vineis, Paolo; Panico, Salvatore; Palli, Domenico; Tumino, Rosario; Grioni, Sara; Quirós, J Ramón; Molina, Esther; Navarro, Carmen; Barricarte, Aurelio; Chamosa, Saioa; Allen, Naomi E; Khaw, Kay-Tee; Bueno-de-Mesquita, H Bas; Siersema, Peter D; Numans, Mattijs E; Trichopoulou, Antonia; Lagiou, Pagona; Trichopoulos, Dimitrios; Kaaks, Rudof; Canzian, Federico; Boeing, Heiner; Meidtner, Karina; Johansson, Mattias; Sund, Malin; Manjer, Jonas; Overvad, Kim; Tjonneland, Anne; Lund, Eiliv; Weiderpass, Elisabete; Jenab, Mazda; Fedirko, Veronika; Offerhaus, G Johan A; Riboli, Elio; González, Carlos A; Jakszyn, Paula

2013-06-01

Hereditary hemochromatosis (HH) is a strong risk factor for hepatocellular cancer, and mutations in the HFE gene associated with HH and iron overload may be related to other tumors, but no studies have been reported for gastric cancer (GC). A nested case-control study was conducted within the European Prospective Investigation into Cancer and Nutrition (EPIC), including 365 incident gastric adenocarcinoma and 1284 controls matched by center, sex, age and date of blood collection. Genotype analysis was performed for two functional polymorphisms (C282Y/rs1800562 and H63D/rs1799945) and seven tagSNPs of the HFE genomic region. Association with all gastric adenocarcinoma, and according to anatomical localization and histological subtype, was assessed by means of the odds ratio (OR) and 95% confidence interval (CI) estimated by unconditional logistic regression adjusted for the matching variables. We observed a significant association for H63D with OR (per rare allele) of 1.32 (CI = 1.03-1.69). In subgroup analyses, the association was stronger for non-cardia anatomical subsite (OR = 1.60, CI = 1.16-2.21) and intestinal histological subtype (OR = 1.82, CI = 1.27-2.62). Among intestinal cases, two tagSNPs (rs1572982 and rs6918586) also showed a significant association that disappeared after adjustment for H63D. No association with tumors located in the cardia or with diffuse subtype was found for any of the nine SNPs analyzed. Our results suggest that H63D variant in HFE gene seems to be associated with GC risk of the non-cardia region and intestinal type, possibly due to its association with iron overload although a role for other mechanisms cannot be entirely ruled out.

Iron overload and HFE gene mutations in Polish patients with liver cirrhosis.

PubMed

Sikorska, Katarzyna; Romanowski, Tomasz; Stalke, Piotr; Iżycka-Świeszewska, Ewa; Bielawski, Krzysztof Piotr

2011-06-01

Increased liver iron stores may contribute to the progression of liver injury and fibrosis, and are associated with a higher risk of hepatocellular carcinoma development. Pre-transplant symptoms of iron overload in patients with liver cirrhosis are associated with higher risk of infectious and malignant complications in liver transplant recipients. HFE gene mutations may be involved in the pathogenesis of liver iron overload and influence the progression of chronic liver diseases of different origins. This study was designed to determine the prevalence of iron overload in relation to HFE gene mutations among Polish patients with liver cirrhosis. Sixty-one patients with liver cirrhosis included in the study were compared with a control group of 42 consecutive patients subjected to liver biopsy because of chronic liver diseases. Liver function tests and serum iron markers were assessed in both groups. All patients were screened for HFE mutations (C282Y, H63D, S65C). Thirty-six of 61 patients from the study group and all controls had liver biopsy performed with semiquantitative assessment of iron deposits in hepatocytes. The biochemical markers of iron overload and iron deposits in the liver were detected with a higher frequency (70% and 47% respectively) in patients with liver cirrhosis. There were no differences in the prevalence of all HFE mutations in both groups. In patients with a diagnosis of hepatocellular carcinoma, no significant associations with iron disorders and HFE gene mutations were found. Iron disorders were detected in patients with liver cirrhosis frequently but without significant association with HFE gene mutations. Only the homozygous C282Y mutation seems to occur more frequently in the selected population of patients with liver cirrhosis. As elevated biochemical iron indices accompanied liver iron deposits more frequently in liver cirrhosis compared to controls with chronic liver disease, there is a need for more extensive studies searching for the possible influence of non-HFE iron homeostasis regulators and their modulation on the course of chronic liver disease and liver cirrhosis.

Mutations in the hereditary haemochromatosis gene HFE in professional endurance athletes

PubMed Central

Chicharro, J; Hoyos, J; Gomez-Gallego, F; Villa, J; Bandres, F; Celaya, P; Jimenez, F; Alonso, J; Cordova, A; Lucia, A

2004-01-01

Background: Hereditary haemochromatosis, a disease that affects iron metabolism, progresses with a greater or lesser tendency to induce iron overload, possibly leading to severe organ dysfunction. Most elite endurance athletes take iron supplements during their active sporting life, which could aggravate this condition. Objective: To determine the prevalence and discuss potential clinical implications of mutations of HFE (the gene responsible for hereditary haemochromatosis) in endurance athletes. Methods: Basal concentrations of iron, ferritin, and transferrin and transferrin saturation were determined in the period before competition in 65 highly trained athletes. Possible mutations in the HFE gene were evaluated in each subject by extracting genomic DNA from peripheral blood. The restriction enzymes SnaBI and BclI were used to detect the mutations 845G→A (C282Y) and 187C→G (H63D). Results: Our findings indicate a high prevalence of HFE gene mutations in this population (49.2%) compared with sedentary controls (33.5%). No association was detected in the athletes between mutations and blood iron markers. Conclusions: The findings support the need to assess regularly iron stores in elite endurance athletes. PMID:15273174

Increased duodenal DMT-1 expression and unchanged HFE mRNA levels in HFE-associated hereditary hemochromatosis and iron deficiency.

PubMed

Byrnes, V; Barrett, S; Ryan, E; Kelleher, T; O'Keane, C; Coughlan, B; Crowe, J

2002-01-01

HFE-associated hereditary hemochromatosis is characterized by imbalances of iron homeostasis and alterations in intestinal iron absorption. The identification of the HFE gene and the apical iron transporter divalent metal transporter-1, DMT-1, provide a direct method to address the mechanisms of iron overload in this disease. The aim of this study was to evaluate the regulation of duodenal HFE and DMT-1 gene expression in HFE-associated hereditary hemochromatosis. Small bowel biopsies and serum iron indices were obtained from a total of 33 patients. The study population comprised 13 patients with hereditary hemochromatosis (C282Y homozygous), 10 patients with iron deficiency anemia, and 10 apparently healthy controls, all of whom were genotyped for the two common mutations in the HFE gene (C282Y and H63D). Total RNA was isolated from tissue and amplified via RT-PCR for HFE, DMT-1, and the internal control GAPDH. DMT-1 protein expression was additionally assessed by immunohistochemistry. Levels of HFE mRNA did not differ significantly between patient groups (P = 0.09), specifically between C282Y homozygotes and iron deficiency anemic patients, when compared to controls (P = 0.09, P = 0.9, respectively). In contrast, DMT-1 mRNA levels were at least twofold greater in patients with hereditary hemochromatosis and iron deficiency anemia when compared to controls (P = 0.02, P = 0.01, respectively). Heightened DMT-1 protein expression correlated with mRNA levels in all patients. Loss of HFE function in hereditary hemochromatosis is not derived from inhibition of its gene expression. DMT-1 expression in C282Y homozygote subjects is consistent with the hypothesis of a "paradoxical" duodenal iron deficiency in hereditary hemochromatosis. The observed twofold upregulation of the DMT-1 is consistent with the slow but steady increase in body iron stores observed in those presenting with clinical features of hereditary hemochromatosis.

Biological variability of transferrin saturation and unsaturated iron binding capacity

PubMed Central

Adams, PC; Reboussin, DM; Press, RD; Barton, JC; Acton, RT; Moses, GC; Leiendecker-Foster, C; McLaren, GD; Dawkins, FW; Gordeuk, VR; Lovato, L; Eckfeldt, JH

2007-01-01

Background Transferrin saturation is widely considered the preferred screening test for hemochromatosis. Unsaturated iron binding capacity has similar performance at lower cost. However, the within-person biological variability of both these tests may limit their ability at commonly used cut points to detect HFE C282Y homozygous patients. Methods The Hemochromatosis and Iron Overload Screening (HEIRS) Study screened 101,168 primary care participants for iron overload using tansferrin saturation, unsaturated iron binding capacity, ferritin and HFE C282Y and H63D genotyping. Transferrin saturation and unsaturated iron binding capacity were performed at initial screening and again when selected participants and controls returned for a clinical examination several months later. A missed case was defined as a C282Y homozygote who had transferrin saturation below cut point (45 % women, 50 % men) or unsaturated iron binding capacity above cut point (150 μmol/L women, 125 μmol/L men) at either the initial screening or clinical examination, or both, regardless of serum ferritin. Results There were 209 C282Y previously undiagnosed homozygotes with transferrin saturation and unsaturated iron binding capacity testing done at initial screening and clinical examination. Sixty-eight C282Y homozygotes (33%) would have been missed at these transferrin saturation cut points (19 men, 49 women, median SF 170 μg/L, first and third quartiles 50 and 474 μg/L), and 58 homozygotes (28 %) would have been missed at the unsaturated iron binding capacity cut points (20 men, 38 women, median SF 168 μg/L, quartiles 38 and 454 μg/L). There was no advantage to using fasting samples. Conclusions The within-person biological variability of transferrin saturation and unsaturated iron binding capacity limit their usefulness as an initial screening test for expressing C282Y homozygotes. PMID:17976429

HLA haplotype map of river valley populations with hemochromatosis traced through five centuries in Central Sweden.

PubMed

Olsson, K Sigvard; Ritter, Bernd; Hansson, Norbeth; Chowdhury, Ruma R

2008-07-01

The hemochromatosis mutation, C282Y of the HFE gene, seems to have originated from a single event which once occurred in a person living in the north west of Europe carrying human leukocyte antigen (HLA)-A3-B7. In descendants of this ancestor also other haplotypes appear probably caused by local recombinations and founder effects. The background of these associations is unknown. Isolated river valley populations may be fruitful for the mapping of genetic disorders such as hemochromatosis. In this study, we try to test this hypothesis in a study from central Sweden where the haplotyope A1-B8 was common. HLA haplotypes and HFE mutations were studied in hemochromatosis patients with present or past parental origin in a sparsely populated (1/km(2)) rural district (n = 8366 in the year of 2005), in central Sweden. Pedigrees were constructed from the Swedish church book registry. Extended haplotypes were studied to evaluate origin of recombinations. There were 87 original probands, 36 females and 51 males identified during 30 yr, of whom 86% carried C282Y/C282Y and 14% C282Y/H63D. Of 32 different HLA haplotypes A1-B8 was the most common (34%), followed by A3-B7 (16%), both in strong linkage disequilibrium with controls, (P < 0.001). Twenty-nine different families with A1-B8 had a common founder origin 15 generations ago in small bottleneck populations of the late 16th century. A second A1-B8 founder born 1655 was of Norwegian origin. Most of the A3 carriers (n = 26) had a common founder origin 16 generations ago in an even smaller nearby river valley. A fourth founder family carrying HLA-A2 seems to have originated from a recombination along the descendant lines from the A3 ancestor supported by extended haplotype studies. A1-haplotypes with alleles at the B locus different from B8 had a similar recombination origin as HLA-A2 alleles and a common founder origin 11 generations ago. The intergenerational time interval averaged 35.5 +/- 7.9 yr in men and 31.9 +/- 5.9 in females. River valley populations may contain HLA haplotypes reflecting their demographic history. This study has demonstrated that the resistance against recombinations between HLA-A and HFE make HLA haplotypes excellent markers for population movements. Founder effects and genetic drift from bottleneck populations (surviving the plague?) may explain the commonness of the mutation in central Scandinavia. The intergenerational time difference >30 yr was greater than expected and means that the age of the original mutation may be underestimated.

[Analysis of H63D mutation in hemochromatosis (HFE) gene in populations of central Eurasia].

PubMed

Khusainova, R I; Khusnutdinova, N N; Litvinov, S S; Khusnutdinova, E K

2013-02-01

An analysis of the frequency of H63D (c. 187C>G) mutations in the HFEgene in 19 populations from Central Eurasia demonstrated that the distribution of the mutation in the region of interest was not uniform and that there were the areas of H63D accumulation. The investigation of three polymorphic variants, c.340+4T>C (rs2071303, IVS2(+4)T>C), c.893-44T>C (rs1800708, IVS4(-44)T>C), and c.1007-47G>A (rs1572982, IVS5(-47)A>G), in the HFE gene in individuals homozygous for H63D mutations in the HFE gene revealed the linkage of H63D with three haplotypes, *CTA, *TG, and *TTA. These findings indicated the partial spread of the mutation in Central Eurasia from Western Europe, as well as the possible repeated appearance of the mutation on the territory on interest.

[Hereditary haemochromatosis].

PubMed

Bismuth, Michael; Aguilar-Martinez, Patricia; Michel, Henri

2003-11-08

EPIDEMIOLOGY ADN PHYSIOPATHOLOGY: Hereditary haemochromatosis is the most common genetic disease in France. Its frequency is on average 1 out of 300 French individuals. It is due to excessive dietary iron absorption, leading to accumulation of iron in the body. Mutations of the HFE1 gene are responsible for the majority of the case of haemochromatosis. FROM A CLINICAL POINT OF VIEW: The first clinical manifestations (weakness, sexual dysfunction, arthralgia, cardiac symptoms, dyspnoea on effort) can occur after the age of 30 years in men and 35 years in women (protected for longer by menstruation, pregnancy and delivery). In the absence of diagnosis, severe complications can develop during the 5th decade: nervous breakdown, arthropathy, heart failure, diabetes mellitus, cirrhosis with risk of progression towards carcinoma, responsible for handicaps and premature death. DIAGNOSTIC ELEMENTS: The diagnosis is evoked in the case of an increase in transferrine saturation (>45%), associated or not with excessive ferritin plasma levels. It is confirmed by the genetic test, showing homozygotes for the C282Y mutation or compound heterozygotes for the C282Y and H63D mutations on the HFE1 gene. RMI quantifies hepatic iron loading and generally avoids the need for a liver biopsy. The differential diagnosis must exclude secondary iron overload due to chronic transfusions in congenital or acquired blood diseases, a polymetabolic syndrome, chronic viral or alcoholic hepatic diseases and porphyria cutanea tarda. EFFICIENT TREATMENT: Today, haemochromatosis is still treated by phlebotomy. This consists in withdrawing 400 to 500ml of blood every week at the initial depletion stage and subsequently a maintenance therapy in order to maintain ferritin levels below 50 ng/ml. Paradoxically and through ignorance, hereditary haemochromatosis remains a serious disease, although its diagnosis is easy and the treatment simple and effective.

Bloodletting therapy in hemochromatosis: Does it affect trace element homeostasis?

PubMed

Bolann, Bjørn J; Distante, Sonia; Mørkrid, Lars; Ulvik, Rune J

2015-01-01

Hemochromatosis is the most common hereditary disorder in the Nordic population, if left untreated it can result in severe parenchymal iron accumulation. Bloodletting is mainstay treatment. Iron and trace elements partially share cellular uptake and transport mechanisms, and the aim of the present study was to see if bloodletting for hemochromatosis affects trace elements homeostasis. We recruited patients referred for diagnosis and treatment of hemochromatosis, four women and 22 men 23-68 years of age. Thirteen were C282Y homozygote, one was C282Y heterozygote, three were H63D homozygote, seven were compound heterozygote and two had none of the mutations above. Iron and liver function tests were performed; serum levels of trace elements were measured using inductively coupled plasma mass spectrometry. Results before the start of treatment and after normalization of iron parameters were compared. On completion of the bloodlettings the following average serum concentrations increased: Co from 5.6 to 11.5 nmol/L, serum Cu 16.2-17.6 μmol/L, Ni increased from 50.0 to 52.6 nmol/L and Sb from 13.2 to 16.3 nmol/L. Average serum Mn concentration declined from 30.2 to 28.3 nmol/L. All changes were statistically significant (by paired t-test). B, Ba, Cs, Mo, Se, Sr and Zn were not significantly changed. We conclude that bloodlettings in hemochromatosis lead to changes in trace element metabolism, including increased absorption of potentially toxic elements. Copyright © 2014 Elsevier GmbH. All rights reserved.

HFE gene mutation and oxidative damage biomarkers in patients with myelodysplastic syndromes and its relation to transfusional iron overload: an observational cross-sectional study.

PubMed

De Souza, Geane Felix; Ribeiro, Howard Lopes; De Sousa, Juliana Cordeiro; Heredia, Fabíola Fernandes; De Freitas, Rivelilson Mendes; Martins, Manoel Ricardo Alves; Gonçalves, Romélia Pinheiro; Pinheiro, Ronald Feitosa; Magalhães, Silvia Maria Meira

2015-04-03

A relation between transfusional IOL (iron overload), HFE status and oxidative damage was evaluated. An observational cross-sectional study involving 87 healthy individuals and 78 patients with myelodysplastic syndromes (MDS) with and without IOL, seen at University Hospital of the Federal University of Ceará, Brazil, between May 2010 and September 2011. IOL was defined using repeated measures of serum ferritin ≥1000 ng/mL. Variations in the HFE gene were investigated using PCR/restriction fragment length polymorphism (RFLP). The biomarkers of oxidative stress (plasmatic malonaldehyde (MDA), glutathione peroxidase (GPx) and superoxide dismutase (SOD)) were determined by spectrophotometry. The HFE gene variations were identified in 24 patients (30.77%) and 5 volunteers (5.74%). The H63D variant was observed in 35% and the C282Y variant as heterozygous in 5% of patients with MDS with IOL. One patient showed double heterozygous variant (C282Y/H63D) and serum ferritin of 11,649 ng/mL. In patients without IOL, the H63D variant was detected in 29.34%. Serum MDA levels were highest in patients with MDS with IOL, with a significant difference when compared with patients without IOL and healthy volunteers, pointing to the relationship between IOL and oxidative stress. The GPx and SOD were also significantly higher in these patients, indicating that lipid peroxidation increase was followed by an increase in antioxidant capacity. Higher ferritin levels were observed in patients with HFE gene variation. 95.7% of patients with MDS with the presence of HFE gene variations had received more of 20 transfusions. We observed a significant increase in MDA levels in patients with MDS and IOL, suggesting an increased lipid peroxidation in these patients. The accumulation of MDA alters the organisation of membrane phospholipids, contributing to the process of cellular degeneration. Results show that excess iron intensifies the process of cell damage through oxidative stress. Local Ethics Committee (licence 150/2009). Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

The evolutionary adaptation of the C282Y mutation to culture and climate during the European Neolithic.

PubMed

Heath, Kathleen M; Axton, Jacob H; McCullough, John M; Harris, Nathan

2016-05-01

The C282Y allele is the major cause of hemochromatosis as a result of excessive iron absorption. The mutation arose in continental Europe no earlier than 6,000 years ago, coinciding with the arrival of the Neolithic agricultural revolution. Here we hypothesize that this new Neolithic diet, which originated in the sunny warm and dry climates of the Middle East, was carried by migrating farmers into the chilly and damp environments of Europe where iron is a critical micronutrient for effective thermoregulation. We argue that the C282Y allele was an adaptation to this novel environment. To address our hypothesis, we compiled C282Y allele frequencies, known Neolithic sites in Europe and climatic data on temperature and rainfall for statistical analysis. Our findings indicate that the geographic cline for C282Y frequency in Europe increases as average temperatures decrease below 16°C, a critical threshold for thermoregulation, with rainy days intensifying the trend. The results indicate that the deleterious C282Y allele, responsible for most cases of hemochromatosis, may have evolved as a selective advantage to culture and climate during the European Neolithic. © 2016 The Authors American Journal of Physical Anthropology Published by Wiley Periodicals, Inc.

Potential nonresponse bias in a clinical examination after initial screening using iron phenotyping and HFE genotyping in the hemochromatosis and iron overload screening study.

PubMed

Speechley, Mark; Barton, James C; Passmore, Leah; Harrison, Helen; Reboussin, David M; Harris, Emily L; Rivers, Charles A; Fadojutimi-Akinsiku, Margaret; Wenzel, Lari; Diaz, Sharmin

2009-12-01

Little is known about the factors affecting participation in clinical assessments after HEmochromatosis and IRon Overload Screening. Initial screening of 101,168 primary care patients in the HEmochromatosis and IRon Overload Screening study was performed using serum iron measures and hemochromatosis gene (HFE) genotyping. Using iron phenotypes and HFE genotypes, we identified 2256 cases and 1232 controls eligible to participate in a clinical examination. To assess the potential for nonresponse bias, we compared the sociodemographic, health status, and attitudinal characteristics of participants and nonparticipants using adjusted odds ratios (ORs) and 95% confidence interval (CI). Overall participation was 74% in cases and 52% in controls; in both groups, participation was highest at a health maintenance organization and lowest among those under 45 years of age (cases: OR = 0.68; 95% CI 0.53, 0.87; controls: OR = 0.59; 95% CI 0.44, 0.78). In controls only, participation was also lower among those over 65 years of age than the reference group aged 46-64 (OR = 0.64; 95% CI 0.47, 0.88). Among cases, participation was higher in HFE C282Y homozygotes (OR = 3.98; 95% CI 2.60, 6.09), H63D homozygotes (OR = 2.79; 95% CI 1.23, 6.32), and C282Y/H63D compound heterozygotes (OR = 1.82; 95% CI 1.03, 3.22) than in other genotypes, and lower among non-Caucasians and those who preferred a non-English language than in Caucasians and those who preferred English (p < 0.0001). Subjects with greatest risk to have iron overload (C282Y homozygotes; cases > or =45 years; Caucasians) were more likely to participate in a postscreening clinical examination than other subjects. We detected no evidence of strong selection bias.

[Molecular genetic analysis and clinical aspects of patients with hereditary hemochromatosis].

PubMed

Lange, U; Teichmann, J; Dischereit, G

2014-08-01

The purpose of the study was to perform a molecular genetic analysis and to document clinical aspects in patients with hereditary hemochromatosis. The study included 33 outpatients (23 males average age 50.6 years and 10 females average age 60.6 years) with a disorder of iron metabolism (transferrin saturation > 75 %) as confirmation of hemochromatosis who were subjected to molecular genetic and clinical analyses. A homozygous mutation of the hemochromatosis (HFE) gene (C282YY) was detected in 63.6 %, a compound heterozygous mutation (C282Y/H63D) in 30.3% and no mutation of the HFE gene was detected in 6.1 %. The following organ manifestations could be objectified: arthralgia (78.8 %), liver disease (39.9 %), skin hyperpigmentation (30.3 %), osteoporosis (24.2 %), diabetes mellitus (24.2 %) and cardiomyopathy (12.1 %). Comparison between patients with heterozygous and homozygous hemochromatosis revealed the following differences: compound heterozygote patients presented less frequently with osteoarthritis of the metacarpophalangeal (MCP) joints and hands (85.7 %/71.4 % homozygotes vs. 60 %/60 % heterozygotes). Osteoarthritis of the shoulder joints and osteoporosis as well as hypothyroidism were more frequent in compound heterozygote patients, whereas osteoarthritis of the knee and hip joints as well as liver disease were more common in homozygote patients. No differences between both groups were seen with respect to the clinical manifestations of cardiomyopathy and diabetes mellitus. Prevalent causes of death in hereditary hemochromatosis are heart failure, liver disease (cirrhosis and hepatocellular carcinoma) and portal hypertension. Therefore, an early diagnosis, adequate therapy and genetic screening of family members are of great importance. Medicinal treatment will only effectively prevent deleterious organ involvement and subsequent complications if initiated at an early stage. Furthermore, an overview of the current data is given.

Clinical expression of haemochromatosis in Irish C282Y homozygotes identified through family screening.

PubMed

Gleeson, F; Ryan, E; Barrett, S; Crowe, J

2004-09-01

In Ireland, the homozygote frequency of the C282Y mutation in the HFE gene is 1/83. The biochemical expression of this mutation is high in haemochromatosis (HH) individuals identified through family screening, but the clinical expression of the mutation in Irish HH subjects to date has not been investigated fully. To determine the clinical, biochemical and histological penetrance of the C282Y mutation in Irish C282Y homozygotes identified through family screening. Two hundred and nine C282Y homozygous individuals comprising of 172 first-degree relatives, 31 second-degree relatives and four unrelated individuals were identified following HFE mutation analysis of 167 families. The following variables were analysed: age at identification, gender, fasting transferrin saturation, fasting serum ferritin, liver enzymes, clinical symptomatology, liver histopathology and histochemical iron staining. An elevated transferrin saturation in combination with an elevated ferritin was present in 43.4% of males and 23.3% of females. Abnormal liver enzymes were found in 32.3% of males. Diabetes, a haemochromatosis-specific association, was noted in 2.8% of males. Of those individuals requiring liver histopathology evaluation, 38% had moderate-to-severe iron staining, and 42% had fibrosis; 2.8% of the biopsied cohort had cirrhosis. Thus, HH cirrhotics were identified in less than 1% of the screened population. Although the homozygote frequency in Ireland is very high, the prevalence of advanced liver disease was less than 1% of the family members screened. Nevertheless, 42% of biopsied patients had histological evidence of iron overload-related architectural change and 2.8% had cirrhosis. This cohort of young people had previously unrecognized biochemical iron overload and histopathological change. This emphasizes the importance and value of both genetic and biochemical screening in first-degree relatives of identified homozygotes.

Hepatitis C Virus Nucleotide Inhibitors PSI-352938 and PSI-353661 Exhibit a Novel Mechanism of Resistance Requiring Multiple Mutations within Replicon RNA▿†

PubMed Central

Lam, Angela M.; Espiritu, Christine; Bansal, Shalini; Micolochick Steuer, Holly M.; Zennou, Veronique; Otto, Michael J.; Furman, Phillip A.

2011-01-01

PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methylguanosine-5′-monophosphate. Both compounds are metabolized to the same active 5′-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2′-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2′-F-2′-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2′-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type. PMID:21957306

High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

PubMed Central

Medintz, Igor; Wong, Wendy W.; Berti, Lorenzo; Shiow, Lawrence; Tom, Jennifer; Scherer, James; Sensabaugh, George; Mathies, Richard A.

2001-01-01

An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population. PMID:11230165

High resolution melting analysis to genotype the most common variants in the HFE gene.

PubMed

Marotta, Roberta V; Turri, Olivia; Morandi, Antonella; Murano, Manuela; d'Eril, Gianlodovico Melzi; Biondi, Maria Luisa

2011-09-01

High resolution melting (HRM) analysis of PCR amplicons was recently introduced as a closed-tube, rapid, and inexpensive method of genotyping. This study evaluated this system as an option for detecting the three most common mutations in the HFE gene (C282Y, H63D, S65C), accounting for the main form of hereditary haemochromatosis. Ninety samples, previously screened by direct sequencing, and 27 controls were used. The analysis were performed on the Rotor Gene Q, using the commercial HRM mix containing the Eva Green dye (Qiagen). Specific primers allowed the amplification of the regions of interest in the HFE gene. Following amplification, a HRM analysis was conducted to detect DNA variants. The thermal denaturation profiles of the samples were compared with those of the controls. One hundred percent of heterozygous and homozygous samples were readily identified. Heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves, but significant differences were also present in the melting curves of the homozygous carries compared with those of the wild-type subjects. HRM analysis is an appealing technology for HFE gene screening. It is a robust technique that can be widely adopted in diagnostic laboratories to facilitate gene mutation screening.

Common founder effects of hereditary hemochromatosis, Wilson´s disease, the long QT syndrome and autosomal recessive deafness caused by two novel mutations in the WHRN and TMC1 genes.

PubMed

Olsson, K Sigvard; Wålinder, Olof; Jansson, Ulf; Wilbe, Maria; Bondeson, Marie-Louise; Stattin, Eva-Lena; Raha-Chowdhury, Ruma; Williams, Roger

2017-01-01

Genealogy and molecular genetic studies of a Swedish river valley population resulted in a large pedigree, showing that the hereditary hemochromatosis (HH) HFE/p. C282Y mutation is inherited with other recessive disorders such as Wilson´s disease (WND), a rare recessive disorder of copper overload. The population also contain individuals with the Swedish long QT syndrome (LQTS1) founder mutation ( KCNQ1 /p.Y111C) which in homozygotes causes the Jervell & Lange Nielsen syndrome (JLNS) and hearing loss (HL).Aims of the study were to test whether the Swedish long QT founder mutation originated in an ancestral HFE family and if carriers had an increased risk for hemochromatosis (HH), a treatable disorder. We also aimed to identify the pathogenic mutation causing the hearing loss disorder segregating in the pedigree. LQTS patients were asked about their ancestry and possible origin in a HH family. They were also offered a predictive testing for the HFE genotype. Church books were screened for families with hearing loss. One HH family had two members with hearing loss, who underwent molecular genetic analysis of the LQTS founder mutation, connexin 26 and thereafter exome sequencing. Another family with hearing loss in repeat generations was also analyzed for connexin 26 and underwent exome sequencing. Of nine LQTS patients studied, four carried a HFE mutation (two p.C282Y, two p.H63D), none was homozygous. Three LQTS patients confirmed origin in a female founder ( b 1694, identical to AJ b 1694, a HFE pedigree member from the Fax river. Her descent of 44 HH families, included also 29 families with hearing loss (HL) suggesting JLNS. Eleven LQTS probands confirmed origin in a second founder couple (b 1614/1605) in which the woman b 1605 was identical to a HFE pedigree member from the Fjällsjö river. In her descent there were not only 64 HH, six WND families, one JLNS, but also 48 hearing loss families. Most hearing loss was non syndromic and caused by founder effects of the late 16 th century. One was of Swedish origin carrying the WHRN, c.1977delC, (p.S660Afs*30) mutation, the other was a TMC1 (NM_138691),c.1814T>C,(p.L605P) mutation, possibly of Finnish origin. Deep human HFE genealogies show HFE to be associated with other genetic disorders like Wilson´s disease, LQTS, JLNS, and autosomal recessive hearing loss. Two new homozygous HL mutations in WHRN /p.S660Afs*30 and TMC1/ p.L605P were identified,none of them previously reported from Scandinavia. The rarity of JLNS was possibly caused by miscarriage or intrauterine death. Most hearing loss (81.7%) was seen after 1844 when first cousin marriages were permitted. However, only 10 (10.3%) came from 1 st cousin unions and only 2 (2.0 %) was born out of wedlock.

HFE Cys282Tyr homozygotes with serum ferritin concentrations below 1000 microg/L are at low risk of hemochromatosis.

PubMed

Allen, Katrina J; Bertalli, Nadine A; Osborne, Nicholas J; Constantine, Clare C; Delatycki, Martin B; Nisselle, Amy E; Nicoll, Amanda J; Gertig, Dorota M; McLaren, Christine E; Giles, Graham G; Hopper, John L; Anderson, Gregory J; Olynyk, John K; Powell, Lawrie W; Gurrin, Lyle C

2010-09-01

Hemochromatosis gene (HFE)-associated hereditary hemochromatosis (HH) is a genetic predisposition to iron overload and subsequent signs and symptoms of disease that potentially affects approximately 80,000 persons in Australia and almost 1 million persons in the United States. Most clinical cases are homozygous for the Cys282Tyr (C282Y) mutation in the HFE gene, with serum ferritin (SF) concentration >1000 microg/L as the strongest predictor of cirrhosis. The optimal treatment regimen for those with SF concentrations above the normal range but <1000 microg/L is unknown. We assessed HFE mutations in a prospective cohort of 31,192 participants of northern European descent, aged 40-69 years. An HFE-stratified random sample of 1438 participants including all C282Y homozygotes with iron studies 12 years apart were examined by physicians blinded to participants' HFE genotype. All previously undiagnosed C282Y homozygotes (35 male, 67 female) and all HFE wild-types (131 male, 160 female) with baseline and follow-up SF concentrations <1000 microg/L were assessed for HH-associated signs and symptoms including abnormal second/third metacarpophalangeal joints (MCP2/3), raised liver enzymes, hepatomegaly, and self-reported liver disease, fatigue, diabetes mellitus, and use of arthritis medication. The prevalence of HH-associated signs and symptoms was similar for C282Y homozygotes and HFE wild-types for both normal and moderately elevated SF concentrations. The maximum prevalence difference between HFE genotype groups with moderately elevated SF was 11% (MCP2/3, 95% confidence interval = -6%, 29%; P = 0.22) and for normal SF was 6% (arthritis medicine use, 95% confidence interval = -3%, 16%; P = 0.11). Previously undiagnosed C282Y homozygotes with SF concentrations that remain below 1000 microg/L are at low risk of developing HH-associated signs and symptoms at an age when disease would be expected to have developed. These observations have implications for the management of C282Y homozygotes.

Diagnosis of hepatic iron overload: a family study illustrating pitfalls in diagnosing hemochromatosis.

PubMed

Schranz, Melanie; Talasz, Heribert; Graziadei, Ivo; Winder, Thomas; Sergi, Consolato; Bogner, Klaus; Vogel, Wolfgang; Zoller, Heinz

2009-03-01

Recent identification of genetic variants in iron storage disease has changed the classification system and diagnostic algorithms for hemochromatosis. Clinical diagnosis of the disease requires phenotypic evidence of iron overload because the commonly disease-associated HFE genotypes have an incomplete penetrance. Furthermore, approximately 20% of patients with a clinical diagnosis of hemochromatosis have no disease-associated genotype, which underlines the importance of clear phenotypic criteria of hemochromatosis. A diagnosis of hemochromatosis cannot be made even in patients with liver cirrhosis simply on the basis of genetic testing that indicates that iron overload is the cause of the disease and not its consequence. Proper diagnosis requires integration of clinical presentation, family history, and the results of biochemical and histopathologic tests. Here we propose a rational diagnostic algorithm for hepatic iron overload syndromes and illustrate potential pitfalls by presenting a family study in a pedigree with rare HFE variants (H63D and E168Q), in cis on the same chromosome. Although the clinical suspicion of hemochromatosis was confirmed by histology, chemical analysis of liver tissue revealed a normal hepatic iron concentration, which is compatible with the genetic finding of 1 normal and 1 doubly mutated allele. In conclusion, clinical suspicion of hemochromatosis and elevated serum iron parameters should prompt HFE genotyping for C282Y and H63D. Should they be uninformative, further genetic tests should be recommended only if iron overload in liver tissue has been confirmed chemically.

An extensive analysis of the hereditary hemochromatosis gene HFE and neighboring histone genes: associations with childhood leukemia.

PubMed

Davis, Charronne F; Dorak, M Tevfik

2010-04-01

The most common mutation of the HFE gene C282Y has shown a risk association with childhood acute lymphoblastic leukemia (ALL) in Welsh and Scottish case-control studies. This finding has not been replicated outside Britain. Here, we present a thorough analysis of the HFE gene in a panel of HLA homozygous reference cell lines and in the original population sample from South Wales (117 childhood ALL cases and 414 newborn controls). The 21 of 24 variants analyzed were from the HFE gene region extending 52 kb from the histone gene HIST1H1C to HIST1H1T. We identified the single-nucleotide polymorphism (SNP) rs807212 as a tagging SNP for the most common HFE region haplotype, which contains wild-type alleles of all HFE variants examined. This intergenic SNP rs807212 yielded a strong male-specific protective association (per allele OR = 0.38, 95% CI = 0.22-0.64, P (trend) = 0.0002; P = 0.48 in females), which accounted for the original C282Y risk association. In the HapMap project data, rs807212 was in strong linkage disequilibrium with 25 other SNPs spanning 151 kb around HFE. Minor alleles of these 26 SNPs characterized the most common haplotype for the HFE region, which lacked all disease-associated HFE variants. The HapMap data suggested positive selection in this region even in populations where the HFE C282Y mutation is absent. These results have implications for the sex-specific associations observed in this region and suggest the inclusion of rs807212 in future studies of the HFE gene and the extended HLA class I region.

HFE interacts with the BMP type I receptor ALK3 to regulate hepcidin expression

PubMed Central

Wu, Xing-gang; Wang, Yang; Wu, Qian; Cheng, Wai-Hang; Liu, Wenjing; Zhao, Yueshui; Mayeur, Claire; Schmidt, Paul J.; Yu, Paul B.; Wang, Fudi

2014-01-01

Mutations in HFE are the most common cause of hereditary hemochromatosis (HH). HFE mutations result in reduced expression of hepcidin, a hepatic hormone, which negatively regulates iron absorption from the duodenum and iron release from macrophages. However, the mechanism by which HFE regulates hepcidin expression in hepatocytes is not well understood. It is known that the bone morphogenetic protein (BMP) pathway plays a central role in controlling hepcidin expression in the liver. Here we show that HFE overexpression increased Smad1/5/8 phosphorylation and hepcidin expression, whereas inhibition of BMP signaling abolished HFE-induced hepcidin expression in Hep3B cells. HFE was found to associate with ALK3, inhibiting ALK3 ubiquitination and proteasomal degradation and increasing ALK3 protein expression and accumulation on the cell surface. The 2 HFE mutants associated with HH, HFE C282Y and HFE H63D, regulated ALK3 protein ubiquitination and trafficking differently, but both failed to increase ALK3 cell-surface expression. Deletion of Hfe in mice resulted in a decrease in hepatic ALK3 protein expression. Our results provide evidence that HFE induces hepcidin expression via the BMP pathway: HFE interacts with ALK3 to stabilize ALK3 protein and increase ALK3 expression at the cell surface. PMID:24904118

[Mutation analysis of the PAH gene in children with phenylketonuria from the Qinghai area of China].

PubMed

He, Jiang; Wang, Hui-Zhen; Xu, Fa-Liang; Yang, Xi; Wang, Rui; Zou, Hong-Yun; Yu, Wu-Zhong

2015-11-01

To study the mutation characteristics of the phenylalanine hydroxylase (PAH) gene in children with phenylketonuria (PKU) from the Qinghai area of China, in order to provide basic information for genetic counseling and prenatal diagnosis. Mutations of the PAH gene were detected in the promoter and exons 1-13 and their flanking intronic sequences of PAH gene by PCR and DNA sequencing in 49 children with PKU and their parents from the Qinghai area of China. A total of 30 different mutations were detected in 80 out of 98 mutant alleles (82%), including 19 missense (63%), 5 nonsense (17%), 3 splice-site (10%) and 3 deletions (10%). Most mutations were detected in exons 3, 6, 7, 11 and intron 4 of PAH gene. The most frequent mutations were p.R243Q (19%), IVS4-1G>A (9%), p.Y356X (7%) and p.EX6-96A>G(5%). Two novel mutations p.N93fsX5 (c.279-282delCATC) and p.G171E (c.512G>A) were found. p.H64fsX9(c.190delC) was documented for the second time in Chinese PAH gene. The mutation spectrum of the gene PAH in the Qinghai population was similar to that in other populations in North China while significantly different from that in the populations from some provinces in southern China, Japan and Europe. The mutations of PAH gene in the Qinghai area of China demonstrate a unique diversity, complexity and specificity.

Coinheritance of hereditary spherocytosis and reversibility of cirrhosis in a young female patient with hereditary hemochromatosis.

PubMed

Höblinger, A; Erdmann, C; Strassburg, C P; Sauerbruch, T; Lammert, F

2009-04-16

Here we report a 33-years-old woman with hereditary spherocytosis and hemochromatosis due to homozygosity for the C282Y mutation of the HFE gene. The coinheritance of both conditions led to severe iron overload and liver cirrhosis at young age. The patient was treated by repeated phlebotomy, and reversibility of cirrhosis was documented by transient elastography. This report discusses the pathophysiology of iron accumulation in patients with hemolytic anemia combined with HFE C282Y homozygosity. The case indicates that patients with hematological disorders characterized by increased erythropoetic activity should be screened for HFE mutations.

Clinical expression of C282Y homozygous HFE haemochromatosis at 14 years of age.

PubMed

Rossi, Enrico; Wallace, Daniel F; Subramaniam, V Nathan; St Pierre, Timothy G; Mews, Catherine; Jeffrey, Gary P

2006-05-01

A 14-year-old boy who presented with debilitating lethargy was shown to have an elevated serum ferritin of 572 microg/L and a C282Y homozygous HFE genotype. Liver iron concentration was measured non-invasively by magnetic resonance imaging, which revealed a liver iron concentration of 59 micromol/g dry weight (children's reference range < 14). The early phenotypic expression was further investigated by screening genomic DNA for the presence of co-inherited mutations in genes responsible for non-HFE haemochromatosis. Coding regions and splice sites in genes encoding hepcidin and haemojuvelin were sequenced and previously described mutations in ferroportin 1 and transferrin receptor 2 genes were screened. Although no mutations were found, the most likely cause for the early expression is the presence of novel mutations or gene(s).

The natural history of serum iron indices for HFE C282Y homozygosity associated with hereditary hemochromatosis.

PubMed

Gurrin, Lyle C; Osborne, Nicholas J; Constantine, Clare C; McLaren, Christine E; English, Dallas R; Gertig, Dorota M; Delatycki, Martin B; Southey, Melissa C; Hopper, John L; Giles, Graham G; Anderson, Gregory J; Olynyk, John K; Powell, Laurie W; Allen, Katrina J

2008-12-01

There are few longitudinal studies of serum ferritin (SF) and transferrin saturation (TS) levels in individuals homozygous for the C282Y mutation. We characterized the development of elevated iron measures in C282Y homozygotes followed for 12 years. From 31,192 people aged 40-69 years at baseline, we identified 203 C282Y homozygotes (95 males), of whom 116 had SF and fasting TS levels measured at baseline (mean age, 55 years) and 86 were untreated and had iron measures at follow-up (mean, 12 years later). The probabilities of SF at follow-up exceeding clinical thresholds were predicted from baseline SF and TS under a multivariate normal model. For C282Y homozygotes, at baseline, 84% of males and 65% of females had elevated SF and 37% of males and 3% of females had SF >1000 microg/L. For males with SF 300-1000 microg/L at baseline, the predicted probability of progressing to SF >1000 microg/L at follow-up was between 13% and 35% and, for females, between 16% and 22%. For C282Y homozygotes with normal baseline SF, <15% were predicted to develop SF >1000 microg/L if left untreated. The majority of C282Y homozygotes who are likely to develop SF levels sufficient to place them at risk of iron overload-related disease will have done so by mean age 55 years. TS >95% at mean age 55 years in males increases the likelihood that SF levels will be elevated at mean age 65 years, but this effect is absent in females, most likely because of physiologic blood loss associated with menstruation.

Mutant HFE H63D Protein Is Associated with Prolonged Endoplasmic Reticulum Stress and Increased Neuronal Vulnerability*

PubMed Central

Liu, Yiting; Lee, Sang Y.; Neely, Elizabeth; Nandar, Wint; Moyo, Mthabisi; Simmons, Zachary; Connor, James R.

2011-01-01

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability. PMID:21349849

Higher ferritin levels, but not serum iron or transferrin saturation, are associated with Type 2 diabetes mellitus in adult men and women free of genetic haemochromatosis.

PubMed

Yeap, Bu B; Divitini, Mark L; Gunton, Jenny E; Olynyk, John K; Beilby, John P; McQuillan, Brendan; Hung, Joseph; Knuiman, Matthew W

2015-04-01

Iron overload predisposes to diabetes and higher ferritin levels have been associated with diabetes. However, it is unclear whether ferritin reflects differences in iron-related parameters between diabetic and nondiabetic persons. We examined associations of serum ferritin, iron and transferrin saturation with Type 2 diabetes in adults without genetic predisposition to iron overload. Cross-sectional analysis of community-dwelling men and women aged 17-97 years from the Busselton Health Survey, Western Australia. Men and women carrying genotypes associated with haemochromatosis (C282Y/C282Y or C282Y/H63D) were excluded. Serum ferritin, iron and transferrin saturation were assayed. There were 1834 men (122 with diabetes, 6·6%) and 2351 women (141 with diabetes, 6%). In men, higher serum ferritin was associated with diabetes after adjusting for age, smoking, alcohol, cardiovascular history, body mass index (BMI), waist, blood pressure, lipids, C-reactive protein (CRP), adiponectin, alanine transaminase (ALT) and gamma-glutamyl transpeptidase (GGT) [odds ratio (OR): 1·29 per 1 unit increase log ferritin, 95% confidence interval (CI) = 1·01-1·65, P = 0·043]. In women, higher serum ferritin was associated with diabetes [fully adjusted OR: 1·31 per 1 unit increase log ferritin, 95% CI = 1·04-1·63, P = 0·020; 1·84 for tertile (T) 3 vs T1, 95% CI = 1·09-3·11]. Neither iron levels nor transferrin saturation were associated with diabetes risk in men or women. Higher ferritin was not associated with insulin resistance in nondiabetic adults. In adults, higher ferritin levels are independently associated with prevalent diabetes while iron and transferrin saturation are not. Ferritin is a robust biomarker for diabetes risk, but further investigation is needed to clarify whether this relationship is mediated via iron metabolism. © 2014 John Wiley & Sons Ltd.

Selectivity in the Use of Gi/o Proteins Is Determined by the DRF Motif in CXCR6 and Is Cell-Type Specific.

PubMed

Singh, Satya P; Foley, John F; Zhang, Hongwei H; Hurt, Darrell E; Richards, Jennifer L; Smith, Craig S; Liao, Fang; Farber, Joshua M

2015-11-01

CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the D(3.49)R(3.50)Y(3.51) sequence, and all but two contain A(3.53) at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein-coupled receptors that contacts G proteins. In CXCR6, H3C contains D(3.49)R(3.50)F(3.51)I(3.52)V(3.53) at positions 126-130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individual Gi/o proteins; and the effects of mutations in H3C on receptor function and selectivity in Gi/o protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments. U.S. Government work not protected by U.S. copyright.

Selectivity in the Use of Gi/o Proteins Is Determined by the DRF Motif in CXCR6 and Is Cell-Type Specific

PubMed Central

Singh, Satya P.; Foley, John F.; Zhang, Hongwei H.; Hurt, Darrell E.; Richards, Jennifer L.; Smith, Craig S.; Liao, Fang

2015-01-01

CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the D3.49R3.50Y3.51 sequence, and all but two contain A3.53 at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein–coupled receptors that contacts G proteins. In CXCR6, H3C contains D3.49R3.50F3.51I3.52V3.53 at positions 126–130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individual Gi/o proteins; and the effects of mutations in H3C on receptor function and selectivity in Gi/o protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments. PMID:26316539

Computational screening and molecular dynamics simulation of disease associated nsSNPs in CENP-E.

PubMed

Kumar, Ambuj; Purohit, Rituraj

2012-01-01

Aneuploidy and chromosomal instability (CIN) are hallmarks of most solid tumors. Mutations in centroemere proteins have been observed in promoting aneuploidy and tumorigenesis. Recent studies reported that Centromere-associated protein-E (CENP-E) is involved in inducing cancers. In this study we investigated the pathogenic effect of 132 nsSNPs reported in CENP-E using computational platform. Y63H point mutation found to be associated with cancer using SIFT, Polyphen, PhD-SNP, MutPred, CanPredict and Dr. Cancer tools. Further we investigated the binding affinity of ATP molecule to the CENP-E motor domain. Complementarity scores obtained from docking studies showed significant loss in ATP binding affinity of mutant structure. Molecular dynamics simulation was carried to examine the structural consequences of Y63H mutation. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (R(g)), solvent accessibility surface area (SASA), energy value, hydrogen bond (NH Bond), eigenvector projection, trace of covariance matrix and atom density analysis results showed notable loss in stability for mutant structure. Y63H mutation was also shown to disrupt the native conformation of ATP binding region in CENP-E motor domain. Docking studies for remaining 18 mutations at 63rd residue position as well as other two computationally predicted disease associated mutations S22L and P69S were also carried to investigate their affect on ATP binding affinity of CENP-E motor domain. Our study provided a promising computational methodology to study the tumorigenic consequences of nsSNPs that have not been characterized and clear clue to the wet lab scientist. Copyright © 2012 Elsevier B.V. All rights reserved.

Pathophysiology of Hereditary Hemochromatosis

PubMed Central

Fleming, Robert E.; Britton, Robert S.; Waheed, Abdul; Sly, William S.; Bacon, Bruce R.

2008-01-01

Hereditary hemochromatosis (HH) encompasses several inherited disorders of iron homeostasis characterized by increased gastrointestinal iron absorption and tissue iron deposition. The most common form of this disorder is HFE-related HH, nearly always caused by homozygosity for the C282Y mutation. A substantial proportion of C282Y homozygotes do not develop clinically significant iron overload, suggesting roles for environmental factors and modifier genes in determining the phenotype. Recent studies have demonstrated that the pathogenesis of nearly all forms of HH involves inappropriately decreased expression of the iron-regulatory hormone hepcidin. Hepcidin serves to decrease the export of iron from reticuloendothelial cells and absorptive enterocytes. Thus, HH patients demonstrate increased iron release from these cell types, elevated circulating iron, and iron deposition in vulnerable tissues. The mechanism by which HFE influences hepcidin expression is an area of current investigation and may offer insights into the phenotypic variability observed in persons with mutations in HFE. PMID:16315135

Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D.

PubMed

Damnjanović, Jasmina; Takahashi, Rie; Suzuki, Atsuo; Nakano, Hideo; Iwasaki, Yugo

2012-08-01

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.

KIT Mutations Are Common in Testicular Seminomas

PubMed Central

Kemmer, Kathleen; Corless, Christopher L.; Fletcher, Jonathan A.; McGreevey, Laura; Haley, Andrea; Griffith, Diana; Cummings, Oscar W.; Wait, Cecily; Town, Ajia; Heinrich, Michael C.

2004-01-01

Expression of KIT tyrosine kinase is critical for normal germ cell development and is observed in the majority of seminomas. Activating mutations in KIT are common in gastrointestinal stromal tumors and mastocytosis. In this study we examined the frequency and spectrum of KIT mutations in 54 testicular seminomas, 1 ovarian dysgerminoma and 37 non-seminomatous germ cell tumors (NSGCT). Fourteen seminomas (25.9%) contained exon 17 point mutations including D816V (6 cases), D816H (3 cases), Y823D (2 cases), and single examples of Y823C, N822K, and T801I. No KIT mutations were found in the ovarian dysgerminoma or the NSGCTs. In transient transfection assays, mutant isoforms D816V, D816H, Y823D, and N822K were constitutively phosphorylated in the absence of the natural ligand for KIT, stem cell factor (SCF). In contrast, activation of T801I and wild-type KIT required SCF. Mutants N822K and Y823D were inhibited by imatinib mesylate (Gleevec, previously STI571) whereas D816V and D816H were both resistant to imatinib mesylate. Biochemical evidence of KIT activation, as assessed by KIT phosphorylation and KIT association with phosphatidylinositol (PI) 3-kinase in tumor cell lysates, was largely confined to seminomas with a genomic KIT mutation. These findings suggest that activating KIT mutations may contribute to tumorigenesis in a subset of seminomas, but are not involved in NSGCT. PMID:14695343

GNPAT p.D519G is independently associated with markedly increased iron stores in HFE p.C282Y homozygotes.

PubMed

Barton, James C; Chen, Wen-Pin; Emond, Mary J; Phatak, Pradyumna D; Subramaniam, V Nathan; Adams, Paul C; Gurrin, Lyle C; Anderson, Gregory J; Ramm, Grant A; Powell, Lawrie W; Allen, Katrina J; Phillips, John D; Parker, Charles J; McLaren, Gordon D; McLaren, Christine E

2017-03-01

GNPAT p.D519G positivity is significantly increased in HFE p.C282Y homozygotes with markedly increased iron stores. We sought to determine associations of p.D519G and iron-related variables with iron stores in p.C282Y homozygotes. We defined markedly increased iron stores as serum ferritin >2247pmol/L (>1000μg/L) and either hepatic iron >236μmol/g dry weight or iron >10g by induction phlebotomy (men and women). We defined normal or mildly elevated iron stores as serum ferritin <674.1pmol/L (<300μg/L) or either age≥40y with iron ≤2.5g iron by induction phlebotomy or age≥50y with ≤3.0g iron by induction phlebotomy (men only). We compared participant subgroups using univariate methods. Using multivariable logistic regression, we evaluated associations of markedly increased iron stores with these variables: age; iron supplement use (dichotomous); whole blood units donated; erythrocyte units received as transfusion; daily alcohol consumption, g; and p.D519G positivity (heterozygosity or homozygosity). The mean age of 56 participants (94.6% men) was 55±10 (SD) y; 41 had markedly increased iron stores. Prevalences of swollen/tender 2nd/3rd metacarpophalangeal joints and elevated aspartate or alanine aminotransferase were significantly greater in participants with markedly increased iron stores. Only participants with markedly increased iron stores had cirrhosis. In multivariable analyses, p.D519G positivity was the only exposure variable significantly associated with markedly increased iron stores (odds ratio 9.9, 95% CI [1.6, 60.3], p=0.0126). GNPAT p.D519G is strongly associated with markedly increased iron stores in p.C282Y homozygotes after correction for age, iron-related variables, and alcohol consumption. Copyright © 2016 Elsevier Inc. All rights reserved.

H63D mutation in hemochromatosis alters cholesterol metabolism and induces memory impairment.

PubMed

Ali-Rahmani, Fatima; Grigson, Patricia S; Lee, Sang; Neely, Elizabeth; Connor, James R; Schengrund, Cara-Lynne

2014-06-01

The H63D variant of the hemochromatosis (HFE) gene, when expressed in carriers of the apolipoprotein E4 allele, is implicated as a risk factor for earlier onset of Alzheimer's disease (AD). We tested the hypothesis that like expression of apolipoprotein E4, expression of H63D-HFE disrupts cholesterol metabolism contributing to an increase in neurodegeneration and memory deficits. Analysis of SH-SY5Y human neuroblastoma cells transfected to stably express either wild type- (WT) or H63D-HFE indicated about a 50% reduction in cholesterol content in cells expressing H63D-HFE. This was accompanied by a significant decrease in expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase, and a significant increase in expression of cholesterol 24-hydroxylase. Consistent with these studies, H67D-HFE (orthologous to human H63D-HFE) knock-in mice, showed a greater age dependent decline in brain cholesterol than WT-HFE animals and changes in expression of proteins regulating cholesterol metabolism. Brains of aged H67D-HFE mice also exhibited a significant decrease in expression of synapse proteins and a significant increase in caspase-3 expression relative to WT-HFE controls. H67D-HFE mice also had a greater reduction in brain volume and poorer recognition and spatial memory than WT-HFE mice, symptoms associated with AD. These results indicate that the alterations in cholesterol metabolism associated with expression of H63D-HFE may contribute to the development of AD. Copyright © 2014 Elsevier Inc. All rights reserved.

Haemochromatosis genotype and iron overload: association with hypertension and left ventricular hypertrophy.

PubMed

Ellervik, C; Tybjaerg-Hansen, A; Appleyard, M; Ibsen, H; Nordestgaard, B G

2010-09-01

We hypothesized that there is an association between haemochromatosis genotype C282Y/C282Y and/or iron overload and risk of hypertension and/or left ventricular hypertrophy (LVH). We analysed data from a cross-sectional study of the general population including 8992 individuals from the Copenhagen City Heart Study (CCHS), a follow-up study of 36,480 individuals from the Copenhagen General Population Study (CGPS), and a case-only study of 3815 Scandinavians from the Losartan Intervention For End-point Reduction in Hypertension Genetic Substudy (LIFEGEN) with LVH and hypertension. In the CCHS, individuals with C282Y/C282Y versus wild type/wild type had an odds ratio for antihypertensive medication use of 4.8 (1.8-13; P = 0.003). In the CGPS, the corresponding hazard ratio was 1.7 (1.0-2.3; P = 0.003). Also, hazard ratios for antihypertensive medication use in the CGPS were 1.6 (1.0-2.6; P = 0.05) for transferrin saturation > or =80% vs. <50%, and 2.3 (1.3-4.2; P = 0.005) for C282Y/C282Y + transferrin saturation > or =80% vs. wild type/wild type + transferrin saturation <50%. These results were most pronounced in men above 55 years of age. We did not find any association between C282Y/C282Y or iron overload and LVH or hypertension (measured as blood pressure at a single occasion or continuous blood pressure), or LVH with hypertension in the CCHS or with severity of LVH in LIFEGEN. We found that haemochromatosis genotype C282Y/C282Y and extremely elevated transferrin saturation either separately or combined were associated with increased risk of antihypertensive medication use. Therefore, testing for haemochromatosis genotype C282Y/C282Y and extreme transferrin saturation could be considered in patients with essential hypertension.

Morbidity and mortality in first-degree relatives of C282Y homozygous probands with clinically detected haemochromatosis compared with the general population: the HEmochromatosis FAmily Study (HEFAS).

PubMed

Jacobs, E M G; Hendriks, J C M; Marx, J J M; van Deursen, C Th B M; Kreeftenberg, H G; de Vries, R A; Stalenhoef, A F H; Verbeek, A L M; Swinkels, D W

2007-12-01

Family screening has been suggested as a sophisticated model for the early detection of HFE-related hereditary haemochromatosis (HH). However, until now, controlled studies on the morbidity and mortality in families with HH are lacking. Data on iron parameters, morbidity and mortality were collected from 224 dutch C282Y-homozygous probands with clinically overt HH and 735 of their first-degree family members, all participating in the HEmochromatosis fAmily study (HEfAs). These data were compared with results obtained from an age- and gender-matched normal population. HEfAs and controls filled in similar questionnaires on demographics, lifestyle factors, health, morbidity and mortality. A significantly higher proportion of the HEfAs first-degree family members reported to be diagnosed with haemochromatosis-related diseases: 45.7 vs 19.4% of the matched normal population (McNemar p<0.001). Mortality among siblings, children and parents in the HEFAS population was similar to that in the relatives of matched control. In this study we show that, morbidity among first-degree family members of C282Y-homozygous probands previously diagnosed with clinically proven HH is higher than that in an age- and gender-matched normal population. Further studies are needed to definitely connect these increase morbidity figures to increase prevalenc of the C282Y mutated HFE-gene and elevated serum iron indices.

A novel homozygous stop-codon mutation in human HFE responsible for nonsense-mediated mRNA decay.

PubMed

Padula, Maria Carmela; Martelli, Giuseppe; Larocca, Marilena; Rossano, Rocco; Olivieri, Attilio

2014-09-01

HFE-hemochromatosis (HH) is an autosomal disease characterized by excessive iron absorption. Homozygotes for H63D variant, and still less H63D heterozygotes, generally do not express HH phenotype. The data collected in our previous study in the province of Matera (Basilicata, Italy) underlined that some H63D carriers showed altered iron metabolism, without additional factors. In this study, we selected a cohort of 10/22 H63D carriers with severe biochemical iron overload (BIO). Additional analysis was performed for studying HFE exons, exon-intron boundaries, and untranslated regions (UTRs) by performing DNA extraction, PCR amplification and sequencing. The results showed a novel substitution (NM_000410.3:c.847C>T) in a patient exon 4 (GenBankJQ478433); it introduces a premature stop-codon (PTC). RNA extraction and reverse-transcription were also performed. Quantitative real-time PCR was carried out for verifying if our aberrant mRNA is targeted for nonsense-mediated mRNA decay (NMD); we observed that patient HFE mRNA was expressed much less than calibrator, suggesting that the mutated HFE protein cannot play its role in iron metabolism regulation, resulting in proband BIO. Our finding is the first evidence of a variation responsible for a PTC in iron cycle genes. The genotype-phenotype correlation observed in our cases could be related to the additional mutation. Copyright © 2014 Elsevier Inc. All rights reserved.

Two strategies to engineer flexible loops for improved enzyme thermostability

PubMed Central

Yu, Haoran; Yan, Yihan; Zhang, Cheng; Dalby, Paul A.

2017-01-01

Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of Escherichia coli transketolase (TK). The first was a “back to consensus mutations” approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes. PMID:28145457

Haplotype analysis of the HFE gene among populations of Northern Eurasia, in patients with metabolic disorders or stomach cancer, and in long-lived people.

PubMed

Mikhailova, S V; Babenko, V N; Ivanoshchuk, D E; Gubina, M A; Maksimov, V N; Solovjova, I G; Voevoda, M I

2016-06-17

Previously, it was shown that the HFE gene (associated with human hereditary hemochromatosis) has several haplotypes of intronic polymorphisms. Some haplotype frequencies are race specific and hence can be used in phylogenetic analysis. We assumed that analysis of Caucasoid patients-living now in Western Siberia and having diseases associated with dietary habits and metabolic rate-will allow us to understand the processes of possible selection during settling of the northern part of Asia. Haplotype analysis of Northern Eurasian native and recently settled ethnic groups was performed on polymorphisms rs1799945, rs1800730, rs1800562, rs2071303, rs1800708, rs1572982, rs2794719, rs807209, and rs2032451 of this gene. The CCA haplotype of the rs2071303, rs1800708, and rs1572982 was found to be associated with HLA-A2 (39 %) in Asian populations. Haplotype analysis for the rs1799945, rs1800730, rs1800562, rs2071303, rs1800708, and rs1572982 was performed on Russian patients with some metabolic disorders or stomach cancer and among long-lived people. Decreased frequencies of the TTA haplotype (T in rs2071303, T in rs1800708, and A in rs1572982) were observed in the groups of patients with diseases associated with overweight (fatty liver disease, type 2 diabetes mellitus, or metabolic syndrome + arterial hypertension) as compared with the control sample. We detected significant differences in this haplotype's frequency between the patients with type 2 diabetes mellitus and Russian adolescents, elderly citizens, and long-lived people (χ(2) P value = 0.003, 0.010, and 0.015, respectively). No significant differences in frequencies of the alleles with mutations in coding regions of the HFE gene (C282Y, H63D, and S65C) were detected between the analyzed patients (with stomach cancer, metabolic syndrome, fatty liver disease, or type 2 diabetes mellitus) and the control Caucasoid sample. Monophyletic origin of H63D (rs1799945) was confirmed in Caucasoids and Northern Asians. The reasons for a sharp increase in the frequency of CCA haplotype of HFE in the Asian race remain unclear.

[CCR5, CCR2, apoe, p53, ITGB3 and HFE gene polymorphism in Western Siberia long-livers].

PubMed

Ivanoshchuk, D E; Mikhaĭlova, S V; Kulikov, I V; Maksimov, V N; Voevoda, M I; Romashchenko, A G

2012-01-01

In order to estimate the distribution of some polymorphisms for the CCR5, CCR2, apoE, p53, ITGB3, and HFE genes in Russian long-livers from Western Siberia, a sample of 271 individuals (range 90-105 years) was examined. It was demonstrated that carriage of the delta32 polymorphism for the CCR5 gene, V64/polymorphism for the CCR2 gene, e2/e3/e4 for the apoE gene, L33P for the ITGB3 gene, as well as H63D and S65C polymorphisms for the HFE gene does not influence on predisposition to the longevity; carriage of the 282 Y allele for the HFE gene negatively influences on the longevity; carriage of the heterozygous genotype for the R72P polymorphism for the p53 gene correlates with the longevity of elderly people.

Association study between four polymorphisms in the HFE, TF and TFR genes and Parkinson's disease in southern Italy.

PubMed

Greco, Valentina; De Marco, Elvira Valeria; Rocca, Francesca Emanuela; Annesi, Ferdinanda; Civitelli, Donatella; Provenzano, Giovanni; Tarantino, Patrizia; Scornaienchi, Vittorio; Pucci, Franco; Salsone, Maria; Novellino, Fabiana; Morelli, Maurizio; Paglionico, Sandra; Gambardella, Antonio; Quattrone, Aldo; Annesi, Grazia

2011-06-01

Iron overload may lead to neurodegenerative disorders such as Parkinson's disease (PD) and alterations of iron-related genes might be involved in the pathogenesis of this disease. The gene of haemochromatosis (HFE) encodes the HFE protein which interacts with the transferrin receptor (TFR), lowering its affinity for iron-bound transferrin (TF). We examined four known polymorphisms, C282Y and H63D in the HFE gene, G258S in the TF gene and S82G in the TFR gene, in 181 sporadic PD patients and 180 controls from Southern Italy to investigate their possible role in susceptibility to PD. No significant differences were found in genotype and allele frequencies between PD and controls for all the polymorphisms studied, suggesting that these variants do not contribute significantly to the risk of PD.

Association between CHEK2 H371Y mutation and response to neoadjuvant chemotherapy in women with breast cancer.

PubMed

Liu, Yin; Xu, Ye; Ouyang, Tao; Li, Jinfeng; Wang, Tianfeng; Fan, Zhaoqing; Fan, Tie; Lin, Benyao; Xie, Yuntao

2015-03-28

Our previous study suggested that the recurrent CHEK2 H371Y mutation is a novel pathogenic mutation that confers an increased risk of breast cancer. The purpose of this study was to investigate whether breast cancer patients with CHEK2 H371Y mutation were more likely to respond to neoadjuvant chemotherapy. We screened a cohort of 2334 Chinese women with operable primary breast cancer who received a neoadjuvant chemotherapy regimen for CHEK2 H371Y germline mutations. Pathologic complete response (pCR) was defined as the absence of tumor cells in the breast after the completion of neoadjuvant chemotherapy. Thirty-nine patients (1.7%) with CHEK2 H371Y germline mutation were identified in this cohort of 2334 patients. CHEK2 H371Y mutation carriers had a significantly higher pCR rate than non-carriers (33.3% versus 19.5%, P = 0.031) in the entire study population, and CHEK2 H371Y mutation-positive status remained an independent favorable predictor of pCR in a multivariate analysis (odds ratio [OR] = 3.01; 95% confidence interval [CI]: 1.34- 6.78, P = 0.008). CHEK2 H371Y carriers had a slightly worse distant recurrence-free survival than non-carriers (adjusted hazard ratio [HR] =1.24, 95% CI: 0.59-2.63). CHEK2 H371Y mutation carriers are more likely to respond to neoadjuvant chemotherapy than are non-carriers.

Mutations in the HFE, TFR2, and SLC40A1 genes in patients with hemochromatosis.

PubMed

Del-Castillo-Rueda, Alejandro; Moreno-Carralero, María-Isabel; Cuadrado-Grande, Nuria; Alvarez-Sala-Walther, Luis-Antonio; Enríquez-de-Salamanca, Rafael; Méndez, Manuel; Morán-Jiménez, María-Josefa

2012-10-15

Hereditary hemochromatosis causes iron overload and is associated with a variety of genetic and phenotypic conditions. Early diagnosis is important so that effective treatment can be administered and the risk of tissue damage avoided. Most patients are homozygous for the c.845G>A (p.C282Y) mutation in the HFE gene; however, rare forms of genetic iron overload must be diagnosed using a specific genetic analysis. We studied the genotype of 5 patients who had hyperferritinemia and an iron overload phenotype, but not classic mutations in the HFE gene. Two patients were undergoing phlebotomy and had no iron overload, 1 with metabolic syndrome and no phlebotomy had mild iron overload, and 2 patients had severe iron overload despite phlebotomy. The patients' first-degree relatives also underwent the analysis. We found 5 not previously published mutations: c.-408_-406delCAA in HFE, c.1118G>A (p.G373D), c.1473G>A (p.E491E) and c.2085G>C (p.S695S) in TFR2; and c.-428_-427GG>TT in SLC40A1. Moreover, we found 3 previously published mutations: c.221C>T (p.R71X) in HFE; c.1127C>A (p.A376D) in TFR2; and c.539T>C (p.I180T) in SLC40A1. Four patients were double heterozygous or compound heterozygous for the mutations mentioned above, and the patient with metabolic syndrome was heterozygous for a mutation in the TFR2 gene. Our findings show that hereditary hemochromatosis is clinically and genetically heterogeneous and that acquired factors may modify or determine the phenotype. Copyright © 2012. Published by Elsevier B.V.

Analysis of human MutS homolog 2 missense mutations in patients with colorectal cancer.

PubMed

Zhang, Xiaomei; Chen, Senqing; Yu, Jun; Zhang, Yuanying; Lv, Min; Zhu, Ming

2018-05-01

Germline mutations of DNA mismatch repair gene human MutS homolog 2 ( hMSH2 ) are associated with hereditary nonpolyposis colorectal cancer (HNPCC). A total of one-third of these mutations are missense mutations. Several hMSH2 missense mutations have been identified in patients in East Asia, although their function has not been evaluated. In the present study, the role of ten hMSH2 missense mutations in the pathogenesis of colorectal cancer was examined. The hMSH2/hMSH6 protein interaction system was established using yeast two-hybrid screening. Next, the missense mutations were analyzed for their ability to affect the protein interaction of hMSH2 with its partner hMSH6. Additionally, the Sorting Intolerant from Tolerant tool was applied to predict the effects of different amino acid substitutions. The results demonstrated that certain hMSH2 mutations (L173R and C199R) caused a significant functional change in the human hMutSα complex and were identified to be pathological mutations. The Y408C, D603Y, P696L and S703Y mutations partially affected interaction and partly affected the function of hMSH2. The remaining four variants, T8M, I169V, A370T and Q419K, may be non-functional polymorphisms or could affect protein function through other molecular mechanisms. The present study evaluated the functional consequences of previously unknown missense mutations in hMSH2 , and may contribute to improved clinical diagnosis and mutation screening of HNPCC.

Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandey, Amit V., E-mail: amit@pandeylab.org; Flueck, Christa E.; Mullis, Primus E.

2010-09-24

Research highlights: {yields} Mutations in POR identified from patients lead to reduced HO-1 activities. {yields} POR mutation Y181D affecting FMN binding results in total loss of HO-1 activity. {yields} POR mutations A287P, C569Y and V608F, lost 50-70% activity. {yields} Mutations in FAD binding domain, R457H, Y459H and V492E lost all HO-1 activity. {yields} POR polymorphisms P228L, R316W, G413S, A503V and G504R have normal activity. -- Abstract: Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare formmore » of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.« less

MicroRNA Targets of Human Androgen Receptor

DTIC Science & Technology

2013-05-01

Clin 62, 10- 29. Siegel R., Naishadham D. & Jemal A. (2013) Cancer statistics, 2013. CA Cancer J Clin 63, 11- 30. Sikand K., Barik S. & Shukla G.C...Wongvipat J ., Arora V.K., Watson P.A., Chen Y., Greene G.L., Shen Y. & Sawyers C.L. (2013) Overcoming mutation-based resistance to antiandrogens...Marzo A.M., Meeker A.K., Zha S., Luo J ., Nakayama M., Platz E.A., Isaacs W.B. & Nelson W.G. (2003) Human prostate cancer precursors and pathobiology

Detection of ESR1 mutations in plasma and tumors from metastatic breast cancer patients using next-generation sequencing.

PubMed

Yanagawa, Takehiro; Kagara, Naofumi; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

2017-06-01

Liquid biopsy using digital PCR (dPCR) has been widely used for the screening of ESR1 mutations, since they are frequently identified in the hotspot. However, dPCR is limited to the known mutations. Therefore, we aimed to analyze the utility of next-generation sequencing (NGS) to discover novel ESR1 mutations. Whole exon sequencing of the ESR1 gene using NGS was performed in 16 primary and 47 recurrent tumor samples and 38 plasma samples from hormone receptor-positive metastatic breast cancer patients. Functional analyses were then performed for the novel mutations we detected. We identified no mutations in primary tumors and six mutations in five recurrent tumors, including three types of known mutations (Y537C, Y537N, and D538G) and two novel mutations (E279V and G557R). We also identified seven mutations in five plasma samples, including three types of known mutations (S463P, Y537S, and D538G) and one mutation not reported in COSMIC database (L536H). All nine patients with ESR1 mutations were treated with aromatase inhibitors (AIs) prior to sampling, and the mutations were frequently detected in patients who received AI treatments in the metastatic setting. Among the three novel mutations (E279V, L536H, and G557R), L536H, but not E279V and G557R, showed ligand-independent activity. All three mutant proteins showed nuclear localization and had no relation with non-genomic ER pathways. Although the molecular mechanisms of the E279V and G557R mutations remain unclear, our data suggest the utility of NGS as a liquid biopsy for metastatic breast cancer patients and the potential to identify novel ESR1 mutations.

Quantitative Analysis of the Mutagenic Potential of 1-Aminopyrene-DNA Adduct Bypass Catalyzed by Y-Family DNA Polymerases

PubMed Central

Sherrer, Shanen M.; Taggart, David J.; Pack, Lindsey R.; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

2012-01-01

N- (deoxyguanosin-8-yl)-1-aminopyrene (dGAP) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dGAP induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dGAP lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dGAP, we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dGAP. Opposite dGAP and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dGAP. Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dGAP bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dGAP bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolkk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dGAP in humans. PMID:22917544

Association between sex, systemic iron variation and probability of Parkinson's disease.

