μ-Calpain (CAPN1), calpastatin (CAST), and growth hormone receptor (GHR) genetic effects on Angus beef heifer performance traits and reproduction.

PubMed

Tait, Richard G; Cushman, Robert A; McNeel, Anthony K; Casas, Eduardo; Smith, Timothy P L; Freetly, Harvey C; Bennett, Gary L

2018-06-01

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to equalize CAPN1 haplotypes, CAST, and GHR genetic marker frequencies. The objective was to estimate the pleiotropic effects of these carcass quality oriented markers for body weight, reproduction, and first calf performance traits in 174 replacement beef females which were managed under 2 post-weaning development protocols. Heifers were weighed at 11-, 12-, and 13-mo, at first breeding season pregnancy evaluation, and prior to first calving season. Pubertal status was determined at 11-, 12-, and 13-mo of age. Antral follicles were counted, reproductive tracts were scored, and tract dimensions were measured at 13-mo. Body condition and hip height were scored and measured at pregnancy evaluation and prior to calving season. Heifer pregnancy and weaning rates and ordinal birth date were recorded. Calf body weights at birth and weaning were analyzed. Single df linear contrasts for recessive effects of the GHR heterozygous genotype showed significant decreases of 2.5-3.6% in 11-, 12-, and 13-mo heifer body weights and heifer weight prior to calving. The additive differences between GHR homozygotes were small and not significant for all body weights measured but a 1 wk difference in calf birth date was significant. For all 13-mo uterine measurements, scores, and antral follicle counts, only the CAST dominance contrast for medium antral follicle count was significant. The CAPN1 haplotype with a strong additive effect for increased beef tenderness also had a significant additive effect on calving date. Heifers homozygous for the tender haplotype calved 7.9 days later than heifers homozygous for the tough haplotype. Most heifer reproductive traits were not significantly affected by CAST and CAPN1 markers that are widely used to improve beef tenderness by selection

A genetic variant of the CAPN10 gene in Mexican subjects with dyslipidemia is associated with increased HDL-cholesterol concentrations after the consumption of a soy protein and soluble fiber dietary portfolio.

PubMed

Guevara-Cruz, Martha; Torres, Nimbe; Tovar, Armando R; Tejero, M Elizabeth; Castellanos-Jankiewicz, Ashley; del Bosque-Plata, Laura

2014-09-01

Dyslipidemia is a major public health problem, and therefore, it is important to develop dietary strategies to diminish the prevalence of this disorder. It was recently reported that diet may play an important role in triggering insulin resistance by interacting with genetic variants at the CAPN10 gene locus in patients with metabolic syndrome. Nonetheless, it remains unknown whether genetic variants of genes involved in the development of type 2 diabetes are associated with variations in high-density lipoprotein cholesterol (HDL-C). The study used a single-center, prospective, cohort design. Here, we assessed the effect of four variants of the CAPN10 gene on HDL-C levels in response to a soy protein and soluble fiber dietary portfolio in subjects with dyslipidemia. In 31 Mexican dyslipidemic individuals, we analyzed four CAPN10 gene variants (rs5030952, rs2975762, rs3792267, and rs2975760) associated with type 2 diabetes. Subjects with the GG genotype of the rs2975762 variant of the CAPN10 gene were better responders to dietary intervention, showing increased HDL-C concentrations from the first month of treatment. HDL-C concentrations in participants with the wild type genotype increased by 17.0%, whereas the HDL-C concentration in subjects with the variant genotypes increased by only 3.22% (p = 0.03); the low-density lipoprotein cholesterol levels of GG carriers tended to decrease (-12.6%). These results indicate that Mexican dyslipidemic carriers of the rs2975762-GG genotype are better responders to this dietary intervention. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

New truncation mutation of the NR2E3 gene in a Japanese patient with enhanced S-cone syndrome.

PubMed

Kuniyoshi, Kazuki; Hayashi, Takaaki; Sakuramoto, Hiroyuki; Mishima, Hiroshi; Tsuneoka, Hiroshi; Tsunoda, Kazushige; Iwata, Takeshi; Shimomura, Yoshikazu

2016-11-01

The enhanced S-cone syndrome (ESCS) is a rare hereditary retinal degeneration that has enhanced short wavelength-sensitive cone (S-cone) functions. The longitudinal clinical course of this disease has been rarely reported, and the genetic aspects of ESCS have not been well investigated in the Japanese population. In this report, we present our clinical and genetic findings for 2 patients with ESCS. The patients were 2 unrelated Japanese men. Standard ophthalmic examinations and mutation screening for the NR2E3 gene were performed. Patient 1 was a 36-year-old man, and his clinical findings were typical of ESCS. His decimal best-corrected visual acuity (BCVA) was 1.0 OD and 0.5 OS after removal of cataracts. Genetic investigations revealed a homozygous truncation frameshift, the p.I307LfsX33 mutation. Patient 2 was an 11-year-old boy when he was first examined by us. His clinical findings were typical of ESCS except for uveitis in the left eye. His decimal BCVA at the age of 39 years was maintained at 1.5 in each eye, although the retinal degeneration and visual field impairments had progressed during the follow-up period. The genetic investigations revealed homozygous mutations of p.R104Q in the NR2E3 gene. The frameshift mutation, p.I307LfsX33, in the NR2E3 gene is a new causative mutation for ESCS. The clinical observations for patient 2 are the longest ever reported. The retinal degeneration caused by this mutation is slowly progressive, and these patients maintained good vision with maintenance of the foveal structure until their late thirties.

µ-Calpain (CAPN1), calpastatin (CAST), and growth hormone receptor (GHR) genetic effects on Angus beef heifer performance traits and reproduction

USDA-ARS?s Scientific Manuscript database

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to equalize CAPN1 haplotypes, CAST, and GHR genetic marker frequencies. The objective was t...

Expression and effect of Calpain9 gene genetic polymorphism on slaughter indicators and intramuscular fat content in chickens.

PubMed

Cui, H X; Wang, S L; Guo, L P; Liu, L; Liu, R R; Li, Q H; Zheng, M Q; Zhao, G P; Wen, J

2018-06-26

Calpain 9 (CAPN9) is expressed in the stomach and small intestine. CAPN9 has regulatory roles in hypertension, heart disease, gastric mucosal defense, and kidney disease. The involvement of CAPN9 has not been reported in the development of chickens. CAPN9 mRNA was found in adipose and muscle tissue in this study. Two linkage single nucleotide polymorphisms (SNP; G7518A and C7542G) in intron 4 were screened from 160 birds of the D2 chicken line. The 2 mutation sites were associated with carcass weight, evisceration weight, abdominal fat weight (AFW), abdominal fat percentage (AFP), and breast muscle percentage (all P < 0.05). Intramuscular fat (IMF) content was not significantly different in the 3 genotypes. But, the AA(7518)/GG(7542) genotype had the highest IMF content, highest breast muscle weight, and lower AFW and AFP. Moreover, the mRNA level of CAPN9 in abdominal fat tissue was significantly different (P < 0.05 or P < 0.01) between any 2 genotypes, consistent with AFW and AFP. In summary, the expression of CAPN9 in adipose and breast muscle tissue is reported for the first time. CAPN9 affected production performance of chickens. As a marker, the linkage G7518A and C7542G polymorphisms in intron 4 of CAPN9 could affect the production traits by regulating mRNA expression. The findings concerning the marker enrich the theoretical foundation for molecular breeding of high-quality broilers.

New evidence for the role of calpain 10 in autosomal recessive intellectual disability: identification of two novel nonsense variants by exome sequencing in Iranian families.

PubMed

Oladnabi, Morteza; Musante, Luciana; Larti, Farzaneh; Hu, Hao; Abedini, Seyedeh Sedigheh; Wienker, Thomas; Ropers, Hans Hilger; Kahrizi, Kimia; Najmabadi, Hossein

2015-03-01

Knowledge of the genes responsible for intellectual disability, particularly autosomal recessive forms, is rapidly expanding. Increasing numbers of the gene show great heterogeneity and supports the hypothesis that human genome may contain over 2000 causative genes with a critical role in brain development. Since 2004, we have applied genome-wide SNP genotyping and next-generation sequencing in large consanguineous Iranian families with intellectual disability, to identify the genes harboring disease-causing mutations. The current study paved the way for identification of responsible genes in two unrelated Iranian families. We found two novel nonsense mutations, p.C77* and p.Q115*, in the calpain catalytic domain of CAPN10, which is a cysteine protease known to be involved in pathogenesis of noninsulin-dependent diabetes mellitus. Another different mutation in this gene (p.S138_R139ins5) has previously been reported in an Iranian family. All of these patients have common clinical features in spite of specific brain structural abnormalities on MRI. Different mutations in CAPN10 have already been found in three independent Iranian families. These results have strongly supported the possible role of CAPN10 in human brain development. Altogether, we proposed CAPN10 as a promising candidate gene for intellectual disability, which should be considered in diagnostic gene panels.

Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.

PubMed

Ivanov, E L; Kovaltzova, S V; Korolev, V G

1989-08-01

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.

Point mutations in the tumor suppressor Smad4/DPC4 enhance its phosphorylation by GSK3 and reversibly inactivate TGF-β signaling

PubMed Central

Demagny, Hadrien; De Robertis, Edward M

2016-01-01

The tumor suppressor Smad4/DPC4 is an essential transcription factor in the TGF-β pathway and is frequently mutated or deleted in prostate, colorectal, and pancreatic carcinomas. We recently discovered that Smad4 activity and stability are regulated by the FGF/EGF and Wnt signaling pathways through a series of MAPK and GSK3 phosphorylation sites located in its linker region. In the present study, we report that loss-of-function associated with 2 point mutations commonly found in colorectal and pancreatic cancers results from enhanced Smad4 phosphorylation by GSK3, generating a phosphodegron that leads to subsequent β-TrCP–mediated polyubiquitination and proteasomal degradation. Using chemical GSK3 inhibitors, we show that Smad4 point mutant proteins can be stabilized and TGF-β signaling restored in cancer cells harboring such mutations. PMID:27308538

[SNP-19 genotypic variants of CAPN10 gene and its relation to diabetes mellitus type 2 in a population of Ciudad Juarez, Mexico].

PubMed

Loya Méndez, Yolanda; Reyes Leal, Gilberto; Sánchez González, Adriana; Portillo Reyes, Verónica; Reyes Ruvalcaba, David; Bojórquez Rangel, Guillermo

2014-09-28

Diabetes Mellitus (DM) type 2 is a common pathology with multifactorial etiology, which exact genetic bases remain unknown. Some studies suggest that single nucleotides polymorphisms (SNPs) in the CAPN10 gene (Locus 2q37.3) could be associated with the development of this disease, including the insertion/deletion polymorphism SNP-19 (2R→3R). The present study determined the association between the SNP-19 and the risk of developing DM type 2 in Ciudad Juarez population. For this study 107 participants were selected: 43 diabetics type 2 (cases) and 64 non diabetics with no family history of DM type 2 in first grade (control). Anthropometric studies were realized as well as lipids, lipoproteins and serum glucose biochemical profiles. The genotypification of SNP-19 was performed using peripheral blood lymphocytes DNA, polymerase chain reactions (PCR), and electrophoretic analysis in agarose gels. Once obtained the genotypic and allelic frequencies, the Hardy-Weinberg equilibrium test (GenAlEx 6.4) was also performed. Using the X² analysis it was identified the genotypic differences between cases and control with higher frequency of the homozygous genotype 3R of SNP- 19 in the cases group (0.418) compared to control group (0.265). Also, it was observed an association between genotype 2R/3R with elevated weight, body mass index, and waist and hip circumferences, but only in the diabetic group (P=< 0.05). The findings in this study suggest that SNP-19 in CAPN10 may participate in the development of DM type 2 in the studied population. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors.

PubMed

Rothwell, Patrick E; Fuccillo, Marc V; Maxeiner, Stephan; Hayton, Scott J; Gokce, Ozgun; Lim, Byung Kook; Fowler, Stephen C; Malenka, Robert C; Südhof, Thomas C

2014-07-03

In humans, neuroligin-3 mutations are associated with autism, whereas in mice, the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse and thereby provide a plausible circuit substrate for autism pathophysiology. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional significance of co-occurring mutations in PIK3CA and MAP3K1 in breast cancer.

PubMed

Avivar-Valderas, Alvaro; McEwen, Robert; Taheri-Ghahfarokhi, Amir; Carnevalli, Larissa S; Hardaker, Elizabeth L; Maresca, Marcello; Hudson, Kevin; Harrington, Elizabeth A; Cruzalegui, Francisco

2018-04-20

The PI3Kα signaling pathway is frequently hyper-activated in breast cancer (BrCa), as a result of mutations/amplifications in oncogenes (e.g. HER2 ), decreased function in tumor suppressors (e.g. PTEN ) or activating mutations in key components of the pathway. In particular, activating mutations of PIK3CA (~45%) are frequently found in luminal A BrCa samples. Genomic studies have uncovered inactivating mutations in MAP3K1 (13-20%) and MAP2K4 (~8%), two upstream kinases of the JNK apoptotic pathway in luminal A BrCa samples. Further, simultaneous mutation of PIK3CA and MAP3K1 are found in ~11% of mutant PIK3CA tumors. How these two alterations may cooperate to elicit tumorigenesis and impact the sensitivity to PI3K and AKT inhibitors is currently unknown. Using CRISPR gene editing we have genetically disrupted MAP3K1 expression in mutant PIK3CA cell lines to specifically create in vitro models reflecting the mutational status of PIK3CA and MAP3K1 in BrCa patients. MAP3K1 deficient cell lines exhibited ~2.4-fold increased proliferation rate and decreased sensitivity to PI3Kα/δ(AZD8835) and AKT (AZD5363) inhibitors (~2.61 and ~5.23-fold IC 50 increases, respectively) compared with parental control cell lines. In addition, mechanistic analysis revealed that MAP3K1 disruption enhances AKT phosphorylation and downstream signaling and reduces sensitivity to AZD5363-mediated pathway inhibition. This appears to be a consequence of deficient MAP3K1-JNK signaling increasing IRS1 stability and therefore promoting IRS1 binding to p85, resulting in enhanced PI3Kα activity. Using 3D-MCF10A-PI3Kα H1047R models, we found that MAP3K1 depletion increased overall acinar volume and counteracted AZD5363-mediated reduction of acinar growth due to enhanced proliferation and reduced apoptosis. Furthermore, in vivo efficacy studies revealed that MAP3K1-deficient MCF7 tumors were less sensitive to AKT inhibitor treatment, compared with parental MCF7 tumors. Our study provides

The STAT3 HIES mutation is a gain-of-function mutation that activates genes via AGG-element carrying promoters.

PubMed

Xu, Li; Ji, Jin-Jun; Le, Wangping; Xu, Yan S; Dou, Dandan; Pan, Jieli; Jiao, Yifeng; Zhong, Tianfei; Wu, Dehong; Wang, Yumei; Wen, Chengping; Xie, Guan-Qun; Yao, Feng; Zhao, Heng; Fan, Yong-Sheng; Chin, Y Eugene

2015-10-15

Cytokine or growth factor activated STAT3 undergoes multiple post-translational modifications, dimerization and translocation into nuclei, where it binds to serum-inducible element (SIE, 'TTC(N3)GAA')-bearing promoters to activate transcription. The STAT3 DNA binding domain (DBD, 320-494) mutation in hyper immunoglobulin E syndrome (HIES), called the HIES mutation (R382Q, R382W or V463Δ), which elevates IgE synthesis, inhibits SIE binding activity and sensitizes genes such as TNF-α for expression. However, the mechanism by which the HIES mutation sensitizes STAT3 in gene induction remains elusive. Here, we report that STAT3 binds directly to the AGG-element with the consensus sequence 'AGG(N3)AGG'. Surprisingly, the helical N-terminal region (1-355), rather than the canonical STAT3 DBD, is responsible for AGG-element binding. The HIES mutation markedly enhances STAT3 AGG-element binding and AGG-promoter activation activity. Thus, STAT3 is a dual specificity transcription factor that promotes gene expression not only via SIE- but also AGG-promoter activity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Surface Enhanced Raman Spectroscopy (SERS) for the Multiplex Detection of Braf, Kras, and Pik3ca Mutations in Plasma of Colorectal Cancer Patients

PubMed Central

Li, Xiaozhou; Yang, Tianyue; Li, Caesar Siqi; Song, Youtao; Lou, Hong; Guan, Dagang; Jin, Lili

2018-01-01

In this paper, we discuss the use of a procedure based on polymerase chain reaction (PCR) and surface enhanced Raman spectroscopy (SERS) (PCR-SERS) to detect DNA mutations. Methods: This method was implemented by first amplifying DNA-containing target mutations, then by annealing probes, and finally by applying SERS detection. The obtained SERS spectra were from a mixture of fluorescence tags labeled to complementary sequences on the mutant DNA. Then, the SERS spectra of multiple tags were decomposed to component tag spectra by multiple linear regression (MLR). Results: The detection limit was 10-11 M with a coefficient of determination (R2) of 0.88. To demonstrate the applicability of this process on real samples, the PCR-SERS method was applied on blood plasma taken from 49 colorectal cancer patients to detect six mutations located at the BRAF, KRAS, and PIK3CA genes. The mutation rates obtained by the PCR-SERS method were in concordance with previous research. Fisher's exact test showed that only two detected mutations at BRAF (V600E) and PIK3CA (E542K) were significantly positively correlated with right-sided colon cancer. No other clinical feature such as gender, age, cancer stage, or differentiation was correlated with mutation (V600E at BRAF, G12C, G12D, G12V, G13D at KRAS, and E542K at PIK3CA). Visually, a dendrogram drawn through hierarchical clustering analysis (HCA) supported the results of Fisher's exact test. The clusters drawn by all six mutations did not conform to the distributions of cancer stages, differentiation or cancer positions. However, the cluster drawn by the two mutations of V600E and E542K showed that all samples with those mutations belonged to the right-sided colon cancer group. Conclusion: The suggested PCR-SERS method is multiplexed, flexible in probe design, easy to incorporate into existing PCR conditions, and was sensitive enough to detect mutations in blood plasma. PMID:29556349

PDE3A mutations cause autosomal dominant hypertension with brachydactyly.

PubMed

Maass, Philipp G; Aydin, Atakan; Luft, Friedrich C; Schächterle, Carolin; Weise, Anja; Stricker, Sigmar; Lindschau, Carsten; Vaegler, Martin; Qadri, Fatimunnisa; Toka, Hakan R; Schulz, Herbert; Krawitz, Peter M; Parkhomchuk, Dmitri; Hecht, Jochen; Hollfinger, Irene; Wefeld-Neuenfeld, Yvette; Bartels-Klein, Eireen; Mühl, Astrid; Kann, Martin; Schuster, Herbert; Chitayat, David; Bialer, Martin G; Wienker, Thomas F; Ott, Jürg; Rittscher, Katharina; Liehr, Thomas; Jordan, Jens; Plessis, Ghislaine; Tank, Jens; Mai, Knut; Naraghi, Ramin; Hodge, Russell; Hopp, Maxwell; Hattenbach, Lars O; Busjahn, Andreas; Rauch, Anita; Vandeput, Fabrice; Gong, Maolian; Rüschendorf, Franz; Hübner, Norbert; Haller, Hermann; Mundlos, Stefan; Bilginturan, Nihat; Movsesian, Matthew A; Klussmann, Enno; Toka, Okan; Bähring, Sylvia

2015-06-01

Cardiovascular disease is the most common cause of death worldwide, and hypertension is the major risk factor. Mendelian hypertension elucidates mechanisms of blood pressure regulation. Here we report six missense mutations in PDE3A (encoding phosphodiesterase 3A) in six unrelated families with mendelian hypertension and brachydactyly type E (HTNB). The syndrome features brachydactyly type E (BDE), severe salt-independent but age-dependent hypertension, an increased fibroblast growth rate, neurovascular contact at the rostral-ventrolateral medulla, altered baroreflex blood pressure regulation and death from stroke before age 50 years when untreated. In vitro analyses of mesenchymal stem cell-derived vascular smooth muscle cells (VSMCs) and chondrocytes provided insights into molecular pathogenesis. The mutations increased protein kinase A-mediated PDE3A phosphorylation and resulted in gain of function, with increased cAMP-hydrolytic activity and enhanced cell proliferation. Levels of phosphorylated VASP were diminished, and PTHrP levels were dysregulated. We suggest that the identified PDE3A mutations cause the syndrome. VSMC-expressed PDE3A deserves scrutiny as a therapeutic target for the treatment of hypertension.

Autism-related neuroligin-3 mutation alters social behavior and spatial learning.

PubMed

Jaramillo, Thomas C; Liu, Shunan; Pettersen, Ami; Birnbaum, Shari G; Powell, Craig M

2014-04-01

Multiple candidate genes have been identified for autism spectrum disorders. While some of these genes reach genome-wide significance, others, such as the R451C point mutation in the synaptic cell adhesion molecule neuroligin-3, appear to be rare. Interestingly, two brothers with the same R451C point mutation in neuroligin-3 present clinically on seemingly disparate sides of the autism spectrum. These clinical findings suggest genetic background may play a role in modifying the penetrance of a particular autism-associated mutation. Animal models may contribute additional support for such mutations as functionally relevant and can provide mechanistic insights. Previously, in collaboration with the Südhof laboratory, we reported that mice with an R451C substitution in neuroligin-3 displayed social deficits and enhanced spatial learning. While some of these behavioral abnormalities have since been replicated independently in the Südhof laboratory, observations from the Crawley laboratory failed to replicate these findings in a similar neuroligin-3 mutant mouse model and suggested that genetic background may contribute to variation in observations across laboratories. Therefore, we sought to replicate our findings in the neuroligin-3 R451C point mutant knock-in mouse model (NL3R451C) in a different genetic background. We backcrossed our NL3R451C mouse line onto a 129S2/SvPasCrl genetic background and repeated a subset of our previous behavioral testing. NL3R451C mice on a 129S2/SvPasCrl displayed social deficits, enhanced spatial learning, and increased locomotor activity. These data extend our previous findings that NL3R451C mice exhibit autism-relevant behavioral abnormalities and further suggest that different genetic backgrounds can modify this behavioral phenotype through epistatic genetic interactions. © 2014 International Society for Autism Research, Wiley Periodicals, Inc.

High frequency of coexistent mutations of PIK3CA and PTEN genes in endometrial carcinoma.

PubMed

Oda, Katsutoshi; Stokoe, David; Taketani, Yuji; McCormick, Frank

2005-12-01

The phosphatidylinositol 3'-kinase (PI3K) pathway is activated in many human cancers. In addition to inactivation of the PTEN tumor suppressor gene, mutations or amplifications of the catalytic subunit alpha of PI3K (PIK3CA) have been reported. However, the coexistence of mutations in these two genes seems exceedingly rare. As PTEN mutations occur at high frequency in endometrial carcinoma, we screened 66 primary endometrial carcinomas for mutations in the helical and catalytic domains of PIK3CA. We identified a total of 24 (36%) mutations in this gene and coexistence of PIK3CA/PTEN mutations at high frequency (26%). PIK3CA mutations were more common in tumors with PTEN mutations (17 of 37, 46%) compared with those without PTEN mutations (7 of 29, 24%). Array comparative genomic hybridization detected 3q24-qter amplification, which covers the PIK3CA gene (3q26.3), in one of nine tumors. Knocking down PTEN expression in the HEC-1B cell line, which possesses both K-Ras and PIK3CA mutations, further enhances phosphorylation of Akt (Ser473), indicating that double mutation of PIK3CA and PTEN has an additive effect on PI3K activation. Our data suggest that the PI3K pathway is extensively activated in endometrial carcinomas, and that combination of PIK3CA/PTEN alterations might play an important role in development of these tumors.

NR2E3 mutations in enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP).

PubMed

Schorderet, Daniel F; Escher, Pascal

2009-11-01

NR2E3, also called photoreceptor-specific nuclear receptor (PNR), is a transcription factor of the nuclear hormone receptor superfamily whose expression is uniquely restricted to photoreceptors. There, its physiological activity is essential for proper rod and cone photoreceptor development and maintenance. Thirty-two different mutations in NR2E3 have been identified in either homozygous or compound heterozygous state in the recessively inherited enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), and clumped pigmentary retinal degeneration (CPRD). The clinical phenotype common to all these patients is night blindness, rudimental or absent rod function, and hyperfunction of the "blue" S-cones. A single p.G56R mutation is inherited in a dominant manner and causes retinitis pigmentosa (RP). We have established a new locus-specific database for NR2E3 (www.LOVD.nl/eye), containing all reported mutations, polymorphisms, and unclassified sequence variants, including novel ones. A high proportion of mutations are located in the evolutionarily-conserved DNA-binding domains (DBDs) and ligand-binding domains (LBDs) of NR2E3. Based on homology modeling of these NR2E3 domains, we propose a structural localization of mutated residues. The high variability of clinical phenotypes observed in patients affected by NR2E3-linked retinal degenerations may be caused by different disease mechanisms, including absence of DNA-binding, altered interactions with transcriptional coregulators, and differential activity of modifier genes.

Enhancement of B-cell receptor signaling by a point mutation of adaptor protein 3BP2 identified in human inherited disease cherubism.

PubMed

Ogi, Kazuhiro; Nakashima, Kenji; Chihara, Kazuyasu; Takeuchi, Kenji; Horiguchi, Tomoko; Fujieda, Shigeharu; Sada, Kiyonao

2011-09-01

Tyrosine phosphorylation of adaptor protein c-Abl-Src homology 3 (SH3) domain-binding protein-2 (3BP2, also referred to SH3BP2) positively regulates the B-cell antigen receptor (BCR)-mediated signal transduction, leading to the activation of nuclear factor of activated T cells (NFAT). Here we showed the effect of the proline to arginine substitution of 3BP2 in which is the most common mutation in patients with cherubism (P418R) on B-cell receptor signaling. Comparing to the wild type, overexpression of the mutant form of 3BP2 (3BP2-P416R, corresponding to P418R in human protein) enhanced BCR-mediated activation of NFAT. 3BP2-P416R increased the signaling complex formation with Syk, phospholipase C-γ2 (PLC-γ2), and Vav1. In contrast, 3BP2-P416R could not change the association with the negative regulator 14-3-3. Loss of the association mutant that was incapable to associate with 14-3-3 could not mimic BCR-mediated NFAT activation in Syk-deficient cells. Moreover, BCR-mediated phosphorylation of extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was not affected by P416R mutation. These results showed that P416R mutation of 3BP2 causes the gain of function in B cells by increasing the interaction with specific signaling molecules. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

An in-silico method for identifying aggregation rate enhancer and mitigator mutations in proteins.

PubMed

Rawat, Puneet; Kumar, Sandeep; Michael Gromiha, M

2018-06-24

Newly synthesized polypeptides must pass stringent quality controls in cells to ensure appropriate folding and function. However, mutations, environmental stresses and aging can reduce efficiencies of these controls, leading to accumulation of protein aggregates, amyloid fibrils and plaques. In-vitro experiments have shown that even single amino acid substitutions can drastically enhance or mitigate protein aggregation kinetics. In this work, we have collected a dataset of 220 unique mutations in 25 proteins and classified them as enhancers or mitigators on the basis of their effect on protein aggregation rate. The data were analyzed via machine learning to identify features capable of distinguishing between aggregation rate enhancers and mitigators. Our initial Support Vector Machine (SVM) model separated such mutations with an overall accuracy of 69%. When local secondary structures at the mutation sites were considered, the accuracies further improved by 13-15%. The machine-learnt features are distinct for each secondary structure class at mutation sites. Protein stability and flexibility changes are important features for mutations in α-helices. β-strand propensity, polarity and charge become important when mutations occur in β-strands and ability to form secondary structure, helical tendency and aggregation propensity are important for mutations lying in coils. These results have been incorporated into a sequence-based algorithm (available at http://www.iitm.ac.in/bioinfo/aggrerate-disc/) capable of predicting whether a mutation will enhance or mitigate a protein's aggregation rate. This algorithm will find several applications towards understanding protein aggregation in human diseases, enable in-silico optimization of biopharmaceuticals and enzymes for improved biophysical attributes and de novo design of bio-nanomaterials. Copyright © 2018. Published by Elsevier B.V.

FGFR3, PIK3CA and RAS mutations in benign lichenoid keratosis.

PubMed

Groesser, L; Herschberger, E; Landthaler, M; Hafner, C

2012-04-01

Benign lichenoid keratoses (BLKs) are solitary skin lesions which have been proposed to represent a regressive form of pre-existent epidermal tumours such as solar lentigo or seborrhoeic keratosis. However, the genetic basis of BLK is unknown. FGFR3, PIK3CA and RAS mutations have been shown to be involved in the pathogenesis of seborrhoeic keratosis and solar lentigo. We thus investigated whether these mutations are also present in BLK. After manual microdissection and DNA isolation, 52 BLKs were screened for FGFR3, PIK3CA and RAS hotspot mutations using SNaPshot(®) multiplex assays. We identified 6/52 (12%) FGFR3 mutations, 10/52 (19%) PIK3CA mutations, 6/52 (12%) HRAS mutations and 2/52 (4%) KRAS mutations. FGFR3 and RAS mutations were mutually exclusive. One BLK showed a simultaneous PIK3CA and HRAS mutation. In nine BLKs with a mutation, nonlesional control tissue from the epidermal margin and the dermal lymphocytic infiltrate were wild-type, indicating that these mutations are somatic. To demonstrate that these findings are specific, 10 samples of lichen planus were analysed without evidence for FGFR3, PIK3CA or RAS mutations. Our results indicate that FGFR3, PIK3CA and RAS mutations are present in approximately 50% of BLKs. These findings support the concept on the molecular genetic level that at least a proportion of BLKs represents regressive variants resulting from former benign epidermal tumours such as seborrhoeic keratosis and solar lentigo. © 2011 The Authors. BJD © 2011 British Association of Dermatologists 2011.

Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function.

PubMed

Etherton, Mark; Földy, Csaba; Sharma, Manu; Tabuchi, Katsuhiko; Liu, Xinran; Shamloo, Mehrdad; Malenka, Robert C; Südhof, Thomas C

2011-08-16

Multiple independent mutations in neuroligin genes were identified in patients with familial autism, including the R451C substitution in neuroligin-3 (NL3). Previous studies showed that NL3(R451C) knock-in mice exhibited modestly impaired social behaviors, enhanced water maze learning abilities, and increased synaptic inhibition in the somatosensory cortex, and they suggested that the behavioral changes in these mice may be caused by a general shift of synaptic transmission to inhibition. Here, we confirm that NL3(R451C) mutant mice behaviorally exhibit social interaction deficits and electrophysiologically display increased synaptic inhibition in the somatosensory cortex. Unexpectedly, however, we find that the NL3(R451C) mutation produced a strikingly different phenotype in the hippocampus. Specifically, in the hippocampal CA1 region, the NL3(R451C) mutation caused an ∼1.5-fold increase in AMPA receptor-mediated excitatory synaptic transmission, dramatically altered the kinetics of NMDA receptor-mediated synaptic responses, induced an approximately twofold up-regulation of NMDA receptors containing NR2B subunits, and enhanced long-term potentiation almost twofold. NL3 KO mice did not exhibit any of these changes. Quantitative light microscopy and EM revealed that the NL3(R451C) mutation increased dendritic branching and altered the structure of synapses in the stratum radiatum of the hippocampus. Thus, in NL3(R451C) mutant mice, a single point mutation in a synaptic cell adhesion molecule causes context-dependent changes in synaptic transmission; these changes are consistent with the broad impact of this mutation on murine and human behaviors, suggesting that NL3 controls excitatory and inhibitory synapse properties in a region- and circuit-specific manner.

Aspirin-induced chemoprevention and response kinetics are enhanced by PIK3CA mutations in colorectal cancer cells

PubMed Central

Zumwalt, Timothy J; Wodarz, Dominik; Komarova, Natalia L; Toden, Shusuke; Turner, Jacob; Cardenas, Jacob; Burn, John; Chan, Andrew T; Boland, C Richard; Goel, Ajay

2017-01-01

This study was designed to determine how aspirin influences the growth kinetics and characteristics of cultured colorectal cancer (CRC) cells that harbor a variety of different mutational backgrounds, including PIK3CA and KRAS activating mutations and the presence or absence of microsatellite instability. CRC cell lines (HCT116, HCT116+Chr3/5, RKO, SW480, HCT15, CACO2, HT29, and SW48) were treated with pharmacologically relevant doses of aspirin (0.5–10 mM) and evaluated for proliferation and cell cycle distribution. These parameters were fitted to a mathematical model to quantify the effects and understand the mechanism(s) by which aspirin modifies growth in CRC cells. We also evaluated the effects of aspirin on key G0/G1 cell cycle genes that are regulated by PI3K-Akt pathway. Aspirin decelerated growth rates and disrupted cell cycle dynamics more profoundly in faster growing CRC cell lines, which tended to be PIK3CA-mutants. Additionally, microarray analysis of 151 CRC cell lines identified important cell cycle regulatory genes downstream targets of PIK3, which were dysregulated by aspirin treatment cycle genes (PCNA and RB1, p<0.01). Our study demonstrated what clinical trials have only speculated, that PIK3CA-mutant CRCs are more sensitive to aspirin. Aspirin inhibited cell growth in all CRC cell lines regardless of mutational background, but the effects were exacerbated in cells with PIK3CA mutations. Mathematical modeling combined with bench science revealed that cells with PIK3CA mutations experience significant G0/G1 arrest and explains why patients with PIK3CA-mutant CRCs may benefit from aspirin use after diagnosis. PMID:28154202

HER3 expression is enhanced during progression of lung adenocarcinoma without EGFR mutation from stage 0 to IA1.

PubMed

Kumagai, Toru; Tomita, Yasuhiko; Nakatsuka, Shin-Ichi; Kimura, Madoka; Kunimasa, Kei; Inoue, Takako; Tamiya, Motohiro; Nishino, Kazumi; Susaki, Yoshiyuki; Kusu, Takashi; Tokunaga, Toshiteru; Okami, Jiro; Higashiyama, Masahiko; Imamura, Fumio

2018-04-01

Activating EGFR mutations, HER2, and HER3 are implicated in lung cancer; however, with the exception of EGFR gene amplification in lung adenocarcinoma harboring EGFR mutations, their involvement in disease progression during the early stages is poorly understood. In this paper, we focused on which receptor is correlated with lung adenocarcinoma progression in the presence or absence of EGFR mutation from stage 0 to IA1. HER2 and HER3 expression and activating EGFR mutations in surgically resected lung adenocarcinoma exhibiting ground glass nodules on chest computed tomography and re-classified to stage 0 and IA1 were examined by immunohistochemistry and peptide nucleic acid-locked nucleic acid PCR clamp method, respectively. HER2 and HER3 expression was detected in 22.2% and 86.1% of samples, respectively. The frequency of EGFR mutation was 45.7% and was not significantly different between stage 0 and IA1 (40.0% and 48.0%, respectively), suggesting that EGFR mutation does not correlate with cancer progression from stage 0 to IA1. HER2 expression also did not correlate to progression. However, not only the frequency, but also the intensity of HER3 expression was increased in stage IA1 lung adenocarcinoma, particularly in lung adenocarcinoma without EGFR mutation. HER3 tends to be intensively expressed during the progression of lung adenocarcinoma without EGFR mutation from carcinoma in situ to invasive carcinoma. © 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Oncogene GAEC1 regulates CAPN10 expression which predicts survival in esophageal squamous cell carcinoma

PubMed Central

Chan, Dessy; Tsoi, Miriam Yuen-Tung; Liu, Christina Di; Chan, Sau-Hing; Law, Simon Ying-Kit; Chan, Kwok-Wah; Chan, Yuen-Piu; Gopalan, Vinod; Lam, Alfred King-Yin; Tang, Johnny Cheuk-On

2013-01-01

AIM: To identify the downstream regulated genes of GAEC1 oncogene in esophageal squamous cell carcinoma and their clinicopathological significance. METHODS: The anti-proliferative effect of knocking down the expression of GAEC1 oncogene was studied by using the RNA interference (RNAi) approach through transfecting the GAEC1-overexpressed esophageal carcinoma cell line KYSE150 with the pSilencer vector cloned with a GAEC1-targeted sequence, followed by MTS cell proliferation assay and cell cycle analysis using flow cytometry. RNA was then extracted from the parental, pSilencer-GAEC1-targeted sequence transfected and pSilencer negative control vector transfected KYSE150 cells for further analysis of different patterns in gene expression. Genes differentially expressed with suppressed GAEC1 expression were then determined using Human Genome U133 Plus 2.0 cDNA microarray analysis by comparing with the parental cells and normalized with the pSilencer negative control vector transfected cells. The most prominently regulated genes were then studied by immunohistochemical staining using tissue microarrays to determine their clinicopathological correlations in esophageal squamous cell carcinoma by statistical analyses. RESULTS: The RNAi approach of knocking down gene expression showed the effective suppression of GAEC1 expression in esophageal squamous cell carcinoma cell line KYSE150 that resulted in the inhibition of cell proliferation and increase of apoptotic population. cDNA microarray analysis for identifying differentially expressed genes detected the greatest levels of downregulation of calpain 10 (CAPN10) and upregulation of trinucleotide repeat containing 6C (TNRC6C) transcripts when GAEC1 expression was suppressed. At the tissue level, the high level expression of calpain 10 protein was significantly associated with longer patient survival (month) of esophageal squamous cell carcinoma compared to the patients with low level of calpain 10 expression (37.73 ± 16

Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern

PubMed Central

Hafner, Christian; López-Knowles, Elena; Luis, Nuno M.; Toll, Agustí; Baselga, Eulàlia; Fernández-Casado, Alex; Hernández, Silvia; Ribé, Adriana; Mentzel, Thomas; Stoehr, Robert; Hofstaedter, Ferdinand; Landthaler, Michael; Vogt, Thomas; Pujol, Ramòn M.; Hartmann, Arndt; Real, Francisco X.

2007-01-01

Activating mutations of the p110 α subunit of PI3K (PIK3CA) oncogene have been identified in a broad spectrum of malignant tumors. However, their role in benign or preneoplastic conditions is unknown. Activating FGF receptor 3 (FGFR3) mutations are common in benign skin lesions, either as embryonic mutations in epidermal nevi (EN) or as somatic mutations in seborrheic keratoses (SK). FGFR3 mutations are also common in low-grade malignant bladder tumors, where they often occur in association with PIK3CA mutations. Therefore, we examined exons 9 and 20 of PIK3CA and FGFR3 hotspot mutations in EN (n = 33) and SK (n = 62), two proliferative skin lesions lacking malignant potential. Nine of 33 (27%) EN harbored PIK3CA mutations; all cases showed the E545G substitution, which is uncommon in cancers. In EN, R248C was the only FGFR3 mutation identified. By contrast, 10 of 62 (16%) SK revealed the typical cancer-associated PIK3CA mutations E542K, E545K, and H1047R. The same lesions displayed a wide range of FGFR3 mutations. Corresponding unaffected tissue was available for four EN and two mutant SK: all control samples displayed a WT sequence, confirming the somatic nature of the mutations found in lesional tissue. Forty of 95 (42%) lesions showed at least one mutation in either gene. PIK3CA and FGFR3 mutations displayed an independent distribution; 5/95 lesions harbored mutations in both genes. Our findings suggest that, in addition to their role in cancer, oncogenic PIK3CA mutations contribute to the pathogenesis of skin tumors lacking malignant potential. The remarkable genotype–phenotype correlation as observed in this study points to a distinct etiopathogenesis of the mutations in keratinocytes occuring either during fetal development or in adult life. PMID:17673550

Differential evolution enhanced with multiobjective sorting-based mutation operators.

PubMed

Wang, Jiahai; Liao, Jianjun; Zhou, Ying; Cai, Yiqiao

2014-12-01

Differential evolution (DE) is a simple and powerful population-based evolutionary algorithm. The salient feature of DE lies in its mutation mechanism. Generally, the parents in the mutation operator of DE are randomly selected from the population. Hence, all vectors are equally likely to be selected as parents without selective pressure at all. Additionally, the diversity information is always ignored. In order to fully exploit the fitness and diversity information of the population, this paper presents a DE framework with multiobjective sorting-based mutation operator. In the proposed mutation operator, individuals in the current population are firstly sorted according to their fitness and diversity contribution by nondominated sorting. Then parents in the mutation operators are proportionally selected according to their rankings based on fitness and diversity, thus, the promising individuals with better fitness and diversity have more opportunity to be selected as parents. Since fitness and diversity information is simultaneously considered for parent selection, a good balance between exploration and exploitation can be achieved. The proposed operator is applied to original DE algorithms, as well as several advanced DE variants. Experimental results on 48 benchmark functions and 12 real-world application problems show that the proposed operator is an effective approach to enhance the performance of most DE algorithms studied.

Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers.

PubMed Central

Ryden, T A; de Mars, M; Beemon, K

1993-01-01

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive. Images PMID:8386280

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

Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities

PubMed Central

Boyden, Lynn M.; Choi, Murim; Choate, Keith A.; Nelson-Williams, Carol J.; Farhi, Anita; Toka, Hakan R.; Tikhonova, Irina R.; Bjornson, Robert; Mane, Shrikant M.; Colussi, Giacomo; Lebel, Marcel; Gordon, Richard D.; Semmekrot, Ben A.; Poujol, Alain; Välimäki, Matti J.; De Ferrari, Maria E.; Sanjad, Sami A.; Gutkin, Michael; Karet, Fiona E.; Tucci, Joseph R.; Stockigt, Jim R.; Keppler-Noreuil, Kim M.; Porter, Craig C.; Anand, Sudhir K.; Whiteford, Margo L.; Davis, Ira D.; Dewar, Stephanie B.; Bettinelli, Alberto; Fadrowski, Jeffrey J.; Belsha, Craig W.; Hunley, Tracy E.; Nelson, Raoul D.; Trachtman, Howard; Cole, Trevor R. P.; Pinsk, Maury; Bockenhauer, Detlef; Shenoy, Mohan; Vaidyanathan, Priya; Foreman, John W.; Rasoulpour, Majid; Thameem, Farook; Al-Shahrouri, Hania Z.; Radhakrishnan, Jai; Gharavi, Ali G.; Goilav, Beatrice; Lifton, Richard P.

2012-01-01

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. A rare Mendelian syndrome, pseudohypoaldosteronism type II (PHAII), featuring hypertension, hyperkalemia, and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption versus K+ and H+ excretion1. We used exome sequencing to identify mutations in Kelch-like 3 (KLHL3) or Cullin 3 (CUL3) in 41 PHAII kindreds. KLHL3 mutations are either recessive or dominant, while CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-Kelch proteins such as KLHL3 are components of Cullin/RING E3 ligase complexes (CRLs) that ubiquitinate substrates bound to Kelch propeller domains2–8. Dominant KLHL3 mutations are clustered in short segments within the Kelch propeller and BTB domains implicated in substrate9 and Cullin5 binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter (NCC) in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3/CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite combined complexities of locus heterogeneity, mixed models of transmission, and frequent de novo mutation, and establish a fundamental role for KLHL3/CUL3 in blood pressure, K+, and pH homeostasis. PMID:22266938

Selective FLT3 inhibitor FI-700 neutralizes Mcl-1 and enhances p53-mediated apoptosis in AML cells with activating mutations of FLT3 through Mcl-1/Noxa axis.

PubMed

Kojima, K; Konopleva, M; Tsao, T; Andreeff, M; Ishida, H; Shiotsu, Y; Jin, L; Tabe, Y; Nakakuma, H

2010-01-01

Treatment using Fms-like tyrosine kinase-3 (FLT3) inhibitors is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. We found that FI-700 immediately reduced antiapoptotic Mcl-1 levels and enhanced Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/internal tandem duplication cells through the Mcl-1/Noxa axis. FI-700 induced proteasome-mediated degradation of Mcl-1, resulting in the reduced ability of Mcl-1 to sequester proapoptotic Bim. Nutlin-3 induced Noxa, which displaced Bim from Mcl-1. The FI-700/Nutlin-3 combination profoundly activated Bax and induced apoptosis. Our findings suggest that FI-700 actively enhances p53 signaling toward mitochondrial apoptosis and that a combination strategy aimed at inhibiting FLT3 and activating p53 signaling could potentially be effective in AML.

DNMT3A mutations in Chinese childhood acute myeloid leukemia.

PubMed

Li, Weijing; Cui, Lei; Gao, Chao; Liu, Shuguang; Zhao, Xiaoxi; Zhang, Ruidong; Zheng, Huyong; Wu, Minyuan; Li, Zhigang

2017-08-01

DNA methyltransferase 3A (DNMT3A) mutations have been found in approximately 20% of adult acute myeloid leukemia (AML) patients and in 0% to 1.4% of children with AML, and the hotspots of mutations are mainly located in the catalytic methyltransferase domain, hereinto, mutation R882 accounts for 60%. Although the negative effect of DNMT3A on treatment outcome is well known, the prognostic significance of other DNMT3A mutations in AML is still unclear. Here, we tried to determine the incidence and prognostic significance of DNMT3A mutations in a large cohort in Chinese childhood AML. We detected the mutations in DNMT3A exon 23 by polymerase chain reaction and direct sequencing in 342 children with AML (0-16 years old) from January 2005 to June 2013, treated on BCH-2003 AML protocol. The correlation of DNMT3A mutations with clinical characteristics, fusion genes, other molecular anomalies (FLT3 internal tandem duplication [FLT3-ITD], Nucleophosmin 1, C-KIT (KIT proto-oncogene receptor tyrosine kinase), and Wilms tumor 1 mutations), and treatment outcome were analyzed. DNMT3A mutations were detected in 4 out of 342 (1.2%) patients. Two patients were PML-RARA positive and 1 patient was FLT3-ITD positive. The mutations in coding sequences included S892S, V912A, R885G, and Q886R. Furthermore, there was 1 intronic mutation (c.2739+55A>C) found in 1 patient. No association of DNMT3A mutations with common clinical features was found. Two patients with DNMT3A mutations died of relapse or complications during treatment. One patient gave up treatment due to remission induction failure in day 33. Only 1 patient achieved continuous complete remission. DNMT3A mutations were rare in Chinese children with AML including PML-RARA positive APL. The mutation positions were different from the hotspots reported in adult AML. DNMT3A mutations may have adverse impact on prognosis of children with AML.

Activating PIK3CA mutations coexist with BRAF or NRAS mutations in a limited fraction of melanomas.

PubMed

Manca, Antonella; Lissia, Amelia; Capone, Mariaelena; Ascierto, Paolo A; Botti, Gerardo; Caracò, Corrado; Stanganelli, Ignazio; Colombino, Maria; Sini, MariaCristina; Cossu, Antonio; Palmieri, Giuseppe

2015-01-28

Activated PI3K-AKT pathway may contribute to decrease sensitivity to inhibitors of key pathogenetic effectors (mutated BRAF, active NRAS or MEK) in melanoma. Functional alterations are deeply involved in PI3K-AKT activation, with a minimal role reported for mutations in PIK3CA, the catalytic subunit of the PI3K gene. We here assessed the prevalence of the coexistence of BRAF/NRAS and PIK3CA mutations in a series of melanoma samples. A total of 245 tumor specimens (212 primary melanomas and 33 melanoma cell lines) was screened for mutations in BRAF, NRAS, and PIK3CA genes by automated direct sequencing. Overall, 110 (44.9%) samples carried mutations in BRAF, 26 (10.6%) in NRAS, and 24 (9.8%) in PIK3CA. All identified PIK3CA mutations have been reported to induce PI3K activation; those detected in cultured melanomas were investigated for their interference with the antiproliferative activity of the BRAF-mutant inhibitor vemurafenib. A reduced suppression in cell growth was observed in treated cells carrying both BRAF and PIK3CA mutations as compared with those presenting a mutated BRAF only. Among the analysed melanomas, 12/245 (4.9%) samples presented the coexistence of PIK3CA and BRAF/NRAS mutations. Our study further suggests that PIK3CA mutations account for a small fraction of PI3K pathway activation and have a limited impact in interfering with the BRAF/NRAS-driven growth in melanoma.

ESR1 Mutations Affect Anti-proliferative Responses to Tamoxifen through Enhanced Cross-Talk with IGF Signaling

PubMed Central

Gelsomino, Luca; Gu, Guowei; Rechoum, Yassine; Beyer, Amanda R; Pejerrey, Sasha M; Tsimelzon, Anna; Wang, Tao; Huffman, Kenneth; Ludlow, Andrew; Ando’, Sebastiano; Fuqua, Suzanne AW

2017-01-01

It is now generally accepted that estrogen receptor (ESR1) mutations occur frequently in metastatic breast cancers, however we do not yet know how to best treat these patients. We have modeled the three most frequent hormone binding ESR1 (HBD-ESR1) mutations (Y537N, Y537S, and D538G) using stable lentiviral transduction in human breast cancer cell lines. Effects on growth were examined in response to hormonal and targeted agents, and mutation-specific changes were studied using microarray and western blot analysis. We determined that the HBD-ESR1 mutations alter anti-proliferative effects to tamoxifen (Tam), due to cell-intrinsic changes in activation of the insulin-like growth factor receptor (IGF1R) signaling pathway and levels of PIK3R1/PIK3R3. The selective estrogen receptor degrader, fulvestrant, significantly reduced the anchorage-independent growth of ESR1 mutant-expressing cells, while combination treatments with the mTOR inhibitor everolimus, or an inhibitor blocking IGF1R and the insulin receptor significantly enhanced anti-proliferative responses. Using digital drop (dd) PCR we identified mutations at high frequencies ranging from 12% for Y537N, 5% for Y537S, and 2% for D538G in archived primary breast tumors from women treated with adjuvant mono-tamoxifen therapy. The HBD-ESR1 mutations were not associated with recurrence-free or overall survival in response in this patient cohort, and suggest that knowledge of other cell-intrinsic factors in combination with ESR1 mutation status will be needed determine anti-proliferative responses to Tam. PMID:27178332

Novel mutations in GALNT3 causing hyperphosphatemic familial tumoral calcinosis.

PubMed

Yancovitch, Alan; Hershkovitz, Dov; Indelman, Margareta; Galloway, Peter; Whiteford, Margo; Sprecher, Eli; Kılıç, Esra

2011-09-01

Hyperphosphatemic familial tumoral calcinosis (HFTC) is known to be caused by mutations in at least three genes: FGF23, GALNT3 and KL. Two families with two affected members suffering from HFTC were scrutinized for mutations in these candidate genes. We identified in both families homozygous missense mutations affecting highly conserved amino acids in GALNT3. One of the mutations is a novel mutation, whereas the second mutation was reported before in a compound heterozygous state. Our data expand the spectrum of known mutations in GALNT3 and contribute to a better understanding of the phenotypic manifestations of mutations in this gene.

Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor suppressor genes

PubMed Central

Chen, Kaifu; Chen, Zhong; Wu, Dayong; Zhang, Lili; Lin, Xueqiu; Su, Jianzhong; Rodriguez, Benjamin; Xi, Yuanxin; Xia, Zheng; Chen, Xi; Shi, Xiaobing; Wang, Qianben; Li, Wei

2016-01-01

Tumor suppressors are mostly defined by inactivating mutations in tumors, yet little is known about their epigenetic features in normal cells. Through integrative analysis of 1,134 genome-wide epigenetic profiles, mutations from >8,200 tumor-normal pairs, and our experimental data from clinical samples, we discovered broad H3K4me3 (wider than 4 kb) as the first epigenetic signature for tumor suppressors in normal cells. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity together leading to exceptionally high gene expression, and is distinct from other broad epigenetic features, such as super-enhancers. Broad H3K4me3 conserved across normal cells may represent pan-cancer tumor suppressors, such as P53 and PTEN, whereas cell-type-specific broad H3K4me3 may indicate cell-identity genes and cell-type-specific tumor suppressors. Furthermore, widespread shortening of broad H3K4me3 in cancers is associated with repression of tumor suppressors. Together, the broad H3K4me3 epigenetic signature provides mutation-independent information for the discovery and characterization of novel tumor suppressors. PMID:26301496

RSPO3 antagonism inhibits growth and tumorigenicity in colorectal tumors harboring common Wnt pathway mutations.

PubMed

Fischer, Marcus M; Yeung, V Pete; Cattaruzza, Fiore; Hussein, Rajaa; Yen, Wan-Ching; Murriel, Christopher; Evans, James W; O'Young, Gilbert; Brunner, Alayne L; Wang, Min; Cain, Jennifer; Cancilla, Belinda; Kapoun, Ann; Hoey, Timothy

2017-11-10

Activating mutations in the Wnt pathway are a characteristic feature of colorectal cancer (CRC). The R-spondin (RSPO) family is a group of secreted proteins that enhance Wnt signaling and RSPO2 and RSPO3 gene fusions have been reported in CRC. We have previously shown that Wnt pathway blockers exhibit potent combinatorial activity with taxanes to inhibit tumor growth. Here we show that RSPO3 antagonism synergizes with paclitaxel based chemotherapies in patient-derived xenograft models (PDX) with RSPO3 fusions and in tumors with common CRC mutations such as APC, β-catenin, or RNF43. In these latter types of tumors that represent over 90% of CRC, RSPO3 is produced by stromal cells in the tumor microenvironment and the activating mutations appear to sensitize the tumors to Wnt-Rspo synergy. The combination of RSPO3 inhibition and taxane treatment provides an approach to effectively target oncogenic WNT signaling in a significant number of patients with colorectal and other intestinal cancers.

