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Sample records for progeria mutation reveals

  1. An inherited LMNA gene mutation in atypical Progeria syndrome.

    PubMed

    Doubaj, Yassamine; De Sandre-Giovannoli, Annachiara; Vera, Esteves-Vieira; Navarro, Claire Laure; Elalaoui, Siham Chafai; Tajir, Mariam; Lévy, Nicolas; Sefiani, Abdelaziz

    2012-11-01

    Hutchinson-Gilford Progeria syndrome (HGPS) is a rare genetic disorder, characterized by several clinical features that begin in early childhood, recalling an accelerated aging process. The diagnosis of HGPS is based on the recognition of common clinical features and detection of the recurrent heterozygous c.1824C>T (p.Gly608Gly) mutation within exon 11 in the Lamin A/C encoding gene (LMNA). Besides "typical HGPS," several "atypical progeria" syndromes (APS) have been described, in a clinical spectrum ranging from mandibuloacral dysplasia to atypical Werner syndrome. These patients's clinical features include progeroid manifestations, such as short stature, prominent nose, premature graying of hair, partial alopecia, skin atrophy, lipodystrophy, skeletal anomalies, such as mandibular hypoplasia and acroosteolyses, and in some cases severe atherosclerosis with metabolic complications. APS are due in several cases to de novo heterozygous LMNA mutations other than the p.Gly608Gly, or due to homozygous BAFN1 mutations in Nestor-Guillermo Progeria syndrome (NGPS). We report here and discuss the observation of a non-consanguineous Moroccan patient presenting with atypical progeria. The molecular studies showed the heterozygous mutation c.412G>A (p.Glu138Lys) of the LMNA gene. This mutation, previously reported as a de novo mutation, was inherited from the apparently healthy father who showed a somatic cell mosaicism.

  2. A novel somatic mutation achieves partial rescue in a child with Hutchinson-Gilford progeria syndrome

    PubMed Central

    Bar, Daniel Z; Arlt, Martin F; Brazier, Joan F; Norris, Wendy E; Campbell, Susan E; Chines, Peter; Larrieu, Delphine; Jackson, Stephen P; Collins, Francis S; Glover, Thomas W; Gordon, Leslie B

    2017-01-01

    Background Hutchinson-Gilford progeria syndrome (HGPS) is a fatal sporadic autosomal dominant premature ageing disease caused by single base mutations that optimise a cryptic splice site within exon 11 of the LMNA gene. The resultant disease-causing protein, progerin, acts as a dominant negative. Disease severity relies partly on progerin levels. Methods and results We report a novel form of somatic mosaicism, where a child possessed two cell populations with different HGPS disease-producing mutations of the same nucleotide—one producing severe HGPS and one mild HGPS. The proband possessed an intermediate phenotype. The mosaicism was initially discovered when Sanger sequencing showed a c.1968+2T>A mutation in blood DNA and a c.1968+2T>C in DNA from cultured fibroblasts. Deep sequencing of DNA from the proband's blood revealed 4.7% c.1968+2T>C mutation, and 41.3% c.1968+2T>A mutation. Conclusions We hypothesise that the germline mutation was c.1968+2T>A, but a rescue event occurred during early development, where the somatic mutation from A to C at 1968+2 provided a selective advantage. This type of mosaicism where a partial phenotypic rescue event results from a second but milder disease-causing mutation in the same nucleotide has not been previously characterised for any disease. PMID:27920058

  3. Induced pluripotent stem cells reveal functional differences between drugs currently investigated in patients with hutchinson-gilford progeria syndrome.

    PubMed

    Blondel, Sophie; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Le Corf, Amelie; Navarro, Claire; Cordette, Véronique; Martinat, Cécile; Laabi, Yacine; Djabali, Karima; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Peschanski, Marc; Nissan, Xavier

    2014-04-01

    Hutchinson-Gilford progeria syndrome is a rare congenital disease characterized by premature aging in children. Identification of the mutation and related molecular mechanisms has rapidly led to independent clinical trials testing different marketed drugs with a preclinically documented impact on those mechanisms. However, the extensive functional effects of those drugs remain essentially unexplored. We have undertaken a systematic comparative study of the three main treatments currently administered or proposed to progeria-affected children, namely, a farnesyltransferase inhibitor, the combination of an aminobisphosphonate and a statin (zoledronate and pravastatin), and the macrolide antibiotic rapamycin. This work was based on the assumption that mesodermal stem cells, which are derived from Hutchinson-Gilford progeria syndrome-induced pluripotent stem cells expressing major defects associated with the disease, may be instrumental to revealing such effects. Whereas all three treatments significantly improved misshapen cell nuclei typically associated with progeria, differences were observed in terms of functional improvement in prelamin A farnesylation, progerin expression, defective cell proliferation, premature osteogenic differentiation, and ATP production. Finally, we have evaluated the effect of the different drug combinations on this cellular model. This study revealed no additional benefit compared with single-drug treatments, whereas a cytostatic effect equivalent to that of a farnesyltransferase inhibitor alone was systematically observed. Altogether, these results reveal the complexity of the modes of action of different drugs, even when they have been selected on the basis of a similar mechanistic hypothesis, and underscore the use of induced pluripotent stem cell derivatives as a critical and powerful tool for standardized, comparative pharmacological studies.

  4. Hutchinson-Gilford progeria syndrome with G608G LMNA mutation.

    PubMed

    Kim, Hui Kwon; Lee, Jong Yoon; Bae, Eun Ju; Oh, Phil Soo; Park, Won Il; Lee, Dong Sung; Kim, Jong-Il; Lee, Hong Jin

    2011-12-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare condition originally described by Hutchinson in 1886. Death result from cardiac complications in the majority of cases and usually occurs at average age of thirteen years. A 4-yr old boy had typical clinical findings such as short stature, craniofacial disproportion, alopecia, prominent scalp veins and sclerodermatous skin. This abnormal appearance began at age of 1 yr. On serological and hormonal evaluation, all values are within normal range. He was neurologically intact with motor and mental development. An echocardiogram showed calcification of aortic and mitral valves. Hypertrophy of internal layer at internal carotid artery suggesting atherosclerosis was found by carotid doppler sonography. He is on low dose aspirin to prevent thromboembolic episodes and on regular follow up. Gene study showed typical G608G (GGC- > GGT) point mutation at exon 11 in LMNA gene. This is a rare case of Hutchinson-Gilford progeria syndrome confirmed by genetic analysis in Korea.

  5. Hutchinson-Gilford progeria syndrome caused by an LMNA mutation: a case report.

    PubMed

    Chu, Yan; Xu, Zi-Gang; Xu, Zhe; Ma, Lin

    2015-01-01

    Hutchinson-Gilford progeria syndrome is a rare genetic disorder characterized by premature aging of the skin, bones, heart, and blood vessels. We report a 6-year-old boy who was born at full term but presented with scleroderma-like appearance at 1 month of age and gradually developed clinical manifestations of progeria. He had characteristic facial features of prominent eyes, scalp, and leg veins; loss of scalp hair, eyebrows, and eyelashes; stunted growth; scleroderma-like changes of the skin; and a premature aged appearance. Metabolic investigations showed transient methylmalonic aciduria, and genetic testing of the peripheral blood identified the c.1824C>T heterozygous LMNA mutation. The present case is reported because of its rarity.

  6. Expression of the Hutchinson-Gilford progeria mutation during osteoblast development results in loss of osteocytes, irregular mineralization, and poor biomechanical properties.

    PubMed

    Schmidt, Eva; Nilsson, Ola; Koskela, Antti; Tuukkanen, Juha; Ohlsson, Claes; Rozell, Björn; Eriksson, Maria

    2012-09-28

    Hutchinson-Gilford progeria syndrome (HGPS) is a very rare genetic disorder that is characterized by multiple features of premature aging and largely affects tissues of mesenchymal origin. In this study, we describe the development of a tissue-specific mouse model that overexpresses the most common HGPS mutation (LMNA, c.1824C>T, p.G608G) in osteoblasts. Already at the age of 5 weeks, HGPS mutant mice show growth retardation, imbalanced gait and spontaneous fractures. Histopathological examination revealed an irregular bone structure, characterized by widespread loss of osteocytes, defects in mineralization, and a hypocellular red bone marrow. Computerized tomography analysis demonstrated impaired skeletal geometry and altered bone structure. The skeletal defects, which resemble the clinical features reported for bone disease in HGPS patients, was associated with an abnormal osteoblast differentiation. The osteoblast-specific expression of the HGPS mutation increased DNA damage and affected Wnt signaling. In the teeth, irregular dentin formation, as was previously demonstrated in human progeria cases, caused severe dental abnormalities affecting the incisors. The observed phenotype also shows similarities to reported bone abnormalities in aging mice and may therefore help to uncover general principles of the aging process.

  7. Hutchinson-Gilford Progeria Syndrome: A premature aging disease caused by LMNA gene mutations.

    PubMed

    Gonzalo, Susana; Kreienkamp, Ray; Askjaer, Peter

    2017-01-01

    Products of the LMNA gene, primarily lamin A and C, are key components of the nuclear lamina, a proteinaceous meshwork that underlies the inner nuclear membrane and is essential for proper nuclear architecture. Alterations in lamin A and C that disrupt the integrity of the nuclear lamina affect a whole repertoire of nuclear functions, causing cellular decline. In humans, hundreds of mutations in the LMNA gene have been identified and correlated with over a dozen degenerative disorders, referred to as laminopathies. These diseases include neuropathies, muscular dystrophies, lipodystrophies, and premature aging diseases. This review focuses on one of the most severe laminopathies, Hutchinson-Gilford Progeria Syndrome (HGPS), which is caused by aberrant splicing of the LMNA gene and expression of a mutant product called progerin. Here, we discuss current views about the molecular mechanisms that contribute to the pathophysiology of this devastating disease, as well as the strategies being tested in vitro and in vivo to counteract progerin toxicity. In particular, progerin accumulation elicits nuclear morphological abnormalities, misregulated gene expression, defects in DNA repair, telomere shortening, and genomic instability, all of which limit cellular proliferative capacity. In patients harboring this mutation, a severe premature aging disease develops during childhood. Interestingly, progerin is also produced in senescent cells and cells from old individuals, suggesting that progerin accumulation might be a factor in physiological aging. Deciphering the molecular mechanisms whereby progerin expression leads to HGPS is an emergent area of research, which could bring us closer to understanding the pathology of aging.

  8. Mechanics in human fibroblasts and progeria: Lamin A mutation E145K results in stiffening of nuclei.

    PubMed

    Apte, Ketaki; Stick, Reimer; Radmacher, Manfred

    2017-02-01

    The lamina is a filamentous meshwork beneath the inner nuclear membrane that confers mechanical stability to nuclei. The E145K mutation in lamin A causes Hutchinson-Gilford progeria syndrome (HGPS). It affects lamin filament assembly and induces profound changes in the nuclear architecture. Expression of wild-type and E145K lamin A in Xenopus oocytes followed by atomic force microscopy (AFM) probing of isolated oocyte nuclei has shown significant changes in the mechanical properties of the lamina. Nuclei of oocytes expressing E145K lamin A are stiffer than those expressing wild-type lamin A. Here we present mechanical measurements by AFM on dermal fibroblasts obtained from a 4-year-old progeria patient bearing the E145K lamin A mutation and compared it to fibroblasts obtained from 2 healthy donors of 10 and 61 years of age, respectively. The abnormal shape of nuclei expressing E145K lamin A was analyzed by fluorescence microscopy. Lamina thickness was measured using electron micrographs. Fluorescence microscopy showed alterations in the actin network of progeria cells. AFM probing of whole dermal fibroblasts did not demonstrate significant differences in the elastic moduli of nuclear and cytoplasmic cell regions. In contrast, AFM measurements of isolated nuclei showed that nuclei of progeria and old person's cells are significantly stiffer than those of the young person, indicating that the process of aging, be it natural or abnormal, increases nuclear stiffness. Our results corroborate AFM data obtained using Xenopus oocyte nuclei and prove that the presence of E145K lamin A abnormally increases nuclear stiffness.

  9. Protein farnesyltransferase inhibitors and progeria.

    PubMed

    Meta, Margarita; Yang, Shao H; Bergo, Martin O; Fong, Loren G; Young, Stephen G

    2006-10-01

    Genetic mutations that lead to an accumulation of farnesyl-prelamin A cause progeroid syndromes, including Hutchinson-Gilford progeria syndrome. It seemed possible that the farnesylated form of prelamin A might be toxic to mammalian cells, accounting for all the disease phenotypes that are characteristic of progeria. This concept led to the hypothesis that protein farnesyltransferase inhibitors (FTIs) might ameliorate the disease phenotypes of progeria in mouse models. Thus far, two different mouse models of progeria have been examined. In both models, FTIs improved progeria-like disease phenotypes. Here, prelamin A post-translational processing is discussed and several mutations underlying human progeroid syndromes are described. In addition, recent data showing that FTIs ameliorate disease phenotypes in a pair of mouse models of progeria are discussed.

  10. Néstor-Guillermo progeria syndrome: a novel premature aging condition with early onset and chronic development caused by BANF1 mutations.

    PubMed

    Cabanillas, Rubén; Cadiñanos, Juan; Villameytide, José A F; Pérez, Mercedes; Longo, Jesús; Richard, José M; Alvarez, Rebeca; Durán, Noelia S; Illán, Rafael; González, Daniel J; López-Otín, Carlos

    2011-11-01

    Progeria syndromes are rare disorders that involve premature aging. Mutations in BANF1 have been recently reported to cause a new hereditary progeroid syndrome that we now propose to call the Néstor-Guillermo progeria syndrome (NGPS). We describe herein the clinical features of the first two NGPS patients, who phenocopy features of classic progerias (i.e., Hutchinson-Gilford progeria syndrome or mandibuloacral dysplasia), such as aged appearance, growth retardation, decreased subcutaneous fat, thin limbs, and stiff joints. However, these NGPS patients have a distinctive phenotype. In their early adulthood (32 and 24 years of age), they have no signs of cardiovascular impairment, diabetes mellitus, or hypertriglyceridemia. In contrast, they suffer profound skeletal abnormalities that affect their quality of life. The observed differences are of utmost importance to patients and their families and palliation of osseous manifestations is a priority, given their relatively long lifespan. We define NGPS as a chronic progeria because of its slow clinical course and relatively long survival, despite its early onset. Understanding the differences between progeria syndromes might contribute to the development of treatment strategies for common skeletal conditions, as well as aging itself.

  11. p.Pro4Arg mutation in LMNA gene: a new atypical progeria phenotype without metabolism abnormalities.

    PubMed

    Guo, Hong; Luo, Na; Hao, Fei; Bai, Yun

    2014-08-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a typical presenile disorder, with mutation in the LMNA gene. Besides HGPS, mutations in LMNA gene have also been reported in atypical progeroid syndrome (APS). The objective of the study was to investigate the phenotype and molecular basis of APS in a Chinese family. LMNA gene mutations were also reviewed to identify the phenotypic and pathogenic differences among APS. Two siblings in a non-consanguineous Chinese family with atypical progeria were reported. The clinical features were observed, including presenile manifestations such as bird-like facial appearance, generalized lipodystrophy involving the extremities and mottled hyperpigmentation on the trunk and extremities. A heterozygous mutation c.11C>G (p.Pro4Arg) of the LMNA gene was detected in the two patients. 28 different variants of the LMNA gene have been reported in APS families, spreading over almost all the 12 exons of the LMNA gene with some hot-spot regions. This is the first detailed description of an APS family without metabolism abnormalities. APS patients share most of the clinical features, but there may be some distinct features in different ethnic groups.

  12. Drug screening on Hutchinson Gilford progeria pluripotent stem cells reveals aminopyrimidines as new modulators of farnesylation.

    PubMed

    Blondel, S; Egesipe, A-L; Picardi, P; Jaskowiak, A-L; Notarnicola, M; Ragot, J; Tournois, J; Le Corf, A; Brinon, B; Poydenot, P; Georges, P; Navarro, C; Pitrez, P R; Ferreira, L; Bollot, G; Bauvais, C; Laustriat, D; Mejat, A; De Sandre-Giovannoli, A; Levy, N; Bifulco, M; Peschanski, M; Nissan, X

    2016-02-18

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by a dramatic appearance of premature aging. HGPS is due to a single-base substitution in exon 11 of the LMNA gene (c.1824C>T) leading to the production of a toxic form of the prelamin A protein called progerin. Because farnesylation process had been shown to control progerin toxicity, in this study we have developed a screening method permitting to identify new pharmacological inhibitors of farnesylation. For this, we have used the unique potential of pluripotent stem cells to have access to an unlimited and relevant biological resource and test 21,608 small molecules. This study identified several compounds, called monoaminopyrimidines, which target two key enzymes of the farnesylation process, farnesyl pyrophosphate synthase and farnesyl transferase, and rescue in vitro phenotypes associated with HGPS. Our results opens up new therapeutic possibilities for the treatment of HGPS by identifying a new family of protein farnesylation inhibitors, and which may also be applicable to cancers and diseases associated with mutations that involve farnesylated proteins.

  13. Progeria: a rare genetic premature ageing disorder.

    PubMed

    Sinha, Jitendra Kumar; Ghosh, Shampa; Raghunath, Manchala

    2014-05-01

    Progeria is characterized by clinical features that mimic premature ageing. Although the mutation responsible for this syndrome has been deciphered, the mechanism of its action remains elusive. Progeria research has gained momentum particularly in the last two decades because of the possibility of revealing evidences about the ageing process in normal and other pathophysiological conditions. Various experimental models, both in vivo and in vitro, have been developed in an effort to understand the cellular and molecular basis of a number of clinically heterogeneous rare genetic disorders that come under the umbrella of progeroid syndromes (PSs). As per the latest clinical trial reports, Lonafarnib, a farnesyltranferase inhibitor, is a potent 'drug of hope' for Hutchinson-Gilford progeria syndrome (HGPS) and has been successful in facilitating weight gain and improving cardiovascular and skeletal pathologies in progeroid children. This can be considered as the dawn of a new era in progeria research and thus, an apt time to review the research developments in this area highlighting the molecular aspects, experimental models, promising drugs in trial and their implications to gain a better understanding of PSs.

  14. Progeria Research Foundation, Inc.

    MedlinePlus

    About Progeria Progeria 101/FAQ The Connection to Other Diseases The Science Behind Progeria The FTI Drug Lonafarnib For School Reports About ... Profile Our Brochure and Logo Quick Facts Chapters Progeria en espanol Meet the Kids Life According to ...

  15. Progeria, rapamycin and normal aging: recent breakthrough.

    PubMed

    Blagosklonny, Mikhail V

    2011-07-01

    A recent discovery that rapamycin suppresses a pro-senescent phenotype in progeric cells not only suggests a non-toxic therapy for progeria but also implies its similarity with normal aging. For one, rapamycin is also known to suppress aging of regular human cells. Here I discuss four potential scenarios, comparing progeria with both normal and accelerated aging. This reveals further indications of rapamycin both for accelerated aging in obese and for progeria.

  16. Targeting protein prenylation in progeria.

    PubMed

    Young, Stephen G; Yang, Shao H; Davies, Brandon S J; Jung, Hea-Jin; Fong, Loren G

    2013-02-06

    A clinical trial of a protein farnesyltransferase inhibitor (lonafarnib) for the treatment of Hutchinson-Gilford progeria syndrome (HGPS) was recently completed. Here, we discuss the mutation that causes HGPS, the rationale for inhibiting protein farnesyltransferase, the potential limitations of this therapeutic approach, and new potential strategies for treating the disease.

  17. Prelamin A causes progeria through cell-extrinsic mechanisms and prevents cancer invasion

    PubMed Central

    de la Rosa, Jorge; Freije, José M. P.; Cabanillas, Rubén; Osorio, Fernando G.; Fraga, Mario F.; Fernández-García, M. Soledad; Rad, Roland; Fanjul, Víctor; Ugalde, Alejandro P.; Liang, Qi; Prosser, Haydn M.; Bradley, Allan; Cadiñanos, Juan; López-Otín, Carlos

    2013-01-01

    Defining the relationship between ageing and cancer is a crucial but challenging task. Mice deficient in Zmpste24, a metalloproteinase mutated in human progeria and involved in nuclear prelamin A maturation, recapitulate multiple features of ageing. However, their short lifespan and serious cell-intrinsic and cell-extrinsic alterations restrict the application and interpretation of carcinogenesis protocols. Here we present Zmpste24 mosaic mice that lack these limitations. Zmpste24 mosaic mice develop normally and keep similar proportions of Zmpste24-deficient (prelamin A accumulating) and Zmpste24-proficient (mature lamin A containing) cells throughout life, revealing that cell-extrinsic mechanisms are preeminent for progeria development. Moreover, prelamin A accumulation does not impair tumour initiation and growth, but it decreases the incidence of infiltrating oral carcinomas. Accordingly, silencing of ZMPSTE24 reduces human cancer cell invasiveness. Our results support the potential of cell-based and systemic therapies for progeria and highlight ZMPSTE24 as a new anticancer target. PMID:23917225

  18. Prelamin A causes progeria through cell-extrinsic mechanisms and prevents cancer invasion.

    PubMed

    de la Rosa, Jorge; Freije, José M P; Cabanillas, Rubén; Osorio, Fernando G; Fraga, Mario F; Fernández-García, M Soledad; Rad, Roland; Fanjul, Víctor; Ugalde, Alejandro P; Liang, Qi; Prosser, Haydn M; Bradley, Allan; Cadiñanos, Juan; López-Otín, Carlos

    2013-01-01

    Defining the relationship between ageing and cancer is a crucial but challenging task. Mice deficient in Zmpste24, a metalloproteinase mutated in human progeria and involved in nuclear prelamin A maturation, recapitulate multiple features of ageing. However, their short lifespan and serious cell-intrinsic and cell-extrinsic alterations restrict the application and interpretation of carcinogenesis protocols. Here we present Zmpste24 mosaic mice that lack these limitations. Zmpste24 mosaic mice develop normally and keep similar proportions of Zmpste24-deficient (prelamin A-accumulating) and Zmpste24-proficient (mature lamin A-containing) cells throughout life, revealing that cell-extrinsic mechanisms are preeminent for progeria development. Moreover, prelamin A accumulation does not impair tumour initiation and growth, but it decreases the incidence of infiltrating oral carcinomas. Accordingly, silencing of ZMPSTE24 reduces human cancer cell invasiveness. Our results support the potential of cell-based and systemic therapies for progeria and highlight ZMPSTE24 as a new anticancer target.

  19. Reprogramming aging and progeria.

    PubMed

    Freije, José M P; López-Otín, Carlos

    2012-12-01

    The aging rate of an organism depends on the ratio of tissue degeneration to tissue repair. As a consequence, molecular alterations that tip this balance toward degeneration cause accelerated aging. Conversely, interventions can be pursued to reduce tissue degeneration or to increase tissue repair with the aim of delaying the onset of age-associated manifestations. Recent studies on the biology of stem cells in aging have revealed the influence of systemic factors on their functionality and demonstrated the feasibility of reprogramming aged and progeroid cells. These results illustrate the reversibility of some aspects of the aging process and encourage the search for new anti-aging and anti-progeria interventions.

  20. Transgene silencing of the Hutchinson-Gilford progeria syndrome mutation results in a reversible bone phenotype, whereas resveratrol treatment does not show overall beneficial effects.

    PubMed

    Strandgren, Charlotte; Nasser, Hasina Abdul; McKenna, Tomás; Koskela, Antti; Tuukkanen, Juha; Ohlsson, Claes; Rozell, Björn; Eriksson, Maria

    2015-08-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder that is most commonly caused by a de novo point mutation in exon 11 of the LMNA gene, c.1824C>T, which results in an increased production of a truncated form of lamin A known as progerin. In this study, we used a mouse model to study the possibility of recovering from HGPS bone disease upon silencing of the HGPS mutation, and the potential benefits from treatment with resveratrol. We show that complete silencing of the transgenic expression of progerin normalized bone morphology and mineralization already after 7 weeks. The improvements included lower frequencies of rib fractures and callus formation, an increased number of osteocytes in remodeled bone, and normalized dentinogenesis. The beneficial effects from resveratrol treatment were less significant and to a large extent similar to mice treated with sucrose alone. However, the reversal of the dental phenotype of overgrown and laterally displaced lower incisors in HGPS mice could be attributed to resveratrol. Our results indicate that the HGPS bone defects were reversible upon suppressed transgenic expression and suggest that treatments targeting aberrant progerin splicing give hope to patients who are affected by HGPS.

  1. A homozygous ZMPSTE24 null mutation in combination with a heterozygous mutation in the LMNA gene causes Hutchinson-Gilford progeria syndrome (HGPS): insights into the pathophysiology of HGPS.

    PubMed

    Denecke, Jonas; Brune, Thomas; Feldhaus, Tobias; Robenek, Horst; Kranz, Christian; Auchus, Richard J; Agarwal, Anil K; Marquardt, Thorsten

    2006-06-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder normally caused by a spontaneous heterozygous mutation in the LMNA gene that codes for the nuclear lamina protein lamin A. Several enzymes are involved in the processing of its precursor, prelamin A, to the mature lamin A. A functional knockout of one of the enzymes involved in prelamin A processing, the zinc metalloprotease ZMPSTE24, causes an even more severe disorder with early neonatal death described as restrictive dermatopathy (RD). This work describes a HGPS patient with a combined defect of a homozygous loss-of-function mutation in the ZMPSTE24 gene and a heterozygous mutation in the LMNA gene that results in a C-terminal elongation of the final lamin A. Whereas the loss of function mutation of ZMPSTE24 normally results in lethal RD, the truncation of LMNA seems to be a salvage alteration alleviating the clinical picture to the HGPS phenotype. The mutations of our patient indicate that farnesylated prelamin A is the deleterious agent leading to the HGPS phenotype, which gives further insights into the pathophysiology of the disorder.

  2. A human iPSC model of Hutchinson Gilford Progeria reveals vascular smooth muscle and mesenchymal stem cell defects.

    PubMed

    Zhang, Jinqiu; Lian, Qizhou; Zhu, Guili; Zhou, Fan; Sui, Lin; Tan, Cindy; Mutalif, Rafidah Abdul; Navasankari, Raju; Zhang, Yuelin; Tse, Hung-Fat; Stewart, Colin L; Colman, Alan

    2011-01-07

    The segmental premature aging disease Hutchinson-Gilford Progeria syndrome (HGPS) is caused by a truncated and farnesylated form of Lamin A called progerin. HGPS affects mesenchymal lineages, including the skeletal system, dermis, and vascular smooth muscle (VSMC). To understand the underlying molecular pathology of HGPS, we derived induced pluripotent stem cells (iPSCs) from HGPS dermal fibroblasts. The iPSCs were differentiated into neural progenitors, endothelial cells, fibroblasts, VSMCs, and mesenchymal stem cells (MSCs). Progerin levels were highest in MSCs, VSMCs, and fibroblasts, in that order, with these lineages displaying increased DNA damage, nuclear abnormalities, and HGPS-VSMC accumulating numerous calponin-staining inclusion bodies. Both HGPS-MSC and -VSMC viability was compromised by stress and hypoxia in vitro and in vivo (MSC). Because MSCs reside in low oxygen niches in vivo, we propose that, in HGPS, this causes additional depletion of the MSC pool responsible for replacing differentiated cells lost to progerin toxicity.

  3. Learning about Progeria

    MedlinePlus

    ... 2008 Inhibiting farnesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson-Gilford progeria syndrome Proceedings of ... That abnormal protein appears to destabilize the cell's nuclear membrane in a way that may be particularly ...

  4. Progeria 101/FAQ

    MedlinePlus

    ... Interview with John Tacket Find the Other 150 Medical Research NEW! Lonafarnib Pre-clinical Drug Supply Program What's ... Progeria gene is a major achievement for the medical research community,” said Dr. Francis Collins, Director of the ...

  5. Effect of progerin on the accumulation of oxidized proteins in fibroblasts from Hutchinson Gilford progeria patients.

    PubMed

    Viteri, Gabriela; Chung, Youn Wook; Stadtman, Earl R

    2010-01-01

    The mutation responsible for Hutchinson Gilford Progeria Syndrome (HGPS) causes abnormal nuclear morphology. Previous studies show that free radicals and reactive oxygen species play major roles in the etiology and/or progression of neurodegenerative diseases and aging. This study compares oxidative stress responses between progeric and normal fibroblasts. Our data revealed higher ROS levels in HGPS cells compared to age-matched controls. In response to oxidative challenge, progeric cells showed increased mRNA levels for mitochondrial superoxide dismutase (SOD) and SOD protein content. However, this did not prevent a drop in the ATP content of progeria fibroblasts. Previous studies have shown that declines in human fibroblast ATP levels interfere with programmed cell death and promote necrotic inflammation. Notably, in our investigations the ATP content of progeria fibroblasts was only approximately 50% of that found in healthy controls. Furthermore, HGPS fibroblast analysis revealed a decrease in total caspase-like proteasome activity and in the levels of two active proteolytic complex subunits (beta(5) and beta(7)). A number of studies indicate that the molecular mechanisms causing accelerated aging in progeric patients also occur in healthy cells of older individuals. Thus, the results of this study may also help explain some of the cellular changes that accompany normal aging.

  6. Neonatal progeria: increased ratio of progerin to lamin A leads to progeria of the newborn.

    PubMed

    Reunert, Janine; Wentzell, Rüdiger; Walter, Michael; Jakubiczka, Sibylle; Zenker, Martin; Brune, Thomas; Rust, Stephan; Marquardt, Thorsten

    2012-09-01

    Hutchinson-Gilford progeria syndrome (HGPS) is an important model disease for premature ageing. Affected children appear healthy at birth, but develop the first symptoms during their first year of life. They die at an average age of 13 years, mostly because of myocardial infarction or stroke. Classical progeria is caused by the heterozygous point mutation c.1824C>T in the LMNA gene, which activates a cryptic splice site. The affected protein cannot be processed correctly to mature lamin A, but is modified into a farnesylated protein truncated by 50 amino acids (progerin). Three more variations in LMNA result in the same mutant protein, but different grades of disease severity. We describe a patient with the heterozygous LMNA mutation c.1821G>A, leading to neonatal progeria with death in the first year of life. Intracellular lamin A was downregulated in the patient's fibroblasts and the ratio of progerin to lamin A was increased when compared with HGPS. It is suggestive that the ratio of farnesylated protein to mature lamin A determines the disease severity in progeria.

  7. LMNA-associated cardiocutaneous progeria: an inherited autosomal dominant premature aging syndrome with late onset.

    PubMed

    Kane, Megan S; Lindsay, Mark E; Judge, Daniel P; Barrowman, Jemima; Ap Rhys, Colette; Simonson, Lisa; Dietz, Harry C; Michaelis, Susan

    2013-07-01

    Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disorder caused by mutations in LMNA, which encodes the nuclear scaffold proteins lamin A and C. In HGPS and related progerias, processing of prelamin A is blocked at a critical step mediated by the zinc metalloprotease ZMPSTE24. LMNA-linked progerias can be grouped into two classes: (1) the processing-deficient, early onset "typical" progerias (e.g., HGPS), and (2) the processing-proficient "atypical" progeria syndromes (APS) that are later in onset. Here we describe a previously unrecognized progeria syndrome with prominent cutaneous and cardiovascular manifestations belonging to the second class. We suggest the name LMNA-associated cardiocutaneous progeria syndrome (LCPS) for this disorder. Affected patients are normal at birth but undergo progressive cutaneous changes in childhood and die in middle age of cardiovascular complications, including accelerated atherosclerosis, calcific valve disease, and cardiomyopathy. In addition, the proband demonstrated cancer susceptibility, a phenotype rarely described for LMNA-based progeria disorders. The LMNA mutation that caused LCPS in this family is a heterozygous c.899A>G (p.D300G) mutation predicted to alter the coiled-coil domain of lamin A/C. In skin fibroblasts isolated from the proband, the processing and levels of lamin A and C are normal. However, nuclear morphology is aberrant and rescued by treatment with farnesyltransferase inhibitors, as is also the case for HGPS and other laminopathies. Our findings advance knowledge of human LMNA progeria syndromes, and raise the possibility that typical and atypical progerias may converge upon a common mechanism to cause premature aging disease.

  8. Labor Market Progeria.

    ERIC Educational Resources Information Center

    Rodeheaver, Dean

    1990-01-01

    Social ambivalence toward women's roles, sexuality, appearance, and aging combine with social standards of attractiveness to create both age and sex discrimination in the workplace. The life expectancy of presentability is shorter among women than men, thus creating an accelerated aging process termed labor market progeria. (SK)

  9. Progeria Research Day at Brunel University.

    PubMed

    Bridger, Joanna M; Eskiw, Christopher H; Makarov, Evgeny M; Tree, David; Kill, Ian R

    2011-01-01

    Hutchinson-Gilford Progeria Syndrome (HGPS) is a severe premature aging syndrome that affects children. These children display characteristics associated with normal aging and die young usually from cardiovascular problems or stroke. Classical HGPS is caused by mutations in the gene encoding the nuclear structural protein lamin A. This mutation leads to a novel version of lamin A that retains a farnesyl group from its processing. This protein is called Progerin and is toxic to cellular function. Pre-lamin A is an immature version of lamin A and also has a farnesylation modification, which is cleaved in the maturation process to create lamin A.