PubMed

Mariani, S; Ventriglia, M; Simonelli, I; Bucossi, S; Siotto, M; Donno, S; Vernieri, F; Squitti, R

2016-01-01

Iron homeostasis appears altered in Parkinson's disease (PD). Recent genetic studies and meta-analyses have produced heterogeneous and inconclusive results. In order to verify the possible role of iron status in PD, we have screened some of the main metal gene variants, evaluated their effects on iron systemic status, and checked for possible interactions with PD. In 92 PD patients and 112 healthy controls, we screened the D544E and R793H variants of the ceruloplasmin gene (CP), the P589S variant of the transferrin gene (TF), and the H63D and C282Y variants of the HFE gene, encoding for homologous proteins, respectively. Furthermore, we analyzed serum concentrations of iron, copper and their related proteins. The genetic investigation revealed no significant differences in allelic and genotype distributions between patients and controls. Two different multivariable forward stepwise logistic models showed that, when the effect of sex is considered, an increase of the probability of having PD is associated with low iron concentration and Tf-saturation. This study provides new evidence of the involvement of iron metabolism in PD pathogenesis and reveals a biological effect of sex.

Transferrin saturation phenotype and HFE genotype screening for hemochromatosis and primary iron overload: predictions from a model based on national, racial, and ethnic group composition in central Alabama.

PubMed

Barton, J C; Acton, R T

2000-01-01

There is interest in general population screening for hemochromatosis and other primary iron overload disorders, although not all persons are at equal risk. We developed a model to estimate the numbers of persons in national, racial, or ethnic population subgroups in Jefferson County, Alabama, who would be detected using transferrin saturation (phenotype) or HFE mutation analysis (genotype) screening. Approximately 62% are Caucasians, 37% are African Americans, and the remainder are Hispanics, Asians, or Native Americans. The predicted phenotype frequencies are greatest in a Caucasian subgroup, ethnicity unspecified, which consists predominantly of persons of Scotch and Irish descent (0.0065 men, 0.0046 women), and in African Americans (0.0089 men, 0.0085 women). Frequencies of the HFE genotype C282Y/C282Y > or = 0.0001 are predicted to occur only among Caucasians; the greatest frequency (0.0080) was predicted to occur in the ethnicity-unspecified Caucasian population. C282Y/C282Y frequency estimates were lower in Italian, Greek, and Jewish subgroups. There is excellent agreement in the numbers of the ethnicity-unspecified Caucasians who would be detected using phenotype and genotype criteria. Our model also indicates that phenotyping would identify more persons with primary iron overload than would genotyping in our Italian Caucasian, Hispanic, and African American subgroups. This is consistent with previous observations that indicate that primary iron overload disorders in persons of southern Italian descent and African Americans are largely attributable to non-HFE alleles. Because the proportions of population subgroups and their genetic constitution may differ significantly in other geographic regions, we suggest that models similar to the present one be constructed to predict optimal screening strategies for primary iron overload disorders.

A mutation in the HFE gene is associated with altered brain iron profiles and increased oxidative stress in mice.

PubMed

Nandar, Wint; Neely, Elizabeth B; Unger, Erica; Connor, James R

2013-06-01

Because of the increasing evidence that H63D HFE polymorphism appears in higher frequency in neurodegenerative diseases, we evaluated the neurological consequences of H63D HFE in vivo using mice that carry H67D HFE (homologous to human H63D). Although total brain iron concentration did not change significantly in the H67D mice, brain iron management proteins expressions were altered significantly. The 6-month-old H67D mice had increased HFE and H-ferritin expression. At 12 months, H67D mice had increased H- and L-ferritin but decreased transferrin expression suggesting increased iron storage and decreased iron mobilization. Increased L-ferritin positive microglia in H67D mice suggests that microglia increase iron storage to maintain brain iron homeostasis. The 6-month-old H67D mice had increased levels of GFAP, increased oxidatively modified protein levels, and increased cystine/glutamate antiporter (xCT) and hemeoxygenase-1 (HO-1) expression indicating increased metabolic and oxidative stress. By 12 months, there was no longer increased astrogliosis or oxidative stress. The decrease in oxidative stress at 12 months could be related to an adaptive response by nuclear factor E2-related factor 2 (Nrf2) that regulates antioxidant enzymes expression and is increased in the H67D mice. These findings demonstrate that the H63D HFE impacts brain iron homeostasis, and promotes an environment of oxidative stress and induction of adaptive mechanisms. These data, along with literature reports on humans with HFE mutations provide the evidence to overturn the traditional paradigm that the brain is protected from HFE mutations. The H67D knock-in mouse can be used as a model to evaluate how the H63D HFE mutation contributes to neurodegenerative diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

CYBRD1 as a modifier gene that modulates iron phenotype in HFE p.C282Y homozygous patients.

PubMed

Pelucchi, Sara; Mariani, Raffaella; Calza, Stefano; Fracanzani, Anna Ludovica; Modignani, Giulia Litta; Bertola, Francesca; Busti, Fabiana; Trombini, Paola; Fraquelli, Mirella; Forni, Gian Luca; Girelli, Domenico; Fargion, Silvia; Specchia, Claudia; Piperno, Alberto

2012-12-01

Most patients with hereditary hemochromatosis in the Caucasian population are homozygous for the p.C282Y mutation in the HFE gene. The penetrance and expression of hereditary hemochromatosis differ largely among cases of homozygous p.C282Y. Genetic factors might be involved in addition to environmental factors. In the present study, we analyzed 50 candidate genes involved in iron metabolism and evaluated the association between 214 single nucleotide polymorphisms in these genes and three phenotypic outcomes of iron overload (serum ferritin, iron removed and transferrin saturation) in a large group of 296 p.C282Y homozygous Italians. Polymorphisms were tested for genetic association with each single outcome using linear regression models adjusted for age, sex and alcohol consumption. We found a series of 17 genetic variants located in different genes with possible additive effects on the studied outcomes. In order to evaluate whether the selected polymorphisms could provide a predictive signature for adverse phenotype, we re-evaluated data by dividing patients in two extreme phenotype classes based on the three phenotypic outcomes. We found that only a small improvement in prediction could be achieved by adding genetic information to clinical data. Among the selected polymorphisms, a significant association was observed between rs3806562, located in the 5'UTR of CYBRD1, and transferrin saturation. This variant belongs to the same haplotype block that contains the CYBRD1 polymorphism rs884409, found to be associated with serum ferritin in another population of p.C282Y homozygotes, and able to modulate promoter activity. A luciferase assay indicated that rs3806562 does not have a significant functional role, suggesting that it is a genetic marker linked to the putative genetic modifier rs884409. While our results support the hypothesis that polymorphisms in genes regulating iron metabolism may modulate penetrance of HFE-hereditary hemochromatosis, with emphasis on CYBRD1, they strengthen the notion that none of these polymorphisms alone is a major modifier of the phenotype of hereditary hemochromatosis.

CYBRD1 as a modifier gene that modulates iron phenotype in HFE p.C282Y homozygous patients

PubMed Central

Pelucchi, Sara; Mariani, Raffaella; Calza, Stefano; Fracanzani, Anna Ludovica; Modignani, Giulia Litta; Bertola, Francesca; Busti, Fabiana; Trombini, Paola; Fraquelli, Mirella; Forni, Gian Luca; Girelli, Domenico; Fargion, Silvia; Specchia, Claudia; Piperno, Alberto

2012-01-01

Background Most patients with hereditary hemochromatosis in the Caucasian population are homozygous for the p.C282Y mutation in the HFE gene. The penetrance and expression of hereditary hemochromatosis differ largely among cases of homozygous p.C282Y. Genetic factors might be involved in addition to environmental factors. Design and Methods: In the present study, we analyzed 50 candidate genes involved in iron metabolism and evaluated the association between 214 single nucleotide polymorphisms in these genes and three phenotypic outcomes of iron overload (serum ferritin, iron removed and transferrin saturation) in a large group of 296 p.C282Y homozygous Italians. Polymorphisms were tested for genetic association with each single outcome using linear regression models adjusted for age, sex and alcohol consumption. Results We found a series of 17 genetic variants located in different genes with possible additive effects on the studied outcomes. In order to evaluate whether the selected polymorphisms could provide a predictive signature for adverse phenotype, we re-evaluated data by dividing patients in two extreme phenotype classes based on the three phenotypic outcomes. We found that only a small improvement in prediction could be achieved by adding genetic information to clinical data. Among the selected polymorphisms, a significant association was observed between rs3806562, located in the 5'UTR of CYBRD1, and transferrin saturation. This variant belongs to the same haplotype block that contains the CYBRD1 polymorphism rs884409, found to be associated with serum ferritin in another population of p.C282Y homozygotes, and able to modulate promoter activity. A luciferase assay indicated that rs3806562 does not have a significant functional role, suggesting that it is a genetic marker linked to the putative genetic modifier rs884409. Conclusions While our results support the hypothesis that polymorphisms in genes regulating iron metabolism may modulate penetrance of HFE-hereditary hemochromatosis, with emphasis on CYBRD1, they strengthen the notion that none of these polymorphisms alone is a major modifier of the phenotype of hereditary hemochromatosis. PMID:22773607

Reduced white matter MRI transverse relaxation rate in cognitively normal H63D-HFE human carriers and H67D-HFE mice.

PubMed

Meadowcroft, Mark D; Wang, Jianli; Purnell, Carson J; Peters, Douglas G; Eslinger, Paul J; Neely, Elizabeth B; Gill, David J; Vasavada, Megha; Ali-Rahmani, Fatima; Yang, Qing X; Connor, James R

2016-12-01

Mutations within the HFE protein gene sequence have been associated with increased risk of developing a number of neurodegenerative disorders. To this effect, an animal model has been created which incorporates the mouse homologue to the human H63D-HFE mutation: the H67D-HFE knock-in mouse. These mice exhibit alterations in iron management proteins, have increased neuronal oxidative stress, and a disruption in cholesterol regulation. However, it remains undetermined how these differences translate to human H63D carriers in regards to white matter (WM) integrity. To this endeavor, MRI transverse relaxation rate (R 2 ) parametrics were employed to test the hypothesis that WM alterations are present in H63D human carriers and are recapitulated in the H67D mice. H63D carriers exhibit widespread reductions in brain R 2 compared to non-carriers within white matter association fibers in the brain. Similar R 2 decreases within white matter tracts were observed in the H67D mouse brain. Additionally, an exacerbation of age-related R 2 decrease is found in the H67D animal model in white matter regions of interest. The decrease in R 2 within white matter tracts of both species is speculated to be multifaceted. The R 2 changes are hypothesized to be due to alterations in axonal biochemical tissue composition. The R 2 changes observed in both the human-H63D and mouse-H67D data suggest that modified white matter myelination is occurring in subjects with HFE mutations, potentially increasing vulnerability to neurodegenerative disorders.

Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flueck, Christa E.; Mullis, Primus E.; Pandey, Amit V., E-mail: amit@pandeylab.org

2010-10-08

Research highlights: {yields} Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). {yields} Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. {yields} We are reporting that mutations in POR may reduce CYP3A4 activity. {yields} POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. {yields} Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizesmore » approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.« less

A Novel Tau Mutation in Exon 12, p.Q336H, Causes Hereditary Pick Disease

PubMed Central

Tacik, Pawel; DeTure, Michael; Hinkle, Kelly M.; Lin, Wen-Lang; Sanchez-Contreras, Monica; Carlomagno, Yari; Pedraza, Otto; Rademakers, Rosa; Ross, Owen A.; Wszolek, Zbigniew K.; Dickson, Dennis W.

2015-01-01

Pick disease (PiD) is a frontotemporal lobar degeneration with distinctive neuronal inclusions (Pick bodies) that are enriched in 3-repeat (3R) tau. Although mostly sporadic, mutations in the tau gene (MAPT) have been reported. We screened 24 cases of neuropathologically confirmed PiD for MAPT mutations and found a novel mutation (c.1008G>C, p.Q336H) in one patient. Pathogenicity was confirmed on microtubule assembly and tau filament formation assays. The patient was compared to sporadic PiD and PiD associated with MAPT mutations from a review of the literature. The patient had behavioral changes at 55 years of age, followed by reduced verbal fluency, parkinsonism and death at 63 years of age. His mother and maternal uncle had similar symptoms. Recombinant tau with p.Q336H mutation formed filaments faster than wild type tau, especially with 3R tau. It also promoted more microtubule assembly than wild type tau. We conclude that mutations in MAPT, including p.Q336H, can be associated with clinical, pathologic, and biochemical features that are similar to those in sporadic PiD. The pathomechanism of p.Q336H, and another previously reported variant at the same codon (p.Q336R), appears to be unique to MAPT mutations in that they not only predispose to abnormal tau filament formation but also facilitate microtubule assembly in a 3R tau-dependent manner. PMID:26426266

HFE gene polymorphism defined by sequence-based typing of the Brazilian population and a standardized nomenclature for HFE allele sequences.

PubMed

Campos, W N; Massaro, J D; Martinelli, A L C; Halliwell, J A; Marsh, S G E; Mendes-Junior, C T; Donadi, E A

2017-10-01

The HFE molecule controls iron uptake from gut, and defects in the molecule have been associated with iron overload, particularly in hereditary hemochromatosis. The HFE gene including both coding and boundary intronic regions were sequenced in 304 Brazilian individuals, encompassing healthy individuals and patients exhibiting hereditary or acquired iron overload. Six sites of variation were detected: (1) H63D C>G in exon 2, (2) IVS2 (+4) T>C in intron 2, (3) a C>G transversion in intron 3, (4) C282Y G>A in exon 4, (5) IVS4 (-44) T>C in intron 4, and (6) a new guanine deletion (G>del) in intron 5, which were used for haplotype inference. Nine HFE alleles were detected and six of these were officially named on the basis of the HLA Nomenclature, defined by the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System, and published via the IPD-IMGT/HLA website. Four alleles, HFE*001, *002, *003, and *004 exhibited variation within their exon sequences. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Novel glucokinase mutations in patients with monogenic diabetes - clinical outline of GCK-MD and potential for founder effect in Slavic population.

PubMed

Borowiec, M; Antosik, K; Fendler, W; Deja, G; Jarosz-Chobot, P; Mysliwiec, M; Zmyslowska, A; Malecki, M; Szadkowska, A; Mlynarski, W

2012-03-01

Glucokinase (GCK) gene mutations are the causative factor of GCK-MD (monogenic diabetes) characterized by a mild clinical phenotype and potential for insulin withdrawal. This study presents the results of a nationwide genetic screening for GCK-MD performed in Poland. A group of 194 patients with clinical suspicion of GCK-MD and 17 patients with neonatal diabetes were subjected to GCK sequencing. Patients negative for GCK mutations were subjected to multiplex ligation-dependent probe amplification (MLPA) to detect deletions or insertions. A total of 44 GCK heterozygous mutations were found in 68 probands (35%). Among those, 20 mutations were novel ones: A282fs, D198V, E158X, G246V, G249R, I348N, L165V, L315Q, M115I, N254S, P284fs, Q338P, R377L, R43C, R46S, S212fs, S212P, T255N, V406A and Y214D. No abnormalities were detected in MLPA analysis. Homozygous D278E mutation was found in one patient with neonatal diabetes. The most frequently observed combinations of symptoms typical for GCK-MD were mild diabetes and/or fasting hyperglycaemia (98.3%), positive C-peptide at diagnosis (76%) and dominant mode of inheritance (59%). This study outlines numerous novel mutations of the GCK gene present in white Caucasians of Slavic origin. Thorough clinical assessment of known factors associated with GCK-MD may facilitate patient selection. © 2011 John Wiley & Sons A/S.

Connection Subdomain Mutations in HIV-1 Subtype-C Treatment-Experienced Patients Enhance NRTI and NNRTI Drug Resistance

PubMed Central

Delviks-Frankenberry, Krista A.; Lengruber, Renan B.; Santos, Andre F.; Silveira, Jussara M.; Soares, Marcelo A.; Kearney, Mary F.; Maldarelli, Frank; Pathak, Vinay K.

2012-01-01

Mutations in the connection subdomain (CN) and RNase H domain (RH) of HIV-1 reverse transcriptase (RT) from subtype B-infected patients enhance nucleoside and nonnucleoside RT inhibitor (NRTI and NNRTI) resistance by affecting the balance between polymerization and RNase H activity. To determine whether CN mutations in subtype C influence drug sensitivity, single genome sequencing was performed on Brazilian subtype C-infected patients failing RTI therapy. CN mutations identified were similar to subtype B, including A376S, A400T, Q334D, G335D, N348I, and A371V, and increased AZT resistance in the presence of thymidine analog mutations. CN mutations also enhanced NNRTI resistance in the presence of classical NNRTI mutations: etravirine resistance was enhanced 6- to 11-fold in the presence of L100I/K103N/Y181C. These results indicate that selection of CN mutations in treatment-experienced patients also occurs in subtype-C-infected patients and are likely to provide valuable information in predicting clinical RTI resistance. PMID:23068886

The Response of Ω-Loop D Dynamics to Truncation of Trimethyllysine 72 of Yeast Iso-1-cytochrome c Depends on the Nature of Loop Deformation

PubMed Central

McClelland, Levi J.; Seagraves, Sean M.; Khan, Khurshid Alam; Cherney, Melisa M.; Bandi, Swati; Culbertson, Justin E.; Bowler, Bruce E.

2015-01-01

Trimethyllysine 72 (tmK72) has been suggested to play a role in sterically constraining the heme crevice dynamics of yeast iso-1-cytochrome c mediated by the Ω-loop D cooperative substructure (residues 70 to 85). A tmK72A mutation causes a gain in peroxidase activity, a function of cytochrome c that is important early in apoptosis. More than one higher energy state is accessible for the Ω-loop D substructure via tier 0 dynamics. Two of these are alkaline conformers mediated by Lys73 and Lys79. In the current work, the effect of the tmK72A mutation on the thermodynamic and kinetic properties of wild type iso-1-cytochrome c (yWT versus WT*) and on variants carrying a K73H mutation (yWT/K73H versus WT*/K73H) is studied. Whereas the tmK72A mutation confers increased peroxidase activity in wild type yeast iso-1-cytochrome c and increased dynamics for formation of a previously studied His79-heme alkaline conformer, the tmK72A mutation speeds return of the His73-heme alkaline conformer to the native state through destabilization of the His73-heme alkaline conformer relative to the native conformer. These opposing behaviors demonstrate that the response of the dynamics of a protein substructure to mutation depends on the nature of the perturbation to the substructure. For a protein substructure which mediates more than one function of a protein through multiple non-native structures, a mutation could change the partitioning between these functions. The current results suggest that the tier 0 dynamics of Ω-loop D that mediates peroxidase activity has similarities to the tier 0 dynamics required to form the His79-heme alkaline conformer. PMID:25948392

Site-directed mutations of thermostable direct hemolysin from Grimontia hollisae alter its arrhenius effect and biophysical properties.

PubMed

Wang, Yu-Kuo; Huang, Sheng-Cih; Wu, Yi-Fang; Chen, Yu-Ching; Lin, Yen-Ling; Nayak, Manoswini; Lin, Yan Ren; Chen, Wen-Hung; Chiu, Yi-Rong; Li, Thomas Tien-Hsiung; Yeh, Bo-Sou; Wu, Tung-Kung

2011-03-31

Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 °C but is reactivated by additional heating above 80 °C. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDH(WT)) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDH(Y53H/T59I) and Gh-rTDH(T59I/S63T) double-mutants and the Gh-rTDH(Y53H/T59I/S63T) triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDH(WT). Circular dichroism measurements of Gh-rTDH(WT) and Gh-rTDH(mut) showed a conspicuous change from a β-sheet to α-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.

Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance.

PubMed

Tian, Yong-Sheng; Xu, Jing; Peng, Ri-He; Xiong, Ai-Sheng; Xu, Hu; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Yao, Quan-Hong

2013-09-01

A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Predictors of haemoconcentration at delivery: association with low birth weight.

PubMed

Aranda, N; Ribot, B; Viteri, F; Cavallé, P; Arija, V

2013-09-01

[corrected] To assess the factors associated with risk of haemoconcentration at delivery, such as initial haemoglobin levels and alterations in the HFE gene, and its effect on low birth weight in pregnant women supplemented with moderate doses of iron. Case-control study nested in a longitudinal study conducted on 217 healthy pregnant women taking moderate iron supplementation and their newborns. Women were classified according to the risk of haemoconcentration at delivery, defined as Hb > 130 g/L. Each subject's obstetric and clinical history, smoking habit, and iron biochemical parameters (haemoglobin (Hb), serum ferritin and transferrin saturation) were recorded at 1st, 2nd and 3rd trimester and at delivery. Polymorphisms of the HFE gene (C282Y, H63D and S65C) were also measured. The average of iron supplementation of all the women was 43.9 mg/dia (geometric mean, 95 % CI: 43.6-44.1). Higher levels of Hb at early gestation and the presence of HFE mutations were associated with greater risk of haemoconcentration at delivery, adjusted odds ratios of 1.14 (95 % CI: 1.05-1.25) and 5.35 (95 % CI: 1.6-17.8). Haemoconcentration at delivery was associated with a greater risk of low birth weight, adjusted odd ratio of 11.48 (95 % CI: 1.13-116.6). Moderate daily doses of supplementary iron may be harmful for foetal growth in women with alterations in HFE gene and who started pregnancy with good haemoglobin levels. Overall, this suggests the importance of determining a woman's iron status early in her pregnancy in order to establish a more appropriate pattern of supplementation.

Molecular analysis of patients with {Beta}-glucuronidase deficiency presenting as hydrops fetalis or as early mucopolysaccharidosis VII

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vervoort, R.; Liebaers, I.; Lissens, W.

1996-03-01

Although not all mucopolysaccharidosis type VII (MPS VII) neonates present with hydrops fetalis or with related symptoms, hydrops fetalis is a common form of presentation of this mucopolysaccharidosis. We used reverse-transcription-PCR-SSCP and direct sequencing to screen for mutations in the human {beta}-glucuronidase cDNA of 17 MPS VII patients with severe presentation of the disease. Mutations resulting in an unstable mRNA were detected in genomic DNA with direct sequencing of the PCR-amplified {beta}-glucuronidase exons. We found extensive genetic heterogeneity in MPS VII alleles: in addition to 6 of 12 previously reported mutations (L176F, R216W, R357X, R382C, W507X, and W627C), we detectedmore » 14 undescribed mutations in the {beta}-glucuronidase coding region that produce MPS VII alleles (G136R, E150K, S312X, Y320S, Y320C, H351Y, R382H, R374C, R435P, R477W, G572D, Y508C, K606N, and 1900{Delta}GA). The mutations in hydropic fetuses were widely scattered in the {beta}-glucuronidase gene. Analysis of three polymorphic sites of the mutant alleles (1766T/C, 1972C/T, and a new 1091+27C/G polymorphism) allowed exclusion of identity by descent for some recurrent mutations. Three of four mutations introducing a premature translation stop codon were found to affect mRNA abundance and/or structure. Expression studies provided evidence for the causal relationship between each of the mutations found in MPS VII alleles and the enzyme deficiency, in that all mutations identified exhibited markedly reduced enzyme activity expressed in COS7 cells following transfection with the mutant cDNA. 52 refs., 4 figs., 5 tabs.« less

Molecular analysis of patients with beta-glucuronidase deficiency presenting as hydrops fetalis or as early mucopolysaccharidosis VII.

PubMed Central

Vervoort, R.; Islam, M. R.; Sly, W. S.; Zabot, M. T.; Kleijer, W. J.; Chabas, A.; Fensom, A.; Young, E. P.; Liebaers, I.; Lissens, W.

1996-01-01

Although not all mucopolysaccharidosis type VII (MPS VII) neonates present with hydrops fetalis or with related symptoms, hydrops fetalis is a common form of presentation of this mucopolysaccharidosis. We used reverse-transcription-PCR-SSCP and direct sequencing to screen for mutations in the human beta-glucuronidase cDNA of 17 MPS VII patients with severe presentation of the disease. Mutations resulting in an unstable mRNA were detected in genomic DNA with direct sequencing of the PCR-amplified beta-glucuronidase exons. We found extensive genetic heterogeneity in MPS VII alleles: in addition to 6 or 12 previously reported mutations (L176F, R216W, R357X, R382C, W507X, and W627C), we detected 14 undescribed mutations in the beta-glucuronidase coding region that produce MPS VII alleles (G136R, E150K, S312X, Y320S, Y320C, H351Y, R382H, R374C, R435P, R477W, G572D, Y508C, K606N and 1900 delta GA). The mutations in hydropic fetuses were widely scattered in the beta-glucuronidase gene. Analysis of three polymorphic sites of the mutant alleles (1766T/C, 1972C/T and a new 1091+27C/G polymorphism) allowed exclusion of identity by descent for some recurrent mutations. Three of four mutations introducing a premature translation stop codon were found to affect mRNA abundance and/or structure. Expression studies provided evidence for the causal relationship between each of the mutations found in MPS VII alleles and the enzyme deficiency, in that all mutations identified exhibited markedly reduced enzyme activity expressed in COS7 cells following transfection with the mutant cDNA. Images Figure 2 Figure 3A Figure 3BC Figure 4 PMID:8644704

C282Y-HFE Gene Variant Affects Cholesterol Metabolism in Human Neuroblastoma Cells

PubMed Central

Ali-Rahmani, Fatima; Huang, Michael A.; Schengrund, C.-L.; Connor, James R.; Lee, Sang Y.

2014-01-01

Although disruptions in the maintenance of iron and cholesterol metabolism have been implicated in several cancers, the association between variants in the HFE gene that is associated with cellular iron uptake and cholesterol metabolism has not been studied. The C282Y-HFE variant is a risk factor for different cancers, is known to affect sphingolipid metabolism, and to result in increased cellular iron uptake. The effect of this variant on cholesterol metabolism and its possible relevance to cancer phenotype was investigated using wild type (WT) and C282Y-HFE transfected human neuroblastoma SH-SY5Y cells. Expression of C282Y-HFE in SH-SY5Y cells resulted in a significant increase in total cholesterol as well as increased transcription of a number of genes involved in its metabolism compared to cells expressing WT-HFE. The marked increase in expression of NPC1L1 relative to that of most other genes, was accompanied by a significant increase in expression of NPC1, a protein that functions in cholesterol uptake by cells. Because inhibitors of cholesterol metabolism have been proposed to be beneficial for treating certain cancers, their effect on the viability of C282Y-HFE neuroblastoma cells was ascertained. C282Y-HFE cells were significantly more sensitive than WT-HFE cells to U18666A, an inhibitor of desmosterol Δ24-reductase the enzyme catalyzing the last step in cholesterol biosynthesis. This was not seen for simvastatin, ezetimibe, or a sphingosine kinase inhibitor. These studies indicate that cancers presenting in carriers of the C282Y-HFE allele might be responsive to treatment designed to selectively reduce cholesterol content in their tumor cells. PMID:24533143

C282Y-HFE gene variant affects cholesterol metabolism in human neuroblastoma cells.

PubMed

Ali-Rahmani, Fatima; Huang, Michael A; Schengrund, C-L; Connor, James R; Lee, Sang Y

2014-01-01

Although disruptions in the maintenance of iron and cholesterol metabolism have been implicated in several cancers, the association between variants in the HFE gene that is associated with cellular iron uptake and cholesterol metabolism has not been studied. The C282Y-HFE variant is a risk factor for different cancers, is known to affect sphingolipid metabolism, and to result in increased cellular iron uptake. The effect of this variant on cholesterol metabolism and its possible relevance to cancer phenotype was investigated using wild type (WT) and C282Y-HFE transfected human neuroblastoma SH-SY5Y cells. Expression of C282Y-HFE in SH-SY5Y cells resulted in a significant increase in total cholesterol as well as increased transcription of a number of genes involved in its metabolism compared to cells expressing WT-HFE. The marked increase in expression of NPC1L1 relative to that of most other genes, was accompanied by a significant increase in expression of NPC1, a protein that functions in cholesterol uptake by cells. Because inhibitors of cholesterol metabolism have been proposed to be beneficial for treating certain cancers, their effect on the viability of C282Y-HFE neuroblastoma cells was ascertained. C282Y-HFE cells were significantly more sensitive than WT-HFE cells to U18666A, an inhibitor of desmosterol Δ24-reductase the enzyme catalyzing the last step in cholesterol biosynthesis. This was not seen for simvastatin, ezetimibe, or a sphingosine kinase inhibitor. These studies indicate that cancers presenting in carriers of the C282Y-HFE allele might be responsive to treatment designed to selectively reduce cholesterol content in their tumor cells.

Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation.

PubMed

de Almeida, Sérgio F; Picarote, Gonçalo; Fleming, John V; Carmo-Fonseca, Maria; Azevedo, Jorge E; de Sousa, Maria

2007-09-21

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.

A recurrent CHEK2 p.H371Y mutation is associated with breast cancer risk in Chinese women.

PubMed

Liu, Yin; Liao, Ji; Xu, Ye; Chen, Weiqiu; Liu, Dongyun; Ouyang, Tao; Li, Jinfeng; Wang, Tianfeng; Fan, Zhaoqing; Fan, Tie; Lin, Benyao; Xu, Xingzhi; Xie, Yuntao

2011-09-01

The association between the CHEK2 and breast cancer risk in Chinese women is unknown. Here, we screened the full CHEK2 coding sequence in 118 Chinese familial breast cancer cases who are negative for mutations in BRCA1 and BRCA2, one recurrent mutation, CHEK2 c.1111C>T (p.H371Y), was identified in five index cases in this cohort. Functional analysis suggested that CHEK2 p.H371Y was a pathogenic mutation that resulted in decreased kinase activity. We further screened the CHEK2 p.H371Y mutation in 909 unselected breast cancer cases and 1,228 healthy individuals. The frequencies of the CHEK2 p.H371Y in familial and unselected breast cancer cases and controls were 4.24% (5/118), 1.76% (16/909), and 0.73% (9/1228), respectively. The p.H371Y mutation was significantly associated with increased breast cancer risk in unselected breast cancer (odds ratio [OR] 2.43, 95% confidence interval [CI] 1.07-5.52, P = 0.034). Our results indicate that the recurrent mutation, p.H371Y, confers a moderate risk of breast cancer in Chinese women. © 2011 Wiley-Liss, Inc.

Involvement of the major histocompatibility complex region in the genetic regulation of circulating CD8 T-cell numbers in humans.

PubMed

Cruz, E; Vieira, J; Gonçalves, R; Alves, H; Almeida, S; Rodrigues, P; Lacerda, R; Porto, G

2004-07-01

Variability in T-lymphocyte numbers is partially explained by a genetic regulation. From studies in animal models, it is known that the Major Histocompatibility Complex (MHC) is involved in this regulation. In humans, this has not been shown yet. The objective of the present study was to test the hypothesis that genes in the MHC region influence the regulation of T-lymphocyte numbers. Two approaches were used. Association studies between T-cell counts (CD4(+) and CD8(+)) or total lymphocyte counts and HLA class I alleles (A and B) or mutations in the HFE (C282Y and H63D), the hemochromatosis gene, in an unrelated population (n = 264). A second approach was a sibpair correlation analysis of the same T-cell counts in relation to HLA-HFE haplotypes in subjects belonging to 48 hemochromatosis families (n = 456 sibpairs). In the normal population, results showed a strong statistically significant association of the HLA-A*01 with high numbers of CD8(+) T cells and a less powerful association with the HLA-A*24 with low numbers of CD8(+) T cells. Sibpair correlations revealed the most significant correlation for CD8(+) T-cell numbers for sibpairs with HLA-HFE-identical haplotypes. This was not observed for CD4(+) T cells. These results show that the MHC region is involved in the genetic regulation of CD8(+) T-cell numbers in humans. Identification of genes responsible for this control may have important biological and clinical implications.

Genome-wide association study identifies TF as a significant modifier gene of iron metabolism in HFE hemochromatosis.

PubMed

de Tayrac, Marie; Roth, Marie-Paule; Jouanolle, Anne-Marie; Coppin, Hélène; le Gac, Gérald; Piperno, Alberto; Férec, Claude; Pelucchi, Sara; Scotet, Virginie; Bardou-Jacquet, Edouard; Ropert, Martine; Bouvet, Régis; Génin, Emmanuelle; Mosser, Jean; Deugnier, Yves

2015-03-01

Hereditary hemochromatosis (HH) is the most common form of genetic iron loading disease. It is mainly related to the homozygous C282Y/C282Y mutation in the HFE gene that is, however, a necessary but not a sufficient condition to develop clinical and even biochemical HH. This suggests that modifier genes are likely involved in the expressivity of the disease. Our aim was to identify such modifier genes. We performed a genome-wide association study (GWAS) using DNA collected from 474 unrelated C282Y homozygotes. Associations were examined for both quantitative iron burden indices and clinical outcomes with 534,213 single nucleotide polymorphisms (SNP) genotypes, with replication analyses in an independent sample of 748 C282Y homozygotes from four different European centres. One SNP met genome-wide statistical significance for association with transferrin concentration (rs3811647, GWAS p value of 7×10(-9) and replication p value of 5×10(-13)). This SNP, located within intron 11 of the TF gene, had a pleiotropic effect on serum iron (GWAS p value of 4.9×10(-6) and replication p value of 3.2×10(-6)). Both serum transferrin and iron levels were associated with serum ferritin levels, amount of iron removed and global clinical stage (p<0.01). Serum iron levels were also associated with fibrosis stage (p<0.0001). This GWAS, the largest one performed so far in unselected HFE-associated HH (HFE-HH) patients, identified the rs3811647 polymorphism in the TF gene as the only SNP significantly associated with iron metabolism through serum transferrin and iron levels. Because these two outcomes were clearly associated with the biochemical and clinical expression of the disease, an indirect link between the rs3811647 polymorphism and the phenotypic presentation of HFE-HH is likely. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study.

PubMed Central

Georis, J.; de Lemos Esteves, F.; Lamotte-Brasseur, J.; Bougnet, V.; Devreese, B.; Giannotta, F.; Granier, B.; Frère, J. M.

2000-01-01

In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional (3D) structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature (70 vs. 60 degrees C) and the apparent Tm were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C. The combined mutations A82R/F168H/N169D/delta170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2). Y11 and Y16 might represent two additional binding subsites (-3, -4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1. PMID:10752608

Comparison of ESR1 Mutations in Tumor Tissue and Matched Plasma Samples from Metastatic Breast Cancer Patients.

PubMed

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Tomiguchi, Mai; Sueta, Aiko; Murakami, Keiichi; Omoto, Yoko; Iwase, Hirotaka

2017-10-01

ESR1 mutation in circulating cell-free DNA (cfDNA) is emerging as a noninvasive biomarker of acquired resistance to endocrine therapy, but there is a paucity of data comparing the status of ESR1 gene in cfDNA with that in its corresponding tumor tissue. The objective of this study is to validate the degree of concordance of ESR1 mutations between plasma and tumor tissue. ESR1 ligand-binding domain mutations Y537S, Y537N, Y537C, and D538G were analyzed using droplet digital PCR in 35 patients with metastatic breast cancer (MBC) (35 tumor tissue samples and 67 plasma samples). Of the 35 paired samples, 26 (74.3%) were concordant: one patient had detectable ESR1 mutations both plasma (ESR1 Y537S/Y537N) and tumor tissue (ESR1 Y537S/Y537C), and 25 had WT ESR1 alleles in both. Nine (25.7%) had discordance between the plasma and tissue results: five had mutations detected only in their tumor tissue (two Y537S, one Y537C, one D538G, and one Y537S/Y537N/D538G), and four had mutations detected only in their plasma (one Y537S, one Y537N, and two Y537S/Y537N/D538G). Furthermore, longitudinal plasma samples from 19 patients were used to assess changes in the presence of ESR1 mutations during treatment. Eleven patients had cfDNA ESR1 mutations over the course of treatment. A total of eight of 11 patients with MBC with cfDNA ESR1 mutations (72.7%) had the polyclonal mutations. We have shown the independent distribution of ESR1 mutations between plasma and tumor tissue in 35 patients with MBC. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Iron loading in HFE p.C282Y homozygotes found by population screening: relationships to HLA-type and T-lymphocyte subsets.

PubMed

Thorstensen, Ketil; Kvitland, Mona A; Irgens, Wenche Ø; Åsberg, Arne; Borch-Iohnsen, Berit; Moen, Torolf; Hveem, Kristian

2017-11-01

Iron loading in p.C282Y homozygous HFE hemochromatosis subjects is highly variable, and it is unclear what factors cause this variability. Finding such factors could aid in predicting which patients are at highest risk and require closest follow-up. The degree of iron loading has previously been associated with certain HLA-types and with abnormally low CD8 + cell counts in peripheral blood. In 183 Norwegian, p.C282Y homozygotes (104 men, 79 women) originally found through population screening we determined HLA type and measured total T-lymphocytes, CD4 + and CD8 + cells, and compared this with data on iron loading. In p.C282Y homozygous men, but not in homozygous women, we found that the presence of two HLA-A*03 alleles increased the iron load on average by approximately 2-fold compared to p.C282Y homozygous men carrying zero or one A*03 allele. On the other hand, the presence of two HLA-A*01 alleles, in male subjects, apparently reduced the iron loading. In p.C282Y homozygous individuals, the iron loading was increased if the CD8 + cell number was below the 25 percentile or if the CD4 + cell number was above the 75 percentile. This effect appeared to be additive to the effect of the number of HLA-A*03 alleles. Our data indicate that homozygosity for the HLA-A*03 allele significantly increases the risk of excessive iron loading in Norwegian p.C282Y homozygous male patients. In addition, low CD8 + cell number or high CD4 + cell number further increases the risk of excessive iron loading.

Ethnic and genetic factors of iron status in women of reproductive age.

PubMed

Gordeuk, Victor R; Brannon, Patsy M

2017-12-01

Background: African Americans are at increased risk of iron deficiency (ID) but also have higher serum ferritin (SF) concentrations than those of the general population. The Hemochromatosis and Iron Overload Screening (HEIRS) Study was a multicenter study of ethnically diverse participants that tested for the hemochromatosis ( HFE ) C282Y genotype and iron status. Objective: We sought to determine the prevalence and predictors of ID (SF concentration ≤15 μg/L) and elevated iron stores (SF concentration >300 μg/L) in HEIRS women of reproductive age (25-44 y). Design: The HEIRS Study was a cross-sectional study of iron status and HFE mutations in primary care patients at 5 centers in the United States and Canada. We analyzed data for women of reproductive age according to whether or not they were pregnant or breastfeeding at the time of the study. Results: ID was present in 12.5% of 20,080 nonpregnant and nonbreastfeeding women compared with 19.2% of 1962 pregnant or breastfeeding women ( P < 0.001). Asian American ethnicity (OR ≤0.9; P ≤ 0.049) and HFE C282Y (OR ≤0.84; P ≤ 0.060) were independently associated with a decreased risk of ID in nonpregnant and nonbreastfeeding women and in pregnant or breastfeeding women. Hispanic ethnicity (OR: 1.8; P < 0.001) and African American ethnicity (OR: 1.6; P < 0.001) were associated with an increased risk of ID in nonpregnant and nonbreastfeeding women. Elevated iron stores were shown in 1.7% of nonpregnant and nonbreastfeeding women compared with 0.7% of pregnant or breastfeeding women ( P = 0.001). HFE C282Y homozygosity had the most marked independent association with elevated iron stores in nonpregnant and nonbreastfeeding women and in pregnant or breastfeeding women (OR >49.0; P < 0.001), but African American ethnicity was also associated with increased iron stores in both groups of women (OR >2.0; P < 0.001). Asian American ethnicity (OR: 1.8; P = 0.001) and HFE C282Y heterozygosity (OR: 1.9; P = 0.003) were associated with increased iron stores in nonpregnant and nonbreastfeeding women. Conclusions: Both ID and elevated iron stores are present in women of reproductive age and are influenced by ethnicity and HFE C282Y. Efforts to optimize iron status should keep these findings in view. This study was registered at clinicaltrials.gov as NCT03276247. © 2017 American Society for Nutrition.

Prevalence of ESR1 E380Q mutation in tumor tissue and plasma from Japanese breast cancer patients.

PubMed

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Sueta, Aiko; Tomiguchi, Mai; Murakami, Keiichi; Omoto, Yoko; Iwase, Hirotaka

2017-11-22

ESR1 mutations have attracted attention as a potentially important marker and treatment target in endocrine therapy-resistant breast cancer patients. The E380Q mutation, which is one of the ESR1 mutations, is associated with estradiol (E2) hypersensitivity, increased DNA binding to the estrogen response element, and E2-independent constitutive trans-activation activity, but its frequency in ESR1 mutations remains unknown. The present study aimed to investigate the E380Q mutation in comparison with the other representative ESR1 mutations. We screened a total of 62 patients (66 tumor tissues and 69 plasma cell-free DNA (cfDNA)) to detect ESR1 mutations (E380Q, Y537S, Y537N, Y537C, and D538G) using droplet-digital polymerase chain reaction. Plasma was collected at more than two points of the clinical course, in whom changes of ESR1 mutations under treatment were investigated. We detected ESR1 mutations in 21% (12/57) of MBCs. The E380Q ESR1 mutation was found in 16% (2/12) and the other ESR1 LBD mutations were five (41.6%) of Y537S, and four each (33.3%) of D538G, Y537N, and Y537C, in 12 ESR1 mutant breast cancer patients. Five tumors had multiple ESR1 mutations: three had double ESR1 mutations; Y537S/E380Q, Y37S/Y537C, and Y537S/D538G, and two had triple ESR1 mutations; Y537S/Y537N/D538G. In plasma cfDNA analysis, the E380Q mutation was not detected, but increases in other ESR1 mutations were detected in 46.2% (6/13) of MBC patients under treatment. We have shown that there are distinct populations of ESR1 mutations in metastatic tissue and plasma. Each ESR1 mutation may have different clinical significance, and it will be necessary to investigate them all.

Molecular basis of mild hyperphenylalaninaemia in Poland.

PubMed Central

Zekanowski, C; Nowacka, M; Cabalska, B; Bal, J

1997-01-01

The major cause of the different forms of hyperphenylalaninaemia (HPA) is mutations in the gene encoding phenylalanine hydroxylase (PAH). The aim of this study was to determine the mutations responsible for mild forms of HPA and to relate different clinical phenotypes of HPA patients to their PAH genotypes. Four "mild" mutations, including the most frequent A403V and R297H mutations, occurred exclusively in mild hyperphenylalaninaemia (MHP). Mutations A104D, R243Q, R241H, and Y414C were detected in patients with mild phenylketonuria (mild PKU) only. These results may be useful in establishing a molecular differential diagnosis for PAH deficiency in Poland. PMID:9429153

Novel mutations of CHST6 in Iranian patients with macular corneal dystrophy

PubMed Central

Salehi, Zivar; Houshmand, Masoud; Mohamadi, Mohamad Javad; Promehr, Leila Azizade; Mozafarzadeh, Zahra

2009-01-01

Purpose To characterize mutations within the carbohydrate sulfotransferase 6 (CHST6) gene in Iranian subjects from 12 families with macular corneal dystrophy (MCD). Methods Genomic DNA was extracted from peripheral blood of 20 affected patients and 60 healthy volunteers followed by polymerase chain reaction (PCR) and direct sequencing of the CHST6 coding region. The observed nucleotide sequences were then compared with those found by investigators in other populations with MCD and in the controls. Results Analysis of CHST6 revealed 11 different mutations. These mutations were comprised of six novel missense mutations (p.F55L, p.P132L, p.S136G, p.C149Y, p.D203Y, and p.H249R), one novel nonsense mutation (p.S48X), one novel frame shift (after P297), and three previously reported missense mutations (p.P31L, p.C165Y, and p.R127C). The majority of the detected MCD mutations are located in the binding sites or the binding pocket, except the p.P31L and p.H249R mutations. Conclusions Nucleotide changes within the coding region of CHST6 are predicted to significantly alter the encoded sulfotransferase within the evolutionary conserved sequences. Our findings show that CHST6 mutations are responsible for the pathogenesis of MCD in Iranian patients. Moreover, the observation that some cases of MCD cannot be explained by mutations in the coding region of CHST6 suggests that MCD may result from possible upstream rearrangements in the CHST6 genomic region. PMID:19223992

Novel mutations of CHST6 in Iranian patients with macular corneal dystrophy.

PubMed

Birgani, Shiva Akbari; Salehi, Zivar; Houshmand, Masoud; Mohamadi, Mohamad Javad; Promehr, Leila Azizade; Mozafarzadeh, Zahra

2009-01-01

To characterize mutations within the carbohydrate sulfotransferase 6 (CHST6) gene in Iranian subjects from 12 families with macular corneal dystrophy (MCD). Genomic DNA was extracted from peripheral blood of 20 affected patients and 60 healthy volunteers followed by polymerase chain reaction (PCR) and direct sequencing of the CHST6 coding region. The observed nucleotide sequences were then compared with those found by investigators in other populations with MCD and in the controls. Analysis of CHST6 revealed 11 different mutations. These mutations were comprised of six novel missense mutations (p.F55L, p.P132L, p.S136G, p.C149Y, p.D203Y, and p.H249R), one novel nonsense mutation (p.S48X), one novel frame shift (after P297), and three previously reported missense mutations (p.P31L, p.C165Y, and p.R127C). The majority of the detected MCD mutations are located in the binding sites or the binding pocket, except the p.P31L and p.H249R mutations. Nucleotide changes within the coding region of CHST6 are predicted to significantly alter the encoded sulfotransferase within the evolutionary conserved sequences. Our findings show that CHST6 mutations are responsible for the pathogenesis of MCD in Iranian patients. Moreover, the observation that some cases of MCD cannot be explained by mutations in the coding region of CHST6 suggests that MCD may result from possible upstream rearrangements in the CHST6 genomic region.

Molecular Diagnostic and Pathogenesis of Hereditary Hemochromatosis

PubMed Central

Santos, Paulo C. J. L.; Krieger, Jose E.; Pereira, Alexandre C.

2012-01-01

Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by enhanced intestinal absorption of dietary iron. Without therapeutic intervention, iron overload leads to multiple organ damage such as liver cirrhosis, cardiomyopathy, diabetes, arthritis, hypogonadism and skin pigmentation. Most HH patients carry HFE mutant genotypes: homozygosity for p.Cys282Tyr or p.Cys282Tyr/p.His63Asp compound heterozygosity. In addition to HFE gene, mutations in the genes that encode hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) have been associated with regulation of iron homeostasis and development of HH. The aim of this review was to identify the main gene mutations involved in the pathogenesis of type 1, 2, 3 and 4 HH and their genetic testing indication. HFE testing for the two main mutations (p.Cys282Tyr and p.His63Asp) should be performed in all patients with primary iron overload and unexplained increased transferrin saturation and/or serum ferritin values. The evaluation of the HJV p.Gly320Val mutation must be the molecular test of choice in suspected patients with juvenile hemochromatosis with less than 30 years and cardiac or endocrine manifestations. In conclusion, HH is an example that genetic testing can, in addition to performing the differential diagnostic with secondary iron overload, lead to more adequate and faster treatment. PMID:22408404

Mechanisms of transcriptional repression of cell-cycle G2/M promoters by p63

PubMed Central

Testoni, Barbara; Mantovani, Roberto

2006-01-01

p63 is a developmentally regulated transcription factor related to p53, which activates and represses specific genes. The human AEC (Ankyloblepharon–Ectodermal dysplasia-Clefting) and EEC (Ectrodactyly–Ectodermal dysplasia–Cleft lip/palate) syndromes are caused by missense mutations of p63, within the DNA-binding domain (EEC) or in the C-terminal sterile alpha motif domain (AEC). We show here that p63 represses transcription of cell-cycle G2/M genes by binding to multiple CCAAT core promoters in immortalized and primary keratinocytes. The CCAAT-activator NF-Y and ΔNp63α are associated in vivo and a conserved α-helix of the NF-YC histone fold is required. p63 AEC mutants, but not an EEC mutant, are incapable to bind NF-Y. ΔNp63α, but not the AEC mutants repress CCAAT-dependent transcription of G2/M genes. Chromatin immunoprecipitation recruitment assays establish that the AEC mutants are not recruited to G2/M promoters, while normally present on 14-3-3σ, which contains a sequence-specific binding site. Surprisingly, the EEC C306R mutant activates transcription. Upon keratinocytes differentiation, NF-Y and p63 remain bound to G2/M promoters, while HDACs are recruited, histones deacetylated, Pol II displaced and transcription repressed. Our data indicate that NF-Y is a molecular target of p63 and that inhibition of growth activating genes upon differentiation is compromised by AEC missense mutations. PMID:16473849

Association of ferroportin Q248H polymorphism with elevated levels of serum ferritin in African-Americans in the Hemochromatosis and Iron Overload Screening (HEIRS) Study

PubMed Central

Rivers, Charles A.; Barton, James C.; Gordeuk, Victor R.; Acton, Ronald T.; Speechley, Mark R.; Snively, Beverly M.; Leiendecker-Foster, Catherine; Press, Richard D.; Adams, Paul C.; McLaren, Gordon D.; Dawkins, Fitzroy W.; McLaren, Christine E.; Reboussin, David M.

2007-01-01

The ferroportin (FPN1) Q248H polymorphism has been associated with increased serum ferritin (SF) levels in sub-Saharan Africans and in African Americans (AA). AA participants of the HEIRS Study who did not have HFE C282Y or H63D who had elevated initial screening SF (≥300 μg/L in men and ≥200 μg/L in women) (defined as cases) were frequency-matched to AA participants with normal SF (defined as controls) to investigate the association of the Q248H with elevated SF. 10.4% of cases and 6.7% of controls were Q248H heterozygotes (P = 0.257). Q248H homozygosity was observed in 0.5% of the cases and none of the controls. The frequency of Q248H was higher among men with elevated SF than among control men (P = 0.047); corresponding differences were not observed among women. This appeared to be unrelated to self-reports of a previous diagnosis of liver disease. Men with elevated SF were three times more likely than women with elevated SF to have Q248H (P = 0.012). There were no significant differences in Q248H frequencies in men and women control participants. We conclude that the frequency of the FPN1 Q248H polymorphism is greater in AA men with elevated SF than in those with normal SF. PMID:17276706

Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling.

PubMed

Qiu, Yue; Ogawa, Haruo; Miyagi, Masaru; Misono, Kunio S

2004-02-13

The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-to-head (hh) and tail-to-tail (tt) dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively. The tt-dimer structure has been proposed previously (van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K. S., and Yee, V. C. (2000) Nature 406, 101-104). However, no direct evidence is available to identify the physiological dimer form. Here we report site-directed mutagenesis studies of residues at the two alternative dimer interfaces in the full-length receptor expressed on COS cells. The Trp74 to Arg mutation (W74R) or D71R at the hh-dimer interface caused partial constitutive guanylate cyclase activation, whereas mutation F96D or H99D caused receptor uncoupling. In contrast, mutation Y196D or L225D at the tt-interface had no such effect. His99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hh-dimer represents the physiological structure. Recently, we determined the crystal structure of ANPR complexed with ANP and proposed a hormone-induced rotation mechanism mediating transmembrane signaling (H. Ogawa, Y. Qiu, C. M. Ogata, and K. S. Misono, submitted for publication). The observed effects of mutations are consistent with the ANP-induced structural change identified from the crystal structures with and without ANP and support the proposed rotation mechanism for ANP receptor signaling.

Different visible colors and green fluorescence were obtained from the mutated purple chromoprotein isolated from sea anemone.

PubMed

Chiang, Cheng-Yi; Chen, Yi-Lin; Tsai, Huai-Jen

2014-08-01

Green fluorescent protein (GFP)-like proteins have been studied with the aim of developing fluorescent proteins. Since the property of color variation is understudied, we isolated a novel GFP-like chromoprotein from the carpet anemone Stichodactyla haddoni, termed shCP. Its maximum absorption wavelength peak (λ(max)) is located at 574 nm, resulting in a purple color. The shCP protein consists of 227 amino acids (aa), sharing 96 % identity with the GFP-like chromoprotein of Heteractis crispa. We mutated aa residues to examine any alteration in color. When E63, the first aa of the chromophore, was replaced by serine (E63S), the λ(max) of the mutated protein shCP-E63S was shifted to 560 nm and exhibited a pink color. When Q39, T194, and I196, which reside in the surrounding 5 Å of the chromophore's microenvironment, were mutated, we found that (1) the λ(max) of the mutated protein shCP-Q39S was shifted to 518 nm and exhibited a red color, (2) shCP-T194I exhibited a purple-blue color, and (3) an additional mutation at I196H of the mutated protein shCP-E63L exhibited green fluorescence. In contrast, when the aa located neither at the chromophore nor within its microenvironment were mutated, the resultant proteins shCP-L122H, -E138G, -S137D, -T95I, -D129N, -T194V, -E138Q, -G75E, -I183V, and -I70V never altered their purple color, suggesting that mutations at the shCP chromophore and the surrounding 5 Å microenvironment mostly control changes in color expression or cause fluorescence to develop. Additionally, we found that the cDNAs of shCP and its mutated varieties are faithfully and stably expressed both in Escherichia coli and zebrafish embryos.

Phosphorylation of cMyBP-C Affects Contractile Mechanisms in a Site-specific Manner

PubMed Central

Wang, Li; Ji, Xiang; Barefield, David; Sadayappan, Sakthivel; Kawai, Masakata

2014-01-01

Cardiac myosin binding protein-C (cMyBP-C) is a cardiac-specific, thick-filament regulatory protein that is differentially phosphorylated at Ser273, Ser282, and Ser302 by various kinases and modulates contraction. In this study, phosphorylation-site-specific effects of cMyBP-C on myocardial contractility and cross-bridge kinetics were studied by sinusoidal analysis in papillary and trabecular muscle fibers isolated from t/t (cMyBP-C-null) mice and in their counterparts in which cMyBP-C contains the ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), and SAS (Ser273-Ala282-Ser302) mutations; the results were compared to those from mice expressing the wild-type (WT) transgene on the t/t background. Under standard activating conditions, DAD fibers showed significant decreases in tension (∼50%), stiffness, the fast apparent rate constant 2πc, and its magnitude C, as well as its magnitude H, but an increase in the medium rate constant 2πb, with respect to WT. The t/t fibers showed a smaller drop in stiffness and a significant decrease in 2πc that can be explained by isoform shift of myosin heavy chain. In the pCa-tension study using the 8 mM phosphate (Pi) solution, there was hardly any difference in Ca2+ sensitivity (pCa50) and cooperativity (nH) between the mutant and WT samples. However, in the solutions without Pi, DAD showed increased nH and slightly decreased pCa50. We infer from these observations that the nonphosphorylatable residue 282 combined with phosphomimetic residues Asp273 and/or Asp302 (in DAD) is detrimental to cardiomyocytes by lowering isometric tension and altering cross-bridge kinetics with decreased 2πc and increased 2πb. In contrast, a single change of residue 282 to nonphosphorylatable Ala (SAS), or to phosphomimetic Asps together with the changes of residues 273 and 302 to nonphosphorylatable Ala (ADA) causes minute changes in fiber mechanics. PMID:24606935

High-resolution Melting Analysis for Gene Scanning of Adenomatous Polyposis Coli (APC) Gene With Oral Squamous Cell Carcinoma Samples.

PubMed

Chang, Ya-Sian; Lin, Chien-Yu; Yang, Shu-Fen; Ho, Cheng Mao; Chang, Jan-Gowth

2016-02-01

There have been many different mutations reported for the large adenomatous polyposis coli (APC) tumor suppressor gene. APC mutations result in inactivation of APC tumor suppressor action, allowing the progression of tumorigenesis. The present study utilized a highly efficient method to identify APC mutations and investigated the association between the APC genetic variants Y486Y, A545A, T1493T, and D1822V and susceptibility to oral squamous cell carcinoma (OSCC). High-resolution melting (HRM) analysis was used to characterize APC mutations. Genomic DNA was extracted from 83 patient specimens of OSCC and 50 blood samples from healthy control subjects. The 14 exons and mutation cluster region of exon 15 were screened by HRM analysis. All mutations were confirmed by direct DNA sequencing. Three mutations and 4 single nucleotide polymorphisms (SNPs) were found in this study. The mutations were c.573T>C (Y191Y) in exon 5, c.1005A>G (L335L) in exon 9, and c.1488A>T (T496T) in exon 11. Two SNPs, c.4479G>A (T1493T) and c.5465A>T (D1822V), were located in exon 15, whereas c.1458T>C (Y486Y) and c.1635G>A (A545A) were located in exon 11 and 13, respectively. There was no observed association between OSCC risk and genotype for any of the 4 APC SNPs. The mutation of APC is rare in Taiwanese patients with OSCC. HRM analysis is a reliable, accurate, and fast screening method for APC mutations.

M.H.D. Diagnostics - Gas Temperature and Emittance

DTIC Science & Technology

1960-04-06

AD-A280 700 DTIC $ELECTE M.H.D. DIAGNOSTICS - GAS TEMPERATURE AND EMITTANCE by W. E. HILL REPORT NO. 60GL63 APRIL 6, 1960t i T i n n e iii, ic ie. n ...Accesion For NTIS CRA&I DTIC TAB Una;Olow;ced Justification Distribution y Availability C,.. Avail ard C.A Dist Special GENERAL* ELECTRIC _ l I SCHOWCTADY...from the Instrumen- tation viewpoint. Some of the instrumentation techniques developed $ n the allied field of combustion instrumentation can be expected

Identification of the critical sites of NNRTI-resistance in reverse transcriptase of HIV-1 CRF_BC strains.

PubMed

Huang, Yang; Li, Zhenpeng; Xing, Hui; Jiao, Yang; Ouyang, Yabo; Liao, Lingjie; Jiang, Shibo; Armstrong, Rebecca; Shao, Yiming; Ma, Liying

2014-01-01

The polymorphisms involved in drug resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) in HIV-1 CRF_BC, the most prevalent HIV-1 strain in China, have been poorly characterized. To reveal the drug resistance mutations, we compared the gene sequences of pol region of HIV-1 CRF_BC from 631 treatment-naïve and 363 treatment-experienced patients using the selection pressure-based method. We calculated an individual Ka/Ks value for each specific amino acid mutation. Result showed that eight polymorphic mutations (W88C, K101Q, I132L, R135L, T139K/R, H221Y and L228R) in RT for treatment-experienced patients were identified, while they, except for R135L, were completely absent in those from treatment-naïve patients. The I132L and T139K/R mutants exhibited high-level resistance to DLV and NVP and moderate resistance to TMC-125 and EFV, while the K101Q and H221Y mutants exhibited an increased resistance to all four NNRTIs tested. The W88C, R135L, and L228R may be RTI-induced adaptive mutations. Y181C+K101Q mutant showed a 2.5-, 4.4-, and 4.7-fold higher resistance to TMC-125, NVP and EFV, respectively, than Y181C alone mutant, while Y181C+H221Y or K103N+H221Y mutants had significantly higher resistance to all four NNRTIs than Y181C or K103N mutants. K103N+T139K and G190A+T139K mutant induce higher resistance (2.0∼14.2-fold and 1.5∼7.2-fold, respectively) to all four NNRTIs than K103N or G190A alone mutation. I132L and T139K/R are rare but critical mutations associated with NNRTI-resistance for some NNRTIs. K101Q, H221Y and T139K can enhance K103N/Y181C/G190A-assocated NNRTI-resistance. Monitoring these mutations will provide useful information for rational design of the NNRTI-based antiretroviral regimen for HIV-1 CRF_BC-infected patients.

Clinical penetrance in hereditary hemochromatosis: estimates of the cumulative incidence of severe liver disease among HFE C282Y homozygotes.

PubMed

Grosse, Scott D; Gurrin, Lyle C; Bertalli, Nadine A; Allen, Katrina J

2018-04-01

Iron overload (hemochromatosis) can cause serious, symptomatic disease that is preventable if detected early and managed appropriately. The leading cause of hemochromatosis in populations of predominantly European ancestry is homozygosity of the C282Y variant in the HFE gene. Screening of adults for iron overload or associated genotypes is controversial, largely because of a belief that severe phenotypes are uncommon, although cascade testing of first-degree relatives of patients is widely endorsed. We contend that severe liver disease (cirrhosis or hepatocellular cancer) is not at all uncommon among older males with hereditary hemochromatosis. Our review of the published data from a variety of empirical sources indicates that roughly 1 in 10 male HFE C282Y homozygotes is likely to develop severe liver disease during his lifetime unless iron overload is detected early and treated. New evidence from a randomized controlled trial of treatment allows for evidence-based management of presymptomatic patients. Although population screening for HFE C282Y homozygosity faces multiple barriers, a potentially effective strategy for increasing the early detection and prevention of clinical iron overload and severe disease is to include HFE C282Y homozygosity in lists of medically actionable gene variants when reporting the results of genome or exome sequencing.

Interfacial Partitioning of a Loop Hinge Residue Contributes to Diacylglycerol Affinity of Conserved Region 1 Domains*

PubMed Central

Stewart, Mikaela D.; Cole, Taylor R.; Igumenova, Tatyana I.

2014-01-01

Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant. The W252Y mutation was previously shown to render C1Bδ less responsive to DAG (Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J. Biol. Chem. 282, 826–830) and thereby emulate the behavior of C1B domains from conventional PKCs that have a conserved Tyr at the equivalent position. Our data revealed that W252Y mutation did not perturb the conformation of C1Bδ in solution but significantly reduced its propensity to partition into a membrane-mimicking environment in the absence of DAG. Using detergent micelles doped with a paramagnetic lipid, we determined that both the residue identity at position 252 and complexation with diacylglycerol influence the geometry of C1Bδ-micelle interactions. In addition, we identified the C-terminal helix α1 of C1Bδ as an interaction site with the head groups of phosphatidylserine, a known activator of PKCδ. Taken together, our studies (i) reveal the identities of C1Bδ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities. PMID:25124034

Analysing the Effect of Mutation on Protein Function and Discovering Potential Inhibitors of CDK4: Molecular Modelling and Dynamics Studies

PubMed Central

N, Nagasundaram; Zhu, Hailong; Liu, Jiming; V, Karthick; C, George Priya Doss; Chakraborty, Chiranjib; Chen, Luonan

2015-01-01

The cyclin-dependent kinase 4 (CDK4)-cyclin D1 complex plays a crucial role in the transition from the G1 phase to S phase of the cell cycle. Among the CDKs, CDK4 is one of the genes most frequently affected by somatic genetic variations that are associated with various forms of cancer. Thus, because the abnormal function of the CDK4-cyclin D1 protein complex might play a vital role in causing cancer, CDK4 can be considered a genetically validated therapeutic target. In this study, we used a systematic, integrated computational approach to identify deleterious nsSNPs and predict their effects on protein-protein (CDK4-cyclin D1) and protein-ligand (CDK4-flavopiridol) interactions. This analysis resulted in the identification of possible inhibitors of mutant CDK4 proteins that bind the conformations induced by deleterious nsSNPs. Using computational prediction methods, we identified five nsSNPs as highly deleterious: R24C, Y180H, A205T, R210P, and R246C. From molecular docking and molecular dynamic studies, we observed that these deleterious nsSNPs affected CDK4-cyclin D1 and CDK4-flavopiridol interactions. Furthermore, in a virtual screening approach, the drug 5_7_DIHYDROXY_ 2_ (3_4_5_TRI HYDROXYPHENYL) _4H_CHROMEN_ 4_ONE displayed good binding affinity for proteins with the mutations R24C or R246C, the drug diosmin displayed good binding affinity for the protein with the mutation Y180H, and the drug rutin displayed good binding affinity for proteins with the mutations A205T and R210P. Overall, this computational investigation of the CDK4 gene highlights the link between genetic variation and biological phenomena in human cancer and aids in the discovery of molecularly targeted therapies for personalized treatment. PMID:26252490

Enantiomer-Selective Photo-Induced Reaction of Protonated Tryptophan with Disaccharides in the Gas Phase

NASA Astrophysics Data System (ADS)

Doan, Thuc N.; Fujihara, Akimasa

2018-03-01

In order to investigate chemical evolution in interstellar molecular clouds, enantiomer-selective photo-induced chemical reactions between an amino acid and disaccharides in the gas phase were examined using a tandem mass spectrometer containing an electrospray ionization source and a cold ion trap. Ultraviolet photodissociation mass spectra of cold gas-phase noncovalent complexes of protonated tryptophan (Trp) enantiomers with disaccharides consisting of two d-glucose units, such as d-maltose or d-cellobiose, were obtained by photoexcitation of the indole ring of Trp. NH2CHCOOH loss via cleavage of the Cα-Cβ bond in Trp induced by hydrogen atom transfer from the NH3 + group of a protonated Trp was observed in a noncovalent heterochiral H+( l-Trp)( d-maltose) complex. In contrast, a photo-induced chemical reaction forming the product ion with m/z 282 occurs in homochiral H+( d-Trp)( d-maltose). For d-cellobiose, both NH2CHCOOH elimination and the m/z 282 product ion were observed, and no enantiomer-selective phenomena occurred. The m/z 282 product ion indicates that the photo-induced C-glycosylation, which links d-glucose residues to the indole moiety of Trp via a C-C bond, can occur in cold gas-phase noncovalent complexes, and its enantiomer-selectivity depends on the structure of the disaccharide.

Rare HFE variants are the most frequent cause of hemochromatosis in non-c282y homozygous patients with hemochromatosis.

PubMed

Hamdi-Rozé, Houda; Beaumont-Epinette, Marie-Pascale; Ben Ali, Zeineb; Le Lan, Caroline; Loustaud-Ratti, Véronique; Causse, Xavier; Loreal, Olivier; Deugnier, Yves; Brissot, Pierre; Jouanolle, Anne-Marie; Bardou-Jacquet, Edouard

2016-12-01

p.Cys282Tyr (C282Y) homozygosity explains most cases of HFE-related hemochromatosis, but a significant number of patients presenting with typical type I hemochromatosis phenotype remain unexplained. We sought to describe the clinical relevance of rare HFE variants in non-C282Y homozygotes. Patients referred for hemochromatosis to the National Reference Centre for Rare Iron Overload Diseases from 2004 to 2010 were studied. Sequencing was performed for coding region and intronic flanking sequences of HFE, HAMP, HFE2, TFR2, and SLC40A1. Nine private HFE variants were identified in 13 of 206 unrelated patients. Among those, five have not been previously described: p.Leu270Argfs*4, p.Ala271Valfs*25, p.Tyr52*, p.Lys166Asn, and p.Asp141Tyr. Our results show that rare HFE variants are identified more frequently than variants in the other genes associated with iron overload. Rare HFE variants are therefore the most frequent cause of hemochromatosis in non-C282Y homozygote HFE patients. Am. J. Hematol. 91:1202-1205, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Missense mutations (p.H371Y, p.D438Y) in gene CHEK2 are associated with breast cancer risk in women of Balochistan origin.

PubMed

Baloch, Abdul Hameed; Daud, Shakeela; Raheem, Nafeesa; Luqman, Muhammad; Ahmad, Adeel; Rehman, Abdul; Shuja, Jameela; Rasheed, Saeeda; Ali, Akhtar; Kakar, Naseebullah; Naseeb, Hafiz Khush; Mengal, Mohammad Alam; Awan, Muhammad Arif; Wasim, Muhammad; Baloch, Dost Mohammad; Ahmad, Jamil

2014-02-01

CHEK2 encodes a serine/threonine-protein kinase which plays a critical role in DNA damage signaling pathways. CHEK2 directly phosphorylates and regulates the functions of p53 and BRCA1. Most women with breast and/or ovarian cancer are not carriers of mutant BRCA1 or BRCA2. Multiple studies have shown that a CHEK2*1100delC confers about a two-fold increased risk of breast cancer in unselected females and a tenfold increase in males. Moreover, studies have shown that first-degree relatives of bilateral breast cancer cases who carried the CHEK2*1100delC allele had an eight-fold increased risk of breast cancer. It has been suggested that CHEK2 functions as a low-penetrance susceptibility gene for cancers and multiplies the risks associated with other gene(s) to increase cancer risk. The main goal of this study was to evaluate and to compare the role of truncating mutations, splice junction mutations and rare missense substitutions in breast cancer susceptibility gene CHEK2. Present study was performed on 140 individuals including 70 breast cancer patients both with and without family history and 70 normal individuals. Written consent was obtained and 3 ml intravenous blood was drawn from all the subjects. DNA was extracted from all the samples through inorganic method published already. Primers were synthesized for all the 14 exons of CHEK2 gene. Coding and adjacent intronic sequences of CHEK2 gene were amplified and sequenced. Two genetic variants (p.H371Y, p.D438Y) were found in exon 10 and exon 11 of gene CHEK2 which were not found in any of the 70 control individuals from same geographical area and ethnic group. The genetic variant c.1312G>T (p.D438Y) identified in a patient with a family history of breast cancer. To our knowledge, this is first mutation scanning study of gene CHEK2 from Balochistan population.

A missense mutation in ALDH18A1, encoding Delta1-pyrroline-5-carboxylate synthase (P5CS), causes an autosomal recessive neurocutaneous syndrome.

PubMed

Bicknell, Louise S; Pitt, James; Aftimos, Salim; Ramadas, Ram; Maw, Marion A; Robertson, Stephen P

2008-10-01

There are several rare syndromes combining wrinkled, redundant skin and neurological abnormalities. Although phenotypic overlap between conditions has suggested that some might be allelic to one another, the aetiology for many of them remains unknown. A consanguineous New Zealand Maori family has been characterised that segregates an autosomal recessive connective tissue disorder (joint dislocations, lax skin) associated with neurological abnormalities (severe global developmental delay, choreoathetosis) without metabolic abnormalities in four affected children. A genome-screen performed under a hypothesis of homozygosity by descent for an ancestral mutation, identified a locus at 10q23 (Z = 3.63). One gene within the candidate interval, ALDH18A1, encoding Delta1-pyrroline-5-carboxylate synthase (P5CS), was considered a plausible disease gene since a missense mutation had previously been shown to cause progressive neurodegeneration, cataracts, skin laxity, joint dislocations and metabolic derangement in a consanguineous Algerian family. A missense mutation, 2350C>T, was identified in ALDH18A1, which predicts the substitution H784Y. H784 is invariant across all phyla and lies within a previously unrecognised, conserved C-terminal motif in P5CS. In an in vivo assay of flux through this metabolic pathway using dermal fibroblasts obtained from an affected individual, proline and ornithine biosynthetic activity of P5CS was not affected by the H784Y substitution. These data suggest that P5CS may possess additional uncharacterised functions that affect connective tissue and central nervous system function.

Serum Ferritin, Insulin Resistance, and Metabolic Syndrome: Clinical and Laboratory Associations in 769 Non-Hispanic Whites Without Diabetes Mellitus in the HEIRS Study

PubMed Central

Barton, J. Clayborn; Barton, James C.

2015-01-01

Abstract Background: In some reports, serum ferritin (SF) has been associated with insulin resistance and metabolic syndrome. Methods: We studied non-Hispanic whites without diabetes mellitus in a postscreening examination. Participants included cases [HFE C282Y homozygosity; and transferrin saturation (TS) >50% and SF >300 μg/L (males) and TS >45% and SF >200 μg/dL (females), regardless of HFE genotype] and controls [HFE wild-type (wt/wt) and TS/SF 25th–75th percentiles]. We excluded participants with overnight fasts <8 hr, cirrhosis, hepatitis B or C, pregnancy, or missing data. Observations were age, sex, C282Y homozygosity, body mass index (BMI), systolic and diastolic blood pressures (SBP, DBP), lymphocytes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), C-reactive protein (CRP), TS, SF, and glucose/insulin. Insulin resistance was defined as homeostasis model assessment of insulin resistance (HOMA-IR) 4th quartile (≥2.70). Results: A total of 407 women and 362 men (mean age 54 years) included 188 C282Y homozygotes and 371 wt/wt. Significant trends across HOMA-IR quartiles included age, male sex, BMI, SBP, DBP, lymphocytes, ALT, CRP >0.5 mg/dL (positive), and TS (negative). Multiple regression on HOMA-IR revealed significant associations with male sex, BMI, SBP, lymphocytes, ALT, CRP>0.5 mg/dL (positive), and DBP and SF (negative). Logistic regression on HOMA-IR 4th quartile revealed significant positive associations with age, male sex, BMI, and lymphocytes. Metabolic syndrome occurred in 53 participants (6.9%). Logistic regression on metabolic syndrome revealed these odds ratios: HOMA-IR 4th quartile [9.1 (4.8, 17.3)] and CRP >0.5 mg/dL [2.9 (1.6, 5.4)]. Conclusions: Age, male sex, BMI, and lymphocytes were positively associated with HOMA-IR after correction for other factors. HOMA-IR 4th quartile and CRP >0.5 mg/dL predicted metabolic syndrome. PMID:25423072

Serum ferritin, insulin resistance, and metabolic syndrome: clinical and laboratory associations in 769 non-hispanic whites without diabetes mellitus in the HEIRS study.