Influence of a Small Fraction of Individuals with Enhanced Mutations on a Population Genetic Pool

NASA Astrophysics Data System (ADS)

Cebrat, S.; Stauffer, D.

It has been observed that a higher mutation load could be introduced into the genomes of children conceived by assisted reproduction technology (fertilization in-vitro). This generates two effects — slightly higher mutational pressure on the whole genetic pool of population and inhomogeneity of mutation distributions in the genetic pool. Computer simulations of the Penna ageing model suggest that already a small fraction of births with enhanced number of new mutations can negatively influence the whole population.

Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin

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

Lee, Yoon Jae; Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul; Vaccine Translational Research Center, Yonsei University, Seoul

In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single aminomore » acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response. - Highlights: • Cold-adaptation process induced four amino acid mutations in the HA of X-31 virus. • The four mutations in the HA also contributed to attenuation of the X-31ca virus • N81K mutation was the most significant marker for the attenuation of X-31ca virus. • Introduction of N81K mutation into H3N2 LAIV further attenuated the vaccine. • This approach provides a useful guideline for enhancing the safety of the LAIVs.« less

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations.

PubMed

Parasuraman, Ponnusamy; Murugan, Veeramani; Selvin, Jeyasigamani F A; Gromiha, M Michael; Fukui, Kazuhiko; Veluraja, Kasinadar

2014-08-01

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.

Loss of Calpain 3 Proteolytic Activity Leads to Muscular Dystrophy and to Apoptosis-Associated Iκbα/Nuclear Factor κb Pathway Perturbation in Mice

PubMed Central

Richard, Isabelle; Roudaut, Carinne; Marchand, Sylvie; Baghdiguian, Stephen; Herasse, Muriel; Stockholm, Daniel; Ono, Yasuko; Suel, Laurence; Bourg, Nathalie; Sorimachi, Hiroyuki; Lefranc, Gérard; Fardeau, Michel; Sébille, Alain; Beckmann, Jacques S.

2000-01-01

Calpain 3 is known as the skeletal muscle–specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IκBα/nuclear factor κB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations. PMID:11134085

Pancreatic Agenesis due to Compound Heterozygosity for a Novel Enhancer and Truncating Mutation in the PTF1A Gene.

PubMed

Gabbay, Monica; Ellard, Sian; De Franco, Elisa; Moisés, Regina S

2017-09-01

Neonatal diabetes, defined as the onset of diabetes within the first six months of life, is very rarely caused by pancreatic agenesis. Homozygous truncating mutations in the PTF1A gene, which encodes a transcriptional factor, have been reported in patients with pancreatic and cerebellar agenesis, whilst mutations located in a distal pancreatic-specific enhancer cause isolated pancreatic agenesis. We report an infant, born to healthy non-consanguineous parents, with neonatal diabetes due to pancreatic agenesis. Initial genetic investigation included sequencing of KCNJ11, ABCC8 and INS genes, but no mutations were found. Following this, 22 neonatal diabetes associated genes were analyzed by a next generation sequencing assay. We found compound heterozygous mutations in the PTF1A gene: A frameshift mutation in exon 1 (c.437_462 del, p.Ala146Glyfs*116) and a mutation affecting a highly conserved nucleotide within the distal pancreatic enhancer (g.23508442A>G). Both mutations were confirmed by Sanger sequencing. Isolated pancreatic agenesis resulting from compound heterozygosity for truncating and enhancer mutations in the PTF1A gene has not been previously reported. This report broadens the spectrum of mutations causing pancreatic agenesis.

Pancreatic Agenesis due to Compound Heterozygosity for a Novel Enhancer and Truncating Mutation in the PTF1A Gene

PubMed Central

Gabbay, Monica; Ellard, Sian; De Franco, Elisa; Moisés, Regina S.

2017-01-01

Neonatal diabetes, defined as the onset of diabetes within the first six months of life, is very rarely caused by pancreatic agenesis. Homozygous truncating mutations in the PTF1A gene, which encodes a transcriptional factor, have been reported in patients with pancreatic and cerebellar agenesis, whilst mutations located in a distal pancreatic-specific enhancer cause isolated pancreatic agenesis. We report an infant, born to healthy non-consanguineous parents, with neonatal diabetes due to pancreatic agenesis. Initial genetic investigation included sequencing of KCNJ11, ABCC8 and INS genes, but no mutations were found. Following this, 22 neonatal diabetes associated genes were analyzed by a next generation sequencing assay. We found compound heterozygous mutations in the PTF1A gene: A frameshift mutation in exon 1 (c.437_462 del, p.Ala146Glyfs*116) and a mutation affecting a highly conserved nucleotide within the distal pancreatic enhancer (g.23508442A>G). Both mutations were confirmed by Sanger sequencing. Isolated pancreatic agenesis resulting from compound heterozygosity for truncating and enhancer mutations in the PTF1A gene has not been previously reported. This report broadens the spectrum of mutations causing pancreatic agenesis. PMID:28663161

Frequent PIK3CA Mutations in Colorectal and Endometrial Cancer with Double Somatic Mismatch Repair Mutations

PubMed Central

Cohen, Stacey A.; Turner, Emily H.; Beightol, Mallory B.; Jacobson, Angela; Gooley, Ted A.; Salipante, Stephen J.; Haraldsdottir, Sigurdis; Smith, Christina; Scroggins, Sheena; Tait, Jonathan F.; Grady, William M.; Lin, Edward H.; Cohn, David E.; Goodfellow, Paul J.; Arnold, Mark W.; de la Chapelle, Albert; Pearlman, Rachel; Hampel, Heather; Pritchard, Colin C.

2016-01-01

Background & Aims Double somatic mutations in mismatch repair (MMR) genes have recently been described in colorectal and endometrial cancers with microsatellite instability (MSI) not attributable to MLH1 hypermethylation or germline mutation. We sought to define the molecular phenotype of this newly recognized tumor subtype. Methods From two prospective Lynch syndrome screening studies, we identified patients with colorectal and endometrial tumors harboring ≥2 somatic MMR mutations, but normal germline MMR testing (“double somatic”). We determined the frequencies of tumor PIK3CA, BRAF, KRAS, NRAS, and PTEN mutations by targeted next-generation sequencing and used logistic-regression models to compare them to: Lynch syndrome, MLH1 hypermethylated, and microsatellite stable (MSS) tumors. We validated our findings using independent datasets from The Cancer Genome Atlas (TCGA). Results Among colorectal cancer cases, we found that 14/21 (67%) of double somatic cases had PIK3CA mutations vs. 4/18 (22%) Lynch syndrome, 2/10 (20%) MLH1 hypermethylated, and 12/78 (15%) MSS tumors; p<0.0001. PIK3CA mutations were detected in 100% of 13 double somatic endometrial cancers (p=0.04). BRAF mutations were absent in double somatic and Lynch syndrome colorectal tumors. We found highly similar results in a validation cohort from TCGA (113 colorectal, 178 endometrial cancer), with 100% of double somatic cases harboring a PIK3CA mutation (p<0.0001). Conclusions PIK3CA mutations are present in double somatic mutated colorectal and endometrial cancers at substantially higher frequencies than other MSI subgroups. PIK3CA mutation status may better define an emerging molecular entity in colorectal and endometrial cancers, with the potential to inform screening and therapeutic decision making. PMID:27302833

Mutations of NOTCH3 in childhood pulmonary arterial hypertension

PubMed Central

Chida, Ayako; Shintani, Masaki; Matsushita, Yoshihisa; Sato, Hiroki; Eitoku, Takahiro; Nakayama, Tomotaka; Furutani, Yoshiyuki; Hayama, Emiko; Kawamura, Yoichi; Inai, Kei; Ohtsuki, Shinichi; Saji, Tsutomu; Nonoyama, Shigeaki; Nakanishi, Toshio

2014-01-01

Mutations of BMPR2 and other TGF-β superfamily genes have been reported in pulmonary arterial hypertension (PAH). However, 60–90% of idiopathic PAH cases have no mutations in these genes. Recently, the expression of NOTCH3 was shown to be increased in the pulmonary artery smooth muscle cells of PAH patients. We sought to investigate NOTCH3 and its target genes in PAH patients and clarify the role of NOTCH3 signaling. We screened for mutations in NOTCH3, HES1, and HES5 in 41 PAH patients who had no mutations in BMPR2, ALK1, endoglin, SMAD1/4/8, BMPR1B, or Caveolin-1. Two novel missense mutations (c.2519 G>A p.G840E, c.2698 A>C p.T900P) in NOTCH3 were identified in two PAH patients. We performed functional analysis using stable cell lines expressing either wild-type or mutant NOTCH3. The protein-folding chaperone GRP78/BiP was colocalized with wild-type NOTCH3 in the endoplasmic reticulum, whereas the majority of GRP78/BiP was translocated into the nuclei of cells expressing mutant NOTCH3. Cell proliferation and viability were higher for cells expressing mutant NOTCH3 than for those expressing wild-type NOTCH3. We identified novel NOTCH3 mutations in PAH patients and revealed that these mutations were involved in cell proliferation and viability. NOTCH3 mutants induced an impairment in NOTCH3-HES5 signaling. The results may contribute to the elucidation of PAH pathogenesis. PMID:24936512

Gamow-Teller Strength Distributions in {sup 48}Sc by the {sup 48}Ca(p,n) and {sup 48}Ti(n,p) Reactions and Two-Neutrino Double-beta Decay Nuclear Matrix Elements

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

Yako, K.; Sasano, M.; Miki, K.

2009-07-03

The double-differential cross sections for the {sup 48}Ca(p,n) and {sup 48}Ti(n,p) reactions were measured at 300 MeV. A multipole decomposition technique was applied to the spectra to extract the Gamow-Teller (GT) components. The integrated GT strengths up to an excitation energy of 30 MeV in {sup 48}Sc are 15.3+-2.2 and 2.8+-0.3 in the (p,n) and (n,p) spectra, respectively. In the (n,p) spectra additional GT strengths were found above 8 MeV where shell models within the fp shell-model space predict almost no GT strengths, suggesting that the present shell-model description of the nuclear matrix element of the two-neutrino double-beta decay ismore » incomplete.« less

Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor-suppressor genes.

PubMed

Chen, Kaifu; Chen, Zhong; Wu, Dayong; Zhang, Lili; Lin, Xueqiu; Su, Jianzhong; Rodriguez, Benjamin; Xi, Yuanxin; Xia, Zheng; Chen, Xi; Shi, Xiaobing; Wang, Qianben; Li, Wei

2015-10-01

Tumor suppressors are mostly defined by inactivating mutations in tumors, yet little is known about their epigenetic features in normal cells. Through integrative analysis of 1,134 genome-wide epigenetic profiles, mutations from >8,200 tumor-normal pairs and our experimental data from clinical samples, we discovered broad peaks for trimethylation of histone H3 at lysine 4 (H3K4me3; wider than 4 kb) as the first epigenetic signature for tumor suppressors in normal cells. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity, which together lead to exceptionally high gene expression, and is distinct from other broad epigenetic features, such as super-enhancers. Genes with broad H3K4me3 peaks conserved across normal cells may represent pan-cancer tumor suppressors, such as TP53 and PTEN, whereas genes with cell type-specific broad H3K4me3 peaks may represent cell identity genes and cell type-specific tumor suppressors. Furthermore, widespread shortening of broad H3K4me3 peaks in cancers is associated with repression of tumor suppressors. Thus, the broad H3K4me3 epigenetic signature provides mutation-independent information for the discovery and characterization of new tumor suppressors.

ESR1 mutations affect anti-proliferative responses to tamoxifen through enhanced cross-talk with IGF signaling.

PubMed

Gelsomino, Luca; Gu, Guowei; Rechoum, Yassine; Beyer, Amanda R; Pejerrey, Sasha M; Tsimelzon, Anna; Wang, Tao; Huffman, Kenneth; Ludlow, Andrew; Andò, Sebastiano; Fuqua, Suzanne A W

2016-06-01

The purpose of this study was to address the role of ESR1 hormone-binding mutations in breast cancer. Soft agar anchorage-independent growth assay, Western blot, ERE reporter transactivation assay, proximity ligation assay (PLA), coimmunoprecipitation assay, silencing assay, digital droplet PCR (ddPCR), Kaplan-Meier analysis, and statistical analysis. It is now generally accepted that estrogen receptor (ESR1) mutations occur frequently in metastatic breast cancers; however, we do not yet know how to best treat these patients. We have modeled the three most frequent hormone-binding ESR1 (HBD-ESR1) mutations (Y537N, Y537S, and D538G) using stable lentiviral transduction in human breast cancer cell lines. Effects on growth were examined in response to hormonal and targeted agents, and mutation-specific changes were studied using microarray and Western blot analysis. We determined that the HBD-ESR1 mutations alter anti-proliferative effects to tamoxifen (Tam), due to cell-intrinsic changes in activation of the insulin-like growth factor receptor (IGF1R) signaling pathway and levels of PIK3R1/PIK3R3. The selective estrogen receptor degrader, fulvestrant, significantly reduced the anchorage-independent growth of ESR1 mutant-expressing cells, while combination treatments with the mTOR inhibitor everolimus, or an inhibitor blocking IGF1R, and the insulin receptor significantly enhanced anti-proliferative responses. Using digital drop (dd) PCR, we identified mutations at high frequencies ranging from 12 % for Y537N, 5 % for Y537S, and 2 % for D538G in archived primary breast tumors from women treated with adjuvant mono-tamoxifen therapy. The HBD-ESR1 mutations were not associated with recurrence-free or overall survival in response in this patient cohort and suggest that knowledge of other cell-intrinsic factors in combination with ESR1 mutation status will be needed determine anti-proliferative responses to Tam.

Disease-associated mutations in CNGB3 promote cytotoxicity in photoreceptor-derived cells

PubMed Central

Liu, Chunming; Sherpa, Tshering

2013-01-01

Purpose To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells. Methods Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3′,5′-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3′,5′-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes. Results Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium. Conclusions These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels. PMID:23805033

Impact of polymorphism of the regulatory subunit of the μ-calpain (CAPN1S) on the proteolysis process and meat tenderness of young cattle

PubMed Central

Iwanowska, Agnieszka; Grześ, Bożena; Mikołajczak, Beata; Iwańska, Ewa; Juszczuk-Kubiak, Edyta; Rosochacki, Stanisław J.

2010-01-01

The objective of this study was to estimate the impact of the polymorphism of μ-calpain (CAPN1S) gene on protein changes of the cattle muscle tissue and its tenderness during 10-day cold storage. The analysis was performed on the longest dorsal and lumbar muscles collected from 76 bulls 6 to 12 months of age. Polymorphism identification of the above-mentioned gene was conducted using the PCR-RFLP technique. Its effect on the course of the proteolysis process was assessed by monitoring changes in proportions of tissue proteins during 10-day process of meat ageing. Special attention was focused on changes in native titin (T1) share and products of its degradation (proteins of molecular weight (m.w.) of 2400 and 200 kDa), α-actinin and protein of 37 kDa as well as myosin heavy chains (MHC). In the case of the last proteins, their polymorphism was evaluated as well. Meat tenderness was estimated measuring the value of shear force and sensorially. The highest tenderness was ascertained for the heterozygote. Its improvement was associated with a significant decrease in proportions of proteins of molecular weight of approximately 37 kDa accompanied by an increase of those with 200 kDa molecular weight. Muscles derived from cattle of CT genotype were characterised by the highest proportions of type 2a MHC isoform. Value differences between proportions determined for the heterozygote and CC and TT homozygotes of the CAPN1S gene were statistically significant. Therefore, it can be presumed that the process of meat tenderisation was especially connected with MHC polymorphism. PMID:20563650

A Novel Founder Mutation in MYBPC3: Phenotypic Comparison With the Most Prevalent MYBPC3 Mutation in Spain.

PubMed

Sabater-Molina, María; Saura, Daniel; García-Molina Sáez, Esperanza; González-Carrillo, Josefa; Polo, Luis; Pérez-Sánchez, Inmaculada; Olmo, María Del Carmen; Oliva-Sandoval, María José; Barriales-Villa, Roberto; Carbonell, Pablo; Pascual-Figal, Domigo; Gimeno, Juan R

2017-02-01

Mutations in MYBPC3 are the cause of hypertrophic cardiomyopathy (HCM). Although most lead to a truncating protein, the severity of the phenotype differs. We describe the clinical phenotype of a novel MYBPC3 mutation, p.Pro108Alafs*9, present in 13 families from southern Spain and compare it with the most prevalent MYBPC3 mutation in this region (c.2308+1 G>A). We studied 107 relatives of 13 index cases diagnosed as HCM carriers of the p.Pro108Alafs*9 mutation. Pedigree analysis, clinical evaluation, and genotyping were performed. A total of 54 carriers of p.Pro108Alafs*9 were identified, of whom 39 had HCM. There were 5 cases of sudden death in the 13 families. Disease penetrance was greater as age increased and HCM patients were more frequently male and developed disease earlier than female patients. The phenotype was similar in p.Pro108Alafs*9 and in c.2308+1 G>A, but differences were found in several risk factors and in survival. There was a trend toward a higher left ventricular mass in p.Pro108Alafs*9 vs c.2308+1G>A. Cardiac magnetic resonance revealed a similar extent and pattern of fibrosis. The p.Pro108Alafs*9 mutation is associated with HCM, high penetrance, and disease onset in middle age. Copyright © 2016 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

The tyrosine kinase receptor Tyro3 enhances lifespan and neuropeptide Y (Npy) neuron survival in the mouse anorexia (anx) mutation.

PubMed

Kim, Dennis Y; Yu, Joanna; Mui, Ryan K; Niibori, Rieko; Taufique, Hamza Bin; Aslam, Rukhsana; Semple, John W; Cordes, Sabine P

2017-05-01

Severe appetite and weight loss define the eating disorder anorexia nervosa, and can also accompany the progression of some neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Although acute loss of hypothalamic neurons that produce appetite-stimulating neuropeptide Y (Npy) and agouti-related peptide (Agrp) in adult mice or in mice homozygous for the anorexia ( anx ) mutation causes aphagia, our understanding of the factors that help maintain appetite regulatory circuitry is limited. Here we identify a mutation (C19T) that converts an arginine to a tryptophan (R7W) in the TYRO3 protein tyrosine kinase 3 ( Tyro3 ) gene, which resides within the anx critical interval, as contributing to the severity of anx phenotypes. Our observation that, like Tyro3 -/- mice, anx/anx mice exhibit abnormal secondary platelet aggregation suggested that the C19T Tyro3 variant might have functional consequences. Tyro3 is expressed in the hypothalamus and other brain regions affected by the anx mutation, and its mRNA localization appeared abnormal in anx/anx brains by postnatal day 19 (P19). The presence of wild-type Tyro3 transgenes, but not an R7W-Tyro3 transgene, doubled the weight and lifespans of anx/anx mice and near-normal numbers of hypothalamic Npy-expressing neurons were present in Tyro3 -transgenic anx/anx mice at P19. Although no differences in R7W-Tyro3 signal sequence function or protein localization were discernible in vitro , distribution of R7W-Tyro3 protein differed from that of Tyro3 protein in the cerebellum of transgenic wild-type mice. Thus, R7W-Tyro3 protein localization deficits are only detectable in vivo Further analyses revealed that the C19T Tyro3 mutation is present in a few other mouse strains, and hence is not the causative anx mutation, but rather an anx modifier. Our work shows that Tyro3 has prosurvival roles in the appetite regulatory circuitry and could also provide useful insights towards the development of interventions targeting

Novel methods to enhance single strand conformation polymorphism (SSCP) senstivity and efficiency: Application to mutation detection in cystic fibrosis (CF)

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

Hagstrom, D.J.; Snow, K.; Yuan, Z.

1994-09-01

For single gene defects in which there are a variety of mutations with significant frequencies, it is a challenge to find an efficient and sensitive method for mutation detection. For example, although 70% to 75% of CF chromosomes in a North American Caucasian population have the mutation {delta}F508, more than 400 mutations (mostly single base pair substitutions) are represented on the remaining chromosomes. SSCP analysis is a relatively straightforward procedure and therefore suitable for routine use in a clinical laboratory. However, previous reports have demonstrated suboptimal sensitivity rates in screening for mutations. We have developed a novel set of conditionsmore » which greatly enhances sensitivity and efficiency of SSCP. Our protocol incorporates multiplex PCR, stepping of wattages during electrophoresis and increased salt concentration at the anode relative to the gel. To screen for mutations in the CFTR gene, three multiplex PCR reactions are performed using identical thermocycler parameters. Sizes of PCR products range from 441 bp to 196 bp: size differences of > 30 bp are necessary to ensure separation during electrophoresis. All PCR products are separated by electrophoresis at room temperature on a single gel containing 8% (37.5:1) polyacrylamide, 5% glycerol and 1x TBE. Using an anode buffer with increased salt (2x TBE) sharpens smaller sized bands, and stepping watts from 5W to 20W during electrophoresis enhances sensitivity. Positive controls were used to demonstrate that mutations could be detected. Other mutations or polymorphisms were verified by cycle sequencing of PCR products or by alternative PCR-based assays for the more common mutations. Thus, using 3 PCR reactions per patient and one gel condition, we are able to achieve a CF mutation detection rate of approximately 90% in a North American Caucasian population.« less

Mutations in eukaryotic release factors 1 and 3 act as general nonsense suppressors in Drosophila.

PubMed Central

Chao, Anna T; Dierick, Herman A; Addy, Tracie M; Bejsovec, Amy

2003-01-01

In a screen for suppressors of the Drosophila wingless(PE4) nonsense allele, we isolated mutations in the two components that form eukaryotic release factor. eRF1 and eRF3 comprise the translation termination complex that recognizes stop codons and catalyzes the release of nascent polypeptide chains from ribosomes. Mutations disrupting the Drosophila eRF1 and eRF3 show a strong maternal-effect nonsense suppression due to readthrough of stop codons and are zygotically lethal during larval stages. We tested nonsense mutations in wg and in other embryonically acting genes and found that different stop codons can be suppressed but only a subset of nonsense alleles are subject to suppression. We suspect that the context of the stop codon is significant: nonsense alleles sensitive to suppression by eRF1 and eRF3 encode stop codons that are immediately followed by a cytidine. Such suppressible alleles appear to be intrinsically weak, with a low level of readthrough that is enhanced when translation termination is disrupted. Thus the eRF1 and eRF3 mutations provide a tool for identifying nonsense alleles that are leaky. Our findings have important implications for assigning null mutant phenotypes and for selecting appropriate alleles to use in suppressor screens. PMID:14573473

Clinical effects of phosphodiesterase 3A mutations in inherited hypertension with brachydactyly.

PubMed

Toka, Okan; Tank, Jens; Schächterle, Carolin; Aydin, Atakan; Maass, Philipp G; Elitok, Saban; Bartels-Klein, Eireen; Hollfinger, Irene; Lindschau, Carsten; Mai, Knut; Boschmann, Michael; Rahn, Gabriele; Movsesian, Matthew A; Müller, Thomas; Doescher, Andrea; Gnoth, Simone; Mühl, Astrid; Toka, Hakan R; Wefeld-Neuenfeld, Yvette; Utz, Wolfgang; Töpper, Agnieszka; Jordan, Jens; Schulz-Menger, Jeanette; Klussmann, Enno; Bähring, Sylvia; Luft, Friedrich C

2015-10-01

Autosomal-dominant hypertension with brachydactyly is a salt-independent Mendelian syndrome caused by activating mutations in the gene encoding phosphodiesterase 3A. These mutations increase the protein kinase A-mediated phosphorylation of phosphodiesterase 3A resulting in enhanced cAMP-hydrolytic affinity and accelerated cell proliferation. The phosphorylated vasodilator-stimulated phosphoprotein is diminished, and parathyroid hormone-related peptide is dysregulated, potentially accounting for all phenotypic features. Untreated patients die prematurely of stroke; however, hypertension-induced target-organ damage is otherwise hardly apparent. We conducted clinical studies of vascular function, cardiac functional imaging, platelet function in affected and nonaffected persons, and cell-based assays. Large-vessel and cardiac functions indeed seem to be preserved. The platelet studies showed normal platelet function. Cell-based studies demonstrated that available phosphodiesterase 3A inhibitors suppress the mutant isoforms. However, increasing cGMP to indirectly inhibit the enzyme seemed to have particular use. Our results shed more light on phosphodiesterase 3A activation and could be relevant to the treatment of severe hypertension in the general population. © 2015 American Heart Association, Inc.

INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia.

PubMed

Jin, Hongjun; Yang, Liyuan; Wang, Lu; Yang, Zailin; Zhan, Qian; Tao, Yao; Zou, Qin; Tang, Yuting; Xian, Jingrong; Zhang, Shuaishuai; Jing, Yipei; Zhang, Ling

2018-01-17

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been recognized as a distinct leukemia entity in the 2016 World Health Organization (WHO) classification. The genetic events underlying oncogenesis in NPM1-mutated AML that is characterized by a normal karyotype remain unclear. Inositol polyphosphate 4-phosphatase type II (INPP4B), a new factor in the phosphoinositide-3 kinase (PI3K) pathway-associated cancers, has been recently found a clinically relevant role in AML. However, little is known about the specific mechanistic function of INPP4B in NPM1-mutated AML. The INPP4B expression levels in NPM1-mutated AML primary blasts and AML OCI-AML3 cell lines were determined by qRT-PCR and western blotting. The effect of INPP4B knockdown on OCI-AML3 leukemia cell proliferation was evaluated, using the Cell Counting Kit-8 and colony formation assay. After INPP4B overexpression or knockdown, the activation of serum and glucocorticoid-regulated kinase 3 (SGK3) and AKT was assessed. The effects of PI3K signaling pathway inhibitors on the levels of p-SGK3 in OCI-AML3 cells were tested. The mass of PI (3,4) P 2 and PI (3) P was analyzed by ELISA upon INPP4B overexpression. Knockdown of SGK3 by RNA interference and a rescue assay were performed to confirm the critical role of SGK3 in INPP4B-mediated cell survival. In addition, the molecular mechanism underlying INPP4B expression in NPM1-mutated leukemia cells was explored. Finally, Kaplan-Meier survival analysis was conducted on the NPM1-mutated AML cohort stratified into quartiles for INPP4B expression in The Cancer Genome Atlas (TCGA) dataset. High expression of INPP4B was observed in NPM1-mutated AML. Knockdown of INPP4B repressed cell proliferation in OCI-AML3 cells, whereas recovered INPP4B rescued this inhibitory effect in vitro. Mechanically, INPP4B enhanced phosphorylated SGK3 (p-SGK3) status, but did not affect AKT activation. SGK3 was required for INPP4B-induced cell proliferation in OCI-AML3 cells

Evidence that the oxygen enhancement ratio for pink somatic mutations in Tradescantia stamen hairs may approach unity at very low x-ray doses

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

Underbrink, A.G.; Woch, B.

1980-11-01

Experimental evidence was found that the oxygen enhancement ratio (OER) for pink somatic mutations in the stamen hairs of Tradescantia clone 02 appears to reach unity at X-ray doses of 2 to 3 rad. There is also a small segment on the dose-response curves from about 3 to 10 rad where the OER appears to be dose-dependent. At higher doses the aerated and hypoxic curves are parallel, and the OER is 3.2 up to doses where the mutation frequency reaches a plateau.

Novel CREB3L3 Nonsense Mutation in a Family With Dominant Hypertriglyceridemia.

PubMed

Cefalù, Angelo B; Spina, Rossella; Noto, Davide; Valenti, Vincenza; Ingrassia, Valeria; Giammanco, Antonina; Panno, Maria D; Ganci, Antonina; Barbagallo, Carlo M; Averna, Maurizio R

2015-12-01

Cyclic AMP responsive element-binding protein 3-like 3 (CREB3L3) is a novel candidate gene for dominant hypertriglyceridemia. To date, only 4 kindred with dominant hypertriglyceridemia have been found to be carriers of 2 nonsense mutations in CREB3L3 gene (245fs and W46X). We investigated a family in which hypertriglyceridemia displayed an autosomal dominant pattern of inheritance. The proband was a 49-year-old woman with high plasma triglycerides (≤1300 mg/dL; 14.68 mmol/L). Her father had a history of moderate hypertriglyceridemia, and her 51-year-old brother had triglycerides levels as high as 1600 mg/dL (18.06 mmol/L). To identify the causal mutation in this family, we analyzed the candidate genes of recessive and dominant forms of primary hypertriglyceridemia by direct sequencing. The sequencing of CREB3L3 gene led to the discovery of a novel minute frame shift mutation in exon 3 of CREB3L3 gene, predicted to result in the formation of a truncated protein devoid of function (c.359delG-p.K120fsX20). Heterozygosity for the c.359delG mutation resulted in a severe phenotype occurring later in life in the proband and her brother and a good response to diet and a hypotriglyceridemic treatment. The same mutation was detected in a 13-year-old daughter who to date is normotriglyceridemic. We have identified a novel pathogenic mutation in CREB3L3 gene in a family with dominant hypertriglyceridemia with a variable pattern of penetrance. © 2015 American Heart Association, Inc.

Mutational analysis of PI3K/AKT and RAS/RAF pathway activation in malignant salivary gland tumours with a new mutation of PIK3CA.

PubMed

Shalmon, B; Drendel, M; Wolf, M; Hirshberg, A; Cohen, Y

2016-06-01

The phosphoinositide 3-kinase (PIK3)/v-akt murine thymoma (AKT) oncogene pathway and the RAS/RAF pathway are involved in regulating the signalling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth. Mutations in the genes within these pathways are frequently found in several tumours. The aim of this study was to investigate the frequency of mutations in the PIK3CA, BRAF, and KRAS genes in cases of malignant salivary gland tumours. Mutational analysis of the PIK3CA, KRAS, and BRAF genes was performed by direct sequencing of material from 21 patients with malignant salivary gland tumours who underwent surgery between 1992 and 2001. No mutations were found in the KRAS exon 2, BRAF exon 15, or PIK3CA exon 9 genes. However, an unpublished mutation of the PIK3CA gene in exon 20 (W1051 stop mutation) was found in one case of adenocarcinoma NOS. The impact of this mutation on the biological behaviour of the tumour has yet to be explored, however the patient with adenocarcinoma NOS harbouring this mutation has survived for over 20 years following surgery despite a high stage at presentation. Further studies with more homogeneous patient cohorts are needed to address whether this mutation reflects a different clinical presentation and may benefit from targeted treatment strategies. Copyright © 2015 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Anaerobically Grown Escherichia coli Has an Enhanced Mutation Rate and Distinct Mutational Spectra

PubMed Central

Shewaramani, Sonal; Finn, Thomas J.; Kassen, Rees; Rainey, Paul B.

2017-01-01

Oxidative stress is a major cause of mutation but little is known about how growth in the absence of oxygen impacts the rate and spectrum of mutations. We employed long-term mutation accumulation experiments to directly measure the rates and spectra of spontaneous mutation events in Escherichia coli populations propagated under aerobic and anaerobic conditions. To detect mutations, whole genome sequencing was coupled with methods of analysis sufficient to identify a broad range of mutational classes, including structural variants (SVs) generated by movement of repetitive elements. The anaerobically grown populations displayed a mutation rate nearly twice that of the aerobic populations, showed distinct asymmetric mutational strand biases, and greater insertion element activity. Consistent with mutation rate and spectra observations, genes for transposition and recombination repair associated with SVs were up-regulated during anaerobic growth. Together, these results define differences in mutational spectra affecting the evolution of facultative anaerobes. PMID:28103245

Mutation analysis of the Smad3 gene in human osteoarthritis.

PubMed

Yao, Jun-Yan; Wang, Yan; An, Jing; Mao, Chun-Ming; Hou, Ning; Lv, Ya-Xin; Wang, You-Liang; Cui, Fang; Huang, Min; Yang, Xiao

2003-09-01

Osteoarthritis (OA) is the most common joint disease worldwide. Recent studies have shown that targeted disruption of Smad3 in mouse results in OA. To reveal the possible association between the Smad3 gene mutation and human OA, we employed polymerase chain reaction-single strand conformation polymorphism and sequencing to screen mutations in all nine exons of the Smad3 gene in 32 patients with knee OA and 50 patients with only bone fracture. A missense mutation of the Smad3 gene was found in one patient. The single base mutation located in the linker region of the SMAD3 protein was A --> T change in the position 2 of codon 197 and resulted in an asparagine to isoleucine amino-acid substitution. The expressions of matrix metalloproteinase 2 (MMP-2) and MMP-9 in sera of the patient carrying the mutation were higher than other OA patients and controls. This is the first report showing that the Smad3 gene mutations could be associated with the pathogenesis of human OA.

Genetic enhancement of cognition in a kindred with cone–rod dystrophy due to RIMS1 mutation

PubMed Central

Sisodiya, Sanjay M; Thompson, Pamela J; Need, Anna; Harris, Sarah E; Weale, Michael E; Wilkie, Susan E; Michaelides, Michel; Free, Samantha L; Walley, Nicole; Gumbs, Curtis; Gerrelli, Dianne; Ruddle, Piers; Whalley, Lawrence J; Starr, John M; Hunt, David M; Goldstein, David B; Deary, Ian J; Moore, Anthony T

2007-01-01

Background The genetic basis of variation in human cognitive abilities is poorly understood. RIMS1 encodes a synapse active‐zone protein with important roles in the maintenance of normal synaptic function: mice lacking this protein have greatly reduced learning ability and memory function. Objective An established paradigm examining the structural and functional effects of mutations in genes expressed in the eye and the brain was used to study a kindred with an inherited retinal dystrophy due to RIMS1 mutation. Materials and methods Neuropsychological tests and high‐resolution MRI brain scanning were undertaken in the kindred. In a population cohort, neuropsychological scores were associated with common variation in RIMS1. Additionally, RIMS1 was sequenced in top‐scoring individuals. Evolution of RIMS1 was assessed, and its expression in developing human brain was studied. Results Affected individuals showed significantly enhanced cognitive abilities across a range of domains. Analysis suggests that factors other than RIMS1 mutation were unlikely to explain enhanced cognition. No association with common variation and verbal IQ was found in the population cohort, and no other mutations in RIMS1 were detected in the highest scoring individuals from this cohort. RIMS1 protein is expressed in developing human brain, but RIMS1 does not seem to have been subjected to accelerated evolution in man. Conclusions A possible role for RIMS1 in the enhancement of cognitive function at least in this kindred is suggested. Although further work is clearly required to explore these findings before a role for RIMS1 in human cognition can be formally accepted, the findings suggest that genetic mutation may enhance human cognition in some cases. PMID:17237123

A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

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

Shan, Chun-Min; Wang, Jiyong; Xu, Ke

2016-09-20

Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of largemore » heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.« less

New insights into genotype–phenotype correlation for GLI3 mutations

PubMed Central

Démurger, Florence; Ichkou, Amale; Mougou-Zerelli, Soumaya; Le Merrer, Martine; Goudefroye, Géraldine; Delezoide, Anne-Lise; Quélin, Chloé; Manouvrier, Sylvie; Baujat, Geneviève; Fradin, Mélanie; Pasquier, Laurent; Megarbané, André; Faivre, Laurence; Baumann, Clarisse; Nampoothiri, Sheela; Roume, Joëlle; Isidor, Bertrand; Lacombe, Didier; Delrue, Marie-Ange; Mercier, Sandra; Philip, Nicole; Schaefer, Elise; Holder, Muriel; Krause, Amanda; Laffargue, Fanny; Sinico, Martine; Amram, Daniel; André, Gwenaelle; Liquier, Alain; Rossi, Massimiliano; Amiel, Jeanne; Giuliano, Fabienne; Boute, Odile; Dieux-Coeslier, Anne; Jacquemont, Marie-Line; Afenjar, Alexandra; Van Maldergem, Lionel; Lackmy-Port-Lis, Marylin; Vincent- Delorme, Catherine; Chauvet, Marie-Liesse; Cormier-Daire, Valérie; Devisme, Louise; Geneviève, David; Munnich, Arnold; Viot, Géraldine; Raoul, Odile; Romana, Serge; Gonzales, Marie; Encha-Razavi, Ferechte; Odent, Sylvie; Vekemans, Michel; Attie-Bitach, Tania

2015-01-01

The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister–Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype–phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype–phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues. PMID:24736735

New insights into genotype-phenotype correlation for GLI3 mutations.

PubMed

Démurger, Florence; Ichkou, Amale; Mougou-Zerelli, Soumaya; Le Merrer, Martine; Goudefroye, Géraldine; Delezoide, Anne-Lise; Quélin, Chloé; Manouvrier, Sylvie; Baujat, Geneviève; Fradin, Mélanie; Pasquier, Laurent; Megarbané, André; Faivre, Laurence; Baumann, Clarisse; Nampoothiri, Sheela; Roume, Joëlle; Isidor, Bertrand; Lacombe, Didier; Delrue, Marie-Ange; Mercier, Sandra; Philip, Nicole; Schaefer, Elise; Holder, Muriel; Krause, Amanda; Laffargue, Fanny; Sinico, Martine; Amram, Daniel; André, Gwenaelle; Liquier, Alain; Rossi, Massimiliano; Amiel, Jeanne; Giuliano, Fabienne; Boute, Odile; Dieux-Coeslier, Anne; Jacquemont, Marie-Line; Afenjar, Alexandra; Van Maldergem, Lionel; Lackmy-Port-Lis, Marylin; Vincent-Delorme, Catherine; Chauvet, Marie-Liesse; Cormier-Daire, Valérie; Devisme, Louise; Geneviève, David; Munnich, Arnold; Viot, Géraldine; Raoul, Odile; Romana, Serge; Gonzales, Marie; Encha-Razavi, Ferechte; Odent, Sylvie; Vekemans, Michel; Attie-Bitach, Tania

2015-01-01

The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype-phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype-phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues.

Mutations in ash1 and trx enhance P-element-dependent silencing in Drosophila melanogaster.

PubMed

McCracken, Allen; Locke, John

2016-08-01

In Drosophila melanogaster, the mini-w(+) transgene in Pci is normally expressed throughout the adult eye; however, when other P or KP elements are present, a variegated-eye phenotype results, indicating random w(+) silencing during development called P-element-dependent silencing (PDS). Mutant Su(var)205 and Su(var)3-7 alleles act as haplo-suppressors/triplo-enhancers of this variegated phenotype, indicating that these heterochromatic modifiers act dose dependently in PDS. Previously, we recovered a spontaneous mutation of P{lacW}ci(Dplac) called P{lacW}ci(DplacE1) (E1) that variegated in the absence of P elements, presumably due to the insertion of an adjacent gypsy element. From a screen for genetic modifiers of E1 variegation, we describe here the isolation of five mutations in ash1 and three in trx that enhance the E1 variegated phenotype in a dose-dependent and cumulative manner. These mutant alleles enhance PDS at E1, and in E1/P{lacW}ci(Dplac), but suppress position effect variegation (PEV) at In(1)w(m)(4). This opposite action is consistent with a model where ASH1 and TRX mark transcriptionally active chromatin domains. If ASH1 or TRX function is lost or reduced, heterochromatin can spread into these domains creating a sink that diverts heterochromatic proteins from other variegating locations, which then may express a suppressed phenotype.

FGFR3 mutation causes abnormal membranous ossification in achondroplasia.

PubMed

Di Rocco, Federico; Biosse Duplan, Martin; Heuzé, Yann; Kaci, Nabil; Komla-Ebri, Davide; Munnich, Arnold; Mugniery, Emilie; Benoist-Lasselin, Catherine; Legeai-Mallet, Laurence

2014-06-01

FGFR3 gain-of-function mutations lead to both chondrodysplasias and craniosynostoses. Achondroplasia (ACH), the most frequent dwarfism, is due to an FGFR3-activating mutation which results in impaired endochondral ossification. The effects of the mutation on membranous ossification are unknown. Fgfr3(Y367C/+) mice mimicking ACH and craniofacial analysis of patients with ACH and FGFR3-related craniosynostoses provide an opportunity to address this issue. Studying the calvaria and skull base, we observed abnormal cartilage and premature fusion of the synchondroses leading to modifications of foramen magnum shape and size in Fgfr3(Y367C/+) mice, ACH and FGFR3-related craniosynostoses patients. Partial premature fusion of the coronal sutures and non-ossified gaps in frontal bones were also present in Fgfr3(Y367C/+) mice and ACH patients. Our data provide strong support that not only endochondral ossification but also membranous ossification is severely affected in ACH. Demonstration of the impact of FGFR3 mutations on craniofacial development should initiate novel pharmacological and surgical therapeutic approaches.

Mutations in STT3A and STT3B cause two congenital disorders of glycosylation

PubMed Central

Shrimal, Shiteshu; Ng, Bobby G.; Losfeld, Marie-Estelle; Gilmore, Reid; Freeze, Hudson H.

2013-01-01

We describe two unreported types of congenital disorders of glycosylation (CDG) which are caused by mutations in different isoforms of the catalytic subunit of the oligosaccharyltransferase (OST). Each isoform is encoded by a different gene (STT3A or STT3B), resides in a different OST complex and has distinct donor and acceptor substrate specificities with partially overlapping functions in N-glycosylation. The two cases from unrelated consanguineous families both show neurologic abnormalities, hypotonia, intellectual disability, failure to thrive and feeding problems. A homozygous mutation (c.1877T > C) in STT3A causes a p.Val626Ala change and a homozygous intronic mutation (c.1539 + 20G > T) in STT3B causes the other disorder. Both mutations impair glycosylation of a GFP biomarker and are rescued with the corresponding cDNA. Glycosylation of STT3A- and STT3B-specific acceptors is decreased in fibroblasts carrying the corresponding mutated gene and expression of the STT3A (p.Val626Ala) allele in STT3A-deficient HeLa cells does not rescue glycosylation. No additional cases were found in our collection or in reviewing various databases. The STT3A mutation significantly impairs glycosylation of the biomarker transferrin, but the STT3B mutation only slightly affects its glycosylation. Additional cases of STT3B-CDG may be missed by transferrin analysis and will require exome or genome sequencing. PMID:23842455

Response of head and neck squamous cell carcinoma cells carrying PIK3CA mutations to selected targeted therapies.

PubMed

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-06-01

The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. (1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy. PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R. Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance. (1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of

Response of Head and Neck Squamous Cell Carcinoma Cells Carrying PIK3CA Mutations to Select Targeted Therapies

PubMed Central

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-01-01

Importance The PIK3CA mutation is one of the most common mutations in Head and Neck Squamous Cell Carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. Objectives 1) To determine the role of oncogene dependence on one of the more common and targetable oncogenes in HNSCC – PIK3CA; 2) To evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. Study Design In vitro study. Setting Academic research laboratory. Participants Cell culture based study assessing the viability of PIK3CA mutated head and neck cell lines when treated with targeted therapy. Exposures PIK3CA mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Main Outcome and Measures Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R Results Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred decreased, rather than increased, sensitivity as measured by IC50 when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared to the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared to control while those expressing E545K showed slightly increased sensitivity of unclear significance. Conclusions and Relevance 1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared to control with dual PI3K/mTOR inhibition. 2) Oncogene addiction to PIK3CA hot spot mutations, if it occurs, is likely to evolve in vivo molecular changes that remain to be identified. Additional study is required to develop new model systems and

Mutations in PIK3CA are infrequent in neuroblastoma

PubMed Central

Dam, Vincent; Morgan, Brian T; Mazanek, Pavel; Hogarty, Michael D

2006-01-01

Background Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessed PIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as these have not been previously reported. Methods Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutations within the C2, helical and kinase domain "hot spots" where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations to assess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. Results We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studied to date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. Conclusion These data

Loss-of-function mutations in APOC3, triglycerides, and coronary disease.

PubMed

Crosby, Jacy; Peloso, Gina M; Auer, Paul L; Crosslin, David R; Stitziel, Nathan O; Lange, Leslie A; Lu, Yingchang; Tang, Zheng-zheng; Zhang, He; Hindy, George; Masca, Nicholas; Stirrups, Kathleen; Kanoni, Stavroula; Do, Ron; Jun, Goo; Hu, Youna; Kang, Hyun Min; Xue, Chenyi; Goel, Anuj; Farrall, Martin; Duga, Stefano; Merlini, Pier Angelica; Asselta, Rosanna; Girelli, Domenico; Olivieri, Oliviero; Martinelli, Nicola; Yin, Wu; Reilly, Dermot; Speliotes, Elizabeth; Fox, Caroline S; Hveem, Kristian; Holmen, Oddgeir L; Nikpay, Majid; Farlow, Deborah N; Assimes, Themistocles L; Franceschini, Nora; Robinson, Jennifer; North, Kari E; Martin, Lisa W; DePristo, Mark; Gupta, Namrata; Escher, Stefan A; Jansson, Jan-Håkan; Van Zuydam, Natalie; Palmer, Colin N A; Wareham, Nicholas; Koch, Werner; Meitinger, Thomas; Peters, Annette; Lieb, Wolfgang; Erbel, Raimund; Konig, Inke R; Kruppa, Jochen; Degenhardt, Franziska; Gottesman, Omri; Bottinger, Erwin P; O'Donnell, Christopher J; Psaty, Bruce M; Ballantyne, Christie M; Abecasis, Goncalo; Ordovas, Jose M; Melander, Olle; Watkins, Hugh; Orho-Melander, Marju; Ardissino, Diego; Loos, Ruth J F; McPherson, Ruth; Willer, Cristen J; Erdmann, Jeanette; Hall, Alistair S; Samani, Nilesh J; Deloukas, Panos; Schunkert, Heribert; Wilson, James G; Kooperberg, Charles; Rich, Stephen S; Tracy, Russell P; Lin, Dan-Yu; Altshuler, David; Gabriel, Stacey; Nickerson, Deborah A; Jarvik, Gail P; Cupples, L Adrienne; Reiner, Alex P; Boerwinkle, Eric; Kathiresan, Sekar

2014-07-03

Plasma triglyceride levels are heritable and are correlated with the risk of coronary heart disease. Sequencing of the protein-coding regions of the human genome (the exome) has the potential to identify rare mutations that have a large effect on phenotype. We sequenced the protein-coding regions of 18,666 genes in each of 3734 participants of European or African ancestry in the Exome Sequencing Project. We conducted tests to determine whether rare mutations in coding sequence, individually or in aggregate within a gene, were associated with plasma triglyceride levels. For mutations associated with triglyceride levels, we subsequently evaluated their association with the risk of coronary heart disease in 110,970 persons. An aggregate of rare mutations in the gene encoding apolipoprotein C3 (APOC3) was associated with lower plasma triglyceride levels. Among the four mutations that drove this result, three were loss-of-function mutations: a nonsense mutation (R19X) and two splice-site mutations (IVS2+1G→A and IVS3+1G→T). The fourth was a missense mutation (A43T). Approximately 1 in 150 persons in the study was a heterozygous carrier of at least one of these four mutations. Triglyceride levels in the carriers were 39% lower than levels in noncarriers (P<1×10(-20)), and circulating levels of APOC3 in carriers were 46% lower than levels in noncarriers (P=8×10(-10)). The risk of coronary heart disease among 498 carriers of any rare APOC3 mutation was 40% lower than the risk among 110,472 noncarriers (odds ratio, 0.60; 95% confidence interval, 0.47 to 0.75; P=4×10(-6)). Rare mutations that disrupt APOC3 function were associated with lower levels of plasma triglycerides and APOC3. Carriers of these mutations were found to have a reduced risk of coronary heart disease. (Funded by the National Heart, Lung, and Blood Institute and others.).

Loss-of-Function Mutations in APOC3, Triglycerides, and Coronary Disease

PubMed Central

2014-01-01

Background Plasma triglyceride levels are heritable and are correlated with the risk of coronary heart disease. Sequencing of the protein-coding regions of the human genome (the exome) has the potential to identify rare mutations that have a large effect on phenotype. Methods We sequenced the protein-coding regions of 18,666 genes in each of 3734 participants of European or African ancestry in the Exome Sequencing Project. We conducted tests to determine whether rare mutations in coding sequence, individually or in aggregate within a gene, were associated with plasma triglyceride levels. For mutations associated with triglyceride levels, we subsequently evaluated their association with the risk of coronary heart disease in 110,970 persons. Results An aggregate of rare mutations in the gene encoding apolipoprotein C3 (APOC3) was associated with lower plasma triglyceride levels. Among the four mutations that drove this result, three were loss-of-function mutations: a nonsense mutation (R19X) and two splice-site mutations (IVS2+1G→A and IVS3+1G→T). The fourth was a missense mutation (A43T). Approximately 1 in 150 persons in the study was a heterozygous carrier of at least one of these four mutations. Triglyceride levels in the carriers were 39% lower than levels in noncarriers (P<1×10−20), and circulating levels of APOC3 in carriers were 46% lower than levels in noncarriers (P = 8×10−10). The risk of coronary heart disease among 498 carriers of any rare APOC3 mutation was 40% lower than the risk among 110,472 noncarriers (odds ratio, 0.60; 95% confidence interval, 0.47 to 0.75; P = 4×10−6). Conclusions Rare mutations that disrupt APOC3 function were associated with lower levels of plasma triglycerides and APOC3. Carriers of these mutations were found to have a reduced risk of coronary heart disease. (Funded by the National Heart, Lung, and Blood Institute and others.) PMID:24941081

Molecular basis of splotch and Waardenburg Pax-3 mutations.

PubMed Central

Chalepakis, G; Goulding, M; Read, A; Strachan, T; Gruss, P

1994-01-01

Pax genes control certain aspects of development, as mutations result in (semi)dominant defects apparent during embryogenesis. Pax-3 has been associated with the mouse mutant splotch (Sp) and the human Waardenburg syndrome type 1 (WS1). We have examined the molecular basis of splotch and WS1 by studying the effect of mutations on DNA binding, using a defined target sequence. Pax-3 contains two different types of functional DNA-binding domains, a paired domain and a homeodomain. Mutational analysis of Pax-3 reveals different modes of DNA binding depending on the presence of these domains. A segment of Pax-3 located between the two DNA-binding domains, including a conserved octapeptide, participates in protein homodimerization. Pax-3 mutations found in splotch alleles and WS1 individuals change DNA binding and, in the case of a protein product of the Sp allele, dimerization. These findings were taken as a basis to define the molecular nature of the mutants. Images PMID:7909605

Dnmt3a deletion cooperates with the Flt3/ITD mutation to drive leukemogenesis in a murine model

PubMed Central

Poitras, Jennifer L.; Heiser, Diane; Li, Li; Nguyen, Bao; Nagai, Kozo; Duffield, Amy S.; Gamper, Christopher; Small, Donald

2016-01-01

Internal tandem duplications of the juxtamembrane domain of FLT3 (FLT3/ITD) are among the most common mutations in Acute Myeloid Leukemia (AML). Resulting in constitutive activation of the kinase, FLT3/ITD portends a particularly poor prognosis, with reduced overall survival and increased rates of relapse. We previously generated a knock-in mouse, harboring an internal tandem duplication at the endogenous Flt3 locus, which develops a fatal myeloproliferative neoplasm (MPN), but fails to develop acute leukemia, suggesting additional mutations are necessary for transformation. To investigate the potential cooperativity of FLT3/ITD and mutant DNMT3A, we bred a conditional Dnmt3a knockout to a substrain of our Flt3/ITD knock-in mice, and found deletion of Dnmt3a significantly reduced median survival of Flt3ITD/+ mice in a dose dependent manner. As expected, pIpC treated Flt3ITD/+ mice solely developed MPN, while Flt3ITD/+;Dnmt3af/f and Flt3ITD/+;Dnmt3af/+ developed a spectrum of neoplasms, including MPN, T-ALL, and AML. Functionally, FLT3/ITD and DNMT3A deletion cooperate to expand LT-HSCs, which exhibit enhanced self-renewal in serial re-plating assays. These results illustrate that DNMT3A loss cooperates with FLT3/ITD to generate hematopoietic neoplasms, including AML. In combination with FLT3/ITD, homozygous Dnmt3a knock-out results in reduced time to disease onset, LT-HSC expansion, and a higher incidence of T-ALL compared with loss of just one allele. The co-occurrence of FLT3 and DNMT3A mutations in AML, as well as subsets of T-ALL, suggests the Flt3ITD/+;Dnmt3af/f model may serve as a valuable resource for delineating effective therapeutic strategies in two clinically relevant contexts. PMID:27636998

CNGA3 mutations in two United Arab Emirates families with achromatopsia.