  10. Hutchinson-Gilford progeria syndrome.

    PubMed

    Ullrich, Nicole J; Gordon, Leslie B

    2015-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare, uniformly fatal, segmental "premature aging" disease in which children exhibit phenotypes that may give us insights into the aging process at both the cellular and organismal levels. Initial presentation in early childhood is primarily based on growth and dermatologic findings. Primary morbidity and mortality for children with HGPS is from atherosclerotic cardiovascular disease and strokes with death occurring at an average age of 14.6 years. There is increasing data to support a unique phenotype of the craniofacial and cerebrovascular anatomy that accompanies the premature aging process. Strokes in HGPS can occur downstream of carotid artery and/or vertebral artery occlusion, stenosis, and calcification, with prominent collateral vessel formation. Both large and small vessel disease are present, and strokes are often clinically silent. Despite the presence of multisystem premature aging, children with HGPS do not appear to have cognitive deterioration, suggesting that some aspects of brain function may be protected from the deleterious effects of progerin, the disease-causing protein. Based on limited autopsy material, there is no pathologic evidence of dementia or Alzheimer-type changes. In a transgenic mouse model of progeria with expression of the most common HGPS mutation in brain, skin, bone, and heart, there are distortions of neuronal nuclei at the ultrastructural level with irregular shape and severe invaginations, but no evidence of inclusions or aberrant tau in brain sections. Importantly, the nuclear distortions did not result in significant changes in gene expression in hippocampal neurons. This chapter will discuss both preclinical and clinical aspects of the genetics, pathobiology, clinical phenotype, clinical care, and treatment of HGPS, with special attention toward neurologic and cutaneous findings.

  11. Progeria, the nucleolus and farnesyltransferase inhibitors.

    PubMed

    Mehta, Ishita S; Bridger, Joanna M; Kill, Ian R

    2010-02-01

    HGPS (Hutchinson-Gilford progeria syndrome) is a rare genetic disease affecting children causing them to age and die prematurely. The disease is typically due to a point mutation in the coding sequence for the nuclear intermediate-type filament protein lamin A and gives rise to a dominant-negative splice variant named progerin. Accumulation of progerin within nuclei causes disruption to nuclear structure, causes and premature replicative senescence and increases apoptosis. Now it appears that accumulation of progerin may have more widespread effects than previously thought since the demonstration that the presence and distribution of some nucleolar proteins are also adversely affected in progeria cells. One of the major breakthroughs both in the lamin field and for this syndrome is that many of the cellular defects observed in HGPS patient cells and model systems can be restored after treatment with a class of compounds known as FTIs (farnesyltransferase inhibitors). Indeed, it is demonstrated that FTI-277 is able to completely restore nucleolar antigen localization in treated progeria cells. This is encouraging news for the HGPS patients who are currently undergoing clinical trials with FTI treatment.

  12. Mutational profiling reveals PIK3CA mutations in gallbladder carcinoma

    PubMed Central

    2011-01-01

    Background The genetics of advanced biliary tract cancers (BTC), which encompass intra- and extra-hepatic cholangiocarcinomas as well as gallbladder carcinomas, are heterogeneous and remain to be fully defined. Methods To better characterize mutations in established known oncogenes and tumor suppressor genes we tested a mass spectrometric based platform to interrogate common cancer associated mutations across a panel of 77 formalin fixed paraffin embedded archived BTC cases. Results Mutations among three genes, KRAS, NRAS and PIK3CA were confirmed in this cohort. Activating mutations in PIK3CA were identified exclusively in GBC (4/32, 12.5%). KRAS mutations were identified in 3 (13%) intra-hepatic cholangiocarcinomas and 1 (33%) perihillar cholangiocarcinoma but were not identified in gallbladder carcinomas and extra-hepatic cholangiocarcinoma. Conclusions The presence of activating mutations in PIK3CA specifically in GBC has clinical implications in both the diagnosis of this cancer type, as well as the potential utility of targeted therapies such as PI3 kinase inhibitors. PMID:21303542

  13. Stem cell depletion in Hutchinson-Gilford progeria syndrome.

    PubMed

    Rosengardten, Ylva; McKenna, Tomás; Grochová, Diana; Eriksson, Maria

    2011-12-01

    Hutchinson-Gilford progeria syndrome (HGPS or progeria) is a very rare genetic disorder with clinical features suggestive of premature aging. Here, we show that induced expression of the most common HGPS mutation (LMNA c.1824C>T, p.G608G) results in a decreased epidermal population of adult stem cells and impaired wound healing in mice. Isolation and growth of primary keratinocytes from these mice demonstrated a reduced proliferative potential and ability to form colonies. Downregulation of the epidermal stem cell maintenance protein p63 with accompanying activation of DNA repair and premature senescence was the probable cause of this loss of adult stem cells. Additionally, upregulation of multiple genes in major inflammatory pathways indicated an activated inflammatory response. This response has also been associated with normal aging, emphasizing the importance of studying progeria to increase the understanding of the normal aging process.

  14. Progeria and the early aging in children: a case report.

    PubMed

    Carvalho, Vania O; Celli, Adriane; Bancke Laverde, Bruno Leonardo; Cunico, Caroline; Santos Piedade, Guilherme; Lucas de Mello, Manuela; Beirao Junior, Paulo Sergio

    2016-02-17

    The Hutchinson-Gilford syndrome or progeria is a rare autosomal dominant syndrome characterized by premature aging and involvement of internal systems, such as the circulatory and locomotor. The diagnosis is essentially clinical and the manifestations become more evident from the first year of life. Long term outcome data from Progeria Research Foundation clinical trials have demonstrated an increase in survival in recent years. Even though new trials are ongoing, the recognition of this syndrome is essential to prevent cardiovascular and cerebrovascular complications. A patient, initially asymptomatic, who developed characteristic signs of the syndrome at the age of 6 months is reported. She was referred for evaluation only when she was two years and eleven months old. The diagnosis of Hutchinson-Gilford syndrome was suspected owing to clinical characteristics. The diagnosis was confirmed by genetic testing. A mutation c.1824C> T in exon 11 of the LMNA gene was detected. She was registered in the Progeria Research Foundation and was invited to participate in the weighing and supplementation program. She was included in the lonafarnib protocol study. This medication is a farnesyl transferase inhibitor that prevents the production of progerina and slows cardiovascular and neurological complications of the syndrome. This case highlights the importance of diagnosing progeria patients because they may be referred to the Progeria Research Foundation, which offers genetic screening and inclusion in clinical and therapeutic follow-up protocols without any costs. Progeria trials and research may also contribute to new drug developments related to prevention of aging and atherosclerosis in the near future.

  15. Hutchinson-Gilford progeria syndrome.

    PubMed

    Agarwal, Uma Shankar; Sitaraman, S; Mehta, Sharad; Panse, Gauri

    2010-01-01

    Progeria is a rare genetic disorder characterized by premature aging, involving the skin, bones, heart, and blood vessels. We report a 4-year-old boy who presented with clinical manifestations of progeria. He had characteristic facies, prominent eyes, scalp and leg veins, senile look, loss of scalp hair, eyebrows and eyelashes, stunted growth, and sclerodermatous changes. The present case is reported due to its rarity.

  16. Genome-Wide Scan Reveals Mutation Associated with Melanoma

    MedlinePlus

    ... historical) Genome-Wide Scan Reveals Mutation Associated with Melanoma A team of international researchers supported by the ... when they divide and grow uncontrollably, develop into melanoma. Also, MITF activity is known to be amplified ...

  17. Cell autonomous and systemic factors in progeria development.

    PubMed

    Osorio, Fernando G; Ugalde, Alejandro P; Mariño, Guillermo; Puente, Xose S; Freije, José M P; López-Otín, Carlos

    2011-12-01

    Progeroid laminopathies are accelerated aging syndromes caused by defects in nuclear envelope proteins. Accordingly, mutations in the LMNA gene and functionally related genes have been described to cause HGPS (Hutchinson-Gilford progeria syndrome), MAD (mandibuloacral dysplasia) or RD (restrictive dermopathy). Functional studies with animal and cellular models of these syndromes have facilitated the identification of the molecular alterations and regulatory pathways involved in progeria development. We have recently described a novel regulatory pathway involving miR-29 and p53 tumour suppressor which has provided valuable information on the molecular components orchestrating the response to nuclear damage stress. Furthermore, by using progeroid mice deficient in ZMPSTE24 (zinc metalloprotease STE24 homologue) involved in lamin A maturation, we have demonstrated that, besides these abnormal cellular responses to stress, dysregulation of the somatotropic axis is responsible for some of the alterations associated with progeria. Consistent with these observations, pharmacological restoration of the somatotroph axis in these mice delays the onset of their progeroid features, significantly extending their lifespan and supporting the importance of systemic alterations in progeria progression. Finally, we have very recently identified a novel progeroid syndrome with distinctive features from HGPS and MAD, which we have designated NGPS (Néstor-Guillermo progeria syndrome) (OMIM #614008). This disorder is caused by a mutation in BANF1, a gene encoding a protein with essential functions in the assembly of the nuclear envelope, further illustrating the importance of the nuclear lamina integrity for human health and providing additional support to the study of progeroid syndromes as a valuable source of information on human aging.

  18. Genetics of aging, progeria and lamin disorders.

    PubMed

    Ghosh, Shrestha; Zhou, Zhongjun

    2014-06-01

    Premature aging disorders, like Werner syndrome, Bloom's syndrome, and Hutchinson-Gilford Progeria Syndrome (HGPS), have been the subjects of immense interest as they recapitulate many of the phenotypes observed in physiological aging. They, therefore, not only provide model systems to study normal aging processes but also give valuable insights into the intricate mechanisms underlying senescence. Recent works on HGPS have revealed alterations in a spectrum of cellular and molecular pathways involved in the maintenance of genomic integrity, thus suggesting a profound impact of the nuclear lamina in nuclear organization, chromatin dynamics, regulation of gene expression and epigenetics.

  19. Ocular manifestations in the Hutchinson-Gilford progeria syndrome.

    PubMed

    Chandravanshi, Shivcharan L; Rawat, Ashok Kumar; Dwivedi, Prem Chand; Choudhary, Pankaj

    2011-01-01

    The Hutchinson-Gilford progeria (HGP) syndrome is an extremely rare genetic condition characterized by an appearance of accelerated aging in children. The word progeria is derived from the Greek word progeros meaning 'prematurely old'. It is caused by de novo dominant mutation in the LMNA gene (gene map locus 1q21.2) and characterized by growth retardation and accelerated degenerative changes of the skin, musculoskeletal and cardiovascular systems. The most common ocular manifestations are prominent eyes, loss of eyebrows and eyelashes, and lagophthalmos. In the present case some additional ocular features such as horizontal narrowing of palpebral fissure, superior sulcus deformity, upper lid retraction, upper lid lag in down gaze, poor pupillary dilatation, were noted. In this case report, a 15-year-old Indian boy with some additional ocular manifestations of the HGP syndrome is described.

  20. [Three cases of Hutchinson-Gilford progeria syndrome].

    PubMed

    Doubaj, Y; Lamzouri, A; Elalaoui, S-C; Laarabi, F-Z; Sefiani, A

    2011-02-01

    Progeria, or Hutchinson-Gilford syndrome, is a rare genetic disease, characterized by several clinical features that develop in childhood, in particular, an accelerated aging aspect. Its incidence is 1-4 per 8 million newborns. Children with progeria syndrome usually appear normal at birth and in early infancy. Profound failure to thrive occurs during the 1st year. Characteristic facies, partial alopecia progressing to total alopecia, loss of subcutaneous fat, stiffness of joints, bone changes, and abnormal tightness of the skin over the abdomen and upper thighs usually become apparent during the 2nd to 3rd years. Motor and mental development is normal. Patients develop severe atherosclerosis. Death occurs as a result of complications of cardiac or cerebrovascular disease (heart attack or stroke) generally between ages 6 and 20 years. The diagnosis of Hutchinson-Gilford progeria syndrome (HGPS) is based on recognition of common clinical features and the detection of the recurrent p.Gly608Gly mutation in exon 11 of the LMNA gene, which is present in almost all individuals with HGPS. We present here 3 patients aged 5, 11, and 12 years referred to genetic consultation for dysmorphic facies and failure to thrive. After careful clinical examination and paraclinical assessment, the diagnosis of progeria syndrome was raised. We performed molecular analysis for the 3 patients by searching for the recurrent mutation c.1824C>T (p.Gly608Gly) of the LMNA gene, which was found only in 1 patient. We discuss the geneticist's role in the diagnosis of rare dysmorphic syndromes and their genetic counseling. We also analyze the clinical spectrum of HGPS by comparing the 3 patients.

  1. Aberrant DNA methylation profiles in the premature aging disorders Hutchinson-Gilford Progeria and Werner syndrome.

    PubMed

    Heyn, Holger; Moran, Sebastian; Esteller, Manel

    2013-01-01

    DNA methylation gradiently changes with age and is likely to be involved in aging-related processes with subsequent phenotype changes and increased susceptibility to certain diseases. The Hutchinson-Gilford Progeria (HGP) and Werner Syndrome (WS) are two premature aging diseases showing features of common natural aging early in life. Mutations in the LMNA and WRN genes were associated to disease onset; however, for a subset of patients the underlying causative mechanisms remain elusive. We aimed to evaluate the role of epigenetic alteration on premature aging diseases by performing comprehensive DNA methylation profiling of HGP and WS patients. We observed profound changes in the DNA methylation landscapes of WRN and LMNA mutant patients, which were narrowed down to a set of aging related genes and processes. Although of low overall variance, non-mutant patients revealed differential DNA methylation at distinct loci. Hence, we propose DNA methylation to have an impact on premature aging diseases.

  2. A novel homozygous p.Arg527Leu LMNA mutation in two unrelated Egyptian families causes overlapping mandibuloacral dysplasia and progeria syndrome.

    PubMed

    Al-Haggar, Mohammad; Madej-Pilarczyk, Agnieszka; Kozlowski, Lukasz; Bujnicki, Janusz M; Yahia, Sohier; Abdel-Hadi, Dina; Shams, Amany; Ahmad, Nermin; Hamed, Sahar; Puzianowska-Kuznicka, Monika

    2012-11-01

    Mandibuloacral dysplasia (MAD) is a rare disease resulting from a mutation of LMNA gene encoding lamins A and C. The most common mutation associated with this disease is a homozygous arginine 527 replacement by histidine. Three female patients originating from two unrelated families from Northeast Egypt were examined. Their growth was retarded; they had microcephaly, widened cranial sutures, prominent eyes and cheeks, micrognathia, dental crowding, hypoplastic mandible, acro-osteolysis of distal phalanges, and joint contractures. In addition, they presented some progeroid features, such as pinched nose, premature loss of teeth, loss of hair, scleroderma-like skin atrophy, spine rigidity, and waddling gait. The clinical presentation of the disease varied between the patient originating from Family 1 and patients from Family 2, suggesting that unknown, possibly epigenetic factors, modify the course of the disease. The first symptoms of the disease appeared at the age of 2.5 (a girl from Family 1), 5, and 3 years (girls from Family 2). All patients had the same, novel homozygous c.1580G>T LMNA mutation, resulting in the replacement of arginine 527 by leucine. Computational predictions of such substitution effects suggested that it might alter protein stability and increase the tendency for protein aggregation, and as a result, might influence its interaction with other proteins. In addition, restriction fragment-length polymorphism analysis performed in 178 unrelated individuals showed that up to 1.12% of inhabitants of Northeast Egypt might be heterozygous carriers of this mutation, suggesting the presence of a founder effect in this area.

  3. Biomedical Impact of Splicing Mutations Revealed through Exome Sequencing

    PubMed Central

    Taneri, Bahar; Asilmaz, Esra; Gaasterland, Terry

    2012-01-01

    Splicing is a cellular mechanism, which dictates eukaryotic gene expression by removing the noncoding introns and ligating the coding exons in the form of a messenger RNA molecule. Alternative splicing (AS) adds a major level of complexity to this mechanism and thus to the regulation of gene expression. This widespread cellular phenomenon generates multiple messenger RNA isoforms from a single gene, by utilizing alternative splice sites and promoting different exon–intron inclusions and exclusions. AS greatly increases the coding potential of eukaryotic genomes and hence contributes to the diversity of eukaryotic proteomes. Mutations that lead to disruptions of either constitutive splicing or AS cause several diseases, among which are myotonic dystrophy and cystic fibrosis. Aberrant splicing is also well established in cancer states. Identification of rare novel mutations associated with splice-site recognition, and splicing regulation in general, could provide further insight into genetic mechanisms of rare diseases. Here, disease relevance of aberrant splicing is reviewed, and the new methodological approach of starting from disease phenotype, employing exome sequencing and identifying rare mutations affecting splicing regulation is described. Exome sequencing has emerged as a reliable method for finding sequence variations associated with various disease states. To date, genetic studies using exome sequencing to find disease-causing mutations have focused on the discovery of nonsynonymous single nucleotide polymorphisms that alter amino acids or introduce early stop codons, or on the use of exome sequencing as a means to genotype known single nucleotide polymorphisms. The involvement of splicing mutations in inherited diseases has received little attention and thus likely occurs more frequently than currently estimated. Studies of exome sequencing followed by molecular and bioinformatic analyses have great potential to reveal the high impact of splicing

  4. Progeria with post-streptococcal glomerulonephritis: a rare case report with differential diagnosis.

    PubMed

    Sebastian, Alphy A; Ahsan, Auswaf K

    2013-02-01

    Hutchinson-Gilford progeria syndrome is a rare autosomal dominant disorder associated with skin fragility. It is characterized by craniofacial disproportion, delayed dentition, micrognathia, and plucked bird appearance. The genetic defect is mainly de nova mutation in the lamin A gene. This report describes a 16-year-old patient with classical features of progeria along with post-streptococcal glomerulonephritis. The symptoms of hepatomegaly were also present in the patient. The differential diagnoses of this lesion are also discussed in detail in this literature.

  5. Gene-rich chromosomal regions are preferentially localized in the lamin B deficient nuclear blebs of atypical progeria cells.

    PubMed

    Bercht Pfleghaar, Katrin; Taimen, Pekka; Butin-Israeli, Veronika; Shimi, Takeshi; Langer-Freitag, Sabine; Markaki, Yolanda; Goldman, Anne E; Wehnert, Manfred; Goldman, Robert D

    2015-01-01

    More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature aging disorder. The major pathognomonic hallmarks of progeria cells are seen as nuclear deformations or blebs that are related to the redistribution of A- and B-type lamins within the nuclear lamina. However, the functional significance of these progeria-associated blebs remains unknown. We have carried out an analysis of the structural and functional consequences of progeria-associated nuclear blebs in dermal fibroblasts from a progeria patient carrying a rare point mutation p.S143F (C428T) in lamin A/C. These blebs form microdomains that are devoid of major structural components of the nuclear envelope (NE)/lamina including B-type lamins and nuclear pore complexes (NPCs) and are enriched in A-type lamins. Using laser capture microdissection and comparative genomic hybridization (CGH) analyses, we show that, while these domains are devoid of centromeric heterochromatin and gene-poor regions of chromosomes, they are enriched in gene-rich chromosomal regions. The active form of RNA polymerase II is also greatly enriched in blebs as well as nascent RNA but the nuclear co-activator SKIP is significantly reduced in blebs compared to other transcription factors. Our results suggest that the p.S143F progeria mutation has a severe impact not only on the structure of the lamina but also on the organization of interphase chromatin domains and transcription. These structural defects are likely to contribute to gene expression changes reported in progeria and other types of laminopathies.

  6. Gene-rich chromosomal regions are preferentially localized in the lamin B deficient nuclear blebs of atypical progeria cells

    PubMed Central

    Bercht Pfleghaar, Katrin; Taimen, Pekka; Butin-Israeli, Veronika; Shimi, Takeshi; Langer-Freitag, Sabine; Markaki, Yolanda; Goldman, Anne E; Wehnert, Manfred; Goldman, Robert D

    2015-01-01

    More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature aging disorder. The major pathognomonic hallmarks of progeria cells are seen as nuclear deformations or blebs that are related to the redistribution of A- and B-type lamins within the nuclear lamina. However, the functional significance of these progeria-associated blebs remains unknown. We have carried out an analysis of the structural and functional consequences of progeria-associated nuclear blebs in dermal fibroblasts from a progeria patient carrying a rare point mutation p.S143F (C428T) in lamin A/C. These blebs form microdomains that are devoid of major structural components of the nuclear envelope (NE)/lamina including B-type lamins and nuclear pore complexes (NPCs) and are enriched in A-type lamins. Using laser capture microdissection and comparative genomic hybridization (CGH) analyses, we show that, while these domains are devoid of centromeric heterochromatin and gene-poor regions of chromosomes, they are enriched in gene-rich chromosomal regions. The active form of RNA polymerase II is also greatly enriched in blebs as well as nascent RNA but the nuclear co-activator SKIP is significantly reduced in blebs compared to other transcription factors. Our results suggest that the p.S143F progeria mutation has a severe impact not only on the structure of the lamina but also on the organization of interphase chromatin domains and transcription. These structural defects are likely to contribute to gene expression changes reported in progeria and other types of laminopathies. PMID:25738644

  7. Genomic profiling reveals mutational landscape in parathyroid carcinomas

    PubMed Central

    Bellizzi, Justin; Lau, Chun Yee; Moe, Aye S.; Strahl, Maya; Newman, Leah C.; Fink, Marc Y.; Antipin, Yevgeniy; Yu, Willie; Stevenson, Mark; Cavaco, Branca M.; Thakker, Rajesh V.; Morreau, Hans; Schadt, Eric E.; Sebra, Robert; Li, Shuyu D.

    2017-01-01

    Parathyroid carcinoma (PC) is an extremely rare malignancy lacking effective therapeutic intervention. We generated and analyzed whole-exome sequencing data from 17 patients to identify somatic and germline genetic alterations. A panel of selected genes was sequenced in a 7-tumor expansion cohort. We show that 47% (8 of 17) of the tumors harbor somatic mutations in the CDC73 tumor suppressor, with germline inactivating variants in 4 of the 8 patients. The PI3K/AKT/mTOR pathway was altered in 21% of the 24 cases, revealing a major oncogenic pathway in PC. We observed CCND1 amplification in 29% of the 17 patients, and a previously unreported recurrent mutation in putative kinase ADCK1. We identified the first sporadic PCs with somatic mutations in the Wnt canonical pathway, complementing previously described epigenetic mechanisms mediating Wnt activation. This is the largest genomic sequencing study of PC, and represents major progress toward a full molecular characterization of this rare malignancy to inform improved and individualized treatments. PMID:28352668

  8. Molecular ageing in progeroid syndromes: Hutchinson-Gilford progeria syndrome as a model

    PubMed Central

    Coutinho, Henrique Douglas M; Falcão-Silva, Vivyanne S; Gonçalves, Gregório Fernandes; da Nóbrega, Raphael Batista

    2009-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder that belongs to a group of conditions called laminopathies which affect nuclear lamins. Mutations in two genes, LMNA and ZMPSTE24, have been found in patients with HGPS. The p.G608G LMNA mutation is the most commonly reported mutation. The aim of this work was to compile a comprehensive literature review of the clinical features and genetic mutations and mechanisms of this syndrome as a contribution to health care workers. This review shows the necessity of a more detailed clinical identification of Hutchinson-Gilford progeria syndrome and the need for more studies on the pharmacologic and pharmacogenomic approach to this syndrome. PMID:19379495

  9. Pluripotent stem cells to model Hutchinson-Gilford progeria syndrome (HGPS): Current trends and future perspectives for drug discovery.

    PubMed

    Lo Cicero, Alessandra; Nissan, Xavier

    2015-11-01

    Progeria, or Hutchinson-Gilford progeria syndrome (HGPS), is a rare, fatal genetic disease characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations in codon 608 (p.G608G) of the LMNA, leading to the production of a mutated form of lamin A precursor called progerin. In HGPS, progerin accumulates in cells causing progressive molecular defects, including nuclear shape abnormalities, chromatin disorganization, damage to DNA and delays in cell proliferation. Here we report how, over the past five years, pluripotent stem cells have provided new insights into the study of HGPS and opened new original therapeutic perspectives to treat the disease.

  10. Replication Study: Melanoma genome sequencing reveals frequent PREX2 mutations

    PubMed Central

    Horrigan, Stephen K; Courville, Pascal; Sampey, Darryl; Zhou, Faren; Cai, Steve

    2017-01-01

    In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Chroscinski et al., 2014) that described how we intended to replicate selected experiments from the paper "Melanoma genome sequencing reveals frequent PREX2 mutations" (Berger et al., 2012). Here we report the results of those experiments. We regenerated cells stably expressing ectopic wild-type and mutant phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2 (PREX2) using the same immortalized human NRASG12D melanocytes as the original study. Evaluation of PREX2 expression in these newly generated stable cells revealed varying levels of expression among the PREX2 isoforms, which was also observed in the stable cells made in the original study (Figure S6A; Berger et al., 2012). Additionally, ectopically expressed PREX2 was found to be at least 5 times above endogenous PREX2 expression. The monitoring of tumor formation of these stable cells in vivo resulted in no statistically significant difference in tumor-free survival driven by PREX2 variants, whereas the original study reported that these PREX2 mutations increased the rate of tumor incidence compared to controls (Figure 3B and S6B; Berger et al., 2012). Surprisingly, the median tumor-free survival was 1 week in this replication attempt, while 70% of the control mice were reported to be tumor-free after 9 weeks in the original study. The rapid tumor onset observed in this replication attempt, compared to the original study, makes the detection of accelerated tumor growth in PREX2 expressing NRASG12D melanocytes extremely difficult. Finally, we report meta-analyses for each result. DOI: http://dx.doi.org/10.7554/eLife.21634.001 PMID:28100394

  11. Progeria of stem cells: stem cell exhaustion in Hutchinson-Gilford progeria syndrome.

    PubMed

    Halaschek-Wiener, Julius; Brooks-Wilson, Angela

    2007-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare, fatal genetic disorder that is characterized by segmental accelerated aging. The major causal mutation associated with HGPS triggers abnormal messenger RNA splicing of the lamin A gene leading to changes in the nuclear architecture. To date, two models have been proposed to explain how mutations in the lamin A gene could lead to HGPS, structural fragility and altered gene expression. We favor a compatible model that links HGPS to stem cell-driven tissue regeneration. In this model, nuclear fragility of lamin A-deficient cells increases apoptotic cell death to levels that exhaust tissues' ability for stem cell-driven regeneration. Tissue-specific differences in cell death or regenerative potential, or both, result in the tissue-specific segmental aging pattern seen in HGPS. We propose that the pattern of aging-related conditions present or absent in HGPS can provide insight into the genetic and environmental factors that contribute to normal aging.

  12. Clinical and radiographic features of Hutchinson-Gilford progeria syndrome: A case report.

    PubMed

    Alves, Daniel Berretta; Silva, Juliana Melo; Menezes, Tatiany Oliveira; Cavaleiro, Rosely Santos; Tuji, Fabrício Mesquita; Lopes, Marcio Ajudarte; Zaia, Alexandre Augusto; Coletta, Ricardo Della

    2014-03-16

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare dysmorphic syndrome characterized by several features of premature aging with clinical involvement of the skin, bones, and cardiovascular system. HGPS has an estimated incidence of one in four million to one in eight million births. The main clinical features of HGPS include short stature, craniofacial dimorphism, alopecia, bone fragility, and cardiovascular disorders. The most frequent cause of death is myocardial infarction at a mean age of 13 years old. Dental manifestations include delayed development and eruption of teeth, discoloration, crowding and rotation of teeth, and displaced teeth. Cone beam computed tomography images revealed the absence of the sphenoid, frontal, and maxillary sinus, flattening of the condyles and glenoid fossa, and bilateral hypoplasia of the mandibular condyles. The disease is caused by mutations in lamin A/C (LMNA). Here, we present a case report of an 11-year-old boy with classical features of HGPS, which was caused by a de novo germ-line mutation (C1824T, G608G) in exon 11 of the LMNA gene. Some uncommon HGPS-associated features in our patient, such as alterations in the facial sinuses and hypoplasia of the condyles, contributed to the expansion of the phenotypic spectrum of this syndrome from a dentomaxillofacial perspective.

  13. Progeria syndrome with cardiac complications.

    PubMed

    Ilyas, Saadia; Ilyas, Hajira; Hameed, Abdul; Ilyas, Muhammad

    2013-09-01

    A case report of 6-year-old boy with progeria syndrome, with marked cardiac complications is presented. The boy had cardiorespiratory failure. Discoloured purpuric skin patches, alopecia, prominent forehead, protuberant eyes, flattened nasal cartilage, malformed mandible, hypodentition, and deformed rigid fingers and toes were observed on examination. The boy was unable to speak. A sclerotic systolic murmur was audible over the mitral and aortic areas. Chest x-rays showed cardiac enlargement and the electrocardiogram (ECG) showed giant peaked P waves (right atrial hypertrophy) and right ventricular hypertrophy. Atherosclerotic dilated ascending aorta, thickened sclerotic aortic, mitral, and tricuspid valves with increased echo texture, left and right atrial and right ventricular dilatation, reduced left ventricular cavity, and thickened speckled atrial and ventricular septa were observed on echocardiography.

  14. Progeria

    MedlinePlus

    ... Read More Failure to thrive Heart attack Stroke Review Date 8/1/2015 Updated by: Chad Haldeman-Englert, MD, FACMG, Fullerton Genetics Center, Asheville, NC. Review provided by VeriMed Healthcare Network. Also reviewed by ...

  15. Mutational screening of the RB1 gene in Italian patients with retinoblastoma reveals 11 novel mutations.

    PubMed

    Sampieri, Katia; Hadjistilianou, Theodora; Mari, Francesca; Speciale, Caterina; Mencarelli, Maria Antonietta; Cetta, Francesco; Manoukian, Siranoush; Peissel, Bernard; Giachino, Daniela; Pasini, Barbara; Acquaviva, Antonio; Caporossi, Aldo; Frezzotti, Renato; Renieri, Alessandra; Bruttini, Mirella

    2006-01-01

    Retinoblastoma (RB, OMIM#180200) is the most common intraocular tumour in infancy and early childhood. Constituent mutations in the RB1 gene predispose individuals to RB development. We performed a mutational screening of the RB1 gene in Italian patients affected by RB referred to the Medical Genetics of the University of Siena. In 35 unrelated patients, we identified germline RB1 mutations in 6 out of 9 familial cases (66%) and in 7 out of 26 with no family history of RB (27%). Using the single-strand conformational polymorphism (SSCP) technique, 11 novel mutations were detected, including 3 nonsense, 5 frameshift and 4 splice-site mutations. Only two of these mutations (1 splice site and 1 missense) were previously reported. The mutation spectrum reflects the published literature, encompassing predominately nonsense or frameshift and splicing mutations. RB1 germline mutation was detected in 37% of our cases. Gross rearrangements outside the investigated region, altered DNA methylation, or mutations in non-coding regions, may be the cause of disease in the remainder of the patients. Some cases, e.g. a case of incomplete penetrance, or variable expressivity ranging from retinoma to multiple tumours, are discussed in detail. In addition, a case of pre-conception genetic counselling resolved by rescue of banked cordonal blood of the affected deceased child is described.

  16. Progenitor genotyping reveals a complex clonal architecture in a subset of CALR-mutated myeloproliferative neoplasms.

    PubMed

    Martin, Sarah; Wright, Casey M; Scott, Linda M

    2017-04-01

    The identification of acquired CALR mutations in patients with essential thrombocythaemia (ET) or myelofibrosis (MF) has meant that disease-initiating mutations can now be detected in about 90% of all patients with a myeloproliferative neoplasm (MPN). Here, we show that only those CALR mutations that cause a +1 frameshift, thereby altering the carboxy-terminus of calreticulin, promote cytokine independence in vitro; in-frame deletions were not functional, and are unlikely to be the pathogenetic mutation underlying some MPN cases. Expression of the thrombopoietin receptor, MPL, was also necessary for factor-independence. Although the CALR mutations are considered to occur only in JAK2 V617F-negative cases and in a heterozygous state, progenitor genotyping revealed that this is not always true. Notably, CALR mutation-positive MPNs can be polyclonal: in one case, two distinct CALR mutation-positive subpopulations could be identified; in another, separate populations of JAK2 V617F-positive and CALR-mutated cells were present. Mitotic recombination involving chromosome 19 in a third instance resulted in the emergence of a CALR mutation-homozygous subclone. Collectively, our studies demonstrate that occasional patients with CALR mutation-positive ET or MF carry other MPN-initiating genetic mutations (including JAK2 V617F), acquire "secondary mutations" before or after the CALR mutation, or evolve over time to being CALR mutation-homozygous.