PubMed

Acton, Ronald T; Barton, J Clayborn; Barton, James C

2015-03-01

In some reports, serum ferritin (SF) has been associated with insulin resistance and metabolic syndrome. We studied non-Hispanic whites without diabetes mellitus in a postscreening examination. Participants included cases [HFE C282Y homozygosity; and transferrin saturation (TS) >50% and SF >300 μg/L (males) and TS >45% and SF >200 μg/dL (females), regardless of HFE genotype] and controls [HFE wild-type (wt/wt) and TS/SF 25th-75th percentiles]. We excluded participants with overnight fasts <8 hr, cirrhosis, hepatitis B or C, pregnancy, or missing data. Observations were age, sex, C282Y homozygosity, body mass index (BMI), systolic and diastolic blood pressures (SBP, DBP), lymphocytes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), C-reactive protein (CRP), TS, SF, and glucose/insulin. Insulin resistance was defined as homeostasis model assessment of insulin resistance (HOMA-IR) 4th quartile (≥2.70). A total of 407 women and 362 men (mean age 54 years) included 188 C282Y homozygotes and 371 wt/wt. Significant trends across HOMA-IR quartiles included age, male sex, BMI, SBP, DBP, lymphocytes, ALT, CRP >0.5 mg/dL (positive), and TS (negative). Multiple regression on HOMA-IR revealed significant associations with male sex, BMI, SBP, lymphocytes, ALT, CRP>0.5 mg/dL (positive), and DBP and SF (negative). Logistic regression on HOMA-IR 4th quartile revealed significant positive associations with age, male sex, BMI, and lymphocytes. Metabolic syndrome occurred in 53 participants (6.9%). Logistic regression on metabolic syndrome revealed these odds ratios: HOMA-IR 4th quartile [9.1 (4.8, 17.3)] and CRP >0.5 mg/dL [2.9 (1.6, 5.4)]. Age, male sex, BMI, and lymphocytes were positively associated with HOMA-IR after correction for other factors. HOMA-IR 4th quartile and CRP >0.5 mg/dL predicted metabolic syndrome.

HFE gene C282Y variant is associated with colorectal cancer in Caucasians: a meta-analysis.

PubMed

Chen, Weidong; Zhao, Hua; Li, Tiegang; Yao, Hongliang

2013-08-01

The HFE gene has been suggested to play an important role in the pathogenesis of colorectal cancer. However, the results have been conflicting. In this study, we performed a meta-analysis to clarify the association of HFE gene C282Y variant with colorectal cancer. PubMed and Embase were retrieved to identify the potential literature. Pooled odds ratio (OR) with 95 % confidence interval (CI) was calculated using fixed- or random-effects model. A total of eight papers including nine studies (7,588 colorectal cancer cases and 81,571 controls) for HFE gene C282Y variant were included in the meta-analysis. The result indicated that HFE gene C282Y variant was significantly associated with colorectal cancer under recessive model (OR = 2.00, 95 % CI = 1.32-3.04), with no evidence of between-study heterogeneity (I (2) = 0.2 %, p = 0.432). Further subgroup analysis by number of cases suggested the effect was significant in studies with more than 500 cases (OR = 2.51, 95 % CI = 1.58-3.98, I (2) = 0.0 %, p = 0.921), but not in studies with less than 500 cases (OR = 0.75, 95 % CI = 0.28-1.97, I (2) = 0.0 %, p = 0.622). The current meta-analysis supported the positive association of HFE gene C282Y variant with colorectal cancer. Further large-scale studies with the consideration for gene-gene/gene-environment interactions should be conducted to investigate the association.

Screening of SOD1, FUS and TARDBP genes in patients with amyotrophic lateral sclerosis in central-southern China.

PubMed

Hou, Lihua; Jiao, Bin; Xiao, Tingting; Zhou, Lu; Zhou, Zhifan; Du, Juan; Yan, Xinxiang; Wang, Junling; Tang, Beisha; Shen, Lu

2016-09-08

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons of the brain, brainstem and spinal cord. To date, mutations in more than 30 genes have been linked to the pathogenesis of ALS. Among them, SOD1, FUS and TARDBP are ranked as the three most common genes associated with ALS. However, no mutation analysis has been reported in central-southern China. In this study, we sequenced SOD1, FUS and TARDBP in a central-southern Chinese cohort of 173 patients with ALS (15 familial ALS and 158 sporadic ALS) to detect mutations. As a result, five missense mutations in SOD1, namely, p.D101N, p.D101G, p.C111Y, p.N86S and p.V87A, were identified in three unrelated familial probands and three sporadic cases; two mutations in FUS were found in two unrelated familial probands, including an insertion mutation (p.P525_Y526insY) and a missense mutation (p.R521H); no variants of TARDBP were observed in patients. Therefore, SOD1 mutations were present in 20.0% of familial ALS patients and 1.9% of sporadic ALS patients, while FUS mutations were responsible for 13.3% of familial ALS cases, and TARDBP mutations were rare in either familial or sporadic ALS cases. This study broadens the known mutational spectrum in patients with ALS and further demonstrates the necessity for genetic screening in ALS patients from central-southern China.

Targeted next generation sequencing identifies novel NOTCH3 gene mutations in CADASIL diagnostics patients.

PubMed

Maksemous, Neven; Smith, Robert A; Haupt, Larisa M; Griffiths, Lyn R

2016-11-24

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic, hereditary, small vessel disease of the brain causing stroke and vascular dementia in adults. CADASIL has previously been shown to be caused by varying mutations in the NOTCH3 gene. The disorder is often misdiagnosed due to its significant clinical heterogeneic manifestation with familial hemiplegic migraine and several ataxia disorders as well as the location of the currently identified causative mutations. The aim of this study was to develop a new, comprehensive and efficient single assay strategy for complete molecular diagnosis of NOTCH3 mutations through the use of a custom next-generation sequencing (NGS) panel for improved routine clinical molecular diagnostic testing. Our custom NGS panel identified nine genetic variants in NOTCH3 (p.D139V, p.C183R, p.R332C, p.Y465C, p.C597W, p.R607H, p.E813E, p.C977G and p.Y1106C). Six mutations were stereotypical CADASIL mutations leading to an odd number of cysteine residues in one of the 34 NOTCH3 gene epidermal growth factor (EGF)-like repeats, including three new typical cysteine mutations identified in exon 11 (p.C597W; c.1791C>G); exon 18 (p.C977G; c.2929T>G) and exon 20 (p.Y1106C; c.3317A>G). Interestingly, a novel missense mutation in the CACNA1A gene was also identified in one CADASIL patient. All variants identified (novel and known) were further investigated using in silico bioinformatic analyses and confirmed through Sanger sequencing. NGS provides an improved and effective methodology for the diagnosis of CADASIL. The NGS approach reduced time and cost for comprehensive genetic diagnosis, placing genetic diagnostic testing within reach of more patients.

A mutation of the p63 gene in non‐syndromic cleft lip

PubMed Central

Leoyklang, P; Siriwan, P; Shotelersuk, V

2006-01-01

Mutations in the p63 gene (TP63) underlie several monogenic malformation syndromes manifesting cleft lip with or without cleft palate (CL/P). We investigated whether p63 mutations also result in non‐syndromic CL/P. Specifically, we performed mutation analysis of the 16 exons of the p63 gene for 100 Thai patients with non‐syndromic CL/P. In total, 21 variant sites were identified. All were single nucleotide changes, with six in coding regions, including three novel non‐synonymous changes: S90L, R313G, and D564H. The R313G was concluded to be pathogenic on the basis of its amino acid change, evolutionary conservation, its occurrence in a functionally important domain, its predicted damaging function, its de novo occurrence, and its absence in 500 control individuals. Our data strongly suggest, for the first time, a causative role of a heterozygous mutation in the p63 gene in non‐syndromic CL/P, highlighting the wide phenotypic spectrum of p63 gene mutations. PMID:16740912

Genomic Instability and Breast Cancer

DTIC Science & Technology

2009-10-01

van Deursen, J., Nussenzweig, A., Paull, T . T ., Alt, F. W., and Chen, J. (2006)Mol. Cell 21, 187–200 7. Huen, M. S., Grant, R ., Manke, I., Minn, K...Science 316, 1198–1202 14. Lechner,M. S., Levitan, I., andDressler, G. R . (2000)Nucleic Acids Res. 28, 2741–2751 15. Cho, Y. W., Hong, T ., Hong, S., Guo...H., Yu, H., Kim, D., Guszczynski, T ., Dressler, G. R ., Copeland, T . D., Kalkum, M., and Ge, K. (2007) J. Biol. Chem. 282, 20395–20406 16. Patel, S. R

Disease-causing mutations C277R and C277Y modify gating of human ClC-1 chloride channels in myotonia congenita

PubMed Central

Weinberger, Sebastian; Wojciechowski, Daniel; Sternberg, Damien; Lehmann-Horn, Frank; Jurkat-Rott, Karin; Becher, Toni; Begemann, Birgit; Fahlke, Christoph; Fischer, Martin

2012-01-01

Myotonia congenita is a genetic condition that is caused by mutations in the muscle chloride channel gene CLCN1 and characterized by delayed muscle relaxation and muscle stiffness. We here investigate the functional consequences of two novel disease-causing missense mutations, C277R and C277Y, using heterologous expression in HEK293T cells and patch clamp recording. Both mutations reduce macroscopic anion currents in transfected cells. Since hClC-1 is a double-barrelled anion channel, this reduction in current amplitude might be caused by altered gating of individual protopores or of joint openings and closing of both protopores. We used non-stationary noise analysis and single channel recordings to separate the mutants’ effects on individual and common gating processes. We found that C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates resulting in decreases of absolute open probabilities of homodimeric channels to values below 3%. In heterodimeric channels, C277R and C277Y also reduce open probabilities and shift the common gate activation curve towards positive potentials. Moreover, C277Y modifies pore properties of hClC-1. It reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence to I− = NO3− > Br− > Cl−. Our findings predict a dramatic reduction of the muscle fibre resting chloride conductance and thus fully explain the disease-causing effects of mutations C277R and C277Y. Moreover, they provide additional insights into the function of C277, a residue recently implicated in common gating of ClC channels. PMID:22641783

Leber's hereditary optic neuropathy is associated with mitochondrial ND1 T3394C mutation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Min; Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang 325003; Guan, Minqiang

2009-06-05

We report here the clinical, genetic and molecular characterization of four Chinese families with Leber's hereditary optic neuropathy (LHON). There were variable severity and age-of-onset in visual impairment among these families. Strikingly, there were extremely low penetrances of visual impairment in these Chinese families. Sequence analysis of complete mitochondrial genomes in these pedigrees showed the homoplasmic T3394C (Y30H) mutation, which localized at a highly conserved tyrosine at position 30 of ND1, and distinct sets of mtDNA polymorphisms belonging to haplogroups D4b and M9a. The occurrence of T3394C mutation in these several genetically unrelated subjects affected by visual impairment strongly indicatesmore » that this mutation is involved in the pathogenesis of visual impairment. However, there was the absence of functionally significant mtDNA mutations in these four Chinese pedigrees carrying the T3394C mutation. Therefore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic expression of the LHON-associated T3394C mutation.« less

Structure-based affinity maturation of a chimeric anti-ricin antibody C4C13.

PubMed

Luo, Longlong; Luo, Qun; Guo, Leiming; Lv, Ming; Lin, Zhou; Geng, Jing; Li, Xinying; Li, Yan; Shen, Beifen; Qiao, Chunxia; Feng, Jiannan

2014-01-01

Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical and physical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (N(H102)F, W(H103)Y); M2 (W(H103)Y) and M3 (R(L90)G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3 nmol/L) than C4C13 (130 nmol/L) and M3 had weaker affinity (234.5 nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future.

Isolation of temperature-sensitive mutations in murC of Staphylococcus aureus.

PubMed

Ishibashi, Mihoko; Kurokawa, Kenji; Nishida, Satoshi; Ueno, Kohji; Matsuo, Miki; Sekimizu, Kazuhisa

2007-09-01

Enzymes in the bacterial peptidoglycan biosynthesis pathway are important targets for novel antibiotics. Of 750 temperature-sensitive (TS) mutants of Gram-positive Staphylococcus aureus, six were complemented by the murC gene, which encodes the UDP-N-acetylmuramic acid:l-alanine ligase. Each mutation resulted in a single amino acid substitution and, in all cases, the TS phenotype was suppressed by high osmotic stress. In mutant strains with the G222E substitution, a decrease in the viable cell number immediately after shift to the restrictive temperature was observed. These results suggest that S. aureus MurC protein is essential for cell growth. The MurC H343Y mutation is located in the putative alanine recognition pocket. Consistent with this, allele-specific suppression was observed of the H343Y mutation by multiple copies of the aapA gene, which encodes an alanine transporter. The results suggest an in vivo role for the H343 residue of S. aureus MurC protein in high-affinity binding to L-alanine.

[Genetic Characterization of Hemagglutinin on Measles Virus Epidemic Strain Genotype H1a in Liaoning Province (China) from 1997 to 2014].

PubMed

Wang, Yan; Ma, Yan; Xu, Xiaoting; Hao, Shuang; Han, Yue; Yao, Wenqing; Zhao, Zhuo

2015-07-01

To wished to characterize the hemagglutinin (H) gene of the measles virus epidemic strain H1a in Liaoning Province (China) from 1997-2014 to provide a basis for the control and elimination of measles. All 63 measles virus strains were the H1a genotype. Fragments of the H gene (1854 nucleotides) and nucleoprotein (N) gene (450 nucleotides) were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the PCR products sequenced and analyzed. Phylogenetic-trees were constructed with reference strains of the genotype-H measles virus downloaded from GenBank, including Chinese measles virus strains isolated in 1993-1994 and the vaccine reference strains S-191 and C-47. Sixty-three strains of the measles virus in 1997-2014 belonged to genotype H1a. The mean evolutionary rate for gene N-450 was higher than that for the H gene. All 63 strains of the measles virus were mutated from: serine (Ser S) to asparagine (Asn N) in the 240th amino acid; arginine (Arg R) to glycine (Gly G) in the 243th; and tyrosine (Tyr Y) to Asn N in the 481th amino acid. All measles virus strains in cluster 2 were mutated from proline (Pro P) to leucine (Leu L) in the 397th amino acid. The other neutralization sites showed no apparent difference when comparing the nucleotides/amino acids of the H gene of S191 vaccine strains.

HFE gene variants and iron-induced oxygen radical generation in idiopathic pulmonary fibrosis.

PubMed

Sangiuolo, Federica; Puxeddu, Ermanno; Pezzuto, Gabriella; Cavalli, Francesco; Longo, Giuliana; Comandini, Alessia; Di Pierro, Donato; Pallante, Marco; Sergiacomi, Gianluigi; Simonetti, Giovanni; Zompatori, Maurizio; Orlandi, Augusto; Magrini, Andrea; Amicosante, Massimo; Mariani, Francesca; Losi, Monica; Fraboni, Daniela; Bisetti, Alberto; Saltini, Cesare

2015-02-01

In idiopathic pulmonary fibrosis (IPF), lung accumulation of excessive extracellular iron and macrophage haemosiderin may suggest disordered iron homeostasis leading to recurring microscopic injury and fibrosing damage. The current study population comprised 89 consistent IPF patients and 107 controls. 54 patients and 11 controls underwent bronchoalveolar lavage (BAL). Haemosiderin was assessed by Perls' stain, BAL fluid malondialdehyde (MDA) by high-performance liquid chromatography, BAL cell iron-dependent oxygen radical generation by fluorimetry and the frequency of hereditary haemochromatosis HFE gene variants by reverse dot blot hybridisation. Macrophage haemosiderin, BAL fluid MDA and BAL cell unstimulated iron-dependent oxygen radical generation were all significantly increased above controls (p<0.05). The frequency of C282Y, S65C and H63D HFE allelic variants was markedly higher in IPF compared with controls (40.4% versus 22.4%, OR 2.35, p=0.008) and was associated with higher iron-dependent oxygen radical generation (HFE variant 107.4±56.0, HFE wild type (wt) 59.4±36.4 and controls 16.7±11.8 fluorescence units per 10(5) BAL cells; p=0.028 HFE variant versus HFE wt, p=0.006 HFE wt versus controls). The data suggest iron dysregulation associated with HFE allelic variants may play an important role in increasing susceptibility to environmental exposures, leading to recurring injury and fibrosis in IPF. Copyright ©ERS 2015.

HFE polymorphisms influence the response to chemotherapeutic agents via induction of p16INK4A.

PubMed

Lee, Sang Y; Liu, Siying; Mitchell, Ryan M; Slagle-Webb, Becky; Hong, Young-Soo; Sheehan, Jonas M; Connor, James R

2011-11-01

HFE is a protein that impacts cellular iron uptake. HFE gene variants are identified as risk factors or modifiers for multiple diseases. Using HFE stably transfected human neuroblastoma cells, we found that cells carrying the C282Y HFE variant do not differentiate when exposed to retinoic acid. Therefore, we hypothesized HFE variants would impact response to therapeutic agents. Both the human neuroblastoma and glioma cells that express the C282Y HFE variant are resistant to Temodar, geldanamycin and γ-radiation. A gene array analysis revealed that p16INK4A (p16) expression was increased in association with C282Y expression. Decreasing p16 protein by siRNA resulted in increased vulnerability to all of the therapeutic agents suggesting that p16 is responsible for the resistance. Decreasing HFE expression by siRNA resulted in a 85% decrease in p16 expression in the neuroblastoma cells but not the astrocytoma cells. These data suggest a potential direct relationship between HFE and p16 that may be cell specific or mediated by different pathways in the different cell types. In conclusion, the C282Y HFE variant impacts the vulnerability of cancer cells to current treatment strategies apparently by increasing expression of p16. Although best known as a tumor suppressor, there are multiple reports that p16 is elevated in some forms of cancer. Given the frequency of the HFE gene variants, as high as 10% of the Caucasian population, these data provide compelling evidence that the C282Y HFE variant should be part of a pharmacogenetic strategy for evaluating treatment efficacy in cancer cells. Copyright © 2011 UICC.

Decreased iron burden in overweight C282Y homozygous women: Putative role of increased hepcidin production.

PubMed

Desgrippes, Romain; Lainé, Fabrice; Morcet, Jeff; Perrin, Michèle; Manet, Ghislain; Jezequel, Caroline; Bardou-Jacquet, Edouard; Ropert, Martine; Deugnier, Yves

2013-05-01

An excess of visceral adipose tissue could be involved as a modulator of the penetrance of HFE hemochromatosis since fat mass is associated with overexpression of hepcidin and low transferrin saturation was found to be associated with being overweight in women. This study was aimed at assessing the relationship between body mass index (BMI), a surrogate marker of insulin resistance, and iron burden in HFE hemochromatosis. In all, 877 patients from a cohort of C282Y homozygotes were included in the study when BMI at diagnosis and amount of iron removed (AIR) by phlebotomy were available. No relationship between AIR and BMI was found in men, whereas 15.1% (52/345) of women with AIR <6 g had BMI ≥28 versus 3.9% (2/51) of women with AIR ≥6 g (P = 0.03). At multivariate analysis, BMI was an independent factor negatively associated with AIR (odds ratio: 0.13; 95% confidence interval [CI]: 0.03-0.71) together with serum ferritin, serum transferrin, transferrin saturation, hemoglobin, and alanine aminotransferase. In a control group of 30 C282Y homozygous women, serum hepcidin was significantly higher in overweight (14.3 mmoL/L ± 7.1) than in lean (7.9 mmoL/L ± 4.3) women (P = 0.0005). In C282Y homozygous women, BMI ≥28 kg/m(2) is independently associated with a lower amount of iron removed by phlebotomy. BMI is likely a modulator factor of the phenotypic expression of C282Y homozygosity, likely through an increase of circulating levels of hepcidin. Copyright © 2013 American Association for the Study of Liver Diseases.

Serum ferritin concentrations and body iron stores in a multicenter, multiethnic primary-care population

PubMed Central

Gordeuk, Victor R.; Reboussin, David M.; McLaren, Christine E.; Barton, James C.; Acton, Ronald T.; McLaren, Gordon D.; Harris, Emily L.; Reiss, Jacob A.; Adams, Paul C.; Speechley, Mark; Phatak, Pradyumna D.; Sholinsky, Phyliss; Eckfeldt, John H.; Chen, Wen-Pin; Passmore, Leah; Dawkins, Fitzroy W.

2013-01-01

How often elevated serum ferritin in primary-care patients reflects increased iron stores (normally 0.8 g in men, 0.4 g in women) is not known. The Hereditary Hemochromatosis and Iron Overload Screening (HEIRS) study screened 101,168 primary-care participants (44% Caucasians, 27% African-Americans, 14% Asians/Pacific Islanders, 13% Hispanics, 2% others). Follow-up clinical evaluation was performed in 302 of 333 HFE C282Y homozygotes regardless of iron measures and 1,375 of 1,920 nonhomozygotes with serum ferritin >300 μg/L (men), >200 μg/L (women) and transferrin saturation >50% (men), >45% (women). Quantitative phlebotomy was conducted in 122 of 175 C282Y homozygotes and 122 of 1,102 nonhomozygotes with non-transfusional serum ferritin elevation at evaluation. The estimated prevalence in the Caucasian population of C282Y homozygotes with serum ferritin >900 μg/L at evaluation was 20 per 10,000 men and 4 per 10,000 women; this constellation was predictive of iron stores >4 g in men and >2 g in women. The estimated prevalence per 10,000 of non-C282Y homozygotes with serum ferritin >900 μg/L at evaluation was 7 among Caucasians, 13 among Hispanics, 20 among African Americans, and 38 among Asians and Pacific Islanders, and this constellation was predictive of iron stores >2 g but <4 g. In conclusion, serum ferritin >900 μg/L after initial elevations of both serum ferritin and transferrin saturation is predictive of mildly increased iron stores in multiple ethnic populations regardless of HFE genotype. Serum ferritin >900 μg/L in male C282Y homozygotes is predictive of moderately increased iron stores. PMID:18429050

Patatin-like phospholipase domain containing-3 gene I148M polymorphism, steatosis, and liver damage in hereditary hemochromatosis

PubMed Central

Valenti, Luca; Maggioni, Paolo; Piperno, Alberto; Rametta, Raffaela; Pelucchi, Sara; Mariani, Raffaella; Dongiovanni, Paola; Fracanzani, Anna Ludovica; Fargion, Silvia

2012-01-01

AIM: To investigate whether the patatin-like phospholipase domain containing-3 gene (PNPLA3) I148M polymorphism is associated with steatosis, fibrosis stage, and cirrhosis in hereditary hemochromatosis (HH). METHODS: We studied 174 consecutive unrelated homozygous for the C282Y HFE mutation of HH (C282Y+/+ HH) patients from Northern Italy, for whom the presence of cirrhosis could be determined based on histological or clinical criteria, without excessive alcohol intake (< 30/20 g/d in males or females) or hepatitis B virus and hepatitis C virus viral hepatitis. Steatosis was evaluated in 123 patients by histology (n = 100) or ultrasound (n = 23). The PNPLA3 rs738409 single nucleotide polymorphism, encoding for the p.148M protein variant, was genotyped by a Taqman assay (assay on demand, Applied Biosystems). The association of the PNPLA3 I148M protein variant (p.I148M) with steatosis, fibrosis stage, and cirrhosis was evaluated by logistic regression analysis. RESULTS: PNPLA3 genotype was not associated with metabolic parameters, including body mass index (BMI), the presence of diabetes, and lipid levels, but the presence of the p.148M variant at risk was independently associated with steatosis [odds ratio (OR) 1.84 per p.148M allele, 95% confidence interval (CI): 1.05-3.31; P = 0.037], independently of BMI and alanine aminotransaminase (ALT) levels. The p.148M variant was also associated with higher aspartate aminotransferase (P = 0.0014) and ALT levels (P = 0.017) at diagnosis, independently of BMI and the severity of iron overload. In patients with liver biopsy, the 148M variant was independently associated with the severity (stage) of fibrosis (estimated coefficient 0.56 ± 0.27, P = 0.041). In the overall series of patients, the p.148M variant was associated with cirrhosis in lean (P = 0.049), but not in overweight patients (P = not significant). At logistic regression analysis, cirrhosis was associated with BMI ≥ 25 (OR 1.82, 95% CI: 1.02-3.55), ferritin > 1000 ng/mL at diagnosis (OR 19.3, 95% CI: 5.3-125), and with the G allele in patients with BMI < 25 (OR 3.26, 95% CI: 1.3-10.3). CONCLUSION: The PNPLA3 I148M polymorphism may represent a permissive factor for fibrosis progression in patients with C282Y+/+ HH. PMID:22719190

The impact of H63D HFE gene carriage on hemoglobin and iron status in children.

PubMed

Barbara, Kaczorowska-Hac; Marcin, Luszczyk; Jedrzej, Antosiewicz; Wieslaw, Ziolkowski; Elzbieta, Adamkiewicz-Drozynska; Malgorzata, Mysliwiec; Ewa, Milosz; Jacek, Kaczor Jan

2016-12-01

The molecular mechanism that regulates iron homeostasis is based on a network of signals, which reflect on the iron requirements of the body. Hereditary hemochromatosis is a heterogenic metabolic syndrome which is due to unchecked transfer of iron into the bloodstream and its toxic effects on parenchymatous organs. It is caused by the mutation of genes that encode proteins that help hepcidin to monitor serum iron. These proteins include the human hemochromatosis protein -HFE, transferrin-receptor 2, hemojuvelin in rare instances, and ferroportin. HFE-related hemochromatosis is the most frequent form of the disease. Interestingly, the low penetrance of polymorphic HFE genes results in rare clinical presentation of the disease, predominantly in middle-aged males. Taking into account the wide dispersion of HFE mutation in our population and also its unknown role in heterozygotes, we analyzed the impact of H63D HFE carriage in the developmental age, with respect to gender, on the iron status and hemoglobin concentration of carriers in comparison to those of wild-type HFE gene (12.7 ± 3.07 years, 42 boys and 41 girls). H63D carriers presented higher blood iron, transferrin saturation, and ferritin concentration than wild-type probands (p < 0.05.) Interestingly, male H63D carriers showed higher hemoglobin concentration than the unburdened children. Moreover, in the H63D carrier group, a positive correlation between iron and hemoglobin was noted. In conclusion, this study demonstrates that changes in iron metabolism occur at a young age in HFE heterozygotes.

Association of HLA alleles and haplotypes with CYP21A2 gene p. V282L mutation in the Croatian population.

PubMed

Grubic, Z; Maskalan, M; Stingl Jankovic, K; Zvecic, S; Dumic Kubat, K; Krnic, N; Zunec, R; Ille, J; Kusec, V; Dumic, M

2016-11-01

The CYP21A2 mutations that are in linkage disequilibrium with particular HLA-A, -B, -DRB1 alleles/haplotypes, cause deficiency of the 21-hydroxylase enzyme (21-OHD) and account for the majority of congenital adrenal hyperplasia (CAH) cases. The aim of this study was to investigate those associations with the p.V282L mutation linked to the non-classical (NC) form of CAH among Croatians. The study included parents of patients with the NC form of CAH, positive for the p.V282L mutation (N = 55) and cadaveric donor samples (N = 231). All subjects were HLA-A, -B, and -DRB1 typed and tested for the presence of the p.V282L mutation. Among parents of patients, 92.73% of subjects were positive for the B*14:02 allele and almost half of them carried the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype. Among cadaveric samples 77 out of 96 subjects positive for the B*14:02 allele had the p.V282L mutation. Among them, 37 were positive for the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype, 23 had the HLA-A*33:01-B*14:02-DRB1*03:01 haplotype, 8 had the B*14:02-DRB1*01:02 combination and 5 were carrying the HLA-A*68:02-B*14:02-DRB1*13:03 haplotype. Only 4 of these subjects were positive for the B*14:02 allele. HLA-B*14:02 was the only single allele with association that reached statistically significant P value (RR = 12.00; P = 0.0024). Haplotypes B*14:02-DRB1*01:02 (P < 0.001) and HLA-A*68:02-B*14:02-DRB1*13:03 (P < 0.001) as well as HLA-A*33:01-B*14:02-DRB1*01:02 and HLA-A*33:01-B*14:02-DRB1*03:01 showed high relative risks (RR = 45.00, RR = 41.63 and RR = 36.96, respectively). Our data support the previously documented association of the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype with the p.V282L mutation, but also point out a high frequency of the p.V282L mutation among Croatians with HLA-A*33:01-B*14:02-DRB1*03:01 and HLA-A*68:02-B*14:02-DRB1*13:03 haplotypes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Spectrum of rhodopsin mutations in Korean patients with retinitis pigmentosa

PubMed Central

Kim, Kwang Joong; Kim, Cinoo; Bok, Jeong; Kim, Kyung-Seon; Lee, Eun-Ju; Park, Sung Pyo; Chung, Hum; Han, Bok-Ghee; Kim, Hyung-Lae; Kimm, Kuchan; Yu, Hyeong Gon

2011-01-01

Purpose To determine the spectrum and frequency of rhodopsin gene (RHO) mutations in Korean patients with retinitis pigmentosa (RP) and to characterize genotype–phenotype correlations in patients with mutations. Methods The RHO mutations were screened by direct sequencing, and mutation prevalence was measured in patients and controls. The impact of missense mutations to RP was predicted by segregation analysis, peptide sequence alignment, and in silico analysis. The severity of disease in patients with the missense mutations was compared by visual acuity, electroretinography, optical coherence tomography, and kinetic visual field testing. Results Five heterozygous mutations were identified in six of 302 probands with RP, including a novel mutation (c.893C>A, p.A298D) and four known mutations (c.50C>T, p.T17M; c.533A>G, p.Y178C; c.888G>T, p.K296N; and c.1040C>T, p.P347L). The allele frequency of missense mutations was measured in 114 ethnically matched controls. p.A298D, newly identified in a sporadic patient, had never been found in controls and was predicted to be pathogenic. Among the patients with the missense mutations, we observed the most severe phenotype in patients with p.P347L, less severe phenotypes in patients with p.Y178C or p.A298D, and a relatively moderate phenotype in a patient with p.T17M. Conclusions The results reveal the spectrum of RHO mutations in Korean RP patients and clinical features that vary according to mutations. Our findings will be useful for understanding these genetic spectra and the genotype–phenotype correlations and will therefore help with predicting disease prognosis and facilitating the development of gene therapy. PMID:21677794

Biochemical Characterization of Mutants in Chaperonin Proteins CCT4 and CCT5 Associated with Hereditary Sensory Neuropathy*

PubMed Central

Sergeeva, Oksana A.; Tran, Meme T.; Haase-Pettingell, Cameron; King, Jonathan A.

2014-01-01

Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin TRiC, a protein machine responsible for folding actin and tubulin in the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, whereas H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in Escherichia coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the WT chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, whereas WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γd-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate β-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations are consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced. PMID:25124038

New insights into thyroglobulin gene: molecular analysis of seven novel mutations associated with goiter and hypothyroidism.

PubMed

Citterio, Cintia E; Machiavelli, Gloria A; Miras, Mirta B; Gruñeiro-Papendieck, Laura; Lachlan, Katherine; Sobrero, Gabriela; Chiesa, Ana; Walker, Joanna; Muñoz, Liliana; Testa, Graciela; Belforte, Fiorella S; González-Sarmiento, Rogelio; Rivolta, Carina M; Targovnik, Héctor M

2013-01-30

The thyroglobulin (TG) gene is organized in 48 exons, spanning over 270 kb on human chromosome 8q24. Up to now, 62 inactivating mutations in the TG gene have been identified in patients with congenital goiter and endemic or non-endemic simple goiter. The purpose of the present study was to identify and characterize new mutations in the TG gene. We report 13 patients from seven unrelated families with goiter, hypothyroidism and low levels of serum TG. All patients underwent clinical, biochemical and imaging evaluation. Single-strand conformation polymorphism (SSCP) analysis, endonuclease restriction analysis, sequencing of DNA, genotyping, population screening, and bioinformatics studies were performed. Molecular analyses revealed seven novel inactivating TG mutations: c.378C>A [p.Y107X], c.2359C>T [p.R768X], c.2736delG [p.R893fsX946], c.3842G>A [p.C1262Y], c.5466delA [p.K1803fsX1833], c.6000C>G [p.C1981W] and c.6605C>G [p.P2183R] and three previously reported mutations: c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.7006C>T [p.R2317X]. Six patients from two families were homozygous for p.R277X mutation, four were compound heterozygous mutations (p.Y107X/p.C1262Y, p.R893fsX946/p.A2215D, p.K1803fsX1832/p.R2317X), one carried three identified mutations (p.R277X/p.C1981W-p.P2183R) together with a hypothetical micro deletion and the remaining two siblings from another family with typical phenotype had a single p.R768X mutated allele. In conclusion, our results confirm the genetic heterogeneity of TG defects and the pathophysiological importance of altered TG folding as a consequency of truncated TG proteins and missense mutations located in ACHE-like domain or that replace cysteine. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

A systematic comparison of all mutations in hereditary sensory neuropathy type I (HSAN I) reveals that the G387A mutation is not disease associated.

PubMed

Hornemann, Thorsten; Penno, Anke; Richard, Stephane; Nicholson, Garth; van Dijk, Fleur S; Rotthier, Annelies; Timmerman, Vincent; von Eckardstein, Arnold

2009-04-01

Hereditary sensory neuropathy type 1 (HSAN I) is an autosomal dominant inherited neurodegenerative disorder of the peripheral nervous system associated with mutations in the SPTLC1 subunit of the serine palmitoyltransferase (SPT). Four missense mutations (C133W, C133Y, V144D and G387A) in SPTLC1 were reported to cause HSAN I. SPT catalyses the condensation of Serine and Palmitoyl-CoA, which is the first and rate-limiting step in the de novo synthesis of ceramides. Earlier studies showed that C133W and C133Y mutants have a reduced activity, whereas the impact of the V144D and G387A mutations on the human enzyme was not tested yet. In this paper, we show that none of the HSAN I mutations interferes with SPT complex formation. We demonstrate that also V144D has a reduced SPT activity, however to a lower extent than C133W and C133Y. In contrast, the G387A mutation showed no influence on SPT activity. Furthermore, the growth phenotype of LY-B cells--a SPTLC1 deficient CHO cell line--could be reversed by expressing either the wild-type SPTLC1 or the G387A mutant, but not the C133W mutant. This indicates that the G387A mutation is most likely not directly associated with HSAN I. These findings were genetically confirmed by the identification of a nuclear HSAN family which showed segregation of the G387A variant as a non-synonymous SNP.

Identifying mutations in Tunisian families with retinal dystrophy.

PubMed

Habibi, Imen; Chebil, Ahmed; Falfoul, Yosra; Allaman-Pillet, Nathalie; Kort, Fedra; Schorderet, Daniel F; El Matri, Leila

2016-11-22

Retinal dystrophies (RD) are a rare genetic disorder with high genetic heterogeneity. This study aimed at identifying disease-causing variants in fifteen consanguineous Tunisian families. Full ophthalmic examination was performed. Index patients were subjected to IROme analysis or whole exome sequencing followed by homozygosity mapping. All detected variations were confirmed by direct Sanger sequencing. Mutation analysis in our patients revealed two compound heterozygous mutations p.(R91W);(V172D) in RPE65, and five novel homozygous mutations: p.R765C in CNGB1, p.H337R in PDE6B, splice site variant c.1129-2A > G and c.678_681delGAAG in FAM161A and c.1133 + 3_1133 + 6delAAGT in CERKL. The latter mutation impacts pre-mRNA splicing of CERKL. The other changes detected were six previously reported mutations in CNGB3 (p.R203*), ABCA4 (p.W782*), NR2E3 (p.R311Q), RPE65 (p.H182Y), PROM1 (c.1354dupT) and EYS (c.5928-2A > G). Segregation analysis in each family showed that all affected individuals were homozygotes and unaffected individuals were either heterozygote carriers or homozygous wild type allele. These results confirm the involvement of a large number of genes in RD in the Tunisian population.

Reduction of Skeletal Muscle Power in Adolescent Males Carrying H63D Mutation in the HFE Gene.

PubMed

Luszczyk, Marcin; Kaczorowska-Hac, Barbara; Milosz, Ewa; Adamkiewicz-Drozynska, Elzbieta; Ziemann, Ewa; Laskowski, Radoslaw; Flis, Damian; Rokicka-Hebel, Magdalena; Antosiewicz, Jedrzej

2017-01-01

Iron overload resulting from the mutation of genes involved in iron metabolism or excess dietary intake has been reported to negatively influence human physical performance. The aim of this study was to test the hypothesis that adolescents bearing a hemochromatosis gene (HFE) mutation in contrast to adults with the same mutation will not experience iron accumulation and their aerobic capacity will be similar to that of age-matched controls. Thirteen boys participated in the study. Seven of them are carriers of H63D mutation in the HFE gene and six were wild type. Fitness levels were assessed using the cardiopulmonary exercise test. In addition, iron status and inflammatory markers were determined. We observed that cardiovascular fitness was significantly lower in the group bearing the HFE mutation compared to the control group. Moreover, the HFE mutation group achieved lower maximal power output compared to the control group. There were no differences in blood ferritin concentrations between the two groups which indicates similar amounts of stored iron. Obtained data do not confirm our hypothesis. On the contrary, it was demonstrated that HFE mutation is associated with a lower level of aerobic capacity, even in the absence of iron accumulation.

Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning.

PubMed

Yamagoshi, Ryohei; Yamamoto, Takenori; Hashimoto, Mitsuru; Sugahara, Ryohei; Shiotsuki, Takahiro; Miyoshi, Hideto; Terada, Hiroshi; Shinohara, Yasuo

2017-01-01

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate "revertants" viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer.

PubMed

Imai, Kazuo; Tarumoto, Norihito; Runtuwene, Lucky Ronald; Sakai, Jun; Hayashida, Kyoko; Eshita, Yuki; Maeda, Ryuichiro; Tuda, Josef; Ohno, Hideaki; Murakami, Takashi; Maesaki, Shigefumi; Suzuki, Yutaka; Yamagishi, Junya; Maeda, Takuya

2018-05-29

The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.

Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum

PubMed Central

Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo

2014-01-01

Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277

Droplet digital polymerase chain reaction assay for screening of ESR1 mutations in 325 breast cancer specimens.

PubMed

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

2015-12-01

Droplet digital polymerase chain reaction (ddPCR), which could perform thousands of PCRs on a nanoliter scale simultaneously, would be an attractive method to massive parallel sequencing for identifying and studying the significance of low-frequency rare mutations. Recent evidence has shown that the key potential mechanisms of the failure of aromatase inhibitors-based therapy involve identifying activating mutations affecting the ligand-binding domain of the ESR1 gene. Therefore, the detection of ESR1 mutations may be useful as a biomarker predicting an effect of the treatment. We aimed to develop a ddPCR-based method for the sensitive detection of ESR1 mutations in 325 breast cancer specimens, in which 270 primary and 55 estrogen receptor-positive (ER+) metastatic breast cancer (MBC) specimens. Our ddPCR assay could detect the ESR1 mutant molecules with low concentration of 0.25 copies/μL. According to the selected cutoff, ESR1 mutations occurred in 7 (2.5%) of 270 primary breast cancer specimens and in 11 (20%) of 55 ER+ MBC specimens. Among the 11 MBC specimens, 5 specimens (45.5%) had the most common ESR1 mutation, Y537S, 4 specimens (36.3%) each had D538G, Y537N, and Y537C. Interestingly, 2 patients had 2 ESR1 mutations, Y537N/D538G and Y537S/Y537C, and 2 patients had 3 ESR1 mutations, Y537S/Y537N/D538G. Biopsy was performed in heterochrony in 8 women twice. In 8 women, 4 women had primary breast cancer and MBC specimens and 4 women had 2 specimens when treatment was failure. Four of these 8 women acquired ESR1 mutation, whereas no ESR1 mutation could be identified at first biopsy. ddPCR technique could be a promising tool for the next-generation sequencing-free precise detection of ESR1 mutations in endocrine therapy resistant cases and may assist in determining the treatment strategy. Copyright © 2015 Elsevier Inc. All rights reserved.

Activating HER2 mutations in HER2 gene amplification negative breast cancer

PubMed Central

Bose, Ron; Kavuri, Shyam M.; Searleman, Adam C.; Shen, Wei; Shen, Dong; Koboldt, Daniel C.; Monsey, John; Goel, Nicholas; Aronson, Adam B.; Li, Shunqiang; Ma, Cynthia X.; Ding, Li; Mardis, Elaine R.; Ellis, Matthew J.

2012-01-01

Data from eight breast cancer genome sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized thirteen HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGFR exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings demonstrate that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment. PMID:23220880

Word Criticality Analysis. MOS: 16B. Skill Levels 1 & 2

DTIC Science & Technology

1981-09-01

43* 2 UI3L, 29.1 2 §CT;:It’:iLL 1.12 2 ACCA 97.1 55.1 43.1 2 2 4,CITUI L 1.1 521 21l 9i 9 P ~ 1.2 2 hA~T)U h 24.1 10-.2 58.1 2.1. 2 A.;aifl U95. 2591...2 17 SOE 16.1 3.tl z J KS ’ f1 . 942.1 4e1 I v/ k Y1 MCS WOR LIT sy rtE AT SM U-2 AG P~~~- .s...:’<.- 14o -!2 7 2~ LVRI4RO LIST 66Y 22G1 140 *17142...71. 4 d1LLY 6? f1 4 NJ51 4l 22#1, 63.1 13.?2 5.2 17,1l 16.1 4 C tM; 4 Cx:v 1::CE. 1 1 7.2 4 Ci ’IF 37. 4 C t .C V1 E It C~C-’ 4:: I! D. 4 C0,; .) 2

CFTR allelic heterogeneity in Mexican patients with cystic fibrosis: implications for molecular screening.

PubMed

Chávez-Saldaña, Margarita; Yokoyama, Emiy; Lezana, José Luis; Carnevale, Alessandra; Macías, Miguel; Vigueras, Rosa M; López, Marisol; Orozco, Lorena

2010-01-01

Cystic fibrosis, the most common autosomal recessive disorder, is caused by defects in the CF transmembrane conductance regulator gene (CFTR) that encodes a chloride channel. To date, over 1,800 mutations have been described related to the causative gene of CF, showing a variable frequency among populations. In a previous extensive analysis of the CFTR locus in 97 Mexican patients, 34 different mutations (75% of CF alleles) were found using several strategies for mutation screening; however, 63% had at least an uncharacterized allele. Despite the combined technologies used, there are still a great number of unknown mutations in the Mexican population. Screening of the CFTR gene to provide additional evidence of the mutational wide spectrum responsible for CF in Mexican patients. In this study, the number of unrelated CF patients was increased to 230, 133 new cases and the 97 previously reported to include 63% with at least an uncharacterized allele. Additional tools were used to improve the detection rate of CF mutations, such as a commercial kit for 36 mutations plus a single chain conformational polymorphism method and DNA sequencing. By using a combination of these strategies we characterized 77.7% of all the CF alleles, resulting in a total of 46 different mutations detected, including the identification of 12 additional mutations (p.R334W, p.A455E, c.3120+1G > A, c.3272-26A > G, c.711+1G > T, p.Q552X, p.W1282X, c.IVS8-5T, p.R1162X and p.R347P, p.D1152H and p.T1036N). Although these 12 mutations have been reported in other populations, they have not yet been reported in Mexican patients. This report shows that Mexico has one of the widest spectra of CFTR mutations worldwide. The knowledge of the ethnic and geographic distribution of CFTR mutations in this population will allow the development of more effective methods for diagnosis and treatment.

HPV-negative penile squamous cell carcinoma: disruptive mutations in the TP53 gene are common.

PubMed

Kashofer, Karl; Winter, Elke; Halbwedl, Iris; Thueringer, Andrea; Kreiner, Marisa; Sauer, Stefan; Regauer, Sigrid

2017-07-01

The majority of penile squamous cell carcinomas is caused by transforming human papilloma virus (HPV) infection. The etiology of HPV-negative cancers is unclear, but TP53 mutations have been implicated. Archival tissues of 108 invasive squamous cell carcinoma from a single pathology institution in a low-incidence area were analyzed for HPV-DNA and p16 ink4a overexpression and for TP53 mutations by ion torrent next-generation sequencing. Library preparation failed in 32/108 squamous cell carcinomas. Institutional review board approval was obtained. Thirty of 76 squamous cell carcinomas (43%; average 63 years) were HPV-negative with 8/33 squamous cell carcinomas being TP53 wild-type (24%; average 63 years). Twenty-five of 33 squamous cell carcinomas (76%; average 65 years) showed 32 different somatic TP53 mutations (23 missense mutations in exons 5-8, 6 nonsense, 1 frameshift and 2 splice-site mutations). Several hotspot mutations were detected multiple times (R175H, R248, R282, and R273). Eighteen of 19 squamous cell carcinomas with TP53 expression in immunohistochemistry had TP53 mutations. Fifty percent of TP53-negative squamous cell carcinomas showed mostly truncating loss-of-function TP53 mutations. Patients without mutations had longer survival (5 years: 86% vs 61%; 10 years: 60% vs 22%), but valid clinically relevant conclusions cannot be drawn due to different tumor stages and heterogeneous treatment of the cases presented in this study. Somatic TP53 mutations are a common feature in HPV-negative penile squamous cell carcinomas and offer an explanation for HPV-independent penile carcinogenesis. About half of HPV-negative penile cancers are driven by oncogenic activation of TP53, while a quarter is induced by loss of TP53 tumor suppressor function. Detection of TP53 mutations should be carried out by sequencing, as immunohistochemical TP53 staining could not identify all squamous cell carcinomas with TP53 mutations.

Lack of integrase inhibitors associated resistance mutations among HIV-1C isolates.

PubMed

Mulu, Andargachew; Maier, Melanie; Liebert, Uwe Gerd

2015-12-01

Although biochemical analysis of HIV-1 integrase enzyme suggested the use of integrase inhibitors (INIs) against HIV-1C, different viral subtypes may favor different mutational pathways potentially leading to varying levels of drug resistance. Thus, the aim of this study was to search for the occurrence and natural evolution of integrase polymorphisms and/or resistance mutations in HIV-1C Ethiopian clinical isolates prior to the introduction of INIs. Plasma samples from chronically infected drug naïve patients (N = 45), of whom the PR and RT sequence was determined previously, were used to generate population based sequences of HIV-1 integrase. HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. Resistance mutations were interpreted according to the Stanford HIV drug resistance database ( http://hivdb.stanford.edu ) and the updated International Antiviral Society (IAS)-USA mutation lists. Moreover, rates of polymorphisms in the current isolates were compared with South African and global HIV-1C isolates. All subjects were infected with HIV-1C concordant to the protease (PR) and reverse transcriptase (RT) regions. Neither major resistance-associated IN mutations (T66I/A/K, E92Q/G, T97A, Y143HCR, S147G, Q148H/R/K, and N155H) nor silent mutations known to change the genetic barrier were observed. Moreover, the DDE-catalytic motif (D64G/D116G/E152 K) and signature HHCC zinc-binding motifs at codon 12, 16, 40 and 43 were found to be highly conserved. However, compared to other South African subtype C isolates, the rate of polymorphism was variable at various positions. Although the sample size is small, the findings suggest that this drug class could be effective in Ethiopia and other southern African countries where HIV-1C is predominantly circulating. The data will contribute to define the importance of integrase polymorphism and to improve resistance interpretation algorithms in HIV-1C isolates.

Rates of Spontaneous Mutation in Bacteriophage T4 Are Independent of Host Fidelity Determinants

PubMed Central

Santos, M. E.; Drake, J. W.

1994-01-01

Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity. PMID:7851754

The pH of activation of the hemagglutinin protein regulates H5N1 influenza virus replication and pathogenesis in mice.

PubMed

Zaraket, Hassan; Bridges, Olga A; Russell, Charles J

2013-05-01

After receptor binding and internalization during influenza virus entry, the hemagglutinin (HA) protein is triggered by low pH to undergo irreversible conformational changes that mediate membrane fusion. To investigate how mutations that alter the activation pH of the HA protein influence the fitness of an avian H5N1 influenza virus in a mammalian model, we infected C57BL/6J or DBA/2J mice and compared the replication and virulence of recombinant A/chicken/Vietnam/C58/04 (H5N1) HA-Y231H mutant, wild-type, and HA-H241Q and HA-K582I mutant viruses that have HA activation pH values of 6.3, 5.9, 5.6, and 5.4, respectively. The HA-Y231H mutant virus was highly susceptible to acid inactivation in vitro and was attenuated for growth and virulence in mice, suggesting that an H5N1 HA protein triggered at pH 6.3 is too unstable for the virus to remain fit. Wild-type and HA-H241Q viruses were similar in pathogenicity and grew to similar levels in mice, ducks, and cell cultures derived from both avian and mammalian tissues, suggesting that H5N1 HA proteins triggered at pH values in the range of 5.9 to 5.6 broadly support replication. The HA-K582I mutant virus had greater growth and virulence in DBA/2J mice than the wild type did, although the mutant virus was highly attenuated in ducks. The data suggest that adaptation of avian H5N1 influenza virus for infection in mammals is supported by a decrease in the HA activation pH to 5.4. Identification of the HA activation pH as a host-specific infectivity factor is expected to aid in the surveillance and risk assessment of currently circulating H5N1 influenza viruses.

Child with RET proto-oncogene codon 634 mutation.

PubMed

İnce, Dilek; Demirağ, Bengü; Ataseven, Eda; Oymak, Yeşim; Tuhan, Hale; Karakuş, Osman Zeki; Hazan, Filiz; Abacı, Ayhan; Özer, Erdener; Mutafoglu, Kamer; Olgun, Nur

2017-01-01

İnce D, Demirağ B, Ataseven E, Oymak Y, Tuhan H, Karakuş OZ, Hazan F, Abacı A, Özer E, Mutafoglu K, Olgun N. Child with RET proto-oncogene codon 634 mutation. Turk J Pediatr 2017; 59: 590-593. Herein we reported a 7-year-old child with RET proto-oncogene c634 mutation. Her mother had been diagnosed with medullary thyroid carcinoma (MTC), and treated six years ago. Heterozygous mutation of the RET proto-oncogene at c634 had been detected in her mother. Genetic analysis showed the presence of the same mutation in our patient. Thyroid functions were normal. Serum calcitonin level was found mildly elevated. Parathormone (PTH) and carcinoembrionic antigen (CEA) levels were normal. Prophylactic thyroidectomy and sampling of cervical lymph nodes were performed. Histopathologic examination revealed hyperplasia in thyroid C cells, and reactive lymphadenopathy. The risk of MTC has been reported 100% through the life of patients with RET proto-oncogene mutation. It has been reported that particularly patients with c634 mutation have more risk of occurence of metastatic and progressive/recurrent MTC. Prophylactic `thyroidectomy, cervical lymph node dissection` before 5-years-of-age should be considered for these patients.

Department of Defense Youth Poll Wave 10 - December 2005. Overview Report

DTIC Science & Technology

2006-07-01

Fishbein , M . & Ajzen , I . (1975). Belief, attitude, intention and behavior: An introduction to theory...a n d E x p e c t e d O u t c o m e s A s s o c ia t e d w it h M il i t a r y S e r v ic e I n f lu e n c e r s M i l i t a r y B e l ie...f s a n d Y o u t h M o t iv a t io n t o C o m p ly Y o u t h A t t i t u d e s N o r m s Y o u t h C o n f id e n c e in S u c c e s

Role of Pfmdr1 in In Vitro Plasmodium falciparum Susceptibility to Chloroquine, Quinine, Monodesethylamodiaquine, Mefloquine, Lumefantrine, and Dihydroartemisinin

PubMed Central

Wurtz, Nathalie; Fall, Bécaye; Pascual, Aurélie; Fall, Mansour; Baret, Eric; Camara, Cheikhou; Nakoulima, Aminata; Diatta, Bakary; Fall, Khadidiatou Ba; Mbaye, Pape Saliou; Diémé, Yaya; Bercion, Raymond; Wade, Boubacar

2014-01-01

The involvement of Pfmdr1 (Plasmodium falciparum multidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms in Pfmdr1 (N86Y, Y184F, S1034C, N1042D, and D1246Y) and Pfcrt (K76T) and in vitro responses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174 Plasmodium falciparum isolates from Dakar, Senegal. The Pfmdr1 86Y mutation was identified in 14.9% of the samples, and the 184F mutation was identified in 71.8% of the isolates. No 1034C, 1042N, or 1246Y mutations were detected. The Pfmdr1 86Y mutation was significantly associated with increased susceptibility to MDAQ (P = 0.0023), LMF (P = 0.0001), DHA (P = 0.0387), and MQ (P = 0.00002). The N86Y mutation was not associated with CQ (P = 0.214) or QN (P = 0.287) responses. The Pfmdr1 184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs (P = 0.168 for CQ, 0.778 for MDAQ, 0.324 for LMF, 0.961 for DHA, 0.084 for QN, and 0.298 for MQ). The Pfmdr1 86Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ (P = 0.0136), LMF (P = 0.0019), and MQ (P = 0.0001). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to MDAQ (P < 0.0001), LMF (P < 0.0001), MQ (P < 0.0001), and QN (P = 0.0026) in wild-type Pfcrt K76 parasites. The additional Pfmdr1 86Y mutation significantly increased the in vitro susceptibility to CQ (P = 0.0179) in Pfcrt 76T CQ-resistant parasites. PMID:25199781

A point mutation in the human Slo1 channel that impairs its sensitivity to omega-3 docosahexaenoic acid

PubMed Central

Xu, Rong; Hou, Shangwei; Heinemann, Stefan H.; Tian, Yutao

2013-01-01

Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA) at nanomolar concentrations reversibly activate human large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels containing auxiliary β1 or β4 subunits in cell-free patches. Here we examined the action of DHA on the Slo1 channel without any auxiliary subunit and sought to elucidate the biophysical mechanism and the molecular determinants of the DHA sensitivity. Measurements of ionic currents through human Slo1 (hSlo1) channels reveal that the stimulatory effect of DHA does not require activation of the voltage or Ca2+ sensors. Unlike gating of the hSlo1 channel, that of the Drosophila melanogaster Slo1 (dSlo1) channel is unaltered by DHA. Our mutagenesis study based on the differential responses of human and dSlo1 channels to DHA pinpoints that Y318 near the cytoplasmic end of S6 in the hSlo1 channel is a critical determinant of the stimulatory action of DHA. The mutation Y318S in hSlo1, which replaces Y with S as found in dSlo1, greatly diminishes the channel’s response to DHA with a 22-carbon chain whether β1 or β4 is absent or present. However, the responses to α-linolenic acid, an omegea-3 fatty acid with an 18-carbon chain, and to arachidonic acid, an omega-6 fatty acid with a 20-carbon chain, remain unaffected by the mutation. Y318 in the S6 segment of hSlo1 is thus an important determinant of the electrophysiological response of the channel to DHA. Furthermore, the mutation Y318S may prove to be useful in dissecting out the complex lipid-mediated modulation of Slo1 BK channels. PMID:24127525

A Developmental Approach to Characterizing the Tissue-Invasion Gene Program in Breast Cancer

DTIC Science & Technology

2001-09-01

OF PAGES Breast Cancer 24 16. PRICE CODE 17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF...induced host response. Am J. Pathol. 149:273-282, 1996. 4. Wolf, c., Rouyer, N., Lutz, Y., Adida , C., Loriot, M., Bellocq, J.P., Chambon, P., and Basset...following a 5 d incubation period. (upper left and right panels). In contrast, MT1-MMP-transfected cells perforated the BM in representative TEM and

Increased risk of death from iron overload among 422 treated probands with HFE hemochromatosis and serum levels of ferritin greater than 1000 μg/L at diagnosis.

PubMed

Barton, James C; Barton, J Clayborn; Acton, Ronald T; So, Jeffrey; Chan, Susanne; Adams, Paul C

2012-04-01

We investigated the risk of death from iron overload among treated hemochromatosis probands who were homozygous for HFE C282Y and had serum levels of ferritin greater than 1000 μg/L at diagnosis. We compared serum levels of ferritin at diagnosis and other conditions with the rate of iron overload-associated death using data from 2 cohorts of probands with hemochromatosis who were homozygous for HFE C282Y (an Alabama cohort, n = 294, 63.9% men and an Ontario cohort, n = 128, 68.8% men). We defined iron overload-associated causes of death as cirrhosis (including hepatic failure and primary liver cancer) caused by iron deposition and cardiomyopathy caused by myocardial siderosis. All probands received phlebotomy and other appropriate therapy. The mean survival times after diagnosis were 13.2 ± 7.3 y and 12.5 ± 8.3 y in Alabama and Ontario probands, respectively. Serum levels of ferritin greater than 1000 μg/L at diagnosis were observed in 30.1% and 47.7% of Alabama and Ontario probands, respectively. In logistic regressions of serum ferritin greater than 1000 μg/L, there were significant positive associations with male sex and cirrhosis in Alabama probands and with age, male sex, increased levels of alanine and aspartate aminotransferases, and cirrhosis in Ontario probands. Of probands with serum levels of ferritin greater than 1000 μg/L at diagnosis, 17.9% of those from Alabama and 14.8% of those from Ontario died of iron overload. Among probands with serum levels of ferritin greater than 1000 μg/L, the relative risk of iron overload-associated death was 5.4 for the Alabama group (95% confidence interval [CI], 2.2-13.1; P = .0002) and 4.9 for the Ontario group (95% CI, 1.1-22.0; P = .0359). In hemochromatosis probands homozygous for HFE C282Y, serum levels of ferritin greater than 1000 μg/L at diagnosis were positively associated with male sex and cirrhosis. Even with treatment, the relative risk of death from iron overload was 5-fold greater in probands with serum levels of ferritin greater than 1000 μg/L. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

A homozygous p53 R282W mutant human embryonic stem cell line generated using TALEN-mediated precise gene editing.

PubMed

Zhou, Ruoji; Xu, An; Wang, Donghui; Zhu, Dandan; Mata, Helen; Huo, Zijun; Tu, Jian; Liu, Mo; Mohamed, Alaa M T; Jewell, Brittany E; Gingold, Julian; Xia, Weiya; Rao, Pulivarthi H; Hung, Mien-Chie; Zhao, Ruiying; Lee, Dung-Fang

2018-03-01

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancers. Many hot-spot mutations of TP53 confer novel functions not found in wild-type p53 and contribute to tumor development and progression. We report on the generation of a H1 human embryonic stem cell line carrying a homozygous TP53 R282W mutation using TALEN-mediated genome editing. The generated cell line demonstrates normal karyotype, maintains a pluripotent state, and is capable of generating a teratoma in vivo containing tissues from all three germ layers. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Strategies for genetic study of hearing loss in the Brazilian northeastern region

PubMed Central

Melo, Uirá S; Santos, Silvana; Cavalcanti, Hannalice G; Andrade, Wagner T; Dantas, Vitor G; Rosa, Marine RD; Mingroni-Netto, Regina C

2014-01-01

The overall aim of this study was to estimate the contribution of genetic factors to the etiology of hearing loss (HL) in two counties in the Brazilian northeastern region. A cross-sectional study, based on the key informant approach (KI) was conducted in Queimadas and Gado Bravo counties (Paraíba, Northeast Brazil). The sample consisted of 182 patients with HL. Genetic screening of the most frequent mutations associated with HL was performed for all samples. DFNB1 mutations were the most frequently found in both counties. The c.35delG mutation was detected in homozygosis in seven non-syndromic probands in Queimadas (7/76, 9.2%) and only a single homozygote with this mutation was found in Gado Bravo (1/44, 2.3%). We also detected the del(GJB6-D13S1854) mutation in non-syndromic probands from Gado Bravo (2/44, 4.5%). The c.189C>A (p.TyrY63*) mutation in the CLRN1 gene was detected in homozygosis in 21/23 Usher syndrome patients from Gado Bravo and it was not found in Queimadas. Cases with probable genetic etiology contributed approximately to half of HL probands in each county (54.6% in Gado Bravo and 45.7% in Queimadas). We confirm the importance of DFNB1 locus to non-syndromic HL but we show that the frequency of mutations in the northeastern region differs somewhat from those reported in southeastern Brazil and other populations. In addition, the extremely high frequency of individuals with Usher syndrome with c.189C>A variation in CLRN1 indicates the need for a specific screening of this mutation. PMID:24596593

EMQN best practice guidelines for the molecular genetic diagnosis of hereditary hemochromatosis (HH).

PubMed

Porto, Graça; Brissot, Pierre; Swinkels, Dorine W; Zoller, Heinz; Kamarainen, Outi; Patton, Simon; Alonso, Isabel; Morris, Michael; Keeney, Steve

2016-04-01

Molecular genetic testing for hereditary hemochromatosis (HH) is recognized as a reference test to confirm the diagnosis of suspected HH or to predict its risk. The vast majority (typically >90%) of patients with clinically characterized HH are homozygous for the p.C282Y variant in the HFE gene, referred to as HFE-related HH. Since 1996, HFE genotyping was implemented in diagnostic algorithms for suspected HH, allowing its early diagnosis and prevention. However, the penetrance of disease in p.C282Y homozygotes is incomplete. Hence, homozygosity for p.C282Y is not sufficient to diagnose HH. Neither is p.C282Y homozygosity required for diagnosis as other rare forms of HH exist, generally referred to as non-HFE-related HH. These pose significant challenges when defining criteria for referral, testing protocols, interpretation of test results and reporting practices. We present best practice guidelines for the molecular genetic diagnosis of HH where recommendations are classified, as far as possible, according to the level and strength of evidence. For clarification, the guidelines' recommendations are preceded by a detailed description of the methodology and results obtained with a series of actions taken in order to achieve a wide expert consensus, namely: (i) a survey on the current practices followed by laboratories offering molecular diagnosis of HH; (ii) a systematic literature search focused on some identified controversial topics; (iii) an expert Best Practice Workshop convened to achieve consensus on the practical recommendations included in the guidelines.

EMQN best practice guidelines for the molecular genetic diagnosis of hereditary hemochromatosis (HH)

PubMed Central

Porto, Graça; Brissot, Pierre; Swinkels, Dorine W; Zoller, Heinz; Kamarainen, Outi; Patton, Simon; Alonso, Isabel; Morris, Michael; Keeney, Steve

2016-01-01

Molecular genetic testing for hereditary hemochromatosis (HH) is recognized as a reference test to confirm the diagnosis of suspected HH or to predict its risk. The vast majority (typically >90%) of patients with clinically characterized HH are homozygous for the p.C282Y variant in the HFE gene, referred to as HFE-related HH. Since 1996, HFE genotyping was implemented in diagnostic algorithms for suspected HH, allowing its early diagnosis and prevention. However, the penetrance of disease in p.C282Y homozygotes is incomplete. Hence, homozygosity for p.C282Y is not sufficient to diagnose HH. Neither is p.C282Y homozygosity required for diagnosis as other rare forms of HH exist, generally referred to as non-HFE-related HH. These pose significant challenges when defining criteria for referral, testing protocols, interpretation of test results and reporting practices. We present best practice guidelines for the molecular genetic diagnosis of HH where recommendations are classified, as far as possible, according to the level and strength of evidence. For clarification, the guidelines' recommendations are preceded by a detailed description of the methodology and results obtained with a series of actions taken in order to achieve a wide expert consensus, namely: (i) a survey on the current practices followed by laboratories offering molecular diagnosis of HH; (ii) a systematic literature search focused on some identified controversial topics; (iii) an expert Best Practice Workshop convened to achieve consensus on the practical recommendations included in the guidelines. PMID:26153218

Stability enhancement of cytochrome c through heme deprotonation and mutations.

PubMed

Sonoyama, Takafumi; Hasegawa, Jun; Uchiyama, Susumu; Nakamura, Shota; Kobayashi, Yuji; Sambongi, Yoshihiro

2009-01-01

The chemical denaturation of Pseudomonas aeruginosa cytochrome c(551) variants was examined at pH 5.0 and 3.6. All variants were stabilized at both pHs compared with the wild-type. Remarkably, the variants carrying the F34Y and/or E43Y mutations were more stabilized than those having the F7A/V13M or V78I ones at pH 5.0 compared with at pH 3.6 by ~3.0-4.6 kJ/mol. Structural analyses predicted that the side chains of introduced Tyr-34 and Tyr-43 become hydrogen donors for the hydrogen bond formation with heme 17-propionate at pH 5.0, but less efficiently at pH 3.6, because the propionate is deprotonated at the higher pH. Our results provide an insight into a stabilization strategy for heme proteins involving variation of the heme electronic state and introduction of appropriate mutations.

Journal of Applied Physics. Volume 63. Number 8. Part 2B. Proceedings of the Annual conference on Magnetism and Magnetic Materials (32nd) Held in Chicago, Illinois on 9-12 November 1987

DTIC Science & Technology

1988-04-15

RBa2 Cu30 7 , compounds S. A. Shaheen, N. Jisrawi , Y. H. Lee, M. Croft, W. L. McLean, H. Zhen, L. Rebelsky, S. Horn 4190 X-ray absorption...Rev. B 36, 226 (1987). ’S. A. Shaheen, N. Jisrawi , Y. H. Lee, Y. Z. Zhang, M. Croft. W. Lvisualizing that superconductivity emerges in the relatively...Sr0 .,,Cu0 4 ,_ and Ba 2 YCu30 7 _,, thin supercon- "D. Dijkkanp, T. Venkatesan, X. D. WuS. A. Shaheen, N. Jisrawi , Y. H. ducting films may be

Absence of association between polymorphisms in the K13 gene and the presence of Plasmodium falciparum parasites at day 3 after treatment with artemisinin derivatives in Senegal.

PubMed

Madamet, Marylin; Kounta, Mame Bou; Wade, Khalifa Ababacar; Lo, Gora; Diawara, Silman; Fall, Mansour; Bercion, Raymond; Nakoulima, Aminata; Fall, Khadidiatou Ba; Benoit, Nicolas; Gueye, Mamadou Wague; Fall, Bécaye; Diatta, Bakary; Pradines, Bruno

2017-06-01

In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy as first-line treatment for uncomplicated malaria. In addition, intravenous (i.v.) injection of artesunate and artemether has gradually replaced quinine for the treatment of severe malaria. Mutations in the propeller domain of the Kelch 13 gene (K13-propeller, PF3D71343700), such as Y493H, R539T, I543T and C580Y, were recently associated with in vivo and in vitro resistance to artemisinin in Southeast Asia. However, these mutations were not identified in Africa. In total, 181 isolates of Plasmodium falciparum from 161 patients from Dakar, Senegal, were collected between August 2015 and January 2016. The K13-propeller gene of the isolates was sequenced. A search for non-synonymous mutations in the propeller region of K13 was performed in the 181 isolates collected from Dakar from 2015 to 2016. Three synonymous mutations were detected (D464D, C469C and R471R). Of 119 patients treated with i.v. artesunate or intramuscular artemether followed by artemether/lumefantrine, 9 patients were still parasitaemic on Day 3. Parasites from these nine patients were wild-type for K13-propeller. None of the polymorphisms known to be involved in artemisinin resistance in Asia were detected. These results suggest that K13 is not the best predictive marker for artemisinin resistance in Africa. More isolates from clinical failure cases or patients with delayed parasite clearance after treatment with artemisinin derivatives are necessary to identify new molecular markers. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Analysis of patients with atypical hemolytic uremic syndrome treated at the Mie University Hospital: concentration of C3 p.I1157T mutation.

PubMed

Matsumoto, Takeshi; Fan, Xinping; Ishikawa, Eiji; Ito, Masaaki; Amano, Keishirou; Toyoda, Hidemi; Komada, Yoshihiro; Ohishi, Kohshi; Katayama, Naoyuki; Yoshida, Yoko; Matsumoto, Masanori; Fujimura, Yoshihiro; Ikejiri, Makoto; Wada, Hideo; Miyata, Toshiyuki

2014-11-01

Atypical hemolytic uremic syndrome (aHUS) is caused by abnormalities of the complement system and has a significantly poor prognosis. The clinical phenotypes of 12 patients in nine families with aHUS with familial or recurrent onset and ADAMTS13 activity of ≥20 % treated at the Mie University Hospital were examined. In seven of the patients, the first episode of aHUS occurred during childhood and ten patients experienced a relapse. All patients had renal dysfunction and three had been treated with hemodialysis. Seven patients experienced probable triggering events including common cold, influenza, bacterial infection and/or vaccination for influenza. All patients had entered remission, and renal function was improved in 11 patients. DNA sequencing of six candidate genes, identified a C3 p.I1157T missense mutation in all eight patients in six families examined and this mutation was causative for aHUS. A causative mutation THBD p.D486Y was also identified in an aHUS patient. Four missense mutations, CFH p.V837I, p.Y1058H, p.V1060L and THBD p.R403K may predispose to aHUS manifestation; the remaining seven missense mutations were likely neutral. In conclusion, the clinical phenotypes of aHUS are various, and there are often trigger factors. The C3 p.I1157T mutation was identified as the causative mutation for aHUS in all patients examined, and may be geographically concentrated in or around the Mie prefecture in central Japan.

Role of Human DNA Polymerase and Its Accessory Proteins in Breast Cancer

DTIC Science & Technology

2002-04-01

the POLD1 gene in breast cancer tissues using a Non-Isotopic RNase Cleavage Assay (NIRCA) and DNA sequencing techniques. Four novel mutations , P327L...M.Y.W.T. Mutational Analysis of the Exo Motif of POLD1 gene in human Breast Cancer cells (in preparation) 9. Jaime, C., Mazloum N., and Lee, M.Y.W. T...Cold Spring Harbor 1999 8. Xu, H., and Lee, M.Y.W.T. Analyzes of POLD1 gene mutation and study of its transcriptional regulation in Breast Cancer Cells

Metabolism of D-[1-3H]glucose, D-[2-3H]glucose, D-[5-3H]glucose, D-[6-3H]glucose and D-[U-14C]glucose by rat and human erythrocytes incubated in the presence of H2O or D2O.

PubMed

Conget, I; Malaisse, W J

1995-02-01

The present study investigates whether heavy water affects the efficiency of 3HOH production from D-[1-3H]glucose, D-[2-3H]glucose, D-[5-3H]glucose and D-[6-3H]glucose relative to the total generation of tritiated metabolites produced by either rat or human erythrocytes. The relative 3HOH yield was close to 95% with D-[5-3H]glucose, 72% with D-[2-3H]glucose, 22-32% with D-[1-3H]glucose, and only 12% with D-[6-3H]glucose. In the latter case, the comparison of the specific radioactivity of intracellular and extracellular acidic metabolites, expressed relative to that of 14C-labelled metabolites produced from D-[U-14C]glucose, indicated that the generation of 3HOH from D-[6-3H]glucose occurs at distal metabolic steps, such as the partial reversion of the pyruvate kinase reaction or the interconversion of pyruvate and L-alanine in the reaction catalysed by glutamate-pyruvate transaminase. As a rule, the substitution of H2O by D2O only caused minor to negligible changes in the relative 3HOH yield. This implies that the unexpectedly high deuteration of 13C-labelled D-glucose metabolites recently documented in erythrocytes exposed to D2O cannot be attributed to any major interference of heavy water with factors regulating both the deuteration and detritiation efficiency, such as the enzyme-to-enzyme tunnelling of specific glycolytic intermediates.

Preliminary investigation of bottlenose dolphins (Tursiops truncatus) for hfe gene-related hemochromatosis.

PubMed

Phillips, Brianne E; Venn-Watson, Stephanie; Archer, Linda L; Nollens, Hendrik H; Wellehan, James F X

2014-10-01

Hemochromatosis (iron storage disease) has been reported in diverse mammals including bottlenose dolphins (Tursiops truncatus). The primary cause of excessive iron storage in humans is hereditary hemochromatosis. Most human hereditary hemochromatosis cases (up to 90%) are caused by a point mutation in the hfe gene, resulting in a C282Y substitution leading to iron accumulation. To evaluate the possibility of a hereditary hemochromatosis-like genetic predisposition in dolphins, we sequenced the bottlenose dolphin hfe gene, using reverse transcriptase-PCR and hfe primers designed from the dolphin genome, from liver of affected and healthy control dolphins. Sample size included two case animals and five control animals. Although isotype diversity was evident, no coding differences were identified in the hfe gene between any of the animals examined. Because our sample size was small, we cannot exclude the possibility that hemochromatosis in dolphins is due to a coding mutation in the hfe gene. Other potential causes of hemochromatosis, including mutations in different genes, diet, primary liver disease, and insulin resistance, should be evaluated.

Analysis of Familial Tendencies in Transferrin Saturation in a Korean Population.