PubMed

Ahuja, Yachna; Kohl, Susanne; Traboulsi, Elias I

2008-07-10

ACHROMATOPSIA RESULTS FROM MUTATIONS IN ONE OF THREE GENES: cyclic nucleotide-gated channel, alpha-3 (CNGA3); cyclic nucleotide-gated channel, beta-3 (CNGB3); and guanine nucleotide-binding protein, alpha-transducing activity polypeptide 2 (GNAT2). We report the responsible mutations in two United Arab Emirates families who have this autosomal recessive disease. Clinical examinations were performed in seven patients from three nuclear families. Molecular genetic testing for common CNGA3 and CNGB3 mutations was undertaken using standard protocols. All patients were extremely light sensitive and had reduced visual acuity and no color perception. Fundus examinations did not show any visible abnormalities. After further pedigree analysis, two of the families were found to be linked through the paternal line. Two mutations in CNGA3 were identified: Arg283Trp and Gly397Val. Family A, the larger pedigree, had one branch in which two sisters and one brother were homozygous for the Gly397Val mutation and another branch in which a brother and sister were compound heterozygous for both aforenamed mutations. Family B, however, only had two brothers who were homozygous for the Arg283Trp mutation. Achromatopsia in these two United Arab Emirates families results from two different mutations in CNGA3. Two branches of the same pedigree had individuals with both homozygous and compound heterozygous disease, demonstrating a complex molecular pathology in this large family.

A novel de novo activating mutation in STAT3 identified in a patient with common variable immunodeficiency (CVID).

PubMed

Russell, Mark A; Pigors, Manuela; Houssen, Maha E; Manson, Ania; Kelsell, David; Longhurst, Hilary; Morgan, Noel G

2018-02-01

Common variable immunodeficiency (CVID) is characterised by repeated infection associated with primary acquired hypogammaglobulinemia. CVID frequently has a complex aetiology but, in certain cases, it has a monogenic cause. Recently, variants within the gene encoding the transcription factor STAT3 were implicated in monogenic CVID. Here, we describe a patient presenting with symptoms synonymous with CVID, who displayed reduced levels of IgG and IgA, repeated viral infections and multiple additional co-morbidities. Whole-exome sequencing revealed a de novo novel missense mutation in the coiled-coil domain of STAT3 (c.870A>T; p.K290N). Accordingly, the K290N variant of STAT3 was generated, and a STAT3 responsive dual-luciferase reporter assay revealed that the variant strongly enhances STAT3 transcriptional activity both under basal and stimulated (with IL-6) conditions. Overall, these data complement earlier studies in which CVID-associated STAT3 mutations are predicted to enhance transcriptional activity, suggesting that such patients may respond favourably to IL-6 receptor antagonists (e.g. tocilizumab). Copyright © 2017 Elsevier Inc. All rights reserved.

[Mutants of the yeast Saccharomyces cerevisiae characterized by enhanced induced mutagenesis. III. Effect of the him mutation on the effectiveness and specificity of UF-induced mutagenesis].

PubMed

Ivanov, E L; Koval'tsova, S V; Korolev, V G

1987-09-01

We have studied the influence of him1-1, him2-1, him3-1 and himX mutations on induction frequency and specificity of UV-induced adenine-dependent mutations in the yeast Saccharomyces cerevisiae. Him mutations do not render haploid cells more sensitive to the lethal action of UV-light; however, in him strains adenine-dependent mutations (ade1, ade2) were induced more frequently (1.5--2-fold), as compared to the HIM strain. An analysis of the molecular nature of ade2 mutants revealed that him1-1, him2-1 and himX mutations increase specifically the yield of transitions (AT----GC and GC----AT), whereas in the him3-1 strain the yield of transversions was enhanced as well. We suggest him mutations analysed to affect specific repair pathway for mismatch correction.

CADASIL with a novel NOTCH3 mutation (Cys478Tyr).

PubMed

Ozaki, Kokoro; Irioka, Takashi; Ishikawa, Kinya; Mizusawa, Hidehiro

2015-03-01

Recently, an increasing number of NOTCH3 mutations have been described to cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Here, we report 2 CADASIL patients from a Japanese family, who were found to possess a novel NOTCH3 mutation. The proband only had chronic headache, and her mother had previously suffered a minor stroke. Although the patients' clinical symptoms were mild, their distinctive magnetic resonance imaging (MRI) features suggested CADASIL. Genetic analysis revealed that both patients had a novel heterozygous NOTCH3 mutation (p.Cys478Tyr) leading to stereotypical cysteine loss. The present finding suggests that genetic testing for NOTCH3 mutations in patients with distinctive MRI features, even if the symptoms are as mild as chronic headache, should help to broaden the mutational and clinical spectrum of CADASIL. Copyright © 2015 National Stroke Association. Published by Elsevier Inc. All rights reserved.

PIK3CA gene mutations in Northwest Chinese esophageal squamous cell carcinoma

PubMed Central

Liu, Shi-Yuan; Chen, Wei; Chughtai, Ehtesham Annait; Qiao, Zhe; Jiang, Jian-Tao; Li, Shao-Min; Zhang, Wei; Zhang, Jin

2017-01-01

AIM To evaluate PIK3CA gene mutational status in Northwest Chinese esophageal squamous cell carcinoma (ESCC) patients, and examine the associations of PIK3CA gene mutations with clinicopathological characteristics and clinical outcome. METHODS A total of 210 patients with ESCC who underwent curative resection were enrolled in this study. Pyrosequencing was applied to investigate mutations in exons 9 and 20 of PIK3CA gene in 210 Northwest Chinese ESCCs. The associations of PIK3CA gene mutations with clinicopathological characteristics and clinical outcome were examined. RESULTS PIK3CA gene mutations in exon 9 were detected in 48 cases (22.9%) of a non-biased database of 210 curatively resected Northwest Chinese ESCCs. PIK3CA gene mutations were not associated with sex, tobacco use, alcohol use, tumor location, stage, or local recurrence. When compared with wild-type PIK3CA gene cases, patients with PIK3CA gene mutations in exons 9 experienced significantly better disease-free survival and overall survival rates. CONCLUSION The results of this study suggest that PIK3CA gene mutations could act as a prognostic biomarker in Northwest Chinese ESCC patients. PMID:28465643

Mutation profiling of 19 candidate genes in acute myeloid leukemia suggests significance of DNMT3A mutations.

PubMed

Shin, Sang-Yong; Lee, Seung-Tae; Kim, Hee-Jin; Cho, Eun Hae; Kim, Jong-Won; Park, Silvia; Jung, Chul Won; Kim, Sun-Hee

2016-08-23

We selected 19 significantly-mutated genes in AMLs, including FLT3, DNMT3A, NPM1, TET2, RUNX1, CEBPA, WT1, IDH1, IDH2, NRAS, ASXL1, SETD2, PTPN11, TP53, KIT, JAK2, KRAS, BRAF and CBL, and performed massively parallel sequencing for 114 patients with acute myeloid leukemias, mainly including those with normal karyotypes (CN-AML). More than 80% of patients had at least one mutation in the genes tested. DNMT3A mutation was significantly associated with adverse outcome in addition to conventional risk stratification such as the European LeukemiaNet (ELN) classification. We observed clinical usefulness of mutation testing on multiple target genes and the association with disease subgroups, clinical features and prognosis in AMLs.

Frequent Nuclear/Cytoplasmic Localization of β-Catenin without Exon 3 Mutations in Malignant Melanoma

PubMed Central

Rimm, David L.; Caca, Karel; Hu, Gang; Harrison, Frank B.; Fearon, Eric R.

1999-01-01

β-Catenin has a critical role in E-cadherin-mediated cell-cell adhesion, and it also functions as a downstream signaling molecule in the wnt pathway. Mutations in the putative glycogen synthase kinase 3β phosphorylation sites near the β-catenin amino terminus have been found in some cancers and cancer cell lines. The mutations render β-catenin resistant to regulation by a complex containing the glycogen synthase kinase 3β, adenomatous polyposis coli, and axin proteins. As a result, β-catenin accumulates in the cytosol and nucleus and activates T-cell factor/lymphoid enhancing factor transcription factors. Previously, 6 of 27 melanoma cell lines were found to have β-catenin exon 3 mutations affecting the N-terminal phosphorylation sites (Rubinfeld B, Robbins P, Elgamil M, Albert I, Porfiri E, Polakis P: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997, 275:1790–1792). To assess the role of β-catenin defects in primary melanomas, we undertook immunohistochemical and DNA sequencing studies in 65 melanoma specimens. Nuclear and/or cytoplasmic localization of β-catenin, a potential indicator of wnt pathway activation, was seen focally within roughly one third of the tumors, though a clonal somatic mutation in β-catenin was found in only one case (codon 45 Ser→Pro). Our findings demonstrate that β-catenin mutations are rare in primary melanoma, in contrast to the situation in melanoma cell lines. Nonetheless, activation of β-catenin, as indicated by its nuclear and/or cytoplasmic localization, appears to be frequent in melanoma, and in some cases, it may reflect focal and transient activation of the wnt pathway within the tumor. PMID:10027390

A KCNJ10 mutation previously identified in the Russell group of terriers also occurs in Smooth-Haired Fox Terriers with hereditary ataxia and in related breeds.

PubMed

Rohdin, Cecilia; Gilliam, Douglas; O'Leary, Caroline A; O'Brien, Dennis P; Coates, Joan R; Johnson, Gary S; Jäderlund, Karin Hultin

2015-05-23

Hereditary ataxias with similar phenotypes were reported in the Smooth-Haired Fox Terrier, the Jack Russell Terrier and the Parson Russell Terrier. However, segregation analyses showed differing inheritance modes in these breeds. Recently, molecular genetic studies on the Russell group of terriers found independent mutations in KCNJ10 and CAPN1, each associated with a specific clinical subtype of inherited ataxia. The aim of this study was to clarify whether or not Smooth-Haired Fox Terriers with hereditary ataxia and dogs of other related breeds harbor either of the same mutations. A sub goal was to update the results of KCNJ10 genotyping in Russell group terriers. Three Smooth-Haired Fox Terriers with hereditary ataxia and two Toy Fox Terriers with a similar phenotype were all homozygous for the KCNJ10 mutation. The same mutation was also found in a heterozygous state in clinically unaffected Tenterfield Terriers (n = 5) and, in agreement with previous studies, in Jack Russell Terriers, Parson Russell Terriers, and Russell Terriers. A KCNJ10 mutation, previously associated with an autosomal recessive spinocerebellar ataxia in Jack Russell Terriers, Parson Russell Terriers, and Russell Terriers segregates in at least three more breeds descended from British hunting terriers. Ataxic members of two of these breeds, the Smooth-Haired Fox Terrier and the Toy Fox Terrier, were homozygous for the mutation, strengthening the likelihood that this genetic defect is indeed the causative mutation for the disease known as "hereditary ataxia" in Fox Terriers and "spinocerebellar ataxia with myokymia, seizures or both" in the Russell group of terriers.

Nutlin‐3a selects for cells harbouring TP 53 mutations

PubMed Central

Hollstein, Monica; Arlt, Volker M.; Phillips, David H.

2016-01-01

TP53 mutations occur in half of all human tumours. Mutagen‐induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock‐in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen‐treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2–5 months) and much effort is expended maintaining TP53‐WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin‐3a, an MDM2 inhibitor that leads to stabilisation and activation of wild‐type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin‐3a to examine the effect on cell growth and p53 activation. Nutlin‐3a induced the p53 pathway in TP53‐WT HUFs and inhibited cell growth, whereas most TP53‐mutated HUFs were resistant to Nutlin‐3a. We then assessed whether Nutlin‐3a treatment could discriminate between TP53‐WT and TP53‐mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin‐3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin‐3a‐resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin‐3a‐sensitive clones were TP53‐WT. These data suggest that including a Nutlin‐3a counter‐screen significantly improves the specificity and efficiency of the HIMA, whereby TP53‐mutated clones are selected prior to sequencing and TP53‐WT clones can be discarded. PMID:27813088

Histone H3.3 mutations drive paediatric glioblastoma through upregulation of MYCN

PubMed Central

Bjerke, Lynn; Mackay, Alan; Nandhabalan, Meera; Burford, Anna; Jury, Alexa; Popov, Sergey; Bax, Dorine A; Carvalho, Diana; Taylor, Kathryn R; Vinci, Maria; Bajrami, Ilirjana; McGonnell, Imelda M; Lord, Christopher J; Reis, Rui M; Hargrave, Darren; Ashworth, Alan; Workman, Paul; Jones, Chris

2013-01-01

Glioblastomas of children and young adults have a median survival of only 12-15months and are clinically and biologically distinct from histologically similar cancers in older adults1. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A2, occurring either at or close to key residues marked by methylation for regulation of transcription – K27 and G34. Here we show that the cerebral hemispheric-specific G34 mutation drives a distinct expression signature through differential genomic binding of the K36 trimethylation mark (H3K36me3). The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem cell maintenance, cell fate decisions and self-renewal. Critically, H3F3A G34 mutations cause profound upregulation of MYCN, a potent oncogene which is causative of glioblastomas when expressed in the correct developmental context. This driving aberration is selectively targetable in this patient population by inhibiting kinases responsible for stabilisation of the protein. PMID:23539269

Calpain 12 Function Revealed through the Study of an Atypical Case of Autosomal Recessive Congenital Ichthyosis.

PubMed

Bochner, Ron; Samuelov, Liat; Sarig, Ofer; Li, Qiaoli; Adase, Christopher A; Isakov, Ofer; Malchin, Natalia; Vodo, Dan; Shayevitch, Ronna; Peled, Alon; Yu, Benjamin D; Fainberg, Gilad; Warshauer, Emily; Adir, Noam; Erez, Noam; Gat, Andrea; Gottlieb, Yehonatan; Rogers, Tova; Pavlovsky, Mor; Goldberg, Ilan; Shomron, Noam; Sandilands, Aileen; Campbell, Linda E; MacCallum, Stephanie; McLean, W H Irwin; Ast, Gil; Gallo, Richard L; Uitto, Jouni; Sprecher, Eli

2017-02-01

Congenital erythroderma is a rare and often life-threatening condition, which has been shown to result from mutations in several genes encoding important components of the epidermal differentiation program. Using whole exome sequencing, we identified in a child with congenital exfoliative erythroderma, hypotrichosis, severe nail dystrophy and failure to thrive, two heterozygous mutations in ABCA12 (c.2956C>T, p.R986W; c.5778+2T>C, p. G1900Mfs*16), a gene known to be associated with two forms of ichthyosis, autosomal recessive congenital ichthyosis, and harlequin ichthyosis. Because the patient displayed an atypical phenotype, including severe hair and nail manifestations, we scrutinized the exome sequencing data for additional potentially deleterious genetic variations in genes of relevance to the cornification process. Two mutations were identified in CAPN12, encoding a member of the calpain proteases: a paternal missense mutation (c.1511C>A; p.P504Q) and a maternal deletion due to activation of a cryptic splice site in exon 9 of the gene (c.1090_1129del; p.Val364Lysfs*11). The calpain 12 protein was found to be expressed in both the epidermis and hair follicle of normal skin, but its expression was dramatically reduced in the patient's skin. The downregulation of capn12 expression in zebrafish was associated with abnormal epidermal morphogenesis. Small interfering RNA knockdown of CAPN12 in three-dimensional human skin models was associated with acanthosis, disorganized epidermal architecture, and downregulation of several differentiation markers, including filaggrin. Accordingly, filaggrin expression was almost absent in the patient skin. Using ex vivo live imaging, small interfering RNA knockdown of calpain 12 in skin from K14-H2B GFP mice led to significant hair follicle catagen transformation compared with controls. In summary, our results indicate that calpain 12 plays an essential role during epidermal ontogenesis and normal hair follicle cycling and that

Hypomorphic NOTCH3 mutation in an Italian family with CADASIL features.

PubMed

Moccia, Marcello; Mosca, Lorena; Erro, Roberto; Cervasio, Mariarosaria; Allocca, Roberto; Vitale, Carmine; Leonardi, Antonio; Caranci, Ferdinando; Del Basso-De Caro, Maria Laura; Barone, Paolo; Penco, Silvana

2015-01-01

The cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is because of NOTCH3 mutations affecting the number of cysteine residues. In this view, the role of atypical NOTCH3 mutations is still debated. Therefore, we investigated a family carrying a NOTCH3 nonsense mutation, with dominantly inherited recurrent cerebrovascular disorders. Among 7 family members, 4 received a clinical diagnosis of CADASIL. A heterozygous truncating mutation in exon 3 (c.307C>T, p.Arg103X) was found in the 4 clinically affected subjects and in one 27-year old lady, only complaining of migraine with aura. Magnetic resonance imaging scans found typical signs of small-vessel disease in the 4 affected subjects, supporting the clinical diagnosis. Skin biopsies did not show the typical granular osmiophilic material, but only nonspecific signs of vascular damage, resembling those previously described in Notch3 knockout mice. Interestingly, messenger RNA (mRNA) analysis supports the hypothesis of an atypical NOTCH3 mutation, suggesting a nonsense-mediated mRNA decay. In conclusion, the present study broadens the spectrum of CADASIL mutations, and, therefore, opens new insights about Notch3 signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulation of hepatitis B virus ENI enhancer activity by hepatocyte-enriched transcription factor HNF3.

PubMed

Chen, M; Hieng, S; Qian, X; Costa, R; Ou, J H

1994-11-15

Hepatitis B virus (HBV) ENI enhancer can activate the expression of HBV and non-HBV genes in a liver-specific manner. By performing the electrophoretic mobility-shift assays, we demonstrated that the three related, liver-enriched, transcription factors, HNF3 alpha, HNF3 beta, and HNF3 gamma could all bind to the 2c site of HBV ENI enhancer. Mutations introduced in the 2c site to abolish the binding by HNF3 reduced the enhancer activity approximately 15-fold. Moreover, expression of HNF3 antisense sequences to suppress the expression of HNF3 in Huh-7 hepatoma cells led to reduction of the ENI enhancer activity. These results indicate that HNF3 positively regulates the ENI enhancer activity and this regulation is most likely mediated through the 2c site. The requirement of HNF3 for the ENI enhancer activity could explain the liver specificity of this enhancer element.

Mutational spectrum of myeloid malignancies with inv(3)/t(3;3) reveals a predominant involvement of RAS/RTK signaling pathways

PubMed Central

Gröschel, Stefan; Sanders, Mathijs A.; Hoogenboezem, Remco; Zeilemaker, Annelieke; Havermans, Marije; Erpelinck, Claudia; Bindels, Eric M. J.; Beverloo, H. Berna; Döhner, Hartmut; Löwenberg, Bob; Döhner, Konstanze; Delwel, Ruud

2015-01-01

Myeloid malignancies bearing chromosomal inv(3)/t(3;3) abnormalities are among the most therapy-resistant leukemias. Deregulated expression of EVI1 is the molecular hallmark of this disease; however, the genome-wide spectrum of cooperating mutations in this disease subset has not been systematically elucidated. Here, we show that 98% of inv(3)/t(3;3) myeloid malignancies harbor mutations in genes activating RAS/receptor tyrosine kinase (RTK) signaling pathways. In addition, hemizygous mutations in GATA2, as well as heterozygous alterations in RUNX1, SF3B1, and genes encoding epigenetic modifiers, frequently co-occur with the inv(3)/t(3;3) aberration. Notably, neither mutational patterns nor gene expression profiles differ across inv(3)/t(3;3) acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome cases, suggesting recognition of inv(3)/t(3;3) myeloid malignancies as a single disease entity irrespective of blast count. The high incidence of activating RAS/RTK signaling mutations may provide a target for a rational treatment strategy in this high-risk patient group. PMID:25381062

Cysteine-sparing CADASIL mutations in NOTCH3 show proaggregatory properties in vitro.

PubMed

Wollenweber, Frank Arne; Hanecker, Patrizia; Bayer-Karpinska, Anna; Malik, Rainer; Bäzner, Hansjörg; Moreton, Fiona; Muir, Keith W; Müller, Susanna; Giese, Armin; Opherk, Christian; Dichgans, Martin; Haffner, Christof; Duering, Marco

2015-03-01

Mutations in NOTCH3 cause cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common monogenic cause of stroke and vascular dementia. Misfolding and aggregation of NOTCH3 proteins triggered by cysteine-affecting mutations are considered to be the key disease mechanisms. However, the significance of cysteine-sparing mutations is still debated. We studied a family with inherited small vessel disease by standardized medical history, clinical examination, MRI, ultrastructural analysis of skin biopsies, and Sanger sequencing of all NOTCH3 exons. In addition, we performed in vitro characterization of NOTCH3 variants using recombinant protein fragments and a single-particle aggregation assay. We identified a novel cysteine-sparing NOTCH3 mutation (D80G) in 4 family members, which was absent in a healthy sibling. All mutation carriers exhibited a CADASIL typical brain imaging and clinical phenotype, whereas skin biopsy showed inconsistent results. In vitro aggregation behavior of the D80G mutant was similar compared with cysteine-affecting mutations. This was reproduced with cysteine-sparing mutations from previously reported families having a phenotype consistent with CADASIL. Our findings support the view that cysteine-sparing mutations, such as D80G, might cause CADASIL with a phenotype largely indistinguishable from cysteine mutations. The in vitro aggregation analysis of atypical NOTCH3 mutations offers novel insights into pathomechanisms and might represent a tool for estimating their clinical significance. © 2015 American Heart Association, Inc.

GATA3 mutation in a family with hypoparathyroidism, deafness and renal dysplasia syndrome.

PubMed

Zhu, Zi-Yang; Zhou, Qiao-Li; Ni, Shi-Ning; Gu, Wei

2014-08-01

The hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is an autosomal dominant disorder primarily caused by GATA3 gene mutation. We report here a case that both of a Chinese boy and his father had HDR syndrome which caused by a novel mutation of GATA3. Polymerase chain reaction and DNA sequencing was performed to detect the exons of the GATA3 gene for mutation analysis. Sequence analysis of GATA3 revealed a heterozygous nonsense mutation in this family: a mutation of GATA3 at exon 2 (c.515C >A) that resulted in a premature stop at codon 172 (p.S172X) with a loss of two zinc finger domains. We identified a novel nonsense mutation which will expand the spectrum of HDR-associated GATA3 mutations.

Failure to up-regulate transcription of genes necessary for muscle adaptation underlies limb girdle muscular dystrophy 2A (calpainopathy)

PubMed Central

Kramerova, Irina; Ermolova, Natalia; Eskin, Ascia; Hevener, Andrea; Quehenberger, Oswald; Armando, Aaron M.; Haller, Ronald; Romain, Nadine; Nelson, Stanley F.; Spencer, Melissa J.

2016-01-01

Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in the Calpain 3 (CAPN3) gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. In Capn3 knock-out mice (C3KO), Ca2+ release and Ca2+/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca2+-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild type (WT) and C3KO mice subjected to endurance exercise. In WT mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscles fail to release fatty acids from stored triacylglycerol. In conjunction with the defects in oxidative metabolism, C3KO mice demonstrate reduced exercise endurance. Failure to up-regulate genes in C3KO muscles is due, in part, to decreased levels of PGC1α, a transcriptional co-regulator that orchestrates the muscle adaptation response. Destabilization of PGC1α is attributable to decreased p38 MAPK activation via diminished CaMKII signaling. Thus, we elucidate a pathway downstream of Ca2+-mediated CaMKII activation that is dysfunctional in C3KO mice, leading to reduced transcription of genes involved in muscle adaptation. These studies identify a novel mechanism of muscular dystrophy: a blunted transcriptional response to muscle loading resulting in chronic failure to adapt and remodel. PMID:27005420

Contribution of SHANK3 Mutations to Autism Spectrum Disorder

PubMed Central

Moessner, Rainald ; Marshall, Christian R. ; Sutcliffe, James S. ; Skaug, Jennifer ; Pinto, Dalila ; Vincent, John ; Zwaigenbaum, Lonnie ; Fernandez, Bridget ; Roberts, Wendy ; Szatmari, Peter ; Scherer, Stephen W. 

2007-01-01

Mutations in SHANK3, which encodes a synaptic scaffolding protein, have been described in subjects with an autism spectrum disorder (ASD). To assess the quantitative contribution of SHANK3 to the pathogenesis of autism, we determined the frequency of DNA sequence and copy-number variants in this gene in 400 ASD-affected subjects ascertained in Canada. One de novo mutation and two gene deletions were discovered, indicating a contribution of 0.75% in this cohort. One additional SHANK3 deletion was characterized in two ASD-affected siblings from another collection, which brings the total number of published mutations in unrelated ASD-affected families to seven. The combined data provide support that haploinsufficiency of SHANK3 can cause a monogenic form of autism in sufficient frequency to warrant consideration in clinical diagnostic testing. PMID:17999366

Rare Noncoding Mutations Extend the Mutational Spectrum in the PGAP3 Subtype of Hyperphosphatasia with Mental Retardation Syndrome

PubMed Central

Knaus, Alexej; Awaya, Tomonari; Helbig, Ingo; Afawi, Zaid; Pendziwiat, Manuela; Abu‐Rachma, Jubran; Thompson, Miles D.; Cole, David E.; Skinner, Steve; Annese, Fran; Canham, Natalie; Schweiger, Michal R.; Robinson, Peter N.; Mundlos, Stefan; Kinoshita, Taroh; Munnich, Arnold

2016-01-01

ABSTRACT HPMRS or Mabry syndrome is a heterogeneous glycosylphosphatidylinositol (GPI) anchor deficiency that is caused by an impairment of synthesis or maturation of the GPI‐anchor. The expressivity of the clinical features in HPMRS varies from severe syndromic forms with multiple organ malformations to mild nonsyndromic intellectual disability. In about half of the patients with the clinical diagnosis of HPMRS, pathogenic mutations can be identified in the coding region in one of the six genes, one among them is PGAP3. In this work, we describe a screening approach with sequence specific baits for transcripts of genes of the GPI pathway that allows the detection of functionally relevant mutations also including introns and the 5′ and 3′ UTR. By this means, we also identified pathogenic noncoding mutations, which increases the diagnostic yield for HPMRS on the basis of intellectual disability and elevated serum alkaline phosphatase. In eight affected individuals from different ethnicities, we found seven novel pathogenic mutations in PGAP3. Besides five missense mutations, we identified an intronic mutation, c.558‐10G>A, that causes an aberrant splice product and a mutation in the 3′UTR, c.*559C>T, that is associated with substantially lower mRNA levels. We show that our novel screening approach is a useful rapid detection tool for alterations in genes coding for key components of the GPI pathway. PMID:27120253

PIK3CA Mutations in Mucinous Cystic Neoplasms of the Pancreas

PubMed Central

Garcia-Carracedo, Dario; Chen, Zong-Ming; Qiu, Wanglong; Huang, Alicia S.; Tang, Sophia M.; Hruban, Ralph H.; Su, Gloria H.

2014-01-01

Objectives Mucinous cystic neoplasms (MCNs) are rare, potentially curable, mucin-producing neoplasms of the pancreas. We have previously reported PIK3CA (phosphoinositide-3-kinase catalytic subunit, p110α) mutations in intraductal papillary mucinous neoplasms, another mucin-producing neoplasm of the pancreas. In this study, we analyzed the presence of PIK3CA and AKT1/PKB (V-akt murine thymoma viral oncogene homolog 1) hot-spot mutations in MCN specimens. Methods Using the genomic DNA sequencing of tumor tissues isolated by laser capture microdissection, we evaluated 15 well-characterized MCNs for the E542K, E545K(exon 9), and H1047R (exon 20) hot-spotmutations in the PIK3CA gene and the E17K mutation in the AKT1 gene. Results A hot-spotmutation (E545K) of the PIK3CA gene was detected in 1 of the 15 MCNs and further confirmed by a mutant-enriched method. Interestingly, this mutation was found to be present only in the high-grade but not in low-grade dysplastic epithelium obtained from this neoplasm and coexisted with a KRASG12D mutation. No mutations were identified in the AKT1 gene. Conclusions Our data, when combined with previous reports on intraductal papillary mucinous neoplasms, indicate that oncogenic activation of the PI3K pathway involving PIK3CA gene mutations can contribute to the progression of mucin-producing neoplasms but not pancreatic intraepithelial neoplasia. PIK3CA status could be useful for understanding their progression to malignancy. PMID:24518503

Clinical spectrum of 4H leukodystrophy caused by POLR3A and POLR3B mutations

PubMed Central

Vanderver, Adeline; van Spaendonk, Rosalina M.L.; Schiffmann, Raphael; Brais, Bernard; Bugiani, Marianna; Sistermans, Erik; Catsman-Berrevoets, Coriene; Kros, Johan M.; Pinto, Pedro Soares; Pohl, Daniela; Tirupathi, Sandya; Strømme, Petter; de Grauw, Ton; Fribourg, Sébastien; Demos, Michelle; Pizzino, Amy; Naidu, Sakkubai; Guerrero, Kether; van der Knaap, Marjo S.; Bernard, Geneviève

2014-01-01

Objective: To study the clinical and radiologic spectrum and genotype–phenotype correlation of 4H (hypomyelination, hypodontia, hypogonadotropic hypogonadism) leukodystrophy caused by mutations in POLR3A or POLR3B. Methods: We performed a multinational cross-sectional observational study of the clinical, radiologic, and molecular characteristics of 105 mutation-proven cases. Results: The majority of patients presented before 6 years with gross motor delay or regression. Ten percent had an onset beyond 10 years. The disease course was milder in patients with POLR3B than in patients with POLR3A mutations. Other than the typical neurologic, dental, and endocrine features, myopia was seen in almost all and short stature in 50%. Dental and hormonal findings were not invariably present. Mutations in POLR3A and POLR3B were distributed throughout the genes. Except for French Canadian patients, patients from European backgrounds were more likely to have POLR3B mutations than other populations. Most patients carried the common c.1568T>A POLR3B mutation on one allele, homozygosity for which causes a mild phenotype. Systematic MRI review revealed that the combination of hypomyelination with relative T2 hypointensity of the ventrolateral thalamus, optic radiation, globus pallidus, and dentate nucleus, cerebellar atrophy, and thinning of the corpus callosum suggests the diagnosis. Conclusions: 4H is a well-recognizable clinical entity if all features are present. Mutations in POLR3A are associated with a more severe clinical course. MRI characteristics are helpful in addressing the diagnosis, especially if patients lack the cardinal non-neurologic features. PMID:25339210

Mutations Enhancing Amino Acid Catabolism Confer a Growth Advantage in Stationary Phase

PubMed Central

Zinser, Erik R.; Kolter, Roberto

1999-01-01

Starved cultures of Escherichia coli undergo successive rounds of population takeovers by mutants of increasing fitness. These mutants express the growth advantage in stationary phase (GASP) phenotype. Previous work identified the rpoS819 allele as a GASP mutation allowing cells to take over stationary-phase cultures after growth in rich media (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757–1760, 1993). Here we have identified three new GASP loci from an aged rpoS819 strain: sgaA, sgaB, and sgaC. Each locus is capable of conferring GASP on the rpoS819 parent, and they can provide successively higher fitnesses for the bacteria in the starved cultures. All four GASP mutations isolated thus far allow for faster growth on both individual and mixtures of amino acids. Each mutation confers a growth advantage on a different subset of amino acids, and these mutations act in concert to increase the overall catabolic capacity of the cell. We present a model whereby this enhanced ability to catabolize amino acids is responsible for the fitness gain during carbon starvation, as it may allow GASP mutants to outcompete the parental cells when growing on the amino acids released by dying cells. PMID:10482523

An Enhanced Differential Evolution Algorithm Based on Multiple Mutation Strategies.

PubMed

Xiang, Wan-li; Meng, Xue-lei; An, Mei-qing; Li, Yin-zhen; Gao, Ming-xia

2015-01-01

Differential evolution algorithm is a simple yet efficient metaheuristic for global optimization over continuous spaces. However, there is a shortcoming of premature convergence in standard DE, especially in DE/best/1/bin. In order to take advantage of direction guidance information of the best individual of DE/best/1/bin and avoid getting into local trap, based on multiple mutation strategies, an enhanced differential evolution algorithm, named EDE, is proposed in this paper. In the EDE algorithm, an initialization technique, opposition-based learning initialization for improving the initial solution quality, and a new combined mutation strategy composed of DE/current/1/bin together with DE/pbest/bin/1 for the sake of accelerating standard DE and preventing DE from clustering around the global best individual, as well as a perturbation scheme for further avoiding premature convergence, are integrated. In addition, we also introduce two linear time-varying functions, which are used to decide which solution search equation is chosen at the phases of mutation and perturbation, respectively. Experimental results tested on twenty-five benchmark functions show that EDE is far better than the standard DE. In further comparisons, EDE is compared with other five state-of-the-art approaches and related results show that EDE is still superior to or at least equal to these methods on most of benchmark functions.

Mutation of Breast Cancer Cell Genomic DNA by APOBEC3B

DTIC Science & Technology

2012-09-01

down Yes, A3B expression increases the steady-state level of genomic uracil Fig. 2a-2c 2) Can A3B mutate a target gene to escape drug...somatic mutation in human cancer genomes. Nature 446, 153-158 (2007). 10 2 Jones, S. et al. Frequent mutations of chromatin remodeling gene ARID1A in...processes molding the genomes of 21 breast cancers. Cell 149, 979-993 (2012). 9 Stephens, P. J. et al. The landscape of cancer genes and mutational

De novo mutations in ATP1A3 cause alternating hemiplegia of childhood

PubMed Central

Heinzen, Erin L.; Swoboda, Kathryn J.; Hitomi, Yuki; Gurrieri, Fiorella; Nicole, Sophie; de Vries, Boukje; Tiziano, F. Danilo; Fontaine, Bertrand; Walley, Nicole M.; Heavin, Sinéad; Panagiotakaki, Eleni; Fiori, Stefania; Abiusi, Emanuela; Di Pietro, Lorena; Sweney, Matthew T.; Newcomb, Tara M.; Viollet, Louis; Huff, Chad; Jorde, Lynn B.; Reyna, Sandra P.; Murphy, Kelley J.; Shianna, Kevin V.; Gumbs, Curtis E.; Little, Latasha; Silver, Kenneth; Ptác̆ek, Louis J.; Haan, Joost; Ferrari, Michel D.; Bye, Ann M.; Herkes, Geoffrey K.; Whitelaw, Charlotte M.; Webb, David; Lynch, Bryan J.; Uldall, Peter; King, Mary D.; Scheffer, Ingrid E.; Neri, Giovanni; Arzimanoglou, Alexis; van den Maagdenberg, Arn M.J.M.; Sisodiya, Sanjay M.; Mikati, Mohamad A.; Goldstein, David B.; Nicole, Sophie; Gurrieri, Fiorella; Neri, Giovanni; de Vries, Boukje; Koelewijn, Stephany; Kamphorst, Jessica; Geilenkirchen, Marije; Pelzer, Nadine; Laan, Laura; Haan, Joost; Ferrari, Michel; van den Maagdenberg, Arn; Zucca, Claudio; Bassi, Maria Teresa; Franchini, Filippo; Vavassori, Rosaria; Giannotta, Melania; Gobbi, Giuseppe; Granata, Tiziana; Nardocci, Nardo; De Grandis, Elisa; Veneselli, Edvige; Stagnaro, Michela; Gurrieri, Fiorella; Neri, Giovanni; Vigevano, Federico; Panagiotakaki, Eleni; Oechsler, Claudia; Arzimanoglou, Alexis; Nicole, Sophie; Giannotta, Melania; Gobbi, Giuseppe; Ninan, Miriam; Neville, Brian; Ebinger, Friedrich; Fons, Carmen; Campistol, Jaume; Kemlink, David; Nevsimalova, Sona; Laan, Laura; Peeters-Scholte, Cacha; van den Maagdenberg, Arn; Casaer, Paul; Casari, Giorgio; Sange, Guenter; Spiel, Georg; Boneschi, Filippo Martinelli; Zucca, Claudio; Bassi, Maria Teresa; Schyns, Tsveta; Crawley, Francis; Poncelin, Dominique; Vavassori, Rosaria

2012-01-01

Alternating hemiplegia of childhood (AHC) is a rare, severe neurodevelopmental syndrome characterized by recurrent hemiplegic episodes and distinct neurologic manifestations. AHC is usually a sporadic disorder with unknown etiology. Using exome sequencing of seven patients with AHC, and their unaffected parents, we identified de novo nonsynonymous mutations in ATP1A3 in all seven AHC patients. Subsequent sequence analysis of ATP1A3 in 98 additional patients revealed that 78% of AHC cases have a likely causal ATP1A3 mutation, including one inherited mutation in a familial case of AHC. Remarkably, six ATP1A3 mutations explain the majority of patients, including one observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset-dystonia-parkinsonism, AHC-causing mutations revealed consistent reductions in ATPase activity without effects on protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC, and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in this gene. PMID:22842232

Oxidative Stress in Dilated Cardiomyopathy Caused by MYBPC3 Mutation

PubMed Central

Lynch, Thomas L.; Sivaguru, Mayandi; Velayutham, Murugesan; Cardounel, Arturo J.; Michels, Michelle; Barefield, David; Govindan, Suresh; dos Remedios, Cristobal; van der Velden, Jolanda; Sadayappan, Sakthivel

2015-01-01

Cardiomyopathies can result from mutations in genes encoding sarcomere proteins including MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). However, whether oxidative stress is augmented due to contractile dysfunction and cardiomyocyte damage in MYBPC3-mutated cardiomyopathies has not been elucidated. To determine whether oxidative stress markers were elevated in MYBPC3-mutated cardiomyopathies, a previously characterized 3-month-old mouse model of dilated cardiomyopathy (DCM) expressing a homozygous MYBPC3 mutation (cMyBP-C(t/t)) was used, compared to wild-type (WT) mice. Echocardiography confirmed decreased percentage of fractional shortening in DCM versus WT hearts. Histopathological analysis indicated a significant increase in myocardial disarray and fibrosis while the second harmonic generation imaging revealed disorganized sarcomeric structure and myocyte damage in DCM hearts when compared to WT hearts. Intriguingly, DCM mouse heart homogenates had decreased glutathione (GSH/GSSG) ratio and increased protein carbonyl and lipid malondialdehyde content compared to WT heart homogenates, consistent with elevated oxidative stress. Importantly, a similar result was observed in human cardiomyopathy heart homogenate samples. These results were further supported by reduced signals for mitochondrial semiquinone radicals and Fe-S clusters in DCM mouse hearts measured using electron paramagnetic resonance spectroscopy. In conclusion, we demonstrate elevated oxidative stress in MYPBC3-mutated DCM mice, which may exacerbate the development of heart failure. PMID:26508994

CREBBP knockdown enhances RAS/RAF/MEK/ERK signaling in Ras pathway mutated acute lymphoblastic leukemia but does not modulate chemotherapeutic response.

PubMed

Dixon, Zach A; Nicholson, Lindsay; Zeppetzauer, Martin; Matheson, Elizabeth; Sinclair, Paul; Harrison, Christine J; Irving, Julie A E

2017-04-01

Relapsed acute lymphoblastic leukemia is the most common cause of cancer-related mortality in young people and new therapeutic strategies are needed to improve outcome. Recent studies have shown that heterozygous inactivating mutations in the histone acetyl transferase, CREBBP , are particularly frequent in relapsed childhood acute lymphoblastic leukemia and associated with a hyperdiploid karyotype and KRAS mutations. To study the functional impact of CREBBP haploinsufficiency in acute lymphoblastic leukemia, RNA interference was used to knock down expression of CREBBP in acute lymphoblastic leukemia cell lines and various primagraft acute lymphoblastic leukemia cells. We demonstrate that attenuation of CREBBP results in reduced acetylation of histone 3 lysine 18, but has no significant impact on cAMP-dependent target gene expression. Impaired induction of glucocorticoid receptor targets was only seen in 1 of 4 CREBBP knockdown models, and there was no significant difference in glucocorticoid-induced apoptosis, sensitivity to other acute lymphoblastic leukemia chemotherapeutics or histone deacetylase inhibitors. Importantly, we show that CREBBP directly acetylates KRAS and that CREBBP knockdown enhances signaling of the RAS/RAF/MEK/ERK pathway in Ras pathway mutated acute lymphoblastic leukemia cells, which are still sensitive to MEK inhibitors. Thus, CREBBP mutations might assist in enhancing oncogenic RAS signaling in acute lymphoblastic leukemia but do not alter response to MEK inhibitors. Copyright© Ferrata Storti Foundation.

Inhibition of FLT3 Expression by Green Tea Catechins in FLT3 Mutated-AML Cells

PubMed Central

Ly, Bui Thi Kim; Chi, Hoang Thanh; Yamagishi, Makoto; Kano, Yasuhiko; Hara, Yukihiko; Nakano, Kazumi; Sato, Yuko; Watanabe, Toshiki

2013-01-01

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by a block in differentiation and uncontrolled proliferation. FLT3 is a commonly mutated gene found in AML patients. In clinical trials, the presence of a FLT3-ITD mutation significantly correlates with an increased risk of relapse and dismal overall survival. Therefore, activated FLT3 is a promising molecular target for AML therapies. In this study, we have shown that green tea polyphenols including (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), and (−)-epicatechin-3-gallate (ECG) suppress the proliferation of AML cells. Interestingly, EGCG, EGC and ECG showed the inhibition of FLT3 expression in cell lines harboring FLT3 mutations. In the THP-1 cells harboring FLT3 wild-type, EGCG showed the suppression of cell proliferation but did not suppress the expression of FLT3 even at the concentration that suppress 100% cell proliferation. Moreover, EGCG-, EGC-and ECG-treated cells showed the suppression of MAPK, AKT and STAT5 phosphorylation. Altogether, we suggest that green tea polyphenols could serve as reagents for treatment or prevention of leukemia harboring FLT3 mutations. PMID:23840454

Novel mutations of CYP3A4 in Chinese.

PubMed

Hsieh, K P; Lin, Y Y; Cheng, C L; Lai, M L; Lin, M S; Siest, J P; Huang, J D

2001-03-01

Human cytochrome P450 3A4 is a major P450 enzyme in the liver and gastrointestinal tract. It plays important roles in the metabolism of a wide variety of drugs, some endogenous steroids, and harmful environmental contaminants. CYP3A4 exhibits a remarkable interindividual activity variation as high as 20-fold. To investigate whether the interindividual variation in CYP3A4 levels can be partly explained by genetic polymorphism, we analyzed DNA samples from 102 Chinese subjects by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis for novel point mutation in the CYP3A4 coding sequence and promoter region. Using PCR and directed sequencing method to establish the complete intron sequence of CYP3A4 from leukocytes, the complete genomic sequence from exon 1 through 13 of CYP3A4 was determined and published in the GenBank database (accession no. AF209389). CYP3A4-specific primers were designed accordingly. After PCR-single-strand conformation polymorphism and restriction fragment length polymorphism screening, we found three novel mutations; two are point mutations and one is insertion. The first variant allele (CYP3A4*4), an Ile118Val change, was found in 3 of 102 Chinese subjects. The next allele (CYP3A4*5), which causes a Pro218Arg amino acid change, was found in 2 of 102 subjects. We found an insertion in A(17776), designated as CYP3A4*6, which causes frame shift and an early stop codon in exon 9, in one heterozygous subject. We also investigated the CYP3A4 activity in these mutant subjects by measuring the morning spot urinary 6beta-hydroxycortisol to free cortisol ratio with the enzyme-linked immunosorbent assay method. When compared with healthy Chinese population data, the 6beta-hydroxycortisol to free cortisol ratio data suggested that these alleles (CYP3A4*4, CYP3A4*5, and CYP3A4*6) may decrease the CYP3A4 activity. Incidences of these mutations in Chinese subjects are rare. The prevalence of these point mutations in other ethnic

IDENTIFICATION OF NOVEL FIBROBLAST GROWTH FACTOR RECEPTOR 3 GENE MUTATIONS IN ACTINIC CHEILITIS

PubMed Central

Chou, Annie; Dekker, Nusi; Jordan, Richard C.K.

2009-01-01

Objective Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several craniosynostosis and chondrodysplasia syndromes as well as some human cancers including bladder and cervical carcinoma. Despite a high frequency in some benign skin disorders, FGFR3 mutations have not been reported in cutaneous malignancies. Actinic cheilitis (AC) is a sun-induced premalignancy affecting the lower lip that frequently progresses to squamous cell carcinoma (SCC). The objective of this study was to determine if FGFR3 gene mutations are present in AC and SCC of the lip. Study Design DNA was extracted and purified from micro-dissected, formalin-fixed, paraffin-embedded tissue sections of 20 cases of AC and SCC arising in AC. Exons 7, 15, and 17 were PCR amplified and direct sequenced. Results Four novel somatic mutations in the FGFR3 gene were identified: exon 7 mutation 742C→T (amino acid change R248C), exon 15 mutations 1850A→G (D617G) and 1888G→A (V630M), and exon 17 mutation 2056G→A (E686K). Grade of dysplasia did not correlate with presence of mutations. Conclusion The frequency of FGFR3 receptor mutations suggests a functional role for the FGFR3 receptor in the development of epithelial disorders and perhaps a change may contribute to the pathogenesis of some AC and SCC. PMID:19327639

Pyrosequencing®-Based Identification of Low-Frequency Mutations Enriched Through Enhanced-ice-COLD-PCR.

PubMed

How-Kit, Alexandre; Tost, Jörg

2015-01-01

A number of molecular diagnostic assays have been developed in the last years for mutation detection. Although these methods have become increasingly sensitive, most of them are incompatible with a sequencing-based readout and require prior knowledge of the mutation present in the sample. Consequently, coamplification at low denaturation (COLD)-PCR-based methods have been developed and combine a high analytical sensitivity due to mutation enrichment in the sample with the identification of known or unknown mutations by downstream sequencing experiments. Among these methods, the recently developed Enhanced-ice-COLD-PCR appeared as the most powerful method as it outperformed the other COLD-PCR-based methods in terms of the mutation enrichment and due to the simplicity of the experimental setup of the assay. Indeed, E-ice-COLD-PCR is very versatile as it can be used on all types of PCR platforms and is applicable to different types of samples including fresh frozen, FFPE, and plasma samples. The technique relies on the incorporation of an LNA containing blocker probe in the PCR reaction followed by selective heteroduplex denaturation enabling amplification of the mutant allele while amplification of the wild-type allele is prevented. Combined with Pyrosequencing(®), which is a very quantitative high-resolution sequencing technology, E-ice-COLD-PCR can detect and identify mutations with a limit of detection down to 0.01 %.

Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer.

PubMed

Rodríguez-Fernández, Lucía; Ferrer-Vicens, Iván; García, Concha; Oltra, Sara S; Zaragozá, Rosa; Viña, Juan R; García-Trevijano, Elena R

2016-09-15

Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca(2+)-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-1 were cleaved as assessed by western blot analysis. Both CAPNs were able to cleave adhesion proteins from lactating mammary gland in vitro Nevertheless, CAPN2 was the only isoform found to co-localize with E-cadherin in cell junctions at the peak of lactation. CAPN2/E-cadherin in vivo interaction, analyzed by proximity ligation assay, was dramatically increased during involution. Calpain inhibitor administration prevented the cytosolic accumulation of truncated E-cadherin cleaved by CAPN2. Conversely, in breast cancer cells, CAPN2 was restricted to the nuclear compartment. The isoform-specific expression of CAPNs and CAPN activity was dependent on the breast cancer subtype. However, CAPN1 and CAPN2 knockdown cells showed that cleavage of adhesion proteins and cell migration was mediated by CAPN1, independently of the breast cancer cell line used. Data presented here suggest that the subcellular distribution of CAPN1 and CAPN2 is a major issue in target-substrate recognition; therefore, it determines the isoform-specific role of CAPNs during disruption of cell adhesion in either a physiological or a pathological context. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Mutation Update and Genotype–Phenotype Correlations of Novel and Previously Described Mutations in TPM2 and TPM3 Causing Congenital Myopathies

PubMed Central

Marttila, Minttu; Lehtokari, Vilma-Lotta; Marston, Steven; Nyman, Tuula A.; Barnerias, Christine; Beggs, Alan H.; Bertini, Enrico; Ceyhan-Birsoy, OÖzge; Cintas, Pascal; Gerard, Marion; Gilbert-Dussardier, Brigitte; Hogue, Jacob S.; Longman, Cheryl; Eymard, Bruno; Frydman, Moshe; Kang, Peter B.; Klinge, Lars; Kolski, Hanna; Lochmüller, Hans; Magy, Laurent; Manel, Véronique; Mayer, Michèle; Mercuri, Eugenio; North, Kathryn N.; Peudenier-Robert, Sylviane; Pihko, Helena; Probst, Frank J.; Reisin, Ricardo; Stewart, Willie; Taratuto, Ana Lia; de Visser, Marianne; Wilichowski, Ekkehard; Winer, John; Nowak, Kristen; Laing, Nigel G.; Winder, Tom L.; Monnier, Nicole; Clarke, Nigel F.; Pelin, Katarina; Grönholm, Mikaela; Wallgren-Pettersson, Carina

2014-01-01

Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core-rod myopathy, congenital fiber-type disproportion, distal arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal-dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca2+ sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non-hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin–actin association or tropomyosin head-to-tail binding. PMID:24692096

The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations.

PubMed

Flores-Rozas, H; Kolodner, R D

1998-10-13

The Saccharomyces cerevisiae genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of MLH3 in mismatch repair. Mutations in MLH3 increased the rate of reversion of the hom3-10 allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3-10 reversion rate, whereas the hom3-10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex.

Mutations in the deubiquitinase gene USP8 cause Cushing's disease.

PubMed

Reincke, Martin; Sbiera, Silviu; Hayakawa, Akira; Theodoropoulou, Marily; Osswald, Andrea; Beuschlein, Felix; Meitinger, Thomas; Mizuno-Yamasaki, Emi; Kawaguchi, Kohei; Saeki, Yasushi; Tanaka, Keiji; Wieland, Thomas; Graf, Elisabeth; Saeger, Wolfgang; Ronchi, Cristina L; Allolio, Bruno; Buchfelder, Michael; Strom, Tim M; Fassnacht, Martin; Komada, Masayuki

2015-01-01

Cushing's disease is caused by corticotroph adenomas of the pituitary. To explore the molecular mechanisms of endocrine autonomy in these tumors, we performed exome sequencing of 10 corticotroph adenomas. We found somatic mutations in the USP8 deubiquitinase gene in 4 of 10 adenomas. The mutations clustered in the 14-3-3 protein binding motif and enhanced the proteolytic cleavage and catalytic activity of USP8. Cleavage of USP8 led to increased deubiqutination of the EGF receptor, impairing its downregulation and sustaining EGF signaling. USP8 mutants enhanced promoter activity of the gene encoding proopiomelanocortin. In summary, our data show that dominant mutations in USP8 cause Cushing's disease via activation of EGF receptor signaling.

Detection of IDH1 mutation in the plasma of patients with glioma.

PubMed

Boisselier, Blandine; Gállego Pérez-Larraya, Jaime; Rossetto, Marta; Labussière, Marianne; Ciccarino, Pietro; Marie, Yannick; Delattre, Jean-Yves; Sanson, Marc

2012-10-16

The IDH1(R132H) mutation is both a strong prognostic predictor and a diagnostic hallmark of gliomas and therefore has major clinical relevance. Here, we developed a new technique to detect the IDH1(R132H) mutation in the plasma of patients with glioma. Small-size DNA (150-250 base pairs) was extracted from the plasma of 31 controls and 80 patients with glioma with known IDH1(R132H) status and correlated with MRI data. The IDH1(R132H) mutation was detected by a combination of coamplification at lower denaturation temperature and digital PCR. The small size DNA concentration was 1.2 ng/mL (range 0.1-6.6) in controls vs 1.2 ng/mL (range 0.1-50.3) in patients with glioma (p = not significant) and 0.9 ng/mL (0.0-3.0) in low-grade gliomas vs 1.5 ng/mL in high-grade gliomas (p < 0.01). The small size DNA concentration correlated with enhancing tumor volume (1.6 ng/mL [0.4-24.9] when <10 cm(3) and 14.0 ng/mL [0.6-50.3] when ≥10 cm(3)). The IDH1(R132H) mutation was detected in 15 out of 25 plasma DNA mixtures (60%) from patients with mutated tumors and in none of the 14 patients with a nonmutated tumor. The sensitivity increased with enhancing tumor volume (3/9 in nonenhancing tumors, 6/10 for enhancing volume <10 cm(3), and 6/6 for enhancing volume ≥10 cm(3)). With a specificity of 100% and a sensitivity related to the tumor volume and contrast enhancement, IDH1(R132H) identification has a valuable diagnostic accuracy in patients not amenable to biopsy.

Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins

PubMed Central

Frederix, Marijke; Edwards, Anne; Swiderska, Anna; Stanger, Andrew; Karunakaran, Ramakrishnan; Williams, Alan; Abbruscato, Pamela; Sanchez-Contreras, Maria; Poole, Philip S; Downie, J Allan

2014-01-01

In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots. PMID:24942546

Etomidate blocks LTP and impairs learning but does not enhance tonic inhibition in mice carrying the N265M point mutation in the beta3 subunit of the GABAA receptor

PubMed Central

Oh, I; Rau, V; Lor, C; Laha, KT; Jurd, R; Rudolph, U; Eger, EI; Pearce, RA

2015-01-01

Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with β3 subunits, we tested the hypothesis that etomidate acts through β3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR β3-subunit (β3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by β3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5β1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 [.proportional]M and below), whereas α5β2γ2L and α5β3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5β3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5β2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons). PMID:25680234

Enhanced hydrogen production of Enterobacter aerogenes mutated by nuclear irradiation.