  17. Immortalization of Werner syndrome and progeria fibroblasts

    SciTech Connect

    Saito, H.; Moses, R.E. )

    1991-02-01

    Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents.

  18. The complete genome sequences, unique mutational spectra and developmental potency of adult neurons revealed by cloning

    PubMed Central

    Rodriguez, Alberto R.; Ferguson, William C.; Shumilina, Svetlana; Clark, Royden A.; Boland, Michael J.; Martin, Greg; Chubukov, Pavel; Tsunemoto, Rachel K.; Torkamani, Ali; Kupriyanov, Sergey; Hall, Ira M.; Baldwin, Kristin K.

    2016-01-01

    Somatic mutation in neurons is linked to neurologic disease and implicated in cell type diversification. However, the origin, extent and patterns of genomic mutation in neurons remain unknown. We established a nuclear transfer method to clonally amplify the genomes of neurons from adult mice for whole genome sequencing. Comprehensive mutation detection and independent validation revealed that individual neurons harbor ~100 unique mutations from all classes, but lack recurrent rearrangements. Most neurons contain at least one gene disrupting mutation and rare (0-2) mobile element insertions. The frequency and gene bias of neuronal mutations differs from other lineages, potentially due to novel mechanisms governing post-mitotic mutation. Fertile mice were cloned from several neurons, establishing the compatibility of mutated adult neuronal genomes with reprogramming to pluripotency and development. PMID:26948891

  19. The Complete Genome Sequences, Unique Mutational Spectra, and Developmental Potency of Adult Neurons Revealed by Cloning.

    PubMed

    Hazen, Jennifer L; Faust, Gregory G; Rodriguez, Alberto R; Ferguson, William C; Shumilina, Svetlana; Clark, Royden A; Boland, Michael J; Martin, Greg; Chubukov, Pavel; Tsunemoto, Rachel K; Torkamani, Ali; Kupriyanov, Sergey; Hall, Ira M; Baldwin, Kristin K

    2016-03-16

    Somatic mutation in neurons is linked to neurologic disease and implicated in cell-type diversification. However, the origin, extent, and patterns of genomic mutation in neurons remain unknown. We established a nuclear transfer method to clonally amplify the genomes of neurons from adult mice for whole-genome sequencing. Comprehensive mutation detection and independent validation revealed that individual neurons harbor ∼100 unique mutations from all classes but lack recurrent rearrangements. Most neurons contain at least one gene-disrupting mutation and rare (0-2) mobile element insertions. The frequency and gene bias of neuronal mutations differ from other lineages, potentially due to novel mechanisms governing postmitotic mutation. Fertile mice were cloned from several neurons, establishing the compatibility of mutated adult neuronal genomes with reprogramming to pluripotency and development.

  20. Dedifferentiation rescues senescence of progeria cells but only while pluripotent.

    PubMed

    Niedernhofer, Laura J; Glorioso, Joseph C; Robbins, Paul D

    2011-06-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease in which children develop pathologies associated with old age. HGPS is caused by a mutation in the LMNA gene, resulting in the formation of a dominant negative form of the intermediate filament, nuclear structural protein lamin A, termed progerin. Expression of progerin alters the nuclear architecture and heterochromatin, affecting cell cycle progression and genomic stability. Two groups recently reported the successful generation and characterization of induced pluripotent stem cells (iPSCs) from HGPS fibroblasts. Remarkably, progerin expression and senescence phenotypes are lost in iPSCs but not in differentiated progeny. These new HGPS iPSCs are valuable for characterizing the role of progerin in driving HGPS and aging and for screening therapeutic strategies to prevent or delay cell senescence.

  1. Integrative analysis of mutational and transcriptional profiles reveals driver mutations of metastatic breast cancers

    PubMed Central

    Lee, Ji-Hyun; Zhao, Xing-Ming; Yoon, Ina; Lee, Jin Young; Kwon, Nam Hoon; Wang, Yin-Ying; Lee, Kyung-Min; Lee, Min-Joo; Kim, Jisun; Moon, Hyeong-Gon; In, Yongho; Hao, Jin-Kao; Park, Kyung-Mii; Noh, Dong-Young; Han, Wonshik; Kim, Sunghoon

    2016-01-01

    Despite the explosion in the numbers of cancer genomic studies, metastasis is still the major cause of cancer mortality. In breast cancer, approximately one-fifth of metastatic patients survive 5 years. Therefore, detecting the patients at a high risk of developing distant metastasis at first diagnosis is critical for effective treatment strategy. We hereby present a novel systems biology approach to identify driver mutations escalating the risk of metastasis based on both exome and RNA sequencing of our collected 78 normal-paired breast cancers. Unlike driver mutations occurring commonly in cancers as reported in the literature, the mutations detected here are relatively rare mutations occurring in less than half metastatic samples. By supposing that the driver mutations should affect the metastasis gene signatures, we develop a novel computational pipeline to identify the driver mutations that affect transcription factors regulating metastasis gene signatures. We identify driver mutations in ADPGK, NUP93, PCGF6, PKP2 and SLC22A5, which are verified to enhance cancer cell migration and prompt metastasis with in vitro experiments. The discovered somatic mutations may be helpful for identifying patients who are likely to develop distant metastasis. PMID:27625789

  2. Integrative analysis of mutational and transcriptional profiles reveals driver mutations of metastatic breast cancers.

    PubMed

    Lee, Ji-Hyun; Zhao, Xing-Ming; Yoon, Ina; Lee, Jin Young; Kwon, Nam Hoon; Wang, Yin-Ying; Lee, Kyung-Min; Lee, Min-Joo; Kim, Jisun; Moon, Hyeong-Gon; In, Yongho; Hao, Jin-Kao; Park, Kyung-Mii; Noh, Dong-Young; Han, Wonshik; Kim, Sunghoon

    2016-01-01

    Despite the explosion in the numbers of cancer genomic studies, metastasis is still the major cause of cancer mortality. In breast cancer, approximately one-fifth of metastatic patients survive 5 years. Therefore, detecting the patients at a high risk of developing distant metastasis at first diagnosis is critical for effective treatment strategy. We hereby present a novel systems biology approach to identify driver mutations escalating the risk of metastasis based on both exome and RNA sequencing of our collected 78 normal-paired breast cancers. Unlike driver mutations occurring commonly in cancers as reported in the literature, the mutations detected here are relatively rare mutations occurring in less than half metastatic samples. By supposing that the driver mutations should affect the metastasis gene signatures, we develop a novel computational pipeline to identify the driver mutations that affect transcription factors regulating metastasis gene signatures. We identify driver mutations in ADPGK, NUP93, PCGF6, PKP2 and SLC22A5, which are verified to enhance cancer cell migration and prompt metastasis with in vitro experiments. The discovered somatic mutations may be helpful for identifying patients who are likely to develop distant metastasis.

  3. Sporadic premature aging in a Japanese monkey: a primate model for progeria.

    PubMed

    Oishi, Takao; Imai, Hiroo; Go, Yasuhiro; Imamura, Masanori; Hirai, Hirohisa; Takada, Masahiko

    2014-01-01

    In our institute, we have recently found a child Japanese monkey who is characterized by deep wrinkles of the skin and cataract of bilateral eyes. Numbers of analyses were performed to identify symptoms representing different aspects of aging. In this monkey, the cell cycle of fibroblasts at early passage was significantly extended as compared to a normal control. Moreover, both the appearance of senescent cells and the deficiency in DNA repair were observed. Also, pathological examination showed that this monkey has poikiloderma with superficial telangiectasia, and biochemical assay confirmed that levels of HbA1c and urinary hyaluronan were higher than those of other (child, adult, and aged) monkey groups. Of particular interest was that our MRI analysis revealed expansion of the cerebral sulci and lateral ventricles probably due to shrinkage of the cerebral cortex and the hippocampus. In addition, the conduction velocity of a peripheral sensory but not motor nerve was lower than in adult and child monkeys, and as low as in aged monkeys. However, we could not detect any individual-unique mutations of known genes responsible for major progeroid syndromes. The present results indicate that the monkey suffers from a kind of progeria that is not necessarily typical to human progeroid syndromes.

  4. Progerin reduces LAP2α-telomere association in Hutchinson-Gilford progeria.

    PubMed

    Chojnowski, Alexandre; Ong, Peh Fern; Wong, Esther S M; Lim, John S Y; Mutalif, Rafidah A; Navasankari, Raju; Dutta, Bamaprasad; Yang, Henry; Liow, Yi Y; Sze, Siu K; Boudier, Thomas; Wright, Graham D; Colman, Alan; Burke, Brian; Stewart, Colin L; Dreesen, Oliver

    2015-08-27

    Hutchinson-Gilford progeria (HGPS) is a premature ageing syndrome caused by a mutation in LMNA, resulting in a truncated form of lamin A called progerin. Progerin triggers loss of the heterochromatic marker H3K27me3, and premature senescence, which is prevented by telomerase. However, the mechanism how progerin causes disease remains unclear. Here, we describe an inducible cellular system to model HGPS and find that LAP2α (lamina-associated polypeptide-α) interacts with lamin A, while its interaction with progerin is significantly reduced. Super-resolution microscopy revealed that over 50% of telomeres localize to the lamina and that LAP2α association with telomeres is impaired in HGPS. This impaired interaction is central to HGPS since increasing LAP2α levels rescues progerin-induced proliferation defects and loss of H3K27me3, whereas lowering LAP2 levels exacerbates progerin-induced defects. These findings provide novel insights into the pathophysiology underlying HGPS, and how the nuclear lamina regulates proliferation and chromatin organization.

  5. Progerin reduces LAP2α-telomere association in Hutchinson-Gilford progeria

    PubMed Central

    Chojnowski, Alexandre; Ong, Peh Fern; Wong, Esther SM; Lim, John SY; Mutalif, Rafidah A; Navasankari, Raju; Dutta, Bamaprasad; Yang, Henry; Liow, Yi Y; Sze, Siu K; Boudier, Thomas; Wright, Graham D; Colman, Alan; Burke, Brian; Stewart, Colin L; Dreesen, Oliver

    2015-01-01

    Hutchinson-Gilford progeria (HGPS) is a premature ageing syndrome caused by a mutation in LMNA, resulting in a truncated form of lamin A called progerin. Progerin triggers loss of the heterochromatic marker H3K27me3, and premature senescence, which is prevented by telomerase. However, the mechanism how progerin causes disease remains unclear. Here, we describe an inducible cellular system to model HGPS and find that LAP2α (lamina-associated polypeptide-α) interacts with lamin A, while its interaction with progerin is significantly reduced. Super-resolution microscopy revealed that over 50% of telomeres localize to the lamina and that LAP2α association with telomeres is impaired in HGPS. This impaired interaction is central to HGPS since increasing LAP2α levels rescues progerin-induced proliferation defects and loss of H3K27me3, whereas lowering LAP2 levels exacerbates progerin-induced defects. These findings provide novel insights into the pathophysiology underlying HGPS, and how the nuclear lamina regulates proliferation and chromatin organization. DOI: http://dx.doi.org/10.7554/eLife.07759.001 PMID:26312502

  6. Progeria: a paradigm for translational medicine.

    PubMed

    Gordon, Leslie B; Rothman, Frank G; López-Otín, Carlos; Misteli, Tom

    2014-01-30

    Rare diseases are powerful windows into biological processes and can serve as models for the development of therapeutic strategies. The progress made on the premature aging disorder Progeria is a shining example of the impact that studies of rare diseases can have.

  7. Functional analysis reveals that RBM10 mutations contribute to lung adenocarcinoma pathogenesis by deregulating splicing

    PubMed Central

    Zhao, Jiawei; Sun, Yue; Huang, Yin; Song, Fan; Huang, Zengshu; Bao, Yufang; Zuo, Ji; Saffen, David; Shao, Zhen; Liu, Wen; Wang, Yongbo

    2017-01-01

    RBM10 is an RNA splicing regulator that is frequently mutated in lung adenocarcinoma (LUAD) and has recently been proposed to be a cancer gene. How RBM10 mutations observed in LUAD affect its normal functions, however, remains largely unknown. Here integrative analysis of RBM10 mutation and RNA expression data revealed that LUAD-associated RBM10 mutations exhibit a mutational spectrum similar to that of tumor suppressor genes. In addition, this analysis showed that RBM10 mutations identified in LUAD patients lacking canonical oncogenes are associated with significantly reduced RBM10 expression. To systematically investigate RBM10 mutations, we developed an experimental pipeline for elucidating their functional effects. Among six representative LUAD-associated RBM10 mutations, one nonsense and one frameshift mutation caused loss-of-function as expected, whereas four missense mutations differentially affected RBM10-mediated splicing. Importantly, changes in proliferation rates of LUAD-derived cells caused by these RBM10 missense mutants correlated with alterations in RNA splicing of RBM10 target genes. Together, our data implies that RBM10 mutations contribute to LUAD pathogenesis, at least in large part, by deregulating splicing. The methods described in this study should be useful for analyzing mutations in additional cancer-associated RNA splicing regulators. PMID:28091594

  8. Exome sequencing reveals AMER1 as a frequently mutated gene in colorectal cancer

    PubMed Central

    Sanz-Pamplona, Rebeca; Lopez-Doriga, Adriana; Paré-Brunet, Laia; Lázaro, Kira; Bellido, Fernando; Alonso, M. Henar; Aussó, Susanna; Guinó, Elisabet; Beltrán, Sergi; Castro-Giner, Francesc; Gut, Marta; Sanjuan, Xavier; Closa, Adria; Cordero, David; Morón-Duran, Francisco D.; Soriano, Antonio; Salazar, Ramón; Valle, Laura; Moreno, Victor

    2015-01-01

    PURPOSE Somatic mutations occur at early stages of adenoma and accumulate throughout colorectal cancer (CRC) progression. The aim of this study was to characterize the mutational landscape of stage II tumors and to search for novel recurrent mutations likely implicated in CRC tumorigenesis. DESIGN The exomic DNA of 42 stage II, microsatellite stable, colon tumors and their paired mucosae were sequenced. Other molecular data available in the discovery dataset (gene expression, methylation, and CNV) was used to further characterize these tumors. Additional datasets comprising 553 CRC samples were used to validate the discovered mutations. RESULTS As a result, 4,886 somatic single nucleotide variants (SNVs) were found. Almost all SNVs were private changes, with few mutations shared by more than one tumor, thus revealing tumor-specific mutational landscapes. Nevertheless, these diverse mutations converged into common cellular pathways such as cell cycle or apoptosis. Among this mutational heterogeneity, variants resulting in early stop-codons in the AMER1 (also known as FAM123B or WTX) gene emerged as recurrent mutations in CRC. Loses of AMER1 by other mechanisms apart from mutations such as methylation and copy number aberrations were also found. Tumors lacking this tumor suppressor gene exhibited a mesenchymal phenotype characterized by inhibition of the canonical Wnt pathway. CONCLUSION In silico and experimental validation in independent datasets confirmed the existence of functional mutations in AMER1 in approximately 10% of analyzed CRC tumors. Moreover, these tumors exhibited a characteristic phenotype. PMID:26071483

  9. Exome sequencing reveals novel IRXI mutation in congenital heart disease.

    PubMed

    Guo, Changlong; Wang, Qidi; Wang, Yuting; Yang, Liping; Luo, Haiyan; Cao, Xiao Fang; An, Lisha; Qiu, Yue; Du, Meng; Ma, Xu; Li, Hui; Lu, Cailing

    2017-03-30

    Genetic variation in specific transcription factors during heart formation may lead to congenital heart disease (CHD) or even miscarriage. The aim of the present study was to identify CHD‑associated genes using next generation sequencing (NGS). The whole exome DNA sequence was obtained from a stillborn fetus diagnosed with tricuspid atresia and complete transposition of the great arteries using high‑throughput sequencing methods. Subsequently, genetic variants of CHD‑associated genes were selected and verified in 215 non‑syndromic CHD patients and 249 healthy control subjects using polymerase chain reaction combined with Sanger sequencing. Genetic variants of previously reported CHD‑inducing genes, such as cysteine rich with EGF like domains 1 and cbp/p300‑interacting transactivator with Glu/Asp rich carboxy‑terminal domain 2, were discovered through the NGS analysis. In addition, a novel non‑synonymous mutation of the iroquois homeobox 1 (IRX1) gene (p.Gln240Glu) was identified. A total of three non‑synonymous mutations (p.Gln240Glu, p.Ser298Asn and p.Ala381Glu) of the IRX1 gene were verified in 215 non‑syndromic CHD patients, but not in 249 healthy volunteers. The results demonstrated that NGS is a powerful tool to study the etiology of CHD. In addition, the results suggest that genetic variants of the IRX1 gene may contribute to the pathogenesis of CHD.

  10. Whole-exome sequencing reveals the mutational spectrum of testicular germ cell tumours.

    PubMed

    Litchfield, Kevin; Summersgill, Brenda; Yost, Shawn; Sultana, Razvan; Labreche, Karim; Dudakia, Darshna; Renwick, Anthony; Seal, Sheila; Al-Saadi, Reem; Broderick, Peter; Turner, Nicholas C; Houlston, Richard S; Huddart, Robert; Shipley, Janet; Turnbull, Clare

    2015-01-22

    Testicular germ cell tumours (TGCTs) are the most common cancer in young men. Here we perform whole-exome sequencing (WES) of 42 TGCTs to comprehensively study the cancer's mutational profile. The mutation rate is uniformly low in all of the tumours (mean 0.5 mutations per Mb) as compared with common cancers, consistent with the embryological origin of TGCT. In addition to expected copy number gain of chromosome 12p and mutation of KIT, we identify recurrent mutations in the tumour suppressor gene CDC27 (11.9%). Copy number analysis reveals recurring amplification of the spermatocyte development gene FSIP2 (15.3%) and a 0.4 Mb region at Xq28 (15.3%). Two treatment-refractory patients are shown to harbour XRCC2 mutations, a gene strongly implicated in defining cisplatin resistance. Our findings provide further insights into genes involved in the development and progression of TGCT.

  11. Whole-exome sequencing reveals the mutational spectrum of testicular germ cell tumours

    PubMed Central

    Litchfield, Kevin; Summersgill, Brenda; Yost, Shawn; Sultana, Razvan; Labreche, Karim; Dudakia, Darshna; Renwick, Anthony; Seal, Sheila; Al-Saadi, Reem; Broderick, Peter; Turner, Nicholas C.; Houlston, Richard S.; Huddart, Robert; Shipley, Janet; Turnbull, Clare

    2015-01-01

    Testicular germ cell tumours (TGCTs) are the most common cancer in young men. Here we perform whole-exome sequencing (WES) of 42 TGCTs to comprehensively study the cancer's mutational profile. The mutation rate is uniformly low in all of the tumours (mean 0.5 mutations per Mb) as compared with common cancers, consistent with the embryological origin of TGCT. In addition to expected copy number gain of chromosome 12p and mutation of KIT, we identify recurrent mutations in the tumour suppressor gene CDC27 (11.9%). Copy number analysis reveals recurring amplification of the spermatocyte development gene FSIP2 (15.3%) and a 0.4 Mb region at Xq28 (15.3%). Two treatment-refractory patients are shown to harbour XRCC2 mutations, a gene strongly implicated in defining cisplatin resistance. Our findings provide further insights into genes involved in the development and progression of TGCT. PMID:25609015

  12. DNA-damage accumulation and replicative arrest in Hutchinson-Gilford progeria syndrome.

    PubMed

    Musich, Phillip R; Zou, Yue

    2011-12-01

    A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.

  13. All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype.

    PubMed

    Pellegrini, Camilla; Columbaro, Marta; Capanni, Cristina; D'Apice, Maria Rosaria; Cavallo, Carola; Murdocca, Michela; Lattanzi, Giovanna; Squarzoni, Stefano

    2015-10-06

    Hutchinson Gilford progeria syndrome is a fatal disorder characterized by accelerated aging, bone resorption and atherosclerosis, caused by a LMNA mutation which produces progerin, a mutant lamin A precursor. Progeria cells display progerin and prelamin A nuclear accumulation, altered histone methylation pattern, heterochromatin loss, increased DNA damage and cell cycle alterations. Since the LMNA promoter contains a retinoic acid responsive element, we investigated if all-trans retinoic acid administration could lower progerin levels in cultured fibroblasts. We also evaluated the effect of associating rapamycin, which induces autophagic degradation of progerin and prelamin A. We demonstrate that all-trans retinoic acid acts synergistically with low-dosage rapamycin reducing progerin and prelamin A, via transcriptional downregulation associated with protein degradation, and increasing the lamin A to progerin ratio. These effects rescue cell dynamics and cellular proliferation through recovery of DNA damage response factor PARP1 and chromatin-associated nuclear envelope proteins LAP2α and BAF. The combined all-trans retinoic acid-rapamycin treatment is dramatically efficient, highly reproducible, represents a promising new approach in Hutchinson-Gilford Progeria therapy and deserves investigation in ageing-associated disorders.

  14. All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype

    PubMed Central

    Pellegrini, Camilla; Columbaro, Marta; Capanni, Cristina; D'Apice, Maria Rosaria; Cavallo, Carola; Murdocca, Michela; Lattanzi, Giovanna; Squarzoni, Stefano

    2015-01-01

    Hutchinson Gilford progeria syndrome is a fatal disorder characterized by accelerated aging, bone resorption and atherosclerosis, caused by a LMNA mutation which produces progerin, a mutant lamin A precursor. Progeria cells display progerin and prelamin A nuclear accumulation, altered histone methylation pattern, heterochromatin loss, increased DNA damage and cell cycle alterations. Since the LMNA promoter contains a retinoic acid responsive element, we investigated if all-trans retinoic acid administration could lower progerin levels in cultured fibroblasts. We also evaluated the effect of associating rapamycin, which induces autophagic degradation of progerin and prelamin A. We demonstrate that all-trans retinoic acid acts synergistically with low-dosage rapamycin reducing progerin and prelamin A, via transcriptional downregulation associated with protein degradation, and increasing the lamin A to progerin ratio. These effects rescue cell dynamics and cellular proliferation through recovery of DNA damage response factor PARP1 and chromatin-associated nuclear envelope proteins LAP2α and BAF. The combined all-trans retinoic acid-rapamycin treatment is dramatically efficient, highly reproducible, represents a promising new approach in Hutchinson-Gilford Progeria therapy and deserves investigation in ageing-associated disorders. PMID:26359359

  15. Sulforaphane enhances progerin clearance in Hutchinson-Gilford progeria fibroblasts.

    PubMed

    Gabriel, Diana; Roedl, Daniela; Gordon, Leslie B; Djabali, Karima

    2015-02-01

    Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare multisystem childhood premature aging disorder linked to mutations in the LMNA gene. The most common HGPS mutation is found at position G608G within exon 11 of the LMNA gene. This mutation results in the deletion of 50 amino acids at the carboxyl-terminal tail of prelamin A, and the truncated protein is called progerin. Progerin only undergoes a subset of the normal post-translational modifications and remains permanently farnesylated. Several attempts to rescue the normal cellular phenotype with farnesyltransferase inhibitors (FTIs) and other compounds have resulted in partial cellular recovery. Using proteomics, we report here that progerin induces changes in the composition of the HGPS nuclear proteome, including alterations to several components of the protein degradation pathways. Consequently, proteasome activity and autophagy are impaired in HGPS cells. To restore protein clearance in HGPS cells, we treated HGPS cultures with sulforaphane (SFN), an antioxidant derived from cruciferous vegetables. We determined that SFN stimulates proteasome activity and autophagy in normal and HGPS fibroblast cultures. Specifically, SFN enhances progerin clearance by autophagy and reverses the phenotypic changes that are the hallmarks of HGPS. Therefore, SFN is a promising therapeutic avenue for children with HGPS.

  16. Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups

    PubMed Central

    Maitra, Arindam; Biswas, Nidhan K.; Amin, Kishore; Kowtal, Pradnya; Kumar, Shantanu; Das, Subrata; Sarin, Rajiv; Majumder, Partha P.; Bagchi, I; Bairagya, B. B.; Basu, A.; Bhan, M. K.; Chaturvedi, P.; Das, D.; D'Cruz, A.; Dhar, R.; Dutta, D.; Ganguli, D.; Gera, P.; Gupta, T.; Mahapatra, S.; Mujawar, M. H. K.; Mukherjee, S.; Nair, S.; Nikam, S.; Nobre, M.; Patil, A.; Patra, S.; Rama-Gowtham, M.; Rao, T. S.; Roy, B.; Roychowdhury, B.; Sarkar, D.; Sarkar, S.; Sarkar-Roy, N.; Sutradhar, D.

    2013-01-01

    Gingivo-buccal oral squamous cell carcinoma (OSCC-GB), an anatomical and clinical subtype of head and neck squamous cell carcinoma (HNSCC), is prevalent in regions where tobacco-chewing is common. Exome sequencing (n=50) and recurrence testing (n=60) reveals that some significantly and frequently altered genes are specific to OSCC-GB (USP9X, MLL4, ARID2, UNC13C and TRPM3), while some others are shared with HNSCC (for example, TP53, FAT1, CASP8, HRAS and NOTCH1). We also find new genes with recurrent amplifications (for example, DROSHA, YAP1) or homozygous deletions (for example, DDX3X) in OSCC-GB. We find a high proportion of C>G transversions among tobacco users with high numbers of mutations. Many pathways that are enriched for genomic alterations are specific to OSCC-GB. Our work reveals molecular subtypes with distinctive mutational profiles such as patients predominantly harbouring mutations in CASP8 with or without mutations in FAT1. Mean duration of disease-free survival is significantly elevated in some molecular subgroups. These findings open new avenues for biological characterization and exploration of therapies. PMID:24292195

  17. Progeria: Medical Aspects, Psychosocial Perspectives, and Intervention Guidelines.

    ERIC Educational Resources Information Center

    Livneh, Hanoch; And Others

    1995-01-01

    Discusses progeria (or Hutchinson-Gilford syndrome), a rare childhood disorder that invariably results in death during adolescence. Describes the major medical aspects of progeria, and discusses the psychosocial implications of the disorder with particular emphasis on grief-triggered reactions. Presents an overview of psychosocial intervention…

  18. Mutational strand asymmetries in cancer genomes reveal mechanisms of DNA damage and repair

    PubMed Central

    Haradhvala, Nicholas J.; Polak, Paz; Stojanov, Petar; Covington, Kyle R.; Shinbrot, Eve; Hess, Julian; Rheinbay, Esther; Kim, Jaegil; Maruvka, Yosef; Braunstein, Lior Z.; Kamburov, Atanas; Hanawalt, Philip C.; Wheeler, David A.; Koren, Amnon; Lawrence, Michael S.; Getz, Gad

    2016-01-01

    Mutational processes constantly shape the somatic genome, leading to immunity, aging, and other diseases. When cancer is the outcome, we are afforded a glimpse into these processes by the clonal expansion of the malignant cell. Here, we characterize a less explored layer of the mutational landscape of cancer: mutational asymmetries between the two DNA strands. Analyzing whole genome sequences of 590 tumors from 14 different cancer types, we reveal widespread asymmetries across mutagenic processes, with transcriptional (“T-class”) asymmetry dominating UV-, smoking-, and liver-cancer-associated mutations, and replicative (“R-class”) asymmetry dominating POLE-, APOBEC-, and MSI-associated mutations. We report a striking phenomenon of Transcription-Coupled Damage (TCD) on the non-transcribed DNA strand, and provide evidence that APOBEC mutagenesis occurs on the lagging-strand template during DNA replication. As more genomes are sequenced, studying and classifying their asymmetries will illuminate the underlying biological mechanisms of DNA damage and repair. PMID:26806129

  19. Study of Alzheimer family case reveals hemochromotosis-associated HFE mutation

    PubMed Central

    Artemov, Artem V; Boulygina, Eugenia S; Tsygankova, Svetlana V; Nedoluzhko, Artem V; Chekanov, Nikolay N; Gruzdeva, Natalia M; Selezneva, Natalia D; Roshchina, Irina F; Gavrilova, Svetlana I; Velichkovsky, Boris B; Skryabin, Konstantin G; Prokhortchouk, Egor B

    2014-01-01

    We report a family case of type II early-onset Alzheimer’s disease (AD) inherited over three generations. None of the patients in the family had mutations in the genes believed to be the major risk factors for AD, such as APP, presenilin 1 or 2. Targeted exome sequencing of 249 genes that were previously reported to be associated with AD revealed a rare mutation in hemochromatosis (HFE) gene known to be associated with hemochromotosis. Compared to previous studies, we show that HFE mutation can possess the risk of AD in transferrin-, APOE- and APP-normal patients. PMID:27081498

  20. Study of Alzheimer family case reveals hemochromotosis-associated HFE mutation.

    PubMed

    Artemov, Artem V; Boulygina, Eugenia S; Tsygankova, Svetlana V; Nedoluzhko, Artem V; Chekanov, Nikolay N; Gruzdeva, Natalia M; Selezneva, Natalia D; Roshchina, Irina F; Gavrilova, Svetlana I; Velichkovsky, Boris B; Skryabin, Konstantin G; Prokhortchouk, Egor B

    2014-01-01

    We report a family case of type II early-onset Alzheimer's disease (AD) inherited over three generations. None of the patients in the family had mutations in the genes believed to be the major risk factors for AD, such as APP, presenilin 1 or 2. Targeted exome sequencing of 249 genes that were previously reported to be associated with AD revealed a rare mutation in hemochromatosis (HFE) gene known to be associated with hemochromotosis. Compared to previous studies, we show that HFE mutation can possess the risk of AD in transferrin-, APOE- and APP-normal patients.

  1. Determinants of spontaneous mutation in the bacterium Escherichia coli as revealed by whole-genome sequencing

    PubMed Central

    Foster, Patricia L.; Lee, Heewook; Popodi, Ellen; Townes, Jesse P.; Tang, Haixu

    2015-01-01

    A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1–2 × 10−3 mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3′ base can affect the mutability of a purine by oxidative damage by as much as eightfold. PMID:26460006

  2. Genomic instability and DNA damage responses in progeria arising from defective maturation of prelamin A.

    PubMed

    Musich, Phillip R; Zou, Yue

    2009-01-01

    Progeria syndromes have in common a premature aging phenotype and increased genome instability. The susceptibility to DNA damage arises from a compromised repair system, either in the repair proteins themselves or in the DNA damage response pathways. The most severe progerias stem from mutations affecting lamin A production, a filamentous protein of the nuclear lamina. Hutchinson-Gilford progeria syndrome (HGPS) patients are heterozygous for aLMNA gene mutation while Restrictive Dermopathy (RD) individuals have a homozygous deficiency in the processing protease Zmpste24. These mutations generate the mutant lamin A proteins progerin and FC-lamina A, respectively, which cause nuclear deformations and chromatin perturbations. Genome instability is observed even though genome maintenance and repair genes appear normal. The unresolved question is what features of the DNA damage response pathways are deficient in HGPS and RD cells. Here we review and discuss recent findings which resolve some mechanistic details of how the accumulation of progerin/FC-lamin A proteins may disrupt DNA damage response pathways in HGPS and RD cells. As the mutant lamin proteins accumulate they sequester replication and repair factors, leading to stalled replication forks which collapse into DNA double-strand beaks (DSBs). In a reaction unique to HGPS and RD cells these accessible DSB termini bind Xeroderma pigmentosum group A (XPA) protein which excludes normal binding by DNA DSB repair proteins. The bound XPA also signals activation of ATM and ATR, arresting cell cycle progression, leading to arrested growth. In addition, the effective sequestration of XPA at these DSB damage sites makes HGPS and RD cells more sensitive to ultraviolet light and other mutagens normally repaired by the nucleotide excision repair pathway of which XPA is a necessary and specific component.

  3. Progeria: pathogenesis and oral manifestation--a review.

    PubMed

    Saigal, S; Bhargava, A

    2012-01-01

    Our life span is genetically programmed and it is possible that a defect in produced proteins encoded by the longevity gene is a cause of aging. Progeria which is a rare, fatal genetic condition which affects between one in four million and one in eight million children of both sexes equally and characterized by premature and accelerated aging. The appearance and physiology of these children resembles to elderly people but they typically have life span to their mid teens. It is also known as the Hutchinson-Gilford syndrome, which was initially reported by Johnathan Hutchinson in 1886 and further described by Hastings Gilford in 1904. It is an autosomal recessive disorder, which means an individual has inherited a mutated gene from both parents. It is added to the expanding catalogue of laminopathies, diseases caused by mutations affecting nuclear lamina proteins known as lamin A (LMNA). In oral manifestation primary finding is micrognathia with delayed tooth eruption and incomplete formation of root of permanent tooth. Presently there are no known cures for this abnormality.