PubMed

Oh, Sung-Hee; Jeong, Tae-Dong; Lee, Woochang; Chun, Sail; Min, Won-Ki

2015-10-01

Despite the high transferrin saturation (TS) level in Koreans, the p.Cys282Tyr and p.His63Asp mutations are markedly less frequent than in Caucasians. We aimed to determine TS levels and their familial tendencies in a Korean population using nationwide data from the Fifth Korea National Health and Nutrition Examination Survey (KNHANES V-1 2010). A total of 4904 subjects without a history of hepatitis B and C virus infection, or liver cirrhosis, and who were negative for anemia and hepatitis B antigen were enrolled. A familial tendency analysis was performed in 260 families. Parents were grouped into four quartiles based on their TS levels. Offspring were categorized according to the mean parental TS four quartile scores (1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0). A familial tendency was evaluated by comparing the mean TS of offspring in seven parental groups. The mean TS was 39.3 ± 15.6% for Korean males and 33.2 ± 12.9% for Korean females, and both were significantly higher than those of Caucasians reported in the HEIRS study (30.6 ± 11.0% for male, 25.6 ± 10.6% for female, P < 0.001). The 260 families showed statistically significant familial tendencies of TS values (P < 0.001). The mean TS of offspring in parental group 1.0, 1.5, 2.0, and 2.5 showed a lower value than that in higher group 3.0, 3.5, and 4.0. In contrast, there were no significant differences in age, daily dietary iron intake, and AST or ALT value among seven groups. These findings suggest unidentified genetic variations on high TS in Koreans beyond the p.Cys282Tyr and p.His63Asp mutations commonly identified in Caucasians.

MUTYH-associated colorectal cancer and adenomatous polyposis.

PubMed

Yamaguchi, Satoru; Ogata, Hideo; Katsumata, Daisuke; Nakajima, Masanobu; Fujii, Takaaki; Tsutsumi, Soichi; Asao, Takayuki; Sasaki, Kinro; Kuwano, Hiroyuki; Kato, Hiroyuki

2014-04-01

MUTYH-associated polyposis (MAP) was first described in 2002. MUTYH is a component of a base excision repair system that protects the genomic information from oxidative damage. When the MUTYH gene product is impaired by bi-allelic germline mutation, it leads to the mutation of cancer-related genes, such as the APC and/or the KRAS genes, via G to T transversion. MAP is a hereditary colorectal cancer syndrome inherited in an autosomal-recessive fashion. The clinical features of MAP include the presence of 10-100 adenomatous polyps in the colon, and early onset of colorectal cancer. Ethnic and geographical differences in the pattern of the MUTYH gene mutations have been suggested. In Caucasian patients, c.536A>G (Y179C) and c.1187G>A (G396D) mutations are frequently detected. In the Asian population, Y179C and G396D are uncommon, whereas other variants are suggested to be the major causes of MAP. We herein review the literature on MUTYH-associated colorectal cancer and adenomatous polyposis.

Enzyme-Catalyzed Mutation in Breast Cancer

DTIC Science & Technology

2014-08-01

from  ATCC)  and  hTERT-­‐HMEC  (a  gift   from  the  lab  of  Dr.   Vitaly  Polunovsky)  to  over-­‐express  A3B...methylation in mouse primordial germ cells is affected by AID deficiency. Nature. 2010;463:1101-5. [10] Bhutani N , Brady JJ, Damian M, Sacco A...Drier Y, Zou L, Ramos AH, Pugh TJ, Stransky N , Helman E, Kim J, Sougnez C, Ambrogio L, Nickerson E, Shefler E, Cortés ML, Auclair D, Saksena G, Voet D

Molecular Surveillance of Antiviral Drug Resistance of Influenza A/H3N2 Virus in Singapore, 2009-2013

PubMed Central

Lee, Hong Kai; Tang, Julian Wei-Tze; Loh, Tze Ping; Hurt, Aeron C.; Oon, Lynette Lin-Ean; Koay, Evelyn Siew-Chuan

2015-01-01

Adamantanes and neuraminidase inhibitors (NAIs) are two classes of antiviral drugs available for the chemoprophylaxis and treatment of influenza infections. To determine the frequency of drug resistance in influenza A/H3N2 viruses in Singapore, large-scale sequencing of neuraminidase (NA) and matrix protein (MP) genes was performed directly without initial culture amplification. 241 laboratory-confirmed influenza A/H3N2 clinical samples, collected between May 2009 and November 2013 were included. In total, 229 NA (95%) and 241 MP (100%) complete sequences were obtained. Drug resistance mutations in the NA and MP genes were interpreted according to published studies. For the NAIs, a visual inspection of the aligned NA sequences revealed no known drug resistant genotypes (DRGs). For the adamantanes, the well-recognised S31N DRG was identified in all 241 MP genes. In addition, there was an increasing number of viruses carrying the combination of D93G+Y155F+D251V (since May 2013) or D93G (since March 2011) mutations in the NA gene. However, in-vitro NAI testing indicated that neither D93G+Y155F+D251V nor D93G alone conferred any changes in NAI susceptibility. Lastly, an I222T mutation in the NA gene that has previously been reported to cause oseltamivir-resistance in influenza A/H1N1/2009, B, and A/H5N1, was detected from a treatment-naïve patient. Further in-vitro NAI testing is required to confirm the effect of this mutation in A/H3N2 virus. PMID:25635767

Improved detection of genetic markers of antimicrobial resistance by hybridization probe-based melting curve analysis using primers to mask proximal mutations: examples include the influenza H275Y substitution.

PubMed

Whiley, David M; Jacob, Kevin; Nakos, Jennifer; Bletchly, Cheryl; Nimmo, Graeme R; Nissen, Michael D; Sloots, Theo P

2012-06-01

Numerous real-time PCR assays have been described for detection of the influenza A H275Y alteration. However, the performance of these methods can be undermined by sequence variation in the regions flanking the codon of interest. This is a problem encountered more broadly in microbial diagnostics. In this study, we developed a modification of hybridization probe-based melting curve analysis, whereby primers are used to mask proximal mutations in the sequence targets of hybridization probes, so as to limit the potential for sequence variation to interfere with typing. The approach was applied to the H275Y alteration of the influenza A (H1N1) 2009 strain, as well as a Neisseria gonorrhoeae mutation associated with antimicrobial resistance. Assay performances were assessed using influenza A and N. gonorrhoeae strains characterized by DNA sequencing. The modified hybridization probe-based approach proved successful in limiting the effects of proximal mutations, with the results of melting curve analyses being 100% consistent with the results of DNA sequencing for all influenza A and N. gonorrhoeae strains tested. Notably, these included influenza A and N. gonorrhoeae strains exhibiting additional mutations in hybridization probe targets. Of particular interest was that the H275Y assay correctly typed influenza A strains harbouring a T822C nucleotide substitution, previously shown to interfere with H275Y typing methods. Overall our modified hybridization probe-based approach provides a simple means of circumventing problems caused by sequence variation, and offers improved detection of the influenza A H275Y alteration and potentially other resistance mechanisms.

Cardiac-Targeted Transgenic Mutant Mitochondrial Enzymes

PubMed Central

Kohler, James J.; Hosseini, Seyed H.; Green, Elgin; Hoying-Brandt, Amy; Cucoranu, Ioan; Haase, Chad P.; Russ, Rodney; Srivastava, Jaya; Ivey, Kristopher; Ludaway, Tomika; Kapoor, Victor; Abuin, Allison; Shapoval, Alexsey; Santoianni, Robert; Saada, Ann; Elpeleg, Orly; Lewis, William

2009-01-01

Mitochondrial (mt) DNA biogenesis is critical to cardiac contractility. DNA polymerase gamma (pol γ) replicates mtDNA, whereas thymidine kinase 2 (TK2) monophosphorylates pyrimidines intramitochondrially. Point mutations in POLG and TK2 result in clinical diseases associated with mtDNA depletion and organ dysfunction. Pyrimidine analogs (NRTIs) inhibit Pol γ and mtDNA replication. Cardiac “dominant negative” murine transgenes (TGs; Pol γ Y955G, and TK2 H121N or I212N) defined the role of each in the heart. mtDNA abundance, histopathological features, histochemistry, mitochondrial protein abundance, morphometry, and echocardiography were determined for TGs in “2 × 2” studies with or without pyrimidine analogs. Cardiac mtDNA abundance decreased in Y955C TGs (∼50%) but increased in H121N and I212N TGs (20-70%). Succinate dehydrogenase (SDH) increased in hearts of all mutants. Ultrastructural changes occurred in Y955C and H121N TGs. Histopathology demonstrated hypertrophy in H121N, LV dilation in I212N, and both hypertrophy and dilation in Y955C TGs. Antiretrovirals increased LV mass (≈50%) for all three TGs which combined with dilation indicates cardiomyopathy. Taken together, these studies demonstrate three manifestations of cardiac dysfunction that depend on the nature of the specific mutation and antiretroviral treatment. Mutations in genes for mtDNA biogenesis increase risk for defective mtDNA replication, leading to LV hypertrophy. PMID:18446447

A variant c-KIT mutation, D816H, fundamental to the sequential development of an ovarian mixed germ cell tumor and systemic mastocytosis with chronic myelomonocytic leukemia.

PubMed

Mitchell, Sarah G; Bunting, Silvia T; Saxe, Debra; Olson, Thomas; Keller, Frank G

2017-04-01

An activating point mutation of the c-KIT tyrosine kinase receptor gene, D816H, has been described in germ cell tumors (GCTs). We report an adolescent diagnosed with an ovarian mixed GCT and systemic mastocytosis with chronic myelomonocytic leukemia (SM-CMML). The teratoma and dysgerminoma differed by copy number aberrations via single nucleotide polymorphism (SNP) microarray, but were inclusive of the same c-KIT D816H point mutation (c.2446G>C) also identified in blood and bone marrow mast cells. These findings indicate not only a clonal origin of the GCT and hematologic malignancy, but also suggest a rare KIT mutation may be playing a fundamental role in malignancy development. © 2016 Wiley Periodicals, Inc.

Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

PubMed

Xuan, Shi-Hai; Zhou, Yu-Gui; Shao, Bo; Cui, Ya-Lin; Li, Jian; Yin, Hong-Bo; Song, Xiao-Ping; Cong, Hui; Jing, Feng-Xiang; Jin, Qing-Hui; Wang, Hui-Min; Zhou, Jie

2009-11-01

Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

The General Base in the Thymidylate Synthase Catalyzed Proton Abstraction

PubMed Central

Ghosh, Ananda K.; Islam, Zahidul; Krueger, Jonathan; Abeysinghe, Don Thelma; Kohen, Amnon

2015-01-01

The enzyme thymidylate synthase (TSase), an important chemotherapeutic drug target, catalyzes the formation of 2′-deoxythymidine-5′-monophosphate (dTMP), a precursor of one of the DNA building blocks. TSase catalyzes a multi-step mechanism that includes the abstraction of a proton from the C5 of the substrate 2′-deoxyuridine-5′-monophosphate (dUMP). Previous studies on ecTSase proposed that an active-site residue, Y94 serves the role of the general base abstracting this proton. However, since Y94 is neither very basic, nor connected to basic residues, nor located close enough to the pyrimidine proton to be abstracted, the actual identity of this base remains enigmatic. Based on crystal structures, an alternative hypothesis is that the nearest potential proton-acceptor of C5 of dUMP is a water molecule that is part of a hydrogen bond (H-bond) network comprised of several water molecules and several protein residues including H147, E58, N177, and Y94. Here, we examine the role of the residue Y94 in the proton abstraction step by removing its hydroxyl group (Y94F mutant). We investigated the effect of the mutation on the temperature dependence of intrinsic kinetic isotope effects (KIEs) and found that these KIEs are more temperature dependent than those of the wild-type enzyme (WT). These results suggest that the phenolic –OH of Y94 is a component of the transition state for the proton abstraction step. The findings further support the hypothesis that no single functional group is the general base, but a network of bases and hydroxyls (from water molecules and tyrosine) sharing H-bonds across the active site can serve the role of the general base to remove the pyrimidine proton. PMID:25912171

Prevalence of etravirine-associated mutations in clinical samples with resistance to nevirapine and efavirenz.

PubMed

Llibre, J M; Santos, J R; Puig, T; Moltó, J; Ruiz, L; Paredes, R; Clotet, B

2008-11-01

To evaluate the expected activity of etravirine in clinical samples, according to mutational patterns associated with decreased virological response (VR). We identified 1586 routine clinical samples with resistance-associated mutations (RAMs) to nevirapine and efavirenz (K103N 60%, Y181C 37%, G190A 27%, V108I 13%). Concerning in vitro identified etravirine mutations, samples with F227C, Y181I, M230L or L100I plus K103N plus Y181C were considered highly resistant. Samples with two RAMs plus Y181C or V179D or K101E or Y188L were considered intermediate. The prevalence of 13 RAMs recently associated with decreased VR to etravirine in the DUET clinical trials was also investigated. Most samples (69%) harboured more than one IAS-USA RAM to first-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs): 42% harboured two RAMs, 21% three RAMs and 6% four or more RAMs. The prevalence of 13 specific etravirine RAMs was V179F 0.12%, G190S 3.9%, Y181V 0.1%, V106I 2.6%, V179D 1.6%, K101P 2.0%, K101E 10.1%, Y181C 36.9%, A98G 5.9%, V90I 6.9%, Y181I 3.6%, G190A 27% and L100I 9.1%. The five RAMs with the most impact on VR (V179F/D, G190S, Y181V and V106I) occurred less often. Overall, 8.2% of the samples had three or more etravirine RAMs and only 1.1% had four or more. In addition, patterns of RAMs previously associated with intermediate etravirine resistance were present in 26.2% of the samples, whereas 4.85% displayed patterns of high-degree resistance. For RAMs associated with decreased VR, etravirine resistance in routine clinical samples was lower than previously reported. High-degree resistance was uncommon, even in patients with resistance to first-generation NNRTIs, whereas low-to-intermediate etravirine resistance was more common.

Nature and Recurrence of AVPR2 Mutations in X-linked Nephrogenic Diabetes Insipidus

PubMed Central

Bichet, Daniel G.; Birnbaumer, Mariel; Lonergan, Michèle; Arthus, Marie-Françoise; Rosenthal, Walter; Goodyer, Paul; Nivet, Hubert; Benoit, Stéphane; Giampietro, Philip; Simonetti, Simonetta; Fish, Alfred; Whitley, Chester B.; Jaeger, Philippe; Gertner, Joseph; New, Maria; DiBona, Francis J.; Kaplan, Bernard S.; Robertson, Gary L.; Hendy, Geoffrey N.; Fujiwara, T. Mary; Morgan, Kenneth

1994-01-01

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine-vasopressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. We analyzed 31 independent NDI families to determine the nature and recurrence of AVPR2 mutations. Twenty-one new putative disease-causing mutations were identified: 113delCT, 253del35, 255del9, 274insG, V88M, R106C, 402delCT, C112R, Y124X, S126F, W164S, S167L, 684delTA, 804insG, W284X, A285P, W293X, R337X, and three large deletions or gene rearrangements. Five other mutations—R113W, Y128S, R137H, R181C, and R202C—that previously had been reported in other families were detected. There was evidence for recurrent mutation for four mutations (R113W, R137H, S167L, and R337X). Eight de novo mutation events were detected (274insG, R106C, Y128S, 167L [twice], R202C, 684delTA, and R337X). The origins were maternal (one), grandmaternal (one), and grandpaternal (six). In the 31 NDI families and 6 families previously reported by us, there is evidence both for mutation hot spots for nucleotide substitutions and for small deletions and insertions. More than half (58%) of the nucleotide substitutions in 26 families could be a consequence of 5-methylcytosine deamination at a CpG dinucleotide. Most of the small deletions and insertions could be attributed to slipped mispairing during DNA replication. PMID:8037205

Secretion of the Streptomyces tyrosinase is mediated through its trans-activator protein, MelC1.

PubMed

Leu, W M; Chen, L Y; Liaw, L L; Lee, Y H

1992-10-05

The tyrosinase of Streptomyces antibioticus is encoded by the second open reading frame, melC2 of the melanin operon (melC). The upstream open reading frame melC1 specifies a 146-amino acid protein with a typical NH2-terminal signal-peptide characteristic of a secretory protein. The MelC1 protein is involved in the transfer of copper ion to apotyrosinase MelC2 via binary complex formation (Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71-81; Chen, L.-Y., Leu, W.-M., Wang, K.-T., and Lee, Y.-H.W. (1992) J. Biol. Chem. 267, 20100-20107). To investigate whether the export of tyrosinase is also dependent on MelC1, a mutational study of its signal-peptide sequence was performed. Four different mutants were obtained. Mutation at the positively charged region (mutant M-6LE, Arg6-Arg7----Leu6-Glu7) or the hydrophobic region (mutant M-16D, Val16----Asp16) led to Mel- phenotypes. These lesions caused a severe 7-10-fold reduction of the export of both the MelC1 and MelC2 proteins and a concomitant accumulation of the two proteins in the cytosolic fraction. The cell-associated tyrosinase activity in M-6LE but not in the M-16D mutant was dramatically reduced to 4% of the activity found in the wild type strain, suggesting that the basic NH2 terminus of MelC1 is also important for the trans-activation function of this protein. Nevertheless, the defects on the trans-activation and/or secretory functions of MelC1 in mutants M-6LE and M-16D are not due to the impairment of the formation of the MelC1.MelC2 complex. The translation of melanin operon genes in these two mutants also decreased. In contrast, the tyrosinase activity and the secretion of MelC2 were not affected if the mutations occurred at the putative cleavage site of the signal peptidase (e.g. mutant M-29SM, Arg29-Ala30----Ser29-Met30 or mutant 29-SMG, Arg29-Ala30-Asp31----Ser29-Med30-Gly31+ ++). Additionally, tyrosinase activity and its export were abolished in a MelC1-negative mutant, M-950. Taken together, these results demonstrate that a functional MelC1 is essential for tyrosinase secretion and activity. Furthermore, the results suggest that like other secretory proteins, basic and hydrophobic residues in the MelC1 signal sequence are an important feature of the signal-peptide and play a pivotal role in the secretion of both the MelC1 and MelC2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Yeast Cells Expressing the Human Mitochondrial DNA Polymerase Reveal Correlations between Polymerase Fidelity and Human Disease Progression*

PubMed Central

Qian, Yufeng; Kachroo, Aashiq H.; Yellman, Christopher M.; Marcotte, Edward M.; Johnson, Kenneth A.

2014-01-01

Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-γ with their physiological consequences, we created “humanized” yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-γ. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-γ suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-γ mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans. PMID:24398692

C incorporation and segregation during Si 1- yC y/Si( 0 0 1 ) gas-source molecular beam epitaxy from Si 2H 6 and CH 3SiH 3

NASA Astrophysics Data System (ADS)

Foo, Y. L.; Bratland, K. A.; Cho, B.; Soares, J. A. N. T.; Desjardins, P.; Greene, J. E.

2002-08-01

We have used in situ D 2 temperature-programmed desorption (TPD) to probe C incorporation and surface segregation kinetics, as well as hydrogen desorption pathways, during Si 1- yC y(0 0 1) gas-source molecular beam epitaxy from Si 2H 6/CH 3SiH 3 mixtures at temperatures Ts between 500 and 650 °C. Parallel D 2 TPD results from C-adsorbed Si(0 0 1) wafers exposed to varying CH 3SiH 3 doses serve as reference data. Si 1- yC y(0 0 1) layer spectra consist of three peaks: first-order β 1 at 515 °C and second-order β 2 at 405 °C, due to D 2 desorption from Si monodeuteride and dideuteride phases, as well as a new second-order C-induced γ 1 peak at 480 °C. C-adsorbed Si(0 0 1) samples with very high CH 3SiH 3 exposures yielded a higher-temperature TPD feature, corresponding to D 2 desorption from surface C atoms, which was never observed in Si 1- yC y(0 0 1) layer spectra. The Si 1- yC y(0 0 1) γ 1 peak arises due to desorption from Si monodeuteride species with C backbonds. γ 1 occurs at a lower temperature than β 1 reflecting the lower D-Si * bond strength, where Si * represents surface Si atoms bonded to second-layer C atoms, as a result of charge transfer from dangling bonds. The total integrated monohydride (β 1+γ 1) intensity, and hence the dangling bond density, remains constant with y indicating that C does not deactivate surface dangling bonds as it segregates to the second-layer during Si 1- yC y(0 0 1) growth. Si * coverages increase with y at constant Ts and with Ts at constant y. The positive Ts-dependence shows that C segregation is kinetically limited at Ts⩽650 °C. D 2 desorption activation energies from β 1, γ 1 and β 2 sites are 2.52, 2.22 and 1.88 eV.

The Southern French registry of genetic hemochromatosis: a tool for determining clinical prevalence of the disorder and genotype penetrance.

PubMed

Aguilar-Martinez, Patricia; Bismuth, Michael; Blanc, François; Blanc, Pierre; Cunat, Severine; Dereure, Olivier; Dujols, Pierre; Giansily-Blaizot, Muriel; Jorgensen, Christian; Konate, Amadou; Larrey, Dominique; Le Quellec, Alain; Mura, Thibault; Raingeard, Isabelle; Ramos, Jeanne; Renard, Eric; Rousseau, Florence; Schved, Jean-François; Picot, Marie-Christine

2010-04-01

Despite great progress in understanding the mechanisms underlying genetic hemochromatosis, data on the prevalence and the penetrance of the disorder are conflicting. A registry of patients with genetic hemochromatosis was established in the South of France and a regional health network was developed to allow the inclusion of all the diagnosed patients. C282Y homozygous patients classified in stages 2 (biological iron overload), 3 and 4 (clinical manifestations of iron overload, stage 4 being the more severe) according to the classification of the French National Authority for Health were included in the registry over a 6-year period. A total of 352 symptomatic C282Y homozygotes were identified, resulting in a total prevalence of 1.83 per 10,000 (95% CI: 1.63 to 2.02) in subjects over 20 years and 2.40 per 10,000 (95% CI, 2.15 to 2.65) among subjects of European descent. Among Europeans, the total calculated penetrance was 15.8% in stage 2 or higher, 12.1% in stage 3 or 4 and 2.9% in stage 4. The penetrance was slightly higher in males (18.7%) than in females (13.2%). It was 19.9% for individuals over 40 years of age (24.1% and 16.3% in males and females, respectively) with a maximum of 31% in subjects between 50 and 54 years old. Among 249 patients with complete records, 24% were in stage 2, the majority (58%) were in stage 3, and 18% in stage 4. There was a higher proportion of males, and excessive alcohol intake was more prevalent in stage 4 than in stages 2 and 3 combined. A French Mediterranean regional hemochromatosis registry with strict inclusion criteria is a useful tool for characterizing the history of this disease, particularly for the most severely affected patients, as defined by the disease severity classification. The total prevalence of symptomatic C282Y homozygotes in the region was found to be low. However, clinical penetrance (stages 3 and 4) was not negligible.

The Southern French registry of genetic hemochromatosis: a tool for determining clinical prevalence of the disorder and genotype penetrance

PubMed Central

Aguilar-Martinez, Patricia; Bismuth, Michael; Blanc, François; Blanc, Pierre; Cunat, Severine; Dereure, Olivier; Dujols, Pierre; Giansily-Blaizot, Muriel; Jorgensen, Christian; Konate, Amadou; Larrey, Dominique; Le Quellec, Alain; Mura, Thibault; Raingeard, Isabelle; Ramos, Jeanne; Renard, Eric; Rousseau, Florence; Schved, Jean-François; Picot, Marie-Christine

2010-01-01

Background Despite great progress in understanding the mechanisms underlying genetic hemochromatosis, data on the prevalence and the penetrance of the disorder are conflicting. Design and Methods A registry of patients with genetic hemochromatosis was established in the South of France and a regional health network was developed to allow the inclusion of all the diagnosed patients. C282Y homozygous patients classified in stages 2 (biological iron overload), 3 and 4 (clinical manifestations of iron overload, stage 4 being the more severe) according to the classification of the French National Authority for Health were included in the registry over a 6-year period. Results A total of 352 symptomatic C282Y homozygotes were identified, resulting in a total prevalence of 1.83 per 10,000 (95% CI: 1.63 to 2.02) in subjects over 20 years and 2.40 per 10,000 (95% CI, 2.15 to 2.65) among subjects of European descent. Among Europeans, the total calculated penetrance was 15.8% in stage 2 or higher, 12.1% in stage 3 or 4 and 2.9% in stage 4. The penetrance was slightly higher in males (18.7%) than in females (13.2%). It was 19.9% for individuals over 40 years of age (24.1% and 16.3% in males and females, respectively) with a maximum of 31% in subjects between 50 and 54 years old. Among 249 patients with complete records, 24% were in stage 2, the majority (58%) were in stage 3, and 18% in stage 4. There was a higher proportion of males, and excessive alcohol intake was more prevalent in stage 4 than in stages 2 and 3 combined. Conclusions A French Mediterranean regional hemochromatosis registry with strict inclusion criteria is a useful tool for characterizing the history of this disease, particularly for the most severely affected patients, as defined by the disease severity classification. The total prevalence of symptomatic C282Y homozygotes in the region was found to be low. However, clinical penetrance (stages 3 and 4) was not negligible. PMID:20145272

Longer survival associated with HLA-A*03, B*14 among 212 hemochromatosis probands with HFE C282Y homozygosity and HLA-A and -B typing and haplotyping.

PubMed

Barton, James C; Barton, J Clayborn; Acton, Ronald T

2010-11-01

Human leukocyte antigen (HLA) haplotypes may influence iron phenotypes in patients with HFE hemochromatosis and could affect survival. We tabulated general characteristics of HLA-A and -B types and haplotypes of HFE C282Y/C282Y probands diagnosed in medical care and analyzed these data to identify HLA survival modifiers. There were 212 probands (130 men, 82 women). Mean follow-up was 12.0 ± 6.4 yr (0.1-41.2 yr; 34 deaths). HLA-A*03 was more prevalent in men (76.9% vs. 61.0% women; P = 0.0129); 35.4% of men and 29.3% of women had A*03, B*07; and 7.7% of men and 8.5% of women had A*03, B*14. Twenty-three probands had cirrhosis; none had A*03, B*14. Positivity for A*03 or A*03, B*07 was not a significant predictor or modifier of survival. In multiple regression analyses, A*03, B*14 predicted longer survival (P = 0.0004). Kaplan-Meier analysis confirmed longer survival in probands with A*03, B*14 (P = 0.0199, log-rank test). After excluding the 23 non-A*03, B*14 probands with cirrhosis, survival of probands with A*03, B*14 was still greater than that of probands without A*03, B*14 (P = 0.0254; log-rank test). Twenty-four years after diagnosis, cumulative survival of probands with and without A*03, B*14 was 100% and 58%, respectively. The percentage of deaths due to iron overload was lower in probands with A*03, B*14 (0% vs. 21.9%; P = 0.0392). In hemochromatosis probands with HFE C282Y/C282Y, survival was longer in those with HLA-A*03, B*14. Earlier age at diagnosis and less severe iron overload in probands with A*03, B*14 could explain this difference. © 2010 John Wiley & Sons A/S.

Proceedings of Advanced Planning Briefing for Industry. Part 1

DTIC Science & Technology

1965-12-01

t h e flow o f in format ion and i d e a s must be both t o you and from you. We thus welcome t h e oppo r tun i t y o f p r e sen t i...megawatt, l i g h t wa te r moderated and c o o l e d , h i g h l y e n r i c h e d uranium , the rmal t y p e t h a t i s c o n s t a n t l y...a l s . Our c u r r e n t programs i n b e r y l l i u m and i n uranium have n o t t h e e x t e n s i v e - n e s s o f t h e e a r l i

A Cys-loop Mutation in the Caenorhabditis elegans Nicotinic Receptor Subunit UNC-63 Impairs but Does Not Abolish Channel Function*

PubMed Central

Jones, Andrew K.; Rayes, Diego; Al-Diwani, Adam; Maynard, Thomas P. R.; Jones, Rachel; Hernando, Guillermina; Buckingham, Steven D.; Bouzat, Cecilia; Sattelle, David B.

2011-01-01

The nematode Caenorhabditis elegans is an established model organism for studying neurobiology. UNC-63 is a C. elegans nicotinic acetylcholine receptor (nAChR) α-subunit. It is an essential component of the levamisole-sensitive muscle nAChR (L-nAChR) and therefore plays an important role in cholinergic transmission at the nematode neuromuscular junction. Here, we show that worms with the unc-63(x26) allele, with its αC151Y mutation disrupting the Cys-loop, have deficient muscle function reflected by impaired swimming (thrashing). Single-channel recordings from cultured muscle cells from the mutant strain showed a 100-fold reduced frequency of opening events and shorter channel openings of L-nAChRs compared with those of wild-type worms. Anti-UNC-63 antibody staining in both cultured adult muscle and embryonic cells showed that L-nAChRs were expressed at similar levels in the mutant and wild-type cells, suggesting that the functional changes in the receptor, rather than changes in expression, are the predominant effect of the mutation. The kinetic changes mimic those reported in patients with fast-channel congenital myasthenic syndromes. We show that pyridostigmine bromide and 3,4-diaminopyridine, which are drugs used to treat fast-channel congenital myasthenic syndromes, partially rescued the motility defect seen in unc-63(x26). The C. elegans unc-63(x26) mutant may therefore offer a useful model to assist in the development of therapies for syndromes produced by altered function of human nAChRs. PMID:20966081

Structural analysis of phospholipase A2 from functional perspective. 1. Functionally relevant solution structure and roles of the hydrogen-bonding network.

PubMed

Yuan, C; Byeon, I J; Li, Y; Tsai, M D

1999-03-09

Bovine pancreatic phospholipase A2 (PLA2), a small (13.8 kDa) Ca2+-dependent lipolytic enzyme, is rich in functional and structural character. In an effort to examine its detailed structure-function relationship, we determined its solution structure by multidimensional nuclear magnetic resonance (NMR) spectroscopy at a functionally relevant pH. An ensemble of 20 structures generated has an average root-mean-square deviation (RMSD) of 0.62 +/- 0.08 A for backbone (N, Calpha, C) atoms and 0.98 +/- 0.09 A for all heavy atoms. The overall structure shows several notable differences from the crystal structure: the first three residues at the N-terminus, the calcium-binding loop (Y25-T36), and the surface loop (V63-N72) appear to be flexible; the alpha-helical conformation of helix B (E17-F22) is absent; helix D appears to be shorter (D59-V63 instead of D59-D66); and the hydrogen-bonding network is less defined. These differences were analyzed in relation to the function of PLA2. We then further examined the H-bonding network, because its functional role or even its existence in solution has been in dispute recently. Our results show that part of the H-bonding network (the portion away from N-terminus) clearly exists in solution, as evidenced by direct observation (at 11.1 ppm) of a strong H-bond between Y73 and D99 and an implicated interaction between D99 and H48. Analyses of a series of mutants indicated that the existence of the Y73.D99 H-bond correlates directly with the conformational stability of the mutant. Loss of this H-bond results in a loss of 2-3 kcal/mol in the conformational stability of PLA2. The unequivocal identification and demonstration of the structural importance of a specific hydrogen bond, and the magnitude of its contribution to conformational stability, are uncommon to the best of our knowledge. Our results also suggest that, while the D99.H48 catalytic diad is the key catalytic machinery of PLA2, it also helps to maintain conformational integrity.

Development of Efficient Charge-Selective Materials for Bulk Heterojunction Polymer Solar Cells

DTIC Science & Technology

2015-01-15

Cho, D. S. Ginger and A. K. Y. Jen, Advanced Materials, 2014, 26, 6262-6267. 17. N. Cho, C.-Z. Li, H.-L. Yip and A. K. Y. Jen, Energy...Solar Cells”, Adv. Funct. Mater. 2015, ASAP. 4. J. H. Kim, P. W. Liang, S. T. Williams, N. C. Cho, C. C. Chueh, M. S. Glaz, D. S. Ginger , A. K.-Y...Kim, P. W. Liang, S. T. Williams, N. C. Cho, C. C. Chueh, M. S. Glaz, D. S. Ginger , A. K.-Y. Jen, 2015, ASAP. 7. P. W. Liang, C. C. Chueh, X. K

The common missense mutation D489N in TRIM32 causing limb girdle muscular dystrophy 2H leads to loss of the mutated protein in knock-in mice resulting in a Trim32-null phenotype.

PubMed

Kudryashova, Elena; Struyk, Arie; Mokhonova, Ekaterina; Cannon, Stephen C; Spencer, Melissa J

2011-10-15

Mutations in tripartite motif protein 32 (TRIM32) are responsible for several hereditary disorders that include limb girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathy (STM) and Bardet Biedl syndrome. Most LGMD2H mutations in TRIM32 are clustered in the NHL β-propeller domain at the C-terminus and are predicted to interfere with homodimerization. To get insight into TRIM32's role in the pathogenesis of LGMD2H and to create an accurate model of disease, we have generated a knock-in mouse (T32KI) carrying the c.1465G > A (p.D489N) mutation in murine Trim32 corresponding to the human LGMD2H/STM pathogenic mutation c.1459G > A (p.D487N). Our data indicate that T32KI mice have both a myopathic and a neurogenic phenotype, very similar to the one described in the Trim32-null mice that we created previously. Analysis of Trim32 gene expression in T32KI mice revealed normal mRNA levels, but a severe reduction in mutant TRIM32 (D489N) at the protein level. Our results suggest that the D489N pathogenic mutation destabilizes the protein, leading to its degradation, and results in the same mild myopathic and neurogenic phenotype as that found in Trim32-null mice. Thus, one potential mechanism of LGMD2H might be destabilization of mutated TRIM32 protein leading to a null phenotype.

Development of the scintillator-based probe for fast-ion losses in the HL-2A tokamak

NASA Astrophysics Data System (ADS)

Zhang, Y. P.; Liu, Yi; Luo, X. B.; Isobe, M.; Yuan, G. L.; Liu, Y. Q.; Hua, Y.; Song, X. Y.; Yang, J. W.; Li, X.; Chen, W.; Li, Y.; Yan, L. W.; Song, X. M.; Yang, Q. W.; Duan, X. R.

2014-05-01

A new scintillator-based lost fast-ion probe (SLIP) has been developed and operated in the HL-2A tokamak [L. W. Yan, X. R. Duan, X. T. Ding, J. Q. Dong, Q. W. Yang, Yi Liu, X. L. Zou, D. Q. Liu, W. M. Xuan, L. Y. Chen, J. Rao, X. M. Song, Y. Huang, W. C. Mao, Q. M. Wang, Q. Li, Z. Cao, B. Li, J. Y. Cao, G. J. Lei, J. H. Zhang, X. D. Li, W. Chen, J. Chen, C. H. Cui, Z. Y. Cui, Z. C. Deng, Y. B. Dong, B. B. Feng, Q. D. Gao, X. Y. Han, W. Y. Hong, M. Huang, X. Q. Ji, Z. H. Kang, D. F. Kong, T. Lan, G. S. Li, H. J. Li, Qing Li, W. Li, Y. G. Li, A. D. Liu, Z. T. Liu, C. W. Luo, X. H. Mao, Y. D. Pan, J. F. Peng, Z. B. Shi, S. D. Song, X. Y. Song, H. J. Sun, A. K. Wang, M. X. Wang, Y. Q. Wang, W. W. Xiao, Y. F. Xie, L. H. Yao, D. L. Yu, B. S. Yuan, K. J. Zhao, G. W. Zhong, J. Zhou, J. C. Yan, C. X. Yu, C. H. Pan, Y. Liu, and the HL-2A Team, Nucl. Fusion 51, 094016 (2011)] to measure the losses of neutral beam ions. The design of the probe is based on the concept of the α-particle detectors on Tokamak Fusion Test Reactor (TFTR) using scintillator plates. The probe is capable of traveling across an equatorial plane port and sweeping the aperture angle rotationally with respect to the axis of the probe shaft by two step motors, in order to optimize the radial position and the collimator angle. The energy and the pitch angle of the lost fast ions can be simultaneously measured if the two-dimensional image of scintillation light intensity due to the impact of the lost fast ions is detected. Measurements of the fast-ion losses using the probe have been performed during HL-2A neutral beam injection discharges. The clear experimental evidence of enhanced losses of beam ions during disruptions has been obtained by means of the SLIP system. A detailed description of the probe system and the first experimental results are reported.

Development of the scintillator-based probe for fast-ion losses in the HL-2A tokamak

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Y. P., E-mail: zhangyp@swip.ac.cn; Liu, Yi; Yuan, G. L.

A new scintillator-based lost fast-ion probe (SLIP) has been developed and operated in the HL-2A tokamak [L. W. Yan, X. R. Duan, X. T. Ding, J. Q. Dong, Q. W. Yang, Yi Liu, X. L. Zou, D. Q. Liu, W. M. Xuan, L. Y. Chen, J. Rao, X. M. Song, Y. Huang, W. C. Mao, Q. M. Wang, Q. Li, Z. Cao, B. Li, J. Y. Cao, G. J. Lei, J. H. Zhang, X. D. Li, W. Chen, J. Chen, C. H. Cui, Z. Y. Cui, Z. C. Deng, Y. B. Dong, B. B. Feng, Q. D. Gao, X. Y.more » Han, W. Y. Hong, M. Huang, X. Q. Ji, Z. H. Kang, D. F. Kong, T. Lan, G. S. Li, H. J. Li, Qing Li, W. Li, Y. G. Li, A. D. Liu, Z. T. Liu, C. W. Luo, X. H. Mao, Y. D. Pan, J. F. Peng, Z. B. Shi, S. D. Song, X. Y. Song, H. J. Sun, A. K. Wang, M. X. Wang, Y. Q. Wang, W. W. Xiao, Y. F. Xie, L. H. Yao, D. L. Yu, B. S. Yuan, K. J. Zhao, G. W. Zhong, J. Zhou, J. C. Yan, C. X. Yu, C. H. Pan, Y. Liu, and the HL-2A Team , Nucl. Fusion 51, 094016 (2011)] to measure the losses of neutral beam ions. The design of the probe is based on the concept of the α-particle detectors on Tokamak Fusion Test Reactor (TFTR) using scintillator plates. The probe is capable of traveling across an equatorial plane port and sweeping the aperture angle rotationally with respect to the axis of the probe shaft by two step motors, in order to optimize the radial position and the collimator angle. The energy and the pitch angle of the lost fast ions can be simultaneously measured if the two-dimensional image of scintillation light intensity due to the impact of the lost fast ions is detected. Measurements of the fast-ion losses using the probe have been performed during HL-2A neutral beam injection discharges. The clear experimental evidence of enhanced losses of beam ions during disruptions has been obtained by means of the SLIP system. A detailed description of the probe system and the first experimental results are reported.« less

FSensitive detection of mono- and polyclonal ESR1 mutations in primary tumors, metastatic lesions and cell free DNA of breast cancer patients

PubMed Central

Wang, Peilu; Bahreini, Amir; Gyanchandani, Rekha; Lucas, Peter C.; Hartmaier, Ryan J.; Watters, Rebecca J.; Jonnalagadda, Amruth R.; Trejo Bittar, Humberto E.; Berg, Aaron; Hamilton, Ronald L.; Kurland, Brenda F.; Weiss, Kurt R.; Mathew, Aju; Leone, Jose Pablo; Davidson, Nancy E; Nikiforova, Marina N.; Brufsky, Adam M.; Ambros, Tadeu F.; Stern, Andrew M.; Puhalla, Shannon L.; Lee, Adrian V.; Oesterreich, Steffi

2015-01-01

Purpose Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell free DNA (cfDNA). Patients and Methods Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n=43), bone (n=12) and brain metastases (n=38), and cfDNA (n=29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. Results ESR1 mutations were detected for D538G (n=13), Y537S (n=3) and Y537C (n=1), and not for K303R, S463P or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3 to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n=5), mutations were detected in both (n=3) or in cfDNA only (n=2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. Conclusions ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1 mutant clones are enriched by endocrine therapy. Further studies should address if sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. PMID:26500237

Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients.

PubMed

Wang, Peilu; Bahreini, Amir; Gyanchandani, Rekha; Lucas, Peter C; Hartmaier, Ryan J; Watters, Rebecca J; Jonnalagadda, Amruth R; Trejo Bittar, Humberto E; Berg, Aaron; Hamilton, Ronald L; Kurland, Brenda F; Weiss, Kurt R; Mathew, Aju; Leone, Jose Pablo; Davidson, Nancy E; Nikiforova, Marina N; Brufsky, Adam M; Ambros, Tadeu F; Stern, Andrew M; Puhalla, Shannon L; Lee, Adrian V; Oesterreich, Steffi

2016-03-01

Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell-free DNA (cfDNA). Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n = 43), bone (n = 12) and brain metastases (n = 38), and cfDNA (n = 29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. ESR1 mutations were detected for D538G (n = 13), Y537S (n = 3), and Y537C (n = 1), and not for K303R, S463P, or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3% to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n = 5), mutations were detected in both (n = 3) or in cfDNA only (n = 2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1-mutant clones are enriched by endocrine therapy. Further studies should address whether sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. See related commentary by Gu and Fuqua, p. 1034. ©2015 American Association for Cancer Research.

Detection of ESR1 mutations in circulating cell-free DNA from patients with metastatic breast cancer treated with palbociclib and letrozole.

PubMed

Gyanchandani, Rekha; Kota, Karthik J; Jonnalagadda, Amruth R; Minteer, Tanya; Knapick, Beth A; Oesterreich, Steffi; Brufsky, Adam M; Lee, Adrian V; Puhalla, Shannon L

2017-09-15

ESR1 mutations are frequently acquired in hormone-resistant metastatic breast cancer (MBC). CDK4/6 inhibition along with endocrine therapy is a promising strategy in hormone receptor-positive MBC. However, the incidence and impact of ESR1 mutations on clinical outcome in patients treated with CDK4/6 inhibitors have not been defined. In this study, we evaluated the frequency of ESR1 mutations in cfDNA from 16 patients with MBC undergoing palbociclib and letrozole therapy. Four common ESR1 mutations (D538G, Y537C, Y537N, and Y537S) were analyzed in serial blood draws using ddPCR. Mutation rate was 31.3% (5/16) (n=3; de novo , n=2; acquired). D538G was the most frequent mutation (n=3), followed by Y537N and Y537S (n=2). One patient showed multiple ESR1 mutations. Mutations were enriched during therapy. Progression-free survival (PFS) and overall survival (OS) were similar in patients with and without mutation detected at any given time during treatment. However, PFS was significantly shorter in patients with ESR1 mutation at initial blood draw (3.3 versus 9.0 months, P-value=0.038). In conclusion, ESR1 mutation prevalence is consistent with recent studies in hormone-refractory breast cancer. Further, treatment with palbociclib and letrozole does not prevent selection of ESR1 mutations in later lines of therapy. Larger studies are warranted to validate these findings.

Detection of ESR1 mutations in circulating cell-free DNA from patients with metastatic breast cancer treated with palbociclib and letrozole

PubMed Central

Gyanchandani, Rekha; Kota, Karthik J.; Jonnalagadda, Amruth R.; Minteer, Tanya; Knapick, Beth A.; Oesterreich, Steffi; Brufsky, Adam M.; Lee, Adrian V.; Puhalla, Shannon L.

2017-01-01

ESR1 mutations are frequently acquired in hormone-resistant metastatic breast cancer (MBC). CDK4/6 inhibition along with endocrine therapy is a promising strategy in hormone receptor-positive MBC. However, the incidence and impact of ESR1 mutations on clinical outcome in patients treated with CDK4/6 inhibitors have not been defined. In this study, we evaluated the frequency of ESR1 mutations in cfDNA from 16 patients with MBC undergoing palbociclib and letrozole therapy. Four common ESR1 mutations (D538G, Y537C, Y537N, and Y537S) were analyzed in serial blood draws using ddPCR. Mutation rate was 31.3% (5/16) (n=3; de novo, n=2; acquired). D538G was the most frequent mutation (n=3), followed by Y537N and Y537S (n=2). One patient showed multiple ESR1 mutations. Mutations were enriched during therapy. Progression-free survival (PFS) and overall survival (OS) were similar in patients with and without mutation detected at any given time during treatment. However, PFS was significantly shorter in patients with ESR1 mutation at initial blood draw (3.3 versus 9.0 months, P-value=0.038). In conclusion, ESR1 mutation prevalence is consistent with recent studies in hormone-refractory breast cancer. Further, treatment with palbociclib and letrozole does not prevent selection of ESR1 mutations in later lines of therapy. Larger studies are warranted to validate these findings. PMID:28978004

Increase in type II collagen turnover after iron depletion in patients with hereditary haemochromatosis.

PubMed

Richette, Pascal; Eymard, Claire; Deberg, Michelle; Vidaud, Dominique; de Kerguenec, Caroline; Valla, Dominique; Vicaut, Eric; Bardin, Thomas; Henrotin, Yves

2010-04-01

To determine the effects of iron depletion on serum levels of joint biomarkers and on joint symptoms in patients with hereditary haemochromatosis (HH). Levels of biomarkers were measured in 18 patients with HH at the time of diagnosis and after iron depletion. The markers were type II collagen degradation (Coll2-1) and its nitrated form (Coll2-1NO(2)), type II procollagen synthesis (CPII), MPO, COMP and HA. For each patient, demographic data were collected and the global joint pain (visual analogue scale) was assessed before and after iron depletion by phlebotomy. A total of 18 patients [10 males; mean (s.d.) age 48 (11) years] were homozygous for the C282Y mutation. No patient had liver dysfunction. Ferritin level before iron removal was 627.5 (range 133-3276) microg/l, and duration of the iron depletion phase was 295 (70-670) days. Serum levels of both Coll2-1 and CPII were significantly increased from diagnosis after iron depletion: 80.1 (55.6-113.5) vs 96.0 (48.8-136.3) nM (P = 0.004) and 731.4 (374.2-1012.3) vs 812.8 (535.8-1165.6) ng/ml (P = 0.03), respectively. Levels of other biomarkers were not modified by iron depletion. Ferritin level, which at baseline was correlated with body iron store (r = 0.63; P = 0.008), was significantly correlated with HA level measured before iron depletion (r = 0.60; P = 0.01). Global joint pain was not correlated with ferritin concentration and did not significantly decrease after iron depletion: 43 (19-73) vs 36 (16-67) mm (P = 0.07). In patients with HH, cartilage homoeostasis is modified by iron excess and an increase in type II collagen turnover occurs after excess iron removal.

Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity.

PubMed

Miller, K W; Schamber, R; Osmanagaoglu, O; Ray, B

1998-06-01

A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to approximately 60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed approximately 2. 8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.

HIV-1 genetic diversity and primary drug resistance mutations before large-scale access to antiretroviral therapy, Republic of Congo.

PubMed

Niama, Fabien Roch; Vidal, Nicole; Diop-Ndiaye, Halimatou; Nguimbi, Etienne; Ahombo, Gabriel; Diakabana, Philippe; Bayonne Kombo, Édith Sophie; Mayengue, Pembe Issamou; Kobawila, Simon-Charles; Parra, Henri Joseph; Toure-Kane, Coumba

2017-07-05

In this work, we investigated the genetic diversity of HIV-1 and the presence of mutations conferring antiretroviral drug resistance in 50 drug-naïve infected persons in the Republic of Congo (RoC). Samples were obtained before large-scale access to HAART in 2002 and 2004. To assess the HIV-1 genetic recombination, the sequencing of the pol gene encoding a protease and partial reverse transcriptase was performed and analyzed with updated references, including newly characterized CRFs. The assessment of drug resistance was conducted according to the WHO protocol. Among the 50 samples analyzed for the pol gene, 50% were classified as intersubtype recombinants, charring complex structures inside the pol fragment. Five samples could not be classified (noted U). The most prevalent subtypes were G with 10 isolates and D with 11 isolates. One isolate of A, J, H, CRF05, CRF18 and CRF37 were also found. Two samples (4%) harboring the mutations M230L and Y181C associated with the TAMs M41L and T215Y, respectively, were found. This first study in the RoC, based on WHO classification, shows that the threshold of transmitted drug resistance before large-scale access to antiretroviral therapy is 4%.

Expression analysis revealing destabilizing mutations in phosphomannomutase 2 deficiency (PMM2-CDG): expression analysis of PMM2-CDG mutations.

PubMed

Vega, Ana Isabel; Pérez-Cerdá, Celia; Abia, David; Gámez, Alejandra; Briones, Paz; Artuch, Rafael; Desviat, Lourdes R; Ugarte, Magdalena; Pérez, Belén

2011-08-01

Deficiency of phosphomannomutase (PMM2, MIM#601785) is the most common congenital disorder of glycosylation. Herein we report the genetic analysis of 22 Spanish PMM2 deficient patients and the functional analysis of 14 nucleotide changes in a prokaryotic expression system in order to elucidate their molecular pathogenesis. PMM2 activity assay revealed the presence of six protein changes with no enzymatic activities (p.R123Q, p.R141H, p.F157S, p.P184T, p.F207S and p.D209G) and seven mild protein changes with residual activities ranging from 16 to 54% (p.L32R, p.V44A p.D65Y, p.P113L p.T118S, p.T237M and p.C241S) and also one variant change with normal activity (p.E197A). The results obtained from Western blot analysis, degradation time courses of 11 protein changes and structural analysis of the PMM2 protein, suggest that the loss-of-function of most mutant proteins is based on their increased susceptibility to degradation or aggregation compared to the wild type protein, considering PMM2 deficiency as a conformational disease. We have identified exclusively catalytic protein change (p.D209G), catalytic protein changes affecting protein stability (p.R123Q and p.R141H), two protein changes disrupting the dimer interface (p.P113L and p.T118S) and several misfolding changes (p.L32R, p.V44A, p.D65Y, p.F157S, p.P184T, p.F207S, p.T237M and p.C241S). Our current work opens a promising therapeutic option using pharmacological chaperones to revert the effect of the characterized misfolding mutations identified in a wide range of PMM2 deficient patients.

Pyrazinamidase, CR-MOX agar, salicin fermentation-esculin hydrolysis, and D-xylose fermentation for identifying pathogenic serotypes of Yersinia enterocolitica.

PubMed Central

Farmer, J J; Carter, G P; Miller, V L; Falkow, S; Wachsmuth, I K

1992-01-01

We evaluated several simple laboratory tests that have been used to identify pathogenic serotypes of Yersinia enterocolitica or to indicate the pathogenic potential of individual strains. A total of 100 strains of Y. enterocolitica were studied, including 25 isolated during five outbreak investigations, 63 from sporadic cases, and 12 from stock cultures. The pyrazinamidase test, which does not depend on the Yersinia virulence plasmid, correctly identified 60 of 63 (95% sensitivity) strains of pathogenic serotypes and 34 of 37 (92% specificity) strains of nonpathogenic serotypes. Salicin fermentation-esculin hydrolysis (25 degrees C, 48 h) correctly identified all 63 (100% sensitivity) strains of the pathogenic serotypes and 34 of 37 (92% specificity) strains of the nonpathogenic serotypes. The results of the pyrazinamidase and salicin-esculin tests disagreed for only 7 of the 100 strains of Y. enterocolitica, and these would require additional testing. Congo red-magnesium oxalate (CR-MOX) agar determines Congo red dye uptake and calcium-dependent growth at 36 degrees C, and small red colonies are present only if the strain contains the Yersinia virulence plasmid. This test has proven to be extremely useful for freshly isolated cultures, but only 15 of 62 strains of pathogenic serotypes that had been stored for 1 to 10 years were CR-MOX positive. None of the 16 strains of Y. enterocolitica serotype O3 fermented D-xylose, so this test easily differentiated strains of this serotype, which now appears to be the most common in the United States. Although antisera that can actually be used to serotype strains of Y. enterocolitica are not readily available, the four simple tests described above can be used to screen for pathogenic serotypes. Images PMID:1400958

Man o' War Mutation in UDP-α-D-Xylose Synthase Favors the Abortive Catalytic Cycle and Uncovers a Latent Potential for Hexamer Formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walsh, Jr., Richard M.; Polizzi, Samuel J.; Kadirvelraj, Renuka

The man o’ war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-D-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ~16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies showmore » that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-D-4-keto-xylose, is not reduced to the final product, UDP-α-D-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.« less

IGF Regulation of Cell Adhesion in Breast Cancer

DTIC Science & Technology

1999-07-01

Abraham, T. Zou, Y. Shi, J. Lei, J. Cottrell, K. Cymes, K. Biden, L. Simms, B. Leggett, P. Lynch, M . Frazier, S. Powell, N. Harpaz, H. Sugimura, J...instability and mutation of DNA mismatch repair genes in gliomas. Am JPathol 153: 1181-1188. 6. C. Oliveira, R. Seruca, M . Seixas, and M . Sobrinho...286-297. 11 11. A. Oates, L. Schumaker, S. Jenkins, A.D. Pearce, SA., B. Arun, and M . Ellis. (1998). The mannose 6-phosphate/insulin-like growth

A Novel Dominant Hyperekplexia Mutation Y705C Alters Trafficking and Biochemical Properties of the Presynaptic Glycine Transporter GlyT2*

PubMed Central

Giménez, Cecilio; Pérez-Siles, Gonzalo; Martínez-Villarreal, Jaime; Arribas-González, Esther; Jiménez, Esperanza; Núñez, Enrique; de Juan-Sanz, Jaime; Fernández-Sánchez, Enrique; García-Tardón, Noemí; Ibáñez, Ignacio; Romanelli, Valeria; Nevado, Julián; James, Victoria M.; Topf, Maya; Chung, Seo-Kyung; Thomas, Rhys H.; Desviat, Lourdes R.; Aragón, Carmen; Zafra, Francisco; Rees, Mark I.; Lapunzina, Pablo; Harvey, Robert J.; López-Corcuera, Beatriz

2012-01-01

Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na+/Cl−-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [3H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311–Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H+ and Zn2+ dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo. PMID:22753417

The phenotype of polycythemia due to Croatian homozygous VHL (571C>G:H191D) mutation is different from that of Chuvash polycythemia (VHL 598C>T:R200W)

PubMed Central

Tomasic, Nikica Ljubas; Piterkova, Lucie; Huff, Chad; Bilic, Ernest; Yoon, Donghoon; Miasnikova, Galina Y.; Sergueeva, Adelina I.; Niu, Xiaomei; Nekhai, Sergei; Gordeuk, Victor; Prchal, Josef T.

2013-01-01

Mutations of VHL (a negative regulator of hypoxia-inducible factors) have position-dependent distinct cancer phenotypes. Only two known inherited homozygous VHL mutations exist and they cause polycythemia: Chuvash R200W and Croatian H191D. We report a second polycythemic Croatian H191D homozygote distantly related to the first propositus. Three generations of both families were genotyped for analysis of shared ancestry. Biochemical and molecular tests were performed to better define their phenotypes, with an emphasis on a comparison with Chuvash polycythemia. The VHL H191D mutation did not segregate in the family defined by the known common ancestors of the two subjects, suggesting a high prevalence in Croatians, but haplotype analysis indicated an undocumented common ancestor ∼six generations ago as the founder of this mutation. We show that erythropoietin levels in homozygous VHL H191D individuals are higher than in VHL R200W patients of similar ages, and their native erythroid progenitors, unlike Chuvash R200W, are not hypersensitive to erythropoietin. This observation contrasts with a report suggesting that polycythemia in VHL R200W and H191D homozygotes is due to the loss of JAK2 regulation from VHL R200W and H191D binding to SOCS1. In conclusion, our studies further define the hematologic phenotype of VHL H191D and provide additional evidence for phenotypic heterogeneity associated with the positional effects of VHL mutations. PMID:23403324

The phenotype of polycythemia due to Croatian homozygous VHL (571C>G:H191D) mutation is different from that of Chuvash polycythemia (VHL 598C>T:R200W).

PubMed

Tomasic, Nikica Ljubas; Piterkova, Lucie; Huff, Chad; Bilic, Ernest; Yoon, Donghoon; Miasnikova, Galina Y; Sergueeva, Adelina I; Niu, Xiaomei; Nekhai, Sergei; Gordeuk, Victor; Prchal, Josef T

2013-04-01

Mutations of VHL (a negative regulator of hypoxia-inducible factors) have position-dependent distinct cancer phenotypes. Only two known inherited homozygous VHL mutations exist and they cause polycythemia: Chuvash R200W and Croatian H191D. We report a second polycythemic Croatian H191D homozygote distantly related to the first propositus. Three generations of both families were genotyped for analysis of shared ancestry. Biochemical and molecular tests were performed to better define their phenotypes, with an emphasis on a comparison with Chuvash polycythemia. The VHL H191D mutation did not segregate in the family defined by the known common ancestors of the two subjects, suggesting a high prevalence in Croatians, but haplotype analysis indicated an undocumented common ancestor ∼six generations ago as the founder of this mutation. We show that erythropoietin levels in homozygous VHL H191D individuals are higher than in VHL R200W patients of similar ages, and their native erythroid progenitors, unlike Chuvash R200W, are not hypersensitive to erythropoietin. This observation contrasts with a report suggesting that polycythemia in VHL R200W and H191D homozygotes is due to the loss of JAK2 regulation from VHL R200W and H191D binding to SOCS1. In conclusion, our studies further define the hematologic phenotype of VHL H191D and provide additional evidence for phenotypic heterogeneity associated with the positional effects of VHL mutations.

A mutation of RNA polymerase β' subunit (RpoC) converts heterogeneously vancomycin-intermediate Staphylococcus aureus (hVISA) into "slow VISA".

PubMed

Matsuo, Miki; Hishinuma, Tomomi; Katayama, Yuki; Hiramatsu, Keiichi

2015-07-01

Various mutations in the rpoB gene, which encodes the RNA polymerase β subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneously VISA (hVISA) strains. We reported that rpoB mutations are also linked to the expression of the recently found "slow VISA" (sVISA) phenotype (M. Saito, Y. Katayama, T. Hishinuma, A. Iwamoto, Y. Aiba, K Kuwahara-Arai, L. Cui, M. Matsuo, N. Aritaka, and K. Hiramatsu, Antimicrob Agents Chemother 58:5024-5035, 2014, http://dx.doi.org/10.1128/AAC.02470-13). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of the rpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (DT) (72 min) and high vancomycin MIC (16 mg/liter). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of low growth rate, cell wall thickness, and reduced autolysis. Whole-genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation, F562L, in rpoC. Introduction of the wild-type (WT) rpoC gene using a multicopy plasmid resolved the sVISA phenotype of V6-5, indicating that the rpoC(P440L) mutant expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated an additional 10 revertants to hVISA and VISA. In subsequent whole-genome analysis, we identified compensatory mutations in the genes of three distinct functional categories: the rpoC gene itself as regulatory mutations, peptidoglycan biosynthesis genes, and relQ, which is involved in the stringent response. It appears that the rpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis and through the control of the stringent response. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Analyzing the Telecommunications Equipment Sector Using a Qualitative Framework

DTIC Science & Technology

2014-10-01

N C E & T E C H N O L O G Y P O L I C Y I N S T I T U T E...I E N C E & T E C H N O L O G Y P O L I C Y I N S T I T U T E IDA Document D-5346 Analyzing the Telecommunications Equipment Sector Using a...Opportunity In ve st m en ts , S m al l B us in es s In no va tio n R es ea rc h (S B IR ) C oo pe ra tiv e R es ea rc h an d Pr od uc tio n

Prevalence of relevant NS5A resistance-associated substitutions to elbasvir in genotype 1a hepatitis C virus patients in Spain.

PubMed

Palladino, Claudia; Esteban-Cartelle, Beatriz; Mate-Cano, Irene; Sánchez-Carrillo, Marta; Resino, Salvador; Briz, Verónica

2018-05-01

Resistance-associated substitutions (RASs) to the new HCV NS5A inhibitor elbasvir may limit its efficacy and lead to virological failure in HCV-GT1a-infected patients. There are no data outside clinical trials evaluating their prevalence and impact in grazoprevir/elbasvir in GT1a-infected patients in Spain. A multicentre cross-sectional study of 632 initial patients was conducted. In 13 of these patients, the sample could not be amplified or a consensus sequence by Sanger sequencing could not be performed. Ultimately, 617 HCV-G1a-infected individuals treated at 84 Spanish hospitals from the 17 autonomous communities plus the 2 autonomous cities of Spain were analysed. HCV population sequencing was used to identify RAS to elbasvir and the mutational pattern and drug sensitivity were confirmed by geno2pheno[HCV]. Viruses bearing RASs to elbasvir were present in 6.2% of HCV-G1a infected patients. The most common RASs were the Y93C/H/N and Q30E/H/R (2.4% and 2.3%, respectively). Only 3.4% of the identified RASs to elbasvir conferred reduced susceptibility to elbasvir by geno2pheno[HCV], which exclusively identified the positions Q30H/R (n=7) and Y93C/H/N (n=8) as single mutations and Q30H+Y93H (n=4) and Q30R+Y93H (n=2) as double mutations as the major RASs to elbasvir. A lower prevalence of RASs to elbasvir was observed in our HCV-G1a Spanish cohort than reported previously in clinical trials evaluating patients from the USA. This information may be essential to guide the implementation of grazoprevir/elbasvir in Spain and to manage G1a-infected patients. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Cell Line Controls for the Genotyping of a Spectrum of Human Single Nucleotide Polymorphisms in the Clinical Laboratory.

PubMed

Kimbacher, Christine; Paar, Christian; Freystetter, Andrea; Berg, Joerg

2018-05-01

Genotyping for clinically important single nucleotide polymorphisms (SNPs) is performed by many clinical routine laboratories. To support testing, quality controls and reference materials are needed. Those may be derived from residual patient samples, left over samples of external quality assurance schemes, plasmid DNA or DNA from cell lines. DNAs from cell lines are commutable and available in large amounts. DNA from 38 cell lines were examined for suitability as controls in 11 SNP assays that are frequently used in a clinical routine laboratory: FV (1691G>A), FII (20210G>A), PAI-1 4G/5G polymorphism, MTHFR (677C>T, 1298A>C), HFE (H63D, S65C, C282Y), APOE (E2, E3, E4), LPH (-13910C>T), UGT1A1 (*28, *36, *37), TPMT (*2, *3A, *3B, *3C), VKORC1 (-1639G>A, 1173C>T), CYP2C9 (*2, *3, *5). Genotyping was performed by real-time PCR with melting curve analysis and confirmed by bi-directional sequencing. We find an almost complete spectrum of genotypic constellations within these 38 cell lines. About 12 cell lines appear sufficient as genotypic controls for the 11 SNP assays by covering almost all of the genotypes. However, hetero- and homozygous genotypes for FII and the alleles TPMT*2, UGT1A1*37 and CYP2C9*5 were not detected in any of the cell lines. DNA from most of the examined cell lines appear suitable as quality controls for these SNP assays in the laboratory routine, as to the implementation of those assays or to prepare samples for quality assurance schemes. Our study may serve as a pilot to further characterize these cell lines to arrive at the status of reference materials.

Serum hepcidin levels, iron status, and HFE gene alterations during the first year of life in healthy Spanish infants.

PubMed

Aranda, Nuria; Bedmar, Cristina; Arija, Victoria; Jardí, Cristina; Jimenez-Feijoo, Rosa; Ferré, Natalia; Tous, Monica

2018-06-01

The aims of this study were to describe hepcidin levels and to assess their associations with iron status and the main variants in the HFE gene in healthy and full-term newborns during the first year of life, as a longitudinal study conducted on 140 infants. Anthropometric and biochemical parameters, hepcidin, hemoglobin (Hb), serum ferritin (SF), transferrin saturation (TS), mean corpuscular volume (MCV), and C-reactive protein (CRP), were assessed in 6- and 12-month-olds. Infants were genotyped for the three main HFE variants: C282Y, H63D, and S65C. Hepcidin levels increased from 6 to 12 months of age (43.7 ± 1.5 to 52.0 ± 1.5 ng/mL; p < 0.001), showing higher levels in infants with better iron status compared to those with iron deficiency (ID) (44.8 ± 1.5 vs 37.9 ± 1.3 ng/mL, p < 0.018, and 54.3 ± 1.5 vs 44.0 ± 1.4 ng/mL, p < 0.038, in 6- and 12-month-olds, respectively). In multivariate linear regression models, iron status was found to be associated with hepcidin levels in infants with wild-type HFE gene (p = 0.046 and p = 0.048 in 6- and 12-month-olds, respectively). However, this association was not found in HFE-alteration-carrying infants. Hepcidin levels increased in healthy infants during the first year of life and were positively associated with iron levels only in infants with wild-type HFE gene, a situation that requires further investigation.

Mutation covariation of HIV-1 CRF07_BC reverse transcriptase during antiretroviral therapy.

PubMed

Li, Zhenpeng; Huang, Yang; Ouyang, Yabo; Xing, Hui; Liao, Lingjie; Jiang, Shibo; Shao, Yiming; Ma, Liying

2013-11-01

To understand the effect of HIV-1 drug resistance mutations in the context of antiretroviral therapy (ART), we compared the prevalence of protease (PR) and reverse transcriptase (RT) mutations in HIV-1 CRF07_BC sequences from blood samples of treatment-naive and ART-treated patients. Mutation covariation in the RT and PR of HIV-1 CRF07_BC viruses from 542 treatment-naive patients and 261 patients treated with lamivudine/zidovudine/nevirapine or lamivudine/zidovudine/efavirenz was analysed. Stratified networks were used to display the mutation covariation. Based on the comparison between treatment-naive and ART-treated patients, three types of featured mutations for RT and PR were initially identified: treatment-associated mutations, treatment-agonistic mutations and overlapping polymorphisms. Twelve significant covariation pairs were found between five treatment-associated mutations (K103N, M184V, Q197K, G190A and Y181C) and nine overlapping polymorphisms (A36E, D39N, Y121H, D123E, R135I, T200A, R277K, L283I and D291E). Meanwhile, three covariation pairs between three treatment-associated mutations (I132L and M184V for RT and I15V for PR) and three overlapping polymorphisms (L10I, L36M and A71V) for PR were also detected. Finally, the overlapping polymorphisms for RT and PR were both found to have significant correlations with treatment-associated mutations, indicating a possible association between polymorphisms and drug resistance. When compared with HIV-1 subtype B under the same regimens as CRF07_BC, the mutation covariations of CRF07_BC showed a distinct pattern of RT and PR mutation covariation. The role of polymorphisms in the development of drug resistance has been widely reported. Here, we found a significant correlation between overlapping polymorphisms for RT and PR and treatment-associated mutations, indicating that polymorphisms exert a global influence on treatment-associated mutations. Polymorphism mutations might therefore be considered before initiating ART to improve the efficacy of drug combinations.

The mutation profile of JAK2 and CALR in Chinese Han patients with Philadelphia chromosome-negative myeloproliferative neoplasms

PubMed Central

2014-01-01

Mutations in JAK2, MPL and CALR are highly relevant to the Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs). We performed high resolution melting analysis and Sanger sequencing together with T-A cloning to elucidate the unique mutation profile of these genes, in Chinese patients with MPNs. Peripheral blood DNA samples were obtained from 80 patients with polycythemia vera (PV), 80 patients with essential thrombocytosis (ET) and 50 patients with primary myelofibrosis (PMF). Ten PV patients were identified with diverse JAK2 exon 12 mutations. Five novel JAK2 Exon 12 mutation patterns (M532V/E543G, N533D, M535I/H538Y/K549I, E543G and D544N) were described. JAK2 V617F was detected in 140 samples (66 PV, 45 ET and 29 PMF). JAK2 Exon 12 mutations were prevalent (13%) and variable in the Chinese patients. Compared with PV patients with JAK2 V617F mutations, PV patients with JAK2 exon 12 mutations had an earlier median onset of disease (P = 0.0013). MPL W515L/K mutations were discerned in 4 ET and 3 PMF patients. Two kinds of CALR mutation, c. 1179_1230del and c. 1234_1235insTTGTC were detected in 20 ET and 16 PMF patients. A novel CALR mutation pattern (c. 1173_1223del/c. 1179_1230del) was identified in 2 PMF samples. In addition, 17 scattered point mutations in CALR c.1153 to c.1255 were also detected in 13 cases with CALR frame-shifting variations and 2 cases without CALR frame-shifting variations. Female patients showed a predisposition to CALR mutations (P = 0.0035). Chinese Ph-negative MPN patients have a unique mutation landscape in the common molecular markers of MPN diagnosis. Validation of the molecular diagnostic pipeline should be emphasized since there is a considerable ethnical diversity in the molecular profiles of Ph-negative MPNs. PMID:25023898

Next-generation sequencing identifies three novel missense variants in ILDR1 and MYO6 genes in an Iranian family with hearing loss with review of the literature.

PubMed

Talebi, Farah; Mardasi, Farideh Ghanbari; Asl, Javad Mohammadi; Sayahi, Masoomeh

2017-12-01

Hearing impairment is the most common sensorineural disorder and is genetically heterogeneous. Identification of the pathogenic mutations underlying hearing impairment is difficult, since causative mutations in 127 different genes have so far been reported. In this study, we performed Next-generation sequencing (NGS) in 2 individuals from a consanguineous family with hearing loss. Three novel mutations in known deafness genes were identified in the family; MYO6-p.R928C and -p.D1223N in heterozygous state and ILDR1-p.Y143C in homozygous state. Sanger sequencing confirmed co-segregation of the three mutations with deafness in the family. The identified mutation in ILDR1 gene is located in the immunoglobulin-type domain of the ILDR1 protein and the detected mutations in MY06 are located in the tail domain of the MYO6 protein. The mutations are predicted to be pathogenic by SIFT, PolyPhen and Mutation Taster. Our results suggest that either the homozygous ILDR1-p.Y143C mutation might be the pathogenic variant for ARNSHL or heterozygous MYO6- p.R928C, -p.D1223N might be involved in these patient's disorder due to compound heterozygousity. To our knowledge, this is the first ILDR1 and MYO6 mutations recognized in the southwest Iran. Our data expands the spectrum of mutations in ILDR1 and MYO6 genes. Copyright © 2017 Elsevier B.V. All rights reserved.

Severity of iron overload of proband determines serum ferritin levels in families with HFE-related hemochromatosis: the HEmochromatosis FAmily Study.

PubMed

Jacobs, Esther M G; Hendriks, Jan C M; van Deursen, Cees Th B M; Kreeftenberg, Herman G; de Vries, Richard A; Marx, Joannes J M; Stalenhoef, Anton F H; Verbeek, André L M; Swinkels, Dorine W

2009-01-01

In families of patients with clinically detected hereditary hemochromatosis (HH) early screening has been suggested to prevent morbidity and mortality. Here, we aim to identify determinants for iron overload in first-degree family members of C282Y homozygous probands with clinically detected HH. Data on HFE-genotype, iron parameters, demographics, lifestyle factors and health, were collected from 224 Dutch C282Y homozygous patients with clinically diagnosed HH and 735 of their first-degree family members (FDFM), all participating in the HEmochromatosis FAmily Study (HEFAS). The best predictive multivariable model forecasted 45% of variation of the serum ferritin levels. In this model severity of iron overload in the proband significantly predicted serum ferritin levels in FDFM. Other significant determinants in this model consisted of C282Y homozygosity, compound heterozygosity, age at testing for serum ferritin and supplemental iron intake, whereas a low body mass index showed a protective effect. This study provides a model to assess the risk of development of iron overload for relatives of probands with HH. These results might be instrumental in the development of an optimal strategy for future family screening programs.

A 10-year follow-up of a child with mild case of xeroderma pigmentosum complementation group D diagnosed by whole-genome sequencing.

PubMed

Ono, Ryusuke; Masaki, Taro; Mayca Pozo, Franklin; Nakazawa, Yuka; Swagemakers, Sigrid M A; Nakano, Eiji; Sakai, Wataru; Takeuchi, Seiji; Kanda, Fumio; Ogi, Tomoo; van der Spek, Peter J; Sugasawa, Kaoru; Nishigori, Chikako

2016-07-01

Most patients with xeroderma pigmentosum complementation group D (XP-D) from Western countries suffer from neurological symptoms, whereas Japanese patients display only skin manifestations without neurological symptoms. We have previously suggested that these differences in clinical manifestations in XP-D patients are attributed partly to a predominant mutation in ERCC2, and the allele frequency of S541R is highest in Japan. We diagnosed a child with mild case of XP-D by the evaluation of DNA repair activity and whole-genome sequencing, and followed her ten years. Skin cancer, mental retardation, and neurological symptoms were not observed. Her minimal erythema dose was 41 mJ/cm(2) , which was slightly lower than that of healthy Japanese volunteers. The patient's cells showed sixfold hypersensitivity to UV in comparison with normal cells. Post-UV unscheduled DNA synthesis was 20.4%, and post-UV recovery of RNA synthesis was 58% of non-irradiated samples, which was lower than that of normal fibroblasts. Genome sequence analysis indicated that the patient harbored a compound heterozygous mutation of c.1621A>C and c.591_594del, resulting in p.S541R and p.Y197* in ERCC2: then, patient was diagnosed with XP-D. Y197* has not been described before. Her mild skin manifestations might be attributed to the mutational site on her genome and daily strict sun protection. c.1621A>C might be a founder mutation of ERCC2 among Japanese XP-D patients, as it was identified most frequently in Japanese XP-D patients and it has not been found elsewhere outside Japan. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

High Resolution Melting Analysis for Rapid Mutation Screening in Gyrase and Topoisomerase IV Genes in Quinolone-Resistant Salmonella enterica

PubMed Central

Thong, Kwai Lin

2014-01-01

The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes. PMID:25371903

High resolution melting analysis for rapid mutation screening in gyrase and Topoisomerase IV genes in quinolone-resistant Salmonella enterica.