PubMed

Cheng, Jun; Liu, Min; Song, Wenlu; Ding, Lingkan; Liu, Jianzhong; Zhang, Li; Cen, Kefa

2017-03-01

Nuclear irradiation was used for the first time to generate efficient mutants of hydrogen-producing bacteria Enterobacter aerogenes, which were screened with larger colour circles of more fermentative acid by-products. E. aerogenes cells were mutated by nuclear irradiation of 60 Co γ-rays. The screened E. aerogenes ZJU1 mutant with larger colour circles enhanced the hydrogenase activity from 89.8 of the wild strain to 157.4mLH 2 /(gDWh). The hereditary stability of the E. aerogenes ZJU1 mutant was certified after over ten generations of cultivation. The hydrogen yield of 301mLH 2 /gglucose with the mutant was higher by 81.8% than that of 166mL/gglucose with the wild strain. The peak hydrogen production rate of 27.2mL/(L·h) with the mutant was higher by 40.9% compared with that of 19.3mL/(L·h) with the wild strain. The mutant produced more acetate and butyrate but less ethanol compared with the wild strain during hydrogen fermentation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts

PubMed Central

Li, S; Li, L; Zhu, Y; Huang, C; Qin, Y; Liu, H; Ren-Heidenreich, L; Shi, B; Ren, H; Chu, X; Kang, J; Wang, W; Xu, J; Tang, K; Yang, H; Zheng, Y; He, J; Yu, G; Liang, N

2014-01-01

Background: Determining the somatic mutations of epidermal growth factor receptor (EGFR)-pathway networks is the key to effective treatment for non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitors (TKIs).The somatic mutation frequencies and their association with gender, smoking history and histology was analysed and reported in this study. Methods: Five thousand one hundred and twenty-five NSCLC patients' pathology samples were collected, and EGFR, KRAS, BRAF and PIK3CA mutations were detected by multiplex testing. The mutation status of EGFR, KRAS, BRAF and PIK3CA and their association with gender, age, smoking history and histological type were evaluated by appropriate statistical analysis. Results: EGFR, KRAS, BRAF and PIK3CA mutation rates revealed 36.2%, 8.4%, 0.5% and 3.3%, respectively, across the 5125 pathology samples. For the first time, evidence of KRAS mutations were detected in two female, non-smoking patients, age 5 and 14, with NSCLC. Furthermore, we identified 153 double and coexisting mutations and 7 triple mutations. Interestingly, the second drug-resistant mutations, T790M or E545K, were found in 44 samples from patients who had never received TKI treatments. Conclusions: EGFR exons 19, 20 and 21, and BRAF mutations tend to happen in females and non-smokers, whereas KRAS mutations were more inclined to males and smokers. Activating and resistant mutations to EGFR-TKI drugs can coexist and ‘second drug-resistant mutations', T790M or E545K, may be primary mutations in some patients. These results will help oncologists to decide candidates for mutation testing and EGFR-TKI treatment. PMID:24743704

Germline LEMD3 mutations are rare in sporadic patients with isolated melorheostosis.

PubMed

Hellemans, Jan; Debeer, Philippe; Wright, Michael; Janecke, Andreas; Kjaer, Klaus W; Verdonk, Peter C M; Savarirayan, Ravi; Basel, Lina; Moss, Celia; Roth, Johannes; David, Albert; De Paepe, Anne; Coucke, Paul; Mortier, Geert R

2006-03-01

To further explore the allelic heterogeneity within the group of LEMD3-related disorders, we have screened a larger series of patients including 5 probands with osteopoikilosis or Buschke-Ollendorff syndrome (BOS), 2 families with the co-occurrence of melorheostosis and BOS, and 12 unrelated patients with isolated melorheostosis. Seven novel LEMD3 mutations were identified, all predicted to result in loss-of-function of the protein. We confirm that loss-of-function mutations in the LEMD3 gene can result in either osteopoikilosis or BOS. However, LEMD3 germline mutations were only found in two melorheostosis patients belonging to a different BOS family and one sporadic patient with melorheostosis. The additional presence of osteopoikilosis lesions in these patients seemed to distinguish them from the group of sporadic melorheostosis patients where no germline LEMD3 mutation was identified. Somatic mosaicism for a LEMD3 mutation in the latter group was also not observed, and therefore we must conclude that the genetic defect in the majority of sporadic and isolated melorheostosis remains unknown. 2006 Wiley-Liss, Inc.

Mutations in the G6PC3 gene cause Dursun syndrome.

PubMed

Banka, Siddharth; Newman, William G; Ozgül, R Koksal; Dursun, Ali

2010-10-01

Dursun syndrome is a triad of familial primary pulmonary hypertension, leucopenia, and atrial septal defect. Here we demonstrate that mutations in G6PC3 cause Dursun syndrome. Mutations in G6PC3 are known to also cause severe congenital neutropenia type 4. Identification of the genetic basis of Dursun syndrome expands the pre-existing knowledge about the phenotypic effects of mutations in G6PC3. We propose that Dursun syndrome should now be considered as a subset of severe congenital neutropenia type 4 with pulmonary hypertension as an important clinical feature. Copyright © 2010 Wiley-Liss, Inc.

Estimation and enhancement of real-time software reliability through mutation analysis

NASA Technical Reports Server (NTRS)

Geist, Robert; Offutt, A. J.; Harris, Frederick C., Jr.

1992-01-01

A simulation-based technique for obtaining numerical estimates of the reliability of N-version, real-time software is presented. An extended stochastic Petri net is employed to represent the synchronization structure of N versions of the software, where dependencies among versions are modeled through correlated sampling of module execution times. Test results utilizing specifications for NASA's planetary lander control software indicate that mutation-based testing could hold greater potential for enhancing reliability than the desirable but perhaps unachievable goal of independence among N versions.

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.

Production and processing studies on calpain-system gene markers for tenderness in Brahman cattle: 2. Objective meat quality.

PubMed

Cafe, L M; McIntyre, B L; Robinson, D L; Geesink, G H; Barendse, W; Pethick, D W; Thompson, J M; Greenwood, P L

2010-09-01

Effects and interactions of calpain-system tenderness gene markers on objective meat quality traits of Brahman (Bos indicus) cattle were quantified within 2 concurrent experiments at different locations. Cattle were selected for study from commercial and research herds at weaning based on their genotype for calpastatin (CAST) and calpain 3 (CAPN3) gene markers for beef tenderness. Gene marker status for mu-calpain (CAPN1-4751 and CAPN1-316) was also determined for inclusion in statistical analyses. Eighty-two heifer and 82 castrated male cattle with 0 or 2 favorable alleles for CAST and CAPN3 were studied in New South Wales (NSW), and 143 castrated male cattle with 0, 1, or 2 favorable alleles for CAST and CAPN3 were studied in Western Australia (WA). The cattle were backgrounded for 6 to 8 mo and grain-fed for 117 d (NSW) or 80 d (WA) before slaughter. One-half the cattle in each experiment were implanted with a hormonal growth promotant during feedlotting. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis (tenderstretch). The M. longissimus lumborum from both sides and the M. semitendinosus from the AT side were collected; then samples of each were aged at 1 degrees C for 1 or 7 d. Favorable alleles for one or more markers reduced shear force, with little effect on other meat quality traits. The size of effects of individual markers varied with site, muscle, method of carcass suspension, and aging period. Individual marker effects were additive as evident in cattle with 4 favorable alleles for CAST and CAPN3 markers, which had shear force reductions of 12.2 N (P < 0.001, NSW) and 9.3 N (P = 0.002, WA) in AT 7 d aged M. longissimus lumborum compared with those with no favorable alleles. There was no evidence (all P > 0.05) of interactions between the gene markers, or between the hormonal growth promotant and gene markers for any meat quality traits. This study provides further evidence that selection based on the

Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins.

PubMed

Frederix, Marijke; Edwards, Anne; Swiderska, Anna; Stanger, Andrew; Karunakaran, Ramakrishnan; Williams, Alan; Abbruscato, Pamela; Sanchez-Contreras, Maria; Poole, Philip S; Downie, J Allan

2014-08-01

In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Novel ENAM and LAMB3 mutations in Chinese families with hypoplastic amelogenesis imperfecta.

PubMed

Wang, Xin; Zhao, Yuming; Yang, Yuan; Qin, Man

2015-01-01

Amelogenesis imperfecta is a group of inherited diseases affecting the quality and quantity of dental enamel. To date, mutations in more than ten genes have been associated with non-syndromic amelogenesis imperfecta (AI). Among these, ENAM and LAMB3 mutations are known to be parts of the etiology of hypoplastic AI in human cases. When both alleles of LAMB3 are defective, it could cause junctional epidermolysis bullosa (JEB), while with only one mutant allele in the C-terminus of LAMB3, it could result in severe hypoplastic AI without skin fragility. We enrolled three Chinese families with hypoplastic autosomal-dominant AI. Despite the diagnosis falling into the same type, the characteristics of their enamel hypoplasia were different. Screening of ENAM and LAMB3 genes was performed by direct sequencing of genomic DNA from blood samples. Disease-causing mutations were identified and perfectly segregated with the enamel defects in three families: a 19-bp insertion mutation in the exon 7 of ENAM (c.406_407insTCAAAAAAGCCGACCACAA, p.K136Ifs*16) in Family 1, a single-base deletion mutation in the exon 5 of ENAM (c. 139delA, p. M47Cfs*11) in Family 2, and a LAMB3 nonsense mutation in the last exon (c.3466C>T, p.Q1156X) in Family 3. Our results suggest that heterozygous mutations in ENAM and LAMB3 genes can cause hypoplastic AI with markedly different phenotypes in Chinese patients. And these findings extend the mutation spectrum of both genes and can be used for mutation screening of AI in the Chinese population.

Mutation and prognostic analyses of PIK3CA in patients with completely resected lung adenocarcinoma.

PubMed

Song, Zhengbo; Yu, Xinmin; Zhang, Yiping

2016-10-01

PIK3CA mutation represents a clinical subset of diverse carcinomas. We explored the status of PIK3CA mutation and evaluated its genetic variability, treatment, and prognosis in patients with lung adenocarcinoma. A total of 810 patients with completely resected lung adenocarcinoma were recruited between 2008 and 2013. The status of PIK3CA mutation and other three genes, that is, EGFR mutation, KRAS mutation and ALK fusion were examined by reverse transcription-polymerase chain reaction (RT-PCR). Survival curves were plotted with the Kaplan-Meier method and log-rank for comparison. Cox proportional hazard model was performed for multivariate analysis. Among the 810 patients, 23 cases of PIK3CA mutation were identified with a frequency of 2.8%. There were 14 men and 9 women with a median age of 61 years. Seventeen tumors revealed concurrent gene abnormalities of EGFR mutation (n = 12), KRAS mutation (n = 3), and ALK fusion (n = 2). Seven patients with EGFR & PIK3CA mutations recurred and administrated of EGFR-TKIs yielded a median progression free-survival of 6.0 months. Among four eviromous-treated patients, stable disease was observed in three patients with a median Progression-free survival (PFS) of 3.5 months. Patients with and without PIK3CA mutation had different overall survivals (32.2 vs. 49.6 months, P = 0.003). Multivariate analysis revealed that PIK3CA mutation was an independent predictor of poor overall survival (HR = 2.37, P = 0.017). The frequency of PIK3CA mutation was around 2.8% in the Chinese patients of lung adenocarcinoma. PIK3CA mutation was associated with reduced PFS of EGFR-TKIs treatment and shorter overall survival. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Consequences of a novel caveolin-3 mutation in a large German family.

PubMed

Fischer, Dirk; Schroers, Anja; Blümcke, Ingmar; Urbach, Horst; Zerres, Klaus; Mortier, Wilhelm; Vorgerd, Matthias; Schröder, Rolf

2003-02-01

Mutations in the human caveolin-3 gene (cav-3) on chromosome 3p25 have been described in limb girdle muscular dystrophy, rippling muscle disease, hyperCKemia, and distal myopathy. Here, we describe the genetic, myopathological, and clinical findings in a large German family harboring a novel heterozygous mutation (GAC-->GAA) in codon 27 of the cav-3 gene. This missense mutation causes an amino acid change from asparagine to glutamate (Asp27Glu) in the N-terminal region of the Cav-3 protein, which leads to a drastic decrease of Cav-3 protein expression in skeletal muscle tissue. In keeping with an autosomal dominant mode of inheritance, this novel cav-3 mutation was found to cosegregate with neuromuscular involvement in the reported family. Ultrastructural analysis of Cav-3-deficient muscle showed an abnormal folding of the plasma membrane as well as multiple vesicular structures in the subsarcolemmal region. Neurological examination of all nine subjects from three generations harboring the novel cav-3 mutation showed clear evidence of rippling muscle disease. However, only two of these nine patients showed isolated signs of rippling muscle disease without muscle weakness or atrophy, whereas five had additional signs of a distal myopathy and two fulfilled the diagnostic criteria of a coexisting limb girdle muscular dystrophy. These findings indicate that mutations in the human cav-3 gene can lead to different and overlapping clinical phenotypes even within the same family. Different clinical phenotypes in caveolinopathies may be attributed to so far unidentified modifying factors/genes in the individual genetic background of affected patients.

Whole-exome sequencing, without prior linkage, identifies a mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta.

PubMed

Poulter, James A; El-Sayed, Walid; Shore, Roger C; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

2014-01-01

The conventional approach to identifying the defective gene in a family with an inherited disease is to find the disease locus through family studies. However, the rapid development and decreasing cost of next generation sequencing facilitates a more direct approach. Here, we report the identification of a frameshift mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta (AI). Whole-exome sequencing of three affected family members and subsequent filtering of shared variants, without prior genetic linkage, sufficed to identify the pathogenic variant. Simultaneous analysis of multiple family members confirms segregation, enhancing the power to filter the genetic variation found and leading to rapid identification of the pathogenic variant. LAMB3 encodes a subunit of Laminin-5, one of a family of basement membrane proteins with essential functions in cell growth, movement and adhesion. Homozygous LAMB3 mutations cause junctional epidermolysis bullosa (JEB) and enamel defects are seen in JEB cases. However, to our knowledge, this is the first report of dominant AI due to a LAMB3 mutation in the absence of JEB.

A novel MKRN3 missense mutation causing familial precocious puberty.

PubMed

de Vries, L; Gat-Yablonski, G; Dror, N; Singer, A; Phillip, M

2014-12-01

Central precocious puberty may be familial in about a quarter of the idiopathic cases. However, little is known about the genetic causes responsible for the disorder. In this report we describe a family with central precocious puberty associated with a mutation in the makorin RING-finger protein 3 (MKRN3) gene. A novel missense mutation (p.H420Q) in the imprinted MKRN3 gene was identified in the four affected siblings, in their unaffected father and in his affected mother. An in silico mutant MKRN3 model predicts that the mutation p.H420Q leads to reduced zinc binding and, subsequently, impaired RNA binding. These findings support the fundamental role of the MKRN3 protein in determining pubertal timing. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mutation analysis of the human CYP3A4 gene 5' regulatory region: population screening using non-radioactive SSCP.

PubMed

Hamzeiy, Hossein; Vahdati-Mashhadian, Nasser; Edwards, Helen J; Goldfarb, Peter S

2002-03-20

Human CYP3A4 is the major cytochrome P450 isoenzyme in adult human liver and is known to metabolise many xenobiotic and endogenous compounds. There is substantial inter-individual variation in the hepatic levels of CYP3A4. Although, polymorphic mutations have been reported in the 5' regulatory region of the CYP3A4 gene, those that have been investigated so far do not appear to have any effect on gene expression. To determine whether other mutations exist in this region of the gene, we have performed a new population screen on a panel of 101 human DNA samples. A 1140 bp section of the 5' proximal regulatory region of the CYP3A4 gene, containing numerous regulatory motifs, was amplified from genomic DNA as three overlapping segments. The 300 bp distal enhancer region at -7.9kb containing additional regulatory motifs was also amplified. Mutation analysis of the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) and confirmatory sequencing of both DNA strands in those samples showing extra SSCP bands. In addition to detection of the previously reported CYP3A4*1B allele in nine subjects, three novel alleles were found: CYP3A4*1E (having a T-->A transversion at -369 in one subject), CYP3A4*1F (having a C-->G tranversion at -747 in 17 subjects) and CYP3A4*15B containing a nine-nucleotide insertion between -845 and -844 linked to an A-->G transition at -392 and a G-->A transition in exon 6 (position 485 in the cDNA) in one subject. All the novel alleles were heterozygous. No mutations were found in the upstream distal enhancer region. Our results clearly indicate that this rapid and simple SSCP approach can reveal mutant alleles in drug metabolising enzyme genes. Detection and determination of the frequency of novel alleles in CYP3A4 will assist investigation of the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.

Gain-of-function STAT1 mutations impair STAT3 activity in patients with chronic mucocutaneous candidiasis (CMC).

PubMed

Zheng, Jie; van de Veerdonk, Frank L; Crossland, Katherine L; Smeekens, Sanne P; Chan, Chun M; Al Shehri, Tariq; Abinun, Mario; Gennery, Andrew R; Mann, Jelena; Lendrem, Dennis W; Netea, Mihai G; Rowan, Andrew D; Lilic, Desa

2015-10-01

Signal transducer and activator of transcription 3 (STAT3) triggered production of Th-17 cytokines mediates protective immunity against fungi. Mutations affecting the STAT3/interleukin 17 (IL-17) pathway cause selective susceptibility to fungal (Candida) infections, a hallmark of chronic mucocutaneous candidiasis (CMC). In patients with autosomal dominant CMC, we and others previously reported defective Th17 responses and underlying gain-of-function (GOF) STAT1 mutations, but how this affects STAT3 function leading to decreased IL-17 is unclear. We also assessed how GOF-STAT1 mutations affect STAT3 activation, DNA binding, gene expression, cytokine production, and epigenetic modifications. We excluded impaired STAT3 phosphorylation, nuclear translocation, and sequestration of STAT3 into STAT1/STAT3 heterodimers and confirm significantly reduced transcription of STAT3-inducible genes (RORC/IL-17/IL-22/IL-10/c-Fos/SOCS3/c-Myc) as likely underlying mechanism. STAT binding to the high affinity sis-inducible element was intact but binding to an endogenous STAT3 DNA target was impaired. Reduced STAT3-dependent gene transcription was reversed by inhibiting STAT1 activation with fludarabine or enhancing histone, but not STAT1 or STAT3 acetylation with histone deacetylase (HDAC) inhibitors trichostatin A or ITF2357. Silencing HDAC1, HDAC2, and HDAC3 indicated a role for HDAC1 and 2. Reduced STAT3-dependent gene transcription underlies low Th-17 responses in GOF-STAT1 CMC, which can be reversed by inhibiting acetylation, offering novel targets for future therapies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The SHOX region and its mutations.

PubMed

Capone, L; Iughetti, L; Sabatini, S; Bacciaglia, A; Forabosco, A

2010-06-01

The short stature homeobox-containing (SHOX) gene lies in the pseudoautosomal region 1 (PAR1) that comprises 2.6 Mb of the short-arm tips of both the X and Y chromosomes. It is known that its heterozygous mutations cause Leri-Weill dyschondrosteosis (LWD) (OMIM #127300), while its homozygous mutations cause a severe form of dwarfism known as Langer mesomelic dysplasia (LMD) (OMIM #249700). The analysis of 238 LWD patients between 1998 and 2007 by multiple authors shows a prevalence of deletions (46.4%) compared to point mutations (21.2%). On the whole, deletions and point mutations account for about 67% of LWD patients. SHOX is located within a 1000 kb desert region without genes. The comparative genomic analysis of this region between genomes of different vertebrates has led to the identification of evolutionarily conserved non-coding DNA elements (CNE). Further functional studies have shown that one of these CNE downstream of the SHOX gene is necessary for the expression of SHOX; this is considered to be typical "enhancer" activity. Including the enhancer, the overall mutation of the SHOX region in LWD patients does not hold in 100% of cases. Various authors have demonstrated the existence of other CNE both downstream and upstream of SHOX regions. The resulting conclusion is that it is necessary to reanalyze all LWD/LMD patients without SHOX mutations for the presence of mutations in the 5'- and 3'-flanking SHOX regions.

Mutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis.

PubMed

Johnson, Janel O; Pioro, Erik P; Boehringer, Ashley; Chia, Ruth; Feit, Howard; Renton, Alan E; Pliner, Hannah A; Abramzon, Yevgeniya; Marangi, Giuseppe; Winborn, Brett J; Gibbs, J Raphael; Nalls, Michael A; Morgan, Sarah; Shoai, Maryam; Hardy, John; Pittman, Alan; Orrell, Richard W; Malaspina, Andrea; Sidle, Katie C; Fratta, Pietro; Harms, Matthew B; Baloh, Robert H; Pestronk, Alan; Weihl, Conrad C; Rogaeva, Ekaterina; Zinman, Lorne; Drory, Vivian E; Borghero, Giuseppe; Mora, Gabriele; Calvo, Andrea; Rothstein, Jeffrey D; Drepper, Carsten; Sendtner, Michael; Singleton, Andrew B; Taylor, J Paul; Cookson, Mark R; Restagno, Gabriella; Sabatelli, Mario; Bowser, Robert; Chiò, Adriano; Traynor, Bryan J

2014-05-01

MATR3 is an RNA- and DNA-binding protein that interacts with TDP-43, a disease protein linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Using exome sequencing, we identified mutations in MATR3 in ALS kindreds. We also observed MATR3 pathology in ALS-affected spinal cords with and without MATR3 mutations. Our data provide more evidence supporting the role of aberrant RNA processing in motor neuron degeneration.

SF3B1 mutations constitute a novel therapeutic target in breast cancer

PubMed Central

Maguire, Sarah L; Leonidou, Andri; Wai, Patty; Marchiò, Caterina; Ng, Charlotte KY; Sapino, Anna; Salomon, Anne-Vincent; Reis-Filho, Jorge S; Weigelt, Britta; Natrajan, Rachael C

2015-01-01

Mutations in genes encoding proteins involved in RNA splicing have been found to occur at relatively high frequencies in several tumour types including myelodysplastic syndromes, chronic lymphocytic leukaemia, uveal melanoma, and pancreatic cancer, and at lower frequencies in breast cancer. To investigate whether dysfunction in RNA splicing is implicated in the pathogenesis of breast cancer, we performed a re-analysis of published exome and whole genome sequencing data. This analysis revealed that mutations in spliceosomal component genes occurred in 5.6% of unselected breast cancers, including hotspot mutations in the SF3B1 gene, which were found in 1.8% of unselected breast cancers. SF3B1 mutations were significantly associated with ER-positive disease, AKT1 mutations, and distinct copy number alterations. Additional profiling of hotspot mutations in a panel of special histological subtypes of breast cancer showed that 16% and 6% of papillary and mucinous carcinomas of the breast harboured the SF3B1 K700E mutation. RNA sequencing identified differentially spliced events expressed in tumours with SF3B1 mutations including the protein coding genes TMEM14C, RPL31, DYNL11, UQCC, and ABCC5, and the long non-coding RNA CRNDE. Moreover, SF3B1 mutant cell lines were found to be sensitive to the SF3b complex inhibitor spliceostatin A and treatment resulted in perturbation of the splicing signature. Albeit rare, SF3B1 mutations result in alternative splicing events, and may constitute drivers and a novel therapeutic target in a subset of breast cancers. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:25424858

FGFR2 Point Mutations in 466 Endometrioid Endometrial Tumors: Relationship with MSI, KRAS, PIK3CA, CTNNB1 Mutations and Clinicopathological Features

PubMed Central

Powell, Matthew A.; Wellens, Candice L.; Gao, Feng; Mutch, David G.; Goodfellow, Paul J.; Pollock, Pamela M.

2012-01-01

Mutations in multiple oncogenes including KRAS, CTNNB1, PIK3CA and FGFR2 have been identified in endometrial cancer. The aim of this study was to provide insight into the clinicopathological features associated with patterns of mutation in these genes, a necessary step in planning targeted therapies for endometrial cancer. 466 endometrioid endometrial tumors were tested for mutations in FGFR2, KRAS, CTNNB1, and PIK3CA. The relationships between mutation status, tumor microsatellite instability (MSI) and clinicopathological features including overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan-Meier survival analysis and Cox proportional hazard models. Mutations were identified in FGFR2 (48/466); KRAS (87/464); CTNNB1 (88/454) and PIK3CA (104/464). KRAS and FGFR2 mutations were significantly more common, and CTNNB1 mutations less common, in MSI positive tumors. KRAS and FGFR2 occurred in a near mutually exclusive pattern (p = 0.05) and, surprisingly, mutations in KRAS and CTNNB1 also occurred in a near mutually exclusive pattern (p = 0.0002). Multivariate analysis revealed that mutation in KRAS and FGFR2 showed a trend (p = 0.06) towards longer and shorter DFS, respectively. In the 386 patients with early stage disease (stage I and II), FGFR2 mutation was significantly associated with shorter DFS (HR = 3.24; 95% confidence interval, CI, 1.35–7.77; p = 0.008) and OS (HR = 2.00; 95% CI 1.09–3.65; p = 0.025) and KRAS was associated with longer DFS (HR = 0.23; 95% CI 0.05–0.97; p = 0.045). In conclusion, although KRAS and FGFR2 mutations share similar activation of the MAPK pathway, our data suggest very different roles in tumor biology. This has implications for the implementation of anti-FGFR or anti-MEK biologic therapies. PMID:22383975

Genetic Correlations Greatly Increase Mutational Robustness and Can Both Reduce and Enhance Evolvability

PubMed Central

Greenbury, Sam F.; Schaper, Steffen; Ahnert, Sebastian E.; Louis, Ard A.

2016-01-01

Mutational neighbourhoods in genotype-phenotype (GP) maps are widely believed to be more likely to share characteristics than expected from random chance. Such genetic correlations should strongly influence evolutionary dynamics. We explore and quantify these intuitions by comparing three GP maps—a model for RNA secondary structure, the HP model for protein tertiary structure, and the Polyomino model for protein quaternary structure—to a simple random null model that maintains the number of genotypes mapping to each phenotype, but assigns genotypes randomly. The mutational neighbourhood of a genotype in these GP maps is much more likely to contain genotypes mapping to the same phenotype than in the random null model. Such neutral correlations can be quantified by the robustness to mutations, which can be many orders of magnitude larger than that of the null model, and crucially, above the critical threshold for the formation of large neutral networks of mutationally connected genotypes which enhance the capacity for the exploration of phenotypic novelty. Thus neutral correlations increase evolvability. We also study non-neutral correlations: Compared to the null model, i) If a particular (non-neutral) phenotype is found once in the 1-mutation neighbourhood of a genotype, then the chance of finding that phenotype multiple times in this neighbourhood is larger than expected; ii) If two genotypes are connected by a single neutral mutation, then their respective non-neutral 1-mutation neighbourhoods are more likely to be similar; iii) If a genotype maps to a folding or self-assembling phenotype, then its non-neutral neighbours are less likely to be a potentially deleterious non-folding or non-assembling phenotype. Non-neutral correlations of type i) and ii) reduce the rate at which new phenotypes can be found by neutral exploration, and so may diminish evolvability, while non-neutral correlations of type iii) may instead facilitate evolutionary exploration and so

A somatic-mutational process recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers in breast cancers

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

Glodzik, Dominik; Morganella, Sandro; Davies, Helen

Somatic rearrangements contribute to the mutagenized landscape of cancer genomes. Here, we systematically interrogated rearrangements in 560 breast cancers by using a piecewise constant fitting approach. We identified 33 hotspots of large (>100 kb) tandem duplications, a mutational signature associated with homologous-recombination-repair deficiency. Notably, these tandem-duplication hotspots were enriched in breast cancer germline susceptibility loci (odds ratio (OR) = 4.28) and breast-specific 'super-enhancer' regulatory elements (OR = 3.54). These hotspots may be sites of selective susceptibility to double-strand-break damage due to high transcriptional activity or, through incrementally increasing copy number, may be sites of secondary selective pressure. Furthermore, the transcriptomicmore » consequences ranged from strong individual oncogene effects to weak but quantifiable multigene expression effects. We thus present a somatic-rearrangement mutational process affecting coding sequences and noncoding regulatory elements and contributing a continuum of driver consequences, from modest to strong effects, thereby supporting a polygenic model of cancer development.« less

A somatic-mutational process recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers in breast cancers

DOE PAGES

Glodzik, Dominik; Morganella, Sandro; Davies, Helen; ...

2017-01-23

Somatic rearrangements contribute to the mutagenized landscape of cancer genomes. Here, we systematically interrogated rearrangements in 560 breast cancers by using a piecewise constant fitting approach. We identified 33 hotspots of large (>100 kb) tandem duplications, a mutational signature associated with homologous-recombination-repair deficiency. Notably, these tandem-duplication hotspots were enriched in breast cancer germline susceptibility loci (odds ratio (OR) = 4.28) and breast-specific 'super-enhancer' regulatory elements (OR = 3.54). These hotspots may be sites of selective susceptibility to double-strand-break damage due to high transcriptional activity or, through incrementally increasing copy number, may be sites of secondary selective pressure. Furthermore, the transcriptomicmore » consequences ranged from strong individual oncogene effects to weak but quantifiable multigene expression effects. We thus present a somatic-rearrangement mutational process affecting coding sequences and noncoding regulatory elements and contributing a continuum of driver consequences, from modest to strong effects, thereby supporting a polygenic model of cancer development.« less

The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

PubMed Central

Julias, J G; Kim, T; Arnold, G; Pathak, V K

1997-01-01

It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis. PMID:9151812

Cryopyrin-associated Periodic Syndrome Caused by a Myeloid-Restricted Somatic NLRP3 Mutation

PubMed Central

Zhou, Qing; Aksentijevich, Ivona; Wood, Geryl M.; Walts, Avram D.; Hoffmann, Patrycja; Remmers, Elaine F.; Kastner, Daniel L.; Ombrello, Amanda K.

2015-01-01

Objective To identify the cause of disease in an adult patient presenting with recent onset fevers, chills, urticaria, fatigue, and profound myalgia, who was negative for cryopyrin-associated periodic syndrome (CAPS) NLRP3 mutations by conventional Sanger DNA sequencing. Methods We performed whole-exome sequencing and targeted deep sequencing using DNA from the patient’s whole blood to identify a possible NLRP3 somatic mutation. We then screened for this mutation in subcloned NLRP3 amplicons from fibroblasts, buccal cells, granulocytes, negatively-selected monocytes, and T and B lymphocytes and further confirmed the somatic mutation by targeted sequencing of exon 3. Results We identified a previously reported CAPS-associated mutation, p.Tyr570Cys, with a mutant allele frequency of 15% based on exome data. Targeted sequencing and subcloning of NLRP3 amplicons confirmed the presence of the somatic mutation in whole blood at a ratio similar to the exome data. The mutant allele frequency was in the range of 13.3%–16.8% in monocytes and 15.2%–18% in granulocytes; Notably, this mutation was either absent or present at a very low frequency in B and T lymphocytes, buccal cells, and in the patient’s cultured fibroblasts. Conclusion These data document the possibility of myeloid-restricted somatic mosaicism in the pathogenesis of CAPS, underscoring the emerging role of massively-parallel sequencing in clinical diagnosis. PMID:25988971

Global profiling of proteolytically modified proteins in human metastatic hepatocellular carcinoma cell lines reveals CAPN2 centered network.

PubMed

Shen, Chengpin; Yu, Yanyan; Li, Hong; Yan, Guoquan; Liu, Mingqi; Shen, Huali; Yang, Pengyuan

2012-06-01

Proteolysis affects every protein at some point in its life cycle. Many biomarkers of disease or cancer are stable proteolytic fragments in biological fluids. There is great interest and a challenge in proteolytically modified protein study to identify physiologic protease-substrate relationships and find potential biomarkers. In this study, two human hepatocellular carcinoma (HCC) cell lines with different metastasis potential, MHCC97L, and HCCLM6, were researched with a high-throughput and sensitive PROTOMAP platform. In total 391 proteins were found to be proteolytically processed and many of them were cleaved into persistent fragments instead of completely degraded. Fragments related to 161 proteins had different expressions in these two cell lines. Through analyzing these significantly changed fragments with bio-informatic tools, several bio-functions such as tumor cell migration and anti-apoptosis were enriched. A proteolysis network was also built up, of which the CAPN2 centered subnetwork, including SPTBN1, ATP5B, and VIM, was more active in highly metastatic HCC cell line. Interestingly, proteolytic modifications of CD44 and FN1 were found to affect their secretion. This work suggests that proteolysis plays an important role in human HCC metastasis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Germinal mosaicism of PAX3 mutation caused Waardenburg syndrome type I.

PubMed

Chen, Kaitian; Zhan, Yuan; Wu, Xuan; Zong, Ling; Jiang, Hongyan

2018-01-01

Waardenburg syndrome mutations are most often recurrent or de novo. The rate of familial recurrence is low and families with several affected children are extremely rare. In this study, we aimed to clarify the underlying hereditary cause of Waardenburg syndrome type I in two siblings in a Chinese family, with a mother affected by prelingual mild hearing loss and a father who was negative for clinical symptoms of Waardenburg syndrome and had a normal hearing threshold. Complete characteristic features of the family members were recorded and genetic sequencing and parent-child relationship analyses were performed. The two probands were found to share double mutations in the PAX3/GJB2 genes that caused concurrent hearing loss in Waardenburg syndrome type I. Their mother carried the GJB2 c.109G > A homozygous mutation; however, neither the novel PAX3 c.592delG mutation, nor the Waardenburg syndrome phenotype, was observed in either parent. These previously unreported digenic mutations in PAX3/GJB2 resulted in deafness associated with Waardenburg syndrome type I in this family. To our knowledge, this is the first report describing germinal mosaicism in Waardenburg syndrome. This concept is important because it complicates genetic counseling of this family regarding the risk of recurrence of the mutations in subsequent pregnancies. Copyright © 2017 Elsevier B.V. All rights reserved.

Mutations in POLR3A and POLR3B Encoding RNA Polymerase III Subunits Cause an Autosomal-Recessive Hypomyelinating Leukoencephalopathy

PubMed Central

Saitsu, Hirotomo; Osaka, Hitoshi; Sasaki, Masayuki; Takanashi, Jun-ichi; Hamada, Keisuke; Yamashita, Akio; Shibayama, Hidehiro; Shiina, Masaaki; Kondo, Yukiko; Nishiyama, Kiyomi; Tsurusaki, Yoshinori; Miyake, Noriko; Doi, Hiroshi; Ogata, Kazuhiro; Inoue, Ken; Matsumoto, Naomichi

2011-01-01

Congenital hypomyelinating disorders are a heterogeneous group of inherited leukoencephalopathies characterized by abnormal myelin formation. We have recently reported a hypomyelinating syndrome characterized by diffuse cerebral hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum (HCAHC). We performed whole-exome sequencing of three unrelated individuals with HCAHC and identified compound heterozygous mutations in POLR3B in two individuals. The mutations include a nonsense mutation, a splice-site mutation, and two missense mutations at evolutionally conserved amino acids. Using reverse transcription-PCR and sequencing, we demonstrated that the splice-site mutation caused deletion of exon 18 from POLR3B mRNA and that the transcript harboring the nonsense mutation underwent nonsense-mediated mRNA decay. We also identified compound heterozygous missense mutations in POLR3A in the remaining individual. POLR3A and POLR3B encode the largest and second largest subunits of RNA Polymerase III (Pol III), RPC1 and RPC2, respectively. RPC1 and RPC2 together form the active center of the polymerase and contribute to the catalytic activity of the polymerase. Pol III is involved in the transcription of small noncoding RNAs, such as 5S ribosomal RNA and all transfer RNAs (tRNA). We hypothesize that perturbation of Pol III target transcription, especially of tRNAs, could be a common pathological mechanism underlying POLR3A and POLR3B mutations. PMID:22036171

Identification and Functional Analysis of ZIC3 Mutations in Heterotaxy and Related Congenital Heart Defects

PubMed Central

Ware, Stephanie M.; Peng, Jianlan; Zhu, Lirong; Fernbach, Susan; Colicos, Suzanne; Casey, Brett; Towbin, Jeffrey; Belmont, John W.

2004-01-01

Mutations in the zinc finger transcription factor ZIC3 cause X-linked heterotaxy and have also been identified in patients with isolated congenital heart disease (CHD). To determine the relative contribution of ZIC3 mutations to both heterotaxy and isolated CHD, we screened the coding region of ZIC3 in 194 unrelated patients, including 61 patients with classic heterotaxy, 93 patients with heart defects characteristic of heterotaxy, and 11 patients with situs inversus totalis. Five novel ZIC3 mutations in three classic heterotaxy kindreds and two sporadic CHD cases were identified. None of these alleles was found in 97 ethnically matched control samples. On the basis of these analyses, we conclude that the phenotypic spectrum of ZIC3 mutations should be expanded to include affected females and CHD not typical for heterotaxy. This screening of a cohort of patients with sporadic heterotaxy indicates that ZIC3 mutations account for ∼1% of affected individuals. Missense and nonsense mutations were found in the highly conserved zinc finger–binding domain and in the N-terminal protein domain. Functional analysis of all currently known ZIC3 point mutations indicates that mutations in the putative zinc finger DNA binding domain and in the N-terminal domain result in loss of reporter gene transactivation. It is surprising that transfection studies demonstrate aberrant cytoplasmic localization resulting from mutations between amino acids 253–323 of the ZIC3 protein, indicating that the pathogenesis of a subset of ZIC3 mutations results at least in part from failure of appropriate nuclear localization. These results further expand the phenotypic and genotypic spectrum of ZIC3 mutations and provide initial mechanistic insight into their functional consequences. PMID:14681828

Mutational characterization of the P3H1/CRTAP/CypB complex in recessive osteogenesis imperfecta.

PubMed

Barbirato, C; Trancozo, M; Almeida, M G; Almeida, L S; Santos, T O; Duarte, J C G; Rebouças, M R G O; Sipolatti, V; Nunes, V R R; Paula, F

2015-12-03

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone deformities and fractures. Most cases are caused by autosomal dominant mutations in the type I collagen genes COL1A1 and COL1A2; however, an increasing number of recessive mutations in other genes have been reported. The LEPRE1, CRTAP, and PPIB genes encode proteins that form the P3H1/CRTAP/CypB complex, which is responsible for posttranslational modifications of type I collagen. In general, mutations in these genes lead to severe and lethal phenotypes of recessive OI. Here, we describe sixteen genetic variations detected in LEPRE1, CRTAP, and PPIB from 25 Brazilian patients with OI. Samples were screened for mutations on single-strand conformation polymorphism gels and variants were determined by automated sequencing. Seven variants were detected in patients but were absent in control samples. LEPRE1 contained the highest number of variants, including the previously described West African allele (c.1080+1G>T) found in one patient with severe OI as well as a previously undescribed p.Trp675Leu change that is predicted to be disease causing. In CRTAP, one patient carried the c.558A>G homozygous mutation, predicted as disease causing through alteration of a splice site. Genetic variations detected in the PPIB gene are probably not pathogenic due to their localization or because of their synonymous effect. This study enhances our knowledge about the mutational pattern of the LEPRE1, CRTAP, and PPIB genes. In addition, the results strengthen the proposition that LEPRE1 should be the first gene analyzed in mutation detection studies in patients with recessive OI.

Familial Cortical Myoclonus with a Mutation in NOL3

PubMed Central

Russell, Jonathan F.; Steckley, Jamie L.; Coppola, Giovanni; Hahn, Angelika F.G.; Howard, MacKenzie A.; Kornberg, Zachary; Huang, Alden; Mirsattari, Seyed M.; Merriman, Barry; Klein, Eric; Choi, Murim; Lee, Hsien-Yang; Kirk, Andrew; Nelson-Williams, Carol; Gibson, Gillian; Baraban, Scott C.; Lifton, Richard P.; Geschwind, Daniel H.; Fu, Ying-Hui; Ptáček, Louis J.

2012-01-01

Objective Myoclonus is characterized by sudden, brief involuntary movements and its presence is debilitating. We identified a family suffering from adult-onset, cortical myoclonus without associated seizures. We performed clinical, electrophysiological, and genetic studies to define this phenotype. Methods A large, four-generation family with history of myoclonus underwent careful questioning, examination, and electrophysiological testing. Thirty-five family members donated blood samples for genetic analysis, which included SNP mapping, microsatellite linkage, targeted massively parallel sequencing, and Sanger sequencing. In silico and in vitro experiments were performed to investigate functional significance of the mutation. Results We identified 11 members of a Canadian Mennonite family suffering from adult-onset, slowly progressive, disabling, multifocal myoclonus. Somatosensory evoked potentials indicated a cortical origin of the myoclonus. There were no associated seizures. Some severely affected individuals developed signs of progressive cerebellar ataxia of variable severity late in the course of their illness. The phenotype was inherited in an autosomal dominant fashion. We demonstrated linkage to chromosome 16q21-22.1. We then sequenced all coding sequence in the critical region, identifying only a single co-segregating, novel, nonsynonymous mutation, which resides in the gene NOL3. Furthermore, this mutation was found to alter post-translational modification of NOL3 protein in vitro. Interpretation We propose that Familial Cortical Myoclonus (FCM) is a novel movement disorder that may be caused by mutation in NOL3. Further investigation of the role of NOL3 in neuronal physiology may shed light on neuronal membrane hyperexcitability and pathophysiology of myoclonus and related disorders. PMID:22926851

Enhanced ε-poly-L-lysine production by inducing double antibiotic-resistant mutations in Streptomyces albulus.

PubMed

Wang, Liang; Chen, Xusheng; Wu, Guangyao; Li, Shu; Zeng, Xin; Ren, Xidong; Tang, Lei; Mao, Zhonggui

2017-02-01

ε-Poly-L-lysine (ε-PL), as a food additive, has been widely used in many countries. However, its production still needs to be improved. We successfully enhanced ε-PL production of Streptomyces albulus FEEL-1 by introducing mutations related to antibiotics, such as streptomycin, gentamicin, and rifampin. Single- and double-resistant mutants (S-88 and SG-31) were finally screened with the improved ε-PL productions of 2.81 and 3.83 g/L, 1.75- to 2.39-fold compared with that of initial strain FEEL-1. Then, the performances of mutants S-88 and SG-31 were compared with the parent strain FEEL-1 in a 5-L bioreactor under the optimal condition for ε-PL production. After 174-h fed-batch fermentation, the ε-PL production and productivity of hyper-strain SG-31 reached the maximum of 59.50 g/L and 8.21 g/L/day, respectively, which was 138 and 105% higher than that of FEEL-1. Analysis of streptomycin-resistant mutants demonstrated that a point mutation occurred in rpsL gene (encoding the ribosomal protein S12). These single and double mutants displayed remarkable increases of the activities and transcriptional levels of key enzymes in ε-PL biosynthesis pathway, which may be responsible for the enhanced mycelia viability, respiratory activity, and ε-PL productions of SG-31. These results showed that the new breeding method, called ribosome engineering, could be a novel and effective breeding strategy for the evolution of ε-PL-producing strains.

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer.

PubMed

Cossu-Rocca, Paolo; Orrù, Sandra; Muroni, Maria Rosaria; Sanges, Francesca; Sotgiu, Giovanni; Ena, Sara; Pira, Giovanna; Murgia, Luciano; Manca, Alessandra; Uras, Maria Gabriela; Sarobba, Maria Giuseppina; Urru, Silvana; De Miglio, Maria Rosaria

2015-01-01

Triple Negative Breast Cancer (TNBC) accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

Hypogonadotropic Hypogonadism due to Novel FGFR1 Mutations.

PubMed

Akkuş, Gamze; Kotan, Leman Damla; Durmaz, Erdem; Mengen, Eda; Turan, İhsan; Ulubay, Ayça; Gürbüz, Fatih; Yüksel, Bilgin; Tetiker, Tamer; Topaloğlu, A Kemal

2017-06-01

The underlying genetic etiology of hypogonadotropic hypogonadism (HH) is heterogeneous. Fibroblast growth factor signaling is pivotal in the ontogeny of gonadotropin-releasing hormone neurons. Loss-of-function mutations in FGFR1 gene cause variable HH phenotypes encompassing pubertal delay to idiopathic HH (IHH) or Kallmann syndrome (KS). As FGFR1 mutations are common, recognizing mutations and associated phenotypes may enhance clinical management. Using a candidate gene approach, we screened 52 IHH/KS patients. We identified three novel (IVS3-1G>C and p.W2X, p.R209C) FGFR1 gene mutations. Despite predictive null protein function, patients from the novel mutation families had normosmic IHH without non-reproductive phenotype. These findings further emphasize the great variability of FGFR1 mutation phenotypes in IHH/KS.

Tumor heterogeneity of fibroblast growth factor receptor 3 (FGFR3) mutations in invasive bladder cancer: implications for perioperative anti-FGFR3 treatment.

PubMed

Pouessel, D; Neuzillet, Y; Mertens, L S; van der Heijden, M S; de Jong, J; Sanders, J; Peters, D; Leroy, K; Manceau, A; Maille, P; Soyeux, P; Moktefi, A; Semprez, F; Vordos, D; de la Taille, A; Hurst, C D; Tomlinson, D C; Harnden, P; Bostrom, P J; Mirtti, T; Horenblas, S; Loriot, Y; Houédé, N; Chevreau, C; Beuzeboc, P; Shariat, S F; Sagalowsky, A I; Ashfaq, R; Burger, M; Jewett, M A S; Zlotta, A R; Broeks, A; Bapat, B; Knowles, M A; Lotan, Y; van der Kwast, T H; Culine, S; Allory, Y; van Rhijn, B W G

2016-07-01

Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer. Preclinical studies show that anti-FGFR3 treatment slows down tumor growth, suggesting that this tyrosine kinase receptor is a candidate for personalized bladder cancer treatment, particularly in patients with mutated FGFR3. We addressed tumor heterogeneity in a large multicenter, multi-laboratory study, as this may have significant impact on therapeutic response. We evaluated possible FGFR3 heterogeneity by the PCR-SNaPshot method in the superficial and deep compartments of tumors obtained by transurethral resection (TUR, n = 61) and in radical cystectomy (RC, n = 614) specimens and corresponding cancer-positive lymph nodes (LN+, n = 201). We found FGFR3 mutations in 13/34 (38%) T1 and 8/27 (30%) ≥T2-TUR samples, with 100% concordance between superficial and deeper parts in T1-TUR samples. Of eight FGFR3 mutant ≥T2-TUR samples, only 4 (50%) displayed the mutation in the deeper part. We found 67/614 (11%) FGFR3 mutations in RC specimens. FGFR3 mutation was associated with pN0 (P < 0.001) at RC. In 10/201 (5%) LN+, an FGFR3 mutation was found, all concordant with the corresponding RC specimen. In the remaining 191 cases, RC and LN+ were both wild type. FGFR3 mutation status seems promising to guide decision-making on adjuvant anti-FGFR3 therapy as it appeared homogeneous in RC and LN+. Based on the results of TUR, the deep part of the tumor needs to be assessed if neoadjuvant anti-FGFR3 treatment is considered. We conclude that studies on the heterogeneity of actionable molecular targets should precede clinical trials with these drugs in the perioperative setting. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Variants within the calpain-10 gene and relationships with type 2 diabetes (T2DM) and T2DM-related traits among Tunisian Arabs.

PubMed

Ezzidi, I; Mtiraoui, N; Nemr, R; Kacem, M; Al-Khateeb, G M; Mahjoub, T; Almawi, W Y

2010-11-01

Common variations in the calpain 10 (CAPN10) gene variants UCSNP-43, UCSNP-19 and UCSNP-63, and the 112/121 diplotype, are associated with an increased risk of type 2 diabetes (T2DM) and T2DM-related traits. The association of UCSNP-43, -19 and -63 CAPN10 SNPs with T2DM was assessed in 917 Tunisian T2DM patients and 748 ethnically matched non-diabetic controls. CAPN10 genotyping was done by PCR-RFLP. Significant differences in UCSNP-19 MAF, but not UCSNP-43 or -63, and genotype distribution were seen between patients and controls. Heterogeneity in UCSNP-19, but not UCSNP-43 and -63, genotype distribution was noted according to geographical origin. Obesity was associated with UCSNP-19, while raised fasting glucose was associated with UCSNP-63, and increased HDL was associated with UCSNP-43. Enrichment of homozygous UCSNP-19 2/2 was seen in overweight and obese compared with lean patients; logistic-regression analyses demonstrated a positive association of the 2/2 genotype with overweight [P=0.003; OR (95% CI)=2.07 (1.28-3.33)] and obese [P=0.021; OR (95% CI)=1.83 (1.10-3.07)] patients. Of the six CAPN10 haplotypes identified, significant enrichment of only haplotype 111 was seen in T2DM patients [Pc=0.034; OR (95% CI)=1.22 (1.06-1.41)], while the frequency of all identified CAPN10 diplotypes, including the high-risk 112/121, was comparable between patients and controls. While CAPN10 UCSNP-19 SNP and haplotype 111 contribute to the risk of T2DM in Tunisian subjects, no significant association between CAPN10 diplotypes and T2DM was demonstrated. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Inherited macular degeneration-associated mutations in CNGB3 increase the ligand sensitivity and spontaneous open probability of cone cyclic nucleotide-gated channels

PubMed Central

Meighan, Peter C.; Peng, Changhong; Varnum, Michael D.

2015-01-01

Cyclic nucleotide gated (CNG) channels are a critical component of the visual transduction cascade in the vertebrate retina. Mutations in the genes encoding these channels have been associated with a spectrum of inherited retinal disorders. To gain insight into their pathophysiological mechanisms, we have investigated the functional consequences of several CNGB3 mutations, previously associated with macular degeneration (Y469D and L595F) or complete achromatopsia (S156F, P309L, and G558C), by expressing these subunits in combination with wild-type CNGA3 in Xenopus oocytes and characterizing them using patch-clamp recordings in the inside-out configuration. These mutations did not prevent the formation of functional heteromeric channels, as indicated by sensitivity to block by L-cis-diltiazem. With the exception of S156F, each of the mutant channels displayed electrophysiological properties reflecting enhanced channel activity at physiological concentrations of cGMP (i.e., a gain-of-function phenotype). The increased channel activity produced by these mutations resulted from either increased functional expression levels, or increased sensitivity to cyclic nucleotides. Furthermore, L595F increased the spontaneous open probability in the absence of activating ligand, signifying a ligand independent gain-of-function change. In addition to the CNGB3 disease-associate mutations, we characterized the effects of several common CNGB3 and CNGA3 single-nucleotide polymorphisms (SNPs) on heteromeric CNGA3+CNGB3 channel function. Two of the SNPs examined (A3-T153M, and B3-W234C) produced decreased ligand sensitivity for heteromeric CNG channels. These changes may contribute to background disease susceptibility when combined with other genetic or non-genetic factors. Together, these studies help to define the underlying molecular phenotype for mutations relating to CNG channel disease pathogenesis. PMID:26106334

Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance

PubMed Central

Tischfield, Max A.; Baris, Hagit N.; Wu, Chen; Rudolph, Guenther; Van Maldergem, Lionel; He, Wei; Chan, Wai-Man; Andrews, Caroline; Demer, Joseph L.; Robertson, Richard L.; Mackey, David A.; Ruddle, Jonathan B.; Bird, Thomas D.; Gottlob, Irene; Pieh, Christina; Traboulsi, Elias I.; Pomeroy, Scott L.; Hunter, David G.; Soul, Janet S.; Newlin, Anna; Sabol, Louise J.; Doherty, Edward J.; de Uzcátegui, Clara E.; de Uzcátegui, Nicolas; Collins, Mary Louise Z.; Sener, Emin C.; Wabbels, Bettina; Hellebrand, Heide; Meitinger, Thomas; de Berardinis, Teresa; Magli, Adriano; Schiavi, Costantino; Pastore-Trossello, Marco; Koc, Feray; Wong, Agnes M.; Levin, Alex V.; Geraghty, Michael T.; Descartes, Maria; Flaherty, Maree; Jamieson, Robyn V.; Møller, H. U.; Meuthen, Ingo; Callen, David F.; Kerwin, Janet; Lindsay, Susan; Meindl, Alfons; Gupta, Mohan L.; Pellman, David; Engle, Elizabeth C.

2011-01-01

We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific β-tubulin isotype III, result in a spectrum of human nervous system disorders we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves, and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate normal TUBB3 is required for axon guidance and maintenance in mammals. PMID:20074521

A single mutation in Securin induces chromosomal instability and enhances cell invasion.