  4. Hutchinson-Gilford progeria syndrome through the lens of transcription.

    PubMed

    Prokocimer, Miron; Barkan, Rachel; Gruenbaum, Yosef

    2013-08-01

    Lamins are nuclear intermediate filaments. In addition to their structural roles, they are implicated in basic nuclear functions such as chromatin organization, DNA replication, transcription, DNA repair, and cell-cycle progression. Mutations in human LMNA gene cause several diseases termed laminopathies. One of the laminopathic diseases is Hutchinson-Gilford progeria syndrome (HGPS), which is caused by a spontaneous mutation and characterized by premature aging. HGPS phenotypes share certain similarities with several apparently comparable medical conditions, such as aging and atherosclerosis, with the conspicuous absence of neuronal degeneration and cancer rarity during the short lifespan of the patients. Cell lines from HGPS patients are characterized by multiple nuclear defects, which include abnormal morphology, altered histone modification patterns, and increased DNA damage. These cell lines provide insight into the molecular pathways including senescence that require lamins A and B1. Here, we review recent data on HGPS phenotypes through the lens of transcriptional deregulation caused by lack of functional lamin A, progerin accumulation, and lamin B1 silencing.

  5. Molecular insights into the premature aging disease progeria.

    PubMed

    Vidak, Sandra; Foisner, Roland

    2016-04-01

    Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare premature aging disease presenting many features resembling the normal aging process. HGPS patients die before the age of 20 years due to cardiovascular problems and heart failure. HGPS is linked to mutations in the LMNA gene encoding the intermediate filament protein lamin A. Lamin A is a major component of the nuclear lamina, a scaffold structure at the nuclear envelope that defines mechanochemical properties of the nucleus and is involved in chromatin organization and epigenetic regulation. Lamin A is also present in the nuclear interior where it fulfills lamina-independent functions in cell signaling and gene regulation. The most common LMNA mutation linked to HGPS leads to mis-splicing of the LMNA mRNA and produces a mutant lamin A protein called progerin that tightly associates with the inner nuclear membrane and affects the dynamic properties of lamins. Progerin expression impairs many important cellular processes providing insight into potential disease mechanisms. These include changes in mechanosignaling, altered chromatin organization and impaired genome stability, and changes in signaling pathways, leading to impaired regulation of adult stem cells, defective extracellular matrix production and premature cell senescence. In this review, we discuss these pathways and their potential contribution to the disease pathologies as well as therapeutic approaches used in preclinical and clinical tests.

  6. Hutchinson-Gilford progeria syndrome: Oral and craniofacial phenotypes

    PubMed Central

    Domingo, D.L.; Trujillo, M.I.; Council, S.E.; Merideth, M.A.; Gordon, L.B.; Wu, T.; Introne, W.J.; Gahl, W.A.; Hart, T.C.

    2008-01-01

    OBJECTIVE Hutchinson-Gilford progeria syndrome (HGPS) is a rare early-onset accelerated senescence syndrome. In HGPS, a recently identified de novo dominant mutation of the lamin A gene (LMNA) produces abnormal lamin A, resulting in compromised nuclear membrane integrity. Clinical features include sclerotic skin, cardiovascular and bone abnormalities, and marked growth retardation. Craniofacial features include “bird-like” facies, alopecia, craniofacial disproportion and dental crowding. Our prospective study describes dental, oral soft tissue, and craniofacial bone features in HGPS. METHODS Fifteen patients with confirmed p.G608G LMNA mutation (1–17 years, 7 males, 8 females) received comprehensive oral evaluations. Anomalies of oral soft tissue, gnathic bones and dentition were identified. RESULTS Radiographic findings included hypodontia (n=7), dysmorphic teeth (n=5), steep mandibular angles (n=11), and thin basal bone (n=11). Soft tissue findings included ogival palatal arch (n=8), median sagittal palatal fissure (n=7), and ankyloglossia (n=7). Calculated dental ages (9months–11y2m) were significantly lower than chronological ages (1y6m–17y8m) (p=0.002). Eleven children manifested a shorter mandibular body, anterior/posterior cranial base and ramus, but a larger gonial angle, compared to age/gender/race norms. CONCLUSION Novel oral-craniofacial phenotypes and quantification of previously reported features are presented. Our findings expand the HGPS phenotype and provide additional insight into the complex pathogenesis of HGPS. PMID:19236595

  7. Clinical imaging findings in a girl with Hutchinson-Gilford progeria syndrome.

    PubMed

    Chen, C P; Lin, S P; Lin, D S; Liu, Y P; Hsu, L J; Wang, W

    2012-01-01

    We report an 82-year-old girl with premature aging, a karyotype of 46,XX and a de novo c.1824C>T mutation encoding p.G608G in the lamin A gene. The clinical features of accelerated aging and the molecular finding were consistent with the diagnosis of Hutchinson-Gilford progeria syndrome (HGPS). In this presentation, we demonstrate the radiological imaging findings of skeletal, oral and craniofacial phenotypes of abnormalities associated with HGPS. The oral and craniofacial abnormalities caused dental caries, severe malocclusion, and swallowing, feeding and speech problems. Dural calcification, and granulation in the ear drum and external ear canal were additionally observed.

  8. Dental and craniofacial characteristics in a patient with Hutchinson-Gilford progeria syndrome.

    PubMed

    Reichert, Christoph; Gölz, Lina; Götz, Werner; Wolf, Michael; Deschner, James; Jäger, Andreas

    2014-07-01

    The Hutchinson-Gilford progeria syndrome (HGPS) is an exceptionally rare medical disorder caused by mutations in the lamin A/C gene. Affected patients display typical features of premature aging. Beside general medical disorders, these patients have several specific features related to the craniofacial phenotype and the oral cavity. In this article, the dental and craniofacial characteristics of a 9-year-old girl with HGPS are presented. It is the first report addressing orthodontic tooth movement and microbiological features in a HGPS patient. We describe and discuss pathologic findings and provide a detailed histology of the teeth which had to be extracted during initial treatment.

  9. Mutational Profiles Reveal an Aberrant TGF-β-CEA Regulated Pathway in Colon Adenomas

    PubMed Central

    Jogunoori, Wilma; Menon, Vipin; Majumdar, Avijit; Chen, Jiun-Sheng; Gi, Young Jin; Jeong, Yun Seong; Phan, Liem; Belkin, Mitchell; Gu, Shoujun; Kundra, Suchin; Mistry, Nipun A.; Zhang, Jianping; Su, Xiaoping; Li, Shulin; Lin, Sue-Hwa; Javle, Milind; McMurray, John S.; Rahlfs, Thomas F.; Mishra, Bibhuti; White, Jon; Rashid, Asif; Beauchemin, Nicole; Weston, Brian R.; Shafi, Mehnaz A.; Stroehlein, John R.; Davila, Marta; Akbani, Rehan; Weinstein, John N.; Wu, Xifeng; Mishra, Lopa

    2016-01-01

    Mutational processes and signatures that drive early tumorigenesis are centrally important for early cancer prevention. Yet, to date, biomarkers and risk factors for polyps (adenomas) that inordinately and rapidly develop into colon cancer remain poorly defined. Here, we describe surprisingly high mutational profiles through whole-genome sequence (WGS) analysis in 2 of 4 pairs of benign colorectal adenoma tissue samples. Unsupervised hierarchical clustered transcriptomic analysis of a further 7 pairs of adenomas reveals distinct mutational signatures regardless of adenoma size. Transitional single nucleotide substitutions of C:G>T:A predominate in the adenoma mutational spectrum. Strikingly, we observe mutations in the TGF-β pathway and CEA-associated genes in 4 out of 11 adenomas, overlapping with the Wnt pathway. Immunohistochemical labeling reveals a nearly 5-fold increase in CEA levels in 23% of adenoma samples with a concomitant loss of TGF-β signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-β signaling. Our study uncovers diverse mutational processes underlying the transition from early adenoma to cancer. This has broad implications for biomarker-driven targeting of CEA/TGF-β in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression. PMID:27100181

  10. Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis.

    PubMed

    Ang, Yen-Sin; Rivas, Renee N; Ribeiro, Alexandre J S; Srivas, Rohith; Rivera, Janell; Stone, Nicole R; Pratt, Karishma; Mohamed, Tamer M A; Fu, Ji-Dong; Spencer, C Ian; Tippens, Nathaniel D; Li, Molong; Narasimha, Anil; Radzinsky, Ethan; Moon-Grady, Anita J; Yu, Haiyuan; Pruitt, Beth L; Snyder, Michael P; Srivastava, Deepak

    2016-12-15

    Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.

  11. Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces

    PubMed Central

    Engin, H. Billur; Kreisberg, Jason F.; Carter, Hannah

    2016-01-01

    Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10−4) and oncogenes (Odds Ratio 1.17, P-value < 10−3). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10−8). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer

  12. Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces.

    PubMed

    Engin, H Billur; Kreisberg, Jason F; Carter, Hannah

    2016-01-01

    Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10(-4)) and oncogenes (Odds Ratio 1.17, P-value < 10(-3)). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10(-8)). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer

  13. Targeted next-generation sequencing of candidate genes reveals novel mutations in patients with dilated cardiomyopathy

    PubMed Central

    ZHAO, YUE; FENG, YUE; ZHANG, YUN-MEI; DING, XIAO-XUE; SONG, YU-ZHU; ZHANG, A-MEI; LIU, LI; ZHANG, HONG; DING, JIA-HUAN; XIA, XUE-SHAN

    2015-01-01

    Dilated cardiomyopathy (DCM) is a major cause of sudden cardiac death and heart failure, and it is characterized by genetic and clinical heterogeneity, even for some patients with a very poor clinical prognosis; in the majority of cases, DCM necessitates a heart transplant. Genetic mutations have long been considered to be associated with this disease. At present, mutations in over 50 genes related to DCM have been documented. This study was carried out to elucidate the characteristics of gene mutations in patients with DCM. The candidate genes that may cause DCM include MYBPC3, MYH6, MYH7, LMNA, TNNT2, TNNI3, MYPN, MYL3, TPM1, SCN5A, DES, ACTC1 and RBM20. Using next-generation sequencing (NGS) and subsequent mutation confirmation with traditional capillary Sanger sequencing analysis, possible causative non-synonymous mutations were identified in ~57% (12/21) of patients with DCM. As a result, 7 novel mutations (MYPN, p.E630K; TNNT2, p.G180A; MYH6, p.R1047C; TNNC1, p.D3V; DES, p.R386H; MYBPC3, p.C1124F; and MYL3, p.D126G), 3 variants of uncertain significance (RBM20, p.R1182H; MYH6, p.T1253M; and VCL, p.M209L), and 2 known mutations (MYH7, p.A26V and MYBPC3, p.R160W) were revealed to be associated with DCM. The mutations were most frequently found in the sarcomere (MYH6, MYBPC3, MYH7, TNNC1, TNNT2 and MYL3) and cytoskeletal (MYPN, DES and VCL) genes. As genetic testing is a useful tool in the clinical management of disease, testing for pathogenic mutations is beneficial to the treatment of patients with DCM and may assist in predicting disease risk for their family members before the onset of symptoms. PMID:26458567

  14. Progeria syndromes and ageing: what is the connection?

    PubMed

    Burtner, Christopher R; Kennedy, Brian K

    2010-08-01

    One of the many debated topics in ageing research is whether progeroid syndromes are really accelerated forms of human ageing. The answer requires a better understanding of the normal ageing process and the molecular pathology underlying these rare diseases. Exciting recent findings regarding a severe human progeria, Hutchinson-Gilford progeria syndrome, have implicated molecular changes that are also linked to normal ageing, such as genome instability, telomere attrition, premature senescence and defective stem cell homeostasis in disease development. These observations, coupled with genetic studies of longevity, lead to a hypothesis whereby progeria syndromes accelerate a subset of the pathological changes that together drive the normal ageing process.

  15. A ceRNA analysis on LMNA gene focusing on the Hutchinson-Gilford progeria syndrome

    PubMed Central

    2013-01-01

    Background Hutchinson-Gilford progeria syndrome is a rare dominant human disease of genetic origin. The average life expectancy is about 20 years, patients’ life quality is still very poor and no efficient therapy has yet been developed. It is caused by mutation of the LMNA gene, which results in accumulation in the nuclear membrane of a particular splicing form of Lamin-A called progerin. The mechanism by which progerin perturbs cellular homeostasis and leads to the symptoms is still under debate. Micro-RNAs are able to negatively regulate transcription by coupling with the 3’ UnTranslated Region of messenger RNAs. Several Micro-RNAs recognize the same 3’ UnTranslated Region and each Micro-RNA can recognize multiple 3’ UnTranslated Regions of different messenger RNAs. When different messenger RNAs are co-regulated via a similar panel of micro-RNAs, these messengers are called Competing Endogenous RNAs, or ceRNAs. The 3’ UnTranslated Region of the longest LMNA transcript was analysed looking for its ceRNAs. The aim of this study was to search for candidate genes and gene ontology functions possibly influenced by LMNA mutations that may exert a role in progeria development. Results 11 miRNAs were isolated as potential LMNA regulators. By computational analysis, the miRNAs pointed to 17 putative LMNA ceRNAs. Gene ontology analysis of isolated ceRNAs showed an enrichment in RNA interference and control of cell cycle functions. Conclusion This study isolated novel genes and functions potentially involved in LMNA network of regulation that could be involved in laminopathies such as the Hutchinson-Gilford progeria syndrome. PMID:23317481

  16. Permanent farnesylation of lamin A mutants linked to progeria impairs its phosphorylation at serine 22 during interphase.

    PubMed

    Moiseeva, Olga; Lopes-Paciencia, Stéphane; Huot, Geneviève; Lessard, Frédéric; Ferbeyre, Gerardo

    2016-02-01

    Mutants of lamin A cause diseases including the Hutchinson-Gilford progeria syndrome (HGPS) characterized by premature aging. Lamin A undergoes a series of processing reactions, including farnesylation and proteolytic cleavage of the farnesylated C-terminal domain. The role of cleavage is unknown but mutations that affect this reaction lead to progeria. Here we show that interphase serine 22 phosphorylation of endogenous mutant lamin A (progerin) is defective in cells from HGPS patients. This defect can be mimicked by expressing progerin in human cells and prevented by inhibition of farnesylation. Furthermore, serine 22 phosphorylation of non-farnesylated progerin was enhanced by a mutation that disrupts lamin A head to tail interactions. The phosphorylation of lamin A or non-farnesylated progerin was associated to the formation of spherical intranuclear lamin A droplets that accumulate protein kinases of the CDK family capable of phosphorylating lamin A at serine 22. CDK inhibitors compromised the turnover of progerin, accelerated senescence of HGPS cells and reversed the effects of FTI on progerin levels. We discuss a model of progeria where faulty serine 22 phosphorylation compromises phase separation of lamin A polymers, leading to accumulation of functionally impaired lamin A structures.

  17. Permanent farnesylation of lamin A mutants linked to progeria impairs its phosphorylation at serine 22 during interphase

    PubMed Central

    Moiseeva, Olga; Lopes-Paciencia, Stéphane; Huot, Geneviève; Lessard, Frédéric; Ferbeyre, Gerardo

    2016-01-01

    Mutants of lamin A cause diseases including the Hutchinson-Gilford progeria syndrome (HGPS) characterized by premature aging. Lamin A undergoes a series of processing reactions, including farnesylation and proteolytic cleavage of the farnesylated C-terminal domain. The role of cleavage is unknown but mutations that affect this reaction lead to progeria. Here we show that interphase serine 22 phosphorylation of endogenous mutant lamin A (progerin) is defective in cells from HGPS patients. This defect can be mimicked by expressing progerin in human cells and prevented by inhibition of farnesylation. Furthermore, serine 22 phosphorylation of non-farnesylated progerin was enhanced by a mutation that disrupts lamin A head to tail interactions. The phosphorylation of lamin A or non-farnesylated progerin was associated to the formation of spherical intranuclear lamin A droplets that accumulate protein kinases of the CDK family capable of phosphorylating lamin A at serine 22. CDK inhibitors compromised the turnover of progerin, accelerated senescence of HGPS cells and reversed the effects of FTI on progerin levels. We discuss a model of progeria where faulty serine 22 phosphorylation compromises phase separation of lamin A polymers, leading to accumulation of functionally impaired lamin A structures. PMID:26922519

  18. Progeria: translational insights from cell biology.

    PubMed

    Gordon, Leslie B; Cao, Kan; Collins, Francis S

    2012-10-01

    Cell biologists love to think outside the box, pursuing many surprising twists and unexpected turns in their quest to unravel the mysteries of how cells work. But can cell biologists think outside the bench? We are certain that they can, and clearly some already do. To encourage more cell biologists to venture into the realm of translational research on a regular basis, we would like to share a handful of the many lessons that we have learned in our effort to develop experimental treatments for Hutchinson-Gilford progeria syndrome (HGPS), an endeavor that many view as a "poster child" for how basic cell biology can be translated to the clinic.

  19. An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria.

    PubMed

    Andressoo, Jaan-Olle; Mitchell, James R; de Wit, Jan; Hoogstraten, Deborah; Volker, Marcel; Toussaint, Wendy; Speksnijder, Ewoud; Beems, Rudolph B; van Steeg, Harry; Jans, Judith; de Zeeuw, Chris I; Jaspers, Nicolaas G J; Raams, Anja; Lehmann, Alan R; Vermeulen, Wim; Hoeijmakers, Jan H J; van der Horst, Gijsbertus T J

    2006-08-01

    Inborn defects in nucleotide excision DNA repair (NER) can paradoxically result in elevated cancer incidence (xeroderma pigmentosum [XP]) or segmental progeria without cancer predisposition (Cockayne syndrome [CS] and trichothiodystrophy [TTD]). We report generation of a knockin mouse model for the combined disorder XPCS with a G602D-encoding mutation in the Xpd helicase gene. XPCS mice are the most skin cancer-prone NER model to date, and we postulate an unusual NER dysfunction that is likely responsible for this susceptibility. XPCS mice also displayed symptoms of segmental progeria, including cachexia and progressive loss of germinal epithelium. Like CS fibroblasts, XPCS and TTD fibroblasts from human and mouse showed evidence of defective repair of oxidative DNA lesions that may underlie these segmental progeroid symptoms.

  20. Whole Exome Sequencing Reveals Novel PHEX Splice Site Mutations in Patients with Hypophosphatemic Rickets

    PubMed Central

    Gillies, Christopher; Sampson, Matthew G.; Kher, Vijay; Sethi, Sidharth K.; Otto, Edgar A.

    2015-01-01

    Objective Hypophosphatemic rickets (HR) is a heterogeneous genetic phosphate wasting disorder. The disease is most commonly caused by mutations in the PHEX gene located on the X-chromosome or by mutations in CLCN5, DMP1, ENPP1, FGF23, and SLC34A3. The aims of this study were to perform molecular diagnostics for four patients with HR of Indian origin (two independent families) and to describe their clinical features. Methods We performed whole exome sequencing (WES) for the affected mother of two boys who also displayed the typical features of HR, including bone malformations and phosphate wasting. B-lymphoblast cell lines were established by EBV transformation and subsequent RT-PCR to investigate an uncommon splice site variant found by WES. An in silico analysis was done to obtain accurate nucleotide frequency occurrences of consensus splice positions other than the canonical sites of all human exons. Additionally, we applied direct Sanger sequencing for all exons and exon/intron boundaries of the PHEX gene for an affected girl from an independent second Indian family. Results WES revealed a novel PHEX splice acceptor mutation in intron 9 (c.1080-3C>A) in a family with 3 affected individuals with HR. The effect on splicing of this mutation was further investigated by RT-PCR using RNA obtained from a patient’s EBV-transformed lymphoblast cell line. RT-PCR revealed an aberrant splice transcript skipping exons 10-14 which was not observed in control samples, confirming the diagnosis of X-linked dominant hypophosphatemia (XLH). The in silico analysis of all human splice sites adjacent to all 327,293 exons across 81,814 transcripts among 20,345 human genes revealed that cytosine is, with 64.3%, the most frequent nucleobase at the minus 3 splice acceptor position, followed by thymidine with 28.7%, adenine with 6.3%, and guanine with 0.8%. We generated frequency tables and pictograms for the extended donor and acceptor splice consensus regions by analyzing all human

  1. Clinical and genetic analyses reveal novel pathogenic ABCA4 mutations in Stargardt disease families

    PubMed Central

    Lin, Bing; Cai, Xue-Bi; Zheng, Zhi-Li; Huang, Xiu-Feng; Liu, Xiao-Ling; Qu, Jia; Jin, Zi-Bing

    2016-01-01

    Stargardt disease (STGD1) is a juvenile macular degeneration predominantly inherited in an autosomal recessive pattern, characterized by decreased central vision in the first 2 decades of life. The condition has a genetic basis due to mutation in the ABCA4 gene, and arises from the deposition of lipofuscin-like substance in the retinal pigmented epithelium (RPE) with secondary photoreceptor cell death. In this study, we describe the clinical and genetic features of Stargardt patients from four unrelated Chinese cohorts. The targeted exome sequencing (TES) was carried out in four clinically confirmed patients and their family members using a gene panel comprising 164 known causative inherited retinal dystrophy (IRD) genes. Genetic analysis revealed eight ABCA4 mutations in all of the four pedigrees, including six mutations in coding exons and two mutations in adjacent intronic areas. All the affected individuals showed typical manifestations consistent with the disease phenotype. We disclose two novel ABCA4 mutations in Chinese patients with STGD disease, which will expand the existing spectrum of disease-causing variants and will further aid in the future mutation screening and genetic counseling, as well as in the understanding of phenotypic and genotypic correlations. PMID:27739528

  2. Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria.

    PubMed

    Xiong, Zheng-Mei; Choi, Ji Young; Wang, Kun; Zhang, Haoyue; Tariq, Zeshan; Wu, Di; Ko, Eunae; LaDana, Christina; Sesaki, Hiromi; Cao, Kan

    2016-04-01

    Hutchinson-Gilford progeria syndrome (HGPS), a fatal premature aging disease, is caused by a single-nucleotide mutation in the LMNA gene. Previous reports have focused on nuclear phenotypes in HGPS cells, yet the potential contribution of the mitochondria, a key player in normal aging, remains unclear. Using high-resolution microscopy analysis, we demonstrated a significantly increased fraction of swollen and fragmented mitochondria and a marked reduction in mitochondrial mobility in HGPS fibroblast cells. Notably, the expression of PGC-1α, a central regulator of mitochondrial biogenesis, was inhibited by progerin. To rescue mitochondrial defects, we treated HGPS cells with a mitochondrial-targeting antioxidant methylene blue (MB). Our analysis indicated that MB treatment not only alleviated the mitochondrial defects but also rescued the hallmark nuclear abnormalities in HGPS cells. Additional analysis suggested that MB treatment released progerin from the nuclear membrane, rescued perinuclear heterochromatin loss and corrected misregulated gene expression in HGPS cells. Together, these results demonstrate a role of mitochondrial dysfunction in developing the premature aging phenotypes in HGPS cells and suggest MB as a promising therapeutic approach for HGPS.

  3. Bilateral stenosis of carotid siphon in Hutchinson-Gilford progeria syndrome.

    PubMed

    Narazaki, Ryo; Makimura, Mika; Sanefuji, Masafumi; Fukamachi, Shigeru; Akiyoshi, Hidetaka; So, Hidenori; Yamamura, Kenichiro; Doisaki, Sayoko; Kojima, Seiji; Ihara, Kenji; Hara, Toshiro; Ohga, Shouichi

    2013-08-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease, caused by a de novo mutation of lamin-A gene, LMNA G608G. Accumulation of abnormal lamin-A (progerin) compromises nuclear membrane integrity and results in the accelerated senescence. Affected patients show a typical feature of birdlike face, alopecia, sclerotic skin, loss of subcutaneous fat, and short stature with advancing years. Neonatal scleroderma is the first presentation, although early diagnosis is challenging. The leading cause of death is cardio-/cerebro-vascular accidents associated with atherosclerosis. However, not all findings may recapitulate the aging process. We herein report a 9-year-old Japanese male with HGPS who developed cerebral infarction. The genetic study of peripheral blood-derived DNA determined a heterozygous c.1824C>T mutation, p.G608G. Telomere length of lymphocytes was normal. Bilateral stenosis of carotid siphons was prominent, while systemic arteriosclerosis was unremarkable assessed by the ankle-brachial index, carotid ultrasound imaging and funduscopic study. HGPS patients have marked loss and functional defects in vascular smooth muscle cells, leading to the vulnerability to circulatory stress. Symmetrical stenosis of siphons might occur as a distinctive cerebral vasculopathy of HGPS, rather than simple vascular senescence. Peripheral blood study on LMNA G608G and telomere length could screen progerias in infancy for early therapeutic intervention.

  4. Interruption of progerin–lamin A/C binding ameliorates Hutchinson-Gilford progeria syndrome phenotype

    PubMed Central

    Lee, Su-Jin; Jung, Youn-Sang; Yoon, Min-Ho; Kang, So-mi; Oh, Ah-Young; Lee, Jee-Hyun; Jun, So-Young; Woo, Tae-Gyun; Chun, Ho-Young; Kim, Sang Kyum; Chung, Kyu Jin; Lee, Ho-Young; Lee, Kyeong; Jin, Guanghai; Na, Min-Kyun; Ha, Nam Chul; Bárcena, Clea; Freije, José M.P.; López-Otín, Carlos; Song, Gyu Yong

    2016-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare autosomal dominant genetic disease that is caused by a silent mutation of the LMNA gene encoding lamins A and C (lamin A/C). The G608G mutation generates a more accessible splicing donor site than does WT and produces an alternatively spliced product of LMNA called progerin, which is also expressed in normal aged cells. In this study, we determined that progerin binds directly to lamin A/C and induces profound nuclear aberrations. Given this observation, we performed a random screening of a chemical library and identified 3 compounds (JH1, JH4, and JH13) that efficiently block progerin–lamin A/C binding. These 3 chemicals, particularly JH4, alleviated nuclear deformation and reversed senescence markers characteristic of HGPS cells, including growth arrest and senescence-associated β-gal (SA–β-gal) activity. We then used microarray-based analysis to demonstrate that JH4 is able to rescue defects of cell-cycle progression in both HGPS and aged cells. Furthermore, administration of JH4 to LmnaG609G/G609G-mutant mice, which phenocopy human HGPS, resulted in a marked improvement of several progeria phenotypes and an extended lifespan. Together, these findings indicate that specific inhibitors with the ability to block pathological progerin–lamin A/C binding may represent a promising strategy for improving lifespan and health in both HGPS and normal aging. PMID:27617860

  5. Interruption of progerin-lamin A/C binding ameliorates Hutchinson-Gilford progeria syndrome phenotype.

    PubMed

    Lee, Su-Jin; Jung, Youn-Sang; Yoon, Min-Ho; Kang, So-Mi; Oh, Ah-Young; Lee, Jee-Hyun; Jun, So-Young; Woo, Tae-Gyun; Chun, Ho-Young; Kim, Sang Kyum; Chung, Kyu Jin; Lee, Ho-Young; Lee, Kyeong; Jin, Guanghai; Na, Min-Kyun; Ha, Nam Chul; Bárcena, Clea; Freije, José M P; López-Otín, Carlos; Song, Gyu Yong; Park, Bum-Joon

    2016-10-03

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare autosomal dominant genetic disease that is caused by a silent mutation of the LMNA gene encoding lamins A and C (lamin A/C). The G608G mutation generates a more accessible splicing donor site than does WT and produces an alternatively spliced product of LMNA called progerin, which is also expressed in normal aged cells. In this study, we determined that progerin binds directly to lamin A/C and induces profound nuclear aberrations. Given this observation, we performed a random screening of a chemical library and identified 3 compounds (JH1, JH4, and JH13) that efficiently block progerin-lamin A/C binding. These 3 chemicals, particularly JH4, alleviated nuclear deformation and reversed senescence markers characteristic of HGPS cells, including growth arrest and senescence-associated β-gal (SA-β-gal) activity. We then used microarray-based analysis to demonstrate that JH4 is able to rescue defects of cell-cycle progression in both HGPS and aged cells. Furthermore, administration of JH4 to LmnaG609G/G609G-mutant mice, which phenocopy human HGPS, resulted in a marked improvement of several progeria phenotypes and an extended lifespan. Together, these findings indicate that specific inhibitors with the ability to block pathological progerin-lamin A/C binding may represent a promising strategy for improving lifespan and health in both HGPS and normal aging.

  6. Exome analysis reveals differentially mutated gene signatures of stage, grade and subtype in breast cancers.

    PubMed

    Li, You; Wang, Xiaosheng; Vural, Suleyman; Mishra, Nitish K; Cowan, Kenneth H; Guda, Chittibabu

    2015-01-01

    Breast cancers exhibit highly heterogeneous molecular profiles. Although gene expression profiles have been used to predict the risks and prognostic outcomes of breast cancers, the high variability of gene expression limits its clinical application. In contrast, genetic mutation profiles would be more advantageous than gene expression profiles because genetic mutations can be stably detected and the mutational heterogeneity widely exists in breast cancer genomes. We analyzed 98 breast cancer whole exome samples that were sorted into three subtypes, two grades and two stages. The sum deleterious effect of all mutations in each gene was scored to identify differentially mutated genes (DMGs) for this case-control study. DMGs were corroborated using extensive published knowledge. Functional consequences of deleterious SNVs on protein structure and function were also investigated. Genes such as ERBB2, ESP8, PPP2R4, KIAA0922, SP4, CENPJ, PRCP and SELP that have been experimentally or clinically verified to be tightly associated with breast cancer prognosis are among the DMGs identified in this study. We also identified some genes such as ARL6IP5, RAET1E, and ANO7 that could be crucial for breast cancer development and prognosis. Further, SNVs such as rs1058808, rs2480452, rs61751507, rs79167802, rs11540666, and rs2229437 that potentially influence protein functions are observed at significantly different frequencies in different comparison groups. Protein structure modeling revealed that many non-synonymous SNVs have a deleterious effect on protein stability, structure and function. Mutational profiling at gene- and SNV-level revealed differential patterns within each breast cancer comparison group, and the gene signatures correlate with expected prognostic characteristics of breast cancer classes. Some of the genes and SNVs identified in this study show high promise and are worthy of further investigation by experimental studies.

  7. Exome Analysis Reveals Differentially Mutated Gene Signatures of Stage, Grade and Subtype in Breast Cancers

    PubMed Central

    Li, You; Wang, Xiaosheng; Vural, Suleyman; Mishra, Nitish K.; Cowan, Kenneth H.; Guda, Chittibabu

    2015-01-01

    Breast cancers exhibit highly heterogeneous molecular profiles. Although gene expression profiles have been used to predict the risks and prognostic outcomes of breast cancers, the high variability of gene expression limits its clinical application. In contrast, genetic mutation profiles would be more advantageous than gene expression profiles because genetic mutations can be stably detected and the mutational heterogeneity widely exists in breast cancer genomes. We analyzed 98 breast cancer whole exome samples that were sorted into three subtypes, two grades and two stages. The sum deleterious effect of all mutations in each gene was scored to identify differentially mutated genes (DMGs) for this case-control study. DMGs were corroborated using extensive published knowledge. Functional consequences of deleterious SNVs on protein structure and function were also investigated. Genes such as ERBB2, ESP8, PPP2R4, KIAA0922, SP4, CENPJ, PRCP and SELP that have been experimentally or clinically verified to be tightly associated with breast cancer prognosis are among the DMGs identified in this study. We also identified some genes such as ARL6IP5, RAET1E, and ANO7 that could be crucial for breast cancer development and prognosis. Further, SNVs such as rs1058808, rs2480452, rs61751507, rs79167802, rs11540666, and rs2229437 that potentially influence protein functions are observed at significantly different frequencies in different comparison groups. Protein structure modeling revealed that many non-synonymous SNVs have a deleterious effect on protein stability, structure and function. Mutational profiling at gene- and SNV-level revealed differential patterns within each breast cancer comparison group, and the gene signatures correlate with expected prognostic characteristics of breast cancer classes. Some of the genes and SNVs identified in this study show high promise and are worthy of further investigation by experimental studies. PMID:25803781

  8. DNA fingerprinting reveals elevated mutation rates in herring gulls inhabiting a genotoxically contaminated site

    SciTech Connect

    Yauk, C.L.; Quinn, J.S.