PubMed

Ngoi, Soo Tein; Thong, Kwai Lin

2014-01-01

The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes.

Characterization of Nucleoside Reverse Transcriptase Inhibitor-Associated Mutations in the RNase H Region of HIV-1 Subtype C Infected Individuals.

PubMed

Ngcapu, Sinaye; Theys, Kristof; Libin, Pieter; Marconi, Vincent C; Sunpath, Henry; Ndung'u, Thumbi; Gordon, Michelle L

2017-11-08

The South African national treatment programme includes nucleoside reverse transcriptase inhibitors (NRTIs) in both first and second line highly active antiretroviral therapy regimens. Mutations in the RNase H domain have been associated with resistance to NRTIs but primarily in HIV-1 subtype B studies. Here, we investigated the prevalence and association of RNase H mutations with NRTI resistance in sequences from HIV-1 subtype C infected individuals. RNase H sequences from 112 NRTI treated but virologically failing individuals and 28 antiretroviral therapy (ART)-naive individuals were generated and analysed. In addition, sequences from 359 subtype C ART-naive sequences were downloaded from Los Alamos database to give a total of 387 sequences from ART-naive individuals for the analysis. Fisher's exact test was used to identify mutations and Bayesian network learning was applied to identify novel NRTI resistance mutation pathways in RNase H domain. The mutations A435L, S468A, T470S, L484I, A508S, Q509L, L517I, Q524E and E529D were more prevalent in sequences from treatment-experienced compared to antiretroviral treatment naive individuals, however, only the E529D mutation remained significant after correction for multiple comparison. Our findings suggest a potential interaction between E529D and NRTI-treatment; however, site-directed mutagenesis is needed to understand the impact of this RNase H mutation.

The Role of Carburization in Gun Barrel Erosion and Cracking

DTIC Science & Technology

1981-07-01

same concentration of active constituents as tests 63 and 64. Test 71 was conducted as an "inert" comparison of tests 63 and 69, since the peak gas...NJ to ^ oo CJl CTJ CJl ( 71 ɟl 3 13 o c bO O U -H nj to C1.-H tD CD 3 P tD O Ci3 X Lp -H u 3 to O C 3 LP...replacing nitrogen with CO. With the exception of the first group of tests, runs 63 through 71 , the total concentration of carbon monoxide and nitrogen

Evaluation of Nanocomposites as Lightweight Electronic Enclosures for Satellites’ Applications

DTIC Science & Technology

2008-06-01

vol. 36, pp. 59-63, 2000. [15] H . Helvajian and E. Y. Robinson, Micro- and Nanotechnology for Space Systems. Los Angeles: The Aerospace Press, 1997...D. Alexander. Nickel NanostrandsTM Expand Nanotechnology Engineering Capabilities. Available: http://www.afrl.af.mil. [2] M. D. Alexander, H . Dowty...Nanofibers and Ni Nanostrands," in NSMMS Meeting, 2006. [3] D. F. Bahr, H . Kung, N. R. Moody and K. J. Wahl, Mechanical Properties Derived from

Genetic diversity of Plasmodium vivax metacaspase 1 and Plasmodium vivax multi-drug resistance 1 genes of field isolates from Mauritania, Sudan and Oman.

PubMed

Sow, Fatimata; Bonnot, Guillaume; Ahmed, Bilal Rabah; Diagana, Sidi Mohamed; Kebe, Hachim; Koita, Mohamedou; Samba, Ba Malado; Al-Mukhaini, Said K; Al-Zadjali, Majed; Al-Abri, Seif S; Ali, Osama A M; Samy, Abdallah M; Hamid, Muzamil Mahdi Abdel; Ali Albsheer, Musab M; Simon, Bruno; Bienvenu, Anne-Lise; Petersen, Eskild; Picot, Stéphane

2017-02-02

Plasmodium vivax is the second most important human malaria parasite, widely spread across the world. This parasite is associated with important issues in the process toward malaria elimination, including potential for relapse and increased resistance to chloroquine. Plasmodium vivax multi-drug resistant (pvmdr1) is suspected to be a marker of resistance although definitive evidence is lacking. Progress has been made in knowledge of biological factors affecting parasite growth, including mechanisms of regulated cell death and the suspected role of metacaspase. Plasmodium vivax metacaspase1 (PvMCA1-cd) has been described with a catalytic domain composed of histidine (H372) and cysteine (C428) residues. The aim of this study was to test for a link between the conserved histidine and cysteine residues in PvMCA1-cd, and the polymorphism of the P. vivax multi-drug resistant gene (pvmdr1). Thirty P. vivax isolates were collected from Mauritania, Sudan, and Oman. Among the 28 P. vivax isolates successfully sequenced, only 4 samples showed the conserved His (372)-Cys (428) residues in PvMCA1-cd. Single nucleotide polymorphisms observed were H372T (46.4%), H372D (39.3%), and C428R (85.7%). A new polymorphic catalytic domain was observed at His (282)-Cys (305) residues. Sequences alignment analysis of pvmdr1 showed SNP in the three codons 958, 976 and 1076. A single SNP was identified at the codon M958Y (60%), 2 SNPs were found at the position 976: Y976F (13%) and Y976V (57%), and 3 SNPs were identified at the position 1076: F1076L (40%), F1076T (53%) and F1076I (3%). Only one isolate was wildtype in all three codons (MYF), 27% were single MYL mutants, and 10% were double MFL mutants. Three new haplotypes were also identified: the triple mutant YVT was most prevalent (53.3%) distributed in the three countries, while triple YFL and YVI mutants (3%), were only found in samples from Sudan and Mauritania. Triple or quadruple mutants for metacaspase genes and double or triple mutants for Pvmdr1 were observed in 24/28 and 19/28 samples. There was no difference in the frequency of mutations between PvMCA1-cd and Pvmdr1 (P > 0.2). Histidine and cysteine residues in PvMCA1-cd are highly polymorphic and linkage disequilibrium with SNPs of Pvmdr1 gene may be expected from these three areas with different patterns of P. vivax transmission.

26 CFR 1.664-3 - Charitable remainder unitrust.

Code of Federal Regulations, 2010 CFR

2010-04-01

... amended by T.D. 8791, 63 FR 68192, Dec. 10, 1998; T.D. 8926, 66 FR 1038, Jan. 5, 2001; T.D. 9102, 69 FR 20.... S establishes Y for her granddaughter, G, when G is 10 years old. The governing instrument of Y... for a combination of methods that satisfies paragraph (a)(1)(i)(c) of this section. Example 10. The...

Activated c-Kit receptor in the heart promotes cardiac repair and regeneration after injury

PubMed Central

Di Siena, S; Gimmelli, R; Nori, S L; Barbagallo, F; Campolo, F; Dolci, S; Rossi, P; Venneri, M A; Giannetta, E; Gianfrilli, D; Feigenbaum, L; Lenzi, A; Naro, F; Cianflone, E; Mancuso, T; Torella, D; Isidori, A M; Pellegrini, M

2016-01-01

The role of endogenous c-Kit receptor activation on cardiac cell homeostasis and repair remains largely unexplored. Transgenic mice carrying an activating point mutation (TgD814Y) in the kinase domain of the c-Kit gene were generated. c-KitTgD814Y receptor was expressed in the heart during embryonic development and postnatal life, in a similar timing and expression pattern to that of the endogenous gene, but not in the hematopoietic compartment allowing the study of a cardiac-specific phenotype. c-KitTgD814Y mutation produced a constitutive active c-Kit receptor in cardiac tissue and cells from transgenic mice as demonstrated by the increased phosphorylation of ERK1/2 and AKT, which are the main downstream molecular effectors of c-Kit receptor signaling. In adult transgenic hearts, cardiac morphology, size and total c-Kit+ cardiac cell number was not different compared with wt mice. However, when c-KitTgD814Y mice were subjected to transmural necrotic heart damage by cryoinjury (CI), all transgenic survived, compared with half of wt mice. In the sub-acute phase after CI, transgenic and wt mice showed similar heart damage. However, 9 days after CI, transgenic mice exhibited an increased number of c-Kit+CD31+ endothelial progenitor cells surrounding the necrotic area. At later follow-up, a consistent reduction of fibrotic area, increased capillary density and increased cardiomyocyte replenishment rate (as established by BrdU incorporation) were observed in transgenic compared with wt mice. Consistently, CD45−c-Kit+ cardiac stem cells isolated from transgenic c-KitTgD814Y mice showed an enhanced endothelial and cardiomyocyte differentiation potential compared with cells isolated from the wt. Constitutive activation of c-Kit receptor in mice is associated with an increased cardiac myogenic and vasculogenic reparative potential after injury, with a significant improvement of survival. PMID:27468693

Novel C617Y mutation in the 7th transmembrane segment of luteinizing hormone/choriogonadotropin receptor in a Japanese boy with peripheral precocious puberty.

PubMed

Nagasaki, Keisuke; Katsumata, Noriyuki; Ogawa, Yohei; Kikuchi, Toru; Uchiyama, Makoto

2010-01-01

Testotoxicosis, also known as familial male-limited precocious puberty, is an autosomal dominant form of gonadotropin-independent precocious puberty caused by heterozygous constitutively activating mutations of the LHCGR gene encoding the luteinizing hormone/choriogonadotropin receptor (LH/CGR). The patient is an 8-year-old boy who started to develop pubic hair and penile enlargement at 6 years of age. The patient had elevated serum testosterone levels, but initially exhibited a prepubertal response of gonadotropins to GnRH, which was followed by central activation of the hypothalamo-pituitary-gonadal axis. The father reported having experienced precocious puberty, and is 158 cm tall. There is no history of short stature and precocious puberty in the family except for the father. The LHCGR gene was analyzed by direct DNA sequencing of amplified PCR products from the patient and his parents. The wild-type and mutant LH/CGRs were transiently expressed in COS-1 cells and cAMP levels in the cells were determined with or without hCG stimulation. Genetic analysis revealed a novel C617Y mutation of the LHCGR gene in the patient and his mother, while his father had no mutations. Functional expression study demonstrated around 15% increase in the basal intracellular cAMP level in cells expressing the mutant LH/CGR compared with that in cells expressing the wild-type receptor. We have reported the first missense C617Y mutation located in the 7th transmembrane segment of LH/CGR causing testotoxicosis. The modest phenotype of our patient may be explained, at least in part, by the modest increase in the intracellular cAMP level caused by the C617Y mutation.

TELNET under Single-Connection TCP Specification

DTIC Science & Technology

1976-02-02

Manager User Oriented Systems International Business Machines Corp. K54-282, Monterey and Cottle Roads San Jose, CA 95193 Dr. Leonard Y. Liu...Manager Computer Science International Business Machines Corp. K51-282, Monterey and Cottle Roads San Jose, CA 95193 Mr. Harry Reinstein... International Business Machines Corp. 1501 California Avenue Palo Alto, Ca 94303 Illinois, University of Mr. John D. Day University of Illinois Center for

Amino acid residue Y196E substitution and C-terminal peptide synergistically alleviate the toxicity of Clostridium perfringens epsilon toxin.

PubMed

Yao, Wenwu; Kang, Lin; Gao, Shan; Zhuang, Xiangjin; Zhang, Tao; Yang, Hao; Ji, Bin; Xin, Wenwen; Wang, Jinglin

2015-06-15

Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains, and is the causative agent of a lethal enterotoxemia in livestock animals and possibly in humans. However, many details of ETX structure and activity are not known. Therefore, it is important to clarify the relationship between ETX structure and activity. To explore the effect and mechanism of ETX amino acid residue Y196E substitution and C-terminal peptide on toxicity, four recombinant proteins, rETX (without 13 N-terminal peptides and 23 C-terminal peptides), rETX-C (rETX with 23 C-terminal peptides), rETX(Y196E) (rETX with an amino acid residue substitution at Y196) and rETX(Y196E)-C (rETX-C with a Y196E mutation), were constructed in this study. Both the amino acid residue Y196E substitution and the C-terminal peptide reduce ETX toxicity to a similar extent, and the two factors synergistically alleviate ETX toxicity. In addition, we demonstrated that the C-terminal peptides and Y196E amino acid mutation reduce the toxin toxicity in two different pathways: the C-terminal peptides inhibit the binding activity of toxins to target cells, and the Y196E amino acid mutation slightly inhibits the pore-forming or heptamer-forming process. Interaction between the two factors was not observed in pore-forming or binding assays but toxicity assays, which demonstrated that the relationship between domains of the toxin is more complicated than previously appreciated. However, the exact mechanism of synergistic action is not yet clarified. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

PubMed

Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

2006-07-01

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

Hypertrophic Cardiomyopathy Cardiac Troponin C Mutations Differentially Affect Slow Skeletal and Cardiac Muscle Regulation

PubMed Central

Veltri, Tiago; Landim-Vieira, Maicon; Parvatiyar, Michelle S.; Gonzalez-Martinez, David; Dieseldorff Jones, Karissa M.; Michell, Clara A.; Dweck, David; Landstrom, Andrew P.; Chase, P. Bryant; Pinto, Jose R.

2017-01-01

Mutations in TNNC1—the gene encoding cardiac troponin C (cTnC)—that have been associated with hypertrophic cardiomyopathy (HCM) and cardiac dysfunction may also affect Ca2+-regulation and function of slow skeletal muscle since the same gene is expressed in both cardiac and slow skeletal muscle. Therefore, we reconstituted rabbit soleus fibers and bovine masseter myofibrils with mutant cTnCs (A8V, C84Y, E134D, and D145E) associated with HCM to investigate their effects on contractile force and ATPase rates, respectively. Previously, we showed that these HCM cTnC mutants, except for E134D, increased the Ca2+ sensitivity of force development in cardiac preparations. In the current study, an increase in Ca2+ sensitivity of isometric force was only observed for the C84Y mutant when reconstituted in soleus fibers. Incorporation of cTnC C84Y in bovine masseter myofibrils reduced the ATPase activity at saturating [Ca2+], whereas, incorporation of cTnC D145E increased the ATPase activity at inhibiting and saturating [Ca2+]. We also tested whether reconstitution of cardiac fibers with troponin complexes containing the cTnC mutants and slow skeletal troponin I (ssTnI) could emulate the slow skeletal functional phenotype. Reconstitution of cardiac fibers with troponin complexes containing ssTnI attenuated the Ca2+ sensitization of isometric force when cTnC A8V and D145E were present; however, it was enhanced for C84Y. In summary, although the A8V and D145E mutants are present in both muscle types, their functional phenotype is more prominent in cardiac muscle than in slow skeletal muscle, which has implications for the protein-protein interactions within the troponin complex. The C84Y mutant warrants further investigation since it drastically alters the properties of both muscle types and may account for the earlier clinical onset in the proband. PMID:28473771

Retigabine, a Kv7.2/Kv7.3-Channel Opener, Attenuates Drug-Induced Seizures in Knock-In Mice Harboring Kcnq2 Mutations.

PubMed

Ihara, Yukiko; Tomonoh, Yuko; Deshimaru, Masanobu; Zhang, Bo; Uchida, Taku; Ishii, Atsushi; Hirose, Shinichi

2016-01-01

The hetero-tetrameric voltage-gated potassium channel Kv7.2/Kv7.3, which is encoded by KCNQ2 and KCNQ3, plays an important role in limiting network excitability in the neonatal brain. Kv7.2/Kv7.3 dysfunction resulting from KCNQ2 mutations predominantly causes self-limited or benign epilepsy in neonates, but also causes early onset epileptic encephalopathy. Retigabine (RTG), a Kv7.2/ Kv7.3-channel opener, seems to be a rational antiepileptic drug for epilepsies caused by KCNQ2 mutations. We therefore evaluated the effects of RTG on seizures in two strains of knock-in mice harboring different Kcnq2 mutations, in comparison to the effects of phenobarbital (PB), which is the first-line antiepileptic drug for seizures in neonates. The subjects were heterozygous knock-in mice (Kcnq2Y284C/+ and Kcnq2A306T/+) bearing the Y284C or A306T Kcnq2 mutation, respectively, and their wild-type (WT) littermates, at 63-100 days of age. Seizures induced by intraperitoneal injection of kainic acid (KA, 12mg/kg) were recorded using a video-electroencephalography (EEG) monitoring system. Effects of RTG on KA-induced seizures of both strains of knock-in mice were assessed using seizure scores from a modified Racine's scale and compared with those of PB. The number and total duration of spike bursts on EEG and behaviors monitored by video recording were also used to evaluate the effects of RTG and PB. Both Kcnq2Y284C/+ and Kcnq2A306T/+ mice showed significantly more KA-induced seizures than WT mice. RTG significantly attenuated KA-induced seizure activities in both Kcnq2Y284C/+ and Kcnq2A306T/+ mice, and more markedly than PB. This is the first reported evidence of RTG ameliorating KA-induced seizures in knock-in mice bearing mutations of Kcnq2, with more marked effects than those observed with PB. RTG or other Kv7.2-channel openers may be considered as first-line antiepileptic treatments for epilepsies resulting from KCNQ2 mutations.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bunge, S.; Steglich, C.; Kleijer, W.J.

Mucopolysaccharidosis type I (MPS I) is caused by alpha-L-iduronidase (IDUA) deficiency. Clinical severity ranges from mild (Scheie) and intermediate (Hurler/Scheie) to severe (Hurler) forms. We investigated 70 patients with various MPS I phenotypes for mutations of the IDUA gene. 28 different mutations on 93% of mutant alleles, defining 90% of the genotypes were characterized. The two common missense mutations Q70X and W402X were found on, respectively, 31% and 28% of mutant alleles. However, Q70X is much more frequent in Scandinavia (64%) than in other European countries (16%). L218P (4.3%) and A327P (6.4%) were also identified in several patients, while allmore » other mutations were found on only one or two alleles each. Of the 11 novel mutations identified in this study, G51D, L218P, D315Y, A327P, R489P, E404X, and R621X were associated with severe phenotypes. Eleven different small deletions and insertions were detected (134del12, 964delC, 1132del6, 1782del11, 1995del11, {Delta}D444/445, 252insC, 396insAC, 682insAC, 974ins12, and 1277ins9), most of them causing severe MPS I. Two novel Hurler/Scheie (M504T and W626R) and two novel Scheie point mutations (R89W and R383H) were also identified. Characterization of the primary genetic defect and establishing genotype/phenotype correlation is important for prognostic predictions, evaluation of therapeutic success, and prenatal diagnosis.« less

Carriers of the Complex Allele HFE c.[187C>G;340+4T>C] Have Increased Risk of Iron Overload in São Miguel Island Population (Azores, Portugal).

PubMed

Branco, Claudia C; Gomes, Cidália T; De Fez, Laura; Bulhões, Sara; Brilhante, Maria José; Pereirinha, Tânia; Cabral, Rita; Rego, Ana Catarina; Fraga, Cristina; Miguel, António G; Brasil, Gracinda; Macedo, Paula; Mota-Vieira, Luisa

2015-01-01

Iron overload is associated with acquired and genetic conditions, the most common being hereditary hemochromatosis (HH) type-I, caused by HFE mutations. Here, we conducted a hospital-based case-control study of 41 patients from the São Miguel Island (Azores, Portugal), six belonging to a family with HH type-I pseudodominant inheritance, and 35 unrelated individuals fulfilling the biochemical criteria of iron overload compatible with HH type-I. For this purpose, we analyzed the most common HFE mutations- c.845G>A [p.Cys282Tyr], c.187C>G [p.His63Asp], and c.193A>T [p.Ser65Cys]. Results revealed that the family's HH pseudodominant pattern is due to consanguineous marriage of HFE-c.845G>A carriers, and to marriage with a genetically unrelated spouse that is a -c.187G carrier. Regarding unrelated patients, six were homozygous for c.845A, and three were c.845A/c.187G compound heterozygous. We then performed sequencing of HFE exons 2, 4, 5 and their intron-flanking regions. No other mutations were observed, but we identified the -c.340+4C [IVS2+4C] splice variant in 26 (74.3%) patients. Functionally, the c.340+4C may generate alternative splicing by HFE exon 2 skipping and consequently, a protein missing the α1-domain essential for HFE/ transferrin receptor-1 interactions. Finally, we investigated HFE mutations configuration with iron overload by determining haplotypes and genotypic profiles. Results evidenced that carriers of HFE-c.187G allele also carry -c.340+4C, suggesting in-cis configuration. This data is corroborated by the association analysis where carriers of the complex allele HFE-c.[187C>G;340+4T>C] have an increased iron overload risk (RR = 2.08, 95% CI = 1.40-2.94, p<0.001). Therefore, homozygous for this complex allele are at risk of having iron overload because they will produce two altered proteins--the p.63Asp [c.187G], and the protein lacking 88 amino acids encoded by exon 2. In summary, we provide evidence that the complex allele HFE-c.[187C>G;340+4T>C] has a role, as genetic predisposition factor, on iron overload in the São Miguel population. Independent replication studies in other populations are needed to confirm this association.

40 CFR Appendix to Subpart Hh of... - Tables

Code of Federal Regulations, 2011 CFR

2011-07-01

... specified in § 63.760(h). § 63.1(c)(3) and (c)(4) No Section reserved. § 63.1(c)(5) Yes. § 63.1(d) No... category and there are additional definitions in subpart HH. § 63.3(a) through (c) Yes. § 63.4(a)(1) through (a)(2) Yes. § 63.4(a)(3) through (a)(5) No Section reserved. § 63.4(b) Yes. § 63.4(c) Yes. § 63.5...

Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability.

PubMed

Denisenko, Yury A; Gusakov, Alexander V; Rozhkova, Aleksandra M; Osipov, Dmitry O; Zorov, Ivan N; Matys, Veronika Yu; Uporov, Igor V; Sinitsyn, Arkady P

2017-11-01

In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50-60°C was 2-2.5-fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1°C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77.

PubMed

Li, Yi-Ping; Ramirez, Santseharay; Mikkelsen, Lotte; Bukh, Jens

2015-01-01

The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10(4.0) focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively. Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

40 CFR 721.10273 - 3′H-Cyclopropa[7,22][5,6]fullerene-C70-D5h(6)-3′-butanoic acid, 3′-phenyl-, methyl ester.

Code of Federal Regulations, 2014 CFR

2014-07-01

... CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10273 3′H-Cyclopropa[7,22][5,6]fullerene-C70-D5h(6)-3′-butanoic acid, 3′-phenyl-, methyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substances identified as 3′H-Cyclopropa[7,22][5,6...

40 CFR 721.10272 - 3′H-Cyclopropa[8,25][5,6]fullerene-C70-D5h(6)-3′-butanoic acid, 3′-phenyl-,methyl ester.

Code of Federal Regulations, 2013 CFR

2013-07-01

... CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10272 3′H-Cyclopropa[8,25][5,6]fullerene-C70-D5h(6)-3′-butanoic acid, 3′-phenyl-,methyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substances identified as 3′H-Cyclopropa[8,25][5,6...

40 CFR 721.10272 - 3′H-Cyclopropa[8,25][5,6]fullerene-C70-D5h(6)-3′-butanoic acid, 3′-phenyl-,methyl ester.

Code of Federal Regulations, 2014 CFR

2014-07-01

... CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10272 3′H-Cyclopropa[8,25][5,6]fullerene-C70-D5h(6)-3′-butanoic acid, 3′-phenyl-,methyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substances identified as 3′H-Cyclopropa[8,25][5,6...

40 CFR 721.10273 - 3′H-Cyclopropa[7,22][5,6]fullerene-C70-D5h(6)-3′-butanoic acid, 3′-phenyl-, methyl ester.

Code of Federal Regulations, 2013 CFR

2013-07-01

... CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10273 3′H-Cyclopropa[7,22][5,6]fullerene-C70-D5h(6)-3′-butanoic acid, 3′-phenyl-, methyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substances identified as 3′H-Cyclopropa[7,22][5,6...

Computational design of variants for cephalosporin C acylase from Pseudomonas strain N176 with improved stability and activity.

PubMed

Tian, Ye; Huang, Xiaoqiang; Li, Qing; Zhu, Yushan

2017-01-01

In this report, redesigning cephalosporin C acylase from the Pseudomonas strain N176 revealed that the loss of stability owing to the introduced mutations at the active site can be recovered by repacking the nearby hydrophobic core regions. Starting from a quadruple mutant M31βF/H57βS/V68βA/H70βS, whose decrease in stability is largely owing to the mutation V68βA at the active site, we employed a computational enzyme design strategy that integrated design both at hydrophobic core regions for stability enhancement and at the active site for activity improvement. Single-point mutations L154βF, Y167βF, L180βF and their combinations L154βF/L180βF and L154βF/Y167βF/L180βF were found to display improved stability and activity. The two-point mutant L154βF/L180βF increased the protein melting temperature (T m ) by 11.7 °C and the catalytic efficiency V max /K m by 57 % compared with the values of the starting quadruple mutant. The catalytic efficiency of the resulting sixfold mutant M31βF/H57βS/V68βA/H70βS/L154βF/L180βF is recovered to become comparable to that of the triple mutant M31βF/H57βS/H70βS, but with a higher T m . Further experiments showed that single-point mutations L154βF, L180βF, and their combination contribute no stability enhancement to the triple mutant M31βF/H57βS/H70βS. These results verify that the lost stability because of mutation V68βA at the active site was recovered by introducing mutations L154βF and L180βF at hydrophobic core regions. Importantly, mutation V68βA in the six-residue mutant provides more space to accommodate the bulky side chain of cephalosporin C, which could help in designing cephalosporin C acylase mutants with higher activities and the practical one-step enzymatic route to prepare 7-aminocephalosporanic acid at industrial-scale levels.

Enhanced vector borne disease surveillance of California Culex mosquito populations reveals spatial and species-specific barriers of infection.

DOE Office of Scientific and Technical Information (OSTI.GOV)

VanderNoot, Victoria A.; Curtis, Deanna Joy; Koh, Chung-Yan

Monitor i ng in f ectio n s in v ect o rs su c h as m osquit o es, s a nd fl i es, tsetse fl i es, a nd ticks to i denti f y hu m a n path o gens m a y s e r v e as a n ear l y w arn i ng det e ction system t o dir e ct loc a l g o v er n ment dise a se pr e v en t i v e m easu r e s . Onemore » major hurdle i n de t ection is the abi l i t y to scre e n l arge n u mbers of v e c t ors for h uman patho g ens w i thout t h e u s e of ge n o t y pe - s p ecific m o lecu l ar tec h nique s . N e x t genera t ion s equ e nc i ng (NG S ) pr o v i des a n unbi a sed p latfo r m capab l e of identi f y i ng k n o w n a n d unk n o w n p ath o ge n s circula t ing w i thin a v e ctor p opul a tion, but utili z ing t h is te c h nolo g y i s tim e - con s u ming a n d cos t l y for v ecto r -b o rne disease su r v e illan c e pr o gra m s. T o addr e s s this w e d e v e lop e d cos t -eff e ct i v e Ilumina(r) R NA- S eq l i bra r y p r epara t ion m e thodol o gies i n con j u n ction w i t h an automa t ed c ompu t at i onal a n a l y sis pipel i n e to ch a racter i ze t h e microbial popula t ions c ircula t i n g in Cu l e x m o squit o e s (Cul e x qui n quef a s c iatu s , C ul e x quinq u efasc i atus / pip i ens co m pl e x h y bri d s, and C u l e x ta r salis ) t hroug h out Californ i a. W e assembled 2 0 n o vel a n d w e l l -do c ume n ted a r b o v i ruses repres e nting mem b e rs of B u n y a v ir i da e , F l a v i virid a e, If a virida e , Meson i v i rida e , Nid o v iri d ae, O rtho m y x o virid a e, Pa r v o v iri d ae, Re o virid a e, R h a b d o v i rid a e, T y m o v iri d ae, a s w ell as s e v e r al u n assi g n e d v irus e s . In addit i o n, w e m app e d mRNA s pecies to d i vergent s peci e s of t r y panos o ma a nd pl a s modium eu k a r yotic parasit e s and cha r a c terized t he p r oka r yot i c microb i al c o mposit i on to i d enti f y bacteri a l tran s c r ipts der i v ed from wolba c hia, clo s tridi u m, m y c oplas m a, fusoba c terium and c am p y l o bacter bac t er i al spec i e s . W e utilized the s e mic r obial transcri p tomes pre s e nt in g e ogra p hical l y defined Cul e x po p ul a tions to defi n e spatial and m osqui t o specie s -spec i fic ba r r iers of i n fecti o n. T he v i r ome and microbi o me c o mpos i tion id e ntified in e ach mosqui t o p o ol pr o v i ded suf f icient resolut i on to dete r m i ne both the mosq u ito species and the g e o graphic regi o n in Californ i a w h e re t h e mosqui t o po o l orig i n ated. T his d a ta pr o v i des ins i ght in t o the compl e x i t y of microb i al spec i es cir c ulati n g in med i cal l y i mport a nt Culex mosqui t oes a nd t h eir potent i al im p act o n t he tran s missi o n of v ector-b o rne human / veter i na r y p a t hogens in C a liforn i a.« less

Activating HER2 mutations in HER2 gene amplification negative breast cancer.

PubMed

Bose, Ron; Kavuri, Shyam M; Searleman, Adam C; Shen, Wei; Shen, Dong; Koboldt, Daniel C; Monsey, John; Goel, Nicholas; Aronson, Adam B; Li, Shunqiang; Ma, Cynthia X; Ding, Li; Mardis, Elaine R; Ellis, Matthew J

2013-02-01

Data from 8 breast cancer genome-sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized 13 HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGF receptor (EGFR) exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings show that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment. We show that the majority of HER2 somatic mutations in breast cancer patients are activating mutations that likely drive tumorigenesis. Several patients had mutations that are resistant to the reversible HER2 inhibitor lapatinib, but are sensitive to the irreversible HER2 inhibitor, neratinib. Our results suggest that patients with HER2 mutation–positive breast cancers could benefit from existing HER2-targeted drugs.

Molecular dynamics studies on the buffalo prion protein.

PubMed

Zhang, Jiapu; Wang, Feng; Chatterjee, Subhojyoti

2016-01-01

It was reported that buffalo is a low susceptibility species resisting to transmissible spongiform encephalopathies (TSEs) (same as rabbits, horses, and dogs). TSEs, also called prion diseases, are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of species (except for rabbits, dogs, horses, and buffalo), manifesting as scrapie in sheep and goats; bovine spongiform encephalopathy (BSE or "mad-cow" disease) in cattle; chronic wasting disease in deer and elk; and Creutzfeldt-Jakob diseases, Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia, and Kulu in humans etc. In molecular structures, these neurodegenerative diseases are caused by the conversion from a soluble normal cellular prion protein (PrP(C)), predominantly with α-helices, into insoluble abnormally folded infectious prions (PrP(Sc)), rich in β-sheets. In this article, we studied the molecular structure and structural dynamics of buffalo PrP(C) (BufPrP(C)), in order to understand the reason why buffalo is resistant to prion diseases. We first did molecular modeling of a homology structure constructed by one mutation at residue 143 from the NMR structure of bovine and cattle PrP(124-227); immediately we found that for BufPrP(C)(124-227), there are five hydrogen bonds (HBs) at Asn143, but at this position, bovine/cattle do not have such HBs. Same as that of rabbits, dogs, or horses, our molecular dynamics studies also revealed there is a strong salt bridge (SB) ASP178-ARG164 (O-N) keeping the β2-α2 loop linked in buffalo. We also found there is a very strong HB SER170-TYR218 linking this loop with the C-terminal end of α-helix H3. Other information, such as (i) there is a very strong SB HIS187-ARG156 (N-O) linking α-helices H2 and H1 (if mutation H187R is made at position 187, then the hydrophobic core of PrP(C) will be exposed (L.H. Zhong (2010). Exposure of hydrophobic core in human prion protein pathogenic mutant H187R. Journal of Biomolecular Structure and Dynamics 28(3), 355-361)), (ii) at D178, there is a HB Y169-D178 and a polar contact R164-D178 for BufPrP(C) instead of a polar contact Q168-D178 for bovine PrP(C) (C.J. Cheng, & V. Daggett. (2014). Molecular dynamics simulations capture the misfolding of the bovine prion protein at acidic pH. Biomolecules 4(1), 181-201), (iii) BufPrP(C) owns three 310 helices at 125-127, 152-156, and in the β2-α2 loop, respectively, and (iv) in the β2-α2 loop, there is a strong π-π stacking and a strong π-cation F175-Y169-R164.(N)NH2, has been discovered.

Dent’s disease: Identification of seven new pathogenic mutations in the CLCN5 gene

PubMed Central

Ramos-Trujillo, Elena; Claverie-Martin, Felix; Garcia-Nieto, Victor; Ariceta, Gema; Vara, Julia; Gonzalez-Acosta, Hilaria; Garcia-Ramirez, Marta; Fons, Jaime; Cordoba-Lanus, Elizabeth; Gonzalez-Paredes, Javier; Valenciano, Blanca; Ramos, Leticia; Muley, Rafael; Caggiani, Marina; Alvarez-Estrada, Pilar; Madrid, Alvaro

2013-01-01

Dent’s disease is an X-linked proximal tubulopathy characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis and progressive renal failure. This disorder is frequently caused by mutations in the CLCN5 gene encoding the electrogenic chloride/proton exchanger ClC-5. Occasionally, Dent’s disease has been associated to atypical cases of asymptomatic proteinuria with focal glomerulosclerosis. Twelve unrelated patients with Dent’s disease, including two who presented with asymptomatic proteinuria and developed glomerulosclerosis, were studied. Mutational analysis of the CLCN5 gene was performed by DNA sequencing. We identified thirteen distinct CLCN5 mutations in the twelve patients. Seven of these mutations, p.P416fsX*17, p.[H107P, V108fs*27], p.G466D, p.G65R, p.G462S, p.Y164* and c.723+1G >T, were novel and possibly pathogenic. In one family, the patient’s mother was not a carrier of the respective mutation. Our results increased the spectrum of CLCN5 disease causing defects with seven new pathogenic mutations and established a de novo origin in one of them. Remarkably, three new missense mutations, p.G466D, p.G65R and p.G462S, affect highly conserved glycine residues located in transmembrane α-helix GxxxG packing motifs. The two atypical cases further support that the diagnosis of Dent’s disease should be considered in children with asymptomatic proteinuria and focal glomerulosclerosis and without evidence of primary glomerular disease. PMID:27625851

Structure and ligand-binding site characteristics of the human P2Y11 nucleotide receptor deduced from computational modelling and mutational analysis

PubMed Central

Zylberg, Jacques; Ecke, Denise; Fischer, Bilha; Reiser, Georg

2007-01-01

The P2Y11-R (P2Y11 receptor) is a less explored drug target. We computed an hP2Y11-R (human P2Y11) homology model with two templates, bovine-rhodopsin (2.6 Å resolution; 1 Å=0.1 nm) and a hP2Y1–ATP complex model. The hP2Y11-R model was refined using molecular dynamics calculations and validated by virtual screening methods, with an enrichment factor of 5. Furthermore, mutational analyses of Arg106, Glu186, Arg268, Arg307 and Ala313 confirmed the adequacy of our hP2Y11-R model and the computed ligand recognition mode. The E186A and R268A mutants reduced the potency of ATP by one and three orders of magnitude respectively. The R106A and R307A mutants were functionally inactive. We propose that residues Arg106, Arg268, Arg307 and Glu186 are involved in ionic interactions with the phosphate moiety of ATP. Arg307 is possibly also H-bonded to N6 of ATP via the backbone carbonyl. Activity of ATP at the F109I mutant revealed that the proposed π-stacking of Phe109 with the adenine ring is a minor interaction. The mutation A313N, which is part of a hydrophobic pocket in the vicinity of the ATP C-2 position, partially explains the high activity of 2-MeS-ATP at P2Y1-R as compared with the negligible activity at the P2Y11-R. Inactivity of ATP at the Y261A mutant implies that Tyr261 acts as a molecular switch, as in other G-protein-coupled receptors. Moreover, analysis of cAMP responses seen with the mutants showed that the efficacy of coupling of the P2Y11-R with Gs is more variable than coupling with Gq. Our model also indicates that Ser206 forms an H-bond with Pγ (the γ-phosphate of the triphosphate chain of ATP) and Met310 interacts with the adenine moiety. PMID:17338680

Preclinical Studies of Signaling Pathways in a Mutant Mouse Model of Hormone-Refractory Prostate Cancer

DTIC Science & Technology

2011-02-01

uro - genital mesenchyme (Lamm et al. 2002; Freestone et al. 2003; Berman et al. 2004); and loss...recombination with rodent embryonic uro - genital mesenchyme and grafting into immunodeficient recipients (Hayward et al. 2001; Gao et al. 2004a; Jiang et al...M y c d ri v en b y A R R 2 P B p ro m o te r; H i- M y c an d lo w -M y c d if fe r in la te n cy . P h en o ty p e: P IN an d ad en o ca rc in o m

DNA mutagenic activity and capacity for HIV-1 restriction of the cytidine deaminase APOBEC3G depend on whether DNA or RNA binds to tyrosine 315

PubMed Central

Polevoda, Bogdan; Joseph, Rebecca; Friedman, Alan E.; Bennett, Ryan P.; Greiner, Rebecca; De Zoysa, Thareendra; Stewart, Ryan A.; Smith, Harold C.

2017-01-01

APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. Bulk RNA and substrate ssDNA bind to the same three A3G tryptic peptides (amino acids 181–194, 314–320, and 345–374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C terminus of A3G to its N terminus. We show here that the A3G tyrosines 181 and 315 directly cross-linked ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an Escherichia coli DNA mutator reporter, whereas Y181A and Y182A mutants retained ∼50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Tyr-315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Tyr-315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity. PMID:28381554

Acid Lipase from Candida viswanathii: Production, Biochemical Properties, and Potential Application

PubMed Central

de Almeida, Alex Fernando; Carmona, Eleonora Cano

2013-01-01

Influences of environmental variables and emulsifiers on lipase production of a Candida viswanathii strain were investigated. The highest lipase activity (101.1 U) was observed at 210 rpm, pH 6.0, and 27.5°C. Other fermentation parameters analyzed showed considerable rates of biomass yield (Y L/S = 1.381 g/g), lipase yield (Y L/S = 6.892 U/g), and biomass productivity (P X = 0.282 g/h). Addition of soybean lecithin increased lipase production in 1.45-fold, presenting lipase yield (Y L/S) of 10.061 U/g. Crude lipase presented optimal activity at acid pH of 3.5, suggesting a new lipolytic enzyme for this genus and yeast in general. In addition, crude lipase presented high stability in acid conditions and temperature between 40 and 45°C, after 24 h of incubation in these temperatures. Lipase remained active in the presence of organic solvents maintaining above 80% activity in DMSO, methanol, acetonitrile, ethanol, acetone, 1-propanol, isopropanol, and 2-propanol. Effectiveness for the hydrolysis of a wide range of natural triglycerides suggests that this new acid lipase has high potential application in the oleochemical and food industries for hydrolysis and/or modification of triacylglycerols to improve the nutritional properties. PMID:24350270

In vitro resistance development for RO-0335, a novel diphenylether nonnucleoside reverse transcriptase inhibitor.

PubMed

Javanbakht, H; Ptak, R G; Chow, E; Yan, J M; Russell, J D; Mankowski, M K; Hogan, P A; Hogg, J H; Vora, H; Hang, J Q; Li, Y; Su, G; Paul, A; Cammack, N; Klumpp, K; Heilek, G

2010-05-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.

Phosphorylation of Mutationally Introduced Tyrosine in the Activation Loop of HER2 Confers Gain-of-Function Activity

PubMed Central

Hu, Zexi; Wan, Xiaobo; Hao, Rui; Zhang, Heng; Li, Li; Li, Lin; Xie, Qiang; Wang, Peng; Gao, Yibo; Chen, She; Wei, Min; Luan, Zhidong; Zhang, Aiqun; Huang, Niu; Chen, Liang

2015-01-01

Amplification, overexpression, and somatic mutation of the HER2 gene have been reported to play a critical role in tumorigenesis of various cancers. The HER2 H878Y mutation was recently reported in 11% of hepatocellular carcinoma (HCC) patients. However, its functional impact on the HER2 protein and its role in tumorigenesis has not been determined. Here, we show that HER2 H878Y is a gain-of-function mutation. Y878 represents a phosphorylation site, and phospho-Y878 interacts with R898 residue to stabilize the active conformation of HER2, thereby enhancing its kinase activity. H878Y mutant is transforming and the transformed cells are sensitive to HER2 kinase inhibitors. Thus, our study reveals the following novel mechanism underlying the tumorigenic function of the HER2 H878Y mutation: the introduction of a tyrosine residue into the kinase activation loop via mutagenesis modulates the conformation of the kinase, thereby enhancing its activity. PMID:25853726

A novel Dutch mutation in UNC13D reveals an essential role of the C2B domain in munc13-4 function.

PubMed

Elstak, Edo D; te Loo, Maroeska; Tesselaar, Kiki; van Kerkhof, Peter; Loeffen, Jan; Grivas, Dimitris; Hennekam, Eric; Boelens, Jaap Jan; Hoogerbrugge, Peter M; van der Sluijs, Peter; van Gijn, Marielle E; van de Corput, Lisette

2012-04-01

UNC13D, encoding the protein munc13-4, is essential in intracellular trafficking and exocytosis of lytic granules. Mutations in this gene are associated with familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a genetically heterogeneous, rare autosomal recessive immune disorder. How mutations affect function of munc13-4 is poorly understood. Since 2006 we genetically identified seven FHL patients with mutations in UNC13D. Here, we report for the first time a c.2695C>T (p.Arg899X) mutation in exon 28 of UNC13D in three young unrelated Dutch patients. The mutation causes a premature stop codon and encodes munc13-4(1-899), which lacks the C-terminal C2 domain. Genealogical research and haplotyping of the patient families demonstrated that a single ancestral founder introduced the mutation in the Netherlands. We then characterized the mutant protein phenotypically in cell biological and immunological assays. Munc13-4(1-899) was correctly targeted to CD63-positive secretory lysosomes, although its stability was reduced and dynamic turnover on the granule membrane became uncoupled from receptor signaling. In accord, and in contrast to wild-type munc13-4, ectopically expressed mutant failed to rescue degranulation in cells with silenced endogenous munc13-4. The functional and clinical data showed that this novel Dutch founder mutation leads to severe early onset of FHL3 due to misfolding and degradation of munc13-4(1-899). Copyright © 2011 Wiley Periodicals, Inc.

A mutagenesis study of a catalytic antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, D.Y.; Prudent, J.R.; Baldwin, E.P.

1991-01-01

The authors have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (V{sub H}) of the phosphocholine-binding antibody S107.S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and fourmore » Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the V{sub H} binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.« less

Split-Hand/Split-Foot Malformation Is Caused by Mutations in the p63 Gene on 3q27

PubMed Central

Ianakiev, Peter; Kilpatrick, Michael W.; Toudjarska, Iva; Basel, Donald; Beighton, Peter; Tsipouras, Petros

2000-01-01

Split-hand/split-foot malformation (SHFM), a limb malformation involving the central rays of the autopod and presenting with syndactyly, median clefts of the hands and feet, and aplasia and/or hypoplasia of the phalanges, metacarpals, and metatarsals, is phenotypically analogous to the naturally occurring murine Dactylaplasia mutant (Dac). Results of recent studies have shown that, in heterozygous Dac embryos, the central segment of the apical ectodermal ridge (AER) degenerates, leaving the anterior and posterior segments intact; this finding suggests that localized failure of ridge maintenance activity is the fundamental developmental defect in Dac and, by inference, in SHFM. Results of gene-targeting studies have demonstrated that p63, a homologue of the cell-cycle regulator TP53, plays a critically important role in regulation of the formation and differentiation of the AER. Two missense mutations, 724A→G, which predicts amino acid substitution K194E, and 982T→C, which predicts amino acid substitution R280C, were identified in exons 5 and 7, respectively, of the p63 gene in two families with SHFM. Two additional mutations (279R→H and 304R→Q) were identified in families with EEC (ectrodactyly, ectodermal dysplasia, and facial cleft) syndrome. All four mutations are found in exons that fall within the DNA-binding domain of p63. The two amino acids mutated in the families with SHFM appear to be primarily involved in maintenance of the overall structure of the domain, in contrast to the p63 mutations responsible for EEC syndrome, which reside in amino acid residues that directly interact with the DNA. PMID:10839977

Loss of actomyosin regulation in distal arthrogryposis myopathy due to mutant myosin binding protein-C slow

PubMed Central

Ackermann, Maegen A.; Patel, Puja D.; Valenti, Jane; Takagi, Yasuharu; Homsher, Earl; Sellers, James R.; Kontrogianni-Konstantopoulos, Aikaterini

2013-01-01

Myosin binding protein C (MyBP-C) is expressed in striated muscles, where it plays key roles in the modulation of actomyosin cross-bridges. Slow MyBP-C (sMyBP-C) consists of multiple variants sharing common domains but also containing unique segments within the NH2 and COOH termini. Two missense mutations in the NH2 terminus (W236R) and COOH terminus (Y856H) of sMyBP-C have been causally linked to the development of distal arthrogryposis-1 (DA-1), a severe skeletal muscle disorder. Using a combination of in vitro binding and motility assays, we show that the COOH terminus mediates binding of sMyBP-C to thick filaments, while the NH2 terminus modulates the formation of actomyosin cross-bridges in a variant-specific manner. Consistent with this, a recombinant NH2-terminal peptide that excludes residues 34–59 reduces the sliding velocity of actin filaments past myosin heads from 9.0 ± 1.3 to 5.7 ± 1.0 μm/s at 0.1 μM, while a recombinant peptide that excludes residues 21–59 fails to do so. Notably, the actomyosin regulatory properties of sMyBP-C are completely abolished by the presence of the DA-1 mutations. In summary, our studies are the first to show that the NH2 and COOH termini of sMyBP-C have distinct functions, which are regulated by differential splicing, and are compromized by the presence of missense point mutations linked to muscle disease.—Ackermann, M. A., Patel, P. D., Valenti, J., Takagi, Y., Homsher, E., Sellers, J. R., Kontrogiannni-Konstantopoulos, A. Loss of actomyosin regulation in distal arthrogryposis myopathy due to mutant myosin binding protein-C slow. PMID:23657818

High-pressure investigations of yttrium(III) oxoarsenate(V): Crystal structure and luminescence properties of Eu3+-doped scheelite-type Y[AsO4] from xenotime-type precursors

NASA Astrophysics Data System (ADS)

Ledderboge, Florian; Nowak, Jan; Massonne, Hans-Joachim; Förg, Katharina; Höppe, Henning A.; Schleid, Thomas

2018-07-01

Colourless, water- and air-stable single crystals of yttrium(III) oxoarsenate(V) Y[AsO4] in the xenotime-type crystal structure were prepared by the reaction of yttrium sesquioxide (Y2O3) dissolved in aqueous nitric acid (13%) with a solution of arsenic(V) oxide hydrate (As2O5·3H2O) and subsequent neutralization with 1 M caustic soda. Y[AsO4] crystallizes tetragonally in the space group I41/amd with the lattice parameters a = 704.63(6) and c = 628.94(5) pm for Z = 4 and is isotypic to the minerals xenotime RE[PO4] (RE: mainly Y and Yb) and chernovite RE[AsO4] (RE: mainly Y and Ce). This xenotime-type yttrium compound was used as precursor in a high-pressure experiment (20 kbar) at 700 °C to create a new tetragonal modification of Y[AsO4]. It shows the scheelite-type structure (space group: I41/a) with the lattice parameters a = 498.23(4) and c = 1120.71(9) pm for Z = 4, named after the mineral scheelite (Ca[WO4]). Both tetragonal structures are characterized by only one crystallographically unique position for each of the Y3+, As5+ and O2- ions with distances of d(Y-O) = 232 and 241 pm (C.N. = 8) as well as d(As-O) = 169 pm (C.N. = 4) in the case of the scheelite-type structure. The xenotime-type compound shows an unexpected slight decrease in average bond lengths for the yttrium to oxygen (d(Y-O) = 230 and 241 pm, C.N. = 8) as well as for the arsenic to oxygen distances (d(As-O) = 168 pm, C.N. = 4), accompanied by a drastic density increase from Dx = 4.85 (xenotime type) to Dx = 5.44 g • cm-3 (scheelite type). Luminescence spectroscopic measurements of the Eu3+-doped Y[AsO4] samples, obtained in experiments at similar conditions as for the pure compounds, show a bright, reddish lighting for the scheelite type, which does not occur for the xenotime type of yttrium(III) oxoarsenate(V).

pfmdr1 amplification and fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum in Venezuela.

PubMed

Griffing, Sean; Syphard, Luke; Sridaran, Sankar; McCollum, Andrea M; Mixson-Hayden, Tonya; Vinayak, Sumiti; Villegas, Leopoldo; Barnwell, John W; Escalante, Ananias A; Udhayakumar, Venkatachalam

2010-04-01

Molecular tools are valuable for determining evolutionary history and the prevalence of drug-resistant malaria parasites. These tools have helped to predict decreased sensitivity to antimalarials and fixation of multidrug resistance genotypes in some regions. In order to assess how historical drug policies impacted Plasmodium falciparum in Venezuela, we examined molecular changes in genes associated with drug resistance. We examined pfmdr1 and pfcrt in samples from Sifontes, Venezuela, and integrated our findings with earlier work describing dhfr and dhps in these samples. We characterized pfmdr1 genotypes and copy number variation, pfcrt genotypes, and proximal microsatellites in 93 samples originating from surveillance from 2003 to 2004. Multicopy pfmdr1 was found in 12% of the samples. Two pfmdr1 alleles, Y184F/N1042D/D1246Y (37%) and Y184F/S1034C/N1042D/D1246Y (63%), were found. These alleles share ancestry, and no evidence of strong selective pressure on mutations was found. pfcrt chloroquine resistance alleles are fixed with two alleles: S(tct)VMNT (91%) and S(agt)VMNT (9%). These alleles are associated with strong selection. There was also an association between pfcrt, pfmdr1, dhfr, and dhps genotypes/haplotypes. Duplication of pfmdr1 suggests a potential shift in mefloquine sensitivity in this region, which warrants further study. A bottleneck occurred in P. falciparum in Sifontes, Venezuela, and multidrug resistance genotypes are present. This population could be targeted for malaria elimination programs to prevent the possible spread of multidrug-resistant parasites.

pfmdr1 Amplification and Fixation of pfcrt Chloroquine Resistance Alleles in Plasmodium falciparum in Venezuela ▿ †

PubMed Central

Griffing, Sean; Syphard, Luke; Sridaran, Sankar; McCollum, Andrea M.; Mixson-Hayden, Tonya; Vinayak, Sumiti; Villegas, Leopoldo; Barnwell, John W.; Escalante, Ananias A.; Udhayakumar, Venkatachalam

2010-01-01

Molecular tools are valuable for determining evolutionary history and the prevalence of drug-resistant malaria parasites. These tools have helped to predict decreased sensitivity to antimalarials and fixation of multidrug resistance genotypes in some regions. In order to assess how historical drug policies impacted Plasmodium falciparum in Venezuela, we examined molecular changes in genes associated with drug resistance. We examined pfmdr1 and pfcrt in samples from Sifontes, Venezuela, and integrated our findings with earlier work describing dhfr and dhps in these samples. We characterized pfmdr1 genotypes and copy number variation, pfcrt genotypes, and proximal microsatellites in 93 samples originating from surveillance from 2003 to 2004. Multicopy pfmdr1 was found in 12% of the samples. Two pfmdr1 alleles, Y184F/N1042D/D1246Y (37%) and Y184F/S1034C/N1042D/D1246Y (63%), were found. These alleles share ancestry, and no evidence of strong selective pressure on mutations was found. pfcrt chloroquine resistance alleles are fixed with two alleles: StctVMNT (91%) and SagtVMNT (9%). These alleles are associated with strong selection. There was also an association between pfcrt, pfmdr1, dhfr, and dhps genotypes/haplotypes. Duplication of pfmdr1 suggests a potential shift in mefloquine sensitivity in this region, which warrants further study. A bottleneck occurred in P. falciparum in Sifontes, Venezuela, and multidrug resistance genotypes are present. This population could be targeted for malaria elimination programs to prevent the possible spread of multidrug-resistant parasites. PMID:20145087

40 CFR Table 9 to Subpart Vvvvvv... - Applicability of General Provisions to Subpart VVVVVV

Code of Federal Regulations, 2010 CFR

2010-07-01

... 63.6 (e)(1)(i) and (ii), (e)(3), and (f)(1) SSM Requirements No 63.6(h)(1)-(h)(4), (h)(5)(i)-(h)(5... Requirements Yes References to SSM in § 63.8(c) do not apply. 63.8(a)(2) Monitoring Requirements No 63.8(a)(3... to SSM in § 63.8(d) do not apply. 63.8(g)(1)-(g)(4) Yes Data reduction requirements apply only if you...

40 CFR Table 9 to Subpart Vvvvvv... - Applicability of General Provisions to Subpart VVVVVV

Code of Federal Regulations, 2011 CFR

2011-07-01

... 63.6 (e)(1)(i) and (ii), (e)(3), and (f)(1) SSM Requirements No 63.6(h)(1)-(h)(4), (h)(5)(i)-(h)(5... Requirements Yes References to SSM in § 63.8(c) do not apply. 63.8(a)(2) Monitoring Requirements No 63.8(a)(3... to SSM in § 63.8(d) do not apply. 63.8(g)(1)-(g)(4) Yes Data reduction requirements apply only if you...

40 CFR Table 9 to Subpart Vvvvvv... - Applicability of General Provisions to Subpart VVVVVV

Code of Federal Regulations, 2012 CFR

2012-07-01

... 63.6 (e)(1)(i) and (ii), (e)(3), and (f)(1) SSM Requirements No 63.6(h)(1)-(h)(4), (h)(5)(i)-(h)(5... Requirements Yes References to SSM in § 63.8(c) do not apply. 63.8(a)(2) Monitoring Requirements No 63.8(a)(3... to SSM in § 63.8(d) do not apply. 63.8(g)(1)-(g)(4) Yes Data reduction requirements apply only if you...

[Construction and characterization of an epitope-mutated Asia 1 type foot-and-mouth disease virus].

PubMed

Zhang, Yan; Hu, Yonghao; Yang, Fan; Yang, Bo; Wang, Songhao; Zhu, Zixiang; Zheng, Haixue

2015-01-01

To generate an epitope-mutated foot-and-mouth disease virus (FMDV) as a marker vaccine, the infectious clone pAsia 1-FMDV containing the complete genomic cDNA of Asia 1 type FMDV was used as backbone, the residues at positions 27 and 31 in the 3D gene were mutated (H27Y and N31R). The resulting plasmid pAsia 1-FMDV-3DM encoding a mutated epitope was transfected into BHK-21 cells and the recombinant virus rAsia 1-3DM was rescued. The recombinant virus showed similar biological characteristics comparable with the parental virus. In serological neutralization test the antisera against recombine virus have a good reactivity with parental virus. The antisera against the mutant virus were shown to be reactive with the mutated epitope but not the wild-type one. The results indicated that the two virus strains could be distinguished by western blotting using synthetic peptides. This epitope-mutated FMDV strain will be evaluated as a potential marker vaccine against FMDV infections.

Effects of Site-Directed Mutagenesis of Escherichia coli Heat-Labile Enterotoxin on ADP-Ribosyltransferase Activity and Interaction with ADP-Ribosylation Factors

PubMed Central

A. Stevens, Linda; Moss, Joel; Vaughan, Martha; Pizza, Mariagrazia; Rappuoli, Rino

1999-01-01

Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsα, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction. PMID:9864224

The C-terminal domain of glyceraldehyde 3-phosphate dehydrogenase plays an important role in suppression of tRNALys3 packaging into human immunodeficiency virus type-1 particles.

PubMed

Kishimoto, Naoki; Onitsuka-Kishimoto, Ayano; Iga, Nozomi; Takamune, Nobutoki; Shoji, Shozo; Misumi, Shogo

2016-12-01

Human immunodeficiency virus type-1 (HIV-1) requires the packaging of human tRNA Lys3 as a primer for effective viral reverse transcription. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) suppresses the packaging efficiency of tRNA Lys3 . Although the binding of GAPDH to Pr55 gag is important for the suppression mechanism, it remains unclear which domain of GAPDH is responsible for the interaction with Pr55 gag . In this study, we show that Asp 256 , Lys 260 , Lys 263 and Glu 267 of GAPDH are important for the suppression of tRNA Lys3 packaging. Yeast two-hybrid analysis demonstrated that the C -terminal domain of GAPDH (151-335) interacts with both the matrix region (MA; 1-132) and capsid N -terminal domain (CA-NTD; 133-282). The D256R, K263E or E267R mutation of GAPDH led to the loss of the ability to bind to wild-type (WT) MA, and the D256R/K260E double mutation of GAPDH resulted in the loss of detectable binding activity to WT CA-NTD. In contrast, R58E, Q59A or Q63A of MA, and E76R or R82E of CA-NTD abrogated the interaction with the C -terminal domain of GAPDH. Multiple-substituted GAPDH mutant (D256R/K260E/K263E/E267R) retained the oligomeric formation with WT GAPDH in HIV-1 producing cells, but the incorporation level of the hetero-oligomer was decreased in viral particles. Furthermore, the viruses produced from cells expressing the D256R/K260E/K263E/E267R mutant restored tRNA Lys3 packaging efficiency because the mutant exerted a dominant negative effect by preventing WT GAPDH from binding to MA and CA-NTD and improved the reverse transcription. These findings indicate that the amino acids Asp 256 , Lys 260 , Lys 263 and Glu 267 of GAPDH is essential for the mechanism of tRNA Lys3 -packaging suppression and the D256R/K260E/K263E/E267R mutant of GAPDH acts in a dominant negative manner to suppress tRNA Lys3 packaging.

Biochemical analysis of human POLG2 variants associated with mitochondrial disease

PubMed Central

Young, Matthew J.; Longley, Matthew J.; Li, Fang-Yuan; Kasiviswanathan, Rajesh; Wong, Lee-Jun; Copeland, William C.

2011-01-01

Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic. PMID:21555342

Mutation in HFE gene decreases manganese accumulation and oxidative stress in the brain after olfactory manganese exposure.

PubMed

Ye, Qi; Kim, Jonghan

2016-06-01

Increased accumulation of manganese (Mn) in the brain is significantly associated with neurobehavioral deficits and impaired brain function. Airborne Mn has a high systemic bioavailability and can be directly taken up into the brain, making it highly neurotoxic. While Mn transport is in part mediated by several iron transporters, the expression of these transporters is altered by the iron regulatory gene, HFE. Mutations in the HFE gene are the major cause of the iron overload disorder, hereditary hemochromatosis, one of the prevalent genetic diseases in humans. However, whether or not HFE mutation modifies Mn-induced neurotoxicity has not been evaluated. Therefore, our goal was to define the role of HFE mutation in Mn deposition in the brain and the resultant neurotoxic effects after olfactory Mn exposure. Mice carrying the H67D HFE mutation, which is homologous to the H63D mutation in humans, and their control, wild-type mice, were intranasally instilled with MnCl2 with different doses (0, 0.2, 1.0 and 5.0 mg kg(-1)) daily for 3 days. Mn levels in the blood, liver and brain were determined using inductively-coupled plasma mass spectrometry (ICP-MS). H67D mutant mice showed significantly lower Mn levels in the blood, liver, and most brain regions, especially in the striatum, while mice fed an iron-overload diet did not. Moreover, mRNA expression of ferroportin, an essential exporter of iron and Mn, was up-regulated in the striatum. In addition, the levels of isoprostane, a marker of lipid peroxidation, were increased in the striatum after Mn exposure in wild-type mice, but were unchanged in H67D mice. Together, our results suggest that the H67D mutation provides decreased susceptibility to Mn accumulation in the brain and neurotoxicity induced by inhaled Mn.

Radiation Resistant Polymer-Based Photonics for Space Applications

DTIC Science & Technology

2006-08-01

M. Flusche", P. Repak", G. Brost’, A. Pirichb, D. CraigC D. Lee’, D. Cardimona’, H. R. Fetterman ’, B. Tsape, C. Castaneda, R. , T. zengh, D. Wood", R...Repak, G. Brost, A. Pirich; D. Le, D. Cardimona, D. Craig; H. Fetterman , B. Tsap; C. Castaneda; R. Barto; T. Zeng, D. Wood, R. Claus, AFRL/SNDP 25...124, 3, 2001. [2] M.-C.Oh, H. Zhang, C. Zhang, H. Erlig, Y. Chang, B. Tsap, D. Chang, A. Szep, W. H. Steier, H. R. Fetterman , and L. R. Dalton,, IEEE

The influence of non-ionic radiation on the chicken hatching.

PubMed

Veterány, Ladislav; Toman, Robert; Jedlicka, Jaroslav

2002-11-01

The study considers the influence of non-ionic radiation (white and monochromatic light) on the hatching of the Hampshire breed chickens. The chicken embryos were most sensitive to the white light (El), reaching the hatching time of 503.63 +/- 3.17 h, the hatchability of 95.12 +/- 3.72% and an average weight of incubated chickens 46.83 +/- 2.82 g. Of the monochromatic lights, the chicken embryos were most sensitive to yellow and green lights (E5, E4) with the hatching time of 505.22 +/- 4.03 and 507.14 +/- 3.95 h, respectively, the hatchability of 94.89 +/- 3.02 and 94.47 +/- 2.93%, respectively and the average weight of incubated chickens 45.72 +/- 1.93 and 45.05 +/- 2.66 g, respectively. The least reaction of chicken was observed with violet light (E2) with the hatching time of 510.04+/- 1.97 h, hatchability of 90.81 +/- 4.05% and the average weight of incubated chickens 42.02 +/- 3.72 g. The effect of violet light brings the same results as we observed in the case of hatching in darkness (control group C), when the hatching time was 510.41 +/- 2.82 h, hatchability 90.42 +/- 3.35% and average weight of incubated chickens 41.98 +/- 3.05 g.

Computational Divided Differencing and Divided Difference Arithmetics

DTIC Science & Technology

2001-01-01

A 1t��923Ã@Zp &+7²; 2³)*[ tD ³ N+³µ´ AG6J@1t@ ´ FD62921t9+F3Ã@1t�B|B@�; G�62923TA衩V3T7+6R_\\ª;T7#�3ÃA"�?3z;¢6J7#�"�:8cb$@f8...3¢6J@ �2;¢�29<� @nPA1¢@�;¢;t��7E9;6d7+ 1q3 ¢6J@=Ft629<�d9@1t�2��@a6H7+1f3¢6J@Wət;73C58J@1t� 0 923¢� 0 @o7<AM@1t923Ã7+1 y v v Computational...92@17C 923Ã@�;@��B3¢6J@1f3¢6J@|8:92371t�^n / ! 7+6;¢�B|B@UC bU4UC 7+ 1q3 ¢6J@ 8¦92371t�^nB. 1 $¦7+6T;¢�B|B@ UC bU4UCA282@BA

Directed evolution of GH43 ß-xylosidase XylBH43 thermal stability and L186 saturation

USDA-ARS?s Scientific Manuscript database

Directed evolution of ß-xylosidase XylBH43 using DNA family shuffling identified three mutations R45K, M69P, and L186Y that affect thermal stability parameter Kt0.5 by -1.8±0.1 º C, 1.7±0.3 º C, and 3.2±0.4 º C, respectively. In addition, a cluster of four mutations near hairpin loop-D83 improved K...

ACVR1 p.Q207E causes classic fibrodysplasia ossificans progressiva and is functionally distinct from the engineered constitutively active ACVR1 p.Q207D variant

PubMed Central

Haupt, Julia; Deichsel, Alexandra; Stange, Katja; Ast, Cindy; Bocciardi, Renata; Ravazzolo, Roberto; Di Rocco, Maja; Ferrari, Paola; Landi, Antonio; Kaplan, Frederick S.; Shore, Eileen M.; Reissner, Carsten; Seemann, Petra

2014-01-01

Fibrodysplasia ossificans progressiva (FOP) is a disabling genetic disorder of progressive heterotopic ossification (HO). Here, we report a patient with an ultra-rare point mutation [c.619C>G, p.Q207E] located in a codon adjacent to the most common FOP mutation [c.617G>A, p.R206H] of Activin A Receptor, type 1 (ACVR1) and that affects the same intracellular amino acid position in the GS activation domain as the engineered constitutively active (c.a.) variant p.Q207D. It was predicted that both mutations at residue 207 have similar functional effects by introducing a negative charge. Transgenic p.Q207D-c.a. mice have served as a model for FOP HO in several in vivo studies. However, we found that the engineered ACVR1Q207D−c.a. is significantly more active than the classic FOP mutation ACVR1R206H when overexpressed in chicken limbs and in differentiation assays of chondrogenesis, osteogenesis and myogenesis. Importantly, our studies reveal that the ACVR1Q207E resembles the classic FOP receptor in these assays, not the engineered ACVR1Q207D−c.a.. Notably, reporter gene assays revealed that both naturally occurring FOP receptors (ACVR1R206H and ACVR1Q207E) were activated by BMP7 and were sensitive to deletion of the ligand binding domain, whereas the engineered ACVR1Q207D−c.a. exhibited ligand independent activity. We performed an in silico analysis and propose a structural model for p.Q207D-c.a. that irreversibly relocates the GS domain into an activating position, where it becomes ligand independent. We conclude that the engineered p.Q207D-c.a. mutation has severe limitations as a model for FOP, whereas the naturally occurring mutations p.R206H and p.Q207E facilitate receptor activation, albeit in a reversible manner. PMID:24852373

Hyperferritinemia increases the risk of hyperuricemia in HFE-hereditary hemochromatosis.

PubMed

Flais, Jérémy; Bardou-Jacquet, Edouard; Deugnier, Yves; Coiffier, Guillaume; Perdriger, Aleth; Chalès, Gérard; Ropert, Martine; Loréal, Olivier; Guggenbuhl, Pascal

2017-05-01

Hyperuricemia is becoming increasingly frequent in the population, and is known to be sometimes the cause of gout. The impact of uric acid is still not clearly understood, however. The iron metabolism may interact with the uric acid metabolism. The aim of this study was to examine the relationship between the serum uric acid and serum ferritin levels in a cohort of hemochromatosis patients who were homozygous for the HFE p.Cys282Tyr mutation. 738 patients with the HFE gene mutation Cys282Tyr in the homozygous state were included in the study. The variables measured during the initial evaluation were compared in univariate analysis by Student's t test. In multivariate analysis, linear stepwise regression was used. In the group of hyperuricemic patients, ferritinemia was significantly higher than in the group of non-hyperuricemic patients (1576.7±1387.4μg/l vs. 1095.63±1319.24μg/l, P<0.005). With multivariate analysis, only ferritin and BMI independently explained the uricemia (R 2 =0.258) after adjustment for age, glycemia and CRP. The correlation between uricemia and log(ferritin) with partial regression correlation coefficients was 0.307 (P<0.01). The increase in uricemia is associated with the increase in ferritin in a population of patients who were homozygous for the HFE gene mutation p.Cys282Tyr and this independently of factors commonly associated with hyperuricemia. The increase in uric acid associated with hyperferritinemia, could be a response to the visceral toxicity of excess non-transferrin bound iron linked to oxidative stress via the antioxidant properties of uric acid. Copyright © 2016 Société française de rhumatologie. Published by Elsevier SAS. All rights reserved.

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase.

PubMed

Huang, Jian-Wen; Cheng, Ya-Shan; Ko, Tzu-Ping; Lin, Cheng-Yen; Lai, Hui-Lin; Chen, Chun-Chi; Ma, Yanhe; Zheng, Yingying; Huang, Chun-Hsiang; Zou, Peijian; Liu, Je-Ruei; Guo, Rey-Ting

2012-04-01

1,3-1,4-β-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.

Prevalence of drug-resistant mutation among drug-treated HIV/AIDS inpatient in Airlangga University teaching hospital, Surabaya, Indonesia

NASA Astrophysics Data System (ADS)

Rachman, B. E.; Khairunisa, S. Q.; Witaningrum, A. M.; Yunifiar, M. Q.; Widiyanti, P.; Nasronudin

2018-03-01

Increased use of antiretroviral therapy did not completely reduce the incidence of HIV/AIDShospitalization. Various factors can be involved. The aim of this study is to examine HIV-1 drug resistance mutations profile in drug-treated HIV/AIDS patients who underwent hospitalization. HIV/AIDS patients who are admitted to hospital who had received ART are included in the study and then examined for the presence of drug resistance-associated mutations. A total of 17 samples were included in the study, but only 11 samples that could be sequence analyzed. On the mutation examination of drug resistance in reverse transcriptase gene, it werefound a major mutation in K103N (9%) and G190A (9%). Most minor mutations were found in A98S (18.1%), followed by M41L, M184V, L210W, T215Y, V108l, Y181C and H221Y at 9% each. Whereas, on examination of drug resistance mutations in protease genes, there is a major mutation in I84V of 9%. Most minor mutations on M36I (45.4%), followed by L10I (36.3%), H69K (36.3%), I93L (27.2%), G16E, L89M, K20R 18.1%, L64V and V771I 9% respectively.A large number of mutated samples pose a challenge in long-term antiretroviral treatment, so a breakthrough policy is needed to minimize the impact.

On understanding proton transfer to the biocatalytic [Fe-Fe](H) sub-cluster in [Fe-Fe]H(2)ases: QM/MM MD simulations.

PubMed

Hong, G; Cornish, A J; Hegg, E L; Pachter, R

2011-05-01

Proton transfer to the [Fe-Fe](H) sub-cluster in the Desulfovibrio desulfuricans (DdH) and Clostridium pasteurianum (CpI) [Fe-Fe] hydrogenases was investigated by a combination of first principles and empirical molecular dynamics simulations. Pathways that can be inferred from the X-ray crystal structures of DdH and CpI, i.e., (Glu159→Ser198→Glu156→water460→Cys178→DTMA([Fe-Fe](H)) and (Glu282→Ser319→Glu279→water612→Cys299), respectively, were considered. Proton transfer from Cys178 to DTMA in the [Fe-Fe](H) sub-cluster in DdH was readily observed in our results, specifically when [Fe-Fe](H) was in the reduced state ([Fe(I)-Fe(I)]) or in the mixed valence state for the protonated distal iron Fe(d) ([Fe(I)-Fe(II)-H(-)](H)). A concerted mechanism is proposed, where proton transfer in DdH from Glu159 to Glu156 via Ser198 and Glu156 to Cys178 via water460 readily occurred, as well as from Glu282 to Glu279 via Ser319 and Glu279 to Cys299 via water612 in CpI. The theoretical prediction of the proton transfer characteristics is consistent with the assumed biocatalytic mechanism of the [Fe-Fe] hydrogenases in which the proton binds at Fe(d), providing confirmation that has not been explored so far. The computational results were qualitatively validated by the agreement with experimental hydrogen production activity data for mutated CpI enzymes, relative to the wild-type protein. Finally, the insight provided by the simulations, combined, in part, with experimental validation, are important for establishing an approach in future exploration of proton transfer to the active site in this class of enzymes, and possibly also for biomimetic analogs. Published by Elsevier B.V.

Spectrum of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies.