PubMed

Mora-Santos, Mar; Castilla, Carolina; Herrero-Ruiz, Joaquín; Giráldez, Servando; Limón-Mortés, M Cristina; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2013-01-01

Pituitary tumour transforming gene (pttg1) encodes Securin, a protein involved in the inhibition of sister chromatid separation binding to Separase until the onset of anaphase. Separase is a cysteine-protease that degrades cohesin to segregate the sister chromatids to opposite poles of the cell. The amount of Securin is strongly regulated because it should allow Separase activation when it is degraded by the anaphase promoting complex/cyclosome, should arrest the cell cycle after DNA damage, when it is degraded through SKP1-CUL1-βTrCP ubiquitin ligase, and its overexpression induces tumour formation and correlates with metastasis in multiple tumours. Securin is a phosphoprotein that contains 32 potentially phosphorylatable residues. We mutated and analysed most of them, and found a single mutant, hSecT60A, that showed enhanced oncogenic properties. Our fluorescence activated cell sorting analysis, fluorescence in situ hybridisation assays, tumour cell migration and invasion experiments and gene expression by microarrays analysis clearly involved hSecT60A in chromosomal instability and cell invasion. These results show, for the first time, that a single mutation in pttg1 is sufficient to trigger the oncogenic properties of Securin. The finding of this point mutation in patients might be used as an effective strategy for early detection of cancer. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mutations in CG8878, a Novel Putative Protein Kinase, Enhance P Element Dependent Silencing (PDS) and Position Effect Variegation (PEV) in Drosophila melanogaster

PubMed Central

McCracken, Allen; Locke, John

2014-01-01

Genes in multicellular organisms are expressed as part of a developmental program that is largely dependent on self-perpetuating higher-order chromatin states. The mechanism of establishing and maintaining these epigenetic events is well studied in Drosophila. The first known example of an epigenetic effect was that of (PEV) in Drosophila, which has been shown to be due to gene silencing via heterochromatin formation. We are investigating a process similar to Position Effect Variegation (PEV) using a mini-w transgene, called Pci, inserted in the upstream regulatory region of ci. The mini-white + transgene in Pci is expressed throughout the adult eye; however, when other P or KP elements are present, a variegated eye phenotype results indicating random w + silencing during development. This P element dependent silencing (PDS) can be modified by the haplo-suppressors/triplo-enhancers, Su(var)205 and Su(var)3–7, indicating that these heterochromatic modifiers also act dose dependently in PDS. Here we use a spontaneous derivative mutation of Pci called PciE1 (E1) that variegates like PDS in the absence of P elements, presumably due to an adjacent gypsy element insertion, to screen for second-site modifier mutations that enhance variable silencing of white + in E1. We isolated 7 mutations in CG8878, an essential gene, that enhance the E1 variegated phenotype. CG8878, a previously uncharacterized gene, potentially encodes a serine/threonine kinase whose closest Drosophila paralogue, ballchen (nhk-1), phosphorylates histones. These mutant alleles enhance both PDS at E1 and Position Effect Variegation (PEV) at wm4, indicating a previously unknown common silencing mechanism between the two. PMID:24614804

CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions

PubMed Central

Dai, Gucan

2013-01-01

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the

CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions.

PubMed

Dai, Gucan; Varnum, Michael D

2013-07-15

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the

The E1784K mutation in SCN5A is associated with mixed clinical phenotype of type 3 long QT syndrome

PubMed Central

Makita, Naomasa; Behr, Elijah; Shimizu, Wataru; Horie, Minoru; Sunami, Akihiko; Crotti, Lia; Schulze-Bahr, Eric; Fukuhara, Shigetomo; Mochizuki, Naoki; Makiyama, Takeru; Itoh, Hideki; Christiansen, Michael; McKeown, Pascal; Miyamoto, Koji; Kamakura, Shiro; Tsutsui, Hiroyuki; Schwartz, Peter J.; George, Alfred L.; Roden, Dan M.

2008-01-01

Phenotypic overlap of type 3 long QT syndrome (LQT3) with Brugada syndrome (BrS) is observed in some carriers of mutations in the Na channel SCN5A. While this overlap is important for patient management, the clinical features, prevalence, and mechanisms underlying such overlap have not been fully elucidated. To investigate the basis for this overlap, we genotyped a cohort of 44 LQT3 families of multiple ethnicities from 7 referral centers and found a high prevalence of the E1784K mutation in SCN5A. Of 41 E1784K carriers, 93% had LQT3, 22% had BrS, and 39% had sinus node dysfunction. Heterologously expressed E1784K channels showed a 15.0-mV negative shift in the voltage dependence of Na channel inactivation and a 7.5-fold increase in flecainide affinity for resting-state channels, properties also seen with other LQT3 mutations associated with a mixed clinical phenotype. Furthermore, these properties were absent in Na channels harboring the T1304M mutation, which is associated with LQT3 without a mixed clinical phenotype. These results suggest that a negative shift of steady-state Na channel inactivation and enhanced tonic block by class IC drugs represent common biophysical mechanisms underlying the phenotypic overlap of LQT3 and BrS and further indicate that class IC drugs should be avoided in patients with Na channels displaying these behaviors. PMID:18451998

The Phenotypic Spectrum of DYT24 Due to ANO3 Mutations

PubMed Central

Stamelou, Maria; Charlesworth, Gavin; Cordivari, Carla; Schneider, Susanne A; Kägi, Georg; Sheerin, Una-Marie; Rubio-Agusti, Ignacio; Batla, Amit; Houlden, Henry; Wood, Nicholas W; Bhatia, Kailash P

2014-01-01

Genes causing primary dystonia are rare. Recently, pathogenic mutations in the anoctamin 3 gene (ANO3) have been identified to cause autosomal dominant craniocervical dystonia and have been assigned to the dystonia locus dystonia-24 (DYT24). Here, we expand on the phenotypic spectrum of DYT24 and provide demonstrative videos. Moreover, tremor recordings were performed, and back-averaged electroencephalography, sensory evoked potentials, and C-reflex studies were carried out in two individuals who carried two different mutations in ANO3. Ten patients from three families are described. The age at onset ranged from early childhood to the forties. Cervical dystonia was the most common site of onset followed by laryngeal dystonia. The characteristic feature in all affected individuals was the presence of tremor, which contrasts DYT24 from the typical DYT6 phenotype. Tremor was the sole initial manifestation in some individuals with ANO3 mutations, leading to misdiagnosis as essential tremor. Electrophysiology in two patients with two different mutations showed co-contraction of antagonist muscles, confirming dystonia, and a 6-Hz arm tremor at rest, which increased in amplitude during action. In one of the studied patients, clinically superimposed myoclonus was observed. The duration of the myoclonus was in the range of 250 msec at about 3 Hz, which is more consistent with subcortical myoclonus. In summary, ANO3 causes a varied phenotype of young-onset or adult-onset craniocervical dystonia with tremor and/or myoclonic jerks. Patients with familial cervical dystonia who also have myoclonus-dystonia as well as patients with prominent tremor and mild dystonia should be tested for ANO3 mutations. © 2014 The Authors. Movement Disorders published by International Parkinson and Movement Disorder Society PMID:24442708

Genotypic and allelic frequencies of gene polymorphisms associated with meat tenderness in Nellore beef cattle.

PubMed

Carvalho, M E; Eler, J P; Bonin, M N; Rezende, F M; Biase, F H; Meirelles, F V; Regitano, L C A; Coutinho, L L; Balieiro, J C C; Ferraz, J B S

2017-02-16

The objectives of this study were to characterize the allelic and genotypic frequencies of polymorphisms in the µ-calpain and calpastatin genes, and to assess their association with meat tenderness and animal growth in Nellore cattle. We evaluated 605 Nellore animals at 24 months of age, on average, at slaughter. The polymorphisms were determined for the molecular markers CAPN316, CAPN530, CAPN4751, CAPN4753, and UOGACAST1. Analyses of meat tenderness at 7, 14, and 21 days of maturation were performed in samples of longissimus thoracis obtained between the 12th and 13th rib and sheared using a Warner Bratzler Shear Force. Significant effects were observed for meat tenderness at days 7, 14, and 21 of maturation for the marker CAPN4751, at day 21 for the marker CAPN4753, and at days 14 and 21 for the marker UOGCAST1. For genotypic combinations of markers, the results were significant for the combination CAPN4751/UOGCAST1 in the three maturation periods and CAPN4753/UOGCAST1 at days 14 and 21 of maturation.

Absence of PITX3 mutation in a Tunisian family with congenital cataract and mental retardation

PubMed Central

Chograni, Manèl; Chaabouni, Myriam; Chelly, Imen; Helayem, Mohamed Bechir

2010-01-01

Purpose The PITX3 (pituitary homeobox 3) gene encodes for a homeobox bicoid-like transcription factor. When one allele is mutated, it leads to dominant cataract and anterior segment mesenchymal dysgenesis in humans. When both copies are mutated, homozygous mutation contributes to microphtalmia with brain malformations. In the current study, a family with autosomal recessive congenital cataract (ARCC) associated with mental retardation (MR) was examined to identify PITX3 mutations. Methods Sequencing of the PITX3 gene was performed on two affected and three unaffected members of the studied Tunisian family. The results were analyzed with Sequencing Analysis 5.2 and SeqScape. Results No mutation in the four exons of PITX3 was revealed. Two substitution polymorphisms, c.439C>T and c.930C>A, were detected in exons 3 and 4, respectively. These alterations did not segregate with the disease. Conclusions Although PITX3 was shown to be essential to normal embryonic eye and brain development in vertebrates, we report the absence of PITX3 mutations in a family presenting congenital cataract and mental retardation. PMID:20376326

[Mutation analysis of FGFR3 gene in a family featuring hereditary dwarfism].

PubMed

Zhang, Qiong; Jiang, Hai-ou; Quan, Qing-li; Li, Jun; He, Ting; Huang, Xue-shuang

2011-12-01

To investigate the clinical symptoms and potential mutation in FGFR3 gene for a family featuring hereditary dwarfism in order to attain diagnosis and provide prenatal diagnosis. Five patients and two unaffected relatives from the family, in addition with 100 healthy controls, were recruited. Genome DNA was extracted. Exons 10 and 13 of the FGFR3 gene were amplified using polymerase chain reaction (PCR). PCR products were sequenced in both directions. All patients had similar features including short stature, short limbs, lumbar hyperlordosis but normal craniofacial features. A heterozygous mutation G1620T (N540K) was identified in the cDNA from all patients but not in the unaffected relatives and 100 control subjects. A heterozygous G380R mutation was excluded. The hereditary dwarfism featured by this family has been caused by hypochondroplasia (HCH) due to a N540K mutation in the FGFR3 gene.

Missense mutations of MLH1 and MSH2 genes detected in patients with gastrointestinal cancer are associated with exonic splicing enhancers and silencers

PubMed Central

ZHU, MING; CHEN, HUI-MEI; WANG, YA-PING

2013-01-01

The MLH1 and MSH2 genes in DNA mismatch repair are important in the pathogenesis of gastrointestinal cancer. Recent studies of normal and alternative splicing suggest that the deleterious effects of missense mutations may in fact be splicing-related when they are located in exonic splicing enhancers (ESEs) or exonic splicing silencers (ESSs). In this study, we used ESE-finder and FAS-ESS software to analyze the potential ESE/ESS motifs of the 114 missense mutations detected in the two genes in East Asian gastrointestinal cancer patients. In addition, we used the SIFT tool to functionally analyze these mutations. The amount of the ESE losses (68) was 51.1% higher than the ESE gains (45) of all the mutations. However, the amount of the ESS gains (27) was 107.7% higher than the ESS losses (13). In total, 56 (49.1%) mutations possessed a potential exonic splicing regulator (ESR) error. Eighty-one mutations (71.1%) were predicted to be deleterious with a lower tolerance index as detected by the Sorting Intolerant from Tolerant (SIFT) tool. Among these, 38 (33.3%) mutations were predicted to be functionally deleterious and possess one potential ESR error, while 18 (15.8%) mutations were predicted to be functionally deleterious and exhibit two potential ESR errors. These may be more likely to affect exon splicing. Our results indicated that there is a strong correlation between missense mutations in MLH1 and MSH2 genes detected in East Asian gastrointestinal cancer patients and ESR motifs. In order to correctly understand the molecular nature of mutations, splicing patterns should be compared between wild-type and mutant samples. PMID:23760103

Common mutations in the fibroblast growth factor receptor 3 (FGFR 3) gene account for achondroplasia, hypochondroplasia, and thanatophoric dwarfism.

PubMed

Bonaventure, J; Rousseau, F; Legeai-Mallet, L; Le Merrer, M; Munnich, A; Maroteaux, P

1996-05-03

The mapping of the achondroplasia locus to the short arm of chromosome 4 and the subsequent identification of a recurrent missense mutation (G380R) in the fibroblast growth factor receptor 3 (FGFR-3) gene has been followed by the detection of common FGFR-3 mutations in two clinically related disorders: thanatophoric dwarfism (types I and II) and hypochondroplasia. The relative clinical homogeneity of achondroplasia was substantiated by demonstration of its genetic homogeneity as more than 98% of all patients hitherto reported exhibit mutations in the transmembrane receptor domain. Although most hypochondroplasia cases were accounted for by a recurrent missense substitution (N540K) in the first tyrosine kinase (TK 1) domain of the receptor, a significant proportion (40%) of our patients did not harbor the N540K mutation and three hypochondroplasia families were not linked to the FGFR-3 locus, thus supporting clinical heterogeneity of this condition. In thanatophoric dwarfism (TD), a recurrent FGFR-3 mutation located in the second tyrosine kinase (TK 2) domain of the receptor was originally detected in 100% of TD II cases, our series seven distinct mutations in three different protein domains were identified in 25 of 26 TD I patients, suggesting that TD, like achondroplasia, is a genetically homogenous skeletal disorder.

PAX3 mutations and clinical characteristics in Chinese patients with Waardenburg syndrome type 1

PubMed Central

Wang, Juan; Li, Shiqiang; Xiao, Xueshan; Wang, Panfeng; Guo, Xiangming

2010-01-01

Purpose To detect paired box gene 3 (PAX3) mutations and associated phenotypes in Chinese patients with Waardenburg syndrome type 1 (WS1). Methods Five unrelated families with suspected WS1 were selected from our Genomic DNA Repository for Hereditary Eye Diseases. The coding and adjacent intronic regions of PAX3 were amplified by polymerase chain reaction and the amplicons were then analyzed by cycle sequencing. Variations detected were further evaluated in available family members as well as one hundred controls with heteroduplex-single strand conformational polymorphism (heteroduplex-SSCP) analysis and/or clone sequencing. Results Three novel and two known mutations in PAX3 were detected in five patients, respectively: c.567_586+17del (p.Asp189_Gln505delinsGluGlyGlyAlaLeuAlaGly), c.456_459dupTTCC (p.Ile154PhefsX162), c.795_800delCTGGTT (p.Trp266_Phe267del), c.799T>A (p.Phe267Ile), and c.667C>T (p.Arg223X). Two novel mutations proved to be de novo as their parents did not carry the mutations. All five patients with PAX3 mutations had dystopia canthorum and different iris color and fundi between their two eyes. However, none had white forelock, skin hypopigmentation, and deafness. Conclusions Our findings expand the frequency and spectrum of PAX3 mutations and ethnic-related phenotypes in Chinese patients with WS1. De novo mutations in PAX3 have not been reported before. PMID:20664692

PAX3 mutations and clinical characteristics in Chinese patients with Waardenburg syndrome type 1.

PubMed

Wang, Juan; Li, Shiqiang; Xiao, Xueshan; Wang, Panfeng; Guo, Xiangming; Zhang, Qingjiong

2010-06-22

To detect paired box gene 3 (PAX3) mutations and associated phenotypes in Chinese patients with Waardenburg syndrome type 1 (WS1). Five unrelated families with suspected WS1 were selected from our Genomic DNA Repository for Hereditary Eye Diseases. The coding and adjacent intronic regions of PAX3 were amplified by polymerase chain reaction and the amplicons were then analyzed by cycle sequencing. Variations detected were further evaluated in available family members as well as one hundred controls with heteroduplex-single strand conformational polymorphism (heteroduplex-SSCP) analysis and/or clone sequencing. Three novel and two known mutations in PAX3 were detected in five patients, respectively: c.567_586+17del (p.Asp189_Gln505delinsGluGlyGlyAlaLeuAlaGly), c.456_459dupTTCC (p.Ile154PhefsX162), c.795_800delCTGGTT (p.Trp266_Phe267del), c.799T>A (p.Phe267Ile), and c.667C>T (p.Arg223X). Two novel mutations proved to be de novo as their parents did not carry the mutations. All five patients with PAX3 mutations had dystopia canthorum and different iris color and fundi between their two eyes. However, none had white forelock, skin hypopigmentation, and deafness. Our findings expand the frequency and spectrum of PAX3 mutations and ethnic-related phenotypes in Chinese patients with WS1. De novo mutations in PAX3 have not been reported before.

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

PubMed Central

Muroni, Maria Rosaria; Sanges, Francesca; Sotgiu, Giovanni; Ena, Sara; Pira, Giovanna; Murgia, Luciano; Manca, Alessandra; Uras, Maria Gabriela; Sarobba, Maria Giuseppina; Urru, Silvana; De Miglio, Maria Rosaria

2015-01-01

Background Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. Materials and Methods PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. Results PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Conclusions Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies. PMID:26540293

SF3B1 and BAP1 mutations in blue nevus-like melanoma.

PubMed

Griewank, Klaus G; Müller, Hansgeorg; Jackett, Louise A; Emberger, Michael; Möller, Inga; van de Nes, Johannes Ap; Zimmer, Lisa; Livingstone, Elisabeth; Wiesner, Thomas; Scholz, Simone L; Cosgarea, Ioana; Sucker, Antje; Schimming, Tobias; Hillen, Uwe; Schilling, Bastian; Paschen, Annette; Reis, Henning; Mentzel, Thomas; Kutzner, Heinz; Rütten, Arno; Murali, Rajmohan; Scolyer, Richard A; Schadendorf, Dirk

2017-07-01

Blue nevi are melanocytic tumors originating in the cutaneous dermis. Malignant tumors may arise in association with or resembling blue nevi, so called 'blue nevus-like melanoma', which can metastasize and result in patient death. Identifying which tumors will behave in a clinically aggressive manner can be challenging. Identifying genetic alterations in such tumors may assist in their diagnosis and prognostication. Blue nevi are known to be genetically related to uveal melanomas (eg, both harboring GNAQ and GNA11 mutations). In this study, we analyzed a large cohort (n=301) of various morphologic variants of blue nevi and related tumors including tumors diagnosed as atypical blue nevi (n=21), and blue nevus-like melanoma (n=12), screening for all gene mutations known to occur in uveal melanoma. Similar to published reports, we found the majority of blue nevi harbored activating mutations in GNAQ (53%) or GNA11 (15%). In addition, rare CYSLTR2 (1%) and PLCB4 (1%) mutations were identified. EIF1AX, SF3B1, and BAP1 mutations were also detected, with BAP1 and SF3B1 R625 mutations being present only in clearly malignant tumors (17% (n=2) and 25% (n=3) of blue nevus-like melanoma, respectively). In sequencing data from a larger cohort of cutaneous melanomas, this genetic profile was also identified in tumors not originally diagnosed as blue nevus-like melanoma. Our findings suggest that the genetic profile of coexistent GNAQ or GNA11 mutations with BAP1 or SF3B1 mutations can aid the histopathological diagnosis of blue nevus-like melanoma and distinguish blue nevus-like melanoma from conventional epidermal-derived melanomas. Future studies will need to further elucidate the prognostic implications and appropriate clinical management for patients with tumors harboring these mutation profiles.

A meta-analysis of the relationship between FGFR3 and TP53 mutations in bladder cancer.

PubMed

Neuzillet, Yann; Paoletti, Xavier; Ouerhani, Slah; Mongiat-Artus, Pierre; Soliman, Hany; de The, Hugues; Sibony, Mathilde; Denoux, Yves; Molinie, Vincent; Herault, Aurélie; Lepage, May-Linda; Maille, Pascale; Renou, Audrey; Vordos, Dimitri; Abbou, Claude-Clément; Bakkar, Ashraf; Asselain, Bernard; Kourda, Nadia; El Gaaied, Amel; Leroy, Karen; Laplanche, Agnès; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Radvanyi, François

2012-01-01

TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications) and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18-0.37], p = 0.0001) or for pT1 tumours alone (OR = 0.47 [0.28-0.79], p = 0.0009). However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23-1.36] (p = 0.12) and OR = 0.99 [0.37-2.7] (p = 0.35), respectively). After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4). These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage.

Enhanced Polyubiquitination of Shank3 and NMDA receptor in a mouse model of Autism

PubMed Central

Bangash, M Ali; Park, Joo Min; Melnikova, Tatiana; Wang, Dehua; Jeon, Soo Kyeong; Lee, Deidre; Syeda, Sbaa; Kim, Juno; Kouser, Mehreen; Schwartz, Joshua; Cui, Yiyuan; Zhao, Xia; Speed, Haley E.; Kee, Sara E.; Tu, Jian Cheng; Hu, Jia-Hua; Petralia, Ronald S.; Linden, David J.; Powell, Craig M.; Savonenko, Alena; Xiao, Bo; Worley, Paul F.

2011-01-01

Summary We have created a mouse genetic model that mimics a human mutation of Shank3 that deletes the C-terminus and is associated with autism. Expressed as a single copy [Shank3(+/ΔC) mice], Shank3ΔC protein interacts with the WT gene product and results in >90 % reduction of Shank3 at synapses. This “gain of function” phenotype is linked to increased polyubiquitination of WT Shank3 and its redistribution into proteasomes. Similarly, the NR1 subunit of the NMDA receptor is reduced at synapses with increased polyubiquitination. Assays of post-synaptic density proteins, spine morphology and synapse number are unchanged in Shank3(+/ΔC) mice, but the amplitude of NMDAR responses is reduced together with reduced NMDAR-dependent LTP and LTD. Reciprocally, mGluR-dependent LTD is markedly enhanced. Shank3(+/ΔC) mice show behavioral deficits suggestive of autism and reduced NMDA receptor function. These studies reveal a mechanism distinct from haploinsufficiency by which mutations of Shank3 can evoke an autism-like disorder. PMID:21565394

Enhanced polyubiquitination of Shank3 and NMDA receptor in a mouse model of autism.

PubMed

Bangash, M Ali; Park, Joo Min; Melnikova, Tatiana; Wang, Dehua; Jeon, Soo Kyeong; Lee, Deidre; Syeda, Sbaa; Kim, Juno; Kouser, Mehreen; Schwartz, Joshua; Cui, Yiyuan; Zhao, Xia; Speed, Haley E; Kee, Sara E; Tu, Jian Cheng; Hu, Jia-Hua; Petralia, Ronald S; Linden, David J; Powell, Craig M; Savonenko, Alena; Xiao, Bo; Worley, Paul F

2011-05-27

We have created a mouse genetic model that mimics a human mutation of Shank3 that deletes the C terminus and is associated with autism. Expressed as a single copy [Shank3(+/ΔC) mice], Shank3ΔC protein interacts with the wild-type (WT) gene product and results in >90% reduction of Shank3 at synapses. This "gain-of-function" phenotype is linked to increased polyubiquitination of WT Shank3 and its redistribution into proteasomes. Similarly, the NR1 subunit of the NMDA receptor is reduced at synapses with increased polyubiquitination. Assays of postsynaptic density proteins, spine morphology, and synapse number are unchanged in Shank3(+/ΔC) mice, but the amplitude of NMDAR responses is reduced together with reduced NMDAR-dependent LTP and LTD. Reciprocally, mGluR-dependent LTD is markedly enhanced. Shank3(+/ΔC) mice show behavioral deficits suggestive of autism and reduced NMDA receptor function. These studies reveal a mechanism distinct from haploinsufficiency by which mutations of Shank3 can evoke an autism-like disorder. Copyright © 2011 Elsevier Inc. All rights reserved.

HOGA1 Gene Mutations of Primary Hyperoxaluria Type 3 in Tunisian Patients.

PubMed

M'dimegh, Saoussen; Aquaviva-Bourdain, Cécile; Omezzine, Asma; Souche, Geneviéve; M'barek, Ibtihel; Abidi, Kamel; Gargah, Tahar; Abroug, Saoussen; Bouslama, Ali

2017-05-01

Primary hyperoxaluria type 3 (PH3) is due to mutations in the recently identified 4-hydroxy-2-oxoglutarate aldolase (HOGA1) gene. PH3 might be the least severe form with a milder phenotype with good preservation of kidney function in most patients. The aim of this study was to report three PH3 cases carrying mutations in HOGA1. Genetic analysis of HOGA1 was performed in patients with a high clinical suspicion of PH after sequencing of AGXT and GRHPR genes, which was negative. Also, a complete AGXT/GRHPR MLPA was performed in these patients in order to detect large deletions/insertions. Two different HOGA1 gene mutations were identified: the p.Pro190Leu in a homozygous state and the p.Gly287Val in two patients in homozygous and heterozygous carriers. The median age at onset of clinical symptoms was 3.93 years. Most of the patients had a positive family history for recurrent urolithiasis. The p.Pro190Leu mutation was reported with impaired renal function at follow-up; however, the p.Gly287Val was presented with normal renal function. All patients were presented with urolithiasis, but only one had a nephrocalcinosis. This study expanded the number of PH3 patients from 63 to 66 cases. The p.Pro190Leu and the p.Gly287Val mutations found in this study can provide a first-line investigation in Tunisian PH1 patients. © 2016 Wiley Periodicals, Inc.

Mutations in POLR3A and POLR3B are a major cause of hypomyelinating leukodystrophies with or without dental abnormalities and/or hypogonadotropic hypogonadism.

PubMed

Daoud, Hussein; Tétreault, Martine; Gibson, William; Guerrero, Kether; Cohen, Ana; Gburek-Augustat, Janina; Synofzik, Matthis; Brais, Bernard; Stevens, Cathy A; Sanchez-Carpintero, Rocio; Goizet, Cyril; Naidu, Sakkubai; Vanderver, Adeline; Bernard, Geneviève

2013-03-01

Leukodystrophies are a heterogeneous group of inherited neurodegenerative disorders characterised by abnormal central nervous system white matter. Mutations in POLR3A and POLR3B genes were recently reported to cause four clinically overlapping hypomyelinating leukodystrophy phenotypes. Our aim was to investigate the presence and frequency of POLR3A and POLR3B mutations in patients with genetically unexplained hypomyelinating leukodystrophies with typical clinical and/or radiologic features of Pol III-related leukodystrophies. The entire coding region and the flanking exon/intron boundaries of POLR3A and/or POLR3B genes were amplified and sequenced in 14 patients. Recessive mutations in POLR3A or POLR3B were uncovered in all 14 patients. Eight novel mutations were identified in POLR3A: six missenses, one nonsense, and one frameshift mutation. Seven patients carried compound heterozygous mutations in POLR3B, of whom six shared the common mutation in exon 15 (p.V523E). Seven novel mutations were identified in POLR3B: four missenses, two splice sites, and one intronic mutation. To date, our group has described 37 patients, of whom 27 have mutations in POLR3A and 10 in POLR3B, respectively. Altogether, our results further support the proposal that POLR3A and POLR3B mutations are a major cause of hypomyelinating leukodystrophies and suggest that POLR3A mutations are more frequent.

Loss of Cbl–PI3K Interaction Enhances Osteoclast Survival due to p21-Ras Mediated PI3K Activation Independent of Cbl-b

PubMed Central

Adapala, Naga Suresh; Barbe, Mary F.; Tsygankov, Alexander Y.; Lorenzo, Joseph A.; Sanjay, Archana

2015-01-01

Cbl family proteins, Cbl and Cbl-b, are E3 ubiquitin ligases and adaptor proteins, which play important roles in bone-resorbing osteoclasts. Loss of Cbl in mice decreases osteoclast migration, resulting in delayed bone development where as absence of Cbl-b decreases bone volume due to hyper-resorptive osteoclasts. A major structural difference between Cbl and Cbl-b is tyrosine 737 (in YEAM motif) only on Cbl, which upon phosphorylation interacts with the p85 subunit of phosphatidylinositol-3 Kinase (PI3K). In contrast to Cbl−/− and Cbl-b−/−, mice lacking Cbl–PI3K interaction due to a Y737F (tyrosine to phenylalanine, YF) mutation showed enhanced osteoclast survival, but defective bone resorption. To investigate whether Cbl–PI3K interaction contributes to distinct roles of Cbl and Cbl-b in osteoclasts, mice bearing CblY737F mutation in the Cbl-b−/− background (YF/YF;Cbl-b−/−) were generated. The differentiation and survival were augmented similarly in YF/YF and YF/YF;Cbl-b−/− osteoclasts, associated with enhanced PI3K signaling suggesting an exclusive role of Cbl–PI3K interaction, independent of Cbl-b. In addition to PI3K, the small GTPase Ras also regulates osteoclast survival. In the absence of Cbl–PI3K interaction, increased Ras GTPase activity and Ras–PI3K binding were observed and inhibition of Ras activation attenuated PI3K mediated osteoclast survival. In contrast to differentiation and survival, increased osteoclast activity observed in Cbl-b−/− mice persisted even after introduction of the resorption-defective YF mutation in YF/YF;Cbl-b−/− mice. Hence, Cbl and Cbl-b play mutually exclusive roles in osteoclasts. Whereas Cbl–PI3K interaction regulates differentiation and survival, bone resorption is predominantly regulated by Cbl-b in osteoclasts. PMID:24470255

Loss of Cbl-PI3K interaction enhances osteoclast survival due to p21-Ras mediated PI3K activation independent of Cbl-b.

PubMed

Adapala, Naga Suresh; Barbe, Mary F; Tsygankov, Alexander Y; Lorenzo, Joseph A; Sanjay, Archana

2014-07-01

Cbl family proteins, Cbl and Cbl-b, are E3 ubiquitin ligases and adaptor proteins, which play important roles in bone-resorbing osteoclasts. Loss of Cbl in mice decreases osteoclast migration, resulting in delayed bone development where as absence of Cbl-b decreases bone volume due to hyper-resorptive osteoclasts. A major structural difference between Cbl and Cbl-b is tyrosine 737 (in YEAM motif) only on Cbl, which upon phosphorylation interacts with the p85 subunit of phosphatidylinositol-3 Kinase (PI3K). In contrast to Cbl(-/-) and Cbl-b(-/-) , mice lacking Cbl-PI3K interaction due to a Y737F (tyrosine to phenylalanine, YF) mutation showed enhanced osteoclast survival, but defective bone resorption. To investigate whether Cbl-PI3K interaction contributes to distinct roles of Cbl and Cbl-b in osteoclasts, mice bearing CblY737F mutation in the Cbl-b(-/-) background (YF/YF;Cbl-b(-/-) ) were generated. The differentiation and survival were augmented similarly in YF/YF and YF/YF;Cbl-b(-/-) osteoclasts, associated with enhanced PI3K signaling suggesting an exclusive role of Cbl-PI3K interaction, independent of Cbl-b. In addition to PI3K, the small GTPase Ras also regulates osteoclast survival. In the absence of Cbl-PI3K interaction, increased Ras GTPase activity and Ras-PI3K binding were observed and inhibition of Ras activation attenuated PI3K mediated osteoclast survival. In contrast to differentiation and survival, increased osteoclast activity observed in Cbl-b(-/-) mice persisted even after introduction of the resorption-defective YF mutation in YF/YF;Cbl-b(-/-) mice. Hence, Cbl and Cbl-b play mutually exclusive roles in osteoclasts. Whereas Cbl-PI3K interaction regulates differentiation and survival, bone resorption is predominantly regulated by Cbl-b in osteoclasts. © 2014 Wiley Periodicals, Inc.

Newly identified CHO ERCC3/XPB mutations and phenotype characterization

PubMed Central

Rybanská, Ivana; Gurský, Ján; Fašková, Miriam; Salazar, Edmund P.; Kimlíčková-Polakovičová, Erika; Kleibl, Karol; Thompson, Larry H.; Piršel, Miroslav

2010-01-01

Nucleotide excision repair (NER) is a complex multistage process involving many interacting gene products to repair a wide range of DNA lesions. Genetic defects in NER cause human hereditary diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and a combined XP/CS overlapping symptom. One key gene product associated with all these disorders is the excision repair cross-complementing 3/xeroderma pigmentosum B (ERCC3/XPB) DNA helicase, a subunit of the transcription factor IIH complex. ERCC3 is involved in initiation of basal transcription and global genome repair as well as in transcription-coupled repair (TCR). The hamster ERCC3 gene shows high degree of homology with the human ERCC3/XPB gene. We identified new mutations in the Chinese hamster ovary cell ERCC3 gene and characterized the role of hamster ERCC3 protein in DNA repair of ultraviolet (UV)-induced and oxidative DNA damage. All but one newly described mutations are located in the protein C-terminal region around the last intron–exon boundary. Due to protein truncations or frameshifts, they lack amino acid Ser751, phosphorylation of which prevents the 5′ incision of the UV-induced lesion during NER. Thus, despite the various locations of the mutations, their phenotypes are similar. All ercc3 mutants are extremely sensitive to UV-C light and lack recovery of RNA synthesis (RRS), confirming a defect in TCR of UV-induced damage. Their limited global genome NER capacity averages ∼8%. We detected modest sensitivity of ercc3 mutants to the photosensitizer Ro19-8022, which primarily introduces 8-oxoguanine lesions into DNA. Ro19-8022-induced damage interfered with RRS, and some of the ercc3 mutants had delayed kinetics. All ercc3 mutants showed efficient base excision repair (BER). Thus, the positions of the mutations have no effect on the sensitivity to, and repair of, Ro19-8022-induced DNA damage, suggesting that the ERCC3 protein is not involved in BER. PMID:19942596

Novel point mutations and mutational complexes in the enhancer II, core promoter and precore regions of hepatitis B virus genotype D1 associated with hepatocellular carcinoma in Saudi Arabia.

PubMed

Khan, Anis; Al Balwi, Mohammed A; Tanaka, Yasuhito; Hajeer, Ali; Sanai, Faisal M; Al Abdulkarim, Ibrahim; Al Ayyar, Latifah; Badri, Motasim; Saudi, Dib; Tamimi, Waleed; Mizokami, Masashi; Al Knawy, Bandar

2013-12-15

In this study, a cohort of 182 patients [55 hepatocellular carcinoma (HCC) and 127 non-HCC] infected with hepatitis B virus (HBV) in Saudi Arabia was investigated to study the relationship between sequence variation in the enhancer II (EnhII), basal core promoter (BCP) and precore regions of HBV genotype D (HBV/D) and the risk of HCC. HBV genotypes were determined by sequencing analysis and/or enzyme-linked immunosorbent assay. Variations in the EnhII, BCP and precore regions were compared between 107 non-HCC and 45 HCC patients infected with HBV/D, followed by age-matched analysis of 40 cases versus equal number of controls. Age and male gender were significantly associated with HCC (p = 0.0001 and p = 0.03, respectively). Serological markers such as aspartate aminotransferase, albumin and anti-HBe were significantly associated with HCC (p = 0.0001 for all), whereas HBeAg positivity was associated with non-HCC (p = 0.0001). The most prevalent HBV genotype was HBV/D (94%), followed by HBV/E (4%), HBV/A (1.6%) and HBV/C (0.5%). For HBV/D1, genomic mutations associated with HCC were T1673/G1679, G1727, C1741, C1761, A1757/T1764/G1766, T1773, T1773/G1775 and C1909. Age- and gender-adjusted stepwise logistic regression analysis indicated that mutations G1727 [odds ratio (OR) = 18.3; 95% confidence interval (CI) = 2.8-118.4; p = 0.002], A1757/T1764/G1766 (OR = 4.7; 95% CI = 1.3-17.2; p = 0.01) and T1773 (OR = 14.06; 95% CI = 2.3-84.8; p = 0.004) are independent predictors of HCC development. These results implicate novel individual and combination patterns of mutations in the X/precore region of HBV/D1 as predictors of HCC. Risk stratification based on these mutation complexes would be useful in determining high-risk patients and improving diagnostic and treatment strategies for HBV/D1. Copyright © 2013 UICC.

Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations

PubMed Central

Vogel, Tiphanie P.; Forbes, Lisa; Ma, Chi A.; Stray-Pedersen, Asbjørg; Niemela, Julie E.; Lyons, Jonathan J.; Engelhardt, Karin R.; Zhang, Yu; Topcagic, Nermina; Roberson, Elisha D. O.; Matthews, Helen; Verbsky, James W.; Dasu, Trivikram; Vargas-Hernandez, Alexander; Varghese, Nidhy; McClain, Kenneth L.; Karam, Lina B.; Nahmod, Karen; Makedonas, George; Mace, Emily M.; Sorte, Hanne S.; Perminow, Gøri; Rao, V. Koneti; O’Connell, Michael P.; Price, Susan; Su, Helen C.; Butrick, Morgan; McElwee, Joshua; Hughes, Jason D.; Willet, Joseph; Swan, David; Xu, Yaobo; Santibanez-Koref, Mauro; Slowik, Voytek; Dinwiddie, Darrell L.; Ciaccio, Christina E.; Saunders, Carol J.; Septer, Seth; Kingsmore, Stephen F.; White, Andrew J.; Cant, Andrew J.; Hambleton, Sophie

2015-01-01

Germline loss-of-function mutations in the transcription factor signal transducer and activator of transcription 3 (STAT3) cause immunodeficiency, whereas somatic gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here, we report on 13 individuals from 10 families with lymphoproliferation and early-onset solid-organ autoimmunity associated with 9 different germline heterozygous mutations in STAT3. Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multiorgan autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections, and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T-cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 led to clinical improvement in 1 patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly described disorder. Some patients for this study were enrolled in a trial registered at www.clinicaltrials.gov as #NCT00001350. PMID:25359994

A novel dominant GJB2 (DFNA3) mutation in a Chinese family

NASA Astrophysics Data System (ADS)

Wang, Hongyang; Wu, Kaiwen; Yu, Lan; Xie, Linyi; Xiong, Wenping; Wang, Dayong; Guan, Jing; Wang, Qiuju

2017-01-01

To decipher the phenotype and genotype of a Chinese family with autosomal dominant non-syndromic hearing loss (ADNSHL) and a novel dominant missense mutation in the GJB2 gene (DFNA3), mutation screening of GJB2 was performed on the propositus from a five-generation ADNSHL family through polymerase chain reaction amplification and Sanger sequencing. The candidate variation and the co-segregation of the phenotype were verified in all ascertained family members. Targeted genes capture and next-generation sequencing (NGS) were performed to explore additional genetic variations. We identified the novel GJB2 mutation c.524C > A (p.P175H), which segregated with high frequency and was involved in progressive sensorineural hearing loss. One subject with an additional c.235delC mutation showed a more severe phenotype than did the other members with single GJB2 dominant variations. Four patients diagnosed with noise-induced hearing loss did not carry this mutation. No other pathogenic variations or modifier genes were identified by NGS. In conclusion, a novel missense mutation in GJB2 (DFNA3), affecting the second extracellular domain of the protein, was identified in a family with ADNSHL.

Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.

PubMed

Renner, Ellen D; Rylaarsdam, Stacey; Anover-Sombke, Stephanie; Rack, Anita L; Reichenbach, Janine; Carey, John C; Zhu, Qili; Jansson, Annette F; Barboza, Julia; Schimke, Lena F; Leppert, Mark F; Getz, Melissa M; Seger, Reinhard A; Hill, Harry R; Belohradsky, Bernd H; Torgerson, Troy R; Ochs, Hans D

2008-07-01

Hyper-IgE syndrome (HIES) is a rare, autosomal-dominant immunodeficiency characterized by eczema, Staphylococcus aureus skin abscesses, pneumonia with pneumatocele formation, Candida infections, and skeletal/connective tissue abnormalities. Recently it was shown that heterozygous signal transducer and activator of transcription 3 (STAT3) mutations cause autosomal-dominant HIES. To determine the spectrum and functional consequences of heterozygous STAT3 mutations in a cohort of patients with HIES. We sequenced the STAT3 gene in 38 patients with HIES (National Institutes of Health score >40 points) from 35 families, quantified T(H)17 cells in peripheral blood, and evaluated tyrosine phosphorylation of STAT3. Most STAT3 mutations in our cohort were in the DNA-binding domain (DBD; 22/35 families) or Src homology 2 (SH2) domain (10/35) and were missense mutations. We identified 2 intronic mutations resulting in exon skipping and in-frame deletions within the DBD. In addition, we identified 2 mutations located in the transactivation domain downstream of the SH2 domain: a 10-amino acid deletion and an amino acid substitution. In 1 patient, we were unable to identify a STAT3 mutation. T(H)17 cells were absent or low in the peripheral blood of all patients who were evaluated (n = 17). IL-6-induced STAT3-phosphorylation was consistently reduced in patients with SH2 domain mutations but comparable to normal controls in patients with mutations in the DBD. Heterozygous STAT3 mutations were identified in 34 of 35 unrelated HIES families. Patients had impaired T(H)17 cell development, and those with SH2 domain mutations had reduced STAT3 phosphorylation.

The anti-neoplastic activity of Vandetanib against high-risk medulloblastoma variants is profoundly enhanced by additional PI3K inhibition

PubMed Central

Velz, Julia; Olschewski, Martin; Goetz, Barbara; Pietsch, Torsten; Dilloo, Dagmar

2017-01-01

Medulloblastoma is comprised of at least four molecular subgroups with distinct clinical outcome (WHO classification 2016). SHH-TP53-mutated as well as MYC-amplified Non-WNT/Non-SHH medulloblastoma show the worst prognosis. Here we present evidence that single application of the multi-kinase inhibitor Vandetanib displays anti-neoplastic efficacy against cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma. The narrow target spectrum of Vandetanib along with a favourable toxicity profile renders this drug ideal for multimodal treatment approaches. In this context our investigation documents that Vandetanib in combination with the clinically available PI3K inhibitor GDC-0941 leads to enhanced cytotoxicity against MYC-amplified and SHH-TP53-mutated medulloblastoma. In line with these findings we show for MYC-amplified medulloblastoma a profound reduction in activity of the oncogenes STAT3 and AKT. Furthermore, we document that Vandetanib and the standard chemotherapeutic Etoposide display additive anti-neoplastic efficacy in the investigated medulloblastoma cell lines that could be further enhanced by PI3K inhibition. Of note, the combination of Vandetanib, GDC-0941 and Etoposide results in MYC-amplified and SHH-TP53-mutated cell lines in complete loss of cell viability. Our findings therefore provide a rational to further evaluate Vandetanib in combination with PI3K inhibitors as well as standard chemotherapeutics in vivo for the treatment of most aggressive medulloblastoma variants. PMID:28159923

The anti-neoplastic activity of Vandetanib against high-risk medulloblastoma variants is profoundly enhanced by additional PI3K inhibition.

PubMed

Craveiro, Rogerio B; Ehrhardt, Michael; Velz, Julia; Olschewski, Martin; Goetz, Barbara; Pietsch, Torsten; Dilloo, Dagmar

2017-07-18

Medulloblastoma is comprised of at least four molecular subgroups with distinct clinical outcome (WHO classification 2016). SHH-TP53-mutated as well as MYC-amplified Non-WNT/Non-SHH medulloblastoma show the worst prognosis.Here we present evidence that single application of the multi-kinase inhibitor Vandetanib displays anti-neoplastic efficacy against cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma. The narrow target spectrum of Vandetanib along with a favourable toxicity profile renders this drug ideal for multimodal treatment approaches. In this context our investigation documents that Vandetanib in combination with the clinically available PI3K inhibitor GDC-0941 leads to enhanced cytotoxicity against MYC-amplified and SHH-TP53-mutated medulloblastoma. In line with these findings we show for MYC-amplified medulloblastoma a profound reduction in activity of the oncogenes STAT3 and AKT. Furthermore, we document that Vandetanib and the standard chemotherapeutic Etoposide display additive anti-neoplastic efficacy in the investigated medulloblastoma cell lines that could be further enhanced by PI3K inhibition. Of note, the combination of Vandetanib, GDC-0941 and Etoposide results in MYC-amplified and SHH-TP53-mutated cell lines in complete loss of cell viability. Our findings therefore provide a rational to further evaluate Vandetanib in combination with PI3K inhibitors as well as standard chemotherapeutics in vivo for the treatment of most aggressive medulloblastoma variants.

A new Gsdma3 mutation affecting anagen phase of first hair cycle

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

Tanaka, Shigekazu; Department of Genetics, School of Life Science, Graduate University for Advanced Studies, 1111 Yata, Mishima, Shizuoka 411-8540; Tamura, Masaru

2007-08-10

Recombination-induced mutation 3 (Rim3) is a spontaneous mouse mutation that exhibits dominant phenotype of hyperkeratosis and hair loss. Fine linkage analysis of Rim3 and sequencing revealed a novel single point mutation, G1124A leading to Ala348Thr, in Gsdma3 in chromosome 11. Transgenesis with BAC DNA harboring the Rim3-type Gsdma3 recaptured the Rim3 phenotype, providing direct evidence that Gsdma3 is the causative gene of Rim3. We examined the spatial expression of Gsdma3 and characterized the Rim3 phenotype in detail. Gsdma3 is expressed in differentiated epidermal cells in the skin, but not in the proliferating epidermal cells. Histological analysis of Rim3 mutant showedmore » hyperplasia of the epidermal cells in the upper hair follicles and abnormal anagen phase at the first hair cycle. Furthermore, immunohistochemical analysis revealed hyperproliferation and misdifferentiation of the upper follicular epidermis in Rim3 mutant. These results suggest that Gsdma3 is involved in the proliferation and differentiation of epidermal stem cells.« less

A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

PubMed Central

Yan, Yu; He, Ying; Boson, Bertrand; Wang, Xuesong; Cosset, François-Loïc

2017-01-01

ABSTRACT The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α0), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α0, which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α0 domain of NS3 that significantly enhanced

ALDH1A3 Mutations Cause Recessive Anophthalmia and Microphthalmia

PubMed Central

Fares-Taie, Lucas; Gerber, Sylvie; Chassaing, Nicolas; Clayton-Smith, Jill; Hanein, Sylvain; Silva, Eduardo; Serey, Margaux; Serre, Valérie; Gérard, Xavier; Baumann, Clarisse; Plessis, Ghislaine; Demeer, Bénédicte; Brétillon, Lionel; Bole, Christine; Nitschke, Patrick; Munnich, Arnold; Lyonnet, Stanislas; Calvas, Patrick; Kaplan, Josseline; Ragge, Nicola; Rozet, Jean-Michel

2013-01-01

Anophthalmia and microphthalmia (A/M) are early-eye-development anomalies resulting in absent or small ocular globes, respectively. A/M anomalies occur in syndromic or nonsyndromic forms. They are genetically heterogeneous, some mutations in some genes being responsible for both anophthalmia and microphthalmia. Using a combination of homozygosity mapping, exome sequencing, and Sanger sequencing, we identified homozygosity for one splice-site and two missense mutations in the gene encoding the A3 isoform of the aldehyde dehydrogenase 1 (ALDH1A3) in three consanguineous families segregating A/M with occasional orbital cystic, neurological, and cardiac anomalies. ALDH1A3 is a key enzyme in the formation of a retinoic acid gradient along the dorso-ventral axis during early eye development. Transitory expression of mutant ALDH1A3 open reading frames showed that both missense mutations reduce the accumulation of the enzyme, potentially leading to altered retinoic acid synthesis. Although the role of retinoic acid signaling in eye development is well established, our findings provide genetic evidence of a direct link between retinoic-acid-synthesis dysfunction and early-eye-development anomalies in humans. PMID:23312594

The genetic spectrum and the evaluation of CADASIL screening scale in Chinese patients with NOTCH3 mutations.

PubMed

Liu, Xiao; Zuo, Yuehuan; Sun, Wei; Zhang, Wei; Lv, He; Huang, Yining; Xiao, Jiangxi; Yuan, Yun; Wang, Zhaoxia

2015-07-15

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small artery disease caused by NOTCH3 gene mutation. Here we report clinical, pathological and genetic profiles of 29 newly-diagnosed CADASIL patients, evaluation of the CADASIL scale in Chinese CADASIL patients, and reanalysis of all reported mainland Chinese patients with identified NOTCH3 gene mutation. We found two novel mutations (p.C134G and p.C291Y) and 13 reported NOTCH3 mutations in the newly-diagnosed group. CADASIL scale score was less than the cutoff score in 19 of 53 Chinese patients with NOTCH3 mutation, generating only a sensitivity of 64.1%. At the time of study, the total number of genetically confirmed CADASIL cases reached 158 from 97 unrelated mainland Chinese families, with 9/97 (9.3%) sporadic patients. The NOTCH3 gene mutation profile showed 43 mutations, with hotspots in exon 4, followed by exon 3. The considerable variability in onset age and CADASIL scale score in patients carrying the same NOTCH3 missense mutation suggested no obvious phenotype-genotype correlation. In conclusion, we report two novel mutations which expand the NOTCH3 mutational spectrum. Exons 4 and 3 are hotspots in mainland Chinese patients with NOTCH3 mutation. The low sensitivity of CADASIL scale in our patients group indicated that the CADASIL scale should be refined according to the clinical characteristics of Chinese CADASIL patients when used in Chinese populations. Copyright © 2015. Published by Elsevier B.V.

Identification of a novel CLRN1 gene mutation in Usher syndrome type 3: two case reports.

PubMed

Yoshimura, Hidekane; Oshikawa, Chie; Nakayama, Jun; Moteki, Hideaki; Usami, Shin-Ichi

2015-05-01

This study examines the CLRN1 gene mutation analysis in Japanese patients who were diagnosed with Usher syndrome type 3 (USH3) on the basis of clinical findings. Genetic analysis using massively parallel DNA sequencing (MPS) was conducted to search for 9 causative USH genes in 2 USH3 patients. We identified the novel pathogenic mutation in the CLRN1 gene in 2 patients. The missense mutation was confirmed by functional prediction software and segregation analysis. Both patients were diagnosed as having USH3 caused by the CLRN1 gene mutation. This is the first report of USH3 with a CLRN1 gene mutation in Asian populations. Validating the presence of clinical findings is imperative for properly differentiating among USH subtypes. In addition, mutation screening using MPS enables the identification of causative mutations in USH. The clinical diagnosis of this phenotypically variable disease can then be confirmed. © The Author(s) 2015.

Acute myeloid leukemia-associated DNMT3A p.Arg882His mutation in a patient with Tatton-Brown-Rahman overgrowth syndrome as a constitutional mutation.

PubMed

Kosaki, Rika; Terashima, Hiroshi; Kubota, Masaya; Kosaki, Kenjiro

2017-01-01

DNA methylation plays a critical role in both embryonic development and tumorigenesis and is mediated through various DNA methyltransferases. Constitutional mutations in the de novo DNA methyltransferase DNMT3A cause a recently identified Tatton-Brown-Rahman overgrowth syndrome (TBRS). Somatically acquired mutations in DNMT3A are causally associated with acute myeloid leukemia (AML), and p.Arg882His represents the most prevalent hotspot. So far, no patients with TBRS have been reported to have subsequently developed AML. Here, we report a live birth and the survival of a female with the TBRS phenotype who had a heterozygous constitutional DNMT3A mutation at the AML somatic mutation hotspot p.Arg882His in her DNA from peripheral blood and buccal tissue. Her characteristic features at birth included hypotonia, narrow palpebral fissures, ventricular septal defect, umbilical hernia, sacral cyst, Chiari type I anomaly. At the age of 6 years, she exhibited overgrowth (> 3 SD) and round face and intellectual disability. This report represents the first documentation of the same variant (DNMT3A p.Arg882His) as both the constitutional mutation associated with TBRS and the somatic mutation hotspot of AML. The observation neither confirms nor denies the notion that mutations responsible for TBRS and those for AML might share the same mode of action. Larger data sets are required to determine whether TBRS patients with constitutional DNMT3A mutations are at an increased risk for AML. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

PIK3CA missense mutation is associated with unfavorable outcome in grade 3 endometrioid carcinoma but not in serous endometrial carcinoma.

PubMed

McIntyre, John B; Nelson, Gregg S; Ghatage, Prafull; Morris, Don; Duggan, Máire A; Lee, Cheng-Han; Doll, Corinne M; Köbel, Martin

2014-01-01

To evaluate the outcome association of PIK3CA mutational status within histological types of rigorously classified high-grade endometrial carcinomas. We assessed PIK3CA mutational status in exon 9 and exon 20 hot spots by Sanger sequencing of DNA derived from formalin fixed paraffin embedded tissue of 57 grade 3 endometrioid, 26 serous, 11 clear cell and 5 dedifferentiated carcinomas. We correlated PIK3CA mutation status with clinicopathological and other molecular parameters. Univariate and multivariate disease specific survival analysis was performed using Kaplan-Meier and Cox regression analyses. PIK3CA exon 9 or exon 20 missense mutations were identified in 20 of 99 (20%) high-grade endometrial carcinomas without significant difference across histological types (p=0.22). Presence of PIK3CA exon 9 or exon 20 missense mutations was associated with shorter disease specific survival within grade 3 endometrioid (p=0.0029) but not endometrial serous (p=0.57) carcinoma based on univariate analysis. Within grade 3 endometrioid carcinoma, PIK3CA exon 9 or exon 20 missense mutations were more commonly observed in cases that were deficient for mismatch repair protein expression (p=0.0058) and showed loss of ARID1A expression (p=0.037). PIK3CA exon 9 or exon 20 missense mutations are present across all histological types of high-grade endometrial carcinomas but a significant outcome association is only seen in grade 3 endometrioid carcinoma, suggesting a greater biological importance in this tumor type. Copyright © 2013 Elsevier Inc. All rights reserved.