    1995-12-31

    The authors used multi-locus DNA fingerprinting to examine families of herring gulls (Larus argentatus) from a genotoxically contaminated site (Hamilton Harbour) and from a pristine location (Kent Island, Bay of Fundy) to show significant differences in mutation rates between the locations. Overall the authors identified 17 mutant bands from 15 individuals of the 35 examined from Hamilton Harbour, and 7 mutant fragments from 7 individuals, of the 43 examined from Kent Island; a mutation frequency of 0.429 per nestling for Hamilton Harbour and 0.163 for Kent Island. The total number of individuals with mutant bands was significantly higher at Hamilton Harbour than at Kent Island (X{sup 2}=6.734; df = 1; P < 0.01). Ongoing analysis of other less contaminated sites also reveals lower mutation rates than those seen in Hamilton Harbour. With multi-locus DNA fingerprinting many regions of the genome can be surveyed simultaneously. The tandemly repeated arrays of nucleotides examined with DNA fingerprinting are known to have elevated rates of mutation. Furthermore, the mutations seen with DNA fingerprinting are predominantly heritable. Other biomarkers currently used in situ are not able to monitor direct and heritable DNA mutation, or measure biological endpoints that frequently result in spontaneous abortion creating difficulty in observing significantly elevated levels in viable offspring. The authors suggest that multilocus DNA fingerprinting can be used as a biomarker to identify potentially heritable risks before the onset of other types of ecological damage. This approach provides a direct measure of mutation in situ and in vivo in a vertebrate species under ambient conditions.

  9. Inherited cobalamin malabsorption. Mutations in three genes reveal functional and ethnic patterns

    PubMed Central

    2012-01-01

    Background Inherited malabsorption of cobalamin (Cbl) causes hematological and neurological abnormalities that can be fatal. Three genes have been implicated in Cbl malabsorption; yet, only about 10% of ~400-500 reported cases have been molecularly studied to date. Recessive mutations in CUBN or AMN cause Imerslund-Gräsbeck Syndrome (IGS), while recessive mutations in GIF cause Intrinsic Factor Deficiency (IFD). IGS and IFD differ in that IGS usually presents with proteinuria, which is not observed in IFD. The genetic heterogeneity and numerous differential diagnoses make clinical assessment difficult. Methods We present a large genetic screening study of 154 families or patients with suspected hereditary Cbl malabsorption. Patients and their families have been accrued over a period spanning >12 years. Systematic genetic testing of the three genes CUBN, AMN, and GIF was accomplished using a combination of single strand conformation polymorphism and DNA and RNA sequencing. In addition, six genes that were contenders for a role in inherited Cbl malabsorption were studied in a subset of these patients. Results Our results revealed population-specific mutations, mutational hotspots, and functionally distinct regions in the three causal genes. We identified mutations in 126/154 unrelated cases (82%). Fifty-three of 126 cases (42%) were mutated in CUBN, 45/126 (36%) were mutated in AMN, and 28/126 (22%) had mutations in GIF. We found 26 undescribed mutations in CUBN, 19 in AMN, and 7 in GIF for a total of 52 novel defects described herein. We excluded six other candidate genes as culprits and concluded that additional genes might be involved. Conclusions Cbl malabsorption is found worldwide and genetically complex. However, our results indicate that population-specific founder mutations are quite common. Consequently, targeted genetic testing has become feasible if ethnic ancestry is considered. These results will facilitate clinical and molecular genetic testing of

  10. Analysis of TP53 mutation spectra reveals the fingerprint of the potent environmental carcinogen, aristolochic acid.

    PubMed

    Hollstein, M; Moriya, M; Grollman, A P; Olivier, M

    2013-01-01

    Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the pieces of evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterized by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbor human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk.

  11. Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

    PubMed

    Shinbrot, Eve; Henninger, Erin E; Weinhold, Nils; Covington, Kyle R; Göksenin, A Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M; Gibbs, Richard A; Sander, Chris; Pursell, Zachary F; Wheeler, David A

    2014-11-01

    Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.

  12. Depleting the methyltransferase Suv39h1 improves DNA repair and extends lifespan in a progeria mouse model.

    PubMed

    Liu, Baohua; Wang, Zimei; Zhang, Le; Ghosh, Shrestha; Zheng, Huiling; Zhou, Zhongjun

    2013-01-01

    A de novo G608G mutation in LMNA gene leads to Hutchinson-Gilford progeria syndrome. Mice lacking the prelamin A-processing metalloprotease, Zmpste24, recapitulate many of the progeroid features of Hutchinson-Gilford progeria syndrome. Here we show that A-type lamins interact with SUV39H1, and prelamin A/progerin exhibits enhanced binding capacity to SUV39H1, protecting it from proteasomal degradation and, consequently, increasing H3K9me3 levels. Depletion of Suv39h1 reduces H3K9me3 levels, restores DNA repair capacity and delays senescence in progeroid cells. Remarkably, loss of Suv39h1 in Zmpste24(-/-) mice delays body weight loss, increases bone mineral density and extends lifespan by ∼60%. Thus, increased H3K9me3 levels, possibly mediated by enhanced Suv39h1 stability in the presence of prelamin A/progerin, compromise genome maintenance, which in turn contributes to accelerated senescence in laminopathy-based premature aging. Our study provides an explanation for epigenetic alterations in Hutchinson-Gilford progeria syndrome and a potential strategy for intervention by targeting SUV39H1-mediated heterochromatin remodelling.

  13. Novel MYH11 and ACTA2 mutations reveal a role for enhanced TGFβ signaling in FTAAD

    PubMed Central

    Renard, Marjolijn; Callewaert, Bert; Baetens, Machteld; Campens, Laurence; MacDermot, Kay; Fryns, Jean-Pierre; Bonduelle, Maryse; Dietz, Hal; Gaspar, Isabel Mendes; Cavaco, Diogo; Stattin, Eva-Lena; Schrander-Stumpel, Constance; Coucke, Paul; Loeys, Bart; De Paepe, Anne; De Backer, Julie

    2011-01-01

    Background Thoracic aortic aneurysm / dissection (TAAD) is a common phenotype that may occur as an isolated manifestation or within the constellation of a defined syndrome. In contrast to syndromic TAAD, the elucidation of the genetic basis of isolated TAAD has only recently started. To date, defects have been found in genes encoding extracellular matrix proteins (fibrillin-1, FBN1; collagen type III alpha 1, COL3A1), proteins involved in transforming growth factor beta (TGFβ) signaling (TGFβ receptor 1 and 2, TGFBR1/2; and SMAD3) or proteins that build up the contractile apparatus of aortic smooth muscle cells (myosin heavy chain 11, MYH11; smooth muscle actin alpha 2, ACTA2; and MYLK). Methods and results In 110 non-syndromic TAAD patients that previously tested negative for FBN1 or TGFBR1/2 mutations, we identified 7 ACTA2 mutations in a cohort of 43 familial TAAD patients, including 2 premature truncating mutations. Sequencing of MYH11 revealed an in frame splice-site alteration in one out of two probands with TAA(D) associated with PDA but none in the series of 22 probands from the cohort of 110 patients with non-syndromic TAAD. Interestingly, immunohistochemical staining of aortic biopsies of a patient and a family member with MYH11 and patients with ACTA2 missense mutations showed upregulation of the TGFβ signaling pathway. Conclusions MYH11 mutations are rare and typically identified in patients with TAAD associated with PDA. ACTA2 mutations were identified in 16% of a cohort presenting familial TAAD. Different molecular defects in TAAD may account for a different pathogenic mechanism of enhanced TGFβ signaling. PMID:21937134

  14. Absence of progeria-like disease phenotypes in knock-in mice expressing a non-farnesylated version of progerin.

    PubMed

    Yang, Shao H; Chang, Sandy Y; Ren, Shuxun; Wang, Yibin; Andres, Douglas A; Spielmann, H Peter; Fong, Loren G; Young, Stephen G

    2011-02-01

    Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutant prelamin A, progerin, that terminates with a farnesylcysteine. HGPS knock-in mice (Lmna(HG/+)) develop severe progeria-like disease phenotypes. These phenotypes can be ameliorated with a protein farnesyltransferase inhibitor (FTI), suggesting that progerin's farnesyl lipid is important for disease pathogenesis and raising the possibility that FTIs could be useful for treating humans with HGPS. Subsequent studies showed that mice expressing non-farnesylated progerin (Lmna(nHG/+) mice, in which progerin's carboxyl-terminal -CSIM motif was changed to -SSIM) also develop severe progeria, raising doubts about whether any treatment targeting protein prenylation would be particularly effective. We suspected that those doubts might be premature and hypothesized that the persistent disease in Lmna(nHG/+) mice could be an unanticipated consequence of the cysteine-to-serine substitution that was used to eliminate farnesylation. To test this hypothesis, we generated a second knock-in allele yielding non-farnesylated progerin (Lmna(csmHG)) in which the carboxyl-terminal -CSIM motif was changed to -CSM. We then compared disease phenotypes in mice harboring the Lmna(nHG) or Lmna(csmHG) allele. As expected, Lmna(nHG/+) and Lmna(nHG/nHG) mice developed severe progeria-like disease phenotypes, including osteolytic lesions and rib fractures, osteoporosis, slow growth and reduced survival. In contrast, Lmna(csmHG/+) and Lmna(csmHG/csmHG) mice exhibited no bone disease and displayed entirely normal body weights and survival. The frequencies of misshapen cell nuclei were lower in Lmna(csmHG/+) and Lmna(csmHG/csmHG) fibroblasts. These studies show that the ability of non-farnesylated progerin to elicit disease depends on the carboxyl-terminal mutation used to eliminate protein prenylation.

  15. DNA repair defects and genome instability in Hutchinson-Gilford Progeria Syndrome.

    PubMed

    Gonzalo, Susana; Kreienkamp, Ray

    2015-06-01

    The integrity of the nuclear lamina has emerged as an important factor in the maintenance of genome stability. In particular, mutations in the LMNA gene, encoding A-type lamins (lamin A/C), alter nuclear morphology and function, and cause genomic instability. LMNA gene mutations are associated with a variety of degenerative diseases and devastating premature aging syndromes such as Hutchinson-Gilford Progeria Syndrome (HGPS) and Restrictive Dermopathy (RD). HGPS is a severe laminopathy, with patients dying in their teens from myocardial infarction or stroke. HGPS patient-derived cells exhibit nuclear shape abnormalities, changes in epigenetic regulation and gene expression, telomere shortening, genome instability, and premature senescence. This review highlights recent advances in identifying molecular mechanisms that contribute to the pathophysiology of HGPS, with a special emphasis on DNA repair defects and genome instability.

  16. DNA repair defects and genome instability in Hutchinson-Gilford Progeria Syndrome

    PubMed Central

    Gonzalo, Susana; Kreienkamp, Ray

    2015-01-01

    The integrity of the nuclear lamina has emerged as an important factor in the maintenance of genome stability. In particular, mutations in the LMNA gene, encoding A-type lamins (lamin A/C), alter nuclear morphology and function, and cause genomic instability. LMNA gene mutations are associated with a variety of degenerative diseases and devastating premature aging syndromes such as Hutchinson-Gilford Progeria Syndrome (HGPS) and Restrictive Dermopathy (RD). HGPS is a severe laminopathy, with patients dying in their teens from myocardial infarction or stroke. HGPS patient-derived cells exhibit nuclear shape abnormalities, changes in epigenetic regulation and gene expression, telomere shortening, genome instability, and premature senescence. This review highlights recent advances in identifying molecular mechanisms that contribute to the pathophysiology of HGPS, with a special emphasis on DNA repair defects and genome instability. PMID:26079711

  17. Progeria: Translational insights from cell biology

    PubMed Central

    Gordon, Leslie B.; Cao, Kan

    2012-01-01

    Cell biologists love to think outside the box, pursuing many surprising twists and unexpected turns in their quest to unravel the mysteries of how cells work. But can cell biologists think outside the bench? We are certain that they can, and clearly some already do. To encourage more cell biologists to venture into the realm of translational research on a regular basis, we would like to share a handful of the many lessons that we have learned in our effort to develop experimental treatments for Hutchinson-Gilford progeria syndrome (HGPS), an endeavor that many view as a “poster child” for how basic cell biology can be translated to the clinic. PMID:23027899

  18. Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.

    PubMed

    Romano, Alessandro; Tessa, Alessandra; Barca, Amilcare; Fattori, Fabiana; de Leva, Maria Fulvia; Terracciano, Alessandra; Storelli, Carlo; Santorelli, Filippo Maria; Verri, Tiziano

    2013-03-01

    Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurological disease with mutations in SACS, encoding sacsin, a multidomain protein of 4,579 amino acids. The large size of SACS and its translated protein has hindered biochemical analysis of ARSACS, and how mutant sacsins lead to disease remains largely unknown. Three repeated sequences, called sacsin repeating region (SRR) supradomains, have been recognized, which contribute to sacsin chaperone-like activity. We found that the three SRRs are much larger (≥1,100 residues) than previously described, and organized in discrete subrepeats. We named the large repeated regions Sacsin Internal RePeaTs (SIRPT1, SIRPT2, and SIRPT3) and the subrepeats sr1, sr2, sr3, and srX. Comparative analysis of vertebrate sacsins in combination with fine positional mapping of a set of human mutations revealed that sr1, sr2, sr3, and srX are functional. Notably, the position of the pathogenic mutations in sr1, sr2, sr3, and srX appeared to be related to the severity of the clinical phenotype, as assessed by defining a severity scoring system. Our results suggest that the relative position of mutations in subrepeats will variably influence sacsin dysfunction. The characterization of the specific role of each repeated region will help in developing a comprehensive and integrated pathophysiological model of function for sacsin.

  19. MonoSeq Variant Caller Reveals Novel Mononucleotide Run Indel Mutations in Tumors with Defective DNA Mismatch Repair

    PubMed Central

    Walker, Christopher J.; Miranda, Mario A.; O’Hern, Matthew J.; Blachly, James S.; Moyer, Cassandra L.; Ivanovich, Jennifer; Kroll, Karl W.; Eisfeld, Ann-Kathrin; Sapp, Caroline E.; Mutch, David G.; Cohn, David E.; Bundschuh, Ralf; Goodfellow, Paul J

    2016-01-01

    Next-generation sequencing has revolutionized cancer genetics, but accurately detecting mutations in repetitive DNA sequences, especially mononucleotide runs, remains a challenge. This is a particular concern for tumors with defective mismatch repair (MMR) that accumulate strand-slippage mutations. We developed MonoSeq to improve indel mutation detection in mononucleotide runs, and used MonoSeq to investigate strand-slippage mutations in endometrial cancers, a tumor type that has frequent loss of MMR. We performed extensive Sanger sequencing to validate both clonal and sub-clonal MonoSeq mutation calls. Eighty-one regions containing mononucleotide runs were sequenced in 542 primary endometrial cancers (223 with defective MMR). Our analyses revealed that the overall mutation rate in MMR-deficient tumors was 20–30-fold higher than in MMR normal tumors. MonoSeq analysis identified several previously unreported mutations, including a novel hotspot in an A7 run in the terminal exon of ARID5B.The ARID5B indel mutations were seen in both MMR-deficient and MMR normal tumors, suggesting biologic selection. Analysis of tumor mRNAs revealed the presence of mutant transcripts that could result in translation of neopeptides. Improved detection of mononucleotide run strand-slippage mutations has clear implications for comprehensive mutation detection in tumors with defective MMR. Indel frameshift mutations and the resultant antigenic peptides could help guide immunotherapy strategies. PMID:27346418

  20. MonoSeq Variant Caller Reveals Novel Mononucleotide Run Indel Mutations in Tumors with Defective DNA Mismatch Repair.

    PubMed

    Walker, Christopher J; Miranda, Mario A; O'Hern, Matthew J; Blachly, James S; Moyer, Cassandra L; Ivanovich, Jennifer; Kroll, Karl W; Eisfeld, Ann-Kathrin; Sapp, Caroline E; Mutch, David G; Cohn, David E; Bundschuh, Ralf; Goodfellow, Paul J

    2016-10-01

    Next-generation sequencing has revolutionized cancer genetics, but accurately detecting mutations in repetitive DNA sequences, especially mononucleotide runs, remains a challenge. This is a particular concern for tumors with defective mismatch repair (MMR) that accumulate strand-slippage mutations. We developed MonoSeq to improve indel mutation detection in mononucleotide runs, and used MonoSeq to investigate strand-slippage mutations in endometrial cancers, a tumor type that has frequent loss of MMR. We performed extensive Sanger sequencing to validate both clonal and subclonal MonoSeq mutation calls. Eighty-one regions containing mononucleotide runs were sequenced in 540 primary endometrial cancers (223 with defective MMR). Our analyses revealed that the overall mutation rate in MMR-deficient tumors was 20-30-fold higher than in MMR-normal tumors. MonoSeq analysis identified several previously unreported mutations, including a novel hotspot in an A7 run in the terminal exon of ARID5B.The ARID5B indel mutations were seen in both MMR-deficient and MMR-normal tumors, suggesting biologic selection. The analysis of tumor mRNAs revealed the presence of mutant transcripts that could result in translation of neopeptides. Improved detection of mononucleotide run strand-slippage mutations has clear implications for comprehensive mutation detection in tumors with defective MMR. Indel frameshift mutations and the resultant antigenic peptides could help guide immunotherapy strategies.

  1. In vitro pathological modelling using patient-specific induced pluripotent stem cells: the case of progeria.

    PubMed

    Nissan, Xavier; Blondel, Sophie; Peschanski, Marc

    2011-12-01

    Progeria, also known as HGPS (Hutchinson-Gilford progeria syndrome), is a rare fatal genetic disease characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations in codon 608 (C1804T) of the gene encoding lamins A and C, LMNA, leading to the production of a truncated form of the protein called progerin. Owing to their unique potential to self-renew and to differentiate into any cell types of the organism, pluripotent stem cells offer a unique tool to study molecular and cellular mechanisms related to this global and systemic disease. Recent studies have exploited this potential by generating human induced pluripotent stem cells from HGPS patients' fibroblasts displaying several phenotypic defects characteristic of HGPS such as nuclear abnormalities, progerin expression, altered DNA-repair mechanisms and premature senescence. Altogether, these findings provide new insights on the use of pluripotent stem cells for pathological modelling and may open original therapeutic perspectives for diseases that lack pre-clinical in vitro human models, such as HGPS.

  2. Hutchinson-Gilford progeria is a skeletal dysplasia.

    PubMed

    Gordon, Catherine M; Gordon, Leslie B; Snyder, Brian D; Nazarian, Ara; Quinn, Nicolle; Huh, Susanna; Giobbie-Hurder, Anita; Neuberg, Donna; Cleveland, Robert; Kleinman, Monica; Miller, David T; Kieran, Mark W

    2011-07-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare segmental premature aging disorder that affects bone and body composition, among other tissues. We sought to determine whether bone density and structural geometry are altered in children with HGPS and whether relationships exist among these parameters and measures of skeletal anthropometry, body composition, and nutrition. We prospectively enrolled 26 children with HGPS (ages 3.1 to 16.2 years). Outcomes included anthropometric data; bone age; areal bone mineral density (aBMD) and body composition by dual-energy X-ray absorptiometry (DXA); volumetric bone mineral density (vBMD), strength-strain index (SSI), and bone structural rigidity calculated from radial transaxial peripheral quantitative computed tomographic (pQCT) images; serum bone biomarkers and hormonal measures; and nutrition assessments. Children with HGPS had low axial aBMD Z-scores by DXA, which improved after adjustment for height age, whereas differences in radial vBMD by pQCT were less striking. However, pQCT revealed distinct abnormalities in both novel measures of bone structural geometry and skeletal strength at the radius compared with healthy controls. Dietary intake was adequate, confirming that HGPS does not represent a model of malnutrition-induced bone loss. Taken together, these findings suggest that the phenotype of HGPS represents a unique skeletal dysplasia.

  3. A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation.

    PubMed

    Potorac, Iulia; Rivero-Müller, Adolfo; Trehan, Ashutosh; Kiełbus, Michał; Jozwiak, Krzysztof; Pralong, Francois; Hafidi, Aicha; Thiry, Albert; Ménagé, Jean-Jacques; Huhtaniemi, Ilpo; Beckers, Albert; Daly, Adrian F

    2016-12-01

    Glycoprotein hormones are complex hormonally active macromolecules. Luteinizing hormone (LH) is essential for the postnatal development and maturation of the male gonad. Inactivating Luteinizing hormone beta (LHB) gene mutations are exceptionally rare and lead to hypogonadism that is particularly severe in males. We describe a family with selective LH deficiency and hypogonadism in two brothers. DNA sequencing of LHB was performed and the effects of genetic variants on hormone function and secretion were characterized by mutagenesis studies, confocal microscopy and functional assays. A 20-year-old male from a consanguineous family had pubertal delay, hypogonadism and undetectable LH. A homozygous c.118_120del (p.Lys40del) mutation was identified in the patient and his brother, who subsequently had the same phenotype. Treatment with hCG led to pubertal development, increased circulating testosterone and spermatogenesis. Experiments in HeLa cells revealed that the mutant LH is retained intracellularly and showed diffuse cytoplasmic distribution. The mutated LHB heterodimerizes with the common alpha-subunit and can activate its receptor. Deletion of flanking glutamic acid residues at positions 39 and 41 impair LH to a similar extent as deletion of Lys40. This region is functionally important across all heterodimeric glycoprotein hormones, because deletion of the corresponding residues in hCG, follicle-stimulating hormone and thyroid-stimulating hormone beta-subunits also led to intracellular hormone retention. This novel LHB mutation results in hypogonadism due to intracellular sequestration of the hormone and reveals a discrete region in the protein that is crucial for normal secretion of all human glycoprotein hormones.

  4. Genetic Background and Environment Influence the Effects of Mutations in pykF and Help Reveal Mechanisms Underlying Their Benefit

    DTIC Science & Technology

    2015-08-01

    authorized to U.S. Government agencies only. Genetic Background and Environment Influence the Effects of Mutations in pykF and Help Reveal Mechanisms...OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. University of Canterbury 20 Kirkwood Ave Ilam - ABSTRACT Genetic Background...were evolved. Differences in mutation × background interactions were found to be driven by different suites of mutations in each genetic background

  5. The mutational spectrum in Treacher Collins syndrome reveals a predominance of mutations that create a premature-termination codon

    SciTech Connect

    Edwards, S.J.; Gladwin, A.J.; Dixon, M.J.

    1997-03-01

    Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. The TCS locus has been mapped to human chromosome 5q31.3-32 and the mutated gene identified. In the current investigation, 25 previously undescribed mutations, which are spread throughout the gene, are presented. This brings the total reported to date to 35, which represents a detection rate of 60%. Of the mutations that have been reported to date, all but one result in the introduction of a premature-termination codon into the predicted protein, treacle. Moreover, the mutations are largely family specific, although a common 5-bp deletion in exon 24 (seven different families) and a recurrent splicing mutation in intron 3 (two different families) have been identified. This mutational spectrum supports the hypothesis that TCS results from haploin-sufficiency. 49 refs., 4 figs., 3 tabs.

  6. History of plastid DNA insertions reveals weak deletion and at mutation biases in angiosperm mitochondrial genomes.

    PubMed

    Sloan, Daniel B; Wu, Zhiqiang

    2014-11-21

    Angiosperm mitochondrial genomes exhibit many unusual properties, including heterogeneous nucleotide composition and exceptionally large and variable genome sizes. Determining the role of nonadaptive mechanisms such as mutation bias in shaping the molecular evolution of these unique genomes has proven challenging because their dynamic structures generally prevent identification of homologous intergenic sequences for comparative analyses. Here, we report an analysis of angiosperm mitochondrial DNA sequences that are derived from inserted plastid DNA (mtpts). The availability of numerous completely sequenced plastid genomes allows us to infer the evolutionary history of these insertions, including the specific nucleotide substitutions and indels that have occurred because their incorporation into the mitochondrial genome. Our analysis confirmed that many mtpts have a complex history, including frequent gene conversion and multiple examples of horizontal transfer between divergent angiosperm lineages. Nevertheless, it is clear that the majority of extant mtpt sequence in angiosperms is the product of recent transfer (or gene conversion) and is subject to rapid loss/deterioration, suggesting that most mtpts are evolving relatively free from functional constraint. The evolution of mtpt sequences reveals a pattern of biased mutational input in angiosperm mitochondrial genomes, including an excess of small deletions over insertions and a skew toward nucleotide substitutions that increase AT content. However, these mutation biases are far weaker than have been observed in many other cellular genomes, providing insight into some of the notable features of angiosperm mitochondrial architecture, including the retention of large intergenic regions and the relatively neutral GC content found in these regions.

  7. Mutational activation of ErbB2 reveals a new protein kinase autoinhibition mechanism.

    PubMed

    Fan, Ying-Xin; Wong, Lily; Ding, Jinhui; Spiridonov, Nikolay A; Johnson, Richard C; Johnson, Gibbes R

    2008-01-18

    Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.

  8. Expression of progerin in aging mouse brains reveals structural nuclear abnormalities without detectible significant alterations in gene expression, hippocampal stem cells or behavior.

    PubMed

    Baek, Jean-Ha; Schmidt, Eva; Viceconte, Nikenza; Strandgren, Charlotte; Pernold, Karin; Richard, Thibaud J C; Van Leeuwen, Fred W; Dantuma, Nico P; Damberg, Peter; Hultenby, Kjell; Ulfhake, Brun; Mugnaini, Enrico; Rozell, Björn; Eriksson, Maria

    2015-03-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a segmental progeroid syndrome with multiple features suggestive of premature accelerated aging. Accumulation of progerin is thought to underlie the pathophysiology of HGPS. However, despite ubiquitous expression of lamin A in all differentiated cells, the HGPS mutation results in organ-specific defects. For example, bone and skin are strongly affected by HGPS, while the brain appears to be unaffected. There are no definite explanations as to the variable sensitivity to progeria disease among different organs. In addition, low levels of progerin have also been found in several tissues from normal individuals, but it is not clear if low levels of progerin contribute to the aging of the brain. In an attempt to clarify the origin of this phenomenon, we have developed an inducible transgenic mouse model with expression of the most common HGPS mutation in brain, skin, bone and heart to investigate how the mutation affects these organs. Ultrastructural analysis of neuronal nuclei after 70 weeks of expression of the LMNA c.1824C>T mutation showed severe distortion with multiple lobulations and irregular extensions. Despite severe distortions in the nuclei of hippocampal neurons of HGPS animals, there were only negligible changes in gene expression after 63 weeks of transgenic expression. Behavioral analysis and neurogenesis assays, following long-term expression of the HGPS mutation, did not reveal significant pathology. Our results suggest that certain tissues are protected from functional deleterious effects of progerin.

  9. Expression of progerin in aging mouse brains reveals structural nuclear abnormalities without detectible significant alterations in gene expression, hippocampal stem cells or behavior

    PubMed Central

    Baek, Jean-Ha; Schmidt, Eva; Viceconte, Nikenza; Strandgren, Charlotte; Pernold, Karin; Richard, Thibaud J. C.; Van Leeuwen, Fred W.; Dantuma, Nico P.; Damberg, Peter; Hultenby, Kjell; Ulfhake, Brun; Mugnaini, Enrico; Rozell, Björn; Eriksson, Maria

    2015-01-01

    Hutchinson–Gilford progeria syndrome (HGPS) is a segmental progeroid syndrome with multiple features suggestive of premature accelerated aging. Accumulation of progerin is thought to underlie the pathophysiology of HGPS. However, despite ubiquitous expression of lamin A in all differentiated cells, the HGPS mutation results in organ-specific defects. For example, bone and skin are strongly affected by HGPS, while the brain appears to be unaffected. There are no definite explanations as to the variable sensitivity to progeria disease among different organs. In addition, low levels of progerin have also been found in several tissues from normal individuals, but it is not clear if low levels of progerin contribute to the aging of the brain. In an attempt to clarify the origin of this phenomenon, we have developed an inducible transgenic mouse model with expression of the most common HGPS mutation in brain, skin, bone and heart to investigate how the mutation affects these organs. Ultrastructural analysis of neuronal nuclei after 70 weeks of expression of the LMNA c.1824C>T mutation showed severe distortion with multiple lobulations and irregular extensions. Despite severe distortions in the nuclei of hippocampal neurons of HGPS animals, there were only negligible changes in gene expression after 63 weeks of transgenic expression. Behavioral analysis and neurogenesis assays, following long-term expression of the HGPS mutation, did not reveal significant pathology. Our results suggest that certain tissues are protected from functional deleterious effects of progerin. PMID:25343989

  10. Whole-genome sequencing of bladder cancers reveals somatic CDKN1A mutations and clinicopathological associations with mutation burden.

    PubMed

    Cazier, J-B; Rao, S R; McLean, C M; Walker, A K; Walker, A L; Wright, B J; Jaeger, E E M; Kartsonaki, C; Marsden, L; Yau, C; Camps, C; Kaisaki, P; Taylor, J; Catto, J W; Tomlinson, I P M; Kiltie, A E; Hamdy, F C

    2014-04-29

    Bladder cancers are a leading cause of death from malignancy. Molecular markers might predict disease progression and behaviour more accurately than the available prognostic factors. Here we use whole-genome sequencing to identify somatic mutations and chromosomal changes in 14 bladder cancers of different grades and stages. As well as detecting the known bladder cancer driver mutations, we report the identification of recurrent protein-inactivating mutations in CDKN1A and FAT1. The former are not mutually exclusive with TP53 mutations or MDM2 amplification, showing that CDKN1A dysfunction is not simply an alternative mechanism for p53 pathway inactivation. We find strong positive associations between higher tumour stage/grade and greater clonal diversity, the number of somatic mutations and the burden of copy number changes. In principle, the identification of sub-clones with greater diversity and/or mutation burden within early-stage or low-grade tumours could identify lesions with a high risk of invasive progression.

  11. Mutational screening of the USH2A gene in Spanish USH patients reveals 23 novel pathogenic mutations

    PubMed Central

    2011-01-01

    Background Usher Syndrome type II (USH2) is an autosomal recessive disorder, characterized by moderate to severe hearing impairment and retinitis pigmentosa (RP). Among the three genes implicated, mutations in the USH2A gene account for 74-90% of the USH2 cases. Methods To identify the genetic cause of the disease and determine the frequency of USH2A mutations in a cohort of 88 unrelated USH Spanish patients, we carried out a mutation screening of the 72 coding exons of this gene by direct sequencing. Moreover, we performed functional minigene studies for those changes that were predicted to affect splicing. Results As a result, a total of 144 DNA sequence variants were identified. Based upon previous studies, allele frequencies, segregation analysis, bioinformatics' predictions and in vitro experiments, 37 variants (23 of them novel) were classified as pathogenic mutations. Conclusions This report provide a wide spectrum of USH2A mutations and clinical features, including atypical Usher syndrome phenotypes resembling Usher syndrome type I. Considering only the patients clearly diagnosed with Usher syndrome type II, and results obtained in this and previous studies, we can state that mutations in USH2A are responsible for 76.1% of USH2 disease in patients of Spanish origin. PMID:22004887

  12. Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy

    PubMed Central

    Correa, Bruna R.; Bettoni, Fabiana; Koyama, Fernanda C.; Navarro, Fabio C.P.; Perez, Rodrigo O.; Mariadason, John; Sieber, Oliver M.; Strausberg, Robert L.; Simpson, Andrew J.G.; Jardim, Denis L.F.; Reis, Luiz Fernando L.; Parmigiani, Raphael B.; Galante, Pedro A.F.; Camargo, Anamaria A.

    2014-01-01

    We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients. PMID:25193853

  13. Defective nuclear import of Tpr in Progeria reflects the Ran sensitivity of large cargo transport.

    PubMed

    Snow, Chelsi J; Dar, Ashraf; Dutta, Anindya; Kehlenbach, Ralph H; Paschal, Bryce M

    2013-05-13

    The RanGTPase acts as a master regulator of nucleocytoplasmic transport by controlling assembly and disassembly of nuclear transport complexes. RanGTP is required in the nucleus to release nuclear localization signal (NLS)-containing cargo from import receptors, and, under steady-state conditions, Ran is highly concentrated in the nucleus. We previously showed the nuclear/cytoplasmic Ran distribution is disrupted in Hutchinson-Gilford Progeria syndrome (HGPS) fibroblasts that express the Progerin form of lamin A, causing a major defect in nuclear import of the protein, translocated promoter region (Tpr). In this paper, we show that Tpr import was mediated by the most abundant import receptor, KPNA2, which binds the bipartite NLS in Tpr with nanomolar affinity. Analyses including NLS swapping revealed Progerin did not cause global inhibition of nuclear import. Rather, Progerin inhibited Tpr import because transport of large protein cargoes was sensitive to changes in the Ran nuclear/cytoplasmic distribution that occurred in HGPS. We propose that defective import of large protein complexes with important roles in nuclear function may contribute to disease-associated phenotypes in Progeria.

  14. Unique preservation of neural cells in Hutchinson- Gilford progeria syndrome is due to the expression of the neural-specific miR-9 microRNA.