PubMed

Yanus, G A; Akhapkina, T A; Ivantsov, A O; Preobrazhenskaya, E V; Aleksakhina, S N; Bizin, I V; Sokolenko, A P; Mitiushkina, N V; Kuligina, E Sh; Suspitsin, E N; Venina, A R; Holmatov, M M; Zaitseva, O A; Yatsuk, O S; Pashkov, D V; Belyaev, A M; Togo, A V; Imyanitov, E N; Iyevleva, A G

2018-05-01

Distribution of cancer-predisposing mutations demonstrates significant interethnic variations. This study aimed to evaluate patterns of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies. APC gene defects were identified in 26/38 (68%) subjects with colon polyposis; 8/26 (31%) APC mutations were associated with 2 known mutational hotspots (p.E1309Dfs*4 [n = 5] and p.Q1062fs* [n = 3]), while 6/26 (23%) mutations were novel (p.K73Nfs*6, p.S254Hfs*12, p.S1072Kfs*9, p.E1547Kfs*11, p.L1564X and p.C1263Wfs*22). Biallelic mutations in MUTYH gene were detected in 3/12 (25%) remaining subjects with polyposis and in 6/90 (6.7%) patients with colorectal cancer (CRC) carrying KRAS p.G12C substitution, but not in 231 early-onset CRC cases negative for KRAS p.G12C allele. In addition to known European founder alleles p.Y179C and p.G396D, this study revealed a recurrent character of MUTYH p.R245H germ-line mutation. Besides that, 3 novel pathogenic MUTYH alleles (p.L111P, p.R245S and p.Q293X) were found. Targeted next-generation sequencing of 7 APC/MUTYH mutation-negative DNA samples identified novel potentially pathogenic POLD1 variant (p.L460R) in 1 patient and known low-penetrant cancer-associated allele CHEK2 p.I157T in 3 patients. The analysis of 1120 healthy subjects revealed 15 heterozygous carriers of recurrent MUTYH mutations, thus the expected incidence of MUTYH-associated polyposis in Russia is likely to be 1:23 000. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

In silico Analysis of Conformational Changes Induced by Mutation of Aromatic Binding Residues: Consequences for Drug Binding in the hERG K+ Channel

PubMed Central

Knape, Kirsten; Linder, Tobias; Wolschann, Peter; Beyer, Anton; Stary-Weinzinger, Anna

2011-01-01

Pharmacological inhibition of cardiac hERG K+ channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652) substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences. PMID:22194911

Shape Representation in V4: Investigating Position-Specific Tuning for Boundary Confirmation with the Standard Model of Object Recognition

DTIC Science & Technology

2004-11-01

s s a c h u s e t t s i n s t ...i t u t e o f t e c h n o l o g y, c a m b r i d g e , m a 0 2 1 3 9 u s a — w w w . c s a i l . m i t . e d u m a ss a c h u se t t s i n...st i t u t e o f t e c h n o l o g y — co m p u t e r sc i e n ce a n d a r t i f ic ia l i n t e l l ig e n ce l a b o ra t o r y

Carbonic anhydrase II deficiency: Single-base deletion in exon 7 is the predominant mutation in Caribbean Hispanic patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, P.Y.; Ernst, A.R.; Sly, W.S.

1994-04-01

To date, three different structural gene mutations have been identified in patients with carbonic anhydrase II deficiency (osteopetrosis with renal tubular acidosis and cerebral calcification). These include a missense mutation (H107Y) in two families, a splice junction mutation in intron 5 in one of these families, and a splice junction mutation in intron 2 for which many Arabic patients are homozygous. The authors report here a novel mutation for which carbonic anhydrase II-deficient patients from seven unrelated Hispanic families were found to be homozygous. The proband was a 2 1/2-year-old Hispanic girl of Puerto Rican ancestry who was unique clinically,more » in that she had no evidence of renal tubular acidosis, even though she did have osteopetrosis, developmental delay, and cerebral calcification. She proved to be homozygous for a single-base deletion in the coding region of exon 7 that produces a frameshift that changes the next 12 amino acids before leading to chain termination and that also introduces a new MaeIII restriction site. The 27-kD truncated enzyme produced when the mutant cDNA was expressed in COS cells was enzymatically inactive, present mainly in insoluble aggregates, and detectable immunologically at only 5% the level of the 29-kD normal carbonic anhydrase II expressed from the wild-type cDNA. Metabolic labeling revealed that this 27-kD mutant protein has an accelerated rate of degradation. Six subsequent Hispanic patients of Caribbean ancestry, all of whom had osteopetrosis and renal tubular acidosis but who varied widely in clinical severity, were found to be homozygous for the same mutation. These findings identify a novel mutation common to Hispanic patients from the Caribbean islands and provide a ready means for PCR-based diagnosis of the [open quotes]Hispanic mutation.[close quotes] The basis for their phenotypic variability is not yet clear. 15 refs., 5 figs., 1 tab.« less

Plasmon Lasers: Coherent Light Source at Molecular Scales

DTIC Science & Technology

2012-01-01

Y . S . Oei, R . Nötzel, C.-Z. Ning, and M.K. Smit, Opt. Express 17, 11107–11112 (2009). [8] M.A. Noginov...Kang, C. Seassal, S .-K. Kim, P. Regreny, Y .-H. Lee, C.M. Lieber, and H.-G. Park Nano, Lett. 10, 3679–3683 (2010). [11] R.M.Ma, R . F. Oulton, V. J... R . T. Hill, J. J. Mock, Y . Urzhumov, D. S . Sebba, S . J. Olden- burg, S .- Y . Chen, A. Lazarides, A. Chilkoti, and D. R . Smith, Nano Lett. 10,

Genetics Home Reference: fumarase deficiency

MedlinePlus

... C, Knape M, Zierz S, Gellerich FN. Molecular and biochemical investigations in fumarase deficiency. Mol Genet Metab. 2006 ... Y, Toulhoat H, de Lonlay P. Clinical and biochemical heterogeneity associated with fumarase deficiency. Hum Mutat. 2011 ...

High penetrance of pheochromocytoma associated with the novel C634Y/Y791F double germline mutation in the RET protooncogene.

PubMed

Toledo, Rodrigo A; Wagner, Simona M; Coutinho, Flavia L; Lourenço, Delmar M; Azevedo, Juliana A; Longuini, Viviane C; Reis, Mariana T A; Siqueira, Sheila A C; Lucon, Antonio M; Tavares, Marcos R; Fragoso, Maria C B V; Pereira, Adelaide A; Dahia, Patricia L M; Mulligan, Lois M; Toledo, Sergio P A

2010-03-01

Previous studies have shown that double RET mutations may be associated with unusual multiple endocrine neoplasia type 2 (MEN 2) phenotypes. Our objective was to report the clinical features of patients harboring a previously unreported double mutation of the RET gene and to characterize this mutation in vitro. Sixteen patients from four unrelated families and harboring the C634Y/Y791F double RET germline mutation were included in the study. Large pheochromocytomas measuring 6.0-14 cm and weighing up to 640 g were identified in the four index cases. Three of the four tumors were bilateral. High penetrance of pheochromocytoma was also seen in the C634Y/Y791F-mutation-positive relatives (seven of nine, 77.7%). Of these, two cases had bilateral tumors, one presented with multifocal tumors, two cases had large tumors (>5 cm), and one case, which was diagnosed with a large (5.5 x 4.5 x 4.0 cm) pheochromocytoma, reported early onset of symptoms of the disease (14 yr old). The overall penetrance of pheochromocytoma was 84.6% (11 of 13). Development of medullary thyroid carcinoma in our patients seemed similar to that observed in patients with codon 634 mutations. Haplotype analysis demonstrated that the mutation did not arise from a common ancestor. In vitro studies showed the double C634Y/Y791F RET receptor was significantly more phosphorylated than either activated wild-type receptor or single C634Y and Y791F RET mutants. Our data suggest that the natural history of the novel C634Y/Y791F double mutation carries a codon 634-like pattern of medullary thyroid carcinoma development, is associated with increased susceptibility to unusually large bilateral pheochromocytomas, and is likely more biologically active than each individual mutation.

Deletion of a dynamic surface loop improves stability and changes kinetic behavior of phosphatidylinositol-synthesizing Streptomyces phospholipase D.

PubMed

Damnjanović, Jasmina; Nakano, Hideo; Iwasaki, Yugo

2014-04-01

Supplementary phosphatidylinositol (PI) was shown to improve lipid metabolism in animals, thus it is interesting for pharmaceutical and nutritional applications. Homogenous PI can be produced in transphosphatidylation of phosphatidylcholine (PC) with myo-inositol catalyzed by phospholipase D (PLD). Only bacterial enzymes able to catalyze PI synthesis are Streptomyces antibioticus PLD (SaPLD) variants, among which DYR (W187D/Y191Y/Y385R) has the best kinetic profile. Increase in PI yield is possible by providing excess of solvated myo-inositol, which is achievable at high temperatures due to its highly temperature-dependent solubility. However, high-temperature PI synthesis requires the thermostable PLD. Previous site-directed combinatorial mutagenesis at the residues of DYR having high B-factor yielded the most improved variant, D40H/T291Y DYR, obtained by the combination of two selected mutations. D40 and T291 are located within dynamic surface loops, D37-G45 (termed D40 loop) and G273-T313. Thus, in this work, thermostabilization of DYR SaPLD was attempted by rational design based on deletion of the D40 loop, generating two variants, Δ37-45 DYR and Δ38-46 DYR PLD. Δ38-46 DYR showed highest thermostability as its activity half-life at 70°C proved 11.7 and 8.0 times longer than that of the DYR and Δ37-45 DYR, respectively. Studies on molecular dynamics predicted Δ38-46 DYR to have the least average RMSD change as temperature dramatically increases. At 60 and 70°C, both mutants synthesized PI in a twofold higher yield compared to the DYR, while at the same time produced less of the hydrolytic side-product, phosphatidic acid. © 2013 Wiley Periodicals, Inc.

HFE p.H63D polymorphism does not influence ALS phenotype and survival.

PubMed

Chiò, Adriano; Mora, Gabriele; Sabatelli, Mario; Caponnetto, Claudia; Lunetta, Christian; Traynor, Bryan J; Johnson, Janel O; Nalls, Mike A; Calvo, Andrea; Moglia, Cristina; Borghero, Giuseppe; Monsurrò, Maria Rosaria; La Bella, Vincenzo; Volanti, Paolo; Simone, Isabella; Salvi, Fabrizio; Logullo, Francesco O; Nilo, Riva; Giannini, Fabio; Mandrioli, Jessica; Tanel, Raffaella; Murru, Maria Rita; Mandich, Paola; Zollino, Marcella; Conforti, Francesca L; Penco, Silvana; Brunetti, Maura; Barberis, Marco; Restagno, Gabriella

2015-10-01

It has been recently reported that the p.His63Asp polymorphism of the HFE gene accelerates disease progression both in the SOD1 transgenic mouse and in amyotrophic lateral sclerosis (ALS) patients. We have evaluated the effect of HFE p.His63Asp polymorphism on the phenotype in 1351 Italian ALS patients (232 of Sardinian ancestry). Patients were genotyped for the HFE p.His63Asp polymorphism (CC, GC, and GG). All patients were also assessed for C9ORF72, TARDBP, SOD1, and FUS mutations. Of the 1351 ALS patients, 363 (29.2%) were heterozygous (GC) for the p.His63Asp polymorphism and 30 (2.2%) were homozygous for the minor allele (GG). Patients with CC, GC, and GG polymorphisms did not significantly differ by age at onset, site of onset of symptoms, and survival; however, in SOD1 patients with CG or GG polymorphism had a significantly longer survival than those with a CC polymorphism. Differently from what observed in the mouse model of ALS, the HFE p.His63Asp polymorphism has no effect on ALS phenotype in this large series of Italian ALS patients. Copyright © 2015 Elsevier Inc. All rights reserved.

MIZEX. A Program for Mesoscale Air-Ice-Ocean Interaction Experiments in Arctic Marginal Ice Zones. III. Modeling the Marginal Ice Zone,

DTIC Science & Technology

1984-04-01

Ii TS C]r.I2 TAB 0] Unzanro’ unoed 0 justi fica ~r: 0 April 1984 vs - ASValabilitY Codes lvyall and/or U.S. Army Cold Regions Research and Engineering...coupled model. Fig. 1. Annual average simulated velocity fields. 3 192 Aloka 190 / 902 190+ WOO S’,. o Ice OnlY Mod" D"’, 55*w F~tth Yea’ Ice Ocean Model...A more precise delinga- inflow boundary conditions. 12 4- a. [ o ll ii traspert 00 0 0- 0e a I " i i , - - I I 1161 63 15 67 69 Ti 73 75 77 1980 *= 4h

Factors Affecting the Vertical Motion of a Zero-Pressure, Polyethylene, Free Balloon

DTIC Science & Technology

1985-05-31

Delle Traiettorie Dei Palloni Stratos- ferici Per Missioni Di Lunga Durata, Instituto di Tecnologia Aerospaziale, Universita di Boma (translation). 19...2) TD=E2-V ( 3D , 2) !%..TI)/DX ~(C.LI.EI) GO0TO 900 Tr (ALIS(rD) LT..01o) Hl=II1*EXFP(-CC*DX/Y(JD,2)) 1)T X 600- ( D rI.eVJD,2)+R1*DX IF (A&OS...MD.GE.U.01) 3P=H1*((Y(JD.?*)/TK)**(CCR)) IF (AL133(TD).LT.0.01) 9P-H14"IXP(-CL’*DXlY( 3D ,2)) c PREFSSUR~E CONVWRGI!NCE CHECK c C:0IRiiCTS MONOTONIC

PCSK 9 gain-of-function mutations (R496W and D374Y) and clinical cardiovascular characteristics in a cohort of Turkish patients with familial hypercholesterolemia.

PubMed

Kaya, Esra; Kayıkçıoğlu, Meral; Tetik Vardarlı, Aslı; Eroğlu, Zuhal; Payzın, Serdar; Can, Levent

2017-10-01

The molecular basis of the mutations in the PCSK9 gene that produces familial hypercholesterolemia (FH) in the Turkish population is unknown. This study was conducted to determine the presence of four different PCSK9 gain-of-function (GOF) mutations (F216L, R496W, S127R, and D374Y) in a group of patients with FH. A total of 80 consecutive patients with FH (mean age: 56±11 years; mean maximum LDL cholesterol: 251±76 mg/dL) were included in the study. Patients with FH were diagnosed according to the Dutch Lipid Clinic Network criteria based on serum cholesterol levels, personal and family histories of cardiovascular disease, tendon xanthomas, and genetic analysis. To identify F216L, R496W, S127R, and D374Y mutations of the PCSK9 gene, high-resolution melting analysis was performed on isolated DNAs. Of the 80 patients, there were 11 patients (13.8%) with PCSK9 GOF mutations. Detected mutations were D374Y mutation in four (5.0%) patients and R496W in seven patients (8.7%). Only one patient was homozygous for R496W mutation. The other two GOF mutations (S127R and F216 variants) were not detected. There was no significant difference with regard to demographic characteristics and CV disease risk factors and clinical course of the disease between the PCSK9 mutation-positive and PCSK9 mutation-negative groups. This is the first study from a Turkish FH cohort, revealing a higher frequency (approximately 14%) of two PCSK9 GOF mutations (D374Y and R496W) and a different disease course compared to the world literature.

Engineering Temperature Sensitive Live Attenuated Influenza Vaccines from Emerging Viruses

PubMed Central

Zhou, Bin; Li, Yan; Speer, Scott D.; Subba, Anju; Lin, Xudong; Wentworth, David E.

2012-01-01

The licensed live attenuated influenza A vaccine (LAIV) in the United States is created by making a reassortant containing six internal genes from a cold-adapted master donor strain (ca A/AA/6/60) and two surface glycoprotein genes from a circulating/emerging strain (e.g., A/CA/7/09 for the 2009/2010 H1N1 pandemic). Technologies to rapidly create recombinant viruses directly from patient specimens were used to engineer alternative LAIV candidates that have genomes composed entirely of vRNAs from pandemic or seasonal strains. Multiple mutations involved in the temperature-sensitive (ts) phenotype of the ca A/AA/6/60 master donor strain were introduced into a 2009 H1N1 pandemic strain rA/New York/1682/2009 (rNY1682-WT) to create rNY1682-TS1, and additional mutations identified in other ts viruses were added to rNY1682-TS1 to create rNY1682-TS2. Both rNY1682-TS1 and rNY1682-TS2 replicated efficiently at 30°C and 33°C. However, rNY1682-TS1 was partially restricted, and rNY1682-TS2 was completely restricted at 39°C. Additionally, engineering the TS1 or TS2 mutations into a distantly related human seasonal H1N1 influenza A virus also resulted pronounced restriction of replication in vitro. Clinical symptoms and virus replication in the lungs of mice showed that although rNY1682-TS2 and the licensed FluMist®-H1N1pdm LAIV that was used to combat the 2009/2010 pandemic were similarly attenuated, the rNY1682-TS2 was more protective upon challenge with a virulent mutant of pandemic H1N1 virus or a heterologous H1N1 (A/PR/8/1934) virus. This study demonstrates that engineering key temperature sensitive mutations (PB1-K391E, D581G, A661T; PB2-P112S, N265S, N556D, Y658H) into the genomes of influenza A viruses attenuates divergent human virus lineages and provides an alternative strategy for the generation of LAIVs. PMID:22449422

Fluorinated Materials for Air-stable and Moisture-resistant Flexible Optoelectronics

DTIC Science & Technology

2014-01-02

Kim Jong , H.; Shin, S.; Yang, H.; An, B.-K.; Yang, L.; Park Soo, Y. Adv Mater 2012, 24, 911-915; (c) Geng, Y.; Li, H.-B.; Wu, S.-X.; Su, Z.-M. J...Park, J.-U.; Shir, D. J.-L.; Nam , Y.-S.; Jeon, S.; Rogers, J. A. Chem. Rev. 2007, 107, 1117-1160; (g) Murphy, A. R.; Frechet, J. M. J. Chem. Rev. 2007...2649-2655; (w) Song, H.-J.; Kim , D.-H.; Lee, E.-J.; Heo, S.-W.; Lee, J.-Y.; Moon, D.-K. Macromolecules (Washington, DC, U. S.) 2012, 45, 7815- 7822; (x

Incidence of cardiac arrhythmias in asymptomatic hereditary hemochromatosis subjects with C282Y homozygosity.

PubMed

Shizukuda, Yukitaka; Tripodi, Dorothy J; Zalos, Gloria; Bolan, Charles D; Yau, Yu-Ying; Leitman, Susan F; Waclawiw, Myron A; Rosing, Douglas R

2012-03-15

It is not well known whether systemic iron overload per se in hereditary hemochromatosis (HH) is associated with cardiac arrhythmias before other signs and symptoms of cardiovascular disease occur. In the present study, we examined the incidence of cardiac arrhythmia in cardiac asymptomatic subjects with HH (New York Heart Association functional class I) and compared it to that in age- and gender-matched normal volunteers. The 42 subjects with HH and the 19 normal control subjects were recruited through the National Heart, Lung, and Blood Institute-sponsored "Heart Study of Hemochromatosis." They completed 48-hour Holter electrocardiography ambulatory monitoring at the baseline evaluation. The subjects with HH were classified as newly diagnosed (group A) and chronically treated (group B) subjects. All subjects with HH had C282Y homozygosity, and the normal volunteers lacked any HFE gene mutations known to cause HH. Although statistically insignificant, the incidence of ventricular and supraventricular ectopy tended to be greater in the combined HH groups than in the controls. Supraventricular ectopy was more frequently noted in group B compared to in the controls (ectopy rate per hour 11.1 ± 29.9 vs 1.5 ± 3.5, p < 0.05, using the Kruskal-Wallis test). No examples of heart block, other than first-degree atrioventricular node block, were seen in any of the subjects. The incidence of cardiac arrhythmias was not significantly reduced after 6 months of intensive iron removal therapy in the group A subjects. No life-threatening arrhythmias were observed in our subjects with HH. In conclusion, our data suggest that the incidence of cardiac arrhythmias is, at most, marginally increased in asymptomatic subjects with HH. A larger clinical study is warranted to further clarify our observation. Published by Elsevier Inc.

Genetic mapping of the cleidocranial dysplasia (CCD) locus on chromosome band 6p21 to include a microdeletion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gelb, B.D.; Desnick, R.J.; Shevell, M.

1995-08-28

Cleidocranial dysplasia (CCD) is a generalized skeletal dysplasia with autosomal dominant inheritance. Recently, the CCD disease locus was localized to 23 and 17 cM regions of chromosome band 6p21 by linkage studies of seven affected families. Of note, the 23 cM region contained a microdeletion detected in one family at D6S459, an interval that was excluded in the 17 cM overlapping region. Here, linkage of CCD to 6p21 was independently confirmed with a maximal two-point LOD score of Z=5.12 with marker D6S452 at {theta}=0.00. Recombinant events in two affected individuals defined a CCD region of 7 cM from D6S465 tomore » D6S282, which overlapped with the CCD region containing the microdeletion but did not overlap with the 17 cM critical region from D6S282 to D6S291. These results suggest the refined localization of the CCD region to 6 cM spanning markers D6S438 to D6S282, thereby reviving the possibility that the CCD gene lies within the microdeletion at D6S459. 13 refs., 2 figs., 1 tab.« less

Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

PubMed

Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

2016-06-28

Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

Relationship of Baseline Hemoglobin Level with Serum Ferritin, Postphlebotomy Hemoglobin Changes, and Phlebotomy Requirements among HFE C282Y Homozygotes

PubMed Central

Mousavi, Seyed Ali; Mahmood, Faiza; Aandahl, Astrid; Knutsen, Teresa Risopatron; Llohn, Abid Hussain

2015-01-01

Objectives. We aimed to examine whether baseline hemoglobin levels in C282Y-homozygous patients are related to the degree of serum ferritin (SF) elevation and whether patients with different baseline hemoglobin have different phlebotomy requirements. Methods. A total of 196 patients (124 males and 72 females) who had undergone therapeutic phlebotomy and had SF and both pre- and posttreatment hemoglobin values were included in the study. Results. Bivariate correlation analysis suggested that baseline SF explains approximately 6 to 7% of the variation in baseline hemoglobin. The results also showed that males who had higher (≥150 g/L) baseline hemoglobin levels had a significantly greater reduction in their posttreatment hemoglobin despite requiring fewer phlebotomies to achieve iron depletion than those who had lower (<150 g/L) baseline hemoglobin, regardless of whether baseline SF was below or above 1000 µg/L. There were no significant differences between hemoglobin subgroups regarding baseline and treatment characteristics, except for transferrin saturation between male subgroups with SF above 1000 µg/L. Similar differences were observed when females with higher (≥138 g/L) baseline hemoglobin were compared with those with lower (<138 g/L) baseline hemoglobin. Conclusion. Dividing C282Y-homozygous patients into just two subgroups according to the degree of baseline SF elevation may obscure important subgroup variations. PMID:26380265

dUTPase (DUT) Is Mutated in a Novel Monogenic Syndrome With Diabetes and Bone Marrow Failure.

PubMed

Dos Santos, Reinaldo Sousa; Daures, Mathilde; Philippi, Anne; Romero, Sophie; Marselli, Lorella; Marchetti, Piero; Senée, Valérie; Bacq, Delphine; Besse, Céline; Baz, Baz; Marroquí, Laura; Ivanoff, Sarah; Masliah-Planchon, Julien; Nicolino, Marc; Soulier, Jean; Socié, Gérard; Eizirik, Decio L; Gautier, Jean-François; Julier, Cécile

2017-04-01

We describe a new syndrome characterized by early-onset diabetes associated with bone marrow failure, affecting mostly the erythrocytic lineage. Using whole-exome sequencing in a remotely consanguineous patient from a family with two affected siblings, we identified a single homozygous missense mutation (chr15.hg19:g.48,626,619A>G) located in the dUTPase ( DUT ) gene (National Center for Biotechnology Information Gene ID 1854), affecting both the mitochondrial (DUT-M p.Y142C) and the nuclear (DUT-N p.Y54C) isoforms. We found the same homozygous mutation in an unrelated consanguineous patient with diabetes and bone marrow aplasia from a family with two affected siblings, whereas none of the >60,000 subjects from the Exome Aggregation Consortium (ExAC) was homozygous for this mutation. This replicated observation probability was highly significant, thus confirming the role of this DUT mutation in this syndrome. DUT is a key enzyme for maintaining DNA integrity by preventing misincorporation of uracil into DNA, which results in DNA toxicity and cell death. We showed that DUT silencing in human and rat pancreatic β-cells results in apoptosis via the intrinsic cell death pathway. Our findings support the importance of tight control of DNA metabolism for β-cell integrity and warrant close metabolic monitoring of patients treated by drugs affecting dUTP balance. © 2017 by the American Diabetes Association.

Functional analysis of 'a' determinant mutations associated with occult HBV in HIV-positive South Africans.

PubMed

Powell, Eleanor A; Boyce, Ceejay L; Gededzha, Maemu P; Selabe, Selokela G; Mphahlele, M Jeffrey; Blackard, Jason T

2016-07-01

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in the absence of hepatitis B surface antigen (HBsAg). Occult HBV is associated with the development of hepatocellular carcinoma, reactivation during immune suppression, and virus transmission. Viral mutations contribute significantly to the occult HBV phenotype. Mutations in the 'a' determinant of HBsAg are of particular interest, as these mutations are associated with immune escape, vaccine escape and diagnostic failure. We examined the effects of selected occult HBV-associated mutations identified in a population of HIV-positive South Africans on HBsAg production in vitro. Mutations were inserted into two different chronic HBV backbones and transfected into a hepatocyte-derived cell line. HBsAg levels were quantified by enzyme-linked immunosorbent assay (ELISA), while the detectability of mutant HBsAg was determined using an HA-tagged HBsAg expression system. Of the seven mutations analysed, four (S132P, C138Y, N146D and C147Y) resulted in decreased HBsAg expression in one viral background but not in the second viral background. One mutation (N146D) led to a decrease in HBsAg detected as compared to HA-tag, indicating that this mutation compromises the ability of the ELISA to detect HBsAg. The contribution of occult-associated mutations to the HBsAg-negative phenotype of occult HBV cannot be determined adequately by testing the effect of the mutation in a single viral background, and rigorous analysis of these mutations is required.

Functional analysis of ‘a’ determinant mutations associated with occult HBV in HIV-positive South Africans

PubMed Central

Powell, Eleanor A.; Boyce, Ceejay L.; Gededzha, Maemu P.; Selabe, Selokela G.; Mphahlele, M. Jeffrey

2016-01-01

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in the absence of hepatitis B surface antigen (HBsAg). Occult HBV is associated with the development of hepatocellular carcinoma, reactivation during immune suppression, and virus transmission. Viral mutations contribute significantly to the occult HBV phenotype. Mutations in the ‘a’ determinant of HBsAg are of particular interest, as these mutations are associated with immune escape, vaccine escape and diagnostic failure. We examined the effects of selected occult HBV-associated mutations identified in a population of HIV-positive South Africans on HBsAg production in vitro. Mutations were inserted into two different chronic HBV backbones and transfected into a hepatocyte-derived cell line. HBsAg levels were quantified by enzyme-linked immunosorbent assay (ELISA), while the detectability of mutant HBsAg was determined using an HA-tagged HBsAg expression system. Of the seven mutations analysed, four (S132P, C138Y, N146D and C147Y) resulted in decreased HBsAg expression in one viral background but not in the second viral background. One mutation (N146D) led to a decrease in HBsAg detected as compared to HA-tag, indicating that this mutation compromises the ability of the ELISA to detect HBsAg. The contribution of occult-associated mutations to the HBsAg-negative phenotype of occult HBV cannot be determined adequately by testing the effect of the mutation in a single viral background, and rigorous analysis of these mutations is required. PMID:27031988

HFE gene variants, iron, and lipids: a novel connection in Alzheimer's disease.

PubMed

Ali-Rahmani, Fatima; Schengrund, Cara-Lynne; Connor, James R

2014-01-01

Iron accumulation and associated oxidative stress in the brain have been consistently found in several neurodegenerative diseases. Multiple genetic studies have been undertaken to try to identify a cause of neurodegenerative diseases but direct connections have been rare. In the iron field, variants in the HFE gene that give rise to a protein involved in cellular iron regulation, are associated with iron accumulation in multiple organs including the brain. There is also substantial epidemiological, genetic, and molecular evidence of disruption of cholesterol homeostasis in several neurodegenerative diseases, in particular Alzheimer's disease (AD). Despite the efforts that have been made to identify factors that can trigger the pathological events associated with neurodegenerative diseases they remain mostly unknown. Because molecular phenotypes such as oxidative stress, synaptic failure, neuronal loss, and cognitive decline, characteristics associated with AD, have been shown to result from disruption of a number of pathways, one can easily argue that the phenotype seen may not arise from a linear sequence of events. Therefore, a multi-targeted approach is needed to understand a complex disorder like AD. This can be achieved only when knowledge about interactions between the different pathways and the potential influence of environmental factors on them becomes available. Toward this end, this review discusses what is known about the roles and interactions of iron and cholesterol in neurodegenerative diseases. It highlights the effects of gene variants of HFE (H63D- and C282Y-HFE) on iron and cholesterol metabolism and how they may contribute to understanding the etiology of complex neurodegenerative diseases.

HFE gene variants, iron, and lipids: a novel connection in Alzheimer’s disease

PubMed Central

Ali-Rahmani, Fatima; Schengrund, Cara-Lynne; Connor, James R.

2014-01-01

Iron accumulation and associated oxidative stress in the brain have been consistently found in several neurodegenerative diseases. Multiple genetic studies have been undertaken to try to identify a cause of neurodegenerative diseases but direct connections have been rare. In the iron field, variants in the HFE gene that give rise to a protein involved in cellular iron regulation, are associated with iron accumulation in multiple organs including the brain. There is also substantial epidemiological, genetic, and molecular evidence of disruption of cholesterol homeostasis in several neurodegenerative diseases, in particular Alzheimer’s disease (AD). Despite the efforts that have been made to identify factors that can trigger the pathological events associated with neurodegenerative diseases they remain mostly unknown. Because molecular phenotypes such as oxidative stress, synaptic failure, neuronal loss, and cognitive decline, characteristics associated with AD, have been shown to result from disruption of a number of pathways, one can easily argue that the phenotype seen may not arise from a linear sequence of events. Therefore, a multi-targeted approach is needed to understand a complex disorder like AD. This can be achieved only when knowledge about interactions between the different pathways and the potential influence of environmental factors on them becomes available. Toward this end, this review discusses what is known about the roles and interactions of iron and cholesterol in neurodegenerative diseases. It highlights the effects of gene variants of HFE (H63D- and C282Y-HFE) on iron and cholesterol metabolism and how they may contribute to understanding the etiology of complex neurodegenerative diseases. PMID:25071582

Crystal chemistry of hydrous phases in the Al2O3-Fe2O3-H2O system: implications for water cycle in the deep lower mantle

NASA Astrophysics Data System (ADS)

Zhang, L.

2016-12-01

Hydrous minerals play an important role in the transportation and storage of water in the Earth's interior. Recently a pyrite-structured iron oxide (FeO2) (P-phase) was found stable at 76 GPa and 1800 K [1] and this discovery has brought new insights into the H2-O2 cycles in the deep mantle. In this study, we perform in situ synchrotron X-ray experiments in the Al2O3-Fe2O3-H2O system in a laser-heated diamond anvil cell (DAC) at P-T conditions in the deep lower mantle. The new results added more complexity to the H2-O2/H2O cycles in the deep lower mantle. The symmetry and unit-cell parameters of each phase in the run products were determined using the multigrain approach [2]. On the other hand, the d-H solid solution AlOOH-MgSiO2(OH)2 is the stable hydrous phase coexisting with bridgmanite or post-perovskite under equilibrium P-T conditions to the deepest lower mantle [3]. The detailed crystal chemistry of the newly found hydrous phases and its relations to the d-H phase have been investigated using both first-principles calculations and experiments, providing new understanding to the hydration mechanism and water storage in the deep mantle. It is worth mentioning that recent development in high pressure multigrain method has realized separation of each individual phase in a multiphase assemblage and even allowed in situ crystal structure determination of a minor phase in the assemblage contained in a DAC [4]. [1] Q. Hu, D. Y. Kim, W. Yang, L. Yang, Y. Meng, L. Zhang, and H. K. Mao, Nature 534, 241 (2016). [2] H. O. Sørensen et al., Zeitschrift für Kristallographie 227, 63 (2012). [3] I. Ohira, E. Ohtani, T. Sakai, M. Miyahara, N. Hirao, Y. Ohishi, and M. Nishijima, Earth and Planetary Science Letters 401, 12 (2014). [4] L. Zhang, D. Popov, Y. Meng, J. Wang, C. Ji, B. Li, and H.-k. Mao, American Mineralogist 101, 231 (2016).

Sensitivity Analysis of Empirical Parameters in the Ionosphere-Plasmasphere Model

DTIC Science & Technology

2011-03-01

IFM output grid (Gardner , 2010). The longitudes have to be fit as well because the IPM flux tubes form a type of ‘ S ...n d 2 0 d a y ru n , a n d th e ri g h t p lo t is th e d iff e re n c e . A ft e r d a y fi v e , th e m o d e l o u tp u t h a s re a ch e d st e a ...lo c a ti o n s * . T h

Proto-oncogene activation in liver tumors of hepatocarcinogenesis-resistant strains of mice.

PubMed

Stanley, L A; Devereux, T R; Foley, J; Lord, P G; Maronpot, R R; Orton, T C; Anderson, M W

1992-12-01

Activation of the ras family of oncogenes occurs frequently in liver tumors of the B6C3F1 mouse, a strain which is highly sensitive to hepatocarcinogenesis. Many other mouse strains are much more resistant to hepatocarcinogenesis; the aim of this study was to determine the frequency and pattern of oncogene activation in spontaneous and chemically induced liver tumors of three such strains, the C57BL/6J, the C57BL/6 x DBA/2 F1 hybrid (B6D2F1) and the C57BL/6 x Balb/c F1 hybrid (B6BCF1). The C57BL/6, DBA/2 and Balb/c strains are all relatively resistant to spontaneous hepatocarcinogenesis (1.5-3.6% of animals develop liver tumors in 2 years); with regard to chemically induced hepatocarcinogenesis the Balb/c is highly resistant, the C57BL/6 has low susceptibility and the DBA/2 has low to moderate susceptibility. The nude mouse tumorigenicity assay was used to search for activated oncogenes in 15 C57BL/6J liver tumors induced by a single neonatal dose of vinyl carbamate (VC, 0.15 mumol/g body weight). Three tumors contained H-ras genes activated by point mutations at codon 61 and one contained a non-ras oncogene. The polymerase chain reaction and allele-specific oligonucleotide hybridization were used to study H-ras mutations in spontaneous and VC-induced tumors from all three strains of mice. The frequency of H-ras codon 61 mutations in tumors induced by 0.15 mumol/g body weight VC in the C57BL/6J mouse (5/37) was similar to that in spontaneous tumors (2/9); surprisingly, tumors induced by a lower dose of VC (0.03 mumol/g body weight) had a higher frequency of H-ras mutations (12/28). The frequencies of H-ras activation detected in VC (0.03 mumol/g body weight)-induced tumors from the two F1 hybrids studied differed markedly. Only one VC-induced B6BCF1 tumor contained a mutated H-ras gene (1/10), whereas the majority of B6D2F1 tumors contained such mutations (23/33). Several spontaneous B6D2F1 liver tumors contained H-ras codon 61 mutations (6/15). Thus, H-ras activation frequency does not determine susceptibility to hepatocarcinogenesis in inbred mice and their F1 hybrids, since a relatively high frequency of H-ras mutations was observed in two resistant strains and a low frequency was found in the other strain.

Navy Nearshore Ocean Prediction Systems

DTIC Science & Technology

2014-09-01

SYSTEMS B Y J AYA R A M V E E R A M O N Y, M A R K D . O R Z E C H , K A C E Y L . E D W A R D S , M I C H A E L G I L L I G A N , J E I K O O...tthor(s) Name(s) (FirstMl est), Code, Afliliation not NR,L / layaram Vet~ramony 7 Mark D 01’%ech 732 Kacey L...Edwards 732{ Michael Gilligan NAVO Eric Terrill Unlv. of CA- Soripp~ rony dePaolo Unlv. of CA. Scripps s intend~ d to offer this paper to the (Nt;~m& of

Characterization and mutational analysis of a nicotinamide mononucleotide deamidase from Agrobacterium tumefaciens showing high thermal stability and catalytic efficiency

PubMed Central

Martínez-Moñino, Ana Belén; Zapata-Pérez, Rubén; García-Saura, Antonio Ginés; Gil-Ortiz, Fernando; Pérez-Gilabert, Manuela

2017-01-01

NAD+ has emerged as a crucial element in both bioenergetic and signaling pathways since it acts as a key regulator of cellular and organismal homeostasis. Among the enzymes involved in its recycling, nicotinamide mononucleotide (NMN) deamidase is one of the key players in the bacterial pyridine nucleotide cycle, where it catalyzes the conversion of NMN into nicotinic acid mononucleotide (NaMN), which is later converted to NAD+ in the Preiss-Handler pathway. The biochemical characteristics of bacterial NMN deamidases have been poorly studied, although they have been investigated in some firmicutes, gamma-proteobacteria and actinobacteria. In this study, we present the first characterization of an NMN deamidase from an alphaproteobacterium, Agrobacterium tumefaciens (AtCinA). The enzyme was active over a broad pH range, with an optimum at pH 7.5. Moreover, the enzyme was quite stable at neutral pH, maintaining 55% of its activity after 14 days. Surprisingly, AtCinA showed the highest optimal (80°C) and melting (85°C) temperatures described for an NMN deamidase. The above described characteristics, together with its high catalytic efficiency, make AtCinA a promising biocatalyst for the production of pure NaMN. In addition, six mutants (C32A, S48A, Y58F, Y58A, T105A and R145A) were designed to study their involvement in substrate binding, and two (S31A and K63A) to determine their contribution to the catalysis. However, only four mutants (C32A, S48A Y58F and T105A) showed activity, although with reduced catalytic efficiency. These results, combined with a thermal and structural analysis, reinforce the Ser/Lys catalytic dyad mechanism as the most plausible among those proposed. PMID:28388636

Enhanced production of d-lactic acid by Sporolactobacillus sp.Y2-8 mutant generated by atmospheric and room temperature plasma.

PubMed

Sun, Jiaduo; Wang, Yan; Wu, Bin; Bai, Zhongzhong; He, Bingfang

2015-01-01

To improve the production of d-lactic acid, atmospheric and room temperature plasma (ARTP) was used to generate mutations in Sporolactobacillus sp. Y2-8. An efficient mutant YBS1-5 was rapidly isolated by implanting ARTP twice with a 100 W radio-frequency power input, 10 standard liters per minute of the helium flow, and a 2 mm treatment distance. Significant improvement of d-lactic acid productivity (1.39 g L(-1) H(-1) ) by YBS1-5 was achieved, and it was 41.84% higher than the productivity (0.98 g L(-1) H(-1) ) of Y2-8. Moreover, the dry cell weight of YBS1-5 was 16.7% higher than that of Y2-8. Metabolic activities of concerned substrates related with key enzymes of d-lactic acid fermentation were analyzed by Biolog approach. Results showed that the activities of the key enzymes glucokinase and d-lactate dehydrogenase in mutant YBS1-5 were increased by approximately 45% and 66%, respectively, in comparison with those of the strain Y2-8. Fed-batch fermentation further improved the productivity; 127 g L(-1) d-lactic acid in 74 H by YBS1-5 with higher productivity (1.72 g L(-1) H(-1) ) was achieved. The subculture experiments indicated that YBS1-5 was genetically stable after eight generations. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

The Y-chromosome haplogroup C3*-F3918, likely attributed to the Mongol Empire, can be traced to a 2500-year-old nomadic group.

PubMed

Zhang, Ye; Wu, Xiyan; Li, Jiawei; Li, Hongjie; Zhao, Yongbin; Zhou, Hui

2018-02-01

The Mongol Empire had a significant role in shaping the landscape of modern populations. Many populations living in Eurasia may have been the product of population mixture between ancient Mongolians and natives following the expansion of Mongol Empire. Geneticists have found that most of these populations carried the Y-haplogroup C3* (C-M217). To trace the history of haplogroup (Hg) C3* and to further understand the origin and development of Mongolians, ancient human remains from the Jinggouzi, Chenwugou and Gangga archaeological sites, which belonged to the Donghu, Xianbei and Shiwei, respectively, were analysed. Our results show that nine of the eleven males of the Gangga site, two of the eight males of Chengwugou site and all of the twelve males of Jinggouzi site were found to have mutations at M130 (Hg C), M217 (Hg C3), L1373 (C2b, ISOGG2015), with the absence of mutations at M93 (Hg C3a), P39 (Hg C3b), M48 (Hg C3c), M407 (Hg C3d) and P62 (Hg C3f). These samples were attributed to the Y-chromosome Hg C3* (Hg C2b, ISOGG2015), and most of them were further typed as Hg C2b1a based on the mutation at F3918. Finally, we inferred that the Y-chromosome Hg C3*-F3918 can trace its origins to the Donghu ancient nomadic group.

Substitutions at Amino Acid Positions 143, 148, and 155 of HIV-1 Integrase Define Distinct Genetic Barriers to Raltegravir Resistance In Vivo

PubMed Central

Fransen, Signe; Gupta, Soumi; Frantzell, Arne; Petropoulos, Christos J.

2012-01-01

Mutations at amino acids 143, 148, and 155 in HIV-1 integrase (IN) define primary resistance pathways in subjects failing raltegravir (RAL)-containing treatments. Although each pathway appears to be genetically distinct, shifts in the predominant resistant virus population have been reported under continued drug pressure. To better understand this dynamic, we characterized the RAL susceptibility of 200 resistant viruses, and we performed sequential clonal analysis for selected cases. Patient viruses containing Y143R, Q148R, or Q148H mutations consistently exhibited larger reductions in RAL susceptibility than patient viruses containing N155H mutations. Sequential analyses of virus populations from three subjects revealed temporal shifts in subpopulations representing N155H, Y143R, or Q148H escape pathways. Evaluation of molecular clones isolated from different time points demonstrated that Y143R and Q148H variants exhibited larger reductions in RAL susceptibility and higher IN-mediated replication capacity (RC) than N155H variants within the same subject. Furthermore, shifts from the N155H pathway to either the Q148R or H pathway or the Y143R pathway were dependent on the amino acid substitution at position 148 and the secondary mutations in Y143R- or Q148R- or H-containing variants and correlated with reductions in RAL susceptibility and restorations in RC. Our observations in patient viruses were confirmed by analyzing site-directed mutations. In summary, viruses that acquire mutations defining the 143 or 148 escape pathways are less susceptible to RAL and exhibit greater RC than viruses containing 155 pathway mutations. These selective pressures result in the displacement of N155H variants by 143 or 148 variants under continued drug exposure. PMID:22553340

Far Infrared, Magnetic and Electronic Studies of High Tc Superconducting Materials

DTIC Science & Technology

1992-09-30

Phys. Rev. Left. 63, 2421(1989). 8. K. H. Fischer and T. Nattermann, Phys. Rev. .43, 10372(1991). 9. R. E. Walstedt, R. F. Bell, and D. B. Mitzi , Phys...Duran, J. Yazyi, F. dela Cruz, D. J. Bishop, D. B. Mitzi , and A. Kapitulnik, Phys. Rev. B 44, 17737(1991). 14. Y. Yeshurun and A. P. Malozemoff, Phys

Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae.

PubMed

Bartish, Galyna; Nygård, Odd

2008-05-01

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, I69M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, I69M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and I69M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to I69) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.

A novel c.1037C > G (p.Ala346Gly) mutation in TP63 as cause of the ectrodactyly-ectodermal dysplasia and cleft lip/palate (EEC) syndrome

PubMed Central

Alves, Leandro Ucela; Pardono, Eliete; Otto, Paulo A.; Mingroni Netto, Regina Célia

2015-01-01

Ectrodactyly – ectodermal dysplasia and cleft lip/palate (EEC) syndrome (OMIM 604292) is a rare disorder determined by mutations in the TP63 gene. Most cases of EEC syndrome are associated to mutations in the DNA binding domain (DBD) region of the p63 protein. Here we report on a three-generation Brazilian family with three individuals (mother, son and grandfather) affected by EEC syndrome, determined by a novel mutation c.1037C > G (p.Ala346Gly). The disorder in this family exhibits a broad spectrum of phenotypes: two individuals were personally examined, one presenting the complete constellation of EEC syndrome manifestations and the other presenting an intermediate phenotype; the third affected, a deceased individual not examined personally and referred to by his daughter, exhibited only the split-hand/foot malformation (SHFM). Our findings contribute to elucidate the complex phenotype-genotype correlations in EEC syndrome and other related TP63-mutation syndromes. The possibility of the mutation c.1037C > G being related both to acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome and SHFM is also raised by the findings here reported. PMID:25983622

Mutation screening of USH3 gene (clarin-1) in Spanish patients with Usher syndrome: low prevalence and phenotypic variability.

PubMed

Aller, E; Jaijo, T; Oltra, S; Alió, J; Galán, F; Nájera, C; Beneyto, M; Millán, J M

2004-12-01

Usher syndrome type III is an autosomal recessive disorder clinically characterized by the association of retinitis pigmentosa (RP), variable presence of vestibular dysfunction and progressive hearing loss, being the progression of the hearing impairment the critical parameter classically used to distinguish this form from Usher syndrome type I and Usher syndrome type II. Usher syndrome type III clinical subtype is the rarest form of Usher syndrome in Spain, accounting only for 6% of all Usher syndrome Spanish cases. The gene responsible for Usher syndrome type III is named clarin-1 and it is thought to be involved in hair cell and photoreceptor cell synapses. Here, we report a screening for mutations in clarin-1 gene among our series of Usher syndrome Spanish patients. Clarin-1 has been found to be responsible for the disease in only two families: the first one is a previously reported family homozygous for Y63X mutation and the second one, described here, is homozygous for C40G. This accounts for 1.7% of Usher syndrome Spanish families. It is noticeable that, whereas C40G family is clinically compatible with Usher syndrome type III due to the progression of the hearing loss, Y63X family could be diagnosed as Usher syndrome type I because the hearing impairment is profound and stable. Thus, we consider that the progression of hearing loss is not the definitive key parameter to distinguish Usher syndrome type III from Usher syndrome type I and Usher syndrome type II.

Chaperone Function in Androgen-Independent Prostate Cancer

DTIC Science & Technology

2012-05-01

differentiation. Proc Natl Acad Sci U S A 92, 11081-11085. Yong, W., Yang, Z., Periyasamy, S., Chen, H., Yucel, S., Li, W., Lin, L. Y., Wolf , I. M., Cohn, M...Biol Chem 282, 5026-5036. Yong, W., Yang, Z., Periyasamy, S., Chen, H., Yucel, S., Li, W., Lin, L. Y., Wolf , I. M., Cohn, M. J., Baskin, L. S., et... Reintroduction of AR into PC-3 cells activates the endogenous FKBP51 gene, an observation that is consistent with AR regulation of FKBP51 expression

A novel pathogenic mutation of the CYP27B1 gene in a patient with vitamin D-dependent rickets type 1: a case report.

PubMed

Babiker, Amir M I; Al Gadi, Iman; Al-Jurayyan, Nasir A M; Al Nemri, Abdulrahman M H; Al Haboob, Ali Abdu N; Al Boukai, Ahmed Amer; Al Zahrani, Ali; Habib, Hanan Ahmed

2014-11-05

Rickets can occur due to Vitamin D deficiency or defects in its metabolism. Three rare genetic types of rickets with different alterations of genes have been reported, including: Vitamin D dependent rickets type 1, Vitamin D dependent rickets type 2 or also known as Vitamin D resistant rickets and 25 hydroxylase deficiency rickets. Vitamin D dependent rickets type 1 is inherited in an autosomal recessive pattern, and is caused by mutations in the CYP27B1 gene encoding the 1α-hydroxylase enzyme. We report here a new mutation in CYP27B1, which lead to Vitamin D dependent rickets type 1. We report on a 13-month-old Arabic Saudi girl with Vitamin D dependent rickets type 1 presented with multiple fractures and classic features of rickets. A whole exome sequencing identified a novel pathogenic missense mutation (CYP27B1:Homozygous c.1510C > T(p.Q504X)) which results in a protein truncating alteration. Both parents are heterozygous carriers of the mutation. Based on data search in Human Gene Mutation Database, 63 CYP27B1 alterations were reported: only 28.6% are protein truncating (5 nonsense, 13 frameshift insertions/deletions, 0 gross deletions), while 61.9% are non-truncating (38 missense, 1 small in-frame insertions/deletion), and 9.5% are possible protein-truncating (5 splice, 1 regulatory). The deleterious effect of this alteration, which was the only mutation detected in the CYP27B1 common gene of Vitamin D dependent rickets type 1 in the proband, and its autosomal recessive inheritance fashion, both support a pathogenic nature of this mutation as the cause of Vitamin D dependent rickets type 1.

Systems Engineering Measurement Primer

DTIC Science & Technology

1998-03-01

Schermerhorn , R ., “Determining metrics for...maintenance process. 12 S e l e c t a n d S p e c i f y M e a s u r e s a n d I n d i c a t o r s C o l l e c t D a t a C a l c u l a t e I n d i c a...t o r s A n a l y z e t h e M e a s u r e s o r I n d i c a t o r s R e p o r t a n d U s e t h e R e s u l t s Issues Goals Risks A

Socio-behavioral considerations in the role of militant extremism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernard, Michael Lewis; Backus, George A.; Beyeler, Walter E.

This paper discusses relevant findings and theories regarding the role of ideology, culture, and context in shaping the behaviors of individuals within violent social movements. Accordingly, this focus concerns the comparative weight placed on ideology and culture (expressed principles and motives) versus external factors as chief influencers for the propensity of individuals to act outside of the norms of society and politics by resorting to violent behaviors. In doing so, we have drawn upon theory from anthropology, behavioral economics, political science, psychology, and sociology to better understand how these variables give birth to and nurture militant social movements. F umore » r t h e r d i s s e m i n a t i o n o n l y a s a u t h o r i z e d t o U . S . G o v e r n m e n t a g e n c i e s a n d t h e i r c o n t r a c t o r s ; o t h e r r e q u e s t s s h a l l b e a p p r o v e d b y t h e o r i g i n a t i n g f a c i l i t y o r h i g h e r D O E p r o g r a m m a t i c a u t h o r i t y .« less

SOCIO-BEHAVIORALCONSIDERATIONS IN THE ROLE OF MILITANT EXTREMISM.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernard, Michael Lewis; Backus, George A.; Beyeler, Walter E.

This paper discusses relevant findings and theories regarding the role of ideology, culture, and context in shaping the behaviors of individuals within violent social movements. Accordingly, this focus concerns the comparative weight placed on ideology and culture (expressed principles and motives) versus external factors as chief influencers for the propensity of individuals to act outside of the norms of society and politics by resorting to violent behaviors. In doing so, we have drawn upon theory from anthropology, behavioral economics, political science, psychology, and sociology to better understand how these variables give birth to and nurture militant social movements. F umore » r t h e r d i s s e m i n a t i o n o n l y a s a u t h o r i z e d t o U . S . G o v e r n m e n t a g e n c i e s a n d t h e i r c o n t r a c t o r s ; o t h e r r e q u e s t s s h a l l b e a p p r o v e d b y t h e o r i g i n a t i n g f a c i l i t y o r h i g h e r D O E p r o g r a m m a t i c a u t h o r i t y .« less

Carriers of the Complex Allele HFE c.[187C>G;340+4T>C] Have Increased Risk of Iron Overload in São Miguel Island Population (Azores, Portugal)

PubMed Central

Bulhões, Sara; Brilhante, Maria José; Pereirinha, Tânia; Cabral, Rita; Rego, Ana Catarina; Fraga, Cristina; Miguel, António G.; Brasil, Gracinda; Macedo, Paula; Mota-Vieira, Luisa

2015-01-01

Iron overload is associated with acquired and genetic conditions, the most common being hereditary hemochromatosis (HH) type-I, caused by HFE mutations. Here, we conducted a hospital-based case-control study of 41 patients from the São Miguel Island (Azores, Portugal), six belonging to a family with HH type-I pseudodominant inheritance, and 35 unrelated individuals fulfilling the biochemical criteria of iron overload compatible with HH type-I. For this purpose, we analyzed the most common HFE mutations– c.845G>A [p.Cys282Tyr], c.187C>G [p.His63Asp], and c.193A>T [p.Ser65Cys]. Results revealed that the family’s HH pseudodominant pattern is due to consanguineous marriage of HFE-c.845G>A carriers, and to marriage with a genetically unrelated spouse that is a -c.187G carrier. Regarding unrelated patients, six were homozygous for c.845A, and three were c.845A/c.187G compound heterozygous. We then performed sequencing of HFE exons 2, 4, 5 and their intron-flanking regions. No other mutations were observed, but we identified the -c.340+4C [IVS2+4C] splice variant in 26 (74.3%) patients. Functionally, the c.340+4C may generate alternative splicing by HFE exon 2 skipping and consequently, a protein missing the α1-domain essential for HFE/ transferrin receptor-1 interactions. Finally, we investigated HFE mutations configuration with iron overload by determining haplotypes and genotypic profiles. Results evidenced that carriers of HFE-c.187G allele also carry -c.340+4C, suggesting in-cis configuration. This data is corroborated by the association analysis where carriers of the complex allele HFE-c.[187C>G;340+4T>C] have an increased iron overload risk (RR = 2.08, 95% CI = 1.40−2.94, p<0.001). Therefore, homozygous for this complex allele are at risk of having iron overload because they will produce two altered proteins—the p.63Asp [c.187G], and the protein lacking 88 amino acids encoded by exon 2. In summary, we provide evidence that the complex allele HFE-c.[187C>G;340+4T>C] has a role, as genetic predisposition factor, on iron overload in the São Miguel population. Independent replication studies in other populations are needed to confirm this association. PMID:26501199

Does oxidative stress modulate left ventricular diastolic function in asymptomatic subjects with hereditary hemochromatosis?

PubMed

Shizukuda, Yukitaka; Bolan, Charles D; Tripodi, Dorothy J; Sachdev, Vandana; Nguyen, Tammy T; Botello, Gilberto; Yau, Yu-Ying; Sidenko, Stanislav; Inez, Ernst; Ali, Mir I; Waclawiw, Myron A; Leitman, Susan F; Rosing, Douglas R

2009-11-01

Little is known about the early mechanisms mediating left ventricular (LV) diastolic dysfunction in patients with hereditary hemochromatosis (HH). However, the increased oxidative stress related to iron overload may be involved in this process, and strain rate (SR), a sensitive echocardiography-derived measure of diastolic function, may detect such changes. we evaluated the relationship between left ventricular diastolic function measured with tissue Doppler SR and oxidative stress in asymptomatic HH subjects and control normal subjects. Ninety-four consecutive visits of 43 HH subjects, age 30-74 (50 +/- 10, mean +/- SD), and 37 consecutive visits of 21 normal volunteers, age 30-63 (48 +/- 8), were evaluated over a 3-year period. SR was obtained from the basal septum in apical four-chamber views. All patients had confirmed C282Y homozygosity, a documented history of iron overload, and were New York Heart Association functional class I. Normal volunteers lacked HFE gene mutations causing HH. In the HH subjects, the SR demonstrated moderate but significant correlations with biomarkers of oxidative stress; however, no correlations were noted in normal subjects. The biomarkers of iron overload per se did not show significant correlations with the SR. Although our study was limited by the relatively small subject number, these results suggest that a possible role of oxidative stress to affect LV diastolic function in asymptomatic HH subjects and SR imaging may be a sensitive measure to detect that effect.

Steroid Biomarkers and Genetic Studies Reveal Inactivating Mutations in Hexose-6-Phosphate Dehydrogenase in Patients with Cortisone Reductase Deficiency

PubMed Central

Lavery, Gareth G.; Walker, Elizabeth A.; Tiganescu, Ana; Ride, Jon P.; Shackleton, Cedric H. L.; Tomlinson, Jeremy W.; Connell, John M. C.; Ray, David W.; Biason-Lauber, Anna; Malunowicz, Ewa M.; Arlt, Wiebke; Stewart, Paul M.

2008-01-01

Context: Cortisone reductase deficiency (CRD) is characterized by a failure to regenerate cortisol from cortisone via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), resulting in increased cortisol clearance, activation of the hypothalamic-pituitary-axis (HPA) and ACTH-mediated adrenal androgen excess. 11β-HSD1 oxoreductase activity requires the reduced nicotinamide adenine dinucleotide phosphate-generating enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the endoplasmic reticulum. CRD manifests with hyperandrogenism resulting in hirsutism, oligo-amenorrhea, and infertility in females and premature pseudopuberty in males. Recent association studies have failed to corroborate findings that polymorphisms in the genes encoding H6PDH (R453Q) and 11β-HSD1 (Intron 3 inserted adenine) interact to cause CRD. Objective: Our objective was to reevaluate the genetics and steroid biochemistry of patients with CRD. Design: We analyzed 24-h urine collection for steroid biomarkers by gas chromatography/mass spectrometry and sequenced the HSD11B1 and H6PD genes in our CRD cohort. Patients: Patients included four cases presenting with hyperandrogenism and biochemical features clearly indicative of CRD. Results: Gas chromatography/mass spectrometry identified steroid biomarkers that correlated with CRD in each case. Three cases were identified as homozygous (R109AfsX3, Y316X, and G359D) and one case identified as compound heterozygous (c.960G→A and D620fsX3) for mutations in H6PD. No mutations affecting enzyme activity were identified in the HSD11B1 gene. Expression and activity assays demonstrate loss of function for all reported H6PDH mutations. Conclusions: CRD is caused by inactivating mutations in the H6PD gene, rendering the 11β-HSD1 enzyme unable to operate as an oxoreductase, preventing local glucocorticoid regeneration. These data highlight the importance of the redox control of cortisol metabolism and the 11β-HSD1-H6PDH pathway in regulating hypothalamic-pituitary-adrenal axis activity. PMID:18628520

Phenotypic and genotypic analysis of clarithromycin-resistant Helicobacter pylori from Bogotá D.C., Colombia.

PubMed

Trespalacios, Alba A; Otero, William; Caminos, Jorge E; Mercado, Marcela M; Avila, Jenny; Rosero, Liliana E; Arévalo, Azucena; Poutou-Piñales, Raúl A; Graham, David Y

2013-08-01

Resistance of Helicobacter pylori to clarithromycin is the most common cause of treatment failure in patients with H. pylori infections. This study describes the MICs and the presence of 23S rRNA mutations of H. pylori isolates from Bogotá, D.C., Colombia. H. pylori were isolated from gastric biopsies from patients with functional dyspepsia. Clarithromycin susceptibility was investigated by agar dilution and strains were considered resistant if the MIC was ≥ 1 μg/ml. DNA sequences of the 23S rRNA gene of strains resistant and sensitive to clarithromycin were determined to identify specific point mutations. Clarithromycin resistance was present in 13.6% of patients by agar dilution. The A2143G, A2142G and A2142C mutations were found in 90.5, 7.1, and 2.4% of H. pylori strains with resistance genotype.The resistant phenotype was associated with 23S rRNA resistance genotype in 85.7% of isolates. The point mutations in 23S rRNA were well correlated with MICs values for clarithromycin.

Mutation screening of INS and KCNJ11 genes in Taiwanese children with type 1B diabetic onset before the age of 5 years.

PubMed

Lo, Fu-Sung

2018-01-17

Type 1 diabetes (T1D) is caused by β-cell destruction, usually leading to absolute insulin deficiency. T1D is a heterogeneous disease and is divided into two subtypes according to the presence or absence of pancreatic autoantibodies: type 1A (immune mediated) and type 1B (idiopathic). Genes such as KCNJ11 or INS, which play key roles in β-cell function, provide some insight into the pathogenesis of type 1B diabetes. In this study, we screened 110 Taiwanese children (61 males and 49 females) with T1D onset before the age of 5 years for mutations of INS and KCNJ11. We identified one missense heterozygous mutation in KCNJ11 (c.989A>G, p.Y330C) and no INS mutations among 28 probands. This is the first study to screen patients with autoantibody-negative T1D diagnosed before the age of 5 years for INS and KCNJ11 mutations in Taiwan. Although KCNJ11 mutations are always reported in patients with permanent neonatal diabetes, this gene mutation can be detected after 6 months of age. Further studies in other patients with type 1B diabetes and their families are required to elucidate the contributions of the KCNJ11 mutation to the T1D phenotype. Copyright © 2018. Published by Elsevier B.V.

Cystic fibrosis presenting with neonatal cholestasis simulating biliary atresia in a patient with a novel mutation.

PubMed

Eminoglu, Tuba Fatma; Polat, Emine; Gökçe, Selim; Ezgü, Fatih Süheyl; Senel, Saliha; Apaydin, Sema

2013-06-01

Neonatal cholestasis is a rare presenting feature of cystic fibrosis which usually cannot be differentiated from other types of cholestasis. Herein, the authors report a 63 d-old boy with cystic fibrosis presenting with neonatal cholestasis mimicking biliary atresia. A new mutation in CFTR gene resulting in severe phenotype has been described. The cystic fibrosis patients with c.3871 G > T mutation may have acholic gaita mimicking biliary atresia in the absence of insipissated bile with minimal histologic findings in the liver.

Properties of Nanocrystalline Cubic Silicon Carbide Thin Films Prepared by Hot-Wire Chemical Vapor Deposition Using SiH4/CH4/H2 at Various Substrate Temperatures

NASA Astrophysics Data System (ADS)

Tabata, Akimori; Komura, Yusuke; Hoshide, Yoshiki; Narita, Tomoki; Kondo, Akihiro

2008-01-01

Silicon carbide (SiC) thin films were prepared by hot-wire chemical vapor deposition from SiH4/CH4/H2 gases, and the influence of substrate temperature, Ts (104 < Ts < 434 °C), on the properties of the SiC thin films was investigated. X-ray diffraction patterns and Raman scattering spectra revealed that nanocrystalline cubic SiC (nc-3C-SiC) films grew at Ts above 187 °C, while completely amorphous films grew at Ts = 104 °C. Fourier transform infrared absorption spectra revealed that the crystallinity of the nc-3C-SiC was improved with increasing Ts up to 282 °C and remained almost unchanged with a further increase in Ts from 282 to 434 °C. The spin density was reduced monotonically with increasing Ts.

Identification and Functional Analysis of a Novel MIP Gene Mutation Associated with Congenital Cataract in a Chinese Family

PubMed Central

Shentu, Xingchao; Miao, Qi; Tang, Xiajing; Yin, Houfa; Zhao, Yingying

2015-01-01

Congenital cataracts are major cause of visual impairment and blindness in children and previous studies have shown about 1/3 of non-syndromic congenital cataracts are inherited. Major intrinsic protein of the lens (MIP), also known as AQP0, plays a critical role in transparency and development of the lens. To date, more than 10 mutations in MIP have been linked to hereditary cataracts in humans. In this study, we investigated the genetic and functional defects underlying a four-generation Chinese family affected with congenital progressive cortical punctate cataract. Mutation screening of the candidate genes revealed a missense mutation at position 448 (c.448G>C) of MIP, which resulted in the substitution of a conserved aspartic acid with histidine at codon 150 (p.D150H). By linkage and haplotype analysis, we obtained positive multipoint logarithm of odds (LOD) scores at microsatellite markers D12S1632 (Zmax = 1.804 at α = 1.000) and D12S1691 (Zmax = 1.806 at α = 1.000), which flanked the candidate locus. The prediction results of PolyPhen-2 and SIFT indicated that the p.D150H mutation was likely to damage to the structure and function of AQP0. The wild type and p.D150H mutant AQP0 were expressed in HeLa cells separately and the immunofluorescence results showed that the WT-AQP0 distributed at the plasma membrane and in cytoplasm, while AQP0-D150H failed to reach the plasma membrane and was mainly retained in the Golgi apparatus. Moreover, protein levels of AQP0-D150H were significantly lower than those of wide type AQP0 in membrane-enriched lysates when the HEK-293T cells were transfected with the same amount of wild type and mutant plasmids individually. Taken together, our data suggest the p.D150H mutation is a novel disease-causing mutation in MIP, which leads to congenital progressive cortical punctate cataract by impairing the trafficking mechanism of AQP0. PMID:25946197

Research in Image Understanding as Applied to 3-D Microwave Tomographic Imaging with Near Optical Resolution.

DTIC Science & Technology

1987-03-01

Oct. 1985. 28. D.L. Jaggard, K. Schultz, Y. Kim and P. Frangos , "Inverse Scattering for Dielectric Media", Annual OSA Meeting, Wash. D.C., Oct. 1985...T.H. Chu - Graduate Student (50%) C.Y. Ho - Graduate Student (50%) Y. Kim - Graduate Student (50%) K S. Lee - Graduate Student (50%) P. Frangos ...1982. 3. P. Frangos (Ph.D.) - "One-Dimensional Inverse Scattering: Exact Methods and Applications". 4. C.L. Werner (Ph.D.) - ŗ-D Imaging of Coherent and

Direct Broad-Range Detection of Alphaviruses in Mosquito Extracts

DTIC Science & Technology

2007-11-25

D.J., Robbins, D.W., Hannis , J.C., Harrell, P.M., Massire, C., Hall, T.A., Jiang, Y., Ranken, R., Drader, J.J., White, N., McNeil, J.A., Crooke, S.T...962. Hofstadler, S.A., Sampath, R., Blyn, L.B., Eshoo, M.W., Hall, T.A., Jiang, Y., Drader, J.J., Hannis , J.C., Sannes-Lowery, K.A., Cummins, L.L...H., Hannis , J., Harrell, P.M., Neuman, B., Buchmeier, M.J., Jiang, Y., Ranken, R., Drader, J., Samant, V., Griffey, R.H., McNeil, J.A., Crooke, S.T

Functional Characterization of CaVα2δ Mutations Associated with Sudden Cardiac Death*

PubMed Central

Bourdin, Benoîte; Shakeri, Behzad; Tétreault, Marie-Philippe; Sauvé, Rémy; Lesage, Sylvie; Parent, Lucie

2015-01-01

L-type Ca2+ channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming CaVα1 with the auxiliary CaVβ and CaVα2δ subunits. CaVα2δ increases the peak current density and improves the voltage-dependent activation gating of CaV1.2 channels without increasing the surface expression of the CaVα1 subunit. The functional impact of genetic variants of CACNA2D1 (the gene encoding for CaVα2δ), associated with shorter repolarization QT intervals (the time interval between the Q and the T waves on the cardiac electrocardiogram), was investigated after recombinant expression of the full complement of L-type CaV1.2 subunits in human embryonic kidney 293 cells. By performing side-by-side high resolution flow cytometry assays and whole-cell patch clamp recordings, we revealed that the surface density of the CaVα2δ wild-type protein correlates with the peak current density. Furthermore, the cell surface density of CaVα2δ mutants S755T, Q917H, and S956T was not significantly different from the cell surface density of the CaVα2δ wild-type protein expressed under the same conditions. In contrast, the cell surface expression of CaVα2δ D550Y, CaVα2δ S709N, and the double mutant D550Y/Q917H was reduced, respectively, by ≈30–33% for the single mutants and by 60% for the latter. The cell surface density of D550Y/Q917H was more significantly impaired than protein stability, suggesting that surface trafficking of CaVα2δ was disrupted by the double mutation. Co-expression with D550Y/Q917H significantly decreased CaV1.2 currents as compared with results obtained with CaVα2δ wild type. It is concluded that D550Y/Q917H reduced inward Ca2+ currents through a defect in the cell surface trafficking of CaVα2δ. Altogether, our results provide novel insight in the molecular mechanism underlying the modulation of CaV1.2 currents by CaVα2δ. PMID:25527503

Genetic evaluations of Chinese patients with odontohypophosphatasia resulting from heterozygosity for mutations in the tissue-non-specific alkaline phosphatase gene.

PubMed

Wan, Jia; Zhang, Li; Liu, Tang; Wang, Yewei

2017-08-01

Hypophosphatasia is a rare heritable metabolic disorder characterized by defective bone and tooth mineralization accompanied by a deficiency of tissue-non-specific (liver/bone/kidney) isoenzyme of alkaline phosphatase activity, caused by a number of loss-of-function mutations in the alkaline phosphatase liver type gene. We seek to explore the clinical manifestations and identify the mutations associated with the disease in a Chinese odonto- hypophosphatasia family. The proband and his younger brother affected with premature loss of primary teeth at their 2-year-old. They have mild abnormal serum alkaline phosphatase and 25-hydroxy vitamin D values, but the serum alkaline phosphatase activity of their father, mother and grandmother, who showed no clinical symptoms of hypophosphatasia, was exhibited significant decreased. In addition to premature loss of primary teeth, the proband and his younger brother showed low bone mineral density, X-rays showed that they had slight metaphyseal osteoporosis changes, but no additional skeletal abnormalities. Deoxyribonucleic acid sequencing and analysis revealed a single nucleotide polymorphism c.787T>C (p.Y263H) in exon 7 and/or a novel mutation c.-92C>T located at 5'UTR were found in the affected individuals. We examined all individuals of an odonto- hypophosphatasia family by clinical and radiographic examinations as well as laboratory assays. Furthermore, all 12 exons and the exon-intron boundaries of the alkaline phosphatase liver type gene were amplified and directly sequenced for further analysis and screened for mutations. Our present findings suggest the single nucleotide polymorphism c.787T>C and c.-92C>T should be responsible for the odonto- hypophosphatasia disorders in this family.

Circadian variations of transferrin saturation levels in iron-overloaded patients: implications for the screening of C282Y-linked haemochromatosis.

PubMed

Guillygomarc'h, Anne; Christian, Jacquelinet; Romain, Moirand; Vincent, Quentin; Véronique, David; Deugnier, Yves

2003-01-01

The phenotypic screening for genetic haemochromatosis (GH) relies upon the determination of transferrin saturation (TS). In large-scale screening programs, the time of blood sampling can be uneasy to control. We studied the circadian variations of TS at 08.00 hours, 12.00 hours, 18.00 hours and 00.00 hours in 46 C282Y homozygous patients (GH) and 47 non-GH patients (NH), to determine whether the time of blood sampling influenced the results of screening. In both groups, there were significant circadian variations in TS, with the highest values at 08.00 hours and the lowest at 00.00 hours. For any given time-point, TS was significantly higher in the GH group when compared with the NH group (P < 0.0001). For both groups, there was a significant decrease in TS between 08.00 hours and 00.00 hours (P < 0.0001) but this decrease was not as significant in GH when compared with NH patients (interaction P < 0.0073). Receiver operating characteristics (ROC) curves generated for TS at 08.00 hours, 12.00 hours, 18.00 hours and 00.00 hours, presented the same efficiency of diagnosis of GH, with TS threshold varying between 64% at 08.00 hours and 36% at 00.00 hours. In conclusion, for screening studies of C282Y homozygosity, determination of transferrin saturation may be performed at any time during the day.

Improved detection of hereditary haemochromatosis.

PubMed

Ogilvie, Catherine; Gaffney, Dairena; Murray, Heather; Kerry, Andrew; Haig, Caroline; Spooner, Richard; Fitzsimons, Edward J

2015-03-01

There is high prevalence of hereditary haemochromatosis (HH) in North European populations, yet the diagnosis is often delayed or missed in primary care. Primary care physicians frequently request serum ferritin (SF) estimation but appear uncertain as how to investigate patients with raised SF values. Our aim was to develop a laboratory algorithm with high predictive value for the diagnosis of HH in patients from primary care with raised SF values. Transferrin saturation (Tsat) was measured on SF samples sent from primary care; 1657 male and 2077 female patients age ≥ 30 years with SF ≥ 200 μg/L. HFE genotyping was performed on all 878 male and 867 female patients with Tsat >30%. This study identified 402 (206 men; 196 women) C282Y carriers and 132 (58 men; 74 women) C282Y homozygotes. Optimal limits for combined SF and Tsat values for HH recognition were established. The detection rate for homozygous C282Y HH for male patients with both SF ≥ 300 μg/L and Tsat >50% was 18.8% (52/272) and 16.3% (68/415) for female patients with both SF ≥ 200 μg/L and Tsat >40%. The large number of SF requests received from primary care should be used as a resource to improve the diagnosis of HH in areas of high prevalence. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Differential effects of human L1CAM mutations on complementing guidance and synaptic defects in Drosophila melanogaster.

PubMed

Kudumala, Sirisha; Freund, Julie; Hortsch, Michael; Godenschwege, Tanja A

2013-01-01

A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin-moesin-radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis.

Differential Effects of Human L1CAM Mutations on Complementing Guidance and Synaptic Defects in Drosophila melanogaster

PubMed Central

Kudumala, Sirisha; Freund, Julie; Hortsch, Michael; Godenschwege, Tanja A.

2013-01-01

A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin–moesin–radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis. PMID:24155914

Discovering, Understanding and Predicting Emergent Phenomena at Strongly Correlated Oxide Interfaces

DTIC Science & Technology

2013-03-31

Orloff, T. Birol, Y. Zhu, L. Fitting Kourkoutis, V. Goian, R. Haislmaier, E. Vlahos , M.D. Biegalski, N. Benedek, M. Bernhagen, Y. Kim, J.D. Brock, R...invited) C.?H. Lee, N.D. Orloff, T. Birol, Y. Zhu, L. Fitting Kourkoutis, V. Goian, R. Haislmaier, E. Vlahos , M.D. Biegalski, N. Benedek, M. Bernhagen, Y

Two novel compound heterozygous mutations in the BCKDHB gene that cause the intermittent form of maple syrup urine disease.