A Meta-Analysis of the Relationship between FGFR3 and TP53 Mutations in Bladder Cancer

PubMed Central

Ouerhani, Slah; Mongiat-Artus, Pierre; Soliman, Hany; de The, Hugues; Sibony, Mathilde; Denoux, Yves; Molinie, Vincent; Herault, Aurélie; Lepage, May-Linda; Maille, Pascale; Renou, Audrey; Vordos, Dimitri; Abbou, Claude-Clément; Bakkar, Ashraf; Asselain, Bernard; Kourda, Nadia; El Gaaied, Amel; Leroy, Karen; Laplanche, Agnès; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Radvanyi, François

2012-01-01

TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications) and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18–0.37], p = 0.0001) or for pT1 tumours alone (OR = 0.47 [0.28–0.79], p = 0.0009). However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23–1.36] (p = 0.12) and OR = 0.99 [0.37–2.7] (p = 0.35), respectively). After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4). These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage. PMID:23272046

Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing.

PubMed

Banaszak, Lauren G; Giudice, Valentina; Zhao, Xin; Wu, Zhijie; Gao, Shouguo; Hosokawa, Kohei; Keyvanfar, Keyvan; Townsley, Danielle M; Gutierrez-Rodrigues, Fernanda; Fernandez Ibanez, Maria Del Pilar; Kajigaya, Sachiko; Young, Neal S

2018-03-01

DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Here, we successfully created four DNMT3A-mutated K562 cell lines with frameshift mutations resulting in truncated DNMT3A proteins. DNMT3A-mutated cell lines exhibited significantly impaired growth and increased apoptotic activity compared to wild-type (WT) cells. Consistent with previous studies, DNMT3A-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of DNMT3A-mutated and WT cells; DNMT3A ablation resulted in downregulation of genes involved in spliceosome function, causing dysfunction of RNA splicing. Unexpectedly, we observed DNMT3A-mutated cells to exhibit marked genomic instability and an impaired DNA damage response compared to WT. CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy. Published by Elsevier Inc.

Systematic Review of Cysteine-Sparing NOTCH3 Missense Mutations in Patients with Clinical Suspicion of CADASIL.

PubMed

Muiño, Elena; Gallego-Fabrega, Cristina; Cullell, Natalia; Carrera, Caty; Torres, Nuria; Krupinski, Jurek; Roquer, Jaume; Montaner, Joan; Fernández-Cadenas, Israel

2017-09-13

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is caused by mutations in the NOTCH3 gene, affecting the number of cysteines in the extracellular domain of the receptor, causing protein misfolding and receptor aggregation. The pathogenic role of cysteine-sparing NOTCH3 missense mutations in patients with typical clinical CADASIL syndrome is unknown. The aim of this article is to describe these mutations to clarify if any could be potentially pathogenic. Articles on cysteine-sparing NOTCH3 missense mutations in patients with clinical suspicion of CADASIL were reviewed. Mutations were considered potentially pathogenic if patients had: (a) typical clinical CADASIL syndrome; (b) diffuse white matter hyperintensities; (c) the 33 NOTCH3 exons analyzed; (d) mutations that were not polymorphisms; and (e) Granular osmiophilic material (GOM) deposits in the skin biopsy. Twenty-five different mutations were listed. Four fulfill the above criteria: p.R61W; p.R75P; p.D80G; and p.R213K. Patients carrying these mutations had typical clinical CADASIL syndrome and diffuse white matter hyperintensities, mostly without anterior temporal pole involvement. Cysteine-sparing NOTCH3 missense mutations are associated with typical clinical CADASIL syndrome and typical magnetic resonance imaging (MRI) findings, although with less involvement of the anterior temporal lobe. Hence, these mutations should be further studied to confirm their pathological role in CADASIL.

Dual PI3K/mTOR Inhibition in Colorectal Cancers with APC and PIK3CA Mutations.

PubMed

Foley, Tyler M; Payne, Susan N; Pasch, Cheri A; Yueh, Alex E; Van De Hey, Dana R; Korkos, Demetra P; Clipson, Linda; Maher, Molly E; Matkowskyj, Kristina A; Newton, Michael A; Deming, Dustin A

2017-02-09

Therapeutic targeting of the PI3K pathway is an active area of research in multiple cancer types, including breast and endometrial cancers. This pathway is commonly altered in cancer and plays an integral role in numerous vital cellular functions. Mutations in the PIK3CA gene, resulting in a constitutively active form of PI3K, often occur in colorectal cancer, though the population of patients who would benefit from targeting this pathway has yet to be identified. In human colorectal cancers, PIK3CA mutations most commonly occur concomitantly with loss of adenomatous polyposis coli (APC). Here, treatment strategies are investigated that target the PI3K pathway in colon cancers with mutations in APC and PIK3CA Colorectal cancer spheroids with Apc and Pik3ca mutations were generated and characterized confirming that these cultures represent the tumors from which they were derived. Pan and alpha isomer-specific PI3K inhibitors did not induce a significant treatment response, whereas the dual PI3K/mTOR inhibitors BEZ235 and LY3023414 induced a dramatic treatment response through decreased cellular proliferation and increased differentiation. The significant treatment responses were confirmed in mice with Apc and Pik3ca -mutant colon cancers as measured using endoscopy with a reduction in median lumen occlusion of 53% with BEZ235 and a 24% reduction with LY3023414 compared with an increase of 53% in controls ( P < 0.001 and P = 0.03, respectively). This response was also confirmed with 18 F-FDG microPET/CT imaging. Implications: Spheroid models and transgenic mice suggest that dual PI3K/mTOR inhibition is a potential treatment strategy for APC and PIK3CA -mutant colorectal cancers. Thus, further clinical studies of dual PI3K/mTOR inhibitors are warranted in colorectal cancers with these mutations. Mol Cancer Res; 15(3); 1-11. ©2016 AACR. ©2016 American Association for Cancer Research.

Unexpected identification of a recurrent mutation in the DLX3 gene causing amelogenesis imperfecta.

PubMed

Kim, Y-J; Seymen, F; Koruyucu, M; Kasimoglu, Y; Gencay, K; Shin, T J; Hyun, H-K; Lee, Z H; Kim, J-W

2016-05-01

To identify the molecular genetic aetiology of a family with autosomal dominant amelogenesis imperfecta (AI). DNA samples were collected from a six-generation family, and the candidate gene approach was used to screen for the enamelin (ENAM) gene. Whole-exome sequencing and linkage analysis with SNP array data identified linked regions, and candidate gene screening was performed. Mutational analysis revealed a mutation (c.561_562delCT and p.Tyr188Glnfs*13) in the DLX3 gene. After finding a recurrent DLX3 mutation, the clinical phenotype of the family members was re-examined. The proband's mother had pulp elongation in the third molars. The proband had not hair phenotype, but her cousin had curly hair at birth. In this study, we identified a recurrent 2-bp deletional DLX3 mutation in a new family. The clinical phenotype was the mildest one associated with the DLX3 mutations. These results will advance the understanding of the functional role of DLX3 in developmental processes. © 2016 The Authors. Oral Diseases Published by John Wiley & Sons Ltd.

Aberrant Receptor Internalization and Enhanced FRS2-dependent Signaling Contribute to the Transforming Activity of the Fibroblast Growth Factor Receptor 2 IIIb C3 Isoform*

PubMed Central

Cha, Jiyoung Y.; Maddileti, Savitri; Mitin, Natalia; Harden, T. Kendall; Der, Channing J.

2009-01-01

Alternative splice variants of fibroblast growth factor receptor 2 (FGFR2) IIIb, designated C1, C2, and C3, possess progressive reduction in their cytoplasmic carboxyl termini (822, 788, and 769 residues, respectively), with preferential expression of the C2 and C3 isoforms in human cancers. We determined that the progressive deletion of carboxyl-terminal sequences correlated with increasing transforming potency. The highly transforming C3 variant lacks five tyrosine residues present in C1, and we determined that the loss of Tyr-770 alone enhanced FGFR2 IIIb C1 transforming activity. Because Tyr-770 may compose a putative YXXL sorting motif, we hypothesized that loss of Tyr-770 in the 770YXXL motif may cause disruption of FGFR2 IIIb C1 internalization and enhance transforming activity. Surprisingly, we found that mutation of Leu-773 but not Tyr-770 impaired receptor internalization and increased receptor stability and activation. Interestingly, concurrent mutations of Tyr-770 and Leu-773 caused 2-fold higher transforming activity than caused by the Y770F or L773A single mutations, suggesting loss of Tyr and Leu residues of the 770YXXL773 motif enhances FGFR2 IIIb transforming activity by distinct mechanisms. We also determined that loss of Tyr-770 caused persistent activation of FRS2 by enhancing FRS2 binding to FGFR2 IIIb. Furthermore, we found that FRS2 binding to FGFR2 IIIb is required for increased FRS2 tyrosine phosphorylation and enhanced transforming activity by Y770F mutation. Our data support a dual mechanism where deletion of the 770YXXL773 motif promotes FGFR2 IIIb C3 transforming activity by causing aberrant receptor recycling and stability and persistent FRS2-dependent signaling. PMID:19103595

Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3.

PubMed

Joensuu, T; Hämäläinen, R; Yuan, B; Johnson, C; Tegelberg, S; Gasparini, P; Zelante, L; Pirvola, U; Pakarinen, L; Lehesjoki, A E; de la Chapelle, A; Sankila, E M

2001-10-01

Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterized by progressive hearing loss, severe retinal degeneration, and variably present vestibular dysfunction, assigned to 3q21-q25. Here, we report on the positional cloning of the USH3 gene. By haplotype and linkage-disequilibrium analyses in Finnish carriers of a putative founder mutation, the critical region was narrowed to 250 kb, of which we sequenced, assembled, and annotated 207 kb. Two novel genes-NOPAR and UCRP-and one previously identified gene-H963-were excluded as USH3, on the basis of mutational analysis. USH3, the candidate gene that we identified, encodes a 120-amino-acid protein. Fifty-two Finnish patients were homozygous for a termination mutation, Y100X; patients in two Finnish families were compound heterozygous for Y100X and for a missense mutation, M44K, whereas patients in an Italian family were homozygous for a 3-bp deletion leading to an amino acid deletion and substitution. USH3 has two predicted transmembrane domains, and it shows no homology to known genes. As revealed by northern blotting and reverse-transcriptase PCR, it is expressed in many tissues, including the retina.

Effect of the G375C and G346E achondroplasia mutations on FGFR3 activation.

PubMed

He, Lijuan; Serrano, Christopher; Niphadkar, Nitish; Shobnam, Nadia; Hristova, Kalina

2012-01-01

Two mutations in FGFR3, G380R and G375C are known to cause achondroplasia, the most common form of human dwarfism. The G380R mutation accounts for 98% of the achondroplasia cases, and thus has been studied extensively. Here we study the effect of the G375C mutation on the phosphorylation and the cross-linking propensity of full-length FGFR3 in HEK 293 cells, and we compare the results to previously published results for the G380R mutant. We observe identical behavior of the two achondroplasia mutants in these experiments, a finding which supports a direct link between the severity of dwarfism phenotypes and the level and mechanism of FGFR3 over-activation. The mutations do not increase the cross-linking propensity of FGFR3, contrary to previous expectations that the achondroplasia mutations stabilize the FGFR3 dimers. Instead, the phosphorylation efficiency within un-liganded FGFR3 dimers is increased, and this increase is likely the underlying cause for pathogenesis in achondroplasia. We further investigate the G346E mutation, which has been reported to cause achondroplasia in one case. We find that this mutation does not increase FGFR3 phosphorylation and decreases FGFR3 cross-linking propensity, a finding which raises questions whether this mutation is indeed a genetic cause for human dwarfism.

Effect of the G375C and G346E Achondroplasia Mutations on FGFR3 Activation

PubMed Central

He, Lijuan; Serrano, Christopher; Niphadkar, Nitish; Shobnam, Nadia; Hristova, Kalina

2012-01-01

Two mutations in FGFR3, G380R and G375C are known to cause achondroplasia, the most common form of human dwarfism. The G380R mutation accounts for 98% of the achondroplasia cases, and thus has been studied extensively. Here we study the effect of the G375C mutation on the phosphorylation and the cross-linking propensity of full-length FGFR3 in HEK 293 cells, and we compare the results to previously published results for the G380R mutant. We observe identical behavior of the two achondroplasia mutants in these experiments, a finding which supports a direct link between the severity of dwarfism phenotypes and the level and mechanism of FGFR3 over-activation. The mutations do not increase the cross-linking propensity of FGFR3, contrary to previous expectations that the achondroplasia mutations stabilize the FGFR3 dimers. Instead, the phosphorylation efficiency within un-liganded FGFR3 dimers is increased, and this increase is likely the underlying cause for pathogenesis in achondroplasia. We further investigate the G346E mutation, which has been reported to cause achondroplasia in one case. We find that this mutation does not increase FGFR3 phosphorylation and decreases FGFR3 cross-linking propensity, a finding which raises questions whether this mutation is indeed a genetic cause for human dwarfism. PMID:22529939

Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn's disease.

PubMed

Mao, Liming; Kitani, Atsushi; Similuk, Morgan; Oler, Andrew J; Albenberg, Lindsey; Kelsen, Judith; Aktay, Atiye; Quezado, Martha; Yao, Michael; Montgomery-Recht, Kim; Fuss, Ivan J; Strober, Warren

2018-05-01

In these studies, we evaluated the contribution of the NLRP3 inflammasome to Crohn's disease (CD) in a kindred containing individuals having a missense mutation in CARD8, a protein known to inhibit this inflammasome. Whole exome sequencing and PCR studies identified the affected individuals as having a V44I mutation in a single allele of the T60 isoform of CARD8. The serum levels of IL-1β in the affected individuals were increased compared with those in healthy controls, and their peripheral monocytes produced increased amounts of IL-1β when stimulated by NLRP3 activators. Immunoblot studies probing the basis of these findings showed that mutated T60 CARD8 failed to downregulate the NLRP3 inflammasome because it did not bind to NLRP3 and inhibit its oligomerization. In addition, these studies showed that mutated T60 CARD8 exerted a dominant-negative effect by its capacity to bind to and form oligomers with unmutated T60 or T48 CARD8 that impeded their binding to NLRP3. Finally, inflammasome activation studies revealed that intact but not mutated CARD8 prevented NLRP3 deubiquitination and serine dephosphorylation. CD due to a CARD8 mutation was not effectively treated by anti-TNF-α, but did respond to IL-1β inhibitors. Thus, patients with anti-TNF-α-resistant CD may respond to this treatment option.

The Metabolome in Finnish Carriers of the MYBPC3-Q1061X Mutation for Hypertrophic Cardiomyopathy.

PubMed

Jørgenrud, Benedicte; Jalanko, Mikko; Heliö, Tiina; Jääskeläinen, Pertti; Laine, Mika; Hilvo, Mika; Nieminen, Markku S; Laakso, Markku; Hyötyläinen, Tuulia; Orešič, Matej; Kuusisto, Johanna

2015-01-01

Mutations in the cardiac myosin-binding protein C gene (MYBPC3) are the most common genetic cause of hypertrophic cardiomyopathy (HCM) worldwide. The molecular mechanisms leading to HCM are poorly understood. We investigated the metabolic profiles of mutation carriers with the HCM-causing MYBPC3-Q1061X mutation with and without left ventricular hypertrophy (LVH) and non-affected relatives, and the association of the metabolome to the echocardiographic parameters. 34 hypertrophic subjects carrying the MYBPC3-Q1061X mutation, 19 non-hypertrophic mutation carriers and 20 relatives with neither mutation nor hypertrophy were examined using comprehensive echocardiography. Plasma was analyzed for molecular lipids and polar metabolites using two metabolomics platforms. Concentrations of branched chain amino acids, triglycerides and ether phospholipids were increased in mutation carriers with hypertrophy as compared to controls and non-hypertrophic mutation carriers, and correlated with echocardiographic LVH and signs of diastolic and systolic dysfunction in subjects with the MYBPC3-Q1061X mutation. Our study implicates the potential role of branched chain amino acids, triglycerides and ether phospholipids in HCM, as well as suggests an association of these metabolites with remodeling and dysfunction of the left ventricle.

Molecular and cellular characteristics of ABCA3 mutations associated with diffuse parenchymal lung diseases in children

PubMed Central

Flamein, Florence; Riffault, Laure; Muselet-Charlier, Céline; Pernelle, Julie; Feldmann, Delphine; Jonard, Laurence; Durand-Schneider, Anne-Marie; Coulomb, Aurore; Maurice, Michèle; Nogee, Lawrence M.; Inagaki, Nobuya; Amselem, Serge; Dubus, Jean Christophe; Rigourd, Virginie; Brémont, François; Marguet, Christophe; Brouard, Jacques; de Blic, Jacques; Clement, Annick; Epaud, Ralph; Guillot, Loïc

2012-01-01

ABCA3 (ATP-binding cassette subfamily A, member 3) is expressed in the lamellar bodies of alveolar type II cells and is crucial to pulmonary surfactant storage and homeostasis. ABCA3 gene mutations have been associated with neonatal respiratory distress (NRD) and pediatric interstitial lung disease (ILD). The objective of this study was to look for ABCA3 gene mutations in patients with severe NRD and/or ILD. The 30 ABCA3 coding exons were screened in 47 patients with severe NRD and/or ILD. ABCA3 mutations were identified in 10 out of 47 patients, including 2 homozygous, 5 compound heterozygous and 3 heterozygous patients. SP-B and SP-C expression patterns varied across patients. Among patients with ABCA3 mutations, five died shortly after birth and five developed ILD (including one without NRD). Functional studies of p.D253H and p.T1173R mutations revealed that p.D253H and p.T1173R induced abnormal lamellar bodies. Additionally, p.T1173R increased IL-8 secretion in vitro. In conclusion, we identified new ABCA3 mutations in patients with life-threatening NRD and/or ILD. Two mutations associated with ILD acted via different pathophysiological mechanisms despite similar clinical phenotypes. PMID:22068586

Clinical Heterogeneity in Patients With FOXP3 Mutations Presenting With Permanent Neonatal Diabetes

PubMed Central

Rubio-Cabezas, Oscar; Minton, Jayne A.L.; Caswell, Richard; Shield, Julian P.; Deiss, Dorothee; Sumnik, Zdenek; Cayssials, Amely; Herr, Mathias; Loew, Anja; Lewis, Vaughan; Ellard, Sian; Hattersley, Andrew T.

2009-01-01

OBJECTIVE—Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by FOXP3 mutations. We aimed to determine the prevalence, genetics, and clinical phenotype of FOXP3 mutations in a large cohort with permanent neonatal diabetes (PNDM). RESEARCH DESIGN AND METHODS—The 11 coding exons and the polyadenylation region of FOXP3 were sequenced in 26 male subjects with diabetes diagnosed before 6 months of age in whom common genetic causes of PNDM had been excluded. Ten subjects had at least one additional immune-related disorder, and the remaining 16 had isolated diabetes. RESULTS—We identified four hemizygous FOXP3 mutations in 6 of 10 patients with associated immune-related disorders and in 0 of 16 patients with isolated diabetes (P = 0.002). Three patients with two novel mutations (R337Q and P339A) and the previously reported L76QfsX53 developed classic IPEX syndrome and died within the first 13 months. The novel mutation V408M was found in three patients from two unrelated families and had a mild phenotype with hypothyroidism and autoimmune enteropathy (n = 2) or nephrotic syndrome (n = 1) and survival to 12–15 years. CONCLUSIONS—FOXP3 mutations result in ∼4% of cases of male patients with permanent diabetes diagnosed before 6 months. Patients not only have classic IPEX syndrome but, unexpectedly, may have a more benign phenotype. FOXP3 sequencing should be performed in any male patient with the diagnosis of diabetes in the first 6 months who develops other possible autoimmune-associated conditions, even in the absence of full IPEX syndrome. PMID:18931102

Avoidance of APOBEC3B-induced mutation by error-free lesion bypass

PubMed Central

Hoopes, James I.; Hughes, Amber L.; Hobson, Lauren A.; Cortez, Luis M.; Brown, Alexander J.

2017-01-01

Abstract APOBEC cytidine deaminases mutate cancer genomes by converting cytidines into uridines within ssDNA during replication. Although uracil DNA glycosylases limit APOBEC-induced mutation, it is unknown if subsequent base excision repair (BER) steps function on replication-associated ssDNA. Hence, we measured APOBEC3B-induced CAN1 mutation frequencies in yeast deficient in BER endonucleases or DNA damage tolerance proteins. Strains lacking Apn1, Apn2, Ntg1, Ntg2 or Rev3 displayed wild-type frequencies of APOBEC3B-induced canavanine resistance (CanR). However, strains without error-free lesion bypass proteins Ubc13, Mms2 and Mph1 displayed respective 4.9-, 2.8- and 7.8-fold higher frequency of APOBEC3B-induced CanR. These results indicate that mutations resulting from APOBEC activity are avoided by deoxyuridine conversion to abasic sites ahead of nascent lagging strand DNA synthesis and subsequent bypass by error-free template switching. We found this mechanism also functions during telomere re-synthesis, but with a diminished requirement for Ubc13. Interestingly, reduction of G to C substitutions in Ubc13-deficient strains uncovered a previously unknown role of Ubc13 in controlling the activity of the translesion synthesis polymerase, Rev1. Our results highlight a novel mechanism for error-free bypass of deoxyuridines generated within ssDNA and suggest that the APOBEC mutation signature observed in cancer genomes may under-represent the genomic damage these enzymes induce. PMID:28334887

Mutational Spectrum of MYO15A and the Molecular Mechanisms of DFNB3 Human Deafness

PubMed Central

Rehman, Atteeq U.; Bird, Jonathan E.; Faridi, Rabia; Shahzad, Mohsin; Shah, Sujay; Lee, Kwanghyuk; Khan, Shaheen N.; Imtiaz, Ayesha; Ahmed, Zubair M.; Riazuddin, Saima; Santos-Cortez, Regie Lyn P.; Ahmad, Wasim; Leal, Suzanne M.; Riazuddin, Sheikh; Friedman, Thomas B.

2016-01-01

Deafness in humans is a common neurosensory disorder and is genetically heterogeneous. Across diverse ethnic groups, mutations of MYO15A at the DFNB3 locus appear to be the third or fourth most common cause of autosomal recessive, nonsyndromic deafness. In 49 of the 67 exons of MYO15A, there are currently 192 recessive mutations identified, including 14 novel mutations reported here. These mutations are distributed uniformly across MYO15A with one enigmatic exception; the alternatively spliced giant exon 2, encoding 1,233 residues, has 17 truncating mutations but no convincing deafness-causing missense mutations. MYO15A encodes three distinct isoform classes, one of which is 395 kDa (3,530 residues), the largest member of the myosin superfamily of molecular motors. Studies of Myo15 mouse models that recapitulate DFNB3 revealed two different pathogenic mechanisms of hearing loss. In the inner ear, myosin 15 is necessary both for the development and the long-term maintenance of stereocilia, mechanosensory sound-transducing organelles that extend from the apical surface of hair cells. The goal of this Mutation Update is to provide a comprehensive review of mutations and functions of MYO15A. PMID:27375115

ALDH1A3 mutations cause recessive anophthalmia and microphthalmia.

PubMed

Fares-Taie, Lucas; Gerber, Sylvie; Chassaing, Nicolas; Clayton-Smith, Jill; Hanein, Sylvain; Silva, Eduardo; Serey, Margaux; Serre, Valérie; Gérard, Xavier; Baumann, Clarisse; Plessis, Ghislaine; Demeer, Bénédicte; Brétillon, Lionel; Bole, Christine; Nitschke, Patrick; Munnich, Arnold; Lyonnet, Stanislas; Calvas, Patrick; Kaplan, Josseline; Ragge, Nicola; Rozet, Jean-Michel

2013-02-07

Anophthalmia and microphthalmia (A/M) are early-eye-development anomalies resulting in absent or small ocular globes, respectively. A/M anomalies occur in syndromic or nonsyndromic forms. They are genetically heterogeneous, some mutations in some genes being responsible for both anophthalmia and microphthalmia. Using a combination of homozygosity mapping, exome sequencing, and Sanger sequencing, we identified homozygosity for one splice-site and two missense mutations in the gene encoding the A3 isoform of the aldehyde dehydrogenase 1 (ALDH1A3) in three consanguineous families segregating A/M with occasional orbital cystic, neurological, and cardiac anomalies. ALDH1A3 is a key enzyme in the formation of a retinoic acid gradient along the dorso-ventral axis during early eye development. Transitory expression of mutant ALDH1A3 open reading frames showed that both missense mutations reduce the accumulation of the enzyme, potentially leading to altered retinoic acid synthesis. Although the role of retinoic acid signaling in eye development is well established, our findings provide genetic evidence of a direct link between retinoic-acid-synthesis dysfunction and early-eye-development anomalies in humans. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

[Rapid detection of hot spot mutations of FGFR3 gene with PCR-high resolution melting assay].

PubMed

Li, Shan; Wang, Han; Su, Hua; Gao, Jinsong; Zhao, Xiuli

2017-08-10

To identify the causative mutations in five individuals affected with dyschondroplasia and develop an efficient procedure for detecting hot spot mutations of the FGFR3 gene. Genomic DNA was extracted from peripheral blood samples with a standard phenol/chloroform method. PCR-Sanger sequencing was used to analyze the causative mutations in the five probands. PCR-high resolution melting (HRM) was developed to detect the identified mutations. A c.1138G>A mutation in exon 8 was found in 4 probands, while a c.1620C>G mutation was found in exon 11 of proband 5 whom had a mild phenotype. All patients were successfully distinguished from healthy controls with the PCR-HRM method. The results of HRM analysis were highly consistent with that of Sanger sequencing. The Gly380Arg and Asn540Lys are hot spot mutations of the FGFR3 gene among patients with ACH/HCH. PCR-HRM analysis is more efficient for detecting hot spot mutations of the FGFR3 gene.

A common FGFR3 gene mutation is present in achondroplasia but not in hypochondroplasia.

PubMed

Stoilov, I; Kilpatrick, M W; Tsipouras, P

1995-01-02

Achondroplasia is the most common type of genetic dwarfism. It is characterized by disproportionate short stature and other skeletal anomalies resulting from a defect in the maturation of the chondrocytes in the growth plate of the cartilage. Recent studies mapped the achondroplasia gene on chromosome region 4p16.3 and identified a common mutation in the gene encoding the fibroblast growth factor receptor 3 (FGFR3). In an analysis of 19 achondroplasia families from a variety of ethnic backgrounds we confirmed the presence of the G380R mutation in 21 of 23 achondroplasia chromosomes studied. In contrast, the G380R mutation was not found in any of the 8 hypochondroplasia chromosomes studied. Furthermore, linkage studies in a 3-generation family with hypochondroplasia show discordant segregation with markers in the 4p16.3 region suggesting that at least some cases of hypochondroplasia are caused by mutations in a gene other than FGFR3.

Novel Mutation of the NOTCH3 Gene in a Chinese Pedigree with CADASIL.

PubMed

Hou, Xiaoxia; He, Chuan; Jin, Qingwen; Niu, Qi; Ren, Guang; Cheng, Hong

2017-01-01

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) results from NOTCH3 gene mutations, which lead to the degeneration of vascular smooth muscle cells (VSMCs). The clinical presentation of CADASIL patients is dependent on the impact of other vascular risk factors and the type of NOTCH3 mutation present. Here, we report a rare pathogenic mutation on exon 14 of the NOTCH3 gene in a Chinese family affected by CADASIL with phenotypic peculiarities. We performed genetic testing, clinical and neuropsychological examination, brain magnetic resonance images (MRI), and electron microscopy (EM) in skin biopsies. NOTCH3 gene analysis revealed a c.2182CT substitution on exon 14, which is the first example of this mutation in a Chinese individual from the Han ancestry. Granular osmiophilic material (GOM) was found in the proband, and all patients had migraine, subcortical ischemic events, and mood disturbances, without progressive cognitive impairment. Cranial MRI further showed white matter hyperintensity, involving bilateral basal ganglia and multiple microbleeds (MBs), in the thalamus and brain stem. This study suggests that different missense mutations in NOTCH3 might contribute to atypical clinical features of CADASIL. This report also indicates that for individuals with a positive family history having clinical and neuroradiological findings suggestive of CADASIL, genetic testing and GOM detection should be performed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Liquid Biopsy Analysis of FGFR3 and PIK3CA Hotspot Mutations for Disease Surveillance in Bladder Cancer.

PubMed

Christensen, Emil; Birkenkamp-Demtröder, Karin; Nordentoft, Iver; Høyer, Søren; van der Keur, Kirstin; van Kessel, Kim; Zwarthoff, Ellen; Agerbæk, Mads; Ørntoft, Torben Falck; Jensen, Jørgen Bjerggaard; Dyrskjøt, Lars

2017-06-01

Disease surveillance in patients with bladder cancer is important for early diagnosis of progression and metastasis and for optimised treatment. To develop urine and plasma assays for disease surveillance for patients with FGFR3 and PIK3CA tumour mutations. Droplet digital polymerase chain reaction (ddPCR) assays were developed and tumour DNA from two patient cohorts was screened for FGFR3 and PIK3CA hotspot mutations. One cohort included 363 patients with non-muscle-invasive bladder cancer (NMIBC). The other cohort included 468 patients with bladder cancer undergoing radical cystectomy (Cx). Urine supernatants (NMIBC n=216, Cx n=27) and plasma samples (NMIBC n=39, Cx n=27) from patients harbouring mutations were subsequently screened using ddPCR assays. Progression-free survival, recurrence-free survival, and overall survival were measured. Fisher's exact test, the Wilcoxon rank-sum test and Cox regression analysis were applied. In total, 36% of the NMIBC patients (129/363) and 11% of the Cx patients (44/403) harboured at least one FGFR3 or PIK3CA mutation. Screening of DNA from serial urine supernatants from the NMIBC cohort revealed that high levels of tumour DNA (tDNA) were associated with later disease progression in NMIBC (p=0.003). Furthermore, high levels of tDNA in plasma samples were associated with recurrence in the Cx cohort (p=0.016). A positive correlation between tDNA levels in urine and plasma was observed (correlation coefficient 0.6). The retrospective study design and low volumes of plasma available for analysis were limitations of the study. Increased levels of FGFR3 and PIK3CA mutated DNA in urine and plasma are indicative of later progression and metastasis in bladder cancer. Urine and plasma from patients with bladder cancer may be monitored for diagnosis of progression and metastasis using mutation assays. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Feature genes predicting the FLT3/ITD mutation in acute myeloid leukemia

PubMed Central

LI, CHENGLONG; ZHU, BIAO; CHEN, JIAO; HUANG, XIAOBING

2016-01-01

In the present study, gene expression profiles of acute myeloid leukemia (AML) samples were analyzed to identify feature genes with the capacity to predict the mutation status of FLT3/ITD. Two machine learning models, namely the support vector machine (SVM) and random forest (RF) methods, were used for classification. Four datasets were downloaded from the European Bioinformatics Institute, two of which (containing 371 samples, including 281 FLT3/ITD mutation-negative and 90 mutation-positive samples) were randomly defined as the training group, while the other two datasets (containing 488 samples, including 350 FLT3/ITD mutation-negative and 138 mutation-positive samples) were defined as the test group. Differentially expressed genes (DEGs) were identified by significance analysis of the micro-array data by using the training samples. The classification efficiency of the SCM and RF methods was evaluated using the following parameters: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the area under the receiver operating characteristic curve. Functional enrichment analysis was performed for the feature genes with DAVID. A total of 585 DEGs were identified in the training group, of which 580 were upregulated and five were downregulated. The classification accuracy rates of the two methods for the training group, the test group and the combined group using the 585 feature genes were >90%. For the SVM and RF methods, the rates of correct determination, specificity and PPV were >90%, while the sensitivity and NPV were >80%. The SVM method produced a slightly better classification effect than the RF method. A total of 13 biological pathways were overrepresented by the feature genes, mainly involving energy metabolism, chromatin organization and translation. The feature genes identified in the present study may be used to predict the mutation status of FLT3/ITD in patients with AML. PMID:27177049

Somatic mutations of GUCY2F, EPHA3, and NTRK3 in human cancers.

PubMed

Wood, Laura D; Calhoun, Eric S; Silliman, Natalie; Ptak, Janine; Szabo, Steve; Powell, Steve M; Riggins, Gregory J; Wang, Tian-Li; Yan, Hai; Gazdar, Adi; Kern, Scott E; Pennacchio, Len; Kinzler, Kenneth W; Vogelstein, Bert; Velculescu, Victor E

2006-10-01

Tyrosine kinases are major regulators of signal transduction cascades involved in cellular proliferation and have important roles in tumorigenesis. We have recently analyzed the tyrosine kinase gene family for alterations in human colorectal cancers and identified somatic mutations in seven members of this gene family. In this study we have used high-throughput sequencing approaches to further evaluate this subset of genes for genetic alterations in other human tumors. We identified somatic mutations in GUCY2F, EPHA3, and NTRK3 in breast, lung, and pancreatic cancers. Our results implicate these tyrosine kinase genes in the pathogenesis of other tumor types and suggest that they may be useful targets for diagnostic and therapeutic intervention in selected patients.

Novel CPVT-Associated Calmodulin Mutation in CALM3 (CALM3-A103V) Activates Arrhythmogenic Ca Waves and Sparks

PubMed Central

Gomez-Hurtado, Nieves; Boczek, Nicole J.; Kryshtal, Dmytro O.; Johnson, Christopher N.; Sun, Jennifer; Nitu, Florentin R.; Cornea, Razvan L.; Chazin, Walter J.; Calvert, Melissa L.; Tester, David J.; Ackerman, Michael J.; Knollmann, Bjorn C.

2016-01-01

Background Calmodulin (CaM) mutations are associated with severe forms of long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently reported that CaM mutations were found in 13% of genotype-negative LQTS patients, but the prevalence of CaM mutations in genotype-negative CPVT patients is unknown. Here, we identify and characterize CaM mutations in 12 patients with genotype-negative but clinically-diagnosed CPVT. Methods and Results Mutational analysis of CALM1, CALM2 and CALM3 coding regions, in vitro measurement of CaM-Ca2+ (Ca) binding affinity, RyR2-CaM binding, Ca handling, L-type Ca current (LTCC) and action potential duration (APD). We identified a novel CaM mutation – A103V – in CALM3 in 1 of 12 patients (8%), a female who experienced episodes of exertion-induced syncope since age 10, had normal QT interval, and displayed ventricular ectopy during stress testing consistent with CPVT. A103V modestly lowered CaM Ca-binding affinity (3-fold reduction vs WT-CaM), but did not alter CaM binding to RyR2. In permeabilized cardiomyocytes, A103V-CaM (100 nM) promoted spontaneous Ca wave and spark activity, a cellular phenotype of RyR2 activation. Even a 1:3 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. Compared to LQTS D96V-CaM, A103V-CaM had significantly less effects on LTCC inactivation and APD, and caused delayed after depolarizations (DADs) and triggered beats in intact cardiomyocytes. Conclusions We discovered a novel CPVT mutation in the CALM3 gene that shares functional characteristics with established CPVT-associated mutations in CALM1. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances via RyR2 dysregulation, which explains the autosomal dominant inheritance. PMID:27516456

Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation.

PubMed

Park, Sujin; Yang, Kyung-Min; Park, Yuna; Hong, Eunji; Hong, Chang Pyo; Park, Jinah; Pang, Kyoungwha; Lee, Jihee; Park, Bora; Lee, Siyoung; An, Haein; Kwak, Mi-Kyung; Kim, Junil; Kang, Jin Muk; Kim, Pyunggang; Xiao, Yang; Nie, Guangjun; Ooshima, Akira; Kim, Seong-Jin

2018-03-01

Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2 , SNAI1 , and ZEB1 in response to TGF-β1 in human pancreatic and prostate cancer cell lines. We identified GADD45B , CTGF , and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B , CTGF , and JUNB genes in various cancers.

Selection enhanced estimates of marker effects on means and variances of beef tenderness

USDA-ARS?s Scientific Manuscript database

Genetic marker associations from surveys of industry cattle populations have low frequencies of rare homozygous animals. Selection for calpain (CAPN1) and calpastatin (CAST) genetic markers was replicated in two cattle populations (Angus and MARC III) at the U.S. Meat Animal Research Center. These...

Nonsense mutations in the PAX3 gene cause Waardenburg syndrome type I in two Chinese patients.

PubMed

Yang, Shu-Zhi; Cao, Ju-Yang; Zhang, Rui-Ning; Liu, Li-Xian; Liu, Xin; Zhang, Xin; Kang, Dong-Yang; Li, Mei; Han, Dong-Yi; Yuan, Hui-Jun; Yang, Wei-Yan

2007-01-05

Waardenburg syndrome type I (WS1) is an autosomal dominant disorder characterized by sensorineural hearing loss, pigmental abnormalities of the eye, hair and skin, and dystopia canthorum. The gene mainly responsible for WS1 is PAX3 which is involved in melanocytic development and survival. Mutations of PAX3 have been reported in familiar or sporadic patients with WS1 in several populations of the world except Chinese. In order to explore the genetic background of Chinese WS1 patients, a mutation screening of PAX3 gene was carried out in four WS1 pedigrees. A questionnaire survey and comprehensive clinical examination were conducted in four Chinese pedigrees of WS1. Genomic DNA from each patient and their family members was extracted and exons of PAX3 were amplified by PCR. PCR fragments were ethanol-purified and sequenced in both directions on an ABI_Prism 3100 DNA sequencer with the BigDye Terminator Cycle Sequencing Ready Reaction Kit. The sequences were obtained and aligned to the wild type sequence of PAX3 with the GeneTool program. Two nonsense PAX3 mutations have been found in the study population. One is heterozygous for a novel nonsense mutation S209X. The other is heterozygous for a previously reported mutation in European population R223X. Both mutations create stop codons leading to truncation of the PAX3 protein. This is the first demonstration of PAX3 mutations in Chinese WS1 patients and one of the few examples of an identical mutation of PAX3 occurred in different populations.

A novel mutation of PAX3 in a Chinese family with Waardenburg syndrome.

PubMed

Qin, Wei; Shu, Anli; Qian, Xueqing; Gao, Jianjun; Xing, Qinghe; Zhang, Juan; Zheng, Yonglan; Li, Xingwang; Li, Sheng; Feng, Guoyin; He, Lin

2006-08-28

The molecular characterization of 34 members of a Chinese family, with 22 members in four generations, affected with Waardenburg syndrome (WS1). A detailed family history and clinical data were collected. A genome-wide scan by two-point linkage analysis using more than 400 microsatellite markers in combination with haplotype analysis was performed. Mutation screening was carried out in the candidate gene by sequencing of amplified products. A maximum two-point lod score of 6.53 at theta = 0.00 was obtained with marker D2S2248. Haplotype analysis placed the WS1 locus to a 45.74 cM region between D2S117 and D2S206, in close proximity to the PAX3 gene on chromosome 2q35. Mutation screening in PAX3 identified a 701T > C mutation which converted a highly conserved Leu to Pro. This nucleotide alteration was neither seen in unaffected members of the family nor found in 50 unrelated control subjects. The present study identified a novel 701T > C mutation in PAX3. The mutation observed in this family highlights the phenotypic heterogeneity of the disorder.

Error-prone PCR mutation of Ls-EPSPS gene from Liriope spicata conferring to its enhanced glyphosate-resistance.

PubMed

Mao, Chanjuan; Xie, Hongjie; Chen, Shiguo; Valverde, Bernal E; Qiang, Sheng

2017-09-01

Liriope spicata (Thunb.) Lour has a unique LsEPSPS structure contributing to the highest-ever-recognized natural glyphosate tolerance. The transformed LsEPSPS confers increased glyphosate resistance to E. coli and A. thaliana. However, the increased glyphosate-resistance level is not high enough to be of commercial value. Therefore, LsEPSPS was subjected to error-prone PCR to screen mutant EPSPS genes capable of endowing higher resistance levels. A mutant designated as ELs-EPSPS having five mutated amino acids (37Val, 67Asn, 277Ser, 351Gly and 422Gly) was selected for its ability to confer improved resistance to glyphosate. Expression of ELs-EPSPS in recombinant E. coli BL21 (DE3) strains enhanced resistance to glyphosate in comparison to both the LsEPSPS-transformed and -untransformed controls. Furthermore, transgenic ELs-EPSPS A. thaliana was about 5.4 fold and 2-fold resistance to glyphosate compared with the wild-type and the Ls-EPSPS-transgenic plants, respectively. Therefore, the mutated ELs-EPSPS gene has potential value for has potential for the development of glyphosate-resistant crops. Copyright © 2016 Elsevier Inc. All rights reserved.

Brief Report: Cryopyrin-Associated Periodic Syndrome Caused by a Myeloid-Restricted Somatic NLRP3 Mutation.

PubMed

Zhou, Qing; Aksentijevich, Ivona; Wood, Geryl M; Walts, Avram D; Hoffmann, Patrycja; Remmers, Elaine F; Kastner, Daniel L; Ombrello, Amanda K

2015-09-01

To identify the cause of disease in an adult patient presenting with recent-onset fevers, chills, urticaria, fatigue, and profound myalgia, who was found to be negative for cryopyrin-associated periodic syndrome (CAPS) NLRP3 mutations by conventional Sanger DNA sequencing. We performed whole-exome sequencing and targeted deep sequencing using DNA from the patient's whole blood to identify a possible NLRP3 somatic mutation. We then screened for this mutation in subcloned NLRP3 amplicons from fibroblasts, buccal cells, granulocytes, negatively selected monocytes, and T and B lymphocytes and further confirmed the somatic mutation by targeted sequencing of exon 3. We identified a previously reported CAPS-associated mutation, p.Tyr570Cys, with a mutant allele frequency of 15% based on exome data. Targeted sequencing and subcloning of NLRP3 amplicons confirmed the presence of the somatic mutation in whole blood at a ratio similar to the exome data. The mutant allele frequency was in the range of 13.3-16.8% in monocytes and 15.2-18% in granulocytes. Notably, this mutation was either absent or present at a very low frequency in B and T lymphocytes, in buccal cells, and in the patient's cultured fibroblasts. Our findings indicate the possibility of myeloid-restricted somatic mosaicism in the pathogenesis of CAPS, underscoring the emerging role of massively parallel sequencing in clinical diagnosis. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

NLGN3/NLGN4 gene mutations are not responsible for autism in the Quebec population.

PubMed

Gauthier, Julie; Bonnel, Anna; St-Onge, Judith; Karemera, Liliane; Laurent, Sandra; Mottron, Laurent; Fombonne, Eric; Joober, Ridha; Rouleau, Guy A

2005-01-05

Jamain [2003: Nat Genet 34:27-29] recently reported mutations in two neuroligin genes in sib-pairs affected with autism. In order to confirm these causative mutations in our autistic population and to determine their frequency we screened 96 individuals affected with autism. We found no mutations in these X-linked genes. These results indicate that mutations in NLGN3 and NLGN4 genes are responsible for at most a small fraction of autism cases and additional screenings in other autistic populations are needed to better determine the frequency with which mutations in NLGN3 and NLGN4 occur in autism. Copyright 2004 Wiley-Liss, Inc.

Gain-of-function mutation in FGFR3 in mice leads to decreased bone mass by affecting both osteoblastogenesis and osteoclastogenesis

PubMed Central

Su, Nan; Sun, Qidi; Li, Can; Lu, Xiumin; Qi, Huabing; Chen, Siyu; Yang, Jing; Du, Xiaolan; Zhao, Ling; He, Qifen; Jin, Min; Shen, Yue; Chen, Di; Chen, Lin

2010-01-01

Achondroplasia (ACH) is a short-limbed dwarfism resulting from gain-of-function mutations in fibroblast growth factor receptor 3 (FGFR3). Previous studies have shown that ACH patients have impaired chondrogenesis, but the effects of FGFR3 on bone formation and bone remodeling at adult stages of ACH have not been fully investigated. Using micro-computed tomography and histomorphometric analyses, we found that 2-month-old Fgfr3G369C/+ mice (mouse model mimicking human ACH) showed decreased bone mass due to reduced trabecular bone volume and bone mineral density, defect in bone mineralization and increased osteoclast numbers and activity. Compared with primary cultures of bone marrow stromal cells (BMSCs) from wild-type mice, Fgfr3G369C/+ cultures showed decreased cell proliferation, increased osteogenic differentiation including up-regulation of alkaline phosphatase activity and expressions of osteoblast marker genes, and reduced bone matrix mineralization. Furthermore, our studies also suggest that decreased cell proliferation and enhanced osteogenic differentiation observed in Fgfr3G369C/+ BMSCs are caused by up-regulation of p38 phosphorylation and that enhanced Erk1/2 activity is responsible for the impaired bone matrix mineralization. In addition, in vitro osteoclast formation and bone resorption assays demonstrated that osteoclast numbers and bone resorption area were increased in cultured bone marrow cells derived from Fgfr3G369C/+ mice. These findings demonstrate that gain-of-function mutation in FGFR3 leads to decreased bone mass by regulating both osteoblast and osteoclast activities. Our studies provide new insight into the mechanism underlying the development of ACH. PMID:20053668

A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding.

PubMed

Yan, Yu; He, Ying; Boson, Bertrand; Wang, Xuesong; Cosset, François-Loïc; Zhong, Jin

2017-03-15

The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α 0 ), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α 0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α 0 , which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α 0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α 0 domain of NS3 that significantly enhanced

Gain of function AMP-activated protein kinase γ3 mutation (AMPKγ3R200Q) in pig muscle increases glycogen storage regardless of AMPK activation.

PubMed

Scheffler, Tracy L; Park, Sungkwon; Roach, Peter J; Gerrard, David E

2016-06-01

Chronic activation of AMP-activated protein kinase (AMPK) increases glycogen content in skeletal muscle. Previously, we demonstrated that a mutation in the ryanodine receptor (RyR1(R615C)) blunts AMPK phosphorylation in longissimus muscle of pigs with a gain of function mutation in the AMPKγ3 subunit (AMPKγ3(R200Q)); this may decrease the glycogen storage capacity of AMPKγ3(R200Q) + RyR1(R615C) muscle. Therefore, our aim in this study was to utilize our pig model to understand how AMPKγ3(R200Q) and AMPK activation contribute to glycogen storage and metabolism in muscle. We selected and bred pigs in order to generate offspring with naturally occurring AMPKγ3(R200Q), RyR1(R615C), and AMPKγ3(R200Q) + RyR1(R615C) mutations, and also retained wild-type littermates (control). We assessed glycogen content and parameters of glycogen metabolism in longissimus muscle. Regardless of RyR1(R615C), AMPKγ3(R200Q) increased the glycogen content by approximately 70%. Activity of glycogen synthase (GS) without the allosteric activator glucose 6-phosphate (G6P) was decreased in AMPKγ3(R200Q) relative to all other genotypes, whereas both AMPKγ3(R200Q) and AMPKγ3(R200Q) + RyR1(R615C) muscle exhibited increased GS activity with G6P. Increased activity of GS with G6P was not associated with increased abundance of GS or hexokinase 2. However, AMPKγ3(R200Q) enhanced UDP-glucose pyrophosphorylase 2 (UGP2) expression approximately threefold. Although UGP2 is not generally considered a rate-limiting enzyme for glycogen synthesis, our model suggests that UGP2 plays an important role in increasing flux to glycogen synthase. Moreover, we have shown that the capacity for glycogen storage is more closely related to the AMPKγ3(R200Q) mutation than activity. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes.

PubMed

Rivière, Jean-Baptiste; Mirzaa, Ghayda M; O'Roak, Brian J; Beddaoui, Margaret; Alcantara, Diana; Conway, Robert L; St-Onge, Judith; Schwartzentruber, Jeremy A; Gripp, Karen W; Nikkel, Sarah M; Worthylake, Thea; Sullivan, Christopher T; Ward, Thomas R; Butler, Hailly E; Kramer, Nancy A; Albrecht, Beate; Armour, Christine M; Armstrong, Linlea; Caluseriu, Oana; Cytrynbaum, Cheryl; Drolet, Beth A; Innes, A Micheil; Lauzon, Julie L; Lin, Angela E; Mancini, Grazia M S; Meschino, Wendy S; Reggin, James D; Saggar, Anand K; Lerman-Sagie, Tally; Uyanik, Gökhan; Weksberg, Rosanna; Zirn, Birgit; Beaulieu, Chandree L; Majewski, Jacek; Bulman, Dennis E; O'Driscoll, Mark; Shendure, Jay; Graham, John M; Boycott, Kym M; Dobyns, William B

2012-06-24

Megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndromes are sporadic overgrowth disorders associated with markedly enlarged brain size and other recognizable features. We performed exome sequencing in 3 families with MCAP or MPPH, and our initial observations were confirmed in exomes from 7 individuals with MCAP and 174 control individuals, as well as in 40 additional subjects with megalencephaly, using a combination of Sanger sequencing, restriction enzyme assays and targeted deep sequencing. We identified de novo germline or postzygotic mutations in three core components of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. These include 2 mutations in AKT3, 1 recurrent mutation in PIK3R2 in 11 unrelated families with MPPH and 15 mostly postzygotic mutations in PIK3CA in 23 individuals with MCAP and 1 with MPPH. Our data highlight the central role of PI3K-AKT signaling in vascular, limb and brain development and emphasize the power of massively parallel sequencing in a challenging context of phenotypic and genetic heterogeneity combined with postzygotic mosaicism.

New localization and function of calpain-2 in nucleoli of colorectal cancer cells in ribosomal biogenesis: effect of KRAS status

PubMed Central

Telechea-Fernández, Marcelino; Rodríguez-Fernández, Lucia; García, Concha; Zaragozá, Rosa; Viña, Juan; Cervantes, Andrés; García-Trevijano, Elena R.

2018-01-01

Calpain-2 belongs to a family of pleiotropic Cys-proteases with modulatory rather than degradative functions. Calpain (CAPN) overexpression has been controversially correlated with poor prognosis in several cancer types, including colorectal carcinoma (CRC). However, the mechanisms of substrate-recognition, calpain-2 regulation/deregulation and specific functions in CRC remain elusive. Herein, calpain subcellular distribution was studied as a key event for substrate-recognition and consequently, for calpain-mediated function. We describe a new localization for calpain-2 in the nucleoli of CRC cells. Calpain-2 nucleolar distribution resulted dependent on its enzymatic activity and on the mutational status of KRAS. In KRASWT/- cells serum-starvation induced CAPN2 expression, nucleolar accumulation and increased binding to the rDNA-core promoter and intergenic spacer (IGS), concomitant with a reduction in pre-rRNA levels. Depletion of calpain-2 by specific siRNA prevented pre-rRNA down-regulation after serum removal. Conversely, ribosomal biogenesis proceeded in the absence of serum in unresponsive KRASG13D/- cells whose CAPN2 expression, nucleolar localization and rDNA-occupancy remained unchanged during the time-course of serum starvation. We propose here that nucleolar calpain-2 might be a KRAS-dependent sensor to repress ribosomal biogenesis in growth limiting conditions. Under constitutive activation of the pathway commonly found in CRC, calpain-2 is deregulated and tumor cells become insensitive to the extracellular microenvironment. PMID:29507677

New localization and function of calpain-2 in nucleoli of colorectal cancer cells in ribosomal biogenesis: effect of KRAS status.

PubMed

Telechea-Fernández, Marcelino; Rodríguez-Fernández, Lucia; García, Concha; Zaragozá, Rosa; Viña, Juan; Cervantes, Andrés; García-Trevijano, Elena R

2018-02-06

Calpain-2 belongs to a family of pleiotropic Cys-proteases with modulatory rather than degradative functions. Calpain (CAPN) overexpression has been controversially correlated with poor prognosis in several cancer types, including colorectal carcinoma (CRC). However, the mechanisms of substrate-recognition, calpain-2 regulation/deregulation and specific functions in CRC remain elusive. Herein, calpain subcellular distribution was studied as a key event for substrate-recognition and consequently, for calpain-mediated function. We describe a new localization for calpain-2 in the nucleoli of CRC cells. Calpain-2 nucleolar distribution resulted dependent on its enzymatic activity and on the mutational status of KRAS. In KRASWT/- cells serum-starvation induced CAPN2 expression, nucleolar accumulation and increased binding to the rDNA-core promoter and intergenic spacer (IGS), concomitant with a reduction in pre-rRNA levels. Depletion of calpain-2 by specific siRNA prevented pre-rRNA down-regulation after serum removal. Conversely, ribosomal biogenesis proceeded in the absence of serum in unresponsive KRASG13D/- cells whose CAPN2 expression, nucleolar localization and rDNA-occupancy remained unchanged during the time-course of serum starvation. We propose here that nucleolar calpain-2 might be a KRAS-dependent sensor to repress ribosomal biogenesis in growth limiting conditions. Under constitutive activation of the pathway commonly found in CRC, calpain-2 is deregulated and tumor cells become insensitive to the extracellular microenvironment.