    PubMed

    Nissan, Xavier; Blondel, Sophie; Navarro, Claire; Maury, Yves; Denis, Cécile; Girard, Mathilde; Martinat, Cécile; De Sandre-Giovannoli, Annachiara; Levy, Nicolas; Peschanski, Marc

    2012-07-26

    One puzzling observation in patients affected with Hutchinson-Gilford progeria syndrome (HGPS), who overall exhibit systemic and dramatic premature aging, is the absence of any conspicuous cognitive impairment. Recent studies based on induced pluripotent stem cells derived from HGPS patient cells have revealed a lack of expression in neural derivatives of lamin A, a major isoform of LMNA that is initially produced as a precursor called prelamin A. In HGPS, defective maturation of a mutated prelamin A induces the accumulation of toxic progerin in patient cells. Here, we show that a microRNA, miR-9, negatively controls lamin A and progerin expression in neural cells. This may bear major functional correlates, as alleviation of nuclear blebbing is observed in nonneural cells after miR-9 overexpression. Our results support the hypothesis, recently proposed from analyses in mice, that protection of neural cells from progerin accumulation in HGPS is due to the physiologically restricted expression of miR-9 to that cell lineage.

  15. Feedback Control of Sex Determination by Dosage Compensation Revealed through Caenorhabditis Elegans Sdc-3 Mutations

    PubMed Central

    DeLong, L.; Plenefisch, J. D.; Klein, R. D.; Meyer, B. J.

    1993-01-01

    In Caenorhabditis elegans, sex determination and dosage compensation are coordinately controlled through a group of genes that respond to the primary sex determination signal. Here we describe a new gene, sdc-3, that also controls these processes. In contrast to previously described genes, the sex determination and dosage compensation activities of sdc-3 are separately mutable, indicating that they function independently. Paradoxically, the sdc-3 null phenotype fails to reveal the role of sdc-3 in sex determination: sdc-3 null mutations that lack both activities disrupt dosage compensation but cause no overt sexual transformation. We demonstrate that the dosage compensation defect of sdc-3 null alleles suppresses their sex determination defect. This self-suppression phenomenon provides a striking example of how a disruption in dosage compensation can affect sexual fate. We propose that the suppression occurs via a feedback mechanism that acts at an early regulatory step in the sex determination pathway to promote proper sexual identity. PMID:8462848

  16. Experimental evolution reveals genome-wide spectrum and dynamics of mutations in the rice blast fungus, Magnaporthe oryzae.

    PubMed

    Jeon, Junhyun; Choi, Jaeyoung; Lee, Gir-Won; Dean, Ralph A; Lee, Yong-Hwan

    2013-01-01

    Knowledge on mutation processes is central to interpreting genetic analysis data as well as understanding the underlying nature of almost all evolutionary phenomena. However, studies on genome-wide mutational spectrum and dynamics in fungal pathogens are scarce, hindering our understanding of their evolution and biology. Here, we explored changes in the phenotypes and genome sequences of the rice blast fungus Magnaporthe oryzae during the forced in vitro evolution by weekly transfer of cultures on artificial media. Through combination of experimental evolution with high throughput sequencing technology, we found that mutations accumulate rapidly prior to visible phenotypic changes and that both genetic drift and selection seem to contribute to shaping mutational landscape, suggesting the buffering capacity of fungal genome against mutations. Inference of mutational effects on phenotypes through the use of T-DNA insertion mutants suggested that at least some of the DNA sequence mutations are likely associated with the observed phenotypic changes. Furthermore, our data suggest oxidative damages and UV as major sources of mutation during subcultures. Taken together, our work revealed important properties of original source of variation in the genome of the rice blast fungus. We believe that these results provide not only insights into stability of pathogenicity and genome evolution in plant pathogenic fungi but also a model in which evolution of fungal pathogens in natura can be comparatively investigated.

  17. Genome evolution in yeast reveals connections between rare mutations in human cancers

    PubMed Central

    Teng, Xinchen; Hardwick, J. M.

    2014-01-01

    Cancer cells are riddled with mutations. Less than one percent of these are thought to be mutations that drive cancer phenotypes. However, a recent study conducted on the yeast knockout collections by Teng et al. [Mol. Cell (2013) 52: 485-494] provides hard evidence that single gene deletions/mutations in most non-essential genes can drive the selection for cancer-like mutations. PMID:28357245

  18. Genetic Code Evolution Reveals the Neutral Emergence of Mutational Robustness, and Information as an Evolutionary Constraint

    PubMed Central

    Massey, Steven E.

    2015-01-01

    The standard genetic code (SGC) is central to molecular biology and its origin and evolution is a fundamental problem in evolutionary biology, the elucidation of which promises to reveal much about the origins of life. In addition, we propose that study of its origin can also reveal some fundamental and generalizable insights into mechanisms of molecular evolution, utilizing concepts from complexity theory. The first is that beneficial traits may arise by non-adaptive processes, via a process of “neutral emergence”. The structure of the SGC is optimized for the property of error minimization, which reduces the deleterious impact of point mutations. Via simulation, it can be shown that genetic codes with error minimization superior to the SGC can emerge in a neutral fashion simply by a process of genetic code expansion via tRNA and aminoacyl-tRNA synthetase duplication, whereby similar amino acids are added to codons related to that of the parent amino acid. This process of neutral emergence has implications beyond that of the genetic code, as it suggests that not all beneficial traits have arisen by the direct action of natural selection; we term these “pseudaptations”, and discuss a range of potential examples. Secondly, consideration of genetic code deviations (codon reassignments) reveals that these are mostly associated with a reduction in proteome size. This code malleability implies the existence of a proteomic constraint on the genetic code, proportional to the size of the proteome (P), and that its reduction in size leads to an “unfreezing” of the codon – amino acid mapping that defines the genetic code, consistent with Crick’s Frozen Accident theory. The concept of a proteomic constraint may be extended to propose a general informational constraint on genetic fidelity, which may be used to explain variously, differences in mutation rates in genomes with differing proteome sizes, differences in DNA repair capacity and genome GC content

  19. Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease

    PubMed Central

    Lewis, Wesley R.; Malarkey, Erik B.; Tritschler, Douglas; Bower, Raqual; Pasek, Raymond C.; Porath, Jonathan D.; Birket, Susan E.; Saunier, Sophie; Antignac, Corinne; Leigh, Margaret W.; Zariwala, Maimoona A.; Drummond, Iain A.; Parant, John M.; Hildebrandt, Friedhelm; Yoder, Bradley K.

    2016-01-01

    Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or ‘primary’ cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants

  20. Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.

    PubMed

    Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz; Petljak, Mia; Alexandrov, Ludmil B; Rahbari, Raheleh; Wedge, David C; Davies, Helen R; Ramakrishna, Manasa; Fullam, Anthony; Martin, Sancha; Alder, Christopher; Patel, Nikita; Gamble, Steve; O'Meara, Sarah; Giri, Dilip D; Sauer, Torril; Pinder, Sarah E; Purdie, Colin A; Borg, Åke; Stunnenberg, Henk; van de Vijver, Marc; Tan, Benita K T; Caldas, Carlos; Tutt, Andrew; Ueno, Naoto T; van 't Veer, Laura J; Martens, John W M; Sotiriou, Christos; Knappskog, Stian; Span, Paul N; Lakhani, Sunil R; Eyfjörd, Jórunn Erla; Børresen-Dale, Anne-Lise; Richardson, Andrea; Thompson, Alastair M; Viari, Alain; Hurles, Matthew E; Nik-Zainal, Serena; Campbell, Peter J; Stratton, Michael R

    2017-03-30

    Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

  1. Preliminary whole-exome sequencing reveals mutations that imply common tumorigenicity pathways in multiple endocrine neoplasia type 1 patients

    PubMed Central

    Arenas, Minerva Angélica Romero; Fowler, Richard G.; Lucas, F. Anthony San; Shen, Jie; Rich, Thereasa A.; Grubbs, Elizabeth G.; Lee, Jeffrey E.; Scheet, Paul; Perrier, Nancy D.; Zhao, Hua

    2016-01-01

    Background Whole-exome sequencing studies have not established definitive somatic mutation patterns among patients with sporadic hyperparathyroidism (HPT). No sequencing has evaluated multiple endocrine neoplasia type 1 (MEN1)-related HPT. We sought to perform whole-exome sequencing in HPT patients to identify somatic mutations and associated biological pathways and tumorigenic networks. Methods Whole-exome sequencing was performed on blood and tissue from HPT patients (MEN1 and sporadic) and somatic single nucleotide variants (SNVs) were identified. Stop-gain and stop-loss SNVs were analyzed with Ingenuity Pathways Analysis (IPA). Loss of heterozygosity (LOH) was also assessed. Results Sequencing was performed on 4 MEN1 and 10 sporadic cases. Eighteen stop-gain/stop-loss SNV mutations were identified in 3 MEN1 patients. One complex network was identified on IPA: Cellular function and maintenance, tumor morphology, and cardiovascular disease (IPA score = 49). A nonsynonymous SNV of TP53 (lysine-to-glutamic acid change at codon 81) identified in a MEN1 patient was suggested to be a driver mutation (Cancer-specific High-throughput Annotation of Somatic Mutations; P = .002). All MEN1 and 3/10 sporadic specimens demonstrated LOH of chromosome 11. Conclusion Whole-exome sequencing revealed somatic mutations in MEN1 associated with a single tumorigenic network, whereas sporadic pathogenesis seemed to be more diverse. A somatic TP53 mutation was also identified. LOH of chromosome 11 was seen in all MEN1 and 3 of 10 sporadic patients. PMID:25456907

  2. Modeling human Coenzyme A synthase mutation in yeast reveals altered mitochondrial function, lipid content and iron metabolism

    PubMed Central

    Berti, Camilla C.; Dallabona, Cristina; Lazzaretti, Mirca; Dusi, Sabrina; Tosi, Elena; Tiranti, Valeria; Goffrini, Paola

    2015-01-01

    Mutations in nuclear genes associated with defective coenzyme A biosynthesis have been identified as responsible for some forms of neurodegeneration with brain iron accumulation (NBIA), namely PKAN and CoPAN. PKAN are defined by mutations in PANK2, encoding the pantothenate kinase 2 enzyme, that account for about 50% of cases of NBIA, whereas mutations in CoA synthase COASY have been recently reported as the second inborn error of CoA synthesis leading to CoPAN. As reported previously, yeast cells expressing the pathogenic mutation exhibited a temperature-sensitive growth defect in the absence of pantothenate and a reduced CoA content. Additional characterization revealed decreased oxygen consumption, reduced activities of mitochondrial respiratory complexes, higher iron content, increased sensitivity to oxidative stress and reduced amount of lipid droplets, thus partially recapitulating the phenotypes found in patients and establishing yeast as a potential model to clarify the pathogenesis underlying PKAN and CoPAN diseases. PMID:28357284

  3. Genome sequencing of normal cells reveals developmental lineages and mutational processes

    PubMed Central

    Behjati, Sam; Wedge, David C; Tamuri, Asif U; Martincorena, Inigo; Petljak, Mia; Alexandrov, Ludmil B; Gundem, Gunes; Tarpey, Patrick S; Roerink, Sophie; Blokker, Joyce; Maddison, Mark; Mudie, Laura; Robinson, Ben; Nik-Zainal, Serena; Campbell, Peter; Goldman, Nick; van de Wetering, Marc; Cuppen, Edwin; Clevers, Hans; Stratton, Michael R

    2014-01-01

    The somatic mutations present in the genome of a cell have been accumulated over the lifetime of a multicellular organism. These mutations can provide insights into the developmental lineage tree1, the number of divisions each cell has undergone and the mutational processes that have been operative2. Here, we conducted whole genome sequencing of clonal lines3 derived from multiple tissues of healthy mice. Using somatic base substitutions, we reconstructed the early cell divisions of each animal demonstrating the contributions of embryonic cells to adult tissues. Differences were observed between tissues in the numbers and types of mutations accumulated by each cell, which likely reflect differences in the number of cell divisions they have undergone and varying contributions of different mutational processes. If somatic mutation rates are similar to those in mice, the results indicate that precise insights into development and mutagenesis of normal human cells will be possible. PMID:25043003

  4. Excess of Deleterious Mutations around HLA Genes Reveals Evolutionary Cost of Balancing Selection

    PubMed Central

    Lenz, Tobias L.; Spirin, Victor; Jordan, Daniel M.; Sunyaev, Shamil R.

    2016-01-01

    Deleterious mutations are expected to evolve under negative selection and are usually purged from the population. However, deleterious alleles segregate in the human population and some disease-associated variants are maintained at considerable frequencies. Here, we test the hypothesis that balancing selection may counteract purifying selection in neighboring regions and thus maintain deleterious variants at higher frequency than expected from their detrimental fitness effect. We first show in realistic simulations that balancing selection reduces the density of polymorphic sites surrounding a locus under balancing selection, but at the same time markedly increases the population frequency of the remaining variants, including even substantially deleterious alleles. To test the predictions of our simulations empirically, we then use whole-exome sequencing data from 6,500 human individuals and focus on the most established example for balancing selection in the human genome, the major histocompatibility complex (MHC). Our analysis shows an elevated frequency of putatively deleterious coding variants in nonhuman leukocyte antigen (non-HLA) genes localized in the MHC region. The mean frequency of these variants declined with physical distance from the classical HLA genes, indicating dependency on genetic linkage. These results reveal an indirect cost of the genetic diversity maintained by balancing selection, which has hitherto been perceived as mostly advantageous, and have implications both for the evolution of recombination and also for the epidemiology of various MHC-associated diseases. PMID:27436009

  5. Defective lamin A-Rb signaling in Hutchinson-Gilford Progeria Syndrome and reversal by farnesyltransferase inhibition.

    PubMed

    Marji, Jackleen; O'Donoghue, Seán I; McClintock, Dayle; Satagopam, Venkata P; Schneider, Reinhard; Ratner, Desiree; Worman, Howard J; Gordon, Leslie B; Djabali, Karima

    2010-06-15

    Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare premature aging disorder caused by a de novo heterozygous point mutation G608G (GGC>GGT) within exon 11 of LMNA gene encoding A-type nuclear lamins. This mutation elicits an internal deletion of 50 amino acids in the carboxyl-terminus of prelamin A. The truncated protein, progerin, retains a farnesylated cysteine at its carboxyl terminus, a modification involved in HGPS pathogenesis. Inhibition of protein farnesylation has been shown to improve abnormal nuclear morphology and phenotype in cellular and animal models of HGPS. We analyzed global gene expression changes in fibroblasts from human subjects with HGPS and found that a lamin A-Rb signaling network is a major defective regulatory axis. Treatment of fibroblasts with a protein farnesyltransferase inhibitor reversed the gene expression defects. Our study identifies Rb as a key factor in HGPS pathogenesis and suggests that its modulation could ameliorate premature aging and possibly complications of physiological aging.

  6. PTT analysis of polyps from FAP patients reveals a great majority of APC truncating mutations

    SciTech Connect

    Luijt, R.B. van der; Khan, P.M.; Tops, C.M.J.

    1994-09-01

    The adenomatous polyposis coli (APC) gene plays an important role in colorectal carcinogenesis. Germline APC mutations are associated with familial adenomatous polyposis (FAP), an autosomal dominantly inherited predisposition to colorectal cancer, characterized by the development of numerous adenomatous polyps in the large intestine. In order to investigate whether somatic inactivation of the remaining APC allele is necessary for adenoma formation, we collected multiple adenomatous polyps from individual FAP patients and investigated the presence of somatic mutations in the APC gene. The analysis of somatic APC mutations in these tumor samples was performed using a rapid and sensitive assay, called the protein truncation test (PTT). Chain-terminating somatic APC mutations were detected in the great majority of the tumor samples investigated. As expected, these mutations were mainly located in the mutation cluster region (MCR) in exon 15. Our results confirm that somatic mutation of the second APC allele is required for adenoma formation in FAP. Interestingly, in the polyps investigated in our study, the second APC allele is somatically inactivated through point mutation leading to a stop codon rather than by loss of heterozygosity. The observation that somatic second hits in APC are required for tumor development in FAP is in apparent accordance with the Knudson hypothesis for classical tumor suppressor genes. However, it is yet unknown whether chain-terminating APC mutations lead to a truncated protein exerting a dominant-negative effect or whether these mutations result in a null allele. Further investigation of this important issue will hopefully provide a better understanding of the mechanism of action of the mutated APC alleles in colorectal carcinogenesis.

  7. Tumor-Derived Suppressor of Fused Mutations Reveal Hedgehog Pathway Interactions.

    PubMed

    Urman, Nicole M; Mirza, Amar; Atwood, Scott X; Whitson, Ramon J; Sarin, Kavita Y; Tang, Jean Y; Oro, Anthony E

    2016-01-01

    The Hedgehog pathway is a potent regulator of cellular growth and plays a central role in the development of many cancers including basal cell carcinoma (BCC). The majority of BCCs arise from mutations in the Patched receptor resulting in constitutive activation of the Hedgehog pathway. Secondary driver mutations promote BCC oncogenesis and occur frequently due to the high mutational burden resulting from sun exposure of the skin. Here, we uncover novel secondary mutations in Suppressor of Fused (SUFU), the major negative regulator of the Hedgehog pathway. SUFU normally binds to a Hedgehog transcriptional activator, GLI1, in order to prevent it from initiating transcription of Hedgehog target genes. We sequenced tumor-normal pairs from patients with early sporadic BCCs. This resulted in the discovery of nine mutations in SUFU, which were functionally investigated to determine whether they help drive BCC formation. Our results show that four of the SUFU mutations inappropriately activate the Hedgehog pathway, suggesting they may act as driver mutations for BCC development. Indeed, all four of the loss of function SUFU variants were found to disrupt its binding to GLI, leading to constitutive pathway activation. Our results from functional characterization of these mutations shed light on SUFU's role in Hedgehog signaling, tumor progression, and highlight a way in which BCCs can arise.

  8. Tumor-Derived Suppressor of Fused Mutations Reveal Hedgehog Pathway Interactions

    PubMed Central

    Urman, Nicole M.; Mirza, Amar; Atwood, Scott X.; Whitson, Ramon J.; Sarin, Kavita Y.; Tang, Jean Y.; Oro, Anthony E.

    2016-01-01

    The Hedgehog pathway is a potent regulator of cellular growth and plays a central role in the development of many cancers including basal cell carcinoma (BCC). The majority of BCCs arise from mutations in the Patched receptor resulting in constitutive activation of the Hedgehog pathway. Secondary driver mutations promote BCC oncogenesis and occur frequently due to the high mutational burden resulting from sun exposure of the skin. Here, we uncover novel secondary mutations in Suppressor of Fused (SUFU), the major negative regulator of the Hedgehog pathway. SUFU normally binds to a Hedgehog transcriptional activator, GLI1, in order to prevent it from initiating transcription of Hedgehog target genes. We sequenced tumor-normal pairs from patients with early sporadic BCCs. This resulted in the discovery of nine mutations in SUFU, which were functionally investigated to determine whether they help drive BCC formation. Our results show that four of the SUFU mutations inappropriately activate the Hedgehog pathway, suggesting they may act as driver mutations for BCC development. Indeed, all four of the loss of function SUFU variants were found to disrupt its binding to GLI, leading to constitutive pathway activation. Our results from functional characterization of these mutations shed light on SUFU’s role in Hedgehog signaling, tumor progression, and highlight a way in which BCCs can arise. PMID:28030567

  9. Transcriptomic Characterization of SF3B1 Mutation Reveals Its Pleiotropic Effects in Chronic Lymphocytic Leukemia.

    PubMed

    Wang, Lili; Brooks, Angela N; Fan, Jean; Wan, Youzhong; Gambe, Rutendo; Li, Shuqiang; Hergert, Sarah; Yin, Shanye; Freeman, Samuel S; Levin, Joshua Z; Fan, Lin; Seiler, Michael; Buonamici, Silvia; Smith, Peter G; Chau, Kevin F; Cibulskis, Carrie L; Zhang, Wandi; Rassenti, Laura Z; Ghia, Emanuela M; Kipps, Thomas J; Fernandes, Stacey; Bloch, Donald B; Kotliar, Dylan; Landau, Dan A; Shukla, Sachet A; Aster, Jon C; Reed, Robin; DeLuca, David S; Brown, Jennifer R; Neuberg, Donna; Getz, Gad; Livak, Kenneth J; Meyerson, Matthew M; Kharchenko, Peter V; Wu, Catherine J

    2016-11-14

    Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch signaling (mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively). SF3B1 mutation leads to diverse changes in CLL-related pathways.

  10. PMD patient mutations reveal a long-distance intronic interaction that regulates PLP1/DM20 alternative splicing

    PubMed Central

    Taube, Jennifer R.; Sperle, Karen; Banser, Linda; Seeman, Pavel; Cavan, Barbra Charina V.; Garbern, James Y.; Hobson, Grace M.

    2014-01-01

    Alternative splicing of the proteolipid protein 1 gene (PLP1) produces two forms, PLP1 and DM20, due to alternative use of 5′ splice sites with the same acceptor site in intron 3. The PLP1 form predominates in central nervous system RNA. Mutations that reduce the ratio of PLP1 to DM20, whether mutant or normal protein is formed, result in the X-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD). We investigated the ability of sequences throughout PLP1 intron 3 to regulate alternative splicing using a splicing minigene construct transfected into the oligodendrocyte cell line, Oli-neu. Our data reveal that the alternative splice of PLP1 is regulated by a long-distance interaction between two highly conserved elements that are separated by 581 bases within the 1071-base intron 3. Further, our data suggest that a base-pairing secondary structure forms between these two elements, and we demonstrate that mutations of either element designed to destabilize the secondary structure decreased the PLP1/DM20 ratio, while swap mutations designed to restore the structure brought the PLP1/DM20 ratio to near normal levels. Sequence analysis of intron 3 in families with clinical symptoms of PMD who did not have coding-region mutations revealed mutations that segregated with disease in three families. We showed that these patient mutations, which potentially destabilize the secondary structure, also reduced the PLP1/DM20 ratio. This is the first report of patient mutations causing disease by disruption of a long-distance intronic interaction controlling alternative splicing. This finding has important implications for molecular diagnostics of PMD. PMID:24890387

  11. Implant supported prosthesis in a patient with progeria: case report.

    PubMed

    Ceylan, Gözlem; Yilmaz, Nergiz; Senyurt, Ozgün; Ergün Kunt, Göknil

    2009-08-01

    Prosthodontic rehabilitation can be accomplished with fixed, overdenture, complete, or implant-retained prostheses. Dental treatment overcomes the patient's functional, psychological, esthetic and phonation problems. Remaining healthy teeth may allow the dentist to fabricate a removable partial overdenture, fixed partial prosthesis or implant - supported prosthesis. The retention of a number of abutments helps maintain a positive ridge form with greater height and volume of the alveolar bone, improving masticatory performance, as well as providing a more stable prostheses. Dental patients who have medical problems need many treatment procedures. Multidisciplinary treatment planning is invaluable for patient's dental health. Progeria is a rare genetic condition where symptoms resembling aspects of aging are manifested at an early age. characteristic clinical findings of Progeria disease include abnormalities of the skin and hair in conjunction with characteristic facial features and skeletal abnormalities. The characteristic facies show protruding ears, beaked nose, thin lips with centrofacial cyanosis, prominent eyes, frontal and parietal bossing with pseudohydrocephaly, midface hypoplasia with micrognathia and large anterior fontanel. The other reported anomalies are dystrophic nails, hypertrophic scars and hypoplastic nipples. The findings that are nearly interested in dentistry are delayed dentition, anodontia, hypodontia, or crowding of teeth. This article presents the multidisciplinary dental treatment planning includes surgical, endodontic and prosthetic treatment of a patient with a history of progeria. In this case complete-arch fixed prostheses in both maxilla and mandible, supported by a combination of implants and teeth are reported.

  12. Exome sequencing reveals germline gain-of-function EGFR mutation in an adult with Lhermitte–Duclos disease

    PubMed Central

    Colby, Samantha; Yehia, Lamis; Niazi, Farshad; Chen, JinLian; Ni, Ying

    2016-01-01

    Lhermitte–Duclos disease (LDD) is a rare cerebellar disorder believed to be pathognomonic for Cowden syndrome. Presently, the only known etiology is germline PTEN mutation. We report a 41-yr-old white female diagnosed with LDD and wild-type for PTEN. Exome sequencing revealed a germline heterozygous EGFR mutation that breaks a disulfide bond in the receptor's extracellular domain, resulting in constitutive activation. Functional studies demonstrate activation of ERK/AKT signaling pathways, mimicking PTEN loss-of-function downstream effects. The identification of EGFR as a candidate LDD susceptibility gene contributes to advancement of molecular diagnosis and targeted therapy for this rare condition with limited treatment options. PMID:27900366

  13. Exome sequencing reveals riboflavin transporter mutations as a cause of motor neuron disease

    PubMed Central

    Johnson, Janel O.; Gibbs, J. Raphael; Megarbane, Andre; Urtizberea, J. Andoni; Hernandez, Dena G.; Foley, A. Reghan; Arepalli, Sampath; Pandraud, Amelie; Simón-Sánchez, Javier; Clayton, Peter; Reilly, Mary M.; Muntoni, Francesco; Abramzon, Yevgeniya; Houlden, Henry

    2012-01-01

    Brown–Vialetto–Van Laere syndrome was first described in 1894 as a rare neurodegenerative disorder characterized by progressive sensorineural deafness in combination with childhood amyotrophic lateral sclerosis. Mutations in the gene, SLC52A3 (formerly C20orf54), one of three known riboflavin transporter genes, have recently been shown to underlie a number of severe cases of Brown–Vialetto–Van Laere syndrome; however, cases and families with this disease exist that do not appear to be caused by SLC52A3 mutations. We used a combination of linkage and exome sequencing to identify the disease causing mutation in an extended Lebanese Brown–Vialetto–Van Laere kindred, whose affected members were negative for SLC52A3 mutations. We identified a novel mutation in a second member of the riboflavin transporter gene family (gene symbol: SLC52A2) as the cause of disease in this family. The same mutation was identified in one additional subject, from 44 screened. Within this group of 44 patients, we also identified two additional cases with SLC52A3 mutations, but none with mutations in the remaining member of this gene family, SLC52A1. We believe this strongly supports the notion that defective riboflavin transport plays an important role in Brown–Vialetto–Van Laere syndrome. Initial work has indicated that patients with SLC52A3 defects respond to riboflavin treatment clinically and biochemically. Clearly, this makes an excellent candidate therapy for the SLC52A2 mutation-positive patients identified here. Initial riboflavin treatment of one of these patients shows promising results. PMID:22740598

  14. Multihost experimental evolution of a plant RNA virus reveals local adaptation and host-specific mutations.

    PubMed

    Bedhomme, Stéphanie; Lafforgue, Guillaume; Elena, Santiago F

    2012-05-01

    For multihost pathogens, adaptation to multiple hosts has important implications for both applied and basic research. At the applied level, it is one of the main factors determining the probability and the severity of emerging disease outbreaks. At the basic level, it is thought to be a key mechanism for the maintenance of genetic diversity both in host and pathogen species. Using Tobacco etch potyvirus (TEV) and four natural hosts, we have designed an evolution experiment whose strength and novelty are the use of complex multicellular host organism as hosts and a high level of replication of different evolutionary histories and lineages. A pattern of local adaptation, characterized by a higher infectivity and virulence on host(s) encountered during the experimental evolution was found. Local adaptation only had a cost in terms of performance on other hosts in some cases. We could not verify the existence of a cost for generalists, as expected to arise from antagonistic pleiotropy and other genetic mechanisms generating a fitness trade-off between hosts. This observation confirms that this classical theoretical prediction lacks empirical support. We discuss the reasons for this discrepancy between theory and experiment in the light of our results. The analysis of full genome consensus sequences of the evolved lineages established that all mutations shared between lineages were host specific. A low degree of parallel evolution was observed, possibly reflecting the various adaptive pathways available for TEV in each host. Altogether, these results reveal a strong adaptive potential of TEV to new hosts without severe evolutionary constraints.

  15. A multigene mutation classification of 468 colorectal cancers reveals a prognostic role for APC

    PubMed Central

    Schell, Michael J.; Yang, Mingli; Teer, Jamie K.; Lo, Fang Yin; Madan, Anup; Coppola, Domenico; Monteiro, Alvaro N. A.; Nebozhyn, Michael V.; Yue, Binglin; Loboda, Andrey; Bien-Willner, Gabriel A.; Greenawalt, Danielle M.; Yeatman, Timothy J.

    2016-01-01

    Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC. PMID:27302369

  16. Alanine-Scanning Mutational Analysis of Durancin GL Reveals Residues Important for Its Antimicrobial Activity.

    PubMed

    Ju, Xingrong; Chen, Xinquan; Du, Lihui; Wu, Xueyou; Liu, Fang; Yuan, Jian

    2015-07-22

    Durancin GL is a novel class IIa bacteriocin with 43 residues produced by Enterococcus durans 41D. This bacteriocin demonstrates narrow inhibition spectrum and potent antimicrobial activity against several Listeria monocytogenes strains, including nisin-resistant L. monocytogenes NR30. A systematic alanine-scanning mutational analysis with site-directed mutagenesis was performed to analyze durancin GL residues important for antimicrobial activity and specificity. Results showed that three mutations lost their antimicrobial activity, ten mutations demonstrated a decreased effect on the activity, and seven mutations exhibited relatively high activity. With regard to inhibitory spectrum, four mutants demonstrated a narrower antimicrobial spectrum than wild-type durancin GL. Another four mutants displayed a broader target cell spectrum and increased potency relative to wild-type durancin GL. These findings broaden our understanding of durancin GL residues important for its antimicrobial activity and contribute to future rational design of variants with increased potency.

  17. Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes

    PubMed Central

    Junager, Nina P. L.; Kongsted, Jacob; Astakhova, Kira

    2016-01-01

    Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the most often used tools for the detection of nucleic acids and in particular, for the detection of mutations. However, multiple parameters must be taken into account in order to create efficient FRET probes that are sensitive to nucleic acid mutations. In this review; we focus on the design principles for such probes and available computational methods that allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cellular heterogeneity to gaining new knowledge of nucleic acid structures directly in living cells. PMID:27472344

  18. Exome Sequencing Reveals Cubilin Mutation as a Single-Gene Cause of Proteinuria

    PubMed Central

    Ovunc, Bugsu; Otto, Edgar A.; Vega-Warner, Virginia; Saisawat, Pawaree; Ashraf, Shazia; Ramaswami, Gokul; Fathy, Hanan M.; Schoeb, Dominik; Chernin, Gil; Lyons, Robert H.; Yilmaz, Engin

    2011-01-01

    In two siblings of consanguineous parents with intermittent nephrotic-range proteinuria, we identified a homozygous deleterious frameshift mutation in the gene CUBN, which encodes cubulin, using exome capture and massively parallel re-sequencing. The mutation segregated with affected members of this family and was absent from 92 healthy individuals, thereby identifying a recessive mutation in CUBN as the single-gene cause of proteinuria in this sibship. Cubulin mutations cause a hereditary form of megaloblastic anemia secondary to vitamin B12 deficiency, and proteinuria occurs in 50% of cases since cubilin is coreceptor for both the intestinal vitamin B12-intrinsic factor complex and the tubular reabsorption of protein in the proximal tubule. In summary, we report successful use of exome capture and massively parallel re-sequencing to identify a rare, single-gene cause of nephropathy. PMID:21903995

  19. Exome sequencing reveals cubilin mutation as a single-gene cause of proteinuria.

    PubMed

    Ovunc, Bugsu; Otto, Edgar A; Vega-Warner, Virginia; Saisawat, Pawaree; Ashraf, Shazia; Ramaswami, Gokul; Fathy, Hanan M; Schoeb, Dominik; Chernin, Gil; Lyons, Robert H; Yilmaz, Engin; Hildebrandt, Friedhelm

    2011-10-01

    In two siblings of consanguineous parents with intermittent nephrotic-range proteinuria, we identified a homozygous deleterious frameshift mutation in the gene CUBN, which encodes cubulin, using exome capture and massively parallel re-sequencing. The mutation segregated with affected members of this family and was absent from 92 healthy individuals, thereby identifying a recessive mutation in CUBN as the single-gene cause of proteinuria in this sibship. Cubulin mutations cause a hereditary form of megaloblastic anemia secondary to vitamin B(12) deficiency, and proteinuria occurs in 50% of cases since cubilin is coreceptor for both the intestinal vitamin B(12)-intrinsic factor complex and the tubular reabsorption of protein in the proximal tubule. In summary, we report successful use of exome capture and massively parallel re-sequencing to identify a rare, single-gene cause of nephropathy.