PubMed

Guo, Yi; Liming, Liu; Jiang, Li

2015-12-01

Intermittent maple syrup urine disease (MSUD) is a potentially life-threatening metabolic disorder caused by a deficiency of branched chain α-ketoacid dehydrogenase (BCKD) complex. In contrast to classic MSUD, children with the intermittent form usually have an atypical clinical manifestation. Here, we describe the presenting symptoms and clinical course of a Chinese boy with intermittent MSUD. Mutation analysis identified two previously unreported mutations in exon 7 of the BCKDHB gene: c.767A > G (p.Y256C) and c.768C > G (p.Y256X); the parents were each heterozygous for one of these mutations. In silico analysis predicted Y256C probably affects protein structure; Y256X leads to a premature stop codon. This case demonstrates intermittent MSUD should be suspected in cases with symptoms of recurrent encephalopathy, especially ataxia or marked drowsiness, which usually present after the neonatal period and in conjunction with infection. symmetrical basal ganglia damage but normal myelination in the posterior limb will assist differential diagnosis; alloisoleucine is a useful diagnostic marker and mutation analysis may be of prognostic value. These novel mutations Y256C and Y256X result in the clinical manifestation of a variant form of MSUD, expanding the mutation spectrum of this disease.

Cleavage site and Ectodomain of HA2 sub-unit sequence of three equine influenza virus isolated in Morocco

PubMed Central

2014-01-01

Background The equine influenza (EI) is an infectious and contagious disease of the upper respiratory tract of horses. Two outbreaks were notified in Morocco during 1997 and 2004 respectively in Nador and Essaouira. The aims of the present study concern the amino acids sequences comparison with reference strain A/equine/Miami/1963(H3N8) of the HA2 subunit including the cleavage site of three equine influenza viruses (H3N8) isolated in Morocco: A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004 (H3N8) and A/equine/Essaouira/3/2004 (H3N8). Results The obtained results demonstrated that the substitutions were located at Ectodomain (ED) and transmembrane domain (TD), and they have only one arginine in cleavage site (HA1-PEKQI-R329-GI-HA2). In the Ectodomain, the mutation N/154 2 /T deleted the NGT glycosylation site at position 154 for both strains A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8). Except for mutation D/1602/Y of the A/equine/Nador/1/1997(H3N8) strain, the other mutations were involved in non conserved sites. While the transmembrane domain (TM) of the strain A/equine/Essaouira/3/2004(H3N8) exhibits a substitution at residue C/199 2 /F. For the A/equine/Nador/1/1997(H3N8) strain the HA2 shows a mutation at residue M/207 2 /L. Three Moroccan strains reveals a common substitution at the residue E/211 2 /Q located between transmembrane domain TM and the cytoplasmic domain (CD). Conclusion The given nature virulence of three Moroccan strains, the identified and reported mutations certainly played a permissive role of infection viral process. PMID:25016480

Lack of robustness of life extension associated with several single-gene P element mutations in Drosophila melanogaster.

PubMed

Mockett, Robin J; Nobles, Amber C

2013-10-01

The hypothesis tested in this study was that single-gene mutations found previously to extend the life span of Drosophila melanogaster could do so consistently in both long-lived y w and standard w (1118) genetic backgrounds. GAL4 drivers were used to express upstream activation sequence (UAS)-responder transgenes globally or in the nervous system. Transgenes associated with oxidative damage prevention (UAS-hSOD1 and UAS-GCLc) or removal (EP-UAS-Atg8a and UAS-dTOR (FRB) ) failed to increase mean life spans in any expression pattern in either genetic background. Flies containing a UAS-EGFP-bMSRA (C) transgene associated with protein repair were found not to exhibit life extension or detectable enhanced green fluorescent protein (EGFP) activity. The presence of UAS-responder transgenes was confirmed by PCR amplification and sequencing at the 5' and 3' end of each insertion. These results cast doubt on the robustness of life extension in flies carrying single-gene mutations and suggest that the effects of all such mutations should be tested independently in multiple genetic backgrounds and laboratory environments.

The E180splice mutation in the GHR gene causing Laron syndrome: witness of a Sephardic Jewish exodus from the Iberian Peninsula to the New World?

PubMed

Gonçalves, Fernanda T; Fridman, Cintia; Pinto, Emília M; Guevara-Aguirre, Jaime; Shevah, Orit; Rosembloom, Arlan L; Hwa, Vivian; Cassorla, Fernando; Rosenfeld, Ron G; Lins, Theresa S S; Damiani, Durval; Arnhold, Ivo J P; Laron, Zvi; Jorge, Alexander A L

2014-05-01

Laron syndrome (LS) is a genetic disorder caused by mutations in the growth hormone receptor (GHR) gene. The most frequent GHR mutation is E180splice (rs121909360), which was initially found in an inbred population of Spanish descent in Ecuador and subsequently in Israel, Brazil, Chile, and the United States. The aim of the present study is to determine if the E180splice mutation arose from a common origin. We studied 22 patients with LS from Ecuador, Israel (of Moroccan origin), Brazil, Chile, and the United States (of Mexican origin) who were homozygous for the E180splice mutation and compared them to control individuals for markers surrounding the GHR, intragenic polymorphisms, and Y-chromosome STR. An identical haplotype was found in all but one of the subjects carrying the E180splice mutation: D5S665: 150/150; D5S2082: 192/192; D5S2087: 246/246; rs6179 G/G; and rs6180 C/C. One patient differed from the others only at D5S2082 (168/192). This haplotype is rare (~1%) in control individuals and confirmed that the E180splice-associated haplotype was not derived from independent origins but represented recombination from a common ancestor. The analysis of paternal lineage markers showed that 50% belong to haplogroup R1b (found in Portugal and Spain) and 40% to haplogroups J and E (typical in the Middle East and in Eastern European Jews). The germline E180Splice mutation appears to have originated from a single common ancestor. The presence of Y-chromosome markers associated with Sephardic populations in persons harboring the E180splice mutation provides genetic evidence in support of the historical tracking of the exodus of this specific population. © 2014 Wiley Periodicals, Inc.

Dynamics of translocation and substrate binding in individual complexes formed with active site mutants of {phi}29 DNA polymerase.

PubMed

Dahl, Joseph M; Wang, Hongyun; Lázaro, José M; Salas, Margarita; Lieberman, Kate R

2014-03-07

The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn(2+) rather than Mg(2+). The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.

Denatured states of yeast cytochrome c induced by heat and guanidinium chloride are structurally and thermodynamically different.

PubMed

Zaidi, Sobia; Haque, Md Anzarul; Ubaid-Ullah, Shah; Prakash, Amresh; Hassan, Md Imtaiyaz; Islam, Asimul; Batra, Janendra K; Ahmad, Faizan

2017-05-01

A sequence alignment of mammalian cytochromes c with yeast iso-1-cytochrome c (y-cyt-c) shows that the yeast protein contains five extra N-terminal residues. We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt-c by sequentially removing these extra residues. During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt-c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state ↔ D state, N state ↔ H state, and H state ↔ D state, was done to obtain Gibbs free energy changes associated with all the three processes. This analysis showed that, for each protein, thermodynamic cycle accommodates Gibbs free energies associated with transitions between N and D states, N and H states, and H and D states, the characteristics required for a thermodynamic function. All these experimental observations have been supported by our molecular dynamics simulation studies.

Cryptography; An Introductory Bibliography of Books and Periodical Articles.

DTIC Science & Technology

1982-06-05

Martin’s H25 Pr., c1976. D810 Johnson, Brian. The Secret War, N.Y., NY: Methuen, c1978. $2J6 D810 Jones, Reginald Victor. The Wizard War: British...Park Pr., c1976. UB290 Langie, Andre. Cryptography, London, Eng.: Constable & L3 Co., Ltd., c1922. D810 Lawson, Don. The Secret World War II, N.Y., NY...Writing, N.Y., NY: W.W. Norton & Co., c1943. Z6724 Smith, Myron J., Jr. The Secret Wars, Vol. 1: Intelligence, I7 Propaganda and Psychological Warfare

Thin-Film Optical Switch.

DTIC Science & Technology

1976-09-01

UNCLASSIFIED St Cu* ITY C LA S S I F I C A T I O N OF tIllS PAGE (W~I. , 0.i. Ent.,.~d) REPODT rVW IIMEIITATIAII PACE READ INSTRUCTIONS...MEASUREMENTS 10 A. Ef fec t ive Channel Guide Width 10 13. Rid ge For mation St udies 10 C. Wave guide Loss Measurement s 10 D. Tape r Coup ling Eff...of less coup l ing e f f i c i e n c y at t h e t aper l e n g t h of 900 ~ rn . Ne l son 2 has c o n s i d e r e d the e f f i c i e n c y of

The WT hemochromatosis protein HFE inhibits CD8⁺ T-lymphocyte activation.

PubMed

Reuben, Alexandre; Phénix, Mikaël; Santos, Manuela M; Lapointe, Réjean

2014-06-01

MHC class I (MHC I) antigen presentation is a ubiquitous process by which cells present endogenous proteins to CD8(+) T lymphocytes during immune surveillance and response. Hereditary hemochromatosis protein, HFE, is involved in cellular iron uptake but, while structurally homologous to MHC I, is unable to bind peptides. However, increasing evidence suggests a role for HFE in the immune system. Here, we investigated the impact of HFE on CD8(+) T-lymphocyte activation. Using transient HFE transfection assays in a model of APCs, we show that WT HFE (HFEWT ), but not C282Y-mutated HFE, inhibits secretion of MIP-1β from antigen-specific CD8(+) T lymphocytes. HFEWT expression also resulted in major decreases in CD8(+) T-lymphocyte activation as measured by 4-1BB expression. We further demonstrate that inhibition of CD8(+) T-lymphocyte activation was independent of MHC I surface levels, β2-m competition, HFE interaction with transferrin receptor, antigen origin, or epitope affinity. Finally, we identified the α1-2 domains of HFEWT as being responsible for inhibiting CD8(+) T-lymphocyte activation. Our data imply a new role for HFEWT in altering CD8(+) T-lymphocyte reactivity, which could modulate antigen immunogenicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HIV-1 drug resistance in antiretroviral-naive individuals with HIV-1-associated tuberculous meningitis initiating antiretroviral therapy in Vietnam.

PubMed

Thao, Vu P; Le, Thuy; Török, Estee M; Yen, Nguyen T B; Chau, Tran T H; Jurriaans, Suzanne; van Doorn, H Rogier; van Doorn, Rogier H; de Jong, Menno D; Farrar, Jeremy J; Dunstan, Sarah J

2012-01-01

Access to antiretroviral therapy (ART) for HIV-infected individuals in Vietnam is rapidly expanding, but there are limited data on HIV drug resistance (HIVDR) to guide ART strategies. We retrospectively conducted HIVDR testing in 220 ART-naive individuals recruited to a randomized controlled trial of immediate versus deferred ART in individuals with HIV-associated tuberculous meningitis in Ho Chi Minh City (HCMC) from 2005-2008. HIVDR mutations were identified by population sequencing of the HIV pol gene and were defined based on 2009 WHO surveillance drug resistance mutations (SDRMs). We successfully sequenced 219/220 plasma samples of subjects prior to ART; 218 were subtype CRF01_AE and 1 was subtype B. SDRMs were identified in 14/219 (6.4%) subjects; 8/14 were resistant to nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs; T69D, L74V, V75M, M184V/I and K219R), 5/14 to non-nucleoside reverse transcriptase inhibitors (NNRTIs; K103N, V106M, Y181C, Y188C and G190A), 1/14 to both NRTIs and NNRTIs (D67N and Y181C) and none to protease inhibitors. After 6 months of ART, eight subjects developed protocol-defined virological failure. HIVDR mutations were identified in 5/8 subjects. All five had mutations with high-level resistance to NNRTIs and three had mutations with high-level resistance to NRTIs. Due to a high early mortality rate (58%), the effect of pre-existing HIVDR mutations on treatment outcome could not be accurately assessed. The prevalence of WHO SDRMs in ART-naive individuals with HIV-associated tuberculous meningitis in HCMC from 2005-2008 is 6.4%. The SDRMs identified conferred resistance to NRTIs and/or NNRTIs, reflecting the standard first-line ART regimens in Vietnam.

Prevalence of ESR1 Mutations in Cell-Free DNA and Outcomes in Metastatic Breast Cancer: A Secondary Analysis of the BOLERO-2 Clinical Trial.

PubMed

Chandarlapaty, Sarat; Chen, David; He, Wei; Sung, Patricia; Samoila, Aliaksandra; You, Daoqi; Bhatt, Trusha; Patel, Parul; Voi, Maurizio; Gnant, Michael; Hortobagyi, Gabriel; Baselga, José; Moynahan, Mary Ellen

2016-10-01

Estrogen receptor α (ESR1) mutations found in metastatic breast cancer (MBC) promote ligand-independent receptor activation and resistance to estrogen-deprivation therapy in laboratory models. The prevalence of these mutations and their potential impact on clinical outcomes has not been established. To determine the prevalence of ESR1 mutations (Y537S and D538G) in estrogen receptor (ER)-positive MBC and determine whether mutation is associated with inferior outcomes. From December 16, 2014, to August 26, 2015, we analyzed cell-free DNA (cfDNA) from baseline plasma samples from participants in the BOLERO-2 double-blind phase 3 study that randomized patients from 189 centers in 24 countries with MBC to exemestane plus placebo or exemestane plus everolimus. The study enrolled postmenopausal women with a diagnosis of MBC and prior exposure to an aromatase inhibitor. Baseline plasma samples were available from 541 of 724 patients (74.7%). We assessed the effect of mutation on overall survival of the population and the effect of mutation on progression-free survival (PFS) by treatment arm. Patients were randomized to treatment with exemestane (25 mg oral daily) together with everolimus (10 mg oral daily) or with placebo. The 2 most frequent mutations in ESR1 (Y537S and D538G) were analyzed from cfDNA using droplet digital polymerase chain reaction and samples scored as wild-type, D538G, Y537S, or double mutant. Cox-proportional hazards model was used to assess PFS in patient subgroups defined by mutations, and the effect of each mutation on overall survival. Of 541 evaluable patients, 156 (28.8%) had ESR1 mutation D538G (21.1%) and/or Y537S (13.3%), and 30 had both. These mutations were associated with shorter overall survival (wild-type, 32.1 months [95% CI, 28.09-36.40 months]; D538G, 25.99 months [95% CI, 19.19-32.36 months]; Y537S, 19.98 months [13.01-29.31 months]; both mutations, 15.15 months [95% CI, 10.87-27.43 months]). The D538G group (hazard ratio, 0.34 [95% CI, 0.02-0.57]) derived a similar PFS benefit as wild type from addition of everolimus to exemestane. ESR1 mutations are prevalent in ER-positive aromatase inhibitor-treated MBC. Both Y537S and D538G mutations are associated with more aggressive disease biology. clinicaltrials.gov Identifier: NCT00863655.

Molecular analysis of congenital goitres with hypothyroidism caused by defective thyroglobulin synthesis. Identification of a novel c.7006C>T [p.R2317X] mutation and expression of minigenes containing nonsense mutations in exon 7.

PubMed

Machiavelli, Gloria A; Caputo, Mariela; Rivolta, Carina M; Olcese, María C; Gruñeiro-Papendieck, Laura; Chiesa, Ana; González-Sarmiento, Rogelio; Targovnik, Héctor M

2010-01-01

Thyroglobulin (TG) deficiency is an autosomal-recessive disorder that results in thyroid dyshormonogenesis. A number of distinct mutations have been identified as causing human hypothyroid goitre. The purpose of this study was to identify and characterize new mutations in the TG gene in an attempt to increase the understanding of the genetic mechanism responsible for this disorder. A total of six patients from four nonconsanguineous families with marked impairment of TG synthesis were studied. Single-strand conformation polymorphism (SSCP) analysis, sequencing of DNA, genotyping, expression of chimeric minigenes and bioinformatic analysis were performed. Four different inactivating TG mutations were identified: one novel mutation (c.7006C>T [p.R2317X]) and three previously reported (c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.6725G>A [p.R2223H]). Consequently, one patient carried a compound heterozygous for p.R2223H/p.R2317X mutations; two brothers showed a homozygous p.A2215D substitution and the remaining three patients, from two families with typical phenotype, had a single p.R277X mutated allele. We also showed functional evidences that premature stop codons inserted at different positions in exon 7, which disrupt exonic splicing enhancer (ESE) sequences, do not interfere with exon definition and processing. In this study, we have identified a novel nonsense mutation p.R2317X in the acetylcholinesterase homology domain of TG. We have also observed that nonsense mutations do not interfere with the pre-mRNA splicing of exon 7. The results are in accordance with previous observations confirming the genetic heterogeneity of TG defects.

Possible nucleus of the Bergman cluster in the Zn-Mg-Y alloy system

NASA Astrophysics Data System (ADS)

Nakayama, Kei; Nakagawa, Masaya; Koyama, Yasumasa

2018-01-01

To understand the formation of the Bergman cluster in the F-type icosahedral quasicrystal (IQ), crystallographic relations between the quasicrystal and the intermetallic-compound H and Zn23Y6 phases in the Zn-Mg-Y alloy system were investigated mainly by transmission electron microscopy. It was found that, although sample rotations of about 1° were required to obtain simple crystallographic relations, the orientation relationship was established among the cubic-Fm?m Zn23Y6 structure, the hexagonal-P63/mmc H structure and the F-type IQ; that is, [? 1 3]c // the five-fold axis in the IQ // N(2 ? 0)H, and [1 1 0]c // the two-fold axis in the IQ // N(0 5 ? 3)H, where N(h k m l)H means the normal direction of the (h k m l)H plane in the H structure. The correspondences between atomic positions in the Bergman cluster and in the Zn23Y6 structure and between those in the cluster and in the H structure were investigated on the basis of the established relationship. As a result, an assembly of six short-penetrated-decagonal columns was identified as an appropriate nucleus in the formation of the Bergman cluster from these two structures.

A molecular dynamics investigation on the crizotinib resistance mechanism of C1156Y mutation in ALK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Hui-Yong; Ji, Feng-Qin, E-mail: fengqinji@mail.hzau.edu.cn; Center for Bioinformatics, Huazhong Agricultural University, Wuhan 430070

2012-06-29

Highlights: Black-Right-Pointing-Pointer The study revealed the detailed resistance mechanism of the non-active mutation C1156Y in ALK. Black-Right-Pointing-Pointer C1156Y leads to crizotinib displacement and conformational changes in the binding cavity. Black-Right-Pointing-Pointer The conformations cause a decline in the vdW and electrostatic energy between crizotinib and ALK. -- Abstract: Crizotinib is an anaplastic lymphoma kinase (ALK) inhibitor that has recently been approved in the US for the treatment of non-small cell lung carcinoma (NSCLC). Despite its outstanding safety and efficacy, several resistant mutations against crizotinib have been detected in the treatment of NSCLC. However, in contrast to the widely accepted mechanism ofmore » steric hindrance by mutations at the active site, the mechanism by which the C1156Y non-active site mutation confers resistance against crizotinib remains unclear. In the present study, the resistance mechanism of C1156Y in ALK was investigated using molecular dynamics simulations. The results suggest that despite the non-active site mutation, C1156Y causes the dislocation of crizotinib as well as the indirect conformational changes in the binding cavity, which results in a marked decrease in the van der Waals and electrostatic interactions between crizotinib and ALK. The obtained results provide a detailed explanation of the resistance caused by C1156Y and may give a vital clue for the design of drugs to combat crizotinib resistance.« less

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Das, Kalyan; Martinez, Sergio E.; Arnold, Eddy

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATPmore » (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.« less

Late onset of Wilson's disease in a family with genetic haemochromatosis.

PubMed

Dib, Nina; Valsesia, Emmanuelle; Malinge, Marie Claire; Mauras, Yves; Misrahi, Micheline; Calès, Paul

2006-01-01

We report the coexistence of Wilson's disease and genetic haemochromatosis in one family. The diagnosis of genetic haemochromatosis was established in a 52-year-old man. Among his siblings, one 57-year-old sister and one 55-year-old brother had decreased copper and ceruloplasmin levels in serum and increased urinary copper excretion. The sister shared the same human leucocyte antigen haplotypes and was homozygous for the HFE mutation C282Y, like the propositus. However, she had normal liver iron content and increased liver copper content. Her dietary copper intake was probably excessive. The association of Wilson's disease and genetic haemochromatosis is rare and has only been described twice. The onset of Wilson's disease after 50 years of age is rare; Wilson's disease should be considered in any patient with unexplained chronic liver disease; an excess in liver copper content might be induced by excessive dietary input in a susceptible individual.

Cancer-associated Isocitrate Dehydrogenase 1 (IDH1) R132H Mutation and d-2-Hydroxyglutarate Stimulate Glutamine Metabolism under Hypoxia*

PubMed Central

Reitman, Zachary J.; Duncan, Christopher G.; Poteet, Ethan; Winters, Ali; Yan, Liang-Jun; Gooden, David M.; Spasojevic, Ivan; Boros, Laszlo G.; Yang, Shao-Hua; Yan, Hai

2014-01-01

Mutations in the cytosolic NADP+-dependent isocitrate dehydrogenase (IDH1) occur in several types of cancer, and altered cellular metabolism associated with IDH1 mutations presents unique therapeutic opportunities. By altering IDH1, these mutations target a critical step in reductive glutamine metabolism, the metabolic pathway that converts glutamine ultimately to acetyl-CoA for biosynthetic processes. While IDH1-mutated cells are sensitive to therapies that target glutamine metabolism, the effect of IDH1 mutations on reductive glutamine metabolism remains poorly understood. To explore this issue, we investigated the effect of a knock-in, single-codon IDH1-R132H mutation on the metabolism of the HCT116 colorectal adenocarcinoma cell line. Here we report the R132H-isobolome by using targeted 13C isotopomer tracer fate analysis to trace the metabolic fate of glucose and glutamine in this system. We show that introduction of the R132H mutation into IDH1 up-regulates the contribution of glutamine to lipogenesis in hypoxia, but not in normoxia. Treatment of cells with a d-2-hydroxyglutarate (d-2HG) ester recapitulated these changes, indicating that the alterations observed in the knocked-in cells were mediated by d-2HG produced by the IDH1 mutant. These studies provide a dynamic mechanistic basis for metabolic alterations observed in IDH1-mutated tumors and uncover potential therapeutic targets in IDH1-mutated cancers. PMID:24986863

Mek1Y130C mice recapitulate aspects of human cardio-facio-cutaneous syndrome

PubMed Central

Aoidi, Rifdat; Houde, Nicolas; Landry-Truchon, Kim; Holter, Michael; Jacquet, Kevin; Charron, Louis; Yu, Benjamin D.; Rauen, Katherine A.; Bisson, Nicolas; Newbern, Jason

2018-01-01

ABSTRACT The RAS/MAPK signaling pathway is one of the most investigated pathways, owing to its established role in numerous cellular processes and implication in cancer. Germline mutations in genes encoding members of the RAS/MAPK pathway also cause severe developmental syndromes collectively known as RASopathies. These syndromes share overlapping characteristics, including craniofacial dysmorphology, cardiac malformations, cutaneous abnormalities and developmental delay. Cardio-facio-cutaneous syndrome (CFC) is a rare RASopathy associated with mutations in BRAF, KRAS, MEK1 (MAP2K1) and MEK2 (MAP2K2). MEK1 and MEK2 mutations are found in ∼25% of the CFC patients and the MEK1Y130C substitution is the most common one. However, little is known about the origins and mechanisms responsible for the development of CFC. To our knowledge, no mouse model carrying RASopathy-linked Mek1 or Mek2 gene mutations has been reported. To investigate the molecular and developmental consequences of the Mek1Y130C mutation, we generated a mouse line carrying this mutation. Analysis of mice from a Mek1 allelic series revealed that the Mek1Y130C allele expresses both wild-type and Y130C mutant forms of MEK1. However, despite reduced levels of MEK1 protein and the lower abundance of MEK1 Y130C protein than wild type, Mek1Y130C mutants showed increased ERK (MAPK) protein activation in response to growth factors, supporting a role for MEK1 Y130C in hyperactivation of the RAS/MAPK pathway, leading to CFC. Mek1Y130C mutant mice exhibited pulmonary artery stenosis, cranial dysmorphia and neurological anomalies, including increased numbers of GFAP+ astrocytes and Olig2+ oligodendrocytes in regions of the cerebral cortex. These data indicate that the Mek1Y130C mutation recapitulates major aspects of CFC, providing a new animal model to investigate the physiopathology of this RASopathy. This article has an associated First Person interview with the first author of the paper. PMID:29590634

40 CFR 1065.703 - Distillate diesel fuel.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 1 Cetane Number 40-50 40-50 40-50 ASTM D613-05. Distillation range: Initial boiling point °C 171-204 171-204 171-204 ASTM D86-07a. 10 pct. point 204-238 204-238 204-238 50 pct. point 243-282 243-282 243...-37 ASTM D4052-96e01. Total sulfur, ultra low sulfur mg/kg 7-15 See 40 CFR 80.580. Total sulfur, low...

Crystal structure refinements of tetragonal (OH,F)-rich spessartine and henritermierite garnets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antao, Sytle M.; Cruickshank, Laura A.

Cubic garnet (space group Ia\\overline 3 d) has the general formulaX 3Y 2Z 3O 12, whereX,YandZare cation sites. In the tetragonal garnet (space groupI4 1/acd), the corresponding cation sites areX1 andX2,Y, andZ1 andZ2. In both space groups only theYsite is the same. The crystal chemistry of a tetragonal (OH,F)-rich spessartine sample from Tongbei, near Yunxiao, Fujian Province, China, with composition X(Mn 2.82Fe^{2+}_{0.14}Ca 0.04) Σ3 Y{Al 1.95Fe^{3+}_{0.05}} Σ2 Z[(SiO 4) 2.61(O 4H 4) 0.28(F 4) 0.11] Σ3(Sps 94Alm 5Grs 1) was studied with single-crystal X-ray diffraction and space groupI4 1/acd. The deviation of the unit-cell parameters from cubic symmetry is smallmore » [a= 11.64463(1),c= 11.65481 (2) Å,c/a= 1.0009]. Point analyses and back-scattered electron images, obtained by electron-probe microanalysis, indicate a homogeneous composition. TheZ2 site is fully occupied, but theZ1 site contains vacancies. The occupiedZ1 andZ2 sites with Si atoms are surrounded by four O atoms, as in anhydrous cubic garnets. Pairs of split sites are O1 with F11 and O2 with O22. When theZ1 site is vacant, a larger [(O 2H 2)F 2] tetrahedron is formed by two OH and two F anions in the O22 and F11 sites, respectively. This [(O 2H 2)F 2] tetrahedron is similar to the O 4H 4tetrahedron in hydrogarnets. These results indicate ^{X}{{\\rm Mn}^ {2+}_{3}}\\,^{Y}{\\rm Al}_{2}^{Z}[({\\rm SiO}_{4})_{2}({\\rm O}_{2}{\\rm H}_{2})_{0.5}({\\rm F}_{2})_{0.5}]_{\\Sigma3} as a possible end member, which is yet unknown. The H atom that is bonded to the O22 site is not located because of the small number of OH groups. In contrast, tetragonal henritermierite, ideally ^{X}{\\rm Ca}_{3}\\,^{Y}{\\rm Mn}^{3+}_{2}\\,^{Z}[({\\rm SiO}_{4})_{2}({\\rm O}_{4}{\\rm H}_{4})_1]_{\\Sigma3}, has a vacantZ2 site that contains the O 4H 4tetrahedron. The H atom is bonded to an O3 atom [O3—H3 = 0.73 (2) Å]. Because of O2—Mn 3+—O2 Jahn–Teller elongation of the Mn 3+O 6octahedron, a weak hydrogen bond is formed to the under-bonded O2 atom. This causes a large deviation from cubic symmetry (c/a= 0.9534).« less

Interaction of CYP2C19, P2Y12, and GPIIIa Variants Associates With Efficacy of Clopidogrel and Adverse Events on Patients With Ischemic Stroke.

PubMed

Yi, Xingyang; Wang, Yanfen; Lin, Jing; Cheng, Wen; Zhou, Qiang; Wang, Chun

2017-10-01

Clopidogrel is a clinically important oral antiplatelet agent for the treatment or prevention of cerebrovascular disease. However, different individuals have different sensitivities to clopidogrel. This study assessed variants of different genes for association with response to clopidogrel, clinical outcome, and side effects in patients with ischemic stroke (IS). We consecutively enrolled 375 patients with IS after they received clopidogrel therapy, and venous blood samples were subjected to genotyping allelic variants of genes modulating clopidogrel absorption (ATP binding cassette subfamily B1, ABCB1), metabolic activation (cytochrome P450[CYP] 3A and CYP2C19), and biologic activity (platelet membrane receptor [ P2Y12, P2Y1)], and glycoprotein IIIa [ GPIIIa]) and statistically analyzing their interactions with clopidogrel sensitivity (CS) and adverse events, risk of IS recurrence, myocardial infarction, and death during 6 months of follow-up. Adverse events occurred in 37 patients (31 had IS recurrence, 4 died, and 2 had myocardial infarction) during the first 6 months of follow-up. Single locus analysis showed that only the CYP2C19*2(rs4244285) variant was independently associated with CS and risk of adverse events after adjusting covariates. However, there was significant gene-gene interaction among CYP2C19*2(rs4244285), P2Y12(rs16863323), and GPIIIa (rs2317676) analyzed by generalized multifactor dimensionality reduction methods. The rate of adverse events among patients with the 3-loci interaction was 2.82 times the rate among those with no interaction (95% confidence interval: 2.04-8.63). Sensitivity of patients with IS to clopidogrel and clopidogrel-induced adverse clinical events may be multifactorial but is not determined by single gene polymorphisms.

Dry-thermophilic anaerobic digestion of organic fraction of municipal solid waste: methane production modeling.

PubMed

Fdez-Güelfo, L A; Alvarez-Gallego, C; Sales, D; García, L I Romero

2012-03-01

The influence of particle size and organic matter content of organic fraction of municipal solid waste (OFMSW) in the overall kinetics of dry (30% total solids) thermophilic (55°C) anaerobic digestion have been studied in a semi-continuous stirred tank reactor (SSTR). Two types of wastes were used: synthetic OFMSW (average particle size of 1mm; 0.71 g Volatile Solids/g waste), and OFMSW coming from a composting full scale plant (average particle size of 30 mm; 0.16 g Volatile Solids/g waste). A modification of a widely-validated product-generation kinetic model has been proposed. Results obtained from the modified-model parameterization at steady-state (that include new kinetic parameters as K, Y(pMAX) and θ(MIN)) indicate that the features of the feedstock strongly influence the kinetics of the process. The overall specific growth rate of microorganisms (μ(max)) with synthetic OFMSW is 43% higher compared to OFMSW coming from a composting full scale plant: 0.238 d(-1) (K=1.391 d(-1); Y(pMAX)=1.167 L CH(4)/gDOC(c); θ(MIN)=7.924 days) vs. 0.135 d(-1) (K=1.282 d(-1); Y(pMAX)=1.150 L CH(4)/gDOC(c); θ(MIN)=9.997 days) respectively. Finally, it could be emphasized that the validation of proposed modified-model has been performed successfully by means of the simulation of non-steady state data for the different SRTs tested with each waste. Copyright © 2011 Elsevier Ltd. All rights reserved.

In vitro emergence of fluoroquinolone resistance in Cutibacterium (formerly Propionibacterium) acnes and molecular characterization of mutations in the gyrA gene.

PubMed

Takoudju, Eve-Marie; Guillouzouic, Aurélie; Kambarev, Stanimir; Pecorari, Frédéric; Corvec, Stéphane

2017-10-01

In vitro occurrence of levofloxacin (LVX) resistance in C. acnes and characterization of its molecular background were investigated. The mutation frequency was determined by inoculation of 10 8  cfu of C. acnes ATCC 11827 (LVX MIC = 0.25 mg/L) on LVX-containing agar plates. The progressive emergence of resistance was studied by a second exposure to increasing LVX concentrations. For mutants, the QRDR regions including the gyrA and parC genes were sequenced and compared to both C. acnes ATCC 11827 and C. acnes KPA171202 reference sequences (NC006085). The importance of the efflux pump system in resistance was investigated by using inhibitors on selected resistant mutants with no mutation in the QRDR. C. acnes growth was observed on LVX-containing plates with mutation frequencies of 3. 8 cfu × 10 -8 (8 × MIC) and 1.6 cfu × 10 -7 (4 × MIC). LVX resistance emerged progressively after one-step or two-step assays. In LVX-resistant isolates, the MIC ranged from 0.75 to >32 mg/L. Mutations were detected exclusively in the gyrA gene. Ten genotypes were identified: G99 C, G99 D, D100N, D100 H, D100 G, S101L, S101W, A102 P, D105 H and A105 G. Mutants S101L and S101W were always associated with a high level of resistance. Mutants with no mutation in the QRDR were more susceptible when incubated with an efflux pump inhibitor (phenyl-arginine β-naphthylamide) only, suggesting, for the first time, the expression of such a system in C. acnes LVX-resistant mutants. Copyright © 2017 Elsevier Ltd. All rights reserved.

40 CFR 408.282 - Effluent limitations guidelines representing the degree of effluent reduction attainable by the...

Code of Federal Regulations, 2010 CFR

2010-07-01

...): (a) Any sardine processing facility which utilizes dry transportation systems from the fish storage... units (kg/kkg of seafood) TSS 36 10 Oil and grease 3.5 1.4 pH (1) (1) English units (lb/1,000 lb of... not exceed— Metric units (kg/kkg of seafood) TSS 48 16 Oil and grease 6.3 2.8 pH (1) (1) English units...

Molecular pathology and haplotype analysis of Wilson disease in Mediterranean populations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Figus, A.; Farcia, A.M.G.; Nurchi, A.

1995-12-01

We analyzed mutations and defined the chromosomal haplotype in 127 patients of Mediterranean descent who were affected in Wilson disease (WD): 39 Sardinians, 49 Italians, 33 Turks, and 6 Albanians. Haplotypes were derived by use of the microsatellite markers D13S301, D13S296, D13S297, and D13S298, which are linked to the WD locus. There were five common haplotypes in Sardinians, three in Italians, and two in Turks, which accounted for 85%, 32%, and 30% of the WD chromosomes, respectively. We identified 16 novel mutations: 8 frameshifts, 7 missense mutations, and 1 splicing defect. In addition, we detected the previously described mutations: 2302insC,more » 3404delC, Arg1320ter, Gly944Ser, and His1070Gin. Of the new mutations detected, two, the 1515insT on haplotype I and 2464delC on haplotype XVI, accounted for 6% and 13%, respectively, of the mutations in WD chromsomes in the Sardinian populations. Mutations H1070Q, 2302insC, and 2533delA represented 13%, 8%, and 8%, respectively, of the mutations in WD chromsomes in other Mediterranean populations. The remaining mutations were rare and limited to one or two patients from different populations. Thus, WD results from some frequent mutations and many rare defects. 28 refs., 1 fig., 3 tabs.« less

29 CFR Appendix V to Part 1918 - Basic Elements of a First Aid Training Program (Non-mandatory)

Code of Federal Regulations, 2010 CFR

2010-07-01

...) Repairs .62(h)(4) Safe Working Load .62(h)(1) Shortened .62(h)(6) Chutes .63(a), (b), (c), (d) Clamps...) Certification .11 Coaming Rollers .52(e) Falls .52(c) Heel Blocks .52(d) Preventers .52(a), .54(d) Safe Working...)). Shackles: Safe Working Loads .62(i)(1) Used Aloft .62(i)(2) Other Decks .34 Slippery Conditions .91(b...

Proton Pump Inhibitors Decrease Phlebotomy Need in HFE Hemochromatosis: Double-Blind Randomized Placebo-Controlled Trial.

PubMed

Vanclooster, Annick; van Deursen, Cees; Jaspers, Reggy; Cassiman, David; Koek, Ger

2017-09-01

Phlebotomy constitutes the established treatment for HFE-related hemochromatosis. Retrospective studies have suggested proton pump inhibitors (PPIs) reduce the need for phlebotomy in this population. We conducted a randomized controlled trial to prove this. Thirty p.C282Y homozygous patients were randomly allocated to PPI (pantoprazole 40 mg/day) or placebo for 12 months. Phlebotomies were performed when serum ferritin was > 100 μg/L. Phlebotomy need turned out to be significantly lower in patients taking PPI (P = .0052). PPI treatment significantly reduces the need for phlebotomies in p.C282Y homozygous patients. In view of the known long-term safety profile of PPI, they can be a valuable addition to standard therapy. Clinicaltrials.gov: NCT01524757. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Prediction of the binding mode and resistance profile for a dual-target pyrrolyl diketo acid scaffold against HIV-1 integrase and reverse-transcriptase-associated ribonuclease H.

PubMed

Yang, Fengyuan; Zheng, Guoxun; Fu, Tingting; Li, Xiaofeng; Tu, Gao; Li, Ying Hong; Yao, Xiaojun; Xue, Weiwei; Zhu, Feng

2018-06-27

The rapid emergence of drug-resistant variants is one of the most common causes of highly active antiretroviral therapeutic (HAART) failure in patients infected with HIV-1. Compared with the existing HAART, the recently developed pyrrolyl diketo acid scaffold targeting both HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) is an efficient approach to counteract the failure of anti-HIV treatment due to drug resistance. However, the binding mode and potential resistance profile of these inhibitors with important mechanistic principles remain poorly understood. To address this issue, an integrated computational method was employed to investigate the binding mode of inhibitor JMC6F with HIV-1 IN and RNase H. By using per-residue binding free energy decomposition analysis, the following residues: Asp64, Thr66, Leu68, Asp116, Tyr143, Gln148 and Glu152 in IN, Asp443, Glu478, Trp536, Lys541 and Asp549 in RNase H were identified as key residues for JMC6F binding. And then computational alanine scanning was carried to further verify the key residues. Moreover, the resistance profile of the currently known major mutations in HIV-1 IN and 2 mutations in RNase H against JMC6F was predicted by in silico mutagenesis studies. The results demonstrated that only three mutations in HIV-1 IN (Y143C, Q148R and N155H) and two mutations in HIV-1 RNase H (Y501R and Y501W) resulted in a reduction of JMC6F potency, thus indicating their potential role in providing resistance to JMC6F. These data provided important insights into the binding mode and resistance profile of the inhibitors with a pyrrolyl diketo acid scaffold in HIV-1 IN and RNase H, which would be helpful for the development of more effective dual HIV-1 IN and RNase H inhibitors.

Solid Phase Synthesis of Polymacromer and Copolymacromer Brushes

DTIC Science & Technology

2012-04-25

between immiscible polymers, which dictates the volume in which the interfacial reaction can occur, depends on the nature of thermodynamic interactions...Y.; Beers , K. L.; Byrd, H. C. M.; VanderHart, D. L.; Washburn, N. R. J. Am. Chem. Soc. 2004, 126, 3472−3476. (4) Merrifield, R. B. J. Am. Chem. Soc...Hartmann, L.; Krause, E.; Antonietti, M.; Boerner, H. G. Biomacromolecules 2006, 7, 1239−1244. (16) Mei, Y.; Beers , K. L.; Byrd, H. C. M.; VanderHart, D. L

Hypothermia-induced dystonia and abnormal cerebellar activity in a mouse model with a single disease-mutation in the sodium-potassium pump

PubMed Central

Isaksen, Toke Jost; Vedovato, Natascia; Vitenzon, Ariel; Gadsby, David C.; Khodakhah, Kamran

2017-01-01

Mutations in the neuron-specific α3 isoform of the Na+/K+-ATPase are found in patients suffering from Rapid onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood, two closely related movement disorders. We show that mice harboring a heterozygous hot spot disease mutation, D801Y (α3+/D801Y), suffer abrupt hypothermia-induced dystonia identified by electromyographic recordings. Single-neuron in vivo recordings in awake α3+/D801Y mice revealed irregular firing of Purkinje cells and their synaptic targets, the deep cerebellar nuclei neurons, which was further exacerbated during dystonia and evolved into abnormal high-frequency burst-like firing. Biophysically, we show that the D-to-Y mutation abolished pump-mediated Na+/K+ exchange, but allowed the pumps to bind Na+ and become phosphorylated. These findings implicate aberrant cerebellar activity in α3 isoform-related dystonia and add to the functional understanding of the scarce and severe mutations in the α3 isoform Na+/K+-ATPase. PMID:28472154

Identification of eight mutations and three sequence variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghanem, N.; Costes, B.; Girodon, E.

1994-05-15

To determine cystic fibrosis (CF) defects in a sample of 224 non-[Delta]F508 CF chromosomes, the authors used denaturing gradient gel multiplex analysis of CF transmembrane conductance regulator gene segments, a strategy based on blind exhaustive analysis rather than a search for known mutations. This process allowed detection of 11 novel variations comprising two nonsense mutations (Q890X and W1204X), a splice defect (405 + 4 A [yields] G), a frameshift (3293delA), four presumed missense mutations (S912L, H949Y, L1065P, Q1071P), and three sequence polymorphisms (R31C or 223 C/T, 3471 T/C, and T1220I or 3791 C/T). The authors describe these variations, together withmore » the associated phenotype when defects on both CF chromosomes were identified. 8 refs., 1 fig., 1 tab.« less

Identification and characterization of novel SNPs in CHEK2 in Ashkenazi Jewish men with prostate cancer

PubMed Central

Tischkowitz, Marc D.; Yilmaz, Ahmet; Chen, Long Q.; Karyadi, Danielle M.; Novak, David; Kirchhoff, Tomas; Hamel, Nancy; Tavtigian, Sean V.; Kolb, Suzanne; Bismar, Tarek A.; Aloyz, Raquel; Nelson, Peter S.; Hood, Lee; Narod, Steven A.; White, Kirsten A.; Ostrander, Elaine A.; Isaacs, William B.; Offit, Kenneth; Cooney, Kathleen A.; Stanford, Janet L.; Foulkes, William D.

2010-01-01

Checkpoint kinase 2 (CHEK2) is a protein involved in arresting cell cycle in response to DNA damage. To investigate whether it plays an important role in the development of prostate cancer (PRCA) in the Ashkenazi Jewish (AJ) population, we sequenced CHEK2 in 75 AJ individuals with prostate, breast, or no cancer (n = 25 each). We identified seven coding SNPs (five are novel) that changed the amino acid sequence, resulting in R3W, E394F, Y424H, S428F, D438Y, P509S, and P509L. We determined the frequency of each variant in 76 AJ families collected by members of the International Consortium for Prostate Cancer Genetics (ICPCG) where ≥ 2 men were affected by PRCA. Only one variant, Y424H in exon 11, was identified in more than two families. Exon 11 was then screened in nine additional AJ ICPCG families (a total of 85 families). The Y424H variant occurred in nine affected cases from four different families; however it did not completely segregate with the disease. We performed bioinformatics analysis, which showed that Y424H is a non-conservative missense substitution that falls at a position that is invariant in vertebrate CHEK2 orthologs. Both SIFT and Align-GVGD predict that Y424H is a loss of function mutation, however the frequency of Y424H was not significantly different between unselected AJ cases from Montreal/Memorial Sloan Kettering Cancer Centre (MSKCC) and AJ controls from Israel/MSKCC (OR 1.18, 95%CI: 0.34–4.61, p=.99). Moreover, functional assays using S. Cerevisae revealed that the Y424H substitution did not alter function of CHEK2 protein. Although we cannot rule out a subtle influence of the CHEK2 variants on PRCA risk, these results suggest that germline CHEK2 mutations have a minor role in PRCA susceptibility in AJ men. PMID:18571837

Establishment of a rat hepatoma-derived cell line proliferating in D-phenylalanine medium and expressing D-amino-acid oxidase.

PubMed

Yoda, N; Konno, R; Nagashima, S

2001-01-01

A cell line (R-Y121B.DF) has been established from a cell line (R-Y121B) derived from a rat hepatoma line (H4-II-E). The R-Y121B.DF cells have been continuously cultured in a serum-free modified Eagle's minimum essential medium in which L-phenylalanine was replaced by D-phenylalanine. They had D-amino-acid oxidase (DAO) activity which is essential for the growth in the medium containing D-amino acids. The enzyme activity of the R-Y121B.DF cells was approximately one-fourth of that of the rat liver. Northern hybridization using a DAO cDNA probe detected a hybridizing signal in the R-Y121B.DF cells and the rat liver but not in the parental R-Y121B and H4-II-E cells. Reverse transcription-polymerase chain reaction using DAO-specific primers amplified a DNA fragment of the expected size in the R-Y121B.DF cells but not in the R-Y121B and H4-II-E cells. This fragment was confirmed to be DAO cDNA by nucleotide sequencing. Western blotting showed that DAO protein was present in the R-Y121B.DF cells and the rat liver but not in the R-Y121B and H4-II-E cells. Southern hybridization showed that the DAO gene structure was not different among the R-Y121B.DF cells, R-Y121B cells, H4-II-E cells, and the rat liver. These results indicate that the R-Y121B.DF is a unique cell line which proliferates in the medium containing D-phenylalanine and explicitly expresses DAO. This line is useful for the study of DAO in vitro.

Role of DNA base excision repair in the mutability and virulence of Streptococcus mutans

PubMed Central

Gonzalez, Kaisha; Faustoferri, Roberta C.; Quivey, Robert G.

2012-01-01

Summary The oral pathogen, Streptococcus mutans, possesses inducible DNA repair defenses for protection against pH fluctuations and production of reactive oxygen metabolites such as hydrogen peroxide (H2O2), which are present in the oral cavity. DNA base excision repair (BER) has a critical role in genome maintenance by preventing the accumulation of mutations associated with environmental factors and normal products of cellular metabolism. In this study, we examined the consequences of compromising the DNA glycosylases (Fpg and MutY) and endonucleases (Smx and Smn) of the BER pathway and their relative role in adaptation and virulence. Enzymatic characterization of the BER system showed that it protects the organism against the effects of the highly mutagenic lesion, 7,8-dihydro-8-oxo-2’-deoxyguanine (8-oxo-dG). S. mutans strains lacking a functional Fpg, MutY, or Smn showed elevated spontaneous mutation frequencies; and, these mutator phenotypes correlated with the ability of the strains to survive killing by acid and oxidative agents. In addition, in the G. mellonella virulence model, strains of S. mutans deficient in Fpg, MutY and Smn showed increased virulence as compared to the parent strain. Our results suggest that, for S. mutans, mutator phenotypes, due to loss of BER enzymes, may confer an advantage to virulence of the organism. PMID:22651851

Menaquinone-7 production from maize meal hydrolysate by Bacillus isolates with diphenylamine and analogue resistance* #

PubMed Central

Xu, Jian-zhong; Zhang, Wei-guo

2017-01-01

A menaquinone-7 (MK-7) high-producing strain needs to be isolated to increase MK-7 production, in order to meet a requirement of MK-7 given the low MK-7 content in food products. This article focuses on developing MK-7 high-producing strains via screening and mutagenesis by an atmospheric and room temperature plasma (ARTP) mutation breeding system. We isolated an MK-7-producing strain Y-2 and identified it as Bacillus amyloliquefaciens, which produced (7.1±0.5) mg/L of MK-7 with maize meal hydrolysate as carbon source. Then, an MK-7 high-producing strain B. amyloliquefaciens H.β.D.R.-5 with resistance to 1-hydroxy-2-naphthoic acid, β-2-thienylalanine, and diphenylamine was obtained from the mutation of the strain Y-2 using an ARTP mutation breeding system. Using strain H.β.D.R.-5, efficient production of MK-7 was achieved ((30.2±2.7) mg/L). In addition, the effects of nitrogen sources, prenyl alcohols, and MgSO4 on MK-7 production were investigated, suggesting that soymeal extract combined with yeast extract, isopentenol, and MgSO4 was beneficial. Under the optimized condition, the MK-7 production and biomass-specific yield reached (61.3±5.2) mg/L and 2.59 mg/L per OD600 unit respectively in a 7-L fermenter. These results demonstrated that strain H.β.D.R.-5 has the capacity to produce MK-7 from maize meal hydrolysate, which could reduce the substrate cost. PMID:28585422

Rifabutin and rifampin resistance levels and associated rpoB mutations in clinical isolates of Mycobacterium tuberculosis complex.

PubMed

Berrada, Zenda L; Lin, Shou-Yean Grace; Rodwell, Timothy C; Nguyen, Duylinh; Schecter, Gisela F; Pham, Lucy; Janda, J Michael; Elmaraachli, Wael; Catanzaro, Antonino; Desmond, Edward

2016-06-01

Cross-resistance in rifamycins has been observed in rifampin (RIF)-resistant Mycobacterium tuberculosis complex isolates; some rpoB mutations do not confer broad in vitro rifamycin resistance. We examined 164 isolates, of which 102 were RIF-resistant, for differential resistance between RIF and rifabutin (RFB). A total of 42 unique single mutations or combinations of mutations were detected. The number of unique mutations identified exceeded that reported in any previous study. RFB and RIF MICs up to 8 μg/mL by MGIT 960 were studied; the cut-off values for susceptibility to RIF and RFB were 1 μg/mL and 0.5 μg/mL, respectively. We identified 31 isolates resistant to RIF but susceptible to RFB with the mutations D516V, D516F, 518 deletion, S522L, H526A, H526C, H526G, H526L, and two dual mutations (S522L + K527R and H526S + K527R). Clinical investigations using RFB to treat multidrug-resistant tuberculosis cases harboring those mutations are recommended. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Association of MTHFR gene C677T mutation with diabetic peripheral neuropathy and diabetic retinopathy

PubMed Central

Yigit, Serbulent; Inanir, Ahmet

2013-01-01

Purpose Diabetic peripheral neuropathy (DPN) is one of the most common diabetic chronic complications. Methylenetetrahydrofolate reductase (MTHFR) gene variants have been associated with vasculopathy that has been linked to diabetic neuropathy. The aim of the present study was to investigate the possible association between MTHFR gene C677T mutation and DPN and evaluate if there is an association with clinical features in a relatively large cohort of Turkish patients. Methods The study included 230 patients affected by DPN and 282 healthy controls. Genomic DNA was isolated and genotyped using the polymerase chain reaction–based restriction fragment length polymorphism assay for the MTHFR gene C677T mutation. Results The genotype and allele frequencies of the C677T mutation showed statistically significant differences between the patients with DPN and the controls (p=0.003 and p=0.002, respectively). After the patients with DPN were stratified according to clinical and demographic characteristics, a significant association was observed between the C677T mutation and history of retinopathy (p=0.039). Conclusions A high association between the MTHFR gene C677T mutation and DPN was observed in the present study. In addition, history of retinopathy was associated with the MTHFR C677T mutation in patients with DPN. PMID:23901246

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morra, Simone; Maurelli, Sara; Chiesa, Mario

A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid caused significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA bymore » CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redox equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics.« less

Understanding the effects on constitutive activation and drug binding of a D130N mutation in the β2 adrenergic receptor via molecular dynamics simulation.

PubMed

Zhu, Yanyan; Yuan, Yuan; Xiao, Xiuchan; Zhang, Liyun; Guo, Yanzhi; Pu, Xuemei

2014-11-01

G-protein-coupled receptors (GPCRs) are currently one of the largest families of drug targets. The constitutive activation induced by mutation of key GPCR residues is associated closely with various diseases. However, the structural basis underlying such activation and its role in drug binding has remained unclear. Herein, we used all-atom molecular dynamics simulations and free energy calculations to study the effects of a D130N mutation on the structure of β2 adrenergic receptor (β2AR) and its binding of the agonist salbutamol. The results indicate that the mutation caused significant changes in some key helices. In particular, the mutation leads to the departure of transmembrane 3 (TM3) from transmembrane 6 (TM6) and marked changes in the NPxxY region as well as the complete disruption of a key ionic lock, all of which contribute to the observed constitutive activation. In addition, the D130N mutation weakens some important H-bonds, leading to structural changes in these regions. Binding free energy calculations indicate that van der Waals and electrostatic interactions are the main driving forces in binding salbutamol; however, binding strength in the mutant β2AR is significantly enhanced mainly through modifying electrostatic interactions. Further analysis revealed that the increase in binding energy upon mutation stems mainly from the H-bonds formed between the hydroxyl group of salbutamol and the serine residues of TM5. This observation suggests that modifications of the H-bond groups of this drug could significantly influence drug efficacy in the treatment of diseases associated with this mutation.

Molecular Biology of STLV-III and HTLV-IV

DTIC Science & Technology

1990-08-22

8217 AT( ACCA CTCAT.(3CA 5 Ci.T G,- .... ; . , P P, ( L D O S H s v H I -U" G r L "S " V " " , 3 E E 7 ’ T - ’ ’ ’ TJ - " t f? I f T G A- C-. C .T ’ CA8C P...R S N T V K N S D’ . Y S T m 2: G ! Y ’) V V G v I L L R 7 V T Y I’ ,9 M 3: i E F M C f7 E e I V C? iT C? e S . (N*’ 1 . Flist 5At(3CTAGC’’TAATGrAG

Characterisation of four novel fibrillin-1 (FBN1) mutations in Marfan syndrome.

PubMed Central

Adès, L C; Haan, E A; Colley, A F; Richard, R I

1996-01-01

Forty-four percent of the fibrillin-1 gene (FBN1) from 19 unrelated families with Marfan syndrome was screened for putative mutations by single strand conformational polymorphism (SSCP) analysis. Four novel mutations were identified and characterised in five people, three with classical Marfan syndrome (two from one family, and one from an unrelated family), one with a more severe phenotype, and one with neonatal Marfan syndrome. The base substitutions G2113A, G2132A, T3163G, and G3458A result in amino acid substitutions A705T, C711Y, C1055G, and C1152Y, respectively. C711Y, C1055G, and C1152Y lead to replacement of a cysteine by another amino acid; the latter two occur within epidermal growth factor-like motifs in exon 25 and 27, respectively. The A705T mutation occurs at exon 16 adjacent to the GT splice site. The A705T and C711Y mutations, at exon 16 and 17, respectively, are the first documented in the second transforming growth factor-beta 1 binding protein-like motif of FBN1. Images PMID:8863159

Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl− channel

PubMed Central

Xu, Weiyi; Sheppard, David N.

2017-01-01

Key points The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP.To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH.We found that short‐lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP‐binding site 1 formed by the nucleotide‐binding domains.Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects. Abstract Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐gated Cl− channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP‐binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single‐channel activity of human CFTR at different intracellular pH (pHi) values. When compared with the control (pHi 7.3), acidifying pHi to 6.3 or alkalinizing pHi to 8.3 and 8.8 caused small reductions in the open‐time constant (τo) of wild‐type CFTR. By contrast, the fast closed‐time constant (τcf), which describes the short‐lived closures that interrupt open bursts, was greatly increased at pHi 5.8 and 6.3. To analyse intraburst kinetics, we used linear three‐state gating schemes. All data were satisfactorily modelled by the C1 ↔ O ↔ C2 kinetic scheme. Changing the intracellular ATP concentration was without effect on τo, τcf and their responses to pHi changes. However, mutations that disrupt the interaction of ATP with ATP‐binding site 1, including K464A, D572N and the CF‐associated mutation G1349D all abolished the prolongation of τcf at pHi 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP‐dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP‐binding site 1 modulates intraburst gating. PMID:27779763

Characterization of active-site residues of the NIa protease from tobacco vein mottling virus.

PubMed

Hwang, D C; Kim, D H; Lee, J S; Kang, B H; Han, J; Kim, W; Song, B D; Choi, K Y

2000-10-31

Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased kcat marginally by about 4.7-fold and increased Km by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in kcat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pKa values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.

Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist.

PubMed

Xiang, Zhimin; Proneth, Bettina; Dirain, Marvin L; Litherland, Sally A; Haskell-Luevano, Carrie

2010-06-08

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.

Antimony-Based Type-II Superlattice Photodetectors

DTIC Science & Technology

2010-09-06

photodetectors, antimony-based mid- infrared detectors Shun L. Chuang, Russell D. Dupuis University of Illinois - Urbana Grants and Contracts Office...Q. Yang, Interband Cascade Detectors , US Patent #7,282,777, October 16, 2007. Graduate Students PERCENT_SUPPORTEDNAME Shin Mou 0.25 Adam Petschke...DD882) Inventions (DD882) Interband Cascade Detectors Patent Filed in US? (5d-1) Y NPatent Filed in Foreign Countries? (5d-2) Was the assignment

Molecular Mechanisms and Treatment Strategies for Obesity-Associated Coronary Artery Disease, an Imminent Military Epidemic

DTIC Science & Technology

2006-12-01

acyltransferase by compound 58-035. J.Biol.Chem. 259:815-819. 5. Li,Y., Gerbod- Giannone ,M.C., Seitz,H., Cui,D., Thorp,E., Tall,A.R., Matsushima,G.K., and...differentiation of human breast cancer through PPAR gamma. Mol.Cell 1:465-470. 9. Li,Y., Schwabe,R.F., DeVries-Seimon,T., Yao,P.M., Gerbod- Giannone ...receptor. J. Cell Biol. 171:61-73. *86. Li, Y., Gerbod- Giannone , M.C., Seitz, H., Cui, D., Thorp, E., Tall, A.R., Matsushima, G.K., and Tabas

Mutation analysis and molecular modeling for the investigation of ligand-binding modes of GPR84.

PubMed

Nikaido, Yoshiaki; Koyama, Yuuta; Yoshikawa, Yasushi; Furuya, Toshio; Takeda, Shigeki

2015-05-01

GPR84 is a G protein-coupled receptor for medium-chain fatty acids. Capric acid and 3,3'-diindolylmethane are specific agonists for GPR84. We built a homology model of a GPR84-capric acid complex to investigate the ligand-binding mode using the crystal structure of human active-state β2-adrenergic receptor. We performed site-directed mutagenesis to subject ligand-binding sites to our model using GPR84-Giα fusion proteins and a [(35)S]GTPγS-binding assay. We compared the activity of the wild type and mutated forms of GPR84 by [(35)S]GTPγS binding to capric acid and diindolylmethane. The mutations L100D `Ballesteros-Weinstein numbering: 3.32), F101Y (3.33) and N104Q (3.36) in the transmembrane helix III and N357D (7.39) in the transmembrane helix VII resulted in reduced capric acid activity but maintained the diindolylmethane responses. Y186F (5.46) and Y186H (5.46) mutations had no characteristic effect on capric acid but with diindolylmethane they significantly affected the G protein activation efficiency. The L100D (3.32) mutant responded to decylamine, a fatty amine, instead of a natural agonist, the fatty acid capric acid, suggesting that we have identified a mutated G protein-coupled receptor-artificial ligand pairing. Our molecular model provides an explanation for these results and interactions between GPR84 and capric acid. Further, from the results of a double stimulation assay, we concluded that diindolylmethane was a positive allosteric modulator for GPR84. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Mechanism of Poliovirus Resistance to Host Phosphatidylinositol-4 Kinase III β Inhibitor.

PubMed

Arita, Minetaro

2016-02-12

Phosphatidylinositol-4 kinase III β (PI4KB) and oxysterol-binding protein (OSBP) family I have been identified as the major targets of anti-enterovirus drug candidates. Resistance mutations in poliovirus (PV) to these inhibitors have been identified in viral 3A protein, represented by a G5318A (3A-Ala70Thr) mutation, but the mechanism of viral resistance to host PI4KB/OSBP inhibitors remained unknown. In this study, we found that a G5318A mutation enhances the basal levels of phosphatidylinositol 4-phosphate (PI4P) and of the 3A protein and decreases the levels of the 3AB protein during PV replication. The 3A protein acted as a major effector responsible for the resistance to PI4KB inhibitor, but did not enhance the PI4KB activity in vitro in contrast to the 2C, 2BC, 3AB, and 3D proteins. The 3AB protein acted as the primary target of a G5318A mutation and also as an effector. We identified novel resistance mutations to a PI4KB inhibitor [C5151U (3A-T14M) and C5366U (3A-H86Y) mutations] and found that there is a positive correlation between the extent of the resistance phenotype and the levels of the 3A proteins. These results suggested that the 3A protein overproduced by enhanced processing of the 3AB protein with the resistance mutations overcomes the inhibitory effect of PI4KB inhibitor on PV replication independently of the hyperactivation of the PI4KB/OSBP pathway.

Superconductor-to-insulator transition and transport properties of underdoped YBa2Cu3O(y) crystals.

PubMed

Semba, K; Matsuda, A

2001-01-15

The carrier-concentration-driven superconductor-to-insulator (SI) transition as well as transport properties in underdoped YBa2Cu3O(y) twinned crystals is studied. The SI transition takes place at y approximately 6.3, carrier concentration n(SI)H approximately 3x10(20) cm(-3), anisotropy rho(c)/rho(ab) approximately 10(3), and the threshold resistivity rho(SI)ab approximately 0.8 mOmega cm which corresponds to a critical sheet resistance h/4e2 approximately 6.5 kOmega per CuO2 bilayer. The evolution of a carrier, nH infiniti y - 6.2, is clearly observed in the underdoped region. The resistivity and Hall coefficient abruptly acquire strong temperature dependence at y approximately 6.5 indicating a radical change in the electronic state.

Energetics of the O-H bond and of intramolecular hydrogen bonding in HOC6H4C(O)Y (Y = H, CH3, CH2CH=CH2, C[triple bond]CH, CH2F, NH2, NHCH3, NO2, OH, OCH3, OCN, CN, F, Cl, SH, and SCH3) compounds.

PubMed

Bernardes, Carlos E S; Minas da Piedade, Manuel E

2008-10-09

The energetics of the phenolic O-H bond in a series of 2- and 4-HOC 6H 4C(O)Y (Y = H, CH3, CH 2CH=CH2, C[triple bond]CH, CH2F, NH2, NHCH 3, NO2, OH, OCH3, OCN, CN, F, Cl, SH, and SCH3) compounds and of the intramolecular O...H hydrogen bond in 2-HOC 6H 4C(O)Y, was investigated by using a combination of experimental and theoretical methods. The standard molar enthalpies of formation of 2-hydroxybenzaldehyde (2HBA), 4-hydroxybenzaldehyde (4HBA), 2'-hydroxyacetophenone (2HAP), 2-hydroxybenzamide (2HBM), and 4-hydroxybenzamide (4HBM), at 298.15 K, were determined by micro- or macrocombustion calorimetry. The corresponding enthalpies of vaporization or sublimation were also measured by Calvet drop-calorimetry and Knudsen effusion measurements. The combination of the obtained experimental data led to Delta f H m (o)(2HBA, g) = -238.3 +/- 2.5 kJ.mol (-1), DeltafHm(o)(4HBA, g) = -220.3 +/- 2.0 kJ.mol(-1), Delta f H m (o)(2HAP, g) = -291.8 +/- 2.1 kJ.mol(-1), DeltafHm(o)(2HBM, g) = -304.8 +/- 1.5 kJ.mol (-1), and DeltafHm(o) (4HBM, g) = -278.4 +/- 2.4 kJ.mol (-1). These values, were used to assess the predictions of the B3LYP/6-31G(d,p), B3LYP/6-311+G(d,p), B3LYP/aug-cc-pVDZ, B3P86/6-31G(d,p), B3P86/6-311+G(d,p), B3P86/aug-cc-pVDZ, and CBS-QB3 methods, for the enthalpies of a series of isodesmic gas phase reactions. In general, the CBS-QB3 method was able to reproduce the experimental enthalpies of reaction within their uncertainties. The B3LYP/6-311+G(d,p) method, with a slightly poorer accuracy than the CBS-QB3 approach, achieved the best performance of the tested DFT models. It was further used to analyze the trends of the intramolecular O...H hydrogen bond in 2-HOC 6H 4C(O)Y evaluated by the ortho-para method and to compare the energetics of the phenolic O-H bond in 2- and 4-HOC 6H 4C(O)Y compounds. It was concluded that the O-H bond "strength" is systematically larger for 2-hydroxybenzoyl than for the corresponding 4-hydroxybenzoyl isomers mainly due to the presence of the intramolecular O...H hydrogen bond in the 2-isomers. The observed differences are, however, significantly dependent on the nature of the substituent Y, in particular, when an intramolecular H-bond can be present in the radical obtained upon cleavage of the O-H bond.

The Influence of Fabrication Process on the Initial Stages of Steam Oxidation Performed on Haynes® 282® Alloy at 760 °C

NASA Astrophysics Data System (ADS)

Dudziak, T.; Boron, L.; Homa, M.; Nowak, R.; Horton, N.; Sheppard, R.; Purgert, R. M.; Siewiorek, A.; Sobczak, N.; Sobczak, J. J.

2017-01-01

This work presents results observed after the first 5 h of oxidation of Haynes® 282® alloy. The steam oxidation tests have been carried out in pure water at 760 °C for 1, 2 and 5 h, respectively, using an accurate thermogravimetric balance technique. The alloy used for comparison in this work was fabricated using three different methods. The initial steam oxidation performance of the commercially wrought alloy Haynes® 282® was compared with a fabricated cast alloy and a HIP/PM alloy. The results show that in terms of corrosion resistance, fabrication techniques appear to have little impact on steam oxidation performance and behavior. The exposed Ni-based alloys all developed the oxide scales consisting mainly of Cr2O3 phase mixed with some TiO2, while internal Al and Ti precipitations along the grain boundaries were observed both in Haynes® 282® wrought and HIP/PM alloy.

Microarray-based mutation detection and phenotypic characterization in Korean patients with retinitis pigmentosa

PubMed Central

Kim, Cinoo; Kim, Kwang Joong; Bok, Jeong; Lee, Eun-Ju; Kim, Dong-Joon; Oh, Ji Hee; Park, Sung Pyo; Shin, Joo Young; Lee, Jong-Young

2012-01-01

Purpose To evaluate microarray-based genotyping technology for the detection of mutations responsible for retinitis pigmentosa (RP) and to perform phenotypic characterization of patients with pathogenic mutations. Methods DNA from 336 patients with RP and 360 controls was analyzed using the GoldenGate assay with microbeads containing 95 previously reported disease-associated mutations from 28 RP genes. Mutations identified by microarray-based genotyping were confirmed by direct sequencing. Segregation analysis and phenotypic characterization were performed in patients with mutations. The disease severity was assessed by visual acuity, electroretinography, optical coherence tomography, and kinetic perimetry. Results Ten RP-related mutations of five RP genes (PRP3 pre-mRNA processing factor 3 homolog [PRPF3], rhodopsin [RHO], phosphodiesterase 6B [PDE6B], peripherin 2 [PRPH2], and retinitis pigmentosa 1 [RP1]) were identified in 26 of the 336 patients (7.7%) and in six of the 360 controls (1.7%). The p.H557Y mutation in PDE6B, which was homozygous in four patients and heterozygous in nine patients, was the most frequent mutation (2.5%). Mutation segregation was assessed in four families. Among the patients with missense mutations, the most severe phenotype occurred in patients with p.D984G in RP1; less severe phenotypes occurred in patients with p.R135W in RHO; a relatively moderate phenotype occurred in patients with p.T494M in PRPF3, p.H557Y in PDE6B, or p.W316G in PRPH2; and a mild phenotype was seen in a patient with p.D190N in RHO. Conclusions The results reveal that the GoldenGate assay may not be an efficient method for molecular diagnosis in RP patients with rare mutations, although it has proven to be reliable and efficient for high-throughput genotyping of single-nucleotide polymorphisms. The clinical features varied according to the mutations. Continuous effort to identify novel RP genes and mutations in a population is needed to improve the efficiency and accuracy of the genetic diagnosis of RP. PMID:23049240

Derivation of the P2T Criterion (in Closed Form) Based on the Frey Shear Band Mechanism

DTIC Science & Technology

1984-01-01

max 6d/d = G (12) max o u Pil < o .tlllilltt, >. X !5 < Q: o W) TJ C rH CU •H ed •P in -H 03 W 4-" FH cd C 03 Cu <U P...C 3 -H 10 T3 cd T3 O H s h ♦J 03 PH If) X3 -d rH fH ^H ed 03 OJ -H (1) •H -t-i Xi MH -H in c X’H <D ■M 43 • H 0 +J O...P o c a> rH X! 0 M 03 w C M •H O 03 u S & K <u TO S 0) +-> FH O ^—^ o3 W) y ^ cd i S: •H -H O -d T3 T3 iH CD *W P O nj

Cloud Climatology Derived from the AFGWC (Air Force Global Weather Central) 3D-Nephanalysis for January and July 1979.

DTIC Science & Technology

1987-01-15

January 1979. 29 Coo E 1979 2~0 C NHOOO. 03 to F *N9 90 E Januar y T i a J d e 1979 ClIou d 20 0 t0i 1 31 " -M No n t h I y 90 w Fig. 14 The monthly...r- 4 -Vto C UovjS 3H 3AOV 30 IJWII H913Hono-4 9__ . bases. The cloud amount from the visual satellite processor is obtained by comparing the 64...satellite analyses are combined to produce a single satellite c .. 10 1n 0 a 0 c ____ ___ ____ ___ ____ ___ a__ _ a0 0 0 "w -4 O *0u mC 07 o v" 0 0. cr. 4" L

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)

PubMed Central

McCabe, Michael T.; Graves, Alan P.; Ganji, Gopinath; Diaz, Elsie; Halsey, Wendy S.; Jiang, Yong; Smitheman, Kimberly N.; Ott, Heidi M.; Pappalardi, Melissa B.; Allen, Kimberly E.; Chen, Stephanie B.; Della Pietra, Anthony; Dul, Edward; Hughes, Ashley M.; Gilbert, Seth A.; Thrall, Sara H.; Tummino, Peter J.; Kruger, Ryan G.; Brandt, Martin; Schwartz, Benjamin; Creasy, Caretha L.

2012-01-01

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 kcat/Km ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 kcat/Km ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 kcat/Km ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2. PMID:22323599

Late-gestation heat stress abatement on performance and behavior of Holstein dairy cows.

PubMed

Karimi, M T; Ghorbani, G R; Kargar, S; Drackley, J K

2015-10-01

The objective of this study was to evaluate cooling to lessen the effects of heat stress during the last 3 wk of gestation on performance and behavior of multiparous Holstein cows. Twenty nonlactating cows were randomly assigned to treatments approximately 21 d before their expected calving date based on mature equivalent milk production and parity. Treatments were only imposed during the last 3 wk of gestation and included heat stress (HT; n=10) and cooling (CL; n=10), both under a similar photoperiod (14 h of light and 10 h of dark). Dry cows were housed in a sand-bedded stall with the stall areas for CL cows equipped with sprinklers and fans that were on from 0700 to 1900 h, whereas those for the HT cows were not. After parturition, all cows were housed in a barn with cooling devices. Rectal temperatures were measured daily at 1400 h and respiration rates were recorded by counting the flank movements for 1 min at 1500 h on odd days over the last 3 wk of gestation to calving. Daily dry matter intake was measured from -21 d relative to expected calving to 21 d after calving and milk production was recorded daily up to 180 d in milk. Behavioral changes of dry cows were studied continuously for 24 h at -10 d relative to expected calving. The average temperature-humidity index during the last 3 wk of gestation was 69.7 and was not significantly different between treatments. Heat-stressed cows exhibited greater rectal temperatures (39.5 vs. 39.2°C), greater respiration rates (70.4 vs. 63.3 breaths/min), and decreased dry matter intake (13.7 vs. 15.5 kg/d) compared with CL cows. Compared with HT cows, CL cows produced more milk during 180 d in milk (40.5 vs. 44.6 kg/d). Heat stress decreased ruminating (243.2 vs. 282.5 min/d) and chewing times (390.6 vs. 448.7 min/d) at -10 d before calving. The CL cows had shorter standing times than their HT counterparts (390.4 vs. 474.0 min/d). These results confirm that heat stress abatement in the late gestation period improves performance of dairy cows in subsequent lactation. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Molecular Epidemiology of Malaria in Cameroon and Côte d'Ivoire. XXXI. Kelch 13 Propeller Sequences in Plasmodium falciparum Isolates before and after Implementation of Artemisinin-Based Combination Therapy.

PubMed

Djaman, Joseph Allico; Olefongo, Dagnogo; Ako, Aristide Berenger; Roman, Jocelyne; Ngane, Vincent Foumane; Basco, Leonardo K; Tahar, Rachida

2017-07-01

Artemisinin-resistant malaria has not been reported from Africa, but resistance can possibly spread from Asia or arise independently in Africa. The emergence of artemisinin resistance in Africa can be monitored by molecular assay of Kelch 13 (K13) propeller sequences. A total of 251 archived DNA samples of Plasmodium falciparum isolates collected in 2002, 2003, and 2006 in Yaounde, Cameroon, and 47 samples collected in 2006 and 2013 in Abidjan, Côte d'Ivoire, were analyzed for K13-propeller sequence polymorphism. Only one isolate carried a mutant K13-propeller allele (E602D). None of the isolates carried the key mutant alleles (Y493H, R539T, I543T, and C580Y) associated with artemisinin resistance in Cambodia. The presence of the mutant allele was not correlated with in vitro response to dihydroartemisinin determined by the classical hypoxanthine incorporation assay. There was no evidence of K13 mutations associated with artemisinin resistance before and soon after the introduction of artemisinin-based combination therapies in Cameroon and Côte d'Ivoire.