Mutation of MSH3 in endometrial cancer and evidence for its functional role in heteroduplex repair.

PubMed

Risinger, J I; Umar, A; Boyd, J; Berchuck, A; Kunkel, T A; Barrett, J C

1996-09-01

Many human tumours have length alterations in repetitive sequence elements. Although this microsatellite instability has been attributed to mutations in four DNA mismatch repair genes in hereditary nonpolyposis colorectal cancer (HNPCC) kindreds, many sporadic tumours exhibit instability but no detectable mutations in these genes. It is therefore of interest to identify other genes that contribute to this instability. In yeast, mutations in several genes, including RTH and MSH3, cause microsatellite instability. Thus, we screened 16 endometrial carcinomas with microsatellite instability for alterations in FEN1 (the human homolog of RTH) and in MSH3 (refs 12-14). Although we found no FEN1 mutations, a frameshift mutation in MSH3 was observed in an endometrial carcinoma and in an endometrial carcinoma cell line. Extracts of the cell line were deficient in repair of DNA substrates containing mismatches or extra nucleotides. Introducing chromosome 5, encoding the MSH3 gene, into the mutant cell line increased the stability of some but not all microsatellites. Extracts of these cells repaired certain substrates containing extra nucleotides, but were deficient in repair of those containing mismatches or other extra nucleotides. A subsequent search revealed a second gene mutation in HHUA cells, a missense mutation in the MSH6 gene. Together the data suggest that the MSH3 gene encodes a product that functions in repair of some but not all pre-mutational intermediates, its mutation in tumours can result in genomic instability and, as in yeast, MSH3 and MSH6 are partially redundant for mismatch repair.

AR mutations in 28 patients with androgen insensitivity syndrome (Prader grade 0-3).

PubMed

Wang, Yi; Gong, Chunxiu; Wang, Xiou; Qin, Miao

2017-07-01

We investigated the androgen receptor (AR) gene mutation profiles of Chinese patients exhibiting severe androgen insensitivity syndrome (AIS) phenotypes. The present study enrolled 28 patients with genetically diagnosed AIS, who presented with severe phenotypes (Prader grade 0-3). Patients and some family members were screened via amplification and sequencing of their AR exons 1-8, including the corresponding intronic flanking regions. Luteinizing (LH), follicle-stimulating (FSH), and testosterone (T) hormone levels were found to be slightly, but not significantly, higher in patients with complete androgen insensitivity syndrome (CAIS) than in patients with partial androgen insensitivity syndrome (PAIS) (P>0.05). We identified 24 different AR mutations, including 12 that were novel. Ten patients (cases 2, 3, 10, 28, 11, 12, 19, 20, 24, and 25) were found to carry five recurrent mutations (p.Y572S, p.P914S, p.S176R, p.Y782N, and p.R841H); of these, p.Y572S, p.S176R, and p.Y782N were novel. Among the mutations identified in patients with CAIS, six (66.7%) were characterized as single-nucleotide missense mutations, and six (66.7%) were found to be located in the AR ligand-binding domain (LBD). Among the mutations identified in patients with PAIS, 15 (93.8%) were found to be missense, and 11 (68.8%) were found to be located in the LBD. Patients 10 and 28 were determined to harbor the same missense mutation (p.P914S), but were diagnosed with CAIS and PAIS, respectively. Sex hormone levels were slightly, but not significantly, elevated in patients with CAIS compared to those with PAIS. Missense mutations spanning AR exons 1-8 were the predominant form of identified mutations, and these were mostly located in the AR LBD. Approximately 50% of the identified mutations were novel, and have enriched the AR gene-mutation database. Patients harboring identical mutations were in some instances found to exhibit divergent phenotypes.

The archetypal R90C CADASIL-NOTCH3 mutation retains NOTCH3 function in vivo.

PubMed

Monet, Marie; Domenga, Valérie; Lemaire, Barbara; Souilhol, Céline; Langa, Francina; Babinet, Charles; Gridley, Thomas; Tournier-Lasserve, Elisabeth; Cohen-Tannoudji, Michel; Joutel, Anne

2007-04-15

Cerebral Autosomal Dominant Arteriopathy with Subcortical infarcts and Leukoencephalopathy (CADASIL) is the most prominent known cause of inherited stroke and vascular dementia in human adult. The disease gene, NOTCH3, encodes a transmembrane receptor primarily expressed in arterial smooth muscle cells (SMC). Pathogenic mutations lead to an odd number of cysteine residues within the NOTCH3 extracellular domain (NOTCH3(ECD)), and are associated with progressive accumulation of NOTCH3(ECD) at the SMC plasma membrane. The murine homolog, Notch3, is dispensable for viability but required post-natally for the elaboration and maintenance of arteries. How CADASIL-associated mutations impact NOTCH3 function remains a fundamental, yet unresolved issue. Particularly, whether NOTCH3(ECD) accumulation may titrate the ligand and inhibit the normal pathway is unknown. Herein, using genetic analyses in the mouse, we assessed the functional significance of an archetypal CADASIL-associated mutation (R90C), in vivo, in brain arteries. We show that transgenic mouse lines expressing either the wild-type human NOTCH3 or the mutant R90C human NOTCH3, at comparable and physiological levels, can rescue the arterial defects of Notch3-/- mice to similar degrees. In vivo assessment of NOTCH3/RBP-Jk activity provides evidence that the mutant NOTCH3 protein exhibits normal level of activity in brain arteries. Remarkably, the mutant NOTCH3 protein remains functional and does not exhibit dominant negative interfering activity, even when NOTCH3(ECD) accumulates. Collectively, these data suggest a model that invokes novel pathogenic roles for the mutant NOTCH3 protein rather than compromised NOTCH3 function as the primary determinant of the CADASIL arteriopathy.

Midostaurin and Decitabine in Treating Older Patients With Newly Diagnosed Acute Myeloid Leukemia and FLT3 Mutation

ClinicalTrials.gov

2017-11-29

Acute Myeloid Leukemia With FLT3/ITD Mutation; Acute Myeloid Leukemia With Gene Mutations; FLT3 Tyrosine Kinase Domain Point Mutation; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Mutational Spectrum of MYO15A and the Molecular Mechanisms of DFNB3 Human Deafness.

PubMed

Rehman, Atteeq U; Bird, Jonathan E; Faridi, Rabia; Shahzad, Mohsin; Shah, Sujay; Lee, Kwanghyuk; Khan, Shaheen N; Imtiaz, Ayesha; Ahmed, Zubair M; Riazuddin, Saima; Santos-Cortez, Regie Lyn P; Ahmad, Wasim; Leal, Suzanne M; Riazuddin, Sheikh; Friedman, Thomas B

2016-10-01

Deafness in humans is a common neurosensory disorder and is genetically heterogeneous. Across diverse ethnic groups, mutations of MYO15A at the DFNB3 locus appear to be the third or fourth most common cause of autosomal-recessive, nonsyndromic deafness. In 49 of the 67 exons of MYO15A, there are currently 192 recessive mutations identified, including 14 novel mutations reported here. These mutations are distributed uniformly across MYO15A with one enigmatic exception; the alternatively spliced giant exon 2, encoding 1,233 residues, has 17 truncating mutations but no convincing deafness-causing missense mutations. MYO15A encodes three distinct isoform classes, one of which is 395 kDa (3,530 residues), the largest member of the myosin superfamily of molecular motors. Studies of Myo15 mouse models that recapitulate DFNB3 revealed two different pathogenic mechanisms of hearing loss. In the inner ear, myosin 15 is necessary both for the development and the long-term maintenance of stereocilia, mechanosensory sound-transducing organelles that extend from the apical surface of hair cells. The goal of this Mutation Update is to provide a comprehensive review of mutations and functions of MYO15A. © 2016 WILEY PERIODICALS, INC.

Hot-Spot Residues to be Mutated Common in G Protein-Coupled Receptors of Class A: Identification of Thermostabilizing Mutations Followed by Determination of Three-Dimensional Structures for Two Example Receptors.

PubMed

Yasuda, Satoshi; Kajiwara, Yuta; Toyoda, Yosuke; Morimoto, Kazushi; Suno, Ryoji; Iwata, So; Kobayashi, Takuya; Murata, Takeshi; Kinoshita, Masahiro

2017-07-06

G protein-coupled receptors (GPCRs), which are indispensable to life and also implicated in a number of diseases, construct important drug targets. For the efficient structure-guided drug design, however, their structural stabilities must be enhanced. An amino-acid mutation is known to possibly lead to the enhancement, but currently available experimental and theoretical methods for identifying stabilizing mutations suffer such drawbacks as the incapability of exploring the whole mutational space with minor effort and the unambiguous physical origin of the enhanced or lowered stability. In general, after the identification is successfully made for a GPCR, the whole procedure must be followed all over again for the identification for another GPCR. Here we report a theoretical strategy by which many different GPCRs can be considered at the same time. The strategy is illustrated for three GPCRs of Class A in the inactive state. We argue that a mutation of the residue at a position of N BW = 3.39 (N BW is the Ballesteros-Weinstein number), a hot-spot residue, leads to substantially higher stability for significantly many GPCRs of Class A in the inactive state. The most stabilizing mutations of the residues with N BW = 3.39 are then identified for two of the three GPCRs, using the improved version of our free-energy function. These identifications are experimentally corroborated, which is followed by the determination of new three-dimensional (3D) structures for the two GPCRs. We expect that on the basis of the strategy, the 3D structures of many GPCRs of Class A can be solved for the first time in succession.

MutationAligner: a resource of recurrent mutation hotspots in protein domains in cancer

PubMed Central

Gauthier, Nicholas Paul; Reznik, Ed; Gao, Jianjiong; Sumer, Selcuk Onur; Schultz, Nikolaus; Sander, Chris; Miller, Martin L.

2016-01-01

The MutationAligner web resource, available at http://www.mutationaligner.org, enables discovery and exploration of somatic mutation hotspots identified in protein domains in currently (mid-2015) more than 5000 cancer patient samples across 22 different tumor types. Using multiple sequence alignments of protein domains in the human genome, we extend the principle of recurrence analysis by aggregating mutations in homologous positions across sets of paralogous genes. Protein domain analysis enhances the statistical power to detect cancer-relevant mutations and links mutations to the specific biological functions encoded in domains. We illustrate how the MutationAligner database and interactive web tool can be used to explore, visualize and analyze mutation hotspots in protein domains across genes and tumor types. We believe that MutationAligner will be an important resource for the cancer research community by providing detailed clues for the functional importance of particular mutations, as well as for the design of functional genomics experiments and for decision support in precision medicine. MutationAligner is slated to be periodically updated to incorporate additional analyses and new data from cancer genomics projects. PMID:26590264

Exome Sequencing Identifies Biallelic MSH3 Germline Mutations as a Recessive Subtype of Colorectal Adenomatous Polyposis.

PubMed

Adam, Ronja; Spier, Isabel; Zhao, Bixiao; Kloth, Michael; Marquez, Jonathan; Hinrichsen, Inga; Kirfel, Jutta; Tafazzoli, Aylar; Horpaopan, Sukanya; Uhlhaas, Siegfried; Stienen, Dietlinde; Friedrichs, Nicolaus; Altmüller, Janine; Laner, Andreas; Holzapfel, Stefanie; Peters, Sophia; Kayser, Katrin; Thiele, Holger; Holinski-Feder, Elke; Marra, Giancarlo; Kristiansen, Glen; Nöthen, Markus M; Büttner, Reinhard; Möslein, Gabriela; Betz, Regina C; Brieger, Angela; Lifton, Richard P; Aretz, Stefan

2016-08-04

In ∼30% of families affected by colorectal adenomatous polyposis, no germline mutations have been identified in the previously implicated genes APC, MUTYH, POLE, POLD1, and NTHL1, although a hereditary etiology is likely. To uncover further genes with high-penetrance causative mutations, we performed exome sequencing of leukocyte DNA from 102 unrelated individuals with unexplained adenomatous polyposis. We identified two unrelated individuals with differing compound-heterozygous loss-of-function (LoF) germline mutations in the mismatch-repair gene MSH3. The impact of the MSH3 mutations (c.1148delA, c.2319-1G>A, c.2760delC, and c.3001-2A>C) was indicated at the RNA and protein levels. Analysis of the diseased individuals' tumor tissue demonstrated high microsatellite instability of di- and tetranucleotides (EMAST), and immunohistochemical staining illustrated a complete loss of nuclear MSH3 in normal and tumor tissue, confirming the LoF effect and causal relevance of the mutations. The pedigrees, genotypes, and frequency of MSH3 mutations in the general population are consistent with an autosomal-recessive mode of inheritance. Both index persons have an affected sibling carrying the same mutations. The tumor spectrum in these four persons comprised colorectal and duodenal adenomas, colorectal cancer, gastric cancer, and an early-onset astrocytoma. Additionally, we detected one unrelated individual with biallelic PMS2 germline mutations, representing constitutional mismatch-repair deficiency. Potentially causative variants in 14 more candidate genes identified in 26 other individuals require further workup. In the present study, we identified biallelic germline MSH3 mutations in individuals with a suspected hereditary tumor syndrome. Our data suggest that MSH3 mutations represent an additional recessive subtype of colorectal adenomatous polyposis. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution

PubMed Central

Timms, Andrew E.; Conti, Valerio; Girisha, Katta M.; Martin, Beth; Olds, Carissa; Collins, Sarah; Park, Kaylee; Carter, Melissa; Krägeloh-Mann, Inge; Chitayat, David; Parikh, Aditi Shah; Bradshaw, Rachael; Torti, Erin; Braddock, Stephen; Burke, Leah; Ghedia, Sondhya; Stephan, Mark; Stewart, Fiona; Prasad, Chitra; Napier, Melanie; Saitta, Sulagna; Straussberg, Rachel; Gabbett, Michael; O’Connor, Bridget C.; Yin, Lim Jiin; Lai, Angeline Hwei Meeng; Martin, Nicole; McKinnon, Margaret; Addor, Marie-Claude; Schwartz, Charles E.; Lanoel, Agustina; Conway, Robert L.; Devriendt, Koenraad; Tatton-Brown, Katrina; Pierpont, Mary Ella; Painter, Michael; Worgan, Lisa; Reggin, James; Hennekam, Raoul; Pritchard, Colin C.; Aracena, Mariana; Gripp, Karen W.; Cordisco, Maria; Van Esch, Hilde; Garavelli, Livia; Curry, Cynthia; Goriely, Anne; Kayserilli, Hulya; Shendure, Jay; Graham, John; Guerrini, Renzo; Dobyns, William B.

2016-01-01

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations. PMID:27631024

Muenke Syndrome Mutation, FgfR3P244R, Causes TMJ Defects

PubMed Central

Yasuda, T.; Nah, H.D.; Laurita, J.; Kinumatsu, T.; Shibukawa, Y.; Shibutani, T.; Minugh-Purvis, N.; Pacifici, M.; Koyama, E.

2012-01-01

Muenke syndrome is characterized by various craniofacial deformities and is caused by an autosomal-dominant activating mutation in fibroblast growth factor receptor 3 (FGFR3P250R). Here, using mice carrying a corresponding mutation (FgfR3P244R), we determined whether the mutation affects temporomandibular joint (TMJ) development and growth. In situ hybridization showed that FgfR3 was expressed in condylar chondroprogenitors and maturing chondrocytes that also expressed the Indian hedgehog (Ihh) receptor and transcriptional target Patched 1(Ptch1). In FgfR3P244R mutants, the condyles displayed reduced levels of Ihh expression, H4C-positive proliferating chondroprogenitors, and collagen type II- and type X-expressing chondrocytes. Primary bone spongiosa formation was also disturbed and was accompanied by increased osteoclastic activity and reduced trabecular bone formation. Treatment of wild-type condylar explants with recombinant FGF2/FGF9 decreased Ptch1 and PTHrP expression in superficial/polymorphic layers and proliferation in chondroprogenitors. We also observed early degenerative changes of condylar articular cartilage, abnormal development of the articular eminence/glenoid fossa in the TMJ, and fusion of the articular disc. Analysis of our data indicates that the activating FgfR3P244R mutation disturbs TMJ developmental processes, likely by reducing hedgehog signaling and endochondral ossification. We suggest that a balance between FGF and hedgehog signaling pathways is critical for the integrity of TMJ development and for the maintenance of cellular organization. PMID:22622662

Mistranslation can enhance fitness through purging of deleterious mutations

PubMed Central

Bratulic, Sinisa; Toll-Riera, Macarena; Wagner, Andreas

2017-01-01

Phenotypic mutations are amino acid changes caused by mistranslation. How phenotypic mutations affect the adaptive evolution of new protein functions is unknown. Here we evolve the antibiotic resistance protein TEM-1 towards resistance on the antibiotic cefotaxime in an Escherichia coli strain with a high mistranslation rate. TEM-1 populations evolved in such strains endow host cells with a general growth advantage, not only on cefotaxime but also on several other antibiotics that ancestral TEM-1 had been unable to deactivate. High-throughput sequencing of TEM-1 populations shows that this advantage is associated with a lower incidence of weakly deleterious genotypic mutations. Our observations show that mistranslation is not just a source of noise that delays adaptive evolution. It could even facilitate adaptive evolution by exacerbating the effects of deleterious mutations and leading to their more efficient purging. The ubiquity of mistranslation and its effects render mistranslation an important factor in adaptive protein evolution. PMID:28524864

The spectrum of epilepsy and electroencephalographic abnormalities due to SHANK3 loss-of-function mutations.

PubMed

Holder, J Lloyd; Quach, Michael M

2016-10-01

The coincidence of autism with epilepsy is 27% in those individuals with intellectual disability. 1 Individuals with loss-of-function mutations in SHANK3 have intellectual disability, autism, and variably, epilepsy. 2-5 The spectrum of seizure semiologies and electroencephalography (EEG) abnormalities has never been investigated in detail. With the recent report that SHANK3 mutations are present in approximately 2% of individuals with moderate to severe intellectual disabilities and 1% of individuals with autism, determining the spectrum of seizure semiologies and electrographic abnormalities will be critical for medical practitioners to appropriately counsel the families of patients with SHANK3 mutations. A retrospective chart review was performed of all individuals treated at the Blue Bird Circle Clinic for Child Neurology who have been identified as having either a chromosome 22q13 microdeletion encompassing SHANK3 or a loss-of-function mutation in SHANK3 identified through whole-exome sequencing. For each subject, the presence or absence of seizures, seizure semiology, frequency, age of onset, and efficacy of therapy were determined. Electroencephalography studies were reviewed by a board certified neurophysiologist. Neuroimaging was reviewed by both a board certified pediatric neuroradiologist and child neurologist. There is a wide spectrum of seizure semiologies, frequencies, and severity in individuals with SHANK3 mutations. There are no specific EEG abnormalities found in our cohort, and EEG abnormalities were present in individuals diagnosed with epilepsy and those without history of a clinical seizure. All individuals with a mutation in SHANK3 should be evaluated for epilepsy due to the high prevalence of seizures in this population. The most common semiology is atypical absence seizure, which can be challenging to identify due to comorbid intellectual disability in individuals with SHANK3 mutations; however, no consistent seizure semiology, neuroimaging

The spectrum of epilepsy and electroencephalographic abnormalities due to SHANK3 loss of function mutations

PubMed Central

Holder, J. Lloyd; Quach, Michael M.

2017-01-01

Summary Objective The coincidence of autism with epilepsy is 27% in those individuals with intellectual disability1. Individuals with loss of function mutations in SHANK3 have intellectual disability, autism and variably, epilepsy2–5. The spectrum of seizure semiologies and electroencephalographic (EEG) abnormalities has never been investigated in detail. With the recent report that SHANK3 mutations are present in approximately two percent of individuals with moderate to severe intellectual disabilities and one percent of individuals with autism, determining the spectrum of seizure semiologies and electrographic abnormalities will be critical for medical practitioners to appropriately counsel the families of patients with SHANK3 mutations. Methods A retrospective chart review was performed of all individuals treated at the Blue Bird Circle Clinic for Child Neurology who have been identified as having either a chromosome 22q13 microdeletion encompassing SHANK3 or a loss of function mutation in SHANK3 identified through whole exome sequencing. For each subject, the presence or absence of seizures, seizure semiology, frequency, age of onset and efficacy of therapy were determined. Electroencephalograms were reviewed by a board certified neurophysiologist. Neuroimaging was reviewed by both a board certified pediatric neuroradiologist and child neurologist. Results There is a wide spectrum of seizure semiologies, frequencies and severity in individuals with SHANK3 mutations. There are no specific electroencephalographic abnormalities found in our cohort, and EEG abnormalities were present in individuals diagnosed with epilepsy and those without history of a clinical seizure. Significance All individuals with a mutation in SHANK3 should be evaluated for epilepsy due to the high prevalence of seizures in this population. The most common semiology is atypical absence seizure which can be challenging to identify due to comorbid intellectual disability in individuals with

De novo mutations in histone modifying genes in congenital heart disease

PubMed Central

Zaidi, Samir; Choi, Murim; Wakimoto, Hiroko; Ma, Lijiang; Jiang, Jianming; Overton, John D.; Romano-Adesman, Angela; Bjornson, Robert D.; Breitbart, Roger E.; Brown, Kerry K.; Carriero, Nicholas J.; Cheung, Yee Him; Deanfield, John; DePalma, Steve; Fakhro, Khalid A.; Glessner, Joseph; Hakonarson, Hakon; Italia, Michael; Kaltman, Jonathan R.; Kaski, Juan; Kim, Richard; Kline, Jennie K.; Lee, Teresa; Leipzig, Jeremy; Lopez, Alexander; Mane, Shrikant M.; Mitchell, Laura E.; Newburger, Jane W.; Parfenov, Michael; Pe'er, Itsik; Porter, George; Roberts, Amy; Sachidanandam, Ravi; Sanders, Stephan J.; Seiden, Howard S.; State, Mathew W.; Subramanian, Sailakshmi; Tikhonova, Irina R.; Wang, Wei; Warburton, Dorothy; White, Peter S.; Williams, Ismee A.; Zhao, Hongyu; Seidman, Jonathan G.; Brueckner, Martina; Chung, Wendy K.; Gelb, Bruce D.; Goldmuntz, Elizabeth; Seidman, Christine E.; Lifton, Richard P.

2013-01-01

Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births1. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. By analysis of exome sequencing of parent-offspring trios, we compared the incidence of de novo mutations in 362 severe CHD cases and 264 controls. CHD cases showed a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging mutations. Similar odds ratios were seen across major classes of severe CHD. We found a marked excess of de novo mutations in genes involved in production, removal or reading of H3K4 methylation (H3K4me), or ubiquitination of H2BK120, which is required for H3K4 methylation2–4. There were also two de novo mutations in SMAD2; SMAD2 signaling in the embryonic left-right organizer induces demethylation of H3K27me5. H3K4me and H3K27me mark `poised' promoters and enhancers that regulate expression of key developmental genes6. These findings implicate de novo point mutations in several hundred genes that collectively contribute to ~10% of severe CHD. PMID:23665959

De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes

PubMed Central

Rivière, Jean-Baptiste; Mirzaa, Ghayda M.; O’Roak, Brian J.; Beddaoui, Margaret; Alcantara, Diana; Conway, Robert L.; St-Onge, Judith; Schwartzentruber, Jeremy A.; Gripp, Karen W.; Nikkel, Sarah M.; Worthylake, Thea; Sullivan, Christopher T.; Ward, Thomas R.; Butler, Hailly E.; Kramer, Nancy A.; Albrecht, Beate; Armour, Christine M.; Armstrong, Linlea; Caluseriu, Oana; Cytrynbaum, Cheryl; Drolet, Beth A.; Innes, A. Micheil; Lauzon, Julie L.; Lin, Angela E.; Mancini, Grazia M. S.; Meschino, Wendy S.; Reggin, James D.; Saggar, Anand K.; Lerman-Sagie, Tally; Uyanik, Gökhan; Weksberg, Rosanna; Zirn, Birgit; Beaulieu, Chandree L.; Majewski, Jacek; Bulman, Dennis E.; O’Driscoll, Mark; Shendure, Jay; Graham, John M.; Boycott, Kym M.; Dobyns, William B.

2012-01-01

Megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndromes are sporadic overgrowth disorders associated with markedly enlarged brain size and other recognizable features1-5. We performed exome sequencing in three families with MCAP or MPPH and confirmed our initial observations in exomes from 7 MCAP and 174 control individuals, as well as in 40 additional megalencephaly subjects using a combination of Sanger sequencing, restriction-enzyme assays, and targeted deep sequencing. We identified de novo germline or postzygotic mutations in three core components of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway. These include two mutations of AKT3, one recurrent mutation of PIK3R2 in 11 unrelated MPPH families, and 15 mostly postzygotic mutations of PIK3CA in 23 MCAP and one MPPH patients. Our data highlight the central role of PI3K/AKT signaling in vascular, limb and brain development, and emphasize the power of massively parallel sequencing in a challenging context of phenotypic and genetic heterogeneity combined with postzygotic mosaicism. PMID:22729224

Deactivating germline mutations in LEMD3 cause osteopoikilosis and Buschke-Ollendorff syndrome, but not sporadic melorheostosis.

PubMed

Mumm, Steven; Wenkert, Deborah; Zhang, Xiafang; McAlister, William H; Mier, Richard J; Whyte, Michael P

2007-02-01

Autosomal dominant OPK and BOS feature widespread foci of osteosclerotic trabeculae without or with skin lesions, respectively. Occasionally, a larger area of dense bone in OPK or BOS resembles MEL, a sporadic sclerosing disorder primarily involving cortical bone. Others, finding deactivating germline LEMD3 mutations in OPK or BOS, concluded such defects explain all three conditions. We found germline LEMD3 mutations in OPK and BOS but not in sporadic MEL. In 2004, others discovered that heterozygous, loss-of-function, germline mutations in the LEMD3 gene (LEMD3 or MAN1) cause both osteopoikilosis (OPK) and Buschke-Ollendorff syndrome (BOS). OPK is an autosomal dominant, usually benign, skeletal dysplasia featuring multiple, small, especially metaphyseal, oval or round, dense trabecular foci distributed symmetrically throughout the skeleton. BOS combines OPK with connective tissue nevi comprised of collagen and elastin. In some OPK and BOS families, an individual may have relatively large, asymmetric areas of dense cortical bone interpreted as melorheostosis (MEL). MEL, however, classically refers to a sporadic, troublesome skeletal dysostosis featuring large, asymmetric, "flowing hyperostosis" of long bone cortices often with overlying, constricting soft tissue abnormalities. However, a heterozygous germline mutation in LEMD3 was offered to explain MEL. We studied 11 unrelated individuals with sclerosing bone disorders where LEMD3 mutation was a potential etiology: familial OPK (1), familial BOS (2), previously reported familial OPK with MEL (1), sporadic MEL (3), sporadic MEL with mixed-sclerosing-bone dystrophy (1), and patients with other unusual sclerosing bone disorders (3). All coding exons and adjacent mRNA splice sites for LEMD3 were amplified by PCR and sequenced using genomic DNA from leukocytes. We did not study lesional tissue from bone or skin. In the OPK family, a heterozygous nonsense mutation (c.1433T>A, p.L478X) was discovered in exon 1. In the

Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability.

PubMed

Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O'Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

2014-04-01

Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

Mutations in the DNA methyltransferase gene, DNMT3A, cause an overgrowth syndrome with intellectual disability

PubMed Central

Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O’Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

2014-01-01

Overgrowth disorders are a heterogeneous group of conditions characterised by increased growth parameters and variable other clinical features, such as intellectual disability and facial dysmorphism1. To identify novel causes of human overgrowth we performed exome sequencing in 10 proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations through DNMT3A sequencing of a further 142 individuals with overgrowth. The mutations were all located in functional DNMT3A domains and protein modelling suggests they interfere with domain-domain interactions and histone binding. No similar mutations were present in 1000 UK population controls (13/152 vs 0/1000; P<0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and increased height. DNMT3A encodes a key methyltransferase essential for establishing the methylation imprint in embryogenesis and is commonly somatically mutated in acute myeloid leukaemia2-4. Thus DNMT3A joins an emerging group of epigenetic DNA and histone modifying genes associated with both developmental growth disorders and haematological malignancies5. PMID:24614070

Prognostic role of tumor PIK3CA mutation in colorectal cancer: a systematic review and meta-analysis.

PubMed

Mei, Z B; Duan, C Y; Li, C B; Cui, L; Ogino, S

2016-10-01

Somatic mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT pathway play a vital role in carcinogenesis. Approximately 15%-20% of colorectal cancers (CRCs) harbor activating mutations in PIK3CA, making it one of the most frequently mutated genes in CRC. We thus carried out a systematic review and meta-analysis investigating the prognostic significance of PIK3CA mutations in CRC. Electronic databases were searched from inception through May 2015. We extracted the study characteristics and prognostic data of each eligible study. The hazard ratio (HR) and 95% confidence interval (CI) were derived and pooled using the random-effects Mantel-Haenszel model. Twenty-eight studies enrolling 12 747 patients were eligible for inclusion. Data on overall survival (OS) and progression-free survival (PFS) were available from 19 and 10 studies, respectively. Comparing PIK3CA-mutated CRC patients with PIK3CA-wild-type CRC patients, the summary HRs for OS and PFS were 0.96 (95% CI 0.83-1.12) and 1.20 (95% CI 0.98-1.46), respectively. The trim-and-fill, Copas model and subgroup analyses stratified by the study characteristics confirmed the robustness of the results. Five studies reported the CRC prognosis for PIK3CA mutations in exons 9 and 20 separately; neither exon 9 mutation nor exon 20 mutation in PIK3CA was significantly associated with patient survival. Our findings suggest that PIK3CA mutation has the neutral prognostic effects on CRC OS and PFS. Evidence was accumulating for the establishment of CRC survival between PIK3CA mutations and patient-specific clinical or molecular profiles. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Functional analysis of Waardenburg syndrome-associated PAX3 and SOX10 mutations: report of a dominant-negative SOX10 mutation in Waardenburg syndrome type II.

PubMed

Zhang, Hua; Chen, Hongsheng; Luo, Hunjin; An, Jing; Sun, Lin; Mei, Lingyun; He, Chufeng; Jiang, Lu; Jiang, Wen; Xia, Kun; Li, Jia-Da; Feng, Yong

2012-03-01

Waardenburg syndrome (WS) is an auditory-pigmentary disorder resulting from melanocyte defects, with varying combinations of sensorineural hearing loss and abnormal pigmentation of the hair, skin, and inner ear. WS is classified into four subtypes (WS1-WS4) based on additional symptoms. PAX3 and SOX10 are two transcription factors that can activate the expression of microphthalmia-associated transcription factor (MITF), a critical transcription factor for melanocyte development. Mutations of PAX3 are associated with WS1 and WS3, while mutations of SOX10 cause WS2 and WS4. Recently, we identified some novel WS-associated mutations in PAX3 and SOX10 in a cohort of Chinese WS patients. Here, we further identified an E248fsX30 SOX10 mutation in a family of WS2. We analyzed the subcellular distribution, expression and in vitro activity of two PAX3 mutations (p.H80D, p.H186fsX5) and four SOX10 mutations (p.E248fsX30, p.G37fsX58, p.G38fsX69 and p.R43X). Except H80D PAX3, which retained partial activity, the other mutants were unable to activate MITF promoter. The H80D PAX3 and E248fsX30 SOX10 were localized in the nucleus as wild type (WT) proteins, whereas the other mutant proteins were distributed in both cytoplasm and nucleus. Furthermore, E248fsX30 SOX10 protein retained the DNA-binding activity and showed dominant-negative effect on WT SOX10. However, E248fsX30 SOX10 protein seems to decay faster than the WT one, which may underlie the mild WS2 phenotype caused by this mutation.

Development of potent in vivo mutagenesis plasmids with broad mutational spectra

PubMed Central

Badran, Ahmed H.; Liu, David R.

2015-01-01

Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms. PMID:26443021

Development of potent in vivo mutagenesis plasmids with broad mutational spectra.

PubMed

Badran, Ahmed H; Liu, David R

2015-10-07

Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms.

Endoplasmic reticulum-associated degradation of the mouse PC1/3-N222D hypomorph and human PCSK1 mutations contributes to obesity.

PubMed

Stijnen, P; Brouwers, B; Dirkx, E; Ramos-Molina, B; Van Lommel, L; Schuit, F; Thorrez, L; Declercq, J; Creemers, J W M

2016-06-01

The proprotein convertase 1/3 (PC1/3), encoded by proprotein convertase subtilisin/kexin type 1 (PCSK1), cleaves and hence activates several orexigenic and anorexigenic proproteins. Congenital inactivation of PCSK1 leads to obesity in human but not in mice. However, a mouse model harboring the hypomorphic mutation N222D is obese. It is not clear why the mouse models differ in phenotype. Gene expression analysis was performed with pancreatic islets from Pcsk1(N222D/N222D) mice. Subsequently, biosynthesis, maturation, degradation and activity were studied in islets, pituitary, hypothalamus and cell lines. Coimmunoprecipitation of PC1/3-N222D and human PC1/3 variants associated with obesity with the endoplasmic reticulum (ER) chaperone BiP was studied in cell lines. Gene expression analysis of islets of Pcsk1(N222D/N222D) mice showed enrichment of gene sets related to the proteasome and the unfolded protein response. Steady-state levels of PC1/3-N222D and in particular the carboxy-terminally processed form were strongly reduced in islets, pituitary and hypothalamus. However, impairment of substrate cleavage was tissue dependent. Proinsulin processing was drastically reduced, while processing of proopiomelanocortin (POMC) to adrenocorticotropic hormone (ACTH) in pituitary was only mildly impaired. Growth hormone expression and IGF-1 levels were normal, indicating near-normal processing of hypothalamic proGHRH. PC1/3-N222D binds to BiP and is rapidly degraded by the proteasome. Analysis of human PC1/3 obesity-associated mutations showed increased binding to BiP and prolonged intracellular retention for all investigated mutations, in particular for PC1/3-T175M, PC1/3-G226R and PC1/3-G593R. This study demonstrates that the hypomorphic mutation in Pcsk1(N222D) mice has an effect on catalytic activity in pancreatic islets, pituitary and hypothalamus. Reduced substrate processing activity in Pcsk1(N222D/N222D) mice is due to enhanced degradation in addition to reduced

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage

PubMed Central

Zygiel, Emily M.; Noren, Karen A.; Adamkiewicz, Marta A.; Aprile, Richard J.; Bowditch, Heather K.; Carroll, Christine L.; Cerezo, Maria Abigail S.; Dagher, Adelle M.; Hebert, Courtney R.; Hebert, Lauren E.; Mahame, Gloria M.; Milne, Stephanie C.; Silvestri, Kelly M.; Sutherland, Sara E.; Sylvia, Alexandria M.; Taveira, Caitlyn N.; VanValkenburgh, David J.; Noren, Christopher J.

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5’-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5’-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries. PMID:28445507

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage.

PubMed

Zygiel, Emily M; Noren, Karen A; Adamkiewicz, Marta A; Aprile, Richard J; Bowditch, Heather K; Carroll, Christine L; Cerezo, Maria Abigail S; Dagher, Adelle M; Hebert, Courtney R; Hebert, Lauren E; Mahame, Gloria M; Milne, Stephanie C; Silvestri, Kelly M; Sutherland, Sara E; Sylvia, Alexandria M; Taveira, Caitlyn N; VanValkenburgh, David J; Noren, Christopher J; Hall, Marilena Fitzsimons

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5'-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5'-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries.

Loss-of-function mutations in APOC3 and risk of ischemic vascular disease.

PubMed

Jørgensen, Anders Berg; Frikke-Schmidt, Ruth; Nordestgaard, Børge G; Tybjærg-Hansen, Anne

2014-07-03

High plasma levels of nonfasting triglycerides are associated with an increased risk of ischemic cardiovascular disease. Whether lifelong low levels of nonfasting triglycerides owing to mutations in the gene encoding apolipoprotein C3 (APOC3) are associated with a reduced risk of ischemic cardiovascular disease in the general population is unknown. Using data from 75,725 participants in two general-population studies, we first tested whether low levels of nonfasting triglycerides were associated with reduced risks of ischemic vascular disease and ischemic heart disease. Second, we tested whether loss-of-function mutations in APOC3, which were associated with reduced levels of nonfasting triglycerides, were also associated with reduced risks of ischemic vascular disease and ischemic heart disease. During follow-up, ischemic vascular disease developed in 10,797 participants, and ischemic heart disease developed in 7557 of these 10,797 participants. Participants with nonfasting triglyceride levels of less than 1.00 mmol per liter (90 mg per deciliter) had a significantly lower incidence of cardiovascular disease than those with levels of 4.00 mmol per liter (350 mg per deciliter) or more (hazard ratio for ischemic vascular disease, 0.43; 95% confidence interval [CI], 0.35 to 0.54; hazard ratio for ischemic heart disease, 0.40; 95% CI, 0.31 to 0.52). Heterozygosity for loss-of-function mutations in APOC3, as compared with no APOC3 mutations, was associated with a mean reduction in nonfasting triglyceride levels of 44% (P<0.001). The cumulative incidences of ischemic vascular disease and ischemic heart disease were reduced in heterozygotes as compared with noncarriers of APOC3 mutations (P=0.009 and P=0.05, respectively), with corresponding risk reductions of 41% (hazard ratio, 0.59; 95% CI, 0.41 to 0.86; P=0.007) and 36% (hazard ratio, 0.64; 95% CI, 0.41 to 0.99; P=0.04). Loss-of-function mutations in APOC3 were associated with low levels of triglycerides and a reduced

APOBEC3B upregulation and genomic mutation patterns in serous ovarian carcinoma

PubMed Central

Leonard, Brandon; Hart, Steven N.; Burns, Michael B.; Carpenter, Michael A.; Temiz, Nuri A.; Rathore, Anurag; Vogel, Rachel Isaksson; Nikas, Jason B.; Law, Emily K.; Brown, William L.; Li, Ying; Zhang, Yuji; Maurer, Matthew J.; Oberg, Ann L.; Cunningham, Julie M.; Shridhar, Viji; Bell, Debra A.; April, Craig; Bentley, David; Bibikova, Marina; Cheetham, R. Keira; Fan, Jian-Bing; Grocock, Russell; Humphray, Sean; Kingsbury, Zoya; Peden, John; Chien, Jeremy; Swisher, Elizabeth M.; Hartmann, Lynn C.; Kalli, Kimberly R.; Goode, Ellen L.; Sicotte, Hugues; Kaufmann, Scott H.; Harris, Reuben S.

2013-01-01

Ovarian cancer is a clinically and molecularly heterogeneous disease. The driving forces behind this variability are unknown. Here we report wide variation in expression of the DNA cytosine deaminase APOBEC3B, with elevated expression in a majority of ovarian cancer cell lines (3 standard deviations above the mean of normal ovarian surface epithelial cells) and high grade primary ovarian cancers. APOBEC3B is active in the nucleus of several ovarian cancer cell lines and elicits a biochemical preference for deamination of cytosines in 5′TC dinucleotides. Importantly, examination of whole-genome sequence from 16 ovarian cancers reveals that APOBEC3B expression correlates with total mutation load as well as elevated levels of transversion mutations. In particular, high APOBEC3B expression correlates with C-to-A and C-to-G transversion mutations within 5′TC dinucleotide motifs in early-stage high grade serous ovarian cancer genomes, suggesting that APOBEC3B-catalyzed genomic uracil lesions are further processed by downstream DNA ‘repair’ enzymes including error-prone translesion polymerases. These data identify a potential role for APOBEC3B in serous ovarian cancer genomic instability. PMID:24154874

A novel LEMD3 mutation common to patients with osteopoikilosis with and without melorheostosis.

PubMed

Couto, Ana R; Bruges-Armas, Jacome; Peach, Chris A; Chapman, Kay; Brown, Matthew A; Wordsworth, B Paul; Zhang, Yun

2007-08-01

Recent studies have reported loss of function mutations in the LEMD3 gene, encoding an inner nuclear membrane protein that influences Smad signaling, as a cause of osteopoikilosis, Buschke-Ollendorff syndrome, and melorheostosis. We investigated LEMD3 in a three-generation family with osteopoikilosis from the Azores, an affected father and daughter from Ireland with osteopoikilosis (the daughter also had melorheostosis), and two other individuals from the UK with isolated melorheostosis. We found a novel C to T substitution at position 2032 bp (cDNA) in exon 8 of LEMD3, resulting in a premature stop codon at amino acid position 678. This mutation co-segregates with the osteopoikilosis phenotype in both the Azorean family and the Irish family. It was not detected in any of the six unaffected family members or in 342 healthy Caucasian individuals. No LEMD3 mutations were detected in the two patients with sporadic melorheostosis. The LEMD3 mutation reported was clearly the cause of osteopoikilosis in the two families but its relationship to melorheostosis in one of the family members is still unclear. Perhaps unsurprisingly in what is a segmental disease, we did not find LEMD3 mutations in peripheral-blood-derived DNA from the two other individuals with sporadic melorheostosis. The nature of the additional genetic and/or environmental influences required for the development of melorheostosis in those with osteopoikilosis requires further investigation.

[NOTCH3 gene mutations in two Chinese families featuring cerebral autosomal dominant arteriopathy with subcortical infarct and leucoencephalopathy].

PubMed

Sun, Qiying; Li, Wenwen; Zhou, Yafang; Yi, Fang; Wang, Jianfeng; Hu, Yacen; Yao, Lingyan; Zhou, Lin; Xu, Hongwei

2017-12-10

To analyze potential mutations of the NOTCH3 gene in two Chinese families featuring cerebral autosomal dominant arteriopathy with subcortical infarct and leucoencephalopathy (CADASIL). The two probands and related family members and 100 healthy controls were recruited. Potential mutations of the NOTCH3 gene were screened by PCR and direct sequencing. PolyPhen-2 and SIFT software were used to predict the protein function. The conditions of both probands were adult-onset, with main clinical features including recurrent transient ischemic attacks and/or strokes, cognitive impairment. MRI findings suggested multiple cerebral infarcts and severe leukoencephalopathy. A heterozygous mutation c.328C>T (p.Arg110Cys), which was located in exon 3 of the NOTCH3 gene and known as a causative mutation, was identified in proband 1. A novel heterozygous mutation c.1013 G>C (p.Cys338Ser) located in exon 6 of the NOTCH3 gene was identified in the proband 2, which was not reported previously. The same mutations were not detected among the 100 unrelated healthy controls. Function analysis suggested that heterozygous mutation c.1013G>C can severely affect the functions of NOTCH3 protein. Two heterozygous missense mutations in the NOTCH3 gene have been identified in two families affected with CADASIL. The novel heterozygous Cys338Ser mutation in exon 6 of the NOTCH3 gene probably underlies the CADASIL.

MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

PubMed

Ma, Wanlong; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; Uyeji, Jennifer; Albitar, Maher

2011-03-01

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

Mutations in the TLR3 signaling pathway and beyond in adult patients with herpes simplex encephalitis.

PubMed

Mørk, N; Kofod-Olsen, E; Sørensen, K B; Bach, E; Ørntoft, T F; Østergaard, L; Paludan, S R; Christiansen, M; Mogensen, T H

2015-12-01

Herpes simplex encephalitis (HSE) in children has previously been linked to defects in type I interferon production downstream of Toll-like receptor (TLR)3. In the present study, we used whole-exome sequencing to investigate the genetic profile of 16 adult patients with a history of HSE. We identified novel mutations in IRF3, TYK2 and MAVS, molecules involved in generating innate antiviral immune responses, which have not previously been associated with HSE. Moreover, data revealed mutations in TLR3, TRIF, TBK1 and STAT1 known to be associated with HSE in children but not previously described in adults. All discovered mutations were heterozygous missense mutations, the majority of which were associated with significantly decreased antiviral responses to HSV-1 infection and/or the TLR3 agonist poly(I:C) in patient peripheral blood mononuclear cells compared with controls. Altogether, this study demonstrates novel mutations in the TLR3 signaling pathway in molecules previously identified in children, suggesting that impaired innate immunity to HSV-1 may also increase susceptibility to HSE in adults. Importantly, the identification of mutations in innate signaling molecules not directly involved in TLR3 signaling suggests the existence of innate immunodeficiencies predisposing to HSE beyond the TLR3 pathway.

In silico analysis of a novel MKRN3 missense mutation in familial central precocious puberty.

PubMed

Neocleous, Vassos; Shammas, Christos; Phelan, Marie M; Nicolaou, Stella; Phylactou, Leonidas A; Skordis, Nicos

2016-01-01

The onset of puberty is influenced by the interplay of stimulating and restraining factors, many of which have a genetic origin. Premature activation of the GnRH secretion in central precocious puberty (CPP) may arise either from gain-of-function mutations of the KISS1 and KISS1R genes or from loss-of-function manner mutations of the MKRN3 gene leading to MKRN3 deficiency. To explore the genetic causes responsible for CPP and the potential role of the RING finger protein 3 (MKRN3) gene. We investigated potential sequence variations in the intronless MKRN3 gene by Sanger sequencing of the entire 507 amino acid coding region of exon 1 in a family with two affected girls presented with CPP at the age of 6 and 5·7 years, respectively. A novel heterozygous g.Gly312Asp missense mutation in the MKRN3 gene was identified in these siblings. The imprinted MKRN3 missense mutation was also identified as expected in the unaffected father and followed as expected an imprinted mode of inheritance. In silico analysis of the altered missense variant using the computational algorithms Polyphen2, SIFT and Mutation Taster predicted a damage and pathogenic alteration causing CPP. The pathogenicity of the alteration at the protein level via an in silico structural model is also explored. A novel mutation in the MKRN3 gene in two sisters with CPP was identified, supporting the fundamental role of this gene in the suppression of the hypothalamic GnRH neurons. © 2015 John Wiley & Sons Ltd.

Evaluation of PIK3CA mutations as a biomarker in Chinese breast carcinomas from Western China.

PubMed

Cheng, Jingliang; Fu, Shangyi; Wei, Chunli; Tania, Mousumi; Khan, Md Asaduzzaman; Imani, Saber; Zhou, Baixu; Chen, Hanchun; Xiao, Xiuli; Wu, Jingbo; Fu, Junjiang

2017-01-01

PIK3CA gene encodes the p110 α catalytic subunit of the oncoprotein phosphatidylinositol 3-kinase (PI3 K) which regulates many biological processes such as cell proliferation, differentiation, migration and survival through the activation of various signaling pathways. In this study, we have investigated the possible somatic mutations in PIK3CA gene in invasive ductal breast carcinomas of Chinese women from Western China. Genomic DNA was extracted from the formalin-fixed paraffin-embedded (FFPE) tissue samples. The hotspot mutations in PIK3CA gene of exon 9 and exon 20 were studied by pyrosequencing. The sequencing identified two hotspot mutations in exon 20 of one cancer samples at p. H1047L (c. 3140A > T) and eight cancer sample at p. H1047R (c. 3140A > G). No mutation in exon 9 of PIK3CA gene was found in these breast cancer tissue samples. PIK3CA mutations showed surprising clinicopathological features in breast cancer patients, as incidence of lymph node invasiveness is increased in the patients with PIK3CA mutation. In addition, all the patients showed tumor size bigger than 3 cm in diameter. It is important that for early detection and early treatment for BC in developing countries or areas like Western China, and for people to provide popularization education using scientific knowledge in cancer fields. This study identified PIK3CA mutations in breast carcinoma patients of Western China that will enable a more rapid molecular diagnosis, and provide a stronger rationale evidence for development of precision therapeutic approaches as well as promising therapeutic targets for breast cancer treatment or patient management.

Gain of Function Mutations of PIK3CD as a Cause of Primary Sclerosing Cholangitis.

PubMed

Hartman, Heather N; Niemela, Julie; Hintermeyer, Mary K; Garofalo, Mary; Stoddard, Jennifer; Verbsky, James W; Rosenzweig, Sergio D; Routes, John M

2015-01-01

Gain of function (GOF) mutation in the p110δ catalytic subunit of the phosphatidylinositol-3-OH kinase (PIK3CD) is the cause of a primary immunodeficiency (PID) characterized by recurrent sinopulmonary infections and lymphoproliferation. We describe a family of two adults and three children with GOF mutation in PIK3CD, all with recurrent sinopulmonary infections and varied infectious and non-infectious complications. The two adults have Primary Sclerosing Cholangitis (PSC) without evidence of Cryptosporidium parvum infection and have required liver transplantation. PSC is a novel phenotype of GOF mutation in PIK3CD.

Mutations in a delta9-Stearoyl-ACP-Desaturase Gene Are Associated with Enhanced Stearic Acid Levels in Soybean Seeds

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

Zhang, P.; Shanklin, J.; Burton, J. W.

2008-11-01

Stearic acid (18:0) is typically a minor component of soybean [Glycine max (L.) Merr.] oil, accounting for only 2 to 4% of the total fatty acid content. Increasing stearic acid levels of soybean oil would lead to enhanced oxidative stability, potentially reducing the need for hydrogenation, a process leading to the formation of undesirable trans fatty acids. Although mutagenesis strategies have been successful in developing soybean germplasm with elevated 18:0 levels in the seed oil, the specific gene mutations responsible for this phenotype were not known. We report a newly identified soybean gene, designated SACPD-C, that encodes a unique isoformmore » of {Delta}{sup 9}-stearoyl-ACP-desaturase, the enzyme responsible for converting stearic acid to oleic acid (18:1). High levels of SACPD-C transcript were only detected in developing seed tissue, suggesting that the encoded desaturase functions to enhance oleic acid biosynthetic capacity as the immature seed is actively engaged in triacylglycerol production and storage. The participation of SACPD-C in storage triacylglycerol synthesis is further supported by the observation of mutations in this gene in two independent sources of elevated 18:0 soybean germplasm, A6 (30% 18:0) and FAM94-41 (9% 18:0). A molecular marker diagnostic for the FAM94-41 SACPD-C gene mutation strictly associates with the elevated 18:0 phenotype in a segregating population, and could thus serve as a useful tool in the development of cultivars with oils possessing enhanced oxidative stability.« less

MutationAligner: a resource of recurrent mutation hotspots in protein domains in cancer.

PubMed

Gauthier, Nicholas Paul; Reznik, Ed; Gao, Jianjiong; Sumer, Selcuk Onur; Schultz, Nikolaus; Sander, Chris; Miller, Martin L

2016-01-04

The MutationAligner web resource, available at http://www.mutationaligner.org, enables discovery and exploration of somatic mutation hotspots identified in protein domains in currently (mid-2015) more than 5000 cancer patient samples across 22 different tumor types. Using multiple sequence alignments of protein domains in the human genome, we extend the principle of recurrence analysis by aggregating mutations in homologous positions across sets of paralogous genes. Protein domain analysis enhances the statistical power to detect cancer-relevant mutations and links mutations to the specific biological functions encoded in domains. We illustrate how the MutationAligner database and interactive web tool can be used to explore, visualize and analyze mutation hotspots in protein domains across genes and tumor types. We believe that MutationAligner will be an important resource for the cancer research community by providing detailed clues for the functional importance of particular mutations, as well as for the design of functional genomics experiments and for decision support in precision medicine. MutationAligner is slated to be periodically updated to incorporate additional analyses and new data from cancer genomics projects. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Toll-like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer

PubMed Central

Maitra, Radhashree; Augustine, Titto; Dayan, Yitzchak; Chandy, Carol; Coffey, Matthew; Goel, Sanjay

2017-01-01

New therapeutic interventions are essential for improved management of patients with metastatic colorectal cancer (mCRC). This is especially critical for those patients whose tumors harbor a mutation in the KRAS oncogene (40-45% of all patients). This patient cohort is excluded from receiving anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Reovirus, a double stranded (ds) RNA virus is in clinical development for patients with chemotherapy refractory KRAS mutated tumors. Toll Like Receptor (TLR) 3, a member of the toll like receptor family of the host innate immune system is the pattern recognition motif for dsRNA pathogens. Using TLR3 expressing commercial HEK-Blue™-hTLR3 cells we confirm that TLR3 is the host pattern recognition motif responsible for the detection of reovirus. Further, our investigation with KRAS mutated HCT116 cell line showed that effective expression of host TLR3 dampens the infection potential of reovirus by mounting a robust innate immune response. Down regulation of TLR3 expression with siRNA improves the anticancer activity of reovirus. In vivo experiments using human CRC cells derived xenografts in athymic mice further demonstrate the beneficial effects of TLR3 knock down by improving tumor response rates to reovirus. Strategies to mitigate the TLR3 response pathway can be utilized as a tool towards improved reovirus efficacy to specifically target the dissemination of KRAS mutated CRC. PMID:28422714

Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation

PubMed Central

Park, Sujin; Yang, Kyung-Min; Park, Yuna; Hong, Eunji; Hong, Chang Pyo; Park, Jinah; Pang, Kyoungwha; Lee, Jihee; Park, Bora; Lee, Siyoung; An, Haein; Kwak, Mi-Kyung; Kim, Junil; Kang, Jin Muk; Kim, Pyunggang; Xiao, Yang; Nie, Guangjun; Ooshima, Akira

2018-01-01

Background Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. Methods We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. Results In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2, SNAI1, and ZEB1 in response to TGF-β1 in human pancreatic and prostate cancer cell lines. We identified GADD45B, CTGF, and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. Conclusions These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B, CTGF, and JUNB genes in various cancers. PMID:29629343

An homozygous mutation in KCNK3 is associated with an aggressive form of hereditary pulmonary arterial hypertension.