  20. Genomic Analyses Reveal Mutational Signatures and Frequently Altered Genes in Esophageal Squamous Cell Carcinoma

    PubMed Central

    Zhang, Ling; Zhou, Yong; Cheng, Caixia; Cui, Heyang; Cheng, Le; Kong, Pengzhou; Wang, Jiaqian; Li, Yin; Chen, Wenliang; Song, Bin; Wang, Fang; Jia, Zhiwu; Li, Lin; Li, Yaoping; Yang, Bin; Liu, Jing; Shi, Ruyi; Bi, Yanghui; Zhang, Yanyan; Wang, Juan; Zhao, Zhenxiang; Hu, Xiaoling; Yang, Jie; Li, Hongyi; Gao, Zhibo; Chen, Gang; Huang, Xuanlin; Yang, Xukui; Wan, Shengqing; Chen, Chao; Li, Bin; Tan, Yongkai; Chen, Longyun; He, Minghui; Xie, Sha; Li, Xiangchun; Zhuang, Xuehan; Wang, Mengyao; Xia, Zhi; Luo, Longhai; Ma, Jie; Dong, Bing; Zhao, Jiuzhou; Song, Yongmei; Ou, Yunwei; Li, Enming; Xu, Liyan; Wang, Jinfen; Xi, Yanfeng; Li, Guodong; Xu, Enwei; Liang, Jianfang; Yang, Xiaofeng; Guo, Jiansheng; Chen, Xing; Zhang, Yanbo; Li, Qingshan; Liu, Lixin; Li, Yingrui; Zhang, Xiuqing; Yang, Huanming; Lin, Dongxin; Cheng, Xiaolong; Guo, Yongjun; Wang, Jun; Zhan, Qimin; Cui, Yongping

    2015-01-01

    Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and the fourth most lethal cancer in China. However, although genomic studies have identified some mutations associated with ESCC, we know little of the mutational processes responsible. To identify genome-wide mutational signatures, we performed either whole-genome sequencing (WGS) or whole-exome sequencing (WES) on 104 ESCC individuals and combined our data with those of 88 previously reported samples. An APOBEC-mediated mutational signature in 47% of 192 tumors suggests that APOBEC-catalyzed deamination provides a source of DNA damage in ESCC. Moreover, PIK3CA hotspot mutations (c.1624G>A [p.Glu542Lys] and c.1633G>A [p.Glu545Lys]) were enriched in APOBEC-signature tumors, and no smoking-associated signature was observed in ESCC. In the samples analyzed by WGS, we identified focal (<100 kb) amplifications of CBX4 and CBX8. In our combined cohort, we identified frequent inactivating mutations in AJUBA, ZNF750, and PTCH1 and the chromatin-remodeling genes CREBBP and BAP1, in addition to known mutations. Functional analyses suggest roles for several genes (CBX4, CBX8, AJUBA, and ZNF750) in ESCC. Notably, high activity of hedgehog signaling and the PI3K pathway in approximately 60% of 104 ESCC tumors indicates that therapies targeting these pathways might be particularly promising strategies for ESCC. Collectively, our data provide comprehensive insights into the mutational signatures of ESCC and identify markers for early diagnosis and potential therapeutic targets. PMID:25839328

  1. Interpatient mutational spectrum of human coronavirus-OC43 revealed by illumina sequencing.

    PubMed

    Gorse, Geoffrey J; Patel, Gira B; Fan, Xiaofeng

    2017-02-12

    Human coronaviruses (HCoV) are RNA viruses that cause respiratory tract infections with viral replication of limited duration. The host and viral population heterogeneity could influence clinical phenotypes. Employing long RT-PCR with Illumina sequencing, we quantified the gene mutation load at 0.5% mutation frequency for the 4,529 bp-domain spanning the Spike gene (4,086 bp) of HCoV-OC43 in four upper respiratory clinical specimens obtained during acute illness. There were a total of 121 mutations for all four HCoV samples with the average number of mutations at 30.3 ± 10.2, which is significantly higher than that expected from the Illumina sequencing error rate. There were two mutation peaks, one at the 5' end and the other near position 1550 in the S1 subunit. Two coronavirus samples were genotype B and two were genotype D, clustering with HCoV-OC43 strain AY391777 in neighbor - joining tree phylogenetic analysis. Nonsynonymous mutations were 76.1 ± 14% of mutation load. Although lower than other RNA viruses such as hepatitis C virus, HCoV-OC43 did exhibit quasi-species. The rate of nonsynonymous mutations was higher in the HCoV-OC43 isolates than in hepatitis C virus genotype 1a isolates analyzed for comparison in this study. These characteristics of HCoV-OC43 may affect viral replication dynamics, receptor binding, antigenicity, evolution, transmission, and clinical illness. This article is protected by copyright. All rights reserved.

  2. CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa.

    PubMed

    Wu, Wen-Hsuan; Tsai, Yi-Ting; Justus, Sally; Lee, Ting-Ting; Zhang, Lijuan; Lin, Chyuan-Sheng; Bassuk, Alexander G; Mahajan, Vinit B; Tsang, Stephen H

    2016-08-01

    Massive parallel sequencing enables identification of numerous genetic variants in mutant organisms, but determining pathogenicity of any one mutation can be daunting. The most commonly studied preclinical model of retinitis pigmentosa called the "rodless" (rd1) mouse is homozygous for two mutations: a nonsense point mutation (Y347X) and an intronic insertion of a leukemia virus (Xmv-28). Distinguishing which mutation causes retinal degeneration is still under debate nearly a century after the discovery of this model organism. Here, we performed gene editing using the CRISPR/Cas9 system and demonstrated that the Y347X mutation is the causative variant of disease. Genome editing in the first generation produced animals that were mosaic for the corrected allele but still showed neurofunction preservation despite low repair frequencies. Furthermore, second-generation CRISPR-repaired mice showed an even more robust rescue and amelioration of the disease. This predicts excellent outcomes for gene editing in diseased human tissue, as Pde6b, the mutated gene in rd1 mice, has an orthologous intron-exon relationship comparable with the human PDE6B gene. Not only do these findings resolve the debate surrounding the source of neurodegeneration in the rd1 model, but they also provide the first example of homology-directed recombination-mediated gene correction in the visual system.

  3. Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events

    PubMed Central

    Liu, Jinfeng; Lee, William; Jiang, Zhaoshi; Chen, Zhongqiang; Jhunjhunwala, Suchit; Haverty, Peter M.; Gnad, Florian; Guan, Yinghui; Gilbert, Houston N.; Stinson, Jeremy; Klijn, Christiaan; Guillory, Joseph; Bhatt, Deepali; Vartanian, Steffan; Walter, Kimberly; Chan, Jocelyn; Holcomb, Thomas; Dijkgraaf, Peter; Johnson, Stephanie; Koeman, Julie; Minna, John D.; Gazdar, Adi F.; Stern, Howard M.; Hoeflich, Klaus P.; Wu, Thomas D.; Settleman, Jeff; de Sauvage, Frederic J.; Gentleman, Robert C.; Neve, Richard M.; Stokoe, David; Modrusan, Zora; Seshagiri, Somasekar; Shames, David S.; Zhang, Zemin

    2012-01-01

    Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology. PMID:23033341

  4. Hutchinson-Gilford progeria syndrome as a model for vascular aging.

    PubMed

    Brassard, Jonathan A; Fekete, Natalie; Garnier, Alain; Hoesli, Corinne A

    2016-02-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by a de novo genetic mutation that leads to the accumulation of a splicing isoform of lamin A termed progerin. Progerin expression alters the organization of the nuclear lamina and chromatin. The life expectancy of HGPS patients is severely reduced due to critical cardiovascular defects. Progerin also accumulates in an age-dependent manner in the vascular cells of adults that do not carry genetic mutations associated with HGPS. The molecular mechanisms that lead to vascular dysfunction in HGPS may therefore also play a role in vascular aging. The vascular phenotypic and molecular changes observed in HGPS are strikingly similar to those seen with age, including increased senescence, altered mechanotransduction and stem cell exhaustion. This article discusses the similarities and differences between age-dependent and HGPS-related vascular aging to highlight the relevance of HGPS as a model for vascular aging. Induced pluripotent stem cells derived from HGPS patients are suggested as an attractive model to study vascular aging in order to develop novel approaches to treat cardiovascular disease.

  5. Molecular cytogenetic insights into the ageing syndrome Hutchinson-Gilford Progeria (HGPS).

    PubMed

    Corso, C; Parry, E M; Faragher, R G A; Seager, A; Green, M H L; Parry, J M

    2005-01-01

    Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder characterized by premature ageing in childhood and serves as a valuable model for the human ageing process in general. Most recently, point mutations in the lamin A (LMNA) gene on chromosome 1q have been associated with the disease, however how these mutations relate to the complex phenotype of HGPS remains to be established. It has been shown that fibroblasts from HGPS patients are frequently resistant to immortalization with telomerase (hTERT), consistent with the idea that the loss of a dominant acting HGPS gene is a pre-requisite for immortalization. In this study we report the first detailed cytogenetic analysis of hTERT-immortalised HGPS cell lines from three patients and one corresponding primary fibroblast culture. Our results provide evidence for a cytogenetic mosaicism in HGPS with a distinctive pattern of chromosome aberrations in all the HGP clones. Chromosome 11 alterations were observed at a high frequency in each immortalised HGPS cell line but were also present at a lower frequency in the corresponding primary cells. Moreover, we were able to identify the 11q13-->q23 region as a potential site of breakage. Our results are therefore consistent with a role of chromosome 11 alterations in the escape from senescence observed in HGPS cells. In addition to this defined rearrangement, we consistently observed complex chromosomal rearrangements, suggesting that HGPS displays features of chromosomal instability.

  6. Increased expression of the Hutchinson-Gilford progeria syndrome truncated lamin A transcript during cell aging.

    PubMed

    Rodriguez, Sofia; Coppedè, Fabio; Sagelius, Hanna; Eriksson, Maria

    2009-07-01

    Most cases of the segmental progeroid syndrome, Hutchinson-Gilford progeria syndrome (HGPS), are caused by a de novo dominant mutation within a single codon of the LMNA gene. This mutation leads to the increased usage of an internal splice site that generates an alternative lamin A transcript with an internal deletion of 150 nucleotides, called lamin A Delta 150. The LMNA gene encodes two major proteins of the inner nuclear lamina, lamins A and C, but not much is known about their expression levels. Determination of the overall expression levels of the LMNA gene transcripts is an important step to further the understanding of the HGPS. In this study, we have performed absolute quantification of the lamins A, C and A Delta 150 transcripts in primary dermal fibroblasts from HGPS patients and unaffected age-matched and parent controls. We show that the lamin A Delta 150 transcript is present in unaffected controls but its expression is >160-fold lower than that in samples from HGPS patients. Analysis of transcript expression during in vitro aging shows that although the levels of lamin A and lamin C transcripts remain unchanged, the lamin A Delta 150 transcript increases in late passage cells from HGPS patients and parental controls. This study provides a new method for LMNA transcript analysis and insights into the expression of the LMNA gene in HGPS and normal cells.

  7. Novel POC1A mutation in primordial dwarfism reveals new insights for centriole biogenesis.

    PubMed

    Koparir, Asuman; Karatas, Omer F; Yuceturk, Betul; Yuksel, Bayram; Bayrak, Ali O; Gerdan, Omer F; Sagiroglu, Mahmut S; Gezdirici, Alper; Kirimtay, Koray; Selcuk, Ece; Karabay, Arzu; Creighton, Chad J; Yuksel, Adnan; Ozen, Mustafa

    2015-10-01

    POC1A encodes a WD repeat protein localizing to centrioles and spindle poles and is associated with short stature, onychodysplasia, facial dysmorphism and hypotrichosis (SOFT) syndrome. These main features are related to the defect in cell proliferation of chondrocytes in growth plate. In the current study, we aimed at identifying the molecular basis of two patients with primordial dwarfism (PD) in a single family through utilization of whole-exome sequencing. A novel homozygous p.T120A missense mutation was detected in POC1A in both patients, a known causative gene of SOFT syndrome, and confirmed using Sanger sequencing. To test the pathogenicity of the detected mutation, primary fibroblast cultures obtained from the patients and a control individual were used. For evaluating the global gene expression profile of cells carrying p.T120A mutation in POC1A, we performed the gene expression array and compared their expression profiles to those of control fibroblast cells. The gene expression array analysis showed that 4800 transcript probes were significantly deregulated in cells with p.T120A mutation in comparison to the control. GO term association results showed that deregulated genes are mostly involved in the extracellular matrix and cytoskeleton. Furthermore, the p.T120A missense mutation in POC1A caused the formation of abnormal mitotic spindle structure, including supernumerary centrosomes, and changes in POC1A were accompanied by alterations in another centrosome-associated WD repeat protein p80-katanin. As a result, we identified a novel mutation in POC1A of patients with PD and showed that this mutation causes the formation of multiple numbers of centrioles and multipolar spindles with abnormal chromosome arrangement.

  8. Integrated genomic sequencing reveals mutational landscape of T-cell prolymphocytic leukemia.

    PubMed

    Kiel, Mark J; Velusamy, Thirunavukkarasu; Rolland, Delphine; Sahasrabuddhe, Anagh A; Chung, Fuzon; Bailey, Nathanael G; Schrader, Alexandra; Li, Bo; Li, Jun Z; Ozel, Ayse B; Betz, Bryan L; Miranda, Roberto N; Medeiros, L Jeffrey; Zhao, Lili; Herling, Marco; Lim, Megan S; Elenitoba-Johnson, Kojo S J

    2014-08-28

    The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

  9. Genomic characterization of pediatric T-cell acute lymphoblastic leukemia reveals novel recurrent driver mutations

    PubMed Central

    Spinella, Jean-François; Cassart, Pauline; Richer, Chantal; Saillour, Virginie; Ouimet, Manon; Langlois, Sylvie; St-Onge, Pascal; Sontag, Thomas; Healy, Jasmine; Minden, Mark D.; Sinnett, Daniel

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with variable prognosis. It represents 15% of diagnosed pediatric ALL cases and has a threefold higher incidence among males. Many recurrent alterations have been identified and help define molecular subgroups of T-ALL, however the full range of events involved in driving transformation remain to be defined. Using an integrative approach combining genomic and transcriptomic data, we molecularly characterized 30 pediatric T-ALLs and identified common recurrent T-ALL targets such as FBXW7, JAK1, JAK3, PHF6, KDM6A and NOTCH1 as well as novel candidate T-ALL driver mutations including the p.R35L missense mutation in splicesome factor U2AF1 found in 3 patients and loss of function mutations in the X-linked tumor suppressor genes MED12 (frameshit mutation p.V167fs, splice site mutation g.chrX:70339329T>C, missense mutation p.R1989H) and USP9X (nonsense mutation p.Q117*). In vitro functional studies further supported the putative role of these novel T-ALL genes in driving transformation. U2AF1 p.R35L was shown to induce aberrant splicing of downstream target genes, and shRNA knockdown of MED12 and USP9X was shown to confer resistance to apoptosis following T-ALL relevant chemotherapy drug treatment in Jurkat leukemia cells. Interestingly, nearly 60% of novel candidate driver events were identified among immature T-ALL cases, highlighting the underlying genomic complexity of pediatric T-ALL, and the need for larger integrative studies to decipher the mechanisms that contribute to its various subtypes and provide opportunities to refine patient stratification and treatment. PMID:27602765

  10. Structural analysis of mitochondrial mutations reveals a role for bigenomic protein interactions in human disease.

    PubMed

    Lloyd, Rhiannon E; McGeehan, John E

    2013-01-01

    Mitochondria are the energy producing organelles of the cell, and mutations within their genome can cause numerous and often severe human diseases. At the heart of every mitochondrion is a set of five large multi-protein machines collectively known as the mitochondrial respiratory chain (MRC). This cellular machinery is central to several processes important for maintaining homeostasis within cells, including the production of ATP. The MRC is unique due to the bigenomic origin of its interacting proteins, which are encoded in the nucleus and mitochondria. It is this, in combination with the sheer number of protein-protein interactions that occur both within and between the MRC complexes, which makes the prediction of function and pathological outcome from primary sequence mutation data extremely challenging. Here we demonstrate how 3D structural analysis can be employed to predict the functional importance of mutations in mtDNA protein-coding genes. We mined the MITOMAP database and, utilizing the latest structural data, classified mutation sites based on their location within the MRC complexes III and IV. Using this approach, four structural classes of mutation were identified, including one underexplored class that interferes with nuclear-mitochondrial protein interactions. We demonstrate that this class currently eludes existing predictive approaches that do not take into account the quaternary structural organization inherent within and between the MRC complexes. The systematic and detailed structural analysis of disease-associated mutations in the mitochondrial Complex III and IV genes significantly enhances the predictive power of existing approaches and our understanding of how such mutations contribute to various pathologies. Given the general lack of any successful therapeutic approaches for disorders of the MRC, these findings may inform the development of new diagnostic and prognostic biomarkers, as well as new drugs and targets for gene therapy.

  11. Next-generation sequencing reveals DGUOK mutations in adult patients with mitochondrial DNA multiple deletions

    PubMed Central

    Garone, Caterina; Bordoni, Andreina; Gutierrez Rios, Purificacion; Calvo, Sarah E.; Ripolone, Michela; Ranieri, Michela; Rizzuti, Mafalda; Villa, Luisa; Magri, Francesca; Corti, Stefania; Bresolin, Nereo; Mootha, Vamsi K.; Moggio, Maurizio; DiMauro, Salvatore; Comi, Giacomo P.; Sciacco, Monica

    2012-01-01

    The molecular diagnosis of mitochondrial disorders still remains elusive in a large proportion of patients, but advances in next generation sequencing are significantly improving our chances to detect mutations even in sporadic patients. Syndromes associated with mitochondrial DNA multiple deletions are caused by different molecular defects resulting in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia to multi-systemic disorders of variable severity. The mutations underlying these conditions remain undisclosed in half of the affected subjects. We applied next-generation sequencing of known mitochondrial targets (MitoExome) to probands presenting with adult-onset mitochondrial myopathy and harbouring mitochondrial DNA multiple deletions in skeletal muscle. We identified autosomal recessive mutations in the DGUOK gene (encoding mitochondrial deoxyguanosine kinase), which has previously been associated with an infantile hepatocerebral form of mitochondrial DNA depletion. Mutations in DGUOK occurred in five independent subjects, representing 5.6% of our cohort of patients with mitochondrial DNA multiple deletions, and impaired both muscle DGUOK activity and protein stability. Clinical presentations were variable, including mitochondrial myopathy with or without progressive external ophthalmoplegia, recurrent rhabdomyolysis in a young female who had received a liver transplant at 9 months of age and adult-onset lower motor neuron syndrome with mild cognitive impairment. These findings reinforce the concept that mutations in genes involved in deoxyribonucleotide metabolism can cause diverse clinical phenotypes and suggest that DGUOK should be screened in patients harbouring mitochondrial DNA deletions in skeletal muscle. PMID:23043144

  12. Point mutation frequency in the FMR1 gene as revealed by fragile X syndrome screening.

    PubMed

    Handt, Maximilian; Epplen, Andrea; Hoffjan, Sabine; Mese, Kemal; Epplen, Jörg T; Dekomien, Gabriele

    2014-01-01

    Fragile X syndrome (FXS) is a common cause of intellectual disability, developmental delay and autism spectrum disorders. This syndrome is due to a functional loss of the FMR1 gene product FMRP, and, in most cases, it is caused by CGG repeat expansion in the FMR1 promoter. Yet, also other FMR1 mutations may cause a FXS-like phenotype. Since standard molecular testing does not include the analysis of the FMR1 coding region, the prevalence of point mutations causing FXS is not well known. Here, high resolution melting (HRM) was used to screen for FMR1 gene mutations in 508 males with clinical signs of mental retardation and developmental delay, but without CGG and GCC repeat expansions in the FMR1 gene and AFF2 genes, respectively. Sequence variations were identified by HRM analysis and verified by direct DNA sequencing. Two novel missense mutations (p.Gly482Ser in one patient and p.Arg534His in two unrelated patients), one intronic and two 3'-untranslated region (UTR) variations were identified in the FMR1 gene. Missense mutations in the FMR1 gene might account for a considerable proportion of cases in male patients with FXS-related symptoms, such as those linked to mental retardation and developmental delay.

  13. Revealing the function of a novel splice-site mutation of CHD7 in CHARGE syndrome.

    PubMed

    Lee, Byeonghyeon; Duz, Mehmet Bugrahan; Sagong, Borum; Koparir, Asuman; Lee, Kyu-Yup; Choi, Jae Young; Seven, Mehmet; Yuksel, Adnan; Kim, Un-Kyung; Ozen, Mustafa

    2016-02-01

    Most cases of CHARGE syndrome are sporadic and autosomal dominant. CHD7 is a major causative gene of CHARGE syndrome. In this study, we screened CHD7 in two Turkish patients demonstrating symptoms of CHARGE syndrome such as coloboma, heart defect, choanal atresia, retarded growth, genital abnomalities and ear anomalies. Two mutations of CHD7 were identified including a novel splice-site mutation (c.2443-2A>G) and a previously known frameshift mutation (c.2504_2508delATCTT). We performed exon trapping analysis to determine the effect of the c.2443-2A>G mutation at the transcriptional level, and found that it caused a complete skip of exon 7 and splicing at a cryptic splice acceptor site. Our current study is the second study demonstrating an exon 7 deficit in CHD7. Results of previous studies suggest that the c.2443-2A>G mutation affects the formation of nasal tissues and the neural retina during early development, resulting in choanal atresia and coloboma, respectively. The findings of the present study will improve our understanding of the genetic causes of CHARGE syndrome.

  14. Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma.

    PubMed

    Mann, Karen M; Ward, Jerrold M; Yew, Christopher Chin Kuan; Kovochich, Anne; Dawson, David W; Black, Michael A; Brett, Benjamin T; Sheetz, Todd E; Dupuy, Adam J; Chang, David K; Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Grimmond, Sean M; Rust, Alistair G; Adams, David J; Jenkins, Nancy A; Copeland, Neal G

    2012-04-17

    Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma.

  15. Massively parallel sequencing of phyllodes tumours of the breast reveals actionable mutations, and TERT promoter hotspot mutations and TERT gene amplification as likely drivers of progression

    PubMed Central

    Piscuoglio, Salvatore; Ng, Charlotte K Y; Murray, Melissa; Burke, Kathleen A; Edelweiss, Marcia; Geyer, Felipe C; Macedo, Gabriel S; Inagaki, Akiko; Papanastasiou, Anastasios D; Martelotto, Luciano G; Marchio, Caterina; Lim, Raymond S; Ioris, Rafael A; Nahar, Pooja K; De Bruijn, Ino; Smyth, Lillian; Akram, Muzaffar; Ross, Dara; Petrini, John H; Norton, Larry; Solit, David B; Baselga, Jose; Brogi, Edi; Ladanyi, Marc; Weigelt, Britta; Reis-Filho, Jorge S

    2015-01-01

    Phyllodes tumours (PTs) are breast fibroepithelial lesions that are graded based on histological criteria as benign, borderline or malignant. PTs may recur locally. Borderline PTs and malignant PTs may metastasize to distant sites. Breast fibroepithelial lesions, including PTs and fibroadenomas, are characterized by recurrent MED12 exon 2 somatic mutations. We sought to define the repertoire of somatic genetic alterations in PTs and whether these may assist in the differential diagnosis of these lesions. We collected 100 fibroadenomas, 40 benign PTs, 14 borderline PTs and 22 malignant PTs. Six, 6 and 13 benign, borderline and malignant PTs respectively and their matched normal tissue were subjected to targeted massively parallel sequencing (MPS) using the MSK-IMPACT sequencing assay. Recurrent MED12 mutations were found in 56% of PTs; in addition, mutations affecting cancer genes (e.g. TP53, RB1, SETD2 and EGFR) were exclusively detected in borderline and malignant PTs. We found a novel recurrent clonal hotspot mutation in the TERT promoter (−124 C>T) in 52% and TERT gene amplification in 4% of PTs. Laser capture microdissection revealed that these mutations were restricted to the mesenchymal component of PTs. Sequencing analysis of the entire cohort revealed that the frequency of TERT alterations increased from benign (18%), to borderline (57%) and to malignant PTs (68%; P<0.01), and TERT alterations were associated with increased levels of TERT mRNA (P<0.001). No TERT alterations were observed in fibroadenomas. An analysis of TERT promoter sequencing and gene amplification distinguished PTs from fibroadenomas with a sensitivity and a positive predictive value of 100% (CI 95.38%–100%) and 100% (CI 85.86%–100%), respectively, and a sensitivity and a negative predictive value of 39% (CI 28.65%–51.36%) and 68% (CI 60.21%–75.78%), respectively. Our results suggest that TERT alterations may drive the progression of PTs, and may assist in the differential

  16. Exome sequencing of osteosarcoma reveals mutation signatures reminiscent of BRCA deficiency.

    PubMed

    Kovac, Michal; Blattmann, Claudia; Ribi, Sebastian; Smida, Jan; Mueller, Nikola S; Engert, Florian; Castro-Giner, Francesc; Weischenfeldt, Joachim; Kovacova, Monika; Krieg, Andreas; Andreou, Dimosthenis; Tunn, Per-Ulf; Dürr, Hans Roland; Rechl, Hans; Schaser, Klaus-Dieter; Melcher, Ingo; Burdach, Stefan; Kulozik, Andreas; Specht, Katja; Heinimann, Karl; Fulda, Simone; Bielack, Stefan; Jundt, Gernot; Tomlinson, Ian; Korbel, Jan O; Nathrath, Michaela; Baumhoer, Daniel

    2015-12-03

    Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which has historically complicated driver gene discovery. Here we sequence exomes of 31 tumours and decipher their evolutionary landscape by inferring clonality of the individual mutation events. Exome findings are interpreted in the context of mutation and SNP array data from a replication set of 92 tumours. We identify 14 genes as the main drivers, of which some were formerly unknown in the context of osteosarcoma. None of the drivers is clearly responsible for the majority of tumours and even TP53 mutations are frequently mapped into subclones. However, >80% of osteosarcomas exhibit a specific combination of single-base substitutions, LOH, or large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours. Our findings imply that multiple oncogenic pathways drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits, which could be therapeutically exploited.

  17. Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations

    PubMed Central

    O’Roak, Brian J.; Vives, Laura; Girirajan, Santhosh; Karakoc, Emre; Krumm, Nik; Coe, Bradley P.; Levy, Roie; Ko, Arthur; Lee, Choli; Smith, Joshua D.; Turner, Emily H.; Stanaway, Ian B.; Vernot, Benjamin; Malig, Maika; Baker, Carl; Reilly, Beau; Akey, Joshua M.; Borenstein, Elhanan; Rieder, Mark J.; Nickerson, Deborah A.; Bernier, Raphael; Shendure, Jay; Eichler, Evan E.

    2012-01-01

    It is well established that autism spectrum disorders (ASD) have a strong genetic component. However, for at least 70% of cases, the underlying genetic cause is unknown1. Under the hypothesis that de novo mutations underlie a substantial fraction of the risk for developing ASD in families with no previous history of ASD or related phenotypes—so-called sporadic or simplex families2,3, we sequenced all coding regions of the genome, i.e. the exome, for parent-child trios exhibiting sporadic ASD, including 189 new trios and 20 previously reported4. Additionally, we also sequenced the exomes of 50 unaffected siblings corresponding to these new (n = 31) and previously reported trios (n = 19)4, for a total of 677 individual exomes from 209 families. Here we show de novo point mutations are overwhelmingly paternal in origin (4:1 bias) and positively correlated with paternal age, consistent with the modest increased risk for children of older fathers to develop ASD5. Moreover, 39% (49/126) of the most severe or disruptive de novo mutations map to a highly interconnected beta-catenin/chromatin remodeling protein network ranked significantly for autism candidate genes. In proband exomes, recurrent protein-altering mutations were observed in two genes, CHD8 and NTNG1. Mutation screening of six candidate genes in 1,703 ASD probands identified additional de novo, protein-altering mutations in GRIN2B, LAMC3, and SCN1A. Combined with copy number variant (CNV) data, these results suggest extreme locus heterogeneity but also provide a target for future discovery, diagnostics, and therapeutics. PMID:22495309

  18. Exome sequencing reveals VCP mutations as a cause of familial ALS

    PubMed Central

    Johnson, Janel O.; Mandrioli, Jessica; Benatar, Michael; Abramzon, Yevgeniya; Van Deerlin, Vivianna M.; Trojanowski, John Q.; Gibbs, J Raphael; Brunetti, Maura; Gronka, Susan; Wuu, Joanne; Ding, Jinhui; McCluskey, Leo; Martinez-Lage, Maria; Falcone, Dana; Hernandez, Dena G.; Arepalli, Sampath; Chong, Sean; Schymick, Jennifer C.; Rothstein, Jeffrey; Landi, Francesco; Wang, Michael; Calvo, Andrea; Mora, Gabriele; Sabatelli, Mario; Monsurrò, Maria Rosaria; Battistini, Stefania; Salvi, Fabrizio; Spataro, Rossella; Sola, Patrizia; Borghero, Giuseppe; Galassi, Giuliana; Scholz, Sonja W.; Taylor, J. Paul; Restagno, Gabriella; Chiò, Adriano; Traynor, Bryan J.

    2010-01-01

    Summary Using exome sequencing, we identified a p.R191Q amino acid change in the valosin-containing protein (VCP) gene in an Italian family with autosomal dominantly inherited amyotrophic lateral sclerosis (ALS). Mutations in VCP have previously been identified in families with Inclusion Body Myopathy, Paget’s disease and Frontotemporal Dementia (IBMPFD). Screening of VCP in a cohort of 210 familial ALS cases and 78 autopsy-proven ALS cases identified four additional mutations including a p.R155H mutation in a pathologically-proven case of ALS. VCP protein is essential for maturation of ubiquitin-containing autophagosomes, and mutant VCP toxicity is partially mediated through its effect on TDP-43 protein, a major constituent of ubiquitin inclusions that neuropathologically characterize ALS. Our data broaden the phenotype of IBMPFD to include motor neuron degeneration, suggest that VCP mutations may account for ~1–2% of familial ALS, and represent the first evidence directly implicating defects in the ubiquitination/protein degradation pathway in motor neuron degeneration. PMID:21145000

  19. Analysis of ultra-deep targeted sequencing reveals mutation burden is associated with gender and clinical outcome in lung adenocarcinoma

    PubMed Central

    Li, Fuqiang; Wu, Kui; Zhang, Xin; He, Jianxing

    2016-01-01

    Gender-associated difference in incidence and clinical outcomes of lung cancer have been established, but the biological mechanisms underlying these gender-associated differences are less studied. Recently we have characterized the genomic landscape of lung adenocarcinoma derived from Chinese population (Reference [1]). In this study we evaluated the clinical significance of mutation burden in lung adenocarcinoma and found that the male tumors harbored statistically greater burden of genetic alterations than female counterparts (Male median 3 (range 0–34) vs female median = 2 (0–24), male to female ratio = 1.636, 95% CI = 1.343–1.992) after adjustment of age at surgery, stage, smoking status. Kaplan-Meier survival analysis revealed that greater burden of genetic alterations was associated with worse overall survival. Moreover, multivariable analysis demonstrated mutation burden was an independent prognostic factor for the patients. Taken together, our analysis demonstrated gender disparity of mutation burden and their prognostic value in lung adenocarcinoma. This gender difference in mutation burden might provide an explanation for the distinct difference in the clinical outcomes between sexes in lung adenocarcinoma. PMID:27009843

  20. The Hutchinson-Gilford Progeria Syndrome: a case report.

    PubMed

    Russo-Menna, I; Arancibias, C

    2010-02-01

    The HGPS (Hutchinson Gilford Progeria Syndrome) is a rare genetic disorder with an incidence of 1 per 8 million live births. Originally described in 1886, less than 100 cases have been reported. The syndrome is characterized by premature aging with changes in many organs. The diagnosis is usually made by age 2, the mean survival age is 13.4 years and the most common cause of death is myocardial infarction. Recent genetic advances have identified the cause as a defect in the LMNA gene of chromosome 1.

  1. Towards a Drosophila model of Hutchinson-Gilford progeria syndrome.

    PubMed

    Beard, Gemma S; Bridger, Joanna M; Kill, Ian R; Tree, David R P

    2008-12-01

    The laminopathy Hutchinson-Gilford progeria syndrome (HGPS) is caused by the mutant lamin A protein progerin and leads to premature aging of affected children. Despite numerous cell biological and biochemical insights into the basis for the cellular abnormalities seen in HGPS, the mechanism linking progerin to the organismal phenotype is not fully understood. To begin to address the mechanism behind HGPS using Drosophila melanogaster, we have ectopically expressed progerin and lamin A. We found that ectopic progerin and lamin A phenocopy several effects of laminopathies in developing and adult Drosophila, but that progerin causes a stronger phenotype than wild-type lamin A.