Further Analysis of the Crouzon Mouse, Effects of the FGFR2C342Y Mutation are Cranial Bone Dependent

PubMed Central

Liu, Jin; Nam, Hwa Kyung; Wang, Estee; Hatch, Nan E.

2013-01-01

Crouzon syndrome is a debilitating congenital disorder involving abnormal craniofacial skeletal development caused by mutations in Fibroblast Growth Factor Receptor-2 (FGFR2). Phenotypic expression in humans exhibits an autosomal dominant pattern that commonly involves premature fusion of the coronal suture (craniosynostosis) and severe midface hypoplasia. To further investigate biologic mechanisms by which the Crouzon syndrome associated FGFR2C342Y mutation leads to abnormal craniofacial skeletal development we created congenic BALB/c FGFR2C342Y/+ mice. Here we show that BALB/c FGFR2C342Y/+ mice have a consistent craniofacial phenotype including partial fusion of the coronal and lambdoid sutures, intersphenoidal synchondrosis and multiple facial bones, with minimal fusion of other craniofacial sutures. This phenotype is similar to the classic and less severe form of Crouzon syndrome that involves significant midface hypoplasia with limited craniosynostosis. Linear and morphometric analyses demonstrate that FGFR2C342Y/+ mice on the BALB/c genetic background differ significantly in form and shape from their wild type littermates, and that in this genetic background the FGFR2C342Y mutation preferentially effects some craniofacial bones and sutures over others. Analysis of cranial bone cells indicates that the FGFR2C342Y mutation promotes aberrant osteoblast differentiation and increased apoptosis that is more severe in frontal than parietal bone cells. Additionally, FGFR2C342Y/+ frontal but not parietal bones exhibit significantly diminished bone volume and density compared to wild type mice. These results confirm that FGFR2-associated craniosynostosis occurs in association with diminished cranial bone tissue and may provide a potential biologic explanation for the clinical finding of phenotype consistency that exists between many Crouzon syndrome patients. PMID:23358860

Investigation of structural dynamics of Thrombocytopenia Cargeeg mutants of human apoptotic cytochrome c: A molecular dynamics simulation approach.

PubMed

Muneeswaran, Gurusamy; Kartheeswaran, Subramanian; Pandiaraj, Manickam; Muthukumar, Kaliappan; Sankaralingam, Muniyandi; Arunachalam, Saravanavadivu

2017-11-01

Naturally occurring mutations to cytochrome c (cyt-c) have been identified recently in patients with mild autosomal dominant thrombocytopenia (low platelet levels), which yield cyt-c mutants with enhanced apoptotic activity. However, the molecular mechanism underlying this low platelet production and enhanced apoptosis remain unclear. Therefore, an attempt is made herein for the first time to investigate the effects of mutations of glycine 41 by serine (G41S) and tyrosine 48 by histidine (Y48H) on the conformational and dynamic changes of apoptotic (Fe 3+ ) cyt-c using all atom molecular dynamics (MD) simulations in explicit water solvent. Our 30ns MD simulations demonstrate considerable structural differences in G41S and Y48H compared to wild type (WT) cyt-c, such as increasing distances between the critical electron transfer residues results in open conformation at the heme active site, large fluctuations in β-turns and α-helices. Additionally, although the β-sheets remain mostly unaffected in all the three cyt-c simulations, the α-helices undergo conformational switch to β-turns in both the mutant simulations. Importantly, this conformational switch of α-helix to β-turn around heme active site should attributes to the loss of intraprotein H-bonds in the mutant simulations especially between NE2 (His26) and O (Pro44) in agreement with the experimental report. Further, essential dynamics analysis reveals that overall motions of WT cyt-c is mainly involved only in the first eigenvector, but in G41S and Y48H the overall motions are mainly in three and two eigenvectors respectively. Overall, the detailed atomistic level information provide a unifying description for the molecular mechanism of structural destabilization, disregulation of platelet formation and enhanced peroxidase activity of the mutant cyt-c's in the pathology of intrinsic apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hypertrimethylation.

PubMed

Ott, Heidi M; Graves, Alan P; Pappalardi, Melissa B; Huddleston, Michael; Halsey, Wendy S; Hughes, Ashley M; Groy, Arthur; Dul, Edward; Jiang, Yong; Bai, Yuchen; Annan, Roland; Verma, Sharad K; Knight, Steven D; Kruger, Ryan G; Dhanak, Dashyant; Schwartz, Benjamin; Tummino, Peter J; Creasy, Caretha L; McCabe, Michael T

2014-12-01

The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell-derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival. ©2014 American Association for Cancer Research.

Cancer-associated isocitrate dehydrogenase 1 (IDH1) R132H mutation and d-2-hydroxyglutarate stimulate glutamine metabolism under hypoxia.

PubMed

Reitman, Zachary J; Duncan, Christopher G; Poteet, Ethan; Winters, Ali; Yan, Liang-Jun; Gooden, David M; Spasojevic, Ivan; Boros, Laszlo G; Yang, Shao-Hua; Yan, Hai

2014-08-22

Mutations in the cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDH1) occur in several types of cancer, and altered cellular metabolism associated with IDH1 mutations presents unique therapeutic opportunities. By altering IDH1, these mutations target a critical step in reductive glutamine metabolism, the metabolic pathway that converts glutamine ultimately to acetyl-CoA for biosynthetic processes. While IDH1-mutated cells are sensitive to therapies that target glutamine metabolism, the effect of IDH1 mutations on reductive glutamine metabolism remains poorly understood. To explore this issue, we investigated the effect of a knock-in, single-codon IDH1-R132H mutation on the metabolism of the HCT116 colorectal adenocarcinoma cell line. Here we report the R132H-isobolome by using targeted (13)C isotopomer tracer fate analysis to trace the metabolic fate of glucose and glutamine in this system. We show that introduction of the R132H mutation into IDH1 up-regulates the contribution of glutamine to lipogenesis in hypoxia, but not in normoxia. Treatment of cells with a d-2-hydroxyglutarate (d-2HG) ester recapitulated these changes, indicating that the alterations observed in the knocked-in cells were mediated by d-2HG produced by the IDH1 mutant. These studies provide a dynamic mechanistic basis for metabolic alterations observed in IDH1-mutated tumors and uncover potential therapeutic targets in IDH1-mutated cancers. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Hemochromatosis and Iron Overload Screening (HEIRS) study design for an evaluation of 100,000 primary care-based adults.

PubMed

McLaren, Christine E; Barton, James C; Adams, Paul C; Harris, Emily L; Acton, Ronald T; Press, Nancy; Reboussin, David M; McLaren, Gordon D; Sholinsky, Phyliss; Walker, Ann P; Gordeuk, Victor R; Leiendecker-Foster, Catherine; Dawkins, Fitzroy W; Eckfeldt, John H; Mellen, Beverly G; Speechley, Mark; Thomson, Elizabeth

2003-02-01

The HEIRS Study will evaluate the prevalence, genetic and environmental determinants, and potential clinical, personal, and societal impact of hemochromatosis and iron overload in a multiethnic, primary care-based sample of 100,000 adults over a 5-year period. Participants are recruited from 5 Field Centers. Laboratory testing and data management and analysis are performed in a Central Laboratory and Coordinating Center, respectively. Participants undergo testing for serum iron measures and common mutations of the hemochromatosis gene ( ) on chromosome 6p and answer questions on demographics, health, and genetic testing attitudes. Participants with elevated values of transferrin saturation and serum ferritin and/or C282Y homozygosity are invited to undergo a comprehensive clinical examination (CCE), as are frequency-matched control subjects. These examinations provide data on personal and family medical history, lifestyle characteristics, physical examination, genetic counseling, and assessment of ethical, legal, and social implications. Primary and secondary causes of iron overload will be distinguished by clinical criteria. Iron overload will be confirmed by quantification of iron stores. Recruiting family members of cases will permit DNA analysis for additional genetic factors that affect iron overload. Of the first 50,520 screened, 51% are white, 24% are African American, 11% are Asian, 11% are Hispanic, and 3% are of other, mixed, or unidentified race; 63% are female and 37% are male. Information from the HEIRS Study will inform policy regarding the feasibility, optimal approach, and potential individual and public health benefits and risks of primary care-based screening for iron overload and hemochromatosis.

Mechanisms of Residual Stress Generation in Mechanical Surface Treatment: the Role of Cyclic Plasticity and Texture

DTIC Science & Technology

2015-08-24

modulus, yield strength, strain hardening exponent, and hardness can be estimated by this technique (Pethica 1982, Newey, Wilkins & Pollock 1982, Stone et...properties of metal alloys: a review", International Journal of Fatigue, vol. 24, no. 10, pp. 1021-1036. Newey, D., Wilkins , M.A. & Pollock, H.M. 1982...Zhang, Y., Zhang, S., Yu, C., Tang , Y., Zhang, H., Wu, H., Guo, D., Wang, S., Xia, X., Chen, M. & Dai, Y. 1997, "Laser shock-processing for

Pyridones as NNRTIs against HIV-1 mutants: 3D-QSAR and protein informatics

NASA Astrophysics Data System (ADS)

Debnath, Utsab; Verma, Saroj; Jain, Surabhi; Katti, Setu B.; Prabhakar, Yenamandra S.

2013-07-01

CoMFA and CoMSIA based 3D-QSAR of HIV-1 RT wild and mutant (K103, Y181C, and Y188L) inhibitory activities of 4-benzyl/benzoyl pyridin-2-ones followed by protein informatics of corresponding non-nucleoside inhibitors' binding pockets from pdbs 2BAN, 3MED, 1JKH, and 2YNF were analysed to discover consensus features of the compounds for broad-spectrum activity. The CoMFA/CoMSIA models indicated that compounds with groups which lend steric-cum-electropositive fields in the vicinity of C5, hydrophobic field in the vicinity of C3 of pyridone region and steric field in aryl region produce broad-spectrum anti-HIV-1 RT activity. Also, a linker rendering electronegative field between pyridone and aryl moieties is common requirement for the activities. The protein informatics showed considerable alteration in residues 181 and 188 characteristics on mutation. Also, mutants' isoelectric points shifted in acidic direction. The study offered fresh avenues for broad-spectrum anti-HIV-1 agents through designing new molecules seeded with groups satisfying common molecular fields and concerns of mutating residues.

A Survey of Serum Bactericidal Antibodies against Neisseria meningitidis Serogroups A, C, W and Y in Adolescents and Adults in the Republic of Korea.

PubMed

Kang, Jin-Han; Miao, Yan; Lee, SooYoung; Kim, Jong-Hyun; Lee, Kyung-Yil; Ma, Sang Hyuk; Jo, Dae Sun; Song, HyoYoung; Haag, Mendel

2016-03-01

This descriptive epidemiological study aimed to assess the prevalence of serum bactericidal antibodies against Neisseria meningitidis serogroups A, C, W and Y in adolescents and adults in the Republic of Korea. In total, 987 subjects aged 11-55 years from five geographical regions of Korea were included in the study. Human serum bactericidal assay (hSBA) was used to measure hSBA titres for serogroups A, C, W and Y. Percentages of subjects with hSBA titres ≥4 and ≥8, geometric mean titres (GMTs), and associated 95% confidence intervals (CIs), were estimated. Analysis was performed for the entire study population and stratified by age group or region. No statistical hypotheses were tested. The highest percentage of subjects with hSBA titres ≥8 was observed for serogroup W (74%), was similar for serogroups C (34%) and Y (36%), and was lowest for serogroup A (9%). The percentages of subjects with hSBA titres ≥4 were similar to those with hSBA titres ≥8 for all serogroups. GMTs were 2.56 µg/mL (serogroup A), 5.14 µg/mL (serogroup C), 22.63 µg/mL (serogroup W) and 5.28 µg/mL (serogroup Y). Similar trends in GMTs across serogroups were seen for individual regions and age groups. The highest GMTs for serogroups A, W and Y were recorded in the >19-29 years group, and for serogroup C in the >49-55 years group. Across all regions, GMTs were very similar for serogroups A, C and Y, while more variation was seen for serogroup W. In the Korean population, among Neisseria meningitidis serogroups A, C, W and Y, serum bactericidal antibodies were most prevalent against serogroup W and least prevalent against serogroup A. These trends were maintained across age groups and regions. The highest GMTs for serogroups A, W and Y were observed in the >19-29 years group. The reasons behind the observed differences in prevalence of bactericidal antibodies against the serogroups are currently not understood, although carriage and cross-reactivity of the assay may be important influences.

Origin and domestication of papaya Yh chromosome.

PubMed

VanBuren, Robert; Zeng, Fanchang; Chen, Cuixia; Zhang, Jisen; Wai, Ching Man; Han, Jennifer; Aryal, Rishi; Gschwend, Andrea R; Wang, Jianping; Na, Jong-Kuk; Huang, Lixian; Zhang, Lingmao; Miao, Wenjing; Gou, Jiqing; Arro, Jie; Guyot, Romain; Moore, Richard C; Wang, Ming-Li; Zee, Francis; Charlesworth, Deborah; Moore, Paul H; Yu, Qingyi; Ming, Ray

2015-04-01

Sex in papaya is controlled by a pair of nascent sex chromosomes. Females are XX, and two slightly different Y chromosomes distinguish males (XY) and hermaphrodites (XY(h)). The hermaphrodite-specific region of the Y(h) chromosome (HSY) and its X chromosome counterpart were sequenced and analyzed previously. We now report the sequence of the entire male-specific region of the Y (MSY). We used a BAC-by-BAC approach to sequence the MSY and resequence the Y regions of 24 wild males and the Y(h) regions of 12 cultivated hermaphrodites. The MSY and HSY regions have highly similar gene content and structure, and only 0.4% sequence divergence. The MSY sequences from wild males include three distinct haplotypes, associated with the populations' geographic locations, but gene flow is detected for other genomic regions. The Y(h) sequence is highly similar to one Y haplotype (MSY3) found only in wild dioecious populations from the north Pacific region of Costa Rica. The low MSY3-Y(h) divergence supports the hypothesis that hermaphrodite papaya is a product of human domestication. We estimate that Y(h) arose only ∼ 4000 yr ago, well after crop plant domestication in Mesoamerica >6200 yr ago but coinciding with the rise of the Maya civilization. The Y(h) chromosome has lower nucleotide diversity than the Y, or the genome regions that are not fully sex-linked, consistent with a domestication bottleneck. The identification of the ancestral MSY3 haplotype will expedite investigation of the mutation leading to the domestication of the hermaphrodite Y(h) chromosome. In turn, this mutation should identify the gene that was affected by the carpel-suppressing mutation that was involved in the evolution of males. © 2015 VanBuren et al.; Published by Cold Spring Harbor Laboratory Press.

Tumor driven by gain-of-function HER2 H878Y mutant is highly sensitive to HER2 inhibitor

PubMed Central

Hu, Zexi; Hu, Yong; Liu, Xicheng; Xi, Rongwen; Zhang, Aiqun; Liu, Deruo; Xie, Qiang; Chen, Liang

2015-01-01

HER2, a well established oncogenic member of EGFR family, is among the most intensely investigated kinase drug targets. In contrast to hotspot mutations of EGFR, few mutations of HER2 locate in activation loop within kinase domain. We previously reported the molecular mechanism underlying hyper kinase activity of HER2H878Y, a mutation located in activation loop. However, its tumorigenicity in vivo and relevant therapeutics remain to be determined. Here, we report for the first time that HER2H878Y was tumorigenic in vivo in lung adenocarcinoma transgenic mouse model. Induced expression of HER2H878Y in lung epithelial compartments resulted in formation of poorly differentiated lung adenocarcinoma with bronchioloalveolar carcinoma (BAC) features. Strikingly, we found that these tumors depended on continuous expression of HER2H878Y for maintenance. Typical HER2 downstream signaling mediators, including PLCγ1, STAT5 and AKT, were hyperactivated in HER2H878Y driven lung tumors. More importantly, administration of HKI-272, a tyrosine kinase inhibitor (TKI), efficiently shrank HER2H878Y driven tumors in transgenic mouse model. Moreover, we found that combinational treatment with HKI272 and mTOR inhibitor, Rapamycin, showed a superior cytotoxicity to H878Y mutant transformed cells and enhanced activity to elicit apoptosis and inhibit growth in situ in tumorous area. Our work therefore showed that HER2H878Y mutant was a reasonable drug target. Hence, our work supported the assessment of HKI-272/rapamycin treatment in clinical trials. PMID:26375550

A virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses.

PubMed

Wu, Kailang; Chen, Lang; Peng, Guiqing; Zhou, Wenbo; Pennell, Christopher A; Mansky, Louis M; Geraghty, Robert J; Li, Fang

2011-06-01

How viruses evolve to select their receptor proteins for host cell entry is puzzling. We recently determined the crystal structures of NL63 coronavirus (NL63-CoV) and SARS coronavirus (SARS-CoV) receptor-binding domains (RBDs), each complexed with their common receptor, human angiotensin-converting enzyme 2 (hACE2), and proposed the existence of a virus-binding hot spot on hACE2. Here we investigated the function of this hypothetical hot spot using structure-guided biochemical and functional assays. The hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. Mutations that disturb the hot spot structure have significant effects on virus/receptor interactions, revealing critical energy contributions from the hot spot structure. The tunnel structure at the NL63-CoV/hACE2 interface is more compact than that at the SARS-CoV/hACE2 interface, and hence RBD/hACE2 binding affinities are decreased either by NL63-CoV mutations decreasing the tunnel space or by SARS-CoV mutations increasing the tunnel space. Furthermore, NL63-CoV RBD inhibits hACE2-dependent transduction by SARS-CoV spike protein, a successful application of the hot spot theory that has the potential to become a new antiviral strategy against SARS-CoV infections. These results suggest that the structural features of the hot spot on hACE2 were among the driving forces for the convergent evolution of NL63-CoV and SARS-CoV.

Electrical Control of Silicon Photonic Crystal Cavity by Graphene

DTIC Science & Technology

2012-01-01

Henriksen, E. A.; Jiang, Z.; Hao, Z.; Martin, M. C.; Kim, P.; Stormer , H. L.; Basov, D. N. Nat. Phys. 2008, 4 (7), 532−535. (4) Liu, M.; Yin, X...Zhang, Y.; Tan, Y.-W.; Stormer , H. L.; Kim, P. Nature 2005, 438 (7065), 201−204. (21) Cho, J. H.; Lee, J.; Xia, Y.; Kim, B.; He, Y.; Renn, M. J

The external pore loop interacts with S6 and S3-S4 linker in domain 4 to assume an essential role in gating control and anticonvulsant action in the Na+ channel

PubMed Central

Yang, Ya-Chin; Hsieh, Jui-Yi

2009-01-01

Carbamazepine, phenytoin, and lamotrigine are widely prescribed anticonvulsants in neurological clinics. These drugs bind to the same receptor site, probably with the diphenyl motif in their structure, to inhibit the Na+ channel. However, the location of the drug receptor remains controversial. In this study, we demonstrate close proximity and potential interaction between an external aromatic residue (W1716 in the external pore loop) and an internal aromatic residue (F1764 in the pore-lining part of the sixth transmembrane segment, S6) of domain 4 (D4), both being closely related to anticonvulsant and/or local anesthetic binding to the Na+ channel. Double-mutant cycle analysis reveals significant cooperativity between the two phenyl residues for anticonvulsant binding. Concomitant F1764C mutation evidently decreases the susceptibility of W1716C to external Cd2+ and membrane-impermeable methanethiosulfonate reagents. Also, the W1716E/F1764R and G1715E/F1764R double mutations significantly alter the selectivity for Na+ over K+ and markedly shift the activation curve, respectively. W1716 and F1764 therefore very likely form a link connecting the outer and inner compartments of the Na+ channel pore (in addition to the selectivity filter). Anticonvulsants and local anesthetics may well traverse this “S6 recess” without trespassing on the selectivity filter. Furthermore, we found that Y1618K, a point mutation in the S3-4 linker (the extracellular extension of D4S4), significantly alters the consequences of carbamazepine binding to the Na+ channel. The effect of Y1618K mutation, however, is abolished by concomitant point mutations in the vicinity of Y1618, but not by those in the internally located inactivation machinery, supporting a direct local rather than a long-range allosteric action. Moreover, Y1618 could interact with D4 pore residues W1716 and L1719 to have a profound effect on both channel gating and anticonvulsant action. We conclude that there are direct interactions among the external S3-4 linker, the external pore loop, and the internal S6 segment in D4, making the external pore loop a pivotal point critically coordinating ion permeation, gating, and anticonvulsant binding in the Na+ channel. PMID:19635852

The external pore loop interacts with S6 and S3-S4 linker in domain 4 to assume an essential role in gating control and anticonvulsant action in the Na(+) channel.

PubMed

Yang, Ya-Chin; Hsieh, Jui-Yi; Kuo, Chung-Chin

2009-08-01

Carbamazepine, phenytoin, and lamotrigine are widely prescribed anticonvulsants in neurological clinics. These drugs bind to the same receptor site, probably with the diphenyl motif in their structure, to inhibit the Na(+) channel. However, the location of the drug receptor remains controversial. In this study, we demonstrate close proximity and potential interaction between an external aromatic residue (W1716 in the external pore loop) and an internal aromatic residue (F1764 in the pore-lining part of the sixth transmembrane segment, S6) of domain 4 (D4), both being closely related to anticonvulsant and/or local anesthetic binding to the Na(+) channel. Double-mutant cycle analysis reveals significant cooperativity between the two phenyl residues for anticonvulsant binding. Concomitant F1764C mutation evidently decreases the susceptibility of W1716C to external Cd(2+) and membrane-impermeable methanethiosulfonate reagents. Also, the W1716E/F1764R and G1715E/F1764R double mutations significantly alter the selectivity for Na(+) over K(+) and markedly shift the activation curve, respectively. W1716 and F1764 therefore very likely form a link connecting the outer and inner compartments of the Na(+) channel pore (in addition to the selectivity filter). Anticonvulsants and local anesthetics may well traverse this "S6 recess" without trespassing on the selectivity filter. Furthermore, we found that Y1618K, a point mutation in the S3-4 linker (the extracellular extension of D4S4), significantly alters the consequences of carbamazepine binding to the Na(+) channel. The effect of Y1618K mutation, however, is abolished by concomitant point mutations in the vicinity of Y1618, but not by those in the internally located inactivation machinery, supporting a direct local rather than a long-range allosteric action. Moreover, Y1618 could interact with D4 pore residues W1716 and L1719 to have a profound effect on both channel gating and anticonvulsant action. We conclude that there are direct interactions among the external S3-4 linker, the external pore loop, and the internal S6 segment in D4, making the external pore loop a pivotal point critically coordinating ion permeation, gating, and anticonvulsant binding in the Na(+) channel.

Mechanisms of Polymer Curing and Thermal Degradation

DTIC Science & Technology

1979-12-01

respectively. 29 0)- Fi . J . IR S e t u f i ( - 3 E hy y p e o y Ph n l S f n .30 z Iii 41 rI2P6i4000 3500 3000 42500 §200010 6010 2010 0 0 0 0...Polymer X Obs.6(_pm) Obs.(ppmA H 7.9,8.0 g,h 7.8 g,h 7.5 I ,e 7.5 i ,c,2 7.3 b,e 6.0-7.2 b,d,f,j,=CH 7.2 d 7.0 f,j 3.1 a HC C =- T O" 7.9 g 7.9 g 7.3 b...e 7.4 b,e 7.1 d 6.0-7.1 c,d,f,=CH 7.0 f,c 3.1 a 3.1 a aFor X 0 H, ggh, f= i b Based on first-order analysis of multiplets. 37 t TABLE 4. SUMMARY OF 13C

Mutation rates at 42 Y chromosomal short tandem repeats in Chinese Han population in Eastern China.

PubMed

Wu, Weiwei; Ren, Wenyan; Hao, Honglei; Nan, Hailun; He, Xin; Liu, Qiuling; Lu, Dejian

2018-01-31

Mutation analysis of 42 Y chromosomal short tandem repeats (Y-STRs) loci was performed using a sample of 1160 father-son pairs from the Chinese Han population in Eastern China. The results showed that the average mutation rate across the 42 Y-STR loci was 0.0041 (95% CI 0.0036-0.0047) per locus per generation. The locus-specific mutation rates varied from 0.000 to 0.0190. No mutation was found at DYS388, DYS437, DYS448, DYS531, and GATA_H4. DYS627, DYS570, DYS576, and DYS449 could be classified as rapidly mutating Y-STRs, with mutation rates higher than 1.0 × 10 -2 . DYS458, DYS630, and DYS518 were moderately mutating Y-STRs, with mutation rates ranging from 8 × 10 -3 to 1 × 10 -2 . Although the characteristics of the Y-STR mutations were consistent with those in previous studies, mutation rate differences between our data and previous published data were found at some rapidly mutating Y-STRs. The single-copy loci located on the short arm of the Y chromosome (Yp) showed relatively higher mutation rates more frequently than the multi-copy loci. These results will not only extend the data for Y-STR mutations but also be important for kinship analysis, paternal lineage identification, and family relationship reconstruction in forensic Y-STR analysis.

Mechanistic Characterization of GS-9190 (Tegobuvir), a Novel Nonnucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase▿

PubMed Central

Shih, I-hung; Vliegen, Inge; Peng, Betty; Yang, Huiling; Hebner, Christy; Paeshuyse, Jan; Pürstinger, Gerhard; Fenaux, Martijn; Tian, Yang; Mabery, Eric; Qi, Xiaoping; Bahador, Gina; Paulson, Matthew; Lehman, Laura S.; Bondy, Steven; Tse, Winston; Reiser, Hans; Lee, William A.; Schmitz, Uli; Neyts, Johan; Zhong, Weidong

2011-01-01

GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the β-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the β-hairpin in the thumb subdomain. PMID:21746939

Evaluating the electronic structure of formal Ln II ions in Ln II(C 5H 4SiMe 3) 3 1– using XANES spectroscopy and DFT calculations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fieser, Megan E.; Ferrier, Maryline Ghislaine; Su, Jing

Here, the isolation of [K(2.2.2-cryptand)][Ln(C 5H 4SiMe 3) 3], formally containing LnII, for all lanthanides (excluding Pm) was surprising given that +2 oxidation states are typically regarded as inaccessible for most 4f-elements. Herein, X-ray absorption near-edge spectroscopy (XANES), ground-state density functional theory (DFT), and transition dipole moment calculations are used to investigate the possibility that Ln(C 5H 4SiMe 3) 3 1– (Ln = Pr, Nd, Sm, Gd, Tb, Dy, Y, Ho, Er, Tm, Yb and Lu) compounds represented molecular Ln II complexes. Results from the ground-state DFT calculations were supported by additional calculations that utilized complete-active-space multi-configuration approach with second-ordermore » perturbation theoretical correction (CASPT2). Through comparisons with standards, Ln(C 5H 4SiMe 3) 3 1– (Ln = Sm, Tm, Yb, Lu, Y) are determined to contain 4f 6 5d 0 (Sm II), 4f 13 5d 0 (Tm II), 4f 14 5d 0 (Yb II), 4f 14 5d 1 (Lu II), and 4d 1 (Y II) electronic configurations. Additionally, our results suggest that Ln(C 5H 4SiMe 3) 3 1– (Ln = Pr, Nd, Gd, Tb, Dy, Ho, and Er) also contain Ln II ions, but with 4f n 5d 1 configurations (not 4f n +1 5d 0). In these 4f n 5d 1 complexes, the C 3h-symmetric ligand environment provides a highly shielded 5d-orbital of a' symmetry that made the 4f n 5d 1 electronic configurations lower in energy than the more typical 4f n+1 5d 0 configuration.« less

Evaluating the electronic structure of formal Ln II ions in Ln II(C 5H 4SiMe 3) 3 1– using XANES spectroscopy and DFT calculations

DOE PAGES

Fieser, Megan E.; Ferrier, Maryline Ghislaine; Su, Jing; ...

2017-06-30

Here, the isolation of [K(2.2.2-cryptand)][Ln(C 5H 4SiMe 3) 3], formally containing LnII, for all lanthanides (excluding Pm) was surprising given that +2 oxidation states are typically regarded as inaccessible for most 4f-elements. Herein, X-ray absorption near-edge spectroscopy (XANES), ground-state density functional theory (DFT), and transition dipole moment calculations are used to investigate the possibility that Ln(C 5H 4SiMe 3) 3 1– (Ln = Pr, Nd, Sm, Gd, Tb, Dy, Y, Ho, Er, Tm, Yb and Lu) compounds represented molecular Ln II complexes. Results from the ground-state DFT calculations were supported by additional calculations that utilized complete-active-space multi-configuration approach with second-ordermore » perturbation theoretical correction (CASPT2). Through comparisons with standards, Ln(C 5H 4SiMe 3) 3 1– (Ln = Sm, Tm, Yb, Lu, Y) are determined to contain 4f 6 5d 0 (Sm II), 4f 13 5d 0 (Tm II), 4f 14 5d 0 (Yb II), 4f 14 5d 1 (Lu II), and 4d 1 (Y II) electronic configurations. Additionally, our results suggest that Ln(C 5H 4SiMe 3) 3 1– (Ln = Pr, Nd, Gd, Tb, Dy, Ho, and Er) also contain Ln II ions, but with 4f n 5d 1 configurations (not 4f n +1 5d 0). In these 4f n 5d 1 complexes, the C 3h-symmetric ligand environment provides a highly shielded 5d-orbital of a' symmetry that made the 4f n 5d 1 electronic configurations lower in energy than the more typical 4f n+1 5d 0 configuration.« less

Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis.

PubMed

Negoro, Seiji; Shibata, Naoki; Tanaka, Yusuke; Yasuhira, Kengo; Shibata, Hiroshi; Hashimoto, Haruka; Lee, Young-Ho; Oshima, Shohei; Santa, Ryuji; Oshima, Shohei; Mochiji, Kozo; Goto, Yuji; Ikegami, Takahisa; Nagai, Keisuke; Kato, Dai-Ichiro; Takeo, Masahiro; Higuchi, Yoshiki

2012-02-10

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T(m) = 52 °C, enhanced the protein thermostability by 36 °C (T(m) = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000-25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500-3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.

Decreased cardiovascular and extrahepatic cancer-related mortality in treated patients with mild HFE hemochromatosis.

PubMed

Bardou-Jacquet, Edouard; Morcet, Jeff; Manet, Ghislain; Lainé, Fabrice; Perrin, Michèle; Jouanolle, Anne-Marie; Guyader, Dominique; Moirand, Romain; Viel, Jean-François; Deugnier, Yves

2015-03-01

Mortality studies in patients with hemochromatosis give conflicting results especially with respect to extrahepatic causes of death. Our objective was to assess mortality and causes of death in a cohort of patients homozygous for the C282Y mutation in the HFE gene, diagnosed since the availability of HFE testing. We studied 1085 C282Y homozygotes, consecutively diagnosed from 1996 to 2009, and treated according to current recommendations. Mortality and causes of death were obtained from death certificates and compared to those of the general population. Standardized mortality ratios (SMRs) were used to assess specific causes of death and the Cox model was used to identify prognostic factors for death. Patients were followed for 8.3±3.9 years. Overall the SMR was the same as in the general population (0.94 CI: 0.71-1.22). Patients with serum ferritin⩾2000 μg/L had increased liver-related deaths (SMR: 23.9 CI: 13.9-38.2), especially due to hepatic cancer (SMR: 49.1 CI: 24.5-87.9). Patients with serum ferritin between normal and 1000 μg/L had a lower mortality than the general population (SMR: 0.27 CI: 0.1-0.5), due to a decreased mortality, related to reduced cardiovascular events and extrahepatic cancers in the absence of increased liver-related mortality. Age, diabetes, alcohol consumption, and hepatic fibrosis were independent prognostic factors of death. In treated HFE hemochromatosis, only patients with serum ferritin higher than 2000 μg/L have an increased mortality, mainly related to liver diseases. Those with mild iron burden have a decreased overall mortality in relation to reduced cardiovascular and extrahepatic cancer-related events. These results support a beneficial effect of early and sustained management of patients with iron excess, even when mild. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Hereditary hemochromatosis is characterized by a clinically definable arthropathy that correlates with iron load.

PubMed

Carroll, G J; Breidahl, W H; Bulsara, M K; Olynyk, J K

2011-01-01

To determine the frequency and character of arthropathy in hereditary hemochromatosis (HH) and to investigate the relationship between this arthropathy, nodal interphalangeal osteoarthritis, and iron load. Participants were recruited from the community by newspaper advertisement and assigned to diagnostic confidence categories for HH (definite/probable or possible/unlikely). Arthropathy was determined by use of a predetermined clinical protocol, radiographs of the hands of all participants, and radiographs of other joints in which clinical criteria were met. An arthropathy considered typical for HH, involving metacarpophalangeal joints 2-5 and bilateral specified large joints, was observed in 10 of 41 patients with definite or probable HH (24%), all of whom were homozygous for the C282Y mutation in the HFE gene, while only 2 of 62 patients with possible/unlikely HH had such an arthropathy (P=0.0024). Arthropathy in definite/probable HH was more common with increasing age and was associated with ferritin concentrations>1,000 μg/liter at the time of diagnosis (odds ratio 14.0 [95% confidence interval 1.30-150.89], P=0.03). A trend toward more episodes requiring phlebotomy was also observed among those with arthropathy, but this was not statistically significant (odds ratio 1.03 [95% confidence interval 0.99-1.06], P=0.097). There was no significant association between arthropathy in definite/probable HH and a history of intensive physical labor (P=0.12). An arthropathy consistent with that commonly attributed to HH was found to occur in 24% of patients with definite/probable HH. The association observed between this arthropathy, homozygosity for C282Y, and serum ferritin concentrations at the time of diagnosis suggests that iron load is likely to be a major determinant of arthropathy in HH and to be more important than occupational factors. Copyright © 2011 by the American College of Rheumatology.

Innovative Methods for Engine Health Monitoring

DTIC Science & Technology

2006-11-30

ELEMENT NUMBER 6. AUTHOR(S) 6d. PROJECT NUMBER C. L. Philip Chen, Z. Pantic-Tanner, H. Millwater , S. Hudak, Y. Huang, P. Cotae, D. Tanner, E. John, K. Chan...Philip Chen’, Z. Pantic-Tanner 2, H. Millwater 3, S. Hudak 4, Y. Huang5, P. Cotae6, D. Tanner 7, E. Johns, K.Chan9, X. Wang𔃺 1 - Professor and Chair... Millwater , R.C. McClung, M.P. Enright, "A new tool for design and certification of aircraft turbine rotors," Journal of Engineering for Gas Turbines

On the Genesis of Reliability Models.

DTIC Science & Technology

1982-07-01

dy1 fRi fI, where I: Y1 H,(yl~y2,t) = ex (tly) Irl(y2)11+ f ,ra(y2*(yl’))dyl’].(.9 The results, dH , = _r(y2*(y))H(y,y,,t)* dt H,(4rfjy 2 t) = eP...Press, 1971. 2. Payne, A. 0.: A reliability approach to the fatigue of structures. ASTM STP 511, pp. 106 - 155, 1972. 3. Diamond, P., and Payne, A. 0...Commonwealth Aircraft Corporation, Library Hawker de Havilland Aust. Pty. Ltd., Bankstown, Library Rolls-Royce of Australia Pty. Ltd., Mr. C. 0. A. Bailey

Altered cellular localization and hemichannel activities of KID syndrome associated connexin26 I30N and D50Y mutations.

PubMed

Aypek, Hande; Bay, Veysel; Meşe, Gülistan

2016-02-02

Gap junctions facilitate exchange of small molecules between adjacent cells, serving a crucial function for the maintenance of cellular homeostasis. Mutations in connexins, the basic unit of gap junctions, are associated with several human hereditary disorders. For example, mutations in connexin26 (Cx26) cause both non-syndromic deafness and syndromic deafness associated with skin abnormalities such as keratitis-ichthyosis-deafness (KID) syndrome. These mutations can alter the formation and function of gap junction channels through different mechanisms, and in turn interfere with various cellular processes leading to distinct disorders. The KID associated Cx26 mutations were mostly shown to result in elevated hemichannel activities. However, the effects of these aberrant hemichannels on cellular processes are recently being deciphered. Here, we assessed the effect of two Cx26 mutations associated with KID syndrome, Cx26I30N and D50Y, on protein biosynthesis and channel function in N2A and HeLa cells. Immunostaining experiments showed that Cx26I30N and D50Y failed to form gap junction plaques at cell-cell contact sites. Further, these mutations resulted in the retention of Cx26 protein in the Golgi apparatus. Examination of hemichannel function by fluorescent dye uptake assays revealed that cells with Cx26I30N and D50Y mutations had increased dye uptake compared to Cx26WT (wild-type) containing cells, indicating abnormal hemichannel activities. Cells with mutant proteins had elevated intracellular calcium levels compared to Cx26WT transfected cells, which were abolished by a hemichannel blocker, carbenoxolone (CBX), as measured by Fluo-3 AM loading and flow cytometry. Here, we demonstrated that Cx26I30N and D50Y mutations resulted in the formation of aberrant hemichannels that might result in elevated intracellular calcium levels, a process which may contribute to the hyperproliferative epidermal phenotypes of KID syndrome.

Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR Yfiler PCR amplification kit.

PubMed

Goedbloed, Miriam; Vermeulen, Mark; Fang, Rixun N; Lembring, Maria; Wollstein, Andreas; Ballantyne, Kaye; Lao, Oscar; Brauer, Silke; Krüger, Carmen; Roewer, Lutz; Lessig, Rüdiger; Ploski, Rafal; Dobosz, Tadeusz; Henke, Lotte; Henke, Jürgen; Furtado, Manohar R; Kayser, Manfred

2009-11-01

The Y-chromosomal short tandem repeat (Y-STR) polymorphisms included in the AmpFlSTR Yfiler polymerase chain reaction amplification kit have become widely used for forensic and evolutionary applications where a reliable knowledge on mutation properties is necessary for correct data interpretation. Therefore, we investigated the 17 Yfiler Y-STRs in 1,730-1,764 DNA-confirmed father-son pairs per locus and found 84 sequence-confirmed mutations among the 29,792 meiotic transfers covered. Of the 84 mutations, 83 (98.8%) were single-repeat changes and one (1.2%) was a double-repeat change (ratio, 1:0.01), as well as 43 (51.2%) were repeat gains and 41 (48.8%) repeat losses (ratio, 1:0.95). Medians from Bayesian estimation of locus-specific mutation rates ranged from 0.0003 for DYS448 to 0.0074 for DYS458, with a median rate across all 17 Y-STRs of 0.0025. The mean age (at the time of son's birth) of fathers with mutations was with 34.40 (+/-11.63) years higher than that of fathers without ones at 30.32 (+/-10.22) years, a difference that is highly statistically significant (p < 0.001). A Poisson-based modeling revealed that the Y-STR mutation rate increased with increasing father's age on a statistically significant level (alpha = 0.0294, 2.5% quantile = 0.0001). From combining our data with those previously published, considering all together 135,212 meiotic events and 331 mutations, we conclude for the Yfiler Y-STRs that (1) none had a mutation rate of >1%, 12 had mutation rates of >0.1% and four of <0.1%, (2) single-repeat changes were strongly favored over multiple-repeat ones for all loci but 1 and (3) considerable variation existed among loci in the ratio of repeat gains versus losses. Our finding of three Y-STR mutations in one father-son pair (and two pairs with two mutations each) has consequences for determining the threshold of allelic differences to conclude exclusion constellations in future applications of Y-STRs in paternity testing and pedigree analyses.

In situ formation of a 3D core-shell and triple-conducting oxygen reduction reaction electrode for proton-conducting SOFCs

NASA Astrophysics Data System (ADS)

Zhang, Zhenbao; Wang, Jian; Chen, Yubo; Tan, Shaozao; Shao, Zongping; Chen, Dengjie

2018-05-01

BaZrxCeyY1-x-yO3-δ are recognized proton-conducting electrolyte materials for proton-conducting solid oxide fuel cells (H+-SOFCs) below 650 °C. Here Co cations are incorporated into the BaZr0.4Ce0.4Y0.2O3-δ (BZCY) scaffold to generate a 3D core-shell and triple-conducting (H+/O2-/e-) electrode in situ via infiltrating and reactive sintering. The core is the bulk BZCY scaffold, while the shell is composed of the cubic Ba(Zr0.4Ce0.4Y0.2)1-xCoxO3-δ, cubic spinel Co3O4 and cubic fluorite (Ce, Zr, Y)O2. The obtained electrode exhibits an excellent compatibility with the BZCY electrolyte, and performs well in yielding a low and stable polarization resistance for oxygen reduction reaction for intermediate-temperature H+-SOFCs. In particular, it achieves polarization resistances as low as 0.094 and 0.198 Ω cm2 at 650 and 600 °C in wet air (3% H2O) when the sintering temperature for the electrode is 900 °C. In addition, a symmetrical cell also exhibits operation stability of 70 h at 650 °C. Furthermore, a fuel cell assembled with the 3D core-shell and triple-conducting electrode delivers a peak power density of ∼330 mW cm-2 at 650 °C. The substantially improved electrochemical performance and high stability are ascribed to the unique core-shell structure and the formation of Ba(Zr0.4Ce0.4Y0.2)1-xCoxO3-δ in the shell.

29 CFR 776.10 - Employees participating in the actual movement of commerce.

Code of Federal Regulations, 2011 CFR

2011-07-01

... goods or services, or plans or other documents across State lines, he comes within the scope of the Act...; (Munic. Ct. N.Y.C.); Phillips v. Meeker Coop. Light & Power Asso., 63 F. Supp. 733 (D. Minn.); Anderson...

Real-Time Radio Wave Propagation for Mobile Ad-Hoc Network Emulation and Simulation Using General Purpose Graphics Processing Units (GPGPUs)

DTIC Science & Technology

2014-05-01

COST-Walfisch- Ikegami model (14) estimates the received power predominantly on the basis of frequency and distance to the transmitter. Ray-optical (15...34 # $ $ # % & ’ ( ) * + , , - . / - 0 1 2 3 4 5 6 7 7 6 8 9 : ; < = > ? @ A B C ? D E F A B G H ? E E I J K L M N O P Q R S T U V W X S Y Z O Y [ Q [ \\ W O P...a b _ c _ d _ e f ` d g h ` i j k l l m n o p k q r s t u v w w x y z

Final Environmental Planning Technical Report

DTIC Science & Technology

1984-01-01

or mildly alkaline (pH 7.3 to 7.8). Soils with a pH of less than 6.3 have been found to be too acidic for bone preservation (Hole and Heizer 1965...230; Heizer and Graham 1967:125). On the other hand, a pH of 6.0 has been said to be the highest level tolerated by pollen (Moore and Webb 1978:15...however, this cutoff point may be as high as 7.0 in arid or semiarid climates (Hole and Heizer 1965:282). The generally dry, neutral, or alkaline soil

Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2'-deoxyguanosine by Human DNA Polymerase η.

PubMed

Patra, Amitraj; Zhang, Qianqian; Guengerich, F Peter; Egli, Martin

2016-11-11

O 6 -Methyl-2'-deoxyguanosine (O 6 -MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the "translesion" Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O 6 -MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O 6 -MeG and 2"-deoxythymidine-5"-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O 6 - MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O 6 -MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O 6 -MeG:dC or O 6 -MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2′-deoxyguanosine by Human DNA Polymerase η*

PubMed Central

Patra, Amitraj; Zhang, Qianqian; Guengerich, F. Peter; Egli, Martin

2016-01-01

O6-Methyl-2′-deoxyguanosine (O6-MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the “translesion” Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O6-MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O6-MeG and 2"-deoxythymidine-5"-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O6- MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O6-MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O6-MeG:dC or O6-MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η. PMID:27694439

Nuclear expression and gain-of-function β-catenin mutation in glomangiopericytoma (sinonasal-type hemangiopericytoma): insight into pathogenesis and a diagnostic marker.

PubMed

Lasota, Jerzy; Felisiak-Golabek, Anna; Aly, F Zahra; Wang, Zeng-Feng; Thompson, Lester D R; Miettinen, Markku

2015-05-01

Glomangiopericytoma (sinonasal-type hemangiopericytoma) is a rare mesenchymal neoplasm with myoid phenotype (smooth muscle actin-positive), which distinguishes this tumor from soft tissue hemangiopericytoma/solitary fibrous tumor. Molecular genetic changes underlying the pathogenesis of glomangiopericytoma are not known. In this study, 13 well-characterized glomangiopericytomas were immunohistochemically evaluated for β-catenin expression. All analyzed tumors showed strong expression and nuclear accumulation of β-catenin. Following this observation, β-catenin glycogen serine kinase-3 beta phosphorylation region, encoded by exon 3, was PCR amplified in all cases and evaluated for mutations using Sanger sequencing. Heterozygous mutations were identified in 12 of 13 tumors. All mutations consisted of single-nucleotide substitutions: three in codon 32 (c.94G>C (n=2) and c.95A>T), four in codon 33 (two each c.98C>G and c.98C>T), two in codon 37 (c.109T>G), one in codon 41 (c.121A>G), and two in codon 45 (c.133T>C). At the protein level, these substitutions would lead to p.D32H, p.D32V, p.S33C, p.S33F, p.S37A, p.T41A, and p.S45L mutations, respectively. Previously, similar mutations have been reported in different types of cancers and shown to trigger activation of β-catenin signaling. All analyzed glomangiopericytomas showed prominent nuclear expression of cyclin D1, as previously shown for tumors with nuclear expression of β-catenin as a sign of oncogenic activation. These results demonstrate that mutational activation of β-catenin and associated cyclin D1 overexpression may be central events in the pathogenesis of glomangiopericytoma. In additon, nuclear accumulation of β-catenin is a diagnostic marker for glomangiopericytoma.

Sperm DNA damage output parameters measured by the alkaline Comet assay and their importance.

PubMed

Simon, L; Aston, K I; Emery, B R; Hotaling, J; Carrell, D T

2017-03-01

The alkaline Comet assay has shown high diagnostic value to determine male reproductive health and prognostic ability to predict ART success. Here, spermatozoon was analysed in 47 fertile donors and 238 patients, including 132 couples undergoing ART [semen was collected: Group I - within 3 months of their treatment (n = 79); and Group II - 3 months prior to their treatment (n = 53)]. We introduce four Comet distribution plots (A, B1, B2 and C) by plotting the level of DNA damage (x-axis) and percentage of comets (y-axis). Fertile donors had low mean DNA damage, olive tail moment and per cent of spermatozoa with damage and increased type A plots. Comet parameters were associated with clinical pregnancies in Group I. About 66% of couples with type A distribution plot were successful after ART, whereas couples with type B1, B2 and C distribution plots achieved 56%, 44% and 33% pregnancies respectively. The efficiency of the Comet assay was due to complete decondensation process, where the compact sperm nuclear DNA (28.2 ± 0.2 μm 3 ) is decondensed to ~63 μm 3 (before lysis) and ~1018 μm 3 (after lysis). A combinational analysis of all the Comet output parameters may provide a comprehensive evaluation of patient's reproductive health as these parameters measure different aspects of DNA damage within the spermatozoa. © 2016 Blackwell Verlag GmbH.

Patients with primary breast and primary female genital tract diffuse large B cell lymphoma have a high frequency of MYD88 and CD79B mutations.

PubMed

Cao, Xin-Xin; Li, Jian; Cai, Hao; Zhang, Wei; Duan, Ming-Hui; Zhou, Dao-Bin

2017-11-01

This study is to retrospectively evaluate the prevalence of MYD88 and CD79B mutations and the clinicopathologic characteristics of patients with primary diffuse large B cell lymphoma (DLBCL) of the female genital tract and breast. The characteristics, treatments, and outcomes of 19 patients diagnosed with primary DLBCL of the female genital tract and breast, who had formalin-fixed and paraffin-embedded tissues obtained from diagnostic samples diagnosed between January 2004 and June 2016, were analyzed retrospectively. Nineteen female patients (7 with primary breast and 12 with primary female genital tract DLBCL) were included in this retrospective study. Eleven patients (57.9%) carried a MYD88 mutation, including 10 with MYD8 L265P and 1 with the MYD88 L265S mutation. Seven patients (36.8%) harbored a CD79B mutation, which included two cases with CD79B Y196H, two cases with CD79B Y196N, one case with CD79B Y196D, one case with CD79B Y196F, and one case with CD79B Y196X. Four cases had both MYD88 and CD79B mutations. The clinicopathologic parameters, progression-free survival (PFS), and overall survival (OS) of the MYD88 mutation-carrying group were not significantly different from those of the MYD88 wild-type group except for higher LDH levels. Six patients received cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP), while 13 patients received rituximab plus CHOP, and 13 patients received central nervous system prophylaxis. The median OS and PFS were 73 and 56 months, respectively. Patients with primary breast and primary female genital tract DLBCL have a high frequency of MYD88 and CD79B mutations. The presence of these mutations does not affect survival but may offer additional therapeutic options.

Cardiac Myosin Binding Protein C Phosphorylation Affects Cross-Bridge Cycle's Elementary Steps in a Site-Specific Manner

PubMed Central

Wang, Li; Sadayappan, Sakthivel; Kawai, Masakata

2014-01-01

Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases. PMID:25420047

Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner.

PubMed

Wang, Li; Sadayappan, Sakthivel; Kawai, Masakata

2014-01-01

Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.

Steam Oxidation Behavior of Advanced Steels and Ni-Based Alloys at 800 °C

NASA Astrophysics Data System (ADS)

Dudziak, T.; Boroń, L.; Deodeshmukh, V.; Sobczak, J.; Sobczak, N.; Witkowska, M.; Ratuszek, W.; Chruściel, K.

2017-03-01

This publication studies the steam oxidation behavior of advanced steels (309S, 310S and HR3C) and Ni-based alloys (Haynes® 230®, alloy 263, alloy 617 and Haynes® 282®) exposed at 800 °C for 2000 h under 1 bar pressure, in a pure water steam system. The results revealed that all exposed materials showed relatively low weight gain, with no spallation of the oxide scale within the 2000 h of exposure. XRD analysis showed that Ni-based alloys developed an oxide scale consisting of four main phases: Cr2O3 (alloy 617, Haynes® 282®, alloy 263 and Haynes® 230®), MnCr2O4 (alloy 617, Haynes® 282® and Haynes® 230®), NiCr2O4 (alloy 617) and TiO2 (alloy 263, Haynes® 282®). In contrast, advanced steels showed the development of Cr2O3, MnCr2O4, Mn7SiO12, FeMn(SiO4) and SiO2 phases. The steel with the highest Cr content showed the formation of Fe3O4 and the thickest oxide scale.

High prevalence of clinical and environmental triazole-resistant Aspergillus fumigatus in Iran: is it a challenging issue?

PubMed

Nabili, Mojtaba; Shokohi, Tahereh; Moazeni, Maryam; Khodavaisy, Sadegh; Aliyali, Masoud; Badiee, Parisa; Zarrinfar, Hossein; Hagen, Ferry; Badali, Hamid

2016-06-01

Triazole antifungal agents are the mainstay of aspergillosis treatment. As highlighted in numerous studies, the global increase in the prevalence of triazole resistance could hamper the management of aspergillosis. In the present three-year study, 513 samples (213 clinical and 300 environmental samples) from 10 provinces of Iran were processed and screened in terms of azole resistance (4 and 1 mg l-1 of itraconazole and voriconazole, respectively), using selective plates. Overall, 150 A. fumigatus isolates (71 clinical and 79 environmental isolates) were detected. The isolates were confirmed by partial sequencing of the β-tubulin gene. Afterwards, in vitro antifungal susceptibility tests against triazole agents were performed, based on the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document. The CYP51A gene was sequenced in order to detect mutations. The MIC of itraconazole against 10 (6.6 %) strains, including clinical (n=3, 4.2 %) and environmental (n=7, 8.8 %) strains, was higher than the breakpoint and epidemiological cut-off value. Based on the findings, the prevalence of azole-resistant A. fumigatus in Iran has increased remarkablyfrom 3.3 % to 6.6 % in comparison with earlier epidemiological research. Among resistant isolates, TR34/L98H mutations in the CYP51A gene were the most prevalent (n=8, 80 %), whereas other point mutations (F46Y, G54W, Y121F, G138C, M172V, F219C, M220I, D255E, T289F, G432C and G448S mutations) were not detected. Although the number of patients affected by azole-resistant A. fumigatus isolates was limited, strict supervision of clinical azole-resistant A. fumigatus isolates and persistent environmental screening of azole resistance are vital to the development of approaches for the management of azole resistance in human pathogenic fungi.

Supramolecular architecture based on [Fe(CN)6]3- metallotectons and melaminium synthons

NASA Astrophysics Data System (ADS)

Krichen, Firas; Walha, Siwar; Lhoste, Jérôme; Bulou, Alain; Kabadou, Ahlem; Goutenoire, François

2017-10-01

Assembly involving [Fe(CN)6]3- metallotectons as building units and melaminium organic cation has been envisioned in order to elaborate a hybrid supramolecular based on ionic H-bonds with formula {(H-mel)4[Fe(CN)6]Cl} (H-mel+: melaminium cation). The compound has been prepared by diffusion method and characterized by single-crystal X-ray diffraction, EDX analysis, and Raman-IR spectroscopies with assignment from ab initio calculations. The melaminium exhibit self cationic coupling with cyclic hydrogen bonds to give a one dimensional {[H-mel]+}∝ synthon. Therefore, these cationic ribbons are inter-linked via hydrogen bonds by the anionic tectons [Fe(CN)6]3- and chlorine anion resulting on a 3D network. Molecular hirshfeld surfaces revealed that the crystal structure has been supported mainly by Nsbnd H⋯N and Nsbnd H⋯Cl intermolecular Hydrogen bonds and by favoured C⋯C and C⋯N weak interactions.

Thermal tolerance characteristics of non-O157 Shiga toxigenic strains of Escherichia coli (STEC) in a beef broth model system are similar to those of O157:H7 STEC.

PubMed

Vasan, Akhila; Leong, Wan Mei; Ingham, Steve C; Ingham, Barbara H

2013-07-01

The non-O157 Shiga toxigenic Escherichia coli (STEC) serogroups most commonly associated with illness are O26, O45, O103, O111, O121, and O145. In the United States, these serogroups are considered adulterants in raw nonintact beef. To begin to understand the behavior of these pathogens in meat systems, we compared the thermal tolerance of acid-adapted cells of non-O157 STEC and O157:H7 STEC in a beef-derived broth. D58°C-values were determined for at least three strains per serogroup, and D54.6°C-values and D63.6°C-values were determined for one strain per serogroup. Each strain was grown to stationary phase in brain heart infusion broth (BHIB; pH 7.0) and inoculated into prewarmed BHIB in a shaking water bath for thermotolerance experiments at 54.6, 58.0, or 63.6°C (three trials per strain). Samples were heated for up to 160 min at 54.6°C, 3 min at 58.0°C, or 45 s at 63.6°C, with periodic sampling followed by rapid cooling and plating on modified Levine's eosin methylene blue agar. For each strain and temperature, the log CFU per milliliter was plotted versus time, and D-values were determined. Across all strains, the least and most heat tolerant STEC serogroups at 58°C were O145 and O157, respectively. D58°C-values in BHIB ranged from 0.44 min for an O145 strain to 1.42 min for an O157:H7 strain. D58°C-values for O157 STEC strains were significantly higher than those for at least one strain in each of the non-O157 STEC serogroups (P < 0.05) except for serogroup O103. At 54.6°C, the most heat-resistant STEC strain belonged to serogroup O103 and was significantly more heat tolerant than the O157:H7 strains (P < 0.05). Grouping the strains, there were no significant differences in heat tolerance between O157 and non-O157 STEC at 63.6°C (P ≥ 0.05). The z-values for non-O157 STEC strains were comparable to those for O157:H7 STEC strains (P ≥ 0.05), ranging from 4.10 to 5.21°C. These results suggest that thermal processing interventions that target destruction of E. coli O157:H7 may have adequate lethality against non-O157 STEC.

Infantile Alexander Disease: Spectrum of GFAP Mutations and Genotype-Phenotype Correlation

PubMed Central

Rodriguez, Diana; Gauthier, Fernande; Bertini, Enrico; Bugiani, Marianna; Brenner, Michael; N'guyen, Sylvie; Goizet, Cyril; Gelot, Antoinette; Surtees, Robert; Pedespan, Jean-Michel; Hernandorena, Xavier; Troncoso, Monica; Uziel, Graziela; Messing, Albee; Ponsot, Gérard; Pham-Dinh, Danielle; Dautigny, André; Boespflug-Tanguy, Odile

2001-01-01

Heterozygous, de novo mutations in the glial fibrillary acidic protein (GFAP) gene have recently been reported in 12 patients affected by neuropathologically proved Alexander disease. We searched for GFAP mutations in a series of patients who had heterogeneous clinical symptoms but were candidates for Alexander disease on the basis of suggestive neuroimaging abnormalities. Missense, heterozygous, de novo GFAP mutations were found in exons 1 or 4 for 14 of the 15 patients analyzed, including patients without macrocephaly. Nine patients carried arginine mutations (four had R79H; four had R239C; and one had R239H) that have been described elsewhere, whereas the other five had one of four novel mutations, of which two affect arginine (2R88C and 1R88S) and two affect nonarginine residues (1L76F and 1N77Y). All mutations were located in the rod domain of GFAP, and there is a correlation between clinical severity and the affected amino acid. These results confirm that GFAP mutations are a reliable molecular marker for the diagnosis of infantile Alexander disease, and they also form a basis for the recommendation of GFAP analysis for prenatal diagnosis to detect potential cases of germinal mosaicism. PMID:11567214

Optical Enhancement of Degraded Fingerprints.

DTIC Science & Technology

1986-05-01

system. The integral 00 g(x,y) = ff f ( Cn)h(x-C,y-n)d dn -00 is the convolution operation g(x,y)=f(x,y)*h(x,y) where h(x,y) is the impulse response...periodic ridges on the left (1-) and right (2--) sides of the fingerprint in Figure 3-1 as periodic impulse -sheets (an impulse -sheet is a one...transformation of uniformly spaced, parallel impulse -sheets is a string of impulses (an impulse is a two-dimensional Dirac delta function, i.e. f(x,y) = 6(x,y

Characterization of chromia scales formed in supercritical carbon dioxide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pint, Bruce A.; Unocic, Kinga A.; Brese, Robert G.

Initial experimental work at 700°–800 °C is in progress to develop a lifetime model for supercritical CO2 (sCO2) compatibility for a 30-year lifetime of a >700 °C concentrated solar power system. Nickel-based alloys 282, 740H and 625 and Fe-based alloy 25 are being evaluated in 500-h cycles at 1 and 300 bar, and 10-h cycles in 1 bar industrial grade CO2. The alloys showed similar low rates of oxidation in 1 and 300 bar CO2 in 500-h cycles at 750 °C. However, in 10-h cycles, alloy 25 showed accelerated attack at 700° and 750 °C. Transmission electron microscopy scale cross-sectionsmore » on alloy 25 after 1000 h at 700 °C in sCO2 and in air only showed a small row of carbides beneath the scale in the former environment. Similar characterisation was performed on alloys 625 and 282 after sCO2 exposure at 750 °C.« less

Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties

PubMed Central

Wang, Yu-Kuo; Huang, Sheng-Cih; Wu, Yi-Fang; Chen, Yu-Ching; Lin, Yen-Ling; Nayak, Manoswini; Lin, Yan Ren; Chen, Wen-Hung; Chiu, Yi-Rong; Li, Thomas Tien-Hsiung; Yeh, Bo-Sou; Wu, Tung-Kung

2011-01-01

Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 oC but is reactivated by additional heating above 80 oC. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDHWT) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDHY53H/T59I and Gh-rTDHT59I/S63T double-mutants and the Gh-rTDHY53H/T59I/S63T triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDHWT. Circular dichroism measurements of Gh-rTDHWT and Gh-rTDHmut showed a conspicuous change from a β-sheet to α-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy. PMID:21494434

Generating mixed morphology BaZrO3 artificial pinning centers for strong and isotropic pinning in BaZrO3-Y2O3 double-doped YBCO thin films

NASA Astrophysics Data System (ADS)

Chen, Shihong; Sebastian, Mary Ann; Gautam, Bibek; Wilt, Jamie; Chen, Yanbin; Sun, Lei; Xing, Zhongwen; Haugan, Timothy; Wu, Judy

2017-12-01

High concentration artificial pinning centers (APCs), such as BaZrO3 nanorods (BZO 1D APCs) aligned along the c-axis of the high temperature superconductor YBa2Cu3O7 (YBCO) can provide strong pinning of magnetic vortices and are desirable for applications in high magnetic fields. Unfortunately, in YBCO films with single-doping (SD) of BZO 1D APCs, a monotonic decreasing superconducting T c and critical current density J c(H) with BZO doping has been observed due to strain field overlap at high-concentration perfectly c-axis aligned BZO 1D APCs. In order to resolve this issue, double-doping (DD) of 2-6 vol% BZO 1D APCs and 3.0 vol% Y2O3 nanoparticles (Y2O3-NPs) in YBCO films has been explored to promote BZO-NR orientation misalignment from the c-axis. Remarkably, a monotonic increasing J c(H) with BZO 1D APCs concentration has been obtained in the BZO DD samples. Such a microstructure change is evidenced in the much smaller c-lattice parameter expansion of 0.103% in the DD samples as opposed to 0.511% in the SD counterparts and reduced c-axis alignment of the BZO 1D APCs as revealed in TEM. This yields a mixed 1D + 2D + 3D APC morphology and enhanced isotropic pinning with respect to the orientation of the H-field in the BZO DD samples.

Analysis of Hairless Corepressor Mutants to Characterize Molecular Cooperation with the Vitamin D Receptor in Promoting the Mammalian Hair Cycle

PubMed Central

Hsieh, Jui-Cheng; Slater, Stephanie A.; Whitfield, G. Kerr; Dawson, Jamie L.; Hsieh, Grace; Sheedy, Craig; Haussler, Carol A.; Haussler, Mark R.

2010-01-01

The mammalian hair cycle requires both the vitamin D receptor (VDR) and the hairless (Hr) corepressor, each of which is expressed in the hair follicle. Hr interacts directly with VDR to repress VDR-targeted transcription. Herein, we further map the VDR-interaction domain to regions in the C-terminal half of Hr that contain two LXXLL-like pairs of motifs known to mediate contact of Hr with the RAR-related orphan receptor alpha and with the thyroid hormone receptor, respectively. Site-directed mutagenesis indicates that all four hydrophobic motifs are required for VDR transrepression by Hr. Point mutation of rat Hr at conserved residues corresponding to natural mutants causing alopecia in mice (G985W and a C-terminal deletion ΔAK) and in humans (P95S, C422Y, E611G, R640Q, C642G, N988S, D1030N, A1040T, V1074M and V1154D), as well as alteration of residues in the C-terminal Jumonji C domain implicated in histone demethylation activity (C1025G/E1027G and H1143G) revealed that all Hr mutants retained VDR association, and that transrepressor activity was selectively abrogated in C642G, G985W, N988S, D1030N, V1074M, H1143G and V1154D. Four of these latter Hr mutants (C642G, N988S, D1030N and V1154D) were found to associate normally with histone deacetylase-3. Finally, we identified three regions of human VDR necessary for association with Hr, namely residues 109–111, 134–201, and 202–303. It is concluded that Hr and VDR interact via multiple protein-protein interfaces, with Hr recruiting histone deacetylases and possibly itself catalyzing histone demethylation to effect chromatin remodeling and repress the transcription of VDR target genes that control the hair cycle. PMID:20512927

Comparative analysis of KRAS codon 12, 13, 18, 61, and 117 mutations using human MCF10A isogenic cell lines

PubMed Central

Stolze, Britta; Reinhart, Stefanie; Bulllinger, Lars; Fröhling, Stefan; Scholl, Claudia

2015-01-01

KRAS mutations occur in one third of human cancers and cluster in several hotspots, with codons 12 and 13 being most commonly affected. It has been suggested that the position and type of amino acid exchange influence the transforming capacity of mutant KRAS proteins. We used MCF10A human mammary epithelial cells to establish isogenic cell lines that express different cancer-associated KRAS mutations (G12C, G12D, G12V, G13C, G13D, A18D, Q61H, K117N) at physiological or elevated levels, and investigated the biochemical and functional consequences of the different variants. The overall effects of low-expressing mutants were moderate compared to overexpressed variants, but allowed delineation of biological functions that were related to specific alleles rather than KRAS expression level. None of the mutations induced morphological changes, migratory abilities, or increased phosphorylation of ERK, PDK1, and AKT. KRAS-G12D, G12V, G13D, and K117N mediated EGF-independent proliferation, whereas anchorage-independent growth was primarily induced by K117N and Q61H. Both codon 13 mutations were associated with increased EGFR expression. Finally, global gene expression analysis of MCF10A-G13D versus MCF10A-G12D revealed distinct transcriptional changes. Together, we describe a useful resource for investigating the function of multiple KRAS mutations and provide insights into the differential effects of these variants in MCF10A cells. PMID:25705018

Fluoroquinolone resistance in Streptococcus pneumoniae: area under the concentration-time curve/MIC ratio and resistance development with gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin.

PubMed

LaPlante, Kerry L; Rybak, Michael J; Tsuji, Brian; Lodise, Thomas P; Kaatz, Glenn W

2007-04-01

The potential for resistance development in Streptococcus pneumoniae secondary to exposure to gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin at various levels was examined at high inoculum (10(8.5) to 10(9) log10 CFU/ml) over 96 h in an in vitro pharmacodynamic (PD) model using two fluoroquinolone-susceptible isolates. The pharmacokinetics of each drug was simulated to provide a range of free areas under the concentration-time curves (fAUC) that correlated with various fluoroquinolone doses. Potential first (parC and parE)- and second-step (gyrA and gyrB) mutations in isolates with raised MICs were identified by sequence analysis. PD models simulating fAUC/MICs of 51 andgatifloxacin>moxifloxacin=gemifloxacin, which may be related to structural differences within the class.

Simulation of Tripod Gaits for a Hexapod Underwater Walking Machine

DTIC Science & Technology

1993-06-01

AquarobotBody class H/ Subclass of RigidBody class fl 170 #iid~dKAQUAROBOTBODY # defint HAQUAROBOTBODY #include <stdio.h> #include *Lia*1.H" #include "AbRis4H...nulU&A4g parameters enuk heme returm (arcsegmnww flag); HI smuit finction ends here i/REVISED: .... /r.71TLE: seito i/INPUT: (x~y)body coordinates of a...kttacqt - foot[1D; d - (x ._inssrq* - fbot(On’x gusrcept - focttI~); todigace= -sqr(c + Y HI orientation we and heme II lieSep=*t izusectionto ten as

The X-linked juvenile retinoschisis protein retinoschisin is a novel regulator of mitogen-activated protein kinase signalling and apoptosis in the retina.

PubMed

Plössl, Karolina; Weber, Bernhard H F; Friedrich, Ulrike

2017-04-01

X-linked juvenile retinoschisis (XLRS) is a hereditary retinal dystrophy in young males, caused by mutations in the RS1 gene. The function of the encoded protein, termed retinoschisin, and the molecular mechanisms underlying XLRS pathogenesis are still unresolved, although a direct interaction partner of the secreted retinoschisin, the retinal Na/K-ATPase, was recently identified. Earlier gene expression studies in retinoschisin-deficient (Rs1h -/Y ) mice provided a first indication of pathological up-regulation of mitogen-activated protein (MAP) kinase signalling in disease pathogenesis. To further investigate the role for retinoschisin in MAP kinase regulation, we exposed Y-79 cells and murine Rs1h -/Y retinae to recombinant retinoschisin and the XLRS-associated mutant RS1-C59S. Although normal retinoschisin stably bound to retinal cells, RS1-C59S exhibited a strongly reduced binding affinity. Simultaneously, exposure to normal retinoschisin significantly reduced phosphorylation of C-RAF and MAP kinases ERK1/2 in Y-79 cells and murine Rs1h -/Y retinae. Expression of MAP kinase target genes C-FOS and EGR1 was also down-regulated in both model systems. Finally, retinoschisin treatment decreased pro-apoptotic BAX-2 transcript levels in Y-79 cells and Rs1h -/Y retinae. Upon retinoschisin treatment, these cells showed increased resistance against apoptosis, reflected by decreased caspase-3 activity (in Y-79 cells) and increased photoreceptor survival (in Rs1h -/Y retinal explants). RS1-C59S did not influence C-RAF or ERK1/2 activation, C-FOS or EGR1 expression, or apoptosis. Our data imply that retinoschisin is a novel regulator of MAP kinase signalling and exerts an anti-apoptotic effect on retinal cells. We therefore discuss that disturbances of MAP kinase signalling by retinoschisin deficiency could be an initial step in XLRS pathogenesis. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Diabetes in first-degree family members: a predictor of type 2 diabetes in 159 nonscreening Alabama hemochromatosis probands with HFE C282Y homozygosity.

PubMed

Barton, James C; Barton, J Clayborn; Acton, Ronald T

2014-01-01

OBJECTIVE We sought to identify predictors of diabetes diagnosed before hemochromatosis. RESEARCH DESIGN AND METHODS We studied these 16 variables in 159 nonscreening hemochromatosis probands with HFE C282Y homozygosity: age; sex; BMI; diabetes reports in first-degree family members (dichotomous); heavy ethanol consumption; cigarette smoking; elevated serum alanine aminotransferase/aspartate aminotransferase levels; nonalcoholic fatty liver; chronic viral hepatitis; cirrhosis; hand arthropathy; iron removed by phlebotomy; and positivity for HLA-A*01, B*08; A*03, B*07; and A*03, B*14 haplotypes. We performed univariable and multivariable analyses. RESULTS Twenty-three probands (14.5%) had diabetes; 19 were men. Each of the 23 probands had type 2 diabetes. Mean BMI was greater in probands with diabetes (31.7 ± 8.5 [SD] kg/m(2) vs. 27.6 ± 5.1 kg/m(2); P = 0.032). Reports of any first-degree family member with diabetes were more prevalent in probands with than in probands without diabetes (69.6 vs. 17.6%; P < 0.0001). In probands with diabetes, the odds ratio (OR) of maternal diabetes was 6.7 (95% CI 2.3-19.7; P = 0.0005) and of sibling diabetes was 11.7 (3.0-45.5; P = 0.0004). In a logistic regression model, predictors of diabetes at hemochromatosis diagnosis in 159 probands were diabetes reports in family members (OR 8.5 [95% CI 2.9-24.8]; P < 0.0001) and BMI (1.1 [1.0-1.2]; P = 0.049). This model explained 26.0% of total deviance contributing to diabetes. CONCLUSIONS In nonscreening hemochromatosis probands with HFE C282Y homozygosity, a heritable factor(s) increases the risk of diabetes diagnosed before hemochromatosis.

The modified iron avidity index: a promising phenotypic predictor in HFE-related haemochromatosis.

PubMed

Verhaegh, Pauline L M; Moris, Wenke; Koek, Ger H; van Deursen, Cees Th B M

2016-10-01

Phenotypes of the HFE-related haemochromatosis vary considerably, making it hard to predict the course of iron accumulation. The aim of this retrospective study was to determine if the Iron Avidity Index (IAI) is a good phenotypic predictor of the number of phlebotomies needed per year during maintenance treatment (NPDMT) in patients with homozygous p.C282Y hereditary haemochromatosis (HH). Patients with HH homozygous for p.C282Y, on maintenance treatment for at least 1 year were included. The IAI (ferritin level at diagnosis/age at diagnosis) was calculated. Ninety-five patients were included in the analysis. Linear regression analysis showed the confounding effect of sex on the relationship between IAI and NPDMT. A modified IAI, adjusted for sex, was calculated. As proton pump inhibitor (PPI) use was independently associated with NPDMT, the group was split in PPI- and non-PPI-users. A positive correlation between the modified IAI and the NPDMT was shown in both groups (PPI r = 0.367, P = 0.023; non-PPI r = 0.453, P < 0.001). An ROC was computed to measure the accuracy of the modified IAI to predict who needed 0-2 vs. ≥3 maintenance treatments per year. The AUROC in the PPI and non-PPI group were respectively 0.576 (0.368-0.784) and 0.752 (0.606-0.899). The modified IAI is a fairly good predictor in non-PPI-using homozygous C282Y HH patients, to differentiate who needs ≥3 maintenance phlebotomies per year. Therefore, this index might help to select patients that benefit from an alternative less frequent therapy, e.g. erythrocytapheresis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.