PubMed

Navas Tejedor, P; Tenorio Castaño, J; Palomino Doza, J; Arias Lajara, P; Gordo Trujillo, G; López Meseguer, M; Román Broto, A; Lapunzina Abadía, P; Escribano Subía, P

2017-03-01

Pulmonary arterial hypertension (PAH) is a rare devastating disease characterized by a high genetic heterogeneity with several related genes recently described, including BMPR2,TBX4 and KCNK3. The association between KCNK3 and PAH has been recently identified, but the prognosis and phenotype associated with these mutations have been poorly described. We studied a series of 136 idiopathic and hereditary PAH Spanish patients for BMPR2, TBX4 and KCNK3 mutations. We report the results of KCNK3 in which we were able to describe two new mutations (p.Gly106Arg and p.Leu214Arg) in three patients. The first one was found in a patient belonging to a consanguineous Romani family, who carried a homozygous mutation in KCNK3 and developed a severe and early form of the disease. To the best of our knowledge, this is the first time that a homozygous mutation in KCNK3 is reported in a PAH patient. The second one was found in a patient who presented at the young adult age a severe form of the disease. The present report supports the contribution of KCNK3 mutations to the genetic etiology of PAH and strongly suggests that mutations in KCNK3 follow incomplete dominance with worsening of the clinical features in homozygous patients. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A single-residue mutation, G203E, causes 3-hydroxy-3-methylglutaric aciduria by occluding the substrate channel in the 3D structural model of HMG-CoA lyase.

PubMed

Mir, C; Lopez-Viñas, E; Aledo, R; Puisac, B; Rizzo, C; Dionisi-Vici, C; Deodato, F; Pié, J; Gomez-Puertas, P; Hegardt, F G; Casals, N

2006-02-01

3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine metabolism. The disease is caused by mutations in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HL). To date 26 different mutations have been described. A (betaalpha)(8) TIM barrel structure has been proposed for the protein, and almost all missense mutations identified so far localize in the beta sheets that define the inside cavity. We report an Italian patient who bears homozygously a novel HL mutation, c.608G > A (p. G203E) in beta sheet six. A structural model of the mutated protein suggests that glutamic acid 203 impedes catalysis by blocking the entrance to the inner cavity of the enzyme. Loss of functionality has been confirmed in expression studies in E. coli, which demonstrate that the G203E mutation completely abolishes enzyme activity. Beta sheet six and beta sheet two are the two protein regions that accumulate most missense mutations, indicating their importance in enzyme functionality. A model for the mechanism of enzyme function is proposed.

Truncating mutations in the last exon of NOTCH3 cause lateral meningocele syndrome.

PubMed

Gripp, Karen W; Robbins, Katherine M; Sobreira, Nara L; Witmer, P Dane; Bird, Lynne M; Avela, Kristiina; Makitie, Outi; Alves, Daniela; Hogue, Jacob S; Zackai, Elaine H; Doheny, Kimberly F; Stabley, Deborah L; Sol-Church, Katia

2015-02-01

Lateral meningocele syndrome (LMS, OMIM%130720), also known as Lehman syndrome, is a very rare skeletal disorder with facial anomalies, hypotonia and meningocele-related neurologic dysfunction. The characteristic lateral meningoceles represent the severe end of the dural ectasia spectrum and are typically most severe in the lower spine. Facial features of LMS include hypertelorism and telecanthus, high arched eyebrows, ptosis, midfacial hypoplasia, micrognathia, high and narrow palate, low-set ears and a hypotonic appearance. Hyperextensibility, hernias and scoliosis reflect a connective tissue abnormality, and aortic dilation, a high-pitched nasal voice, wormian bones and osteolysis may be present. Lateral meningocele syndrome has phenotypic overlap with Hajdu-Cheney syndrome. We performed exome resequencing in five unrelated individuals with LMS and identified heterozygous truncating NOTCH3 mutations. In an additional unrelated individual Sanger sequencing revealed a deleterious variant in the same exon 33. In total, five novel de novo NOTCH3 mutations were identified in six unrelated patients. One had a 26 bp deletion (c.6461_6486del, p.G2154fsTer78), two carried the same single base pair insertion (c.6692_93insC, p.P2231fsTer11), and three individuals had a nonsense point mutation at c.6247A > T (pK2083*), c.6663C > G (p.Y2221*) or c.6732C > A, (p.Y2244*). All mutations cluster into the last coding exon, resulting in premature termination of the protein and truncation of the negative regulatory proline-glutamate-serine-threonine rich PEST domain. Our results suggest that mutant mRNA products escape nonsense mediated decay. The truncated NOTCH3 may cause gain-of-function through decreased clearance of the active intracellular product, resembling NOTCH2 mutations in the clinically related Hajdu-Cheney syndrome and contrasting the NOTCH3 missense mutations causing CADASIL. © 2014 Wiley Periodicals, Inc.

PIK3CA mutations in non-small cell lung cancer (NSCLC): genetic heterogeneity, prognostic impact and incidence of prior malignancies.

PubMed

Scheffler, Matthias; Bos, Marc; Gardizi, Masyar; König, Katharina; Michels, Sebastian; Fassunke, Jana; Heydt, Carina; Künstlinger, Helen; Ihle, Michaela; Ueckeroth, Frank; Albus, Kerstin; Serke, Monika; Gerigk, Ulrich; Schulte, Wolfgang; Töpelt, Karin; Nogova, Lucia; Zander, Thomas; Engel-Riedel, Walburga; Stoelben, Erich; Ko, Yon-Dschun; Randerath, Winfried; Kaminsky, Britta; Panse, Jens; Becker, Carolin; Hellmich, Martin; Merkelbach-Bruse, Sabine; Heukamp, Lukas C; Büttner, Reinhard; Wolf, Jürgen

2015-01-20

Somatic mutations of the PIK3CA gene have been described in non-small cell lung cancer (NSCLC), but limited data is available on their biological relevance. This study was performed to characterize PIK3CA-mutated NSCLC clinically and genetically. Tumor tissue collected consecutively from 1144 NSCLC patients within a molecular screening network between March 2010 and March 2012 was analyzed for PIK3CA mutations using dideoxy-sequencing and next-generation sequencing (NGS). Clinical, pathological, and genetic characteristics of PIK3CA-mutated patients are described and compared with a control group of PIK3CA-wildtype patients. Among the total cohort of 1144 patients we identified 42 (3.7%) patients with PIK3CA mutations in exon 9 and exon 20. These mutations were found with a higher frequency in sqamous cell carcinoma (8.9%) compared to adenocarcinoma (2.9%, p<0.001). The most common PIK3CA mutation was exon 9 E545K. The majority of patients (57.1%) had additional oncogenic driver aberrations. With the exception of EGFR-mutated patients, non of the genetically defined subgroups in this cohort had a significantly better median overall survival. Further, PIK3CA-mutated patients had a significantly higher incidence of malignancy prior to lung cancer (p<0.001). PIK3CA-mutated NSCLC represents a clinically and genetically heterogeneous subgroup in adenocarcinomas as well as in squamous cell carcinomas with a higher prevalence of these mutations in sqamous cell carcinoma. PIK3CA mutations have no negative impact on survival after surgery or systemic therapy. However, PIK3CA mutated lung cancer frequently develops in patients with prior malignancies.

Feature genes predicting the FLT3/ITD mutation in acute myeloid leukemia.

PubMed

Li, Chenglong; Zhu, Biao; Chen, Jiao; Huang, Xiaobing

2016-07-01

In the present study, gene expression profiles of acute myeloid leukemia (AML) samples were analyzed to identify feature genes with the capacity to predict the mutation status of FLT3/ITD. Two machine learning models, namely the support vector machine (SVM) and random forest (RF) methods, were used for classification. Four datasets were downloaded from the European Bioinformatics Institute, two of which (containing 371 samples, including 281 FLT3/ITD mutation-negative and 90 mutation‑positive samples) were randomly defined as the training group, while the other two datasets (containing 488 samples, including 350 FLT3/ITD mutation-negative and 138 mutation-positive samples) were defined as the test group. Differentially expressed genes (DEGs) were identified by significance analysis of the microarray data by using the training samples. The classification efficiency of the SCM and RF methods was evaluated using the following parameters: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the area under the receiver operating characteristic curve. Functional enrichment analysis was performed for the feature genes with DAVID. A total of 585 DEGs were identified in the training group, of which 580 were upregulated and five were downregulated. The classification accuracy rates of the two methods for the training group, the test group and the combined group using the 585 feature genes were >90%. For the SVM and RF methods, the rates of correct determination, specificity and PPV were >90%, while the sensitivity and NPV were >80%. The SVM method produced a slightly better classification effect than the RF method. A total of 13 biological pathways were overrepresented by the feature genes, mainly involving energy metabolism, chromatin organization and translation. The feature genes identified in the present study may be used to predict the mutation status of FLT3/ITD in patients with AML.

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy

PubMed Central

Fang, Xi; Wu, Tongbin; Liu, Canzhao; Veevers, Jennifer; Stroud, Matthew J.; Zhang, Zhiyuan; Ma, Xiaolong; Mu, Yongxin; Lao, Dieu-Hung; Dalton, Nancy D.; Gu, Yusu; Wang, Celine; Wang, Michael; Liang, Yan; Ouyang, Kunfu; Peterson, Kirk L.; Evans, Sylvia M.

2017-01-01

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2–associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure. PMID:28737513

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy.

PubMed

Fang, Xi; Bogomolovas, Julius; Wu, Tongbin; Zhang, Wei; Liu, Canzhao; Veevers, Jennifer; Stroud, Matthew J; Zhang, Zhiyuan; Ma, Xiaolong; Mu, Yongxin; Lao, Dieu-Hung; Dalton, Nancy D; Gu, Yusu; Wang, Celine; Wang, Michael; Liang, Yan; Lange, Stephan; Ouyang, Kunfu; Peterson, Kirk L; Evans, Sylvia M; Chen, Ju

2017-08-01

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2-associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

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.

Mutations in ALDH1A3 represent a frequent cause of microphthalmia/anophthalmia in consanguineous families.

PubMed

Abouzeid, Hana; Favez, Tatiana; Schmid, Angélique; Agosti, Céline; Youssef, Mohammed; Marzouk, Iman; El Shakankiry, Nihal; Bayoumi, Nader; Munier, Francis L; Schorderet, Daniel F

2014-08-01

Anophthalmia or microphthalmia (A/M), characterized by absent or small eye, can be unilateral or bilateral and represent developmental anomalies due to the mutations in several genes. Recently, mutations in aldehyde dehydrogenase family 1, member A3 (ALDH1A3) also known as retinaldehyde dehydrogenase 3, have been reported to cause A/M. Here, we screened a cohort of 75 patients with A/M and showed that mutations in ALDH1A3 occurred in six families. Based on this series, we estimate that mutations in ALDH1A3 represent a major cause of A/M in consanguineous families, and may be responsible for approximately 10% of the cases. Screening of this gene should be performed in a first line of investigation, together with SOX2. © 2014 WILEY PERIODICALS, INC.

Mutational Analysis of the Adaptor Protein 2 Sigma Subunit (AP2S1) Gene: Search for Autosomal Dominant Hypocalcemia Type 3 (ADH3)

PubMed Central

Rogers, Angela; Nesbit, M. Andrew; Hannan, Fadil M.; Howles, Sarah A.; Gorvin, Caroline M.; Cranston, Treena; Allgrove, Jeremy; Bevan, John S.; Bano, Gul; Brain, Caroline; Datta, Vipan; Grossman, Ashley B.; Hodgson, Shirley V.; Izatt, Louise; Millar-Jones, Lynne; Pearce, Simon H.; Robertson, Lisa; Selby, Peter L.; Shine, Brian; Snape, Katie; Warner, Justin

2014-01-01

Context: Autosomal dominant hypocalcemia (ADH) types 1 and 2 are due to calcium-sensing receptor (CASR) and G-protein subunit-α11 (GNA11) gain-of-function mutations, respectively, whereas CASR and GNA11 loss-of-function mutations result in familial hypocalciuric hypercalcemia (FHH) types 1 and 2, respectively. Loss-of-function mutations of adaptor protein-2 sigma subunit (AP2σ 2), encoded by AP2S1, cause FHH3, and we therefore sought for gain-of-function AP2S1 mutations that may cause an additional form of ADH, which we designated ADH3. Objective: The objective of the study was to investigate the hypothesis that gain-of-function AP2S1 mutations may cause ADH3. Design: The sample size required for the detection of at least one mutation with a greater than 95% likelihood was determined by binomial probability analysis. Nineteen patients (including six familial cases) with hypocalcemia in association with low or normal serum PTH concentrations, consistent with ADH, but who did not have CASR or GNA11 mutations, were ascertained. Leukocyte DNA was used for sequence and copy number variation analysis of AP2S1. Results: Binomial probability analysis, using the assumption that AP2S1 mutations would occur in hypocalcemic patients at a prevalence of 20%, which is observed in FHH patients without CASR or GNA11 mutations, indicated that the likelihood of detecting at least one AP2S1 mutation was greater than 95% and greater than 98% in sample sizes of 14 and 19 hypocalcemic patients, respectively. AP2S1 mutations and copy number variations were not detected in the 19 hypocalcemic patients. Conclusion: The absence of AP2S1 abnormalities in hypocalcemic patients, suggests that ADH3 may not occur or otherwise represents a rare hypocalcemic disorder. PMID:24708097

Liquid biopsy of PIK3CA mutations in cervical cancer in Hong Kong Chinese women.

PubMed

Chung, Tony K H; Cheung, Tak Hong; Yim, So Fan; Yu, Mei Yun; Chiu, Rossa W K; Lo, Keith W K; Lee, Ida P C; Wong, Raymond R Y; Lau, Kitty K M; Wang, Vivian W; Worley, Michael J; Elias, Kevin M; Fiascone, Stephen J; Smith, David I; Berkowitz, Ross S; Wong, Yick Fu

2017-08-01

Cervical cancer is the fourth most common female cancer worldwide. The prognosis for women with advanced-stage or recurrent cervical cancer remains poor and response to treatment is variable. Standardized management protocols leave little room for individualization. We report on a novel blood-based liquid biopsy for specific PIK3CA mutations as a clinically useful biomarker in patients with invasive cervical cancer. One hundred seventeen Hong Kong Chinese women with primary invasive cervical cancer and their pre-treatment plasma samples were investigated. Two PIK3CA mutations, p.E542K and p.E545K were measured in cell free DNA (cfDNA) extracted from plasma using droplet digital PCR. This liquid biopsy of PIK3CA in cervical cancer was correlated to clinico-pathological features to verify the potential of PIK3CA as a clinically useful molecular biomarker for predicting disease prognosis and monitoring for progression. PIK3CA mutations, either p.E542K or p.E545K, were detected in plasma cfDNA from 22.2% of the patients. PIK3CA mutation status was significantly correlated to median tumor size (p<0.01). PIK3CA mutations detected in the plasma were significantly associated with decreased disease-free survival and overall survival (p<0.05). As a liquid molecular biopsy, analysis of circulating PIK3CA mutations shows promise as a way to refine risk stratification of individual patients with cervical cancer, and provides a platform for further research to offer individualized therapy with the purpose of improving outcomes. Copyright © 2017 Elsevier Inc. All rights reserved.

Arsenic Exposure and Calpain-10 Polymorphisms Impair the Function of Pancreatic Beta-Cells in Humans: A Pilot Study of Risk Factors for T2DM

PubMed Central

Díaz-Villaseñor, Andrea; Cruz, Laura; Cebrián, Arturo; Hernández-Ramírez, Raúl U.; Hiriart, Marcia; García-Vargas, Gonzálo; Bassol, Susana; Sordo, Monserrat; Gandolfi, A. Jay; Klimecki, Walter T.; López-Carillo, Lizbeth; Cebrián, Mariano E.; Ostrosky-Wegman, Patricia

2013-01-01

The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide and diverse environmental and genetic risk factors are well recognized. Single nucleotide polymorphisms (SNPs) in the calpain-10 gene (CAPN-10), which encodes a protein involved in the secretion and action of insulin, and chronic exposure to inorganic arsenic (iAs) through drinking water have been independently associated with an increase in the risk for T2DM. In the present work we evaluated if CAPN-10 SNPs and iAs exposure jointly contribute to the outcome of T2DM. Insulin secretion (beta-cell function) and insulin sensitivity were evaluated indirectly through validated indexes (HOMA2) in subjects with and without T2DM who have been exposed to a gradient of iAs in their drinking water in northern Mexico. The results were analyzed taking into account the presence of the risk factor SNPs SNP-43 and -44 in CAPN-10. Subjects with T2DM had significantly lower beta-cell function and insulin sensitivity. An inverse association was found between beta-cell function and iAs exposure, the association being more pronounced in subjects with T2DM. Subjects without T2DM who were carriers of the at-risk genotype SNP-43 or -44, also had significantly lower beta-cell function. The association of SNP-43 with beta-cell function was dependent on iAs exposure, age, gender and BMI, whereas the association with SNP-44 was independent of all of these factors. Chronic exposure to iAs seems to be a risk factor for T2DM in humans through the reduction of beta-cell function, with an enhanced effect seen in the presence of the at-risk genotype of SNP-43 in CAPN-10. Carriers of CAPN-10 SNP-44 have also shown reduced beta-cell function. PMID:23349674

Comprehensive mutation profiling of mucinous gastric carcinoma.

PubMed

Rokutan, Hirofumi; Hosoda, Fumie; Hama, Natsuko; Nakamura, Hiromi; Totoki, Yasushi; Furukawa, Eisaku; Arakawa, Erika; Ohashi, Shoko; Urushidate, Tomoko; Satoh, Hironori; Shimizu, Hiroko; Igarashi, Keiko; Yachida, Shinichi; Katai, Hitoshi; Taniguchi, Hirokazu; Fukayama, Masashi; Shibata, Tatsuhiro

2016-10-01

Mucinous gastric carcinoma (MGC) is a unique subtype of gastric cancer with a poor survival outcome. Comprehensive molecular profiles and putative therapeutic targets of MGC remain undetermined. We subjected 16 tumour-normal tissue pairs to whole-exome sequencing (WES) and an expanded set of 52 tumour-normal tissue pairs to subsequent targeted sequencing. The latter focused on 114 genes identified by WES. Twenty-two histologically differentiated MGCs (D-MGCs) and 46 undifferentiated MGCs (U-MGCs) were analysed. Chromatin modifier genes, including ARID1A (21%), MLL2 (19%), MLL3 (15%), and KDM6A (7%), were frequently mutated (47%) in MGC. We also identified mutations in potential therapeutic target genes, including MTOR (9%), BRCA2 (9%), BRCA1 (7%), and ERBB3 (6%). RHOA mutation was detected only in 4% of U-MGCs and in no D-MGCs. MYH9 was recurrently (13%) mutated in MGC, with all these being of the U-MGC subtype (p = 0.023). Three U-MGCs harboured MYH9 nonsense mutations. MYH9 knockdown enhanced cell migration and induced intracytoplasmic mucin and cellular elongation. BCOR mutation was associated with improved survival. In U-MGCs, the MLH1 expression status and combined mutation status (TP53/BCL11B or TP53/MLL2) were prognostic factors. A comparative analysis of driver genes revealed that the mutation profile of D-MGC was similar to that of intestinal-type gastric cancer, whereas U-MGC was a distinct entity, harbouring a different mutational profile to intestinal- and diffuse-type gastric cancers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genetic Screening for OPA1 and OPA3 Mutations in Patients with Suspected Inherited Optic Neuropathies

PubMed Central

Yu-Wai-Man, Patrick; Shankar, Suma P.; Biousse, Valérie; Miller, Neil R.; Bean, Lora J.H.; Coffee, Bradford; Hegde, Madhuri; Newman, Nancy J.

2010-01-01

Purpose Autosomal-dominant optic atrophy (DOA) is one of the most common inherited optic neuropathies, and it is genetically heterogeneous, with mutations in both OPA1 and OPA3 known to cause disease. About 60% of cases harbor OPA1 mutations, whereas OPA3 mutations have only been reported in two pedigrees with DOA and premature cataracts. The aim of this study was to determine the yield of OPA1 and OPA3 screening in a cohort of presumed DOA cases referred to a tertiary diagnostic laboratory. Design Retrospective case series. Participants One hundred and eighty-eight probands with bilateral optic atrophy referred for molecular genetic investigations at a tertiary diagnostic facility: 38 patients with an autosomal-dominant pattern of inheritance and 150 sporadic cases. Methods OPA1 and OPA3 genetic testing was initially performed using PCR-based sequencing methods. The presence of large-scale OPA1 and OPA3 genomic rearrangements was further assessed with a targeted comparative genomic hybridization (CGH) microarray platform. The three primary Leber hereditary optic neuropathy (LHON) mutations, m.3460G>A, m.11778G>A, and m.14484T>C, were also screened in all patients. Main Outcome Measures The proportion of patients with OPA1 and OPA3 pathogenic mutations. The clinical profile observed in molecularly confirmed DOA cases. Results We found 21 different OPA1 mutations in 27 of the 188 (14.4%) probands screened. The mutations included six novel pathogenic variants and the first reported OPA1 initiation codon mutation at c.1A>T. An OPA1 missense mutation, c.239A>G (p.Y80C), was identified in an 11-year-old African-American girl with optic atrophy and peripheral sensori-motor neuropathy in her lower limbs. The OPA1 detection rate was significantly higher among individuals with a positive family history of visual failure (50.0%) compared with sporadic cases (5.3%). The primary LHON screen was negative in our patient cohort, and additional molecular investigations did not

Expanding spectrum of neurologic manifestations in patients with NLRP3 low-penetrance mutations

PubMed Central

Schuh, Elisabeth; Lohse, Peter; Ertl-Wagner, Birgit; Witt, Matthias; Krumbholz, Markus; Frankenberger, Marion; Gerdes, Lisa-Ann; Hohlfeld, Reinhard

2015-01-01

Objective: To evaluate the frequency of the cryoporin/NLRP3 low-penetrance mutations V198M and Q703K in patients who reported at least 2 symptoms compatible with cryopyrin-associated periodic syndromes (CAPS) and to characterize the phenotype in mutation-positive patients. Methods: The frequency of the V198M and Q703K mutations was investigated in a selected cohort of 108 patients from our neuroimmunology department. We describe the clinical, neurologic, immunologic, and neuroradiologic features of the mutation carriers. Results: Seventeen patients (16%) tested positive for either of the 2 mutations (V198M: n = 2; Q703K: n = 15). Eleven patients (65%) had severe headache syndromes. Six of these 11 patients were diagnosed with migraine. Nine patients (53%) had a concomitant diagnosis of multiple sclerosis (MS). In 3 patients, we identified additional family members with the respective mutation as well as the diagnosis of MS. Severe recurrent cranial nerve (CN) affection was the hallmark feature in 7 of the 8 (88%) non-MS mutation carriers. Brain MRI showed abnormalities in all but 2 patients (88%) and detected CN inflammation in 4 patients. Interleukin-6 was elevated in the CSF of 2 patients in the non-MS cohort during acute CAPS episodes with severe CNS inflammation. 5 of 9 treated patients (56%) responded to anti–interleukin-1 therapy. Conclusion: CAPS constitute rare but treatable and commonly misdiagnosed autoinflammatory syndromes. Our data expand the spectrum of CAPS-associated neurologic manifestations. They also broaden our concept of autoimmunity and autoinflammation by linking CAPS and MS. PMID:26020059

Two truncating USH3A mutations, including one novel, in a German family with Usher syndrome.

PubMed

Ebermann, Inga; Wilke, Robert; Lauhoff, Thomas; Lübben, Dirk; Zrenner, Eberhart; Bolz, Hanno Jörn

2007-08-30

To identify the genetic defect in a German family with Usher syndrome (USH) and linkage to the USH3A locus. DNA samples of five family members (both parents and the three patients) were genotyped with polymorphic microsatellite markers specific for eight USH genes. Three affected family members underwent detailed ocular and audiologic characterization. Symptoms in the patients were compatible with Usher syndrome and show intrafamilial variation, for both hearing loss (ranging from severe to profound with non-linear progression) and vision. Genotyping of microsatellite markers for the different USH loci was in line with a defect in the USH3A gene on chromosome 3q25. Sequence analysis of the USH3A gene revealed two truncating mutations; c.149_152delCAGGinsTGTCCAAT, which has been described previously, and a novel mutation, c.502_503insA, segregating with the phenotype. To date, only 11 USH3A mutations have been described. This is the first description of a German family with USH due to USH3A mutations, including one novel. Our findings indicate that also in the Central European population, USH3A mutations should be considered in cases of USH.

Screening for MPL mutations in essential thrombocythemia and primary myelofibrosis: normal Mpl expression and absence of constitutive STAT3 and STAT5 activation in MPLW515L-positive platelets.

PubMed

Glembotsky, Ana C; Korin, Laura; Lev, Paola R; Chazarreta, Carlos D; Marta, Rosana F; Molinas, Felisa C; Heller, Paula G

2010-05-01

To evaluate the frequency of MPL W515L, W515K and S505N mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO). Mutation detection was performed by allele-specific PCR and sequencing. Platelet Mpl expression was evaluated by flow cytometry, immunoblotting and real-time RT-PCR. Activation of STAT3 and STAT5 before and after stimulation with increasing concentrations of TPO was studied by immunoblotting. Plasma TPO was measured by ELISA. MPLW515L was detected in 1 of 100 patients with ET and 1 of 11 with PMF. Platelets from the PMF patient showed 100% mutant allele, which was <50% in platelets from the ET patient, who also showed the mutation in granulocytes, monocytes and B cells. Mpl surface and total protein expression were normal, and TPO levels were mildly increased in the MPLW515L-positive ET patient, while MPL transcripts did not differ from controls in both MPLW515L-positive patients. Constitutive STAT3 and STAT5 phosphorylation was absent and dose response to TPO-induced phosphorylation was not enhanced. The low frequency of MPL mutations in this cohort is in agreement with previous studies. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for myeloproliferative neoplasms. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models.

Cell-Specific Actions of a Human LHX3 Gene Enhancer During Pituitary and Spinal Cord Development

PubMed Central

Park, Soyoung; Mullen, Rachel D.

2013-01-01

The LIM class of homeodomain protein 3 (LHX3) transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie complex pediatric syndromes featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. Here we characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in α-glycoprotein subunit -expressing cells secreting the TSHβ, LHβ, or FSHβ hormones and expressing the GATA2 and steroidogenic factor 1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module active in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression. Furthermore, these studies revealed significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice also provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. PMID:24100213

Cell Cycle Reprogramming for PI3K Inhibition Overrides Relapse-Specific C481S BTK Mutation Revealed by Longitudinal Functional Genomics in Mantle Cell Lymphoma

PubMed Central

Chiron, David; Di Liberto, Maurizio; Martin, Peter; Huang, Xiangao; Sharman, Jeff; Blecua, Pedro; Mathew, Susan; Vijay, Priyanka; Eng, Ken; Ali, Siraj; Johnson, Amy; Chang, Betty; Ely, Scott; Elemento, Olivier; Mason, Christopher E.; Leonard, John P.; Chen-Kiang, Selina

2014-01-01

Despite the unprecedented clinical activity of the Bruton’s tyrosine kinase inhibitor ibrutinib in MCL, acquired-resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib-binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary-resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired-ibrutinib resistance in MCL and suggest a strategy to override both primary- and acquired-ibrutinib resistance. PMID:25082755

Application of marker selection to enhance estimation of genetic effects and gene interaction in cattle

USDA-ARS?s Scientific Manuscript database

Selection on important genetic markers can improve estimates of additive and dominance association effects. A composite population of beef cattle was selected for intermediate frequencies of myostatin (GDF8) F94L and µ-calpain (CAPN1) polymorphisms. Important additive associations of the GDF8 locu...

Two Novel HOGA1 Splicing Mutations Identified in a Chinese Patient with Primary Hyperoxaluria Type 3.

PubMed

Wang, Xinsheng; Zhao, Xiangzhong; Wang, Xiaoling; Yao, Jian; Zhang, Feifei; Lang, Yanhua; Tuffery-Giraud, Sylvie; Bottillo, Irene; Shao, Leping

2015-01-01

Twenty-six HOGA1 mutations have been reported in primary hyperoxaluria (PH) type 3 (PH3) patients with c.700 + 5G>T accounting for about 50% of the total alleles. However, PH3 has never been described in Asians. A Chinese child with early-onset nephrolithiasis was suspected of having PH. We searched for AGXT, GRHPR and HOGA1 gene mutations in this patient and his parents. All coding regions, including intron-exon boundaries, were analyzed using PCR followed by direct sequence analysis. Two heterozygous mutations not previously described in the literature about HOGA1 were identified (compound heterozygous). One mutation was a successive 2 bp substitution at the last nucleotide of exon 6 and at the first nucleotide of intron 6, respectively (c.834_834 + 1GG>TT), while the other one was a guanine to adenine substitution of the last nucleotide of exon 6 (c.834G>A). Direct sequencing analysis failed to find these mutations in 100 unrelated healthy subjects and the functional role on splicing of both variants found in this study was confirmed by a minigene assay based on the pSPL3 exon trapping vector. In addition, we found a SNP in this family (c.715G>A, p.V239I). There were no mutations detected in AGXT and GRHPR. Two novel HOGA1 mutations were identified in association with PH3. This is the first description and investigation on mutant gene analysis of PH3 in an Asian. © 2015 S. Karger AG, Basel

Identification of a novel insertion mutation in FGFR3 that causes thanatophoric dysplasia type 1.

PubMed

Lindy, Amanda S; Basehore, Monica J; Munisha, Mumingjiang; Williams, Aimee Leanne; Friez, Michael J; Writzl, Karin; Willems, Patrick; Dougan, Scott T

2016-06-01

Thanatophoric dysplasia is a type of short-limbed neonatal dwarfism that is usually lethal in the perinatal period. It is characterized by short limbs, a narrow, bell-shaped thorax, macrocephaly with a prominent forehead, and flattened vertebral bodies. These malformations result from autosomal dominant mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. In this report, we describe a novel FGFR3 insertion mutation in a fetus with shortened limbs, curved femurs, and a narrow thorax. The diagnosis of thanatophoric dysplasia type 1 was suspected clinically, and FGFR3 sequencing showed a c.742_743insTGT variant, which predicts p.R248delinsLC. In vivo studies in zebrafish demonstrated that this mutation resulted in the overexpression of zebrafish Fgfr3, leading to the over-activation of downstream signaling and dorsalized embryos. To date, no insertions or deletions in FGFR3 have been reported to cause thanatophoric dysplasia types 1 or 2; therefore, this represents the first report to describe such a mutation. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

AIP mutations impair AhR signaling in pituitary adenoma patients fibroblasts and in GH3 cells.

PubMed

Lecoq, Anne-Lise; Viengchareun, Say; Hage, Mirella; Bouligand, Jérôme; Young, Jacques; Boutron, Audrey; Zizzari, Philippe; Lombès, Marc; Chanson, Philippe; Kamenický, Peter

2016-05-01

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas through unknown molecular mechanisms. The best-known interacting partner of AIP is the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the effects of xenobiotics implicated in carcinogenesis. As 75% of AIP mutations disrupt the physical and/or functional interaction with AhR, we postulated that the tumorigenic potential of AIP mutations might result from altered AhR signaling. We evaluated the impact of AIP mutations on the AhR signaling pathway, first in fibroblasts from AIP-mutated patients with pituitary adenomas, by comparison with fibroblasts from healthy subjects, then in transfected pituitary GH3 cells. The AIP protein level in mutated fibroblasts was about half of that in cells from healthy subjects, but AhR expression was unaffected. Gene expression analyses showed significant modifications in the expression of the AhR target genes CYP1B1 and AHRR in AIP-mutated fibroblasts, both before and after stimulation with the endogenous AhR ligand kynurenine. Kynurenine increased Cyp1b1 expression to a greater extent in GH3 cells overexpressing wild type compared with cells expressing mutant AIP Knockdown of endogenous Aip in these cells attenuated Cyp1b1 induction by the AhR ligand. Both mutant AIP expression and knockdown of endogenous Aip affected the kynurenine-dependent GH secretion of GH3 cells. This study of human fibroblasts bearing endogenous heterozygous AIP mutations and transfected pituitary GH3 cells shows that AIP mutations affect the AIP protein level and alter AhR transcriptional activity in a gene- and tissue-dependent manner. © 2016 Society for Endocrinology.

Favorable outcome of patients with acute myeloid leukemia harboring a low-allelic burden FLT3-ITD mutation and concomitant NPM1 mutation: relevance to post-remission therapy.

PubMed

Pratcorona, Marta; Brunet, Salut; Nomdedéu, Josep; Ribera, Josep Maria; Tormo, Mar; Duarte, Rafael; Escoda, Lourdes; Guàrdia, Ramon; Queipo de Llano, M Paz; Salamero, Olga; Bargay, Joan; Pedro, Carmen; Martí, Josep Maria; Torrebadell, Montserrat; Díaz-Beyá, Marina; Camós, Mireia; Colomer, Dolors; Hoyos, Montserrat; Sierra, Jorge; Esteve, Jordi

2013-04-04

Risk associated to FLT3 internal tandem duplication (FLT3-ITD) in patients with acute myeloid leukemia (AML) may depend on mutational burden and its interaction with other mutations. We analyzed the effect of FLT3-ITD/FLT3 wild-type (FLT3wt) ratio depending on NPM1 mutation (NPM1mut) in 303 patients with intermediate-risk cytogenetics AML treated with intensive chemotherapy. Among NPM1mut patients, FLT3wt and low ratio (<0.5) subgroups showed similar overall survival, relapse risk, and leukemia-free survival, whereas high ratio (≥0.5) patients had a worse outcome. In NPM1wt AML, FLT3-ITD subgroups showed a comparable outcome, with higher risk of relapse and shortened overall survival than FLT3wt patients. Allogeneic stem cell transplantation in CR1 was associated with a reduced relapse risk in all molecular subgroups with the exception of NPM1mut AML with absent or low ratio FLT3-ITD. In conclusion, effect of FLT3 burden is modulated by NPM1 mutation, especially in patients with a low ratio.

PTH 1-34 Ameliorates the Osteopenia and Delayed Healing of Stabilized Tibia Fracture in Mice with Achondroplasia Resulting from Gain-Of-Function Mutation of FGFR3

PubMed Central

Chen, Hangang; Sun, Xianding; Yin, Liangjun; Chen, Shuai; Zhu, Ying; Huang, Junlan; Jiang, Wanling; Chen, Bo; Zhang, Ruobin; Chen, Lin; Nie, Mao; Xie, Yangli; Deng, Zhongliang

2017-01-01

Bone fracture healing is processed through multiple stages including the cartilaginous callus formation and its transition to bony callus. FGFR3 negatively regulates chondrogenesis and enhances osteogenesis during skeleton development. We previously found in mice carrying gain-of-function mutation of FGFR3 that FGFR3 delays the healing of un-stabilized fracture that heals mainly through endochondral ossification. Since fracture is regularly treated in clinics with rigid fixation, and stabilized fracture is healed largely through intramembranous ossification, we asked whether FGFR3, a key regulator of osteogenesis, also affect the regeneration of stabilized fracture. We found that gain-of-function mutation of FGFR3 inhibits the initiation of chondrogenesis and the subsequent bone formation. We further studied whether PTH1-34 can improve the osteopenia and delayed healing of the stabilized tibia fracture in mice with achondroplasia. Fracture healing was evaluated by radiography, micro-CT, biomechanical tests, histology, and real-time polymerase chain reaction (RT-PCR) analysis. We found that PTH 1-34 can alleviate the decreased bone mass and compromised architecture in ACH mice. Histological analysis revealed that administration of PTH1-34 increased the size of both the total callus and cartilaginous callus at 14 days after the surgery in ACH mice. RT-PCR data suggested that systemic PTH1-34 accelerated the initiation of chondrogenesis and chondrocyte maturation (earlier and higher levels of expression of chondrogenesis related markers) and enhanced the osteogenic differentiation in the fracture callus in ACH mice. These results indicate that the PTH1-34 administration resulted in an enhanced callus formation during bone fracture healing in ACH mice, which is at least in part mediated by an increase of cartilaginous callus at early stage and the promotion of bone formation in bony callus. In summary, in this study we revealed that FGFR3 delays the regeneration of

Calpain-5 gene variants are associated with diastolic blood pressure and cholesterol levels

PubMed Central

Sáez, María E; Martínez-Larrad, María T; Ramírez-Lorca, Reposo; González-Sánchez, José L; Zabena, Carina; Martinez-Calatrava, María J; González, Alejandro; Morón, Francisco J; Ruiz, Agustín; Serrano-Ríos, Manuel

2007-01-01

Background Genes implicated in common complex disorders such as obesity, type 2 diabetes mellitus (T2DM) or cardiovascular diseases are not disease specific, since clinically related disorders also share genetic components. Cysteine protease Calpain 10 (CAPN10) has been associated with T2DM, hypertension, hypercholesterolemia, increased body mass index (BMI) and polycystic ovary syndrome (PCOS), a reproductive disorder of women in which isunlin resistance seems to play a pathogenic role. The calpain 5 gene (CAPN5) encodes a protein homologue of CAPN10. CAPN5 has been previously associated with PCOS by our group. In this new study, we have analysed the association of four CAPN5 gene variants(rs948976A>G, rs4945140G>A, rs2233546C>T and rs2233549G>A) with several cardiovascular risk factors related to metabolic syndrome in general population. Methods Anthropometric measurements, blood pressure, insulin, glucose and lipid profiles were determined in 606 individuals randomly chosen from a cross-sectional population-based epidemiological survey in the province of Segovia in Central Spain (Castille), recruited to investigate the prevalence of anthropometric and physiological parameters related to obesity and other components of the metabolic syndrome. Genotypes at the four polymorphic loci in CAPN5 gene were detected by polymerase chain reaction (PCR). Results Genotype association analysis was significant for BMI (p ≤ 0.041), diastolic blood pressure (p = 0.015) and HDL-cholesterol levels (p = 0.025). Different CAPN5 haplotypes were also associated with diastolic blood pressure (DBP) (0.0005 ≤ p ≤ 0.006) and total cholesterol levels (0.001 ≤ p ≤ 0.029). In addition, the AACA haplotype, over-represented in obese individuals, is also more frequent in individuals with metabolic syndrome defined by ATPIII criteria (p = 0.029). Conclusion As its homologue CAPN10, CAPN5 seems to influence traits related to increased risk for cardiovascular diseases. Our results also

The BAG3 gene variants in Polish patients with dilated cardiomyopathy: four novel mutations and a genotype-phenotype correlation.

PubMed

Franaszczyk, Maria; Bilinska, Zofia T; Sobieszczańska-Małek, Małgorzata; Michalak, Ewa; Sleszycka, Justyna; Sioma, Agnieszka; Małek, Łukasz A; Kaczmarska, Dorota; Walczak, Ewa; Włodarski, Paweł; Hutnik, Łukasz; Milanowska, Blanka; Dzielinska, Zofia; Religa, Grzegorz; Grzybowski, Jacek; Zieliński, Tomasz; Ploski, Rafal

2014-07-09

BAG3 gene mutations have been recently implicated as a novel cause of dilated cardiomyopathy (DCM). Our aim was to evaluate the prevalence of BAG3 mutations in Polish patients with DCM and to search for genotype-phenotype correlations. We studied 90 unrelated probands by direct sequencing of BAG3 exons and splice sites. Large deletions/insertions were screened for by quantitative real time polymerase chain reaction (qPCR). We found 5 different mutations in 6 probands and a total of 21 mutations among their relatives: the known p.Glu455Lys mutation (2 families), 4 novel mutations: p.Gln353ArgfsX10 (c.1055delC), p.Gly379AlafsX45 (c.1135delG), p.Tyr451X (c.1353C>A) and a large deletion of 17,990 bp removing BAG3 exons 3-4. Analysis of mutation positive relatives of the probands from this study pooled with those previously reported showed higher DCM prevalence among those with missense vs. truncating mutations (OR = 8.33, P = 0.0058) as well as a difference in age at disease onset between the former and the latter in Kaplan-Meier survival analysis (P = 0.006). Clinical data from our study suggested that in BAG3 mutation carriers acute onset DCM with hemodynamic compromise may be triggered by infection. BAG3 point mutations and large deletions are relatively frequent cause of DCM. Delayed DCM onset associated with truncating vs. non-truncating mutations may be important for genetic counseling.

ESR1 and PIK3CA mutational status in serum and plasma from metastatic breast cancer patients: A comparative study.

PubMed

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Tomiguchi, Mai; Sueta, Aiko; Iwase, Hirotaka

2018-04-07

Plasma and serum cell-free DNA (cfDNA) are useful sources of tumor DNA, but comparative investigations of the tumor mutational status between them are rare. we performed droplet digital PCR assay for representative hotspot mutations in metastatic breast cancer (MBC) (ESR1 and PIK3CA) in serum and plasma cfDNA concurrently extracted from the blood of 33 estrogen receptor-positive MBC patients. ESR1 mutations in plasma cfDNA were found in 7 of the 33 patients; ESR1 mutations in serum cfDNA were detected in only one out of 7 patients with ESR1 mutations in plasma cfDNA. PIK3CA exon 9 and exon 20 mutations in plasma cfDNA were found in 3 and 7 out of the 33 patients, respectively; PIK3CA exon 9 mutations in serum cfDNA were detected in 2 out of 3 patients with PIK3CA exon 9 mutations in plasma cfDNA; PIK3CA exon 20 mutations in serum cfDNA were detected in 2 out of 7 patients with PIK3CA exon 20 mutations in plasma cfDNA. Here we show the higher frequency of ESR1 and PIK3CA mutations in the plasma than in the serum in 33 MBC patients; therefore, serum samples should not be considered the preferred source of cfDNA.

a/alpha-specific effect on the mms3 mutation on ultraviolet mutagenesis in Saccharomyces cerevisiae.

PubMed

Martin, P; Prakash, L; Prakash, S

1981-05-01

A new gene involved in error-prone repair of ultraviolet (UV) damage has been identified in Saccharomyces cerevisiae by the mms3-1 mutation. UV-induced reversion is reduced in diploids that are homozygous for mms3-1, only if they are also heterozygous (MATa/MAT alpha) at the mating type locus. The mms3-1 mutation has no effect on UV-induced reversion either in haploids or MATa/MATa or MAT alpha/MAT alpha diploids. The mutation confers sensitivity to UV and methyl methane sulfonate in both haploids and diploids. Even though mutation induction by UV is restored to wild-type levels in MATa/MATa mms3-1/mms3-1 or MAT alpha/MAT alpha mms3-1/mms3-1 diploids, such strains still retain sensitivity to the lethal effects of UV. Survival after UV irradiation in mms3-1 rad double mutant combinations indicates that mms3-1 is epistatic to rad6-1 whereas non-epistatic interactions are observed with rad3 and rad52 mutants. When present in the homozygous state in MATa/MAT alpha his1-1/his1-315 heteroallelic diploids, mms3-1 was found to lower UV-induced mitotic recombination.

A novel PAX3 mutation in a Japanese boy with Waardenburg syndrome type 1.

PubMed

Yoshida, Yu; Doi, Rieko; Adachi, Kaori; Nanba, Eiji; Kodani, Isamu; Ryoke, Kazuo

2016-01-01

Waardenburg syndrome type 1 (WS1) is a rare autosomal dominant disorder characterized by hair hypopigmentation, abnormal iris pigmentation, and congenital hearing loss. WS1 is caused by mutations in paired box gene 3 (PAX3). We identified a novel PAX3 mutation (c.1107 C>G, p.Ser369Arg) in a Japanese WS1 patient showing abnormal right iris pigmentation, right-sided congenital hearing loss, synophrys, incomplete left cleft lip, and cryptorchidism.

Phosphatidyl inositol-3 kinase (PIK3CA) E545K mutation confers cisplatin resistance and a migratory phenotype in cervical cancer cells.

PubMed

Arjumand, Wani; Merry, Cole D; Wang, Chen; Saba, Elias; McIntyre, John B; Fang, Shujuan; Kornaga, Elizabeth; Ghatage, Prafull; Doll, Corinne M; Lees-Miller, Susan P

2016-12-13

The phosphatidylinositol-3 kinase (PI3K)/Akt/mTOR signaling pathway is activated in many human cancers. Previously, we reported that patients with early stage cervical cancer whose tumours harbour PIK3CA exon 9 or 20 mutations have worse overall survival in response to treatment with radiation and cisplatin than patients with wild-type PIK3CA. The purpose of this study was to determine whether PIK3CA-E545K mutation renders cervical cancer cells more resistant to cisplatin and/or radiation, and whether PI3K inhibition reverses the phenotype. We found that CaSki cells that are heterozygous for the PIK3CA-E545K mutation are more resistant to cisplatin or cisplatin plus radiation than either HeLa or SiHa cells that express only wild-type PIK3CA. Similarly, HeLa cells engineered to stably express PIK3CA-E545K were more resistant to cisplatin or cisplatin plus radiation than cells expressing only wild-type PIK3CA or with PIK3CA depleted. Cells expressing the PIK3CA-E545K mutation also had constitutive PI3K pathway activation and increased cellular migration and each of these phenotypes was reversed by treatment with the PI3K inhibitor GDC-0941/Pictilisib. Our results suggests that cervical cancer patients whose tumours are positive for the PIK3CA-E545K mutation may benefit from PI3K inhibitor therapy in concert with standard cisplatin and radiation therapy.

Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene.

PubMed

Martorell, Sara; Palanca, Sarai; Maquieira, Ángel; Tortajada-Genaro, Luis A

2018-03-01

A blocked recombinase polymerase amplification (blocked-RPA) approach has been developed for the enrichment of mutated templates in heterogeneous specimens as tumor tissues. This isothermal amplification technique opens alternative solutions for meeting the technological demand of physician office laboratories. Herein, the detection of mutations in PIK3CA gene, such as p.E545K, and p.H1047L, is presented. The main element was an oligonucleotide (dideoxycytidine functionalized at 3'-end) which matched with wild-type sequence in the target locus. The amplification was performed operating at 37 °C during 40 min. The results demonstrated that the competition between the upstream primer and the blocker reduced the percentage of amplified wild-type allele, making the detection of the present mutation easier. For mutation discrimination, a fast hybridization assay was performed in microarray format on plastic chip and colorimetric detection. This approach enabled the reliable discrimination of specific mutations against a background of up to 95% wild-type DNA. The applicability of the method, based on the combination of blocked-RPA and low-cost chip hybridization, was successfully proven for the genotyping of various cancer cell lines as well as tumor tissues. The assignations agreed with those provided by next-generation sequencing. Therefore, these investigations would support a personalized approach to patient care based on the molecular signature of human cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

Population growth enhances the mean fixation time of neutral mutations and the persistence of neutral variation.

PubMed

Waxman, D

2012-06-01

A fundamental result of population genetics states that a new mutation, at an unlinked neutral locus in a randomly mating diploid population, has a mean time of fixation of ∼4N(e) generations, where N(e) is the effective population size. This result is based on an assumption of fixed population size, which does not universally hold in natural populations. Here, we analyze such neutral fixations in populations of changing size within the framework of the diffusion approximation. General expressions are derived for the mean and variance of the fixation time in changing populations. Some explicit results are given for two cases: (i) the effective population size undergoes a sudden change, representing a sudden population expansion or a sudden bottleneck; (ii) the effective population changes linearly for a limited period of time and then remains constant. Additionally, a lower bound for the mean time of fixation is obtained for an effective population size that increases with time, and this is applied to exponentially growing populations. The results obtained in this work show, among other things, that for populations that increase in size, the mean time of fixation can be enhanced, sometimes substantially so, over 4N(e,0) generations, where N(e,0) is the effective population size at the time the mutation arises. Such an enhancement is associated with (i) an increased probability of neutral polymorphism in a population and (ii) an enhanced persistence of high-frequency neutral variation, which is the variation most likely to be observed.

Dual mechanism of integrin αIIbβ3 closure in procoagulant platelets.

PubMed

Mattheij, Nadine J A; Gilio, Karen; van Kruchten, Roger; Jobe, Shawn M; Wieschhaus, Adam J; Chishti, Athar H; Collins, Peter; Heemskerk, Johan W M; Cosemans, Judith M E M

2013-05-10

Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation. Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbβ3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbβ3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbβ3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbβ3 inactivation. Integrin αIIbβ3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbβ3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbβ3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbβ3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

Common polymorphisms of calpain-10 and the risk of Type 2 Diabetes in a Tunisian Arab population: a case-control study

PubMed Central

2010-01-01

Background Genetic variations in the calpain-10 gene (CAPN10), in particular the at-risk diplotype (112/121), were previously implicated with increased risk of type 2 diabetes (T2D). Methods We examined the association of CAPN10 UCSNP-43 (rs3792267), UCSNP-19 (rs3842570), and UCSNP-63 (rs5030952) SNPs with T2D in 917 Tunisian T2D patients and 748 non-diabetic controls. CAPN10 genotyping was done by PCR-RFLP. Results Enrichment of UCSNP-19 2R (minor) allele and 2R/2R genotype was found in T2D patients; the allele and genotype distribution of UCSNP-43 and UCSNP-63 alleles and genotypes were not significantly different between patient groups and non-diabetic control subjects. Regression analysis demonstrated progressive increases in T2D risk in 3R/2R [OR (95% CI) = 1.35 (1.08 - 1.68)] and 2R/2R [OR (95% CI) = 1.61 (1.20 - 2.18)] genotypes. Of the six haplotypes detected, enrichment of haplotype 111 (UCSNP-43/UCSNP-19/UCSNP-63) was seen in patients (Pc = 0.034); the distribution of the other haplotypes was comparable between patients and control subjects; neither haplotype 211 nor haplotype 212 was observed. Furthermore, the frequency of all CAPN10 diplotypes identified, including the "high-risk diplotype (112/121) reported for Mexican-Americans and Northern Europeans, were comparable between patients and controls. Conclusions CAPN10 UCSNP-19 variant, and the 111 haplotype contribute to the risk of T2D in Tunisian subjects; no significant associations between CAPN10 diplotypes and T2D were demonstrated for Tunisians. PMID:20470430

Analysis of ESR1 and PIK3CA mutations in plasma cell-free DNA from ER-positive breast cancer patients.

PubMed

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

2017-08-08

The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients. The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients. cfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations. We have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations.

Characterization of New Otic Enhancers of the Pou3f4 Gene Reveal Distinct Signaling Pathway Regulation and Spatio-Temporal Patterns

PubMed Central

Robert-Moreno, Àlex; Naranjo, Silvia; de la Calle-Mustienes, Elisa; Gómez-Skarmeta, José Luis; Alsina, Berta

2010-01-01

POU3F4 is a member of the POU-homedomain transcription factor family with a prominent role in inner ear development. Mutations in the human POU3F4 coding unit leads to X-linked deafness type 3 (DFN3), characterized by conductive hearing loss and progressive sensorineural deafness. Microdeletions found 1 Mb 5′ upstream of the coding region also displayed the same phenotype, suggesting that cis-regulatory elements might be present in that region. Indeed, we and others have recently identified several enhancers at the 1 Mb 5′ upstream interval of the pou3f4 locus. Here we characterize the spatio-temporal patterns of these regulatory elements in zebrafish transgenic lines. We show that the most distal enhancer (HCNR 81675) is activated earlier and drives GFP reporter expression initially to a broad ear domain to progressively restrict to the sensory patches. The proximal enhancer (HCNR 82478) is switched later during development and promotes expression, among in other tissues, in sensory patches from its onset. The third enhancer (HCNR 81728) is also active at later stages in the otic mesenchyme and in the otic epithelium. We also characterize the signaling pathways regulating these enhancers. While HCNR 81675 is regulated by very early signals of retinoic acid, HCNR 82478 is regulated by Fgf activity at a later stage and the HCNR 81728 enhancer is under the control of Hh signaling. Finally, we show that Sox2 and Pax2 transcription factors are bound to HCNR 81675 genomic region during otic development and specific mutations to these transcription factor binding sites abrogates HCNR 81675 enhancer activity. Altogether, our results suggest that pou3f4 expression in inner ear might be under the control of distinct regulatory elements that fine-tune the spatio-temporal activity of this gene and provides novel data on the signaling mechanisms controlling pou3f4 function. PMID:21209840