  2. Mutation in a primate-conserved retrotransposon reveals a noncoding RNA as a mediator of infantile encephalopathy

    PubMed Central

    Cartault, François; Munier, Patrick; Benko, Edgar; Desguerre, Isabelle; Hanein, Sylvain; Boddaert, Nathalie; Bandiera, Simonetta; Vellayoudom, Jeanine; Krejbich-Trotot, Pascale; Bintner, Marc; Hoarau, Jean-Jacques; Girard, Muriel; Génin, Emmanuelle; de Lonlay, Pascale; Fourmaintraux, Alain; Naville, Magali; Rodriguez, Diana; Feingold, Josué; Renouil, Michel; Munnich, Arnold; Westhof, Eric; Fähling, Michael; Lyonnet, Stanislas; Henrion-Caude, Alexandra

    2012-01-01

    The human genome is densely populated with transposons and transposon-like repetitive elements. Although the impact of these transposons and elements on human genome evolution is recognized, the significance of subtle variations in their sequence remains mostly unexplored. Here we report homozygosity mapping of an infantile neurodegenerative disease locus in a genetic isolate. Complete DNA sequencing of the 400-kb linkage locus revealed a point mutation in a primate-specific retrotransposon that was transcribed as part of a unique noncoding RNA, which was expressed in the brain. In vitro knockdown of this RNA increased neuronal apoptosis, consistent with the inappropriate dosage of this RNA in vivo and with the phenotype. Moreover, structural analysis of the sequence revealed a small RNA-like hairpin that was consistent with the putative gain of a functional site when mutated. We show here that a mutation in a unique transposable element-containing RNA is associated with lethal encephalopathy, and we suggest that RNAs that harbor evolutionarily recent repetitive elements may play important roles in human brain development. PMID:22411793

  3. A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function.

    PubMed

    Okray, Zeynep; de Esch, Celine E F; Van Esch, Hilde; Devriendt, Koen; Claeys, Annelies; Yan, Jiekun; Verbeeck, Jelle; Froyen, Guy; Willemsen, Rob; de Vrij, Femke M S; Hassan, Bassem A

    2015-04-01

    Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

  4. Cardiomyopathy mutations reveal variable region of myosin converter as major element of cross-bridge compliance.

    PubMed

    Seebohm, B; Matinmehr, F; Köhler, J; Francino, A; Navarro-Lopéz, F; Perrot, A; Ozcelik, C; McKenna, W J; Brenner, B; Kraft, T

    2009-08-05

    The ability of myosin to generate motile forces is based on elastic distortion of a structural element of the actomyosin complex (cross-bridge) that allows strain to develop before filament sliding. Addressing the question, which part of the actomyosin complex experiences main elastic distortion, we suggested previously that the converter domain might be the most compliant region of the myosin head domain. Here we test this proposal by studying functional effects of naturally occurring missense mutations in the beta-myosin heavy chain, 723Arg --> Gly (R723G) and 736Ile --> Thr (I736T), in comparison to 719Arg --> Trp (R719W). All three mutations are associated with hypertrophic cardiomyopathy and are located in the converter region of the myosin head domain. We determined several mechanical parameters of single skinned slow fibers isolated from Musculus soleus biopsies of hypertrophic cardiomyopathy patients and healthy controls. Major findings of this study for mutation R723G were i), a >40% increase in fiber stiffness in rigor with a 2.9-fold increase in stiffness per myosin head (S( *)(rigor R723G) = 0.84 pN/nm S( *)(rigor WT) = 0.29 pN/nm); and ii), a significant increase in force per head (F( *)(10 degrees C), 1.99 pN vs. 1.49 pN = 1.3-fold increase; F( *)(20 degrees C), 2.56 pN vs. 1.92 pN = 1.3-fold increase) as well as stiffness per head during isometric steady-state contraction (S( *)(active10 degrees C), 0.52 pN/nm vs. 0.28 pN/nm = 1.9-fold increase). Similar changes were found for mutation R719W (2.6-fold increase in S( *)(rigor); 1.8-fold increase in F( *)(10 degrees C), 1.6-fold in F( *)(20 degrees C); twofold increase in S( *)(active10 degrees C)). Changes in active cross-bridge cycling kinetics could not account for the increase in force and active stiffness. For the above estimates the previously determined fraction of mutated myosin in the biopsies was taken into account. Data for wild-type myosin of slow soleus muscle fibers support previous

  5. MtDNA analysis reveals enriched pathogenic mutations in Tibetan highlanders

    PubMed Central

    Kang, Longli; Zheng, Hong-Xiang; Zhang, Menghan; Yan, Shi; Li, Lei; Liu, Lijun; Liu, Kai; Hu, Kang; Chen, Feng; Ma, Lifeng; Qin, Zhendong; Wang, Yi; Wang, Xiaofeng; Jin, Li

    2016-01-01

    Tibetan highlanders, including Tibetans, Monpas, Lhobas, Dengs and Sherpas, are considered highly adaptive to severe hypoxic environments. Mitochondrial DNA (mtDNA) might be important in hypoxia adaptation given its role in coding core subunits of oxidative phosphorylation. In this study, we employed 549 complete highlander mtDNA sequences (including 432 random samples) to obtain a comprehensive view of highlander mtDNA profile. In the phylogeny of a total of 36,914 sequences, we identified 21 major haplogroups representing founding events of highlanders, most of which were coalesced in 10 kya. Through founder analysis, we proposed a three-phase model of colonizing the plateau, i.e., pre-LGM Time (30 kya, 4.68%), post-LGM Paleolithic Time (16.8 kya, 29.31%) and Neolithic Time (after 8 kya, 66.01% in total). We observed that pathogenic mutations occurred far more frequently in 22 highlander-specific lineages (five lineages carrying two pathogenic mutations and six carrying one) than in the 6,857 haplogroups of all the 36,914 sequences (P = 4.87 × 10−8). Furthermore, the number of possible pathogenic mutations carried by highlanders (in average 3.18 ± 1.27) were significantly higher than that in controls (2.82 ± 1.40) (P = 1.89 × 10−4). Considering that function-altering and pathogenic mutations are enriched in highlanders, we therefore hypothesize that they may have played a role in hypoxia adaptation. PMID:27498855

  6. Mutational and fitness landscapes of an RNA virus revealed through population sequencing.

    PubMed

    Acevedo, Ashley; Brodsky, Leonid; Andino, Raul

    2014-01-30

    RNA viruses exist as genetically diverse populations. It is thought that diversity and genetic structure of viral populations determine the rapid adaptation observed in RNA viruses and hence their pathogenesis. However, our understanding of the mechanisms underlying virus evolution has been limited by the inability to accurately describe the genetic structure of virus populations. Next-generation sequencing technologies generate data of sufficient depth to characterize virus populations, but are limited in their utility because most variants are present at very low frequencies and are thus indistinguishable from next-generation sequencing errors. Here we present an approach that reduces next-generation sequencing errors and allows the description of virus populations with unprecedented accuracy. Using this approach, we define the mutation rates of poliovirus and uncover the mutation landscape of the population. Furthermore, by monitoring changes in variant frequencies on serially passaged populations, we determined fitness values for thousands of mutations across the viral genome. Mapping of these fitness values onto three-dimensional structures of viral proteins offers a powerful approach for exploring structure-function relationships and potentially uncovering new functions. To our knowledge, our study provides the first single-nucleotide fitness landscape of an evolving RNA virus and establishes a general experimental platform for studying the genetic changes underlying the evolution of virus populations.

  7. Biophysical analysis of a lethal laminin alpha-1 mutation reveals altered self-interaction.

    PubMed

    Patel, Trushar R; Nikodemus, Denise; Besong, Tabot M D; Reuten, Raphael; Meier, Markus; Harding, Stephen E; Winzor, Donald J; Koch, Manuel; Stetefeld, Jörg

    2016-01-01

    Laminins are key basement membrane molecules that influence several biological activities and are linked to a number of diseases. They are secreted as heterotrimeric proteins consisting of one α, one β, and one γ chain, followed by their assembly into a polymer-like sheet at the basement membrane. Using sedimentation velocity, dynamic light scattering, and surface plasmon resonance experiments, we studied self-association of three laminin (LM) N-terminal fragments α-1 (hLM α-1N), α-5 (hLM α-5N) and β-3 (hLM β-3N) originating from the short arms of the human laminin αβγ heterotrimer. Corresponding studies of the hLM α-1N C49S mutant, equivalent to the larval lethal C56S mutant in zebrafish, have shown that this mutation causes enhanced self-association behavior, an observation that provides a plausible explanation for the inability of laminin bearing this mutation to fulfill functional roles in vivo, and hence for the deleterious pathological consequences of the mutation on lens function.

  8. Exome sequencing reveals HINT1 mutations as a cause of distal hereditary motor neuropathy

    PubMed Central

    Zhao, Hui; Race, Valérie; Matthijs, Gert; De Jonghe, Peter; Robberecht, Wim; Lambrechts, Diether; Van Damme, Philip

    2014-01-01

    Distal hereditary motor neuropathies (dHMNs) are a heterogenous group of genetic disorders with length-dependent degeneration of motor axons. Obtaining a genetic diagnosis in patients with dHMN remains challenging. We performed exome sequencing in a diagnostic setting in 12 patients with a clinical diagnosis of dHMN. Potential disease-causing variants in genes associated with dHMN and other forms of inherited neuropathies/motor neuron diseases were validated using Sequenom. The coverage in the genes studied was >95% with an average coverage of >50 times. In none of the patients a mutations was found in genes previously reported to be associated with dHMN. However, in 2/12 patients a recessive mutation in histidine triad nucleotide binding protein 1 (HINT1, recently discovered as a cause of axonal neuropathy with neuromyotonia) was identified. Our results demonstrate the diagnostic value of exome sequencing for patients with inherited neuropathies. The phenotypic spectrum of recessive mutations in HINT1 includes dHMN. HINT1 should be added to the list of genes to check for in dHMN. PMID:24105373

  9. In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase.

    PubMed Central

    Geslain, R; Martin, F; Delagoutte, B; Cavarelli, J; Gangloff, J; Eriani, G

    2000-01-01

    Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination. PMID:10744027

  10. Mechanisms controlling the smooth muscle cell death in progeria via down-regulation of poly(ADP-ribose) polymerase 1.

    PubMed

    Zhang, Haoyue; Xiong, Zheng-Mei; Cao, Kan

    2014-06-03

    Hutchinson-Gilford progeria syndrome (HGPS) is a severe human premature aging disorder caused by a lamin A mutant named progerin. Death occurs at a mean age of 13 y from cardiovascular problems. Previous studies revealed loss of vascular smooth muscle cells (SMCs) in the media of large arteries in a patient with HGPS and two mouse models, suggesting a causal connection between the SMC loss and cardiovascular malfunction. However, the mechanisms of how progerin leads to massive SMC loss are unknown. In this study, using SMCs differentiated from HGPS induced pluripotent stem cells, we show that HGPS SMCs exhibit a profound proliferative defect, which is primarily caused by caspase-independent cell death. Importantly, progerin accumulation stimulates a powerful suppression of PARP1 and consequently triggers an activation of the error-prone nonhomologous end joining response. As a result, most HGPS SMCs exhibit prolonged mitosis and die of mitotic catastrophe. This study demonstrates a critical role of PARP1 in mediating SMC loss in patients with HGPS and elucidates a molecular pathway underlying the progressive SMC loss in progeria.

  11. Mouse models and aging: longevity and progeria.

    PubMed

    Liao, Chen-Yu; Kennedy, Brian K

    2014-01-01

    Aging is a complex, multifactorial process that is likely influenced by the activities of a range of biological pathways. Genetic approaches to identify genes modulating longevity have been highly successful and recent efforts have extended these studies to mammalian aging. A variety of genetic models have been reported to have enhanced lifespan and, similarly, many genetic interventions lead to progeroid phenotypes. Here, we detail and evaluate both sets of models, focusing on the insights they provide about the molecular processes modulating aging and the extent to which mutations conferring progeroid pathologies really phenocopy accelerated aging.

  12. Exome and deep sequencing of clinically aggressive neuroblastoma reveal somatic mutations that affect key pathways involved in cancer progression

    PubMed Central

    Lasorsa, Vito Alessandro; Formicola, Daniela; Pignataro, Piero; Cimmino, Flora; Calabrese, Francesco Maria; Mora, Jaume; Esposito, Maria Rosaria; Pantile, Marcella; Zanon, Carlo; De Mariano, Marilena; Longo, Luca; Hogarty, Michael D.; de Torres, Carmen; Tonini, Gian Paolo; Iolascon, Achille; Capasso, Mario

    2016-01-01

    The spectrum of somatic mutation of the most aggressive forms of neuroblastoma is not completely determined. We sought to identify potential cancer drivers in clinically aggressive neuroblastoma. Whole exome sequencing was conducted on 17 germline and tumor DNA samples from high-risk patients with adverse events within 36 months from diagnosis (HR-Event3) to identify somatic mutations and deep targeted sequencing of 134 genes selected from the initial screening in additional 48 germline and tumor pairs (62.5% HR-Event3 and high-risk patients), 17 HR-Event3 tumors and 17 human-derived neuroblastoma cell lines. We revealed 22 significantly mutated genes, many of which implicated in cancer progression. Fifteen genes (68.2%) were highly expressed in neuroblastoma supporting their involvement in the disease. CHD9, a cancer driver gene, was the most significantly altered (4.0% of cases) after ALK. Other genes (PTK2, NAV3, NAV1, FZD1 and ATRX), expressed in neuroblastoma and involved in cell invasion and migration were mutated at frequency ranged from 4% to 2%. Focal adhesion and regulation of actin cytoskeleton pathways, were frequently disrupted (14.1% of cases) thus suggesting potential novel therapeutic strategies to prevent disease progression. Notably BARD1, CHEK2 and AXIN2 were enriched in rare, potentially pathogenic, germline variants. In summary, whole exome and deep targeted sequencing identified novel cancer genes of clinically aggressive neuroblastoma. Our analyses show pathway-level implications of infrequently mutated genes in leading neuroblastoma progression. PMID:27009842

  13. Homozygosity mapping and whole exome sequencing reveal a novel homozygous COL18A1 mutation causing Knobloch syndrome.

    PubMed

    Haghighi, Alireza; Tiwari, Amit; Piri, Niloofar; Nürnberg, Gudrun; Saleh-Gohari, Nasrollah; Haghighi, Amirreza; Neidhardt, John; Nürnberg, Peter; Berger, Wolfgang

    2014-01-01

    The aim of this study was to identify the genetic basis of a chorioretinal dystrophy with high myopia of unknown origin in a child of a consanguineous marriage. The proband and ten family members of Iranian ancestry participated in this study. Linkage analysis was carried out with DNA samples of the proband and her parents by using the Human SNP Array 6.0. Whole exome sequencing (WES) was performed with the patients' DNA. Specific sequence alterations within the homozygous regions identified by whole exome sequencing were verified by Sanger sequencing. Upon genetic analysis, a novel homozygous frameshift mutation was found in exon 42 of the COL18A1 gene in the patient. Both parents were heterozygous for this sequence variation. Mutations in COL18A1 are known to cause Knobloch syndrome (KS). Retrospective analysis of clinical records of the patient revealed surgical removal of a meningocele present at birth. The clinical features shown by our patient were typical of KS with the exception of chorioretinal degeneration which is a rare manifestation. This is the first case of KS reported in a family of Iranian ancestry. We identified a novel disease-causing (deletion) mutation in the COL18A1 gene leading to a frameshift and premature stop codon in the last exon. The mutation was not present in SNP databases and was also not found in 192 control individuals. Its localization within the endostatin domain implicates a functional relevance of endostatin in KS. A combined approach of linkage analysis and WES led to a rapid identification of the disease-causing mutation even though the clinical description was not completely clear at the beginning.

  14. Homozygosity Mapping and Whole Exome Sequencing Reveal a Novel Homozygous COL18A1 Mutation Causing Knobloch Syndrome

    PubMed Central

    Piri, Niloofar; Nürnberg, Gudrun; Saleh-Gohari, Nasrollah; Haghighi, Amirreza; Neidhardt, John; Nürnberg, Peter; Berger, Wolfgang

    2014-01-01

    The aim of this study was to identify the genetic basis of a chorioretinal dystrophy with high myopia of unknown origin in a child of a consanguineous marriage. The proband and ten family members of Iranian ancestry participated in this study. Linkage analysis was carried out with DNA samples of the proband and her parents by using the Human SNP Array 6.0. Whole exome sequencing (WES) was performed with the patients’ DNA. Specific sequence alterations within the homozygous regions identified by whole exome sequencing were verified by Sanger sequencing. Upon genetic analysis, a novel homozygous frameshift mutation was found in exon 42 of the COL18A1 gene in the patient. Both parents were heterozygous for this sequence variation. Mutations in COL18A1 are known to cause Knobloch syndrome (KS). Retrospective analysis of clinical records of the patient revealed surgical removal of a meningocele present at birth. The clinical features shown by our patient were typical of KS with the exception of chorioretinal degeneration which is a rare manifestation. This is the first case of KS reported in a family of Iranian ancestry. We identified a novel disease-causing (deletion) mutation in the COL18A1 gene leading to a frameshift and premature stop codon in the last exon. The mutation was not present in SNP databases and was also not found in 192 control individuals. Its localization within the endostatin domain implicates a functional relevance of endostatin in KS. A combined approach of linkage analysis and WES led to a rapid identification of the disease-causing mutation even though the clinical description was not completely clear at the beginning. PMID:25392994

  15. Single-Nucleotide Mutations in FMR1 Reveal Novel Functions and Regulatory Mechanisms of the Fragile X Syndrome Protein FMRP

    PubMed Central

    Suhl, Joshua A.; Warren, Stephen T.

    2015-01-01

    Fragile X syndrome is a monogenic disorder and a common cause of intellectual disability. Despite nearly 25 years of research on FMR1, the gene underlying the syndrome, very few pathological mutations other than the typical CGG-repeat expansion have been reported. This is in contrast to other X-linked, monogenic, intellectual disability disorders, such as Rett syndrome, where many point mutations have been validated as causative of the disorder. As technology has improved and significantly driven down the cost of sequencing, allowing for whole genes to be sequenced with relative ease, in-depth sequencing studies on FMR1 have recently been performed. These studies have led to the identification of novel variants in FMR1, where some of which have been functionally evaluated and are likely pathogenic. In this review, we discuss recently identified FMR1 variants, the ways these novel variants cause dysfunction, and how they reveal new regulatory mechanisms and functionalities of the gene. PMID:26819560

  16. Mutations in TRAF3IP1/IFT54 reveal a new role for IFT proteins in microtubule stabilization.

    PubMed

    Bizet, Albane A; Becker-Heck, Anita; Ryan, Rebecca; Weber, Kristina; Filhol, Emilie; Krug, Pauline; Halbritter, Jan; Delous, Marion; Lasbennes, Marie-Christine; Linghu, Bolan; Oakeley, Edward J; Zarhrate, Mohammed; Nitschké, Patrick; Garfa-Traore, Meriem; Serluca, Fabrizio; Yang, Fan; Bouwmeester, Tewis; Pinson, Lucile; Cassuto, Elisabeth; Dubot, Philippe; Elshakhs, Neveen A Soliman; Sahel, José A; Salomon, Rémi; Drummond, Iain A; Gubler, Marie-Claire; Antignac, Corinne; Chibout, Salahdine; Szustakowski, Joseph D; Hildebrandt, Friedhelm; Lorentzen, Esben; Sailer, Andreas W; Benmerah, Alexandre; Saint-Mezard, Pierre; Saunier, Sophie

    2015-10-21

    Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF Receptor-Associated Factor Interacting Protein 1) in eight patients from five families with nephronophthisis (NPH) and retinal degeneration, two of the most common manifestations of ciliopathies. TRAF3IP1 encodes IFT54, a subunit of the IFT-B complex required for ciliogenesis. The identified mutations result in mild ciliary defects in patients but also reveal an unexpected role of IFT54 as a negative regulator of microtubule stability via MAP4 (microtubule-associated protein 4). Microtubule defects are associated with altered epithelialization/polarity in renal cells and with pronephric cysts and microphthalmia in zebrafish embryos. Our findings highlight the regulation of cytoplasmic microtubule dynamics as a role of the IFT54 protein beyond the cilium, contributing to the development of NPH-related ciliopathies.

  17. Clinical trial of a farnesyltransferase inhibitor in children with Hutchinson–Gilford progeria syndrome

    PubMed Central

    Gordon, Leslie B.; Kleinman, Monica E.; Miller, David T.; Neuberg, Donna S.; Giobbie-Hurder, Anita; Gerhard-Herman, Marie; Smoot, Leslie B.; Gordon, Catherine M.; Cleveland, Robert; Snyder, Brian D.; Fligor, Brian; Bishop, W. Robert; Statkevich, Paul; Regen, Amy; Sonis, Andrew; Riley, Susan; Ploski, Christine; Correia, Annette; Quinn, Nicolle; Ullrich, Nicole J.; Nazarian, Ara; Liang, Marilyn G.; Huh, Susanna Y.; Schwartzman, Armin; Kieran, Mark W.

    2012-01-01

    Hutchinson–Gilford progeria syndrome (HGPS) is an extremely rare, fatal, segmental premature aging syndrome caused by a mutation in LMNA that produces the farnesylated aberrant lamin A protein, progerin. This multisystem disorder causes failure to thrive and accelerated atherosclerosis leading to early death. Farnesyltransferase inhibitors have ameliorated disease phenotypes in preclinical studies. Twenty-five patients with HGPS received the farnesyltransferase inhibitor lonafarnib for a minimum of 2 y. Primary outcome success was predefined as a 50% increase over pretherapy in estimated annual rate of weight gain, or change from pretherapy weight loss to statistically significant on-study weight gain. Nine patients experienced a ≥50% increase, six experienced a ≥50% decrease, and 10 remained stable with respect to rate of weight gain. Secondary outcomes included decreases in arterial pulse wave velocity and carotid artery echodensity and increases in skeletal rigidity and sensorineural hearing within patient subgroups. All patients improved in one or more of these outcomes. Results from this clinical treatment trial for children with HGPS provide preliminary evidence that lonafarnib may improve vascular stiffness, bone structure, and audiological status. PMID:23012407

  18. Clinical trial of a farnesyltransferase inhibitor in children with Hutchinson-Gilford progeria syndrome.

    PubMed

    Gordon, Leslie B; Kleinman, Monica E; Miller, David T; Neuberg, Donna S; Giobbie-Hurder, Anita; Gerhard-Herman, Marie; Smoot, Leslie B; Gordon, Catherine M; Cleveland, Robert; Snyder, Brian D; Fligor, Brian; Bishop, W Robert; Statkevich, Paul; Regen, Amy; Sonis, Andrew; Riley, Susan; Ploski, Christine; Correia, Annette; Quinn, Nicolle; Ullrich, Nicole J; Nazarian, Ara; Liang, Marilyn G; Huh, Susanna Y; Schwartzman, Armin; Kieran, Mark W

    2012-10-09

    Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare, fatal, segmental premature aging syndrome caused by a mutation in LMNA that produces the farnesylated aberrant lamin A protein, progerin. This multisystem disorder causes failure to thrive and accelerated atherosclerosis leading to early death. Farnesyltransferase inhibitors have ameliorated disease phenotypes in preclinical studies. Twenty-five patients with HGPS received the farnesyltransferase inhibitor lonafarnib for a minimum of 2 y. Primary outcome success was predefined as a 50% increase over pretherapy in estimated annual rate of weight gain, or change from pretherapy weight loss to statistically significant on-study weight gain. Nine patients experienced a ≥50% increase, six experienced a ≥50% decrease, and 10 remained stable with respect to rate of weight gain. Secondary outcomes included decreases in arterial pulse wave velocity and carotid artery echodensity and increases in skeletal rigidity and sensorineural hearing within patient subgroups. All patients improved in one or more of these outcomes. Results from this clinical treatment trial for children with HGPS provide preliminary evidence that lonafarnib may improve vascular stiffness, bone structure, and audiological status.

  19. Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome.

    PubMed

    Liu, Guang-Hui; Barkho, Basam Z; Ruiz, Sergio; Diep, Dinh; Qu, Jing; Yang, Sheng-Lian; Panopoulos, Athanasia D; Suzuki, Keiichiro; Kurian, Leo; Walsh, Christopher; Thompson, James; Boue, Stephanie; Fung, Ho Lim; Sancho-Martinez, Ignacio; Zhang, Kun; Yates, John; Izpisua Belmonte, Juan Carlos

    2011-04-14

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs, progerin and its ageing-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.

  20. Increased mechanosensitivity and nuclear stiffness in Hutchinson-Gilford progeria cells: effects of farnesyltransferase inhibitors.

    PubMed

    Verstraeten, Valerie L R M; Ji, Julie Y; Cummings, Kiersten S; Lee, Richard T; Lammerding, Jan

    2008-06-01

    Hutchinson-Gilford progeria syndrome (HGPS), reportedly a model for normal aging, is a genetic disorder in children marked by dramatic signs suggestive for premature aging. It is usually caused by de novo mutations in the nuclear envelope protein lamin A. Lamins are essential to maintaining nuclear integrity, and loss of lamin A/C results in increased cellular sensitivity to mechanical strain and defective mechanotransduction signaling. Since increased mechanical sensitivity in vascular cells could contribute to loss of smooth muscle cells and the development of arteriosclerosis--the leading cause of death in HGPS patients--we investigated the effect of mechanical stress on cells from HGPS patients. We found that skin fibroblasts from HGPS patients developed progressively stiffer nuclei with increasing passage number. Importantly, fibroblasts from HGPS patients had decreased viability and increased apoptosis under repetitive mechanical strain, as well as attenuated wound healing, and these defects preceded changes in nuclear stiffness. Treating fibroblasts with farnesyltransferase inhibitors restored nuclear stiffness in HGPS cells and accelerated the wound healing response in HGPS and healthy control cells by increasing the directional persistence of migrating cells. However, farnesyltransferase inhibitors did not improve cellular sensitivity to mechanical strain. These data suggest that increased mechanical sensitivity in HGPS cells is unrelated to changes in nuclear stiffness and that increased biomechanical sensitivity could provide a potential mechanism for the progressive loss of vascular smooth muscle cells under physiological strain in HGPS patients.

  1. Distinct structural and mechanical properties of the nuclear lamina in Hutchinson-Gilford progeria syndrome.

    PubMed

    Dahl, Kris Noel; Scaffidi, Paola; Islam, Mohammad F; Yodh, Arjun G; Wilson, Katherine L; Misteli, Tom

    2006-07-05

    The nuclear lamina is a network of structural filaments, the A and B type lamins, located at the nuclear envelope and throughout the nucleus. Lamin filaments provide the nucleus with mechanical stability and support many basic activities, including gene regulation. Mutations in LMNA, the gene encoding A type lamins, cause numerous human diseases, including the segmental premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here we show that structural and mechanical properties of the lamina are altered in HGPS cells. We demonstrate by live-cell imaging and biochemical analysis that lamins A and C become trapped at the nuclear periphery in HGPS patient cells. Using micropipette aspiration, we show that the lamina in HGPS cells has a significantly reduced ability to rearrange under mechanical stress. Based on polarization microscopy results, we suggest that the lamins are disordered in the healthy nuclei, whereas the lamins in HGPS nuclei form orientationally ordered microdomains. The reduced deformability of the HGPS nuclear lamina possibly could be due to the inability of these orientationally ordered microdomains to dissipate mechanical stress. Surprisingly, intact HGPS cells exhibited a degree of resistance to acute mechanical stress similar to that of cells from healthy individuals. Thus, in contrast to the nuclear fragility seen in lmna null cells, the lamina network in HGPS cells has unique mechanical properties that might contribute to disease phenotypes by affecting responses to mechanical force and misregulation of mechanosensitive gene expression.

  2. Adhesion of Escherichia coli under flow conditions reveals potential novel effects of FimH mutations.

    PubMed

    Feenstra, T; Thøgersen, M S; Wieser, E; Peschel, A; Ball, M J; Brandes, R; Satchell, S C; Stockner, T; Aarestrup, F M; Rees, A J; Kain, R

    2017-03-01

    FimH-mediated adhesion of Escherichia coli to bladder epithelium is a prerequisite for urinary tract infections. FimH is also essential for blood-borne bacterial dissemination, but the mechanisms are poorly understood. The purpose of this study was to assess the influence of different FimH mutations on bacterial adhesion using a novel adhesion assay, which models the physiological flow conditions bacteria are exposed to. We introduced 12 different point mutations in the mannose binding pocket of FimH in an E. coli strain expressing type 1 fimbriae only (MSC95-FimH). We compared the bacterial adhesion of each mutant across several commonly used adhesion assays, including agglutination of yeast, adhesion to mono- and tri-mannosylated substrates, and static adhesion to bladder epithelial and endothelial cells. We performed a comparison of these assays to a novel method that we developed to study bacterial adhesion to mammalian cells under flow conditions. We showed that E. coli MSC95-FimH adheres more efficiently to microvascular endothelium than to bladder epithelium, and that only endothelium supports adhesion at physiological shear stress. The results confirmed that mannose binding pocket mutations abrogated adhesion. We demonstrated that FimH residues E50 and T53 are crucial for adhesion under flow conditions. The coating of endothelial cells on biochips and modelling of physiological flow conditions enabled us to identify FimH residues crucial for adhesion. These results provide novel insights into screening methods to determine the effect of FimH mutants and potentially FimH antagonists.

  3. Combining Natural Sequence Variation with High Throughput Mutational Data to Reveal Protein Interaction Sites

    PubMed Central

    Melamed, Daniel; Young, David L.; Miller, Christina R.; Fields, Stanley

    2015-01-01

    Many protein interactions are conserved among organisms despite changes in the amino acid sequences that comprise their contact sites, a property that has been used to infer the location of these sites from protein homology. In an inter-species complementation experiment, a sequence present in a homologue is substituted into a protein and tested for its ability to support function. Therefore, substitutions that inhibit function can identify interaction sites that changed over evolution. However, most of the sequence differences within a protein family remain unexplored because of the small-scale nature of these complementation approaches. Here we use existing high throughput mutational data on the in vivo function of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein, Pab1, to analyze its sites of interaction. Of 197 single amino acid differences in 52 Pab1 homologues, 17 reduce the function of Pab1 when substituted into the yeast protein. The majority of these deleterious mutations interfere with the binding of the RRM2 domain to eIF4G1 and eIF4G2, isoforms of a translation initiation factor. A large-scale mutational analysis of the RRM2 domain in a two-hybrid assay for eIF4G1 binding supports these findings and identifies peripheral residues that make a smaller contribution to eIF4G1 binding. Three single amino acid substitutions in yeast Pab1 corresponding to residues from the human orthologue are deleterious and eliminate binding to the yeast eIF4G isoforms. We create a triple mutant that carries these substitutions and other humanizing substitutions that collectively support a switch in binding specificity of RRM2 from the yeast eIF4G1 to its human orthologue. Finally, we map other deleterious substitutions in Pab1 to inter-domain (RRM2–RRM1) or protein-RNA (RRM2–poly(A)) interaction sites. Thus, the combined approach of large-scale mutational data and evolutionary conservation can be used to characterize interaction sites at single

  4. Initial cutaneous manifestations of Hutchinson-Gilford progeria syndrome.

    PubMed

    Rork, Jillian F; Huang, Jennifer T; Gordon, Leslie B; Kleinman, Monica; Kieran, Mark W; Liang, Marilyn G

    2014-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare, uniformly fatal, premature aging disease with distinct dermatologic features. We sought to identify and describe the initial skin and hair findings as potential diagnostic signs of the disease. We performed a chart review of the structured initial intake histories of 39 individuals with HGPS enrolled in clinical trials from 2007 to 2010 at Boston Children's Hospital, limited to cutaneous history from birth to 24 months. Medical photographs were provided through the clinical trials and the Progeria Research Foundation Medical and Research Database at Brown University Center for Gerontology and Healthcare Research. All 39 patients reported skin and hair abnormalities within the first 24 months of life. Pathologies included sclerodermoid change, prominent superficial veins, dyspigmentation, and alopecia. The mean age of presentation for each finding was <12 months. The most frequently reported skin feature was sclerodermoid change, which commonly involved the abdomen and bilateral lower extremities. Prominent superficial vasculature manifested as circumoral cyanosis and pronounced veins on the scalp and body. Hypo- and hyperpigmentation were observed over areas of sclerodermoid change. Scalp alopecia progressed in a distinct pattern, with preservation of the hair over the midscalp and vertex areas for the longest period of time. HGPS has distinct cutaneous manifestations during the first 2 years of life that may be the first signs of disease. Awareness of these findings could expedite diagnosis.

  5. Hutchinson–Gilford progeria mutant lamin A primarily targets human vascular cells as detected by an anti-Lamin A G608G antibody

    PubMed Central

    McClintock, Dayle; Gordon, Leslie B.; Djabali, Karima

    2006-01-01

    Hutchinson–Gilford progeria syndrome (HGPS; Online Mendelian Inheritance in Man accession no. 176670) is a rare disorder that is characterized by segmental premature aging and death between 7 and 20 years of age from severe premature atherosclerosis. Mutations in the LMNA gene are responsible for this syndrome. Approximately 80% of HGPS cases are caused by a G608 (GGC→GGT) mutation within exon 11 of LMNA, which elicits a deletion of 50 aa near the C terminus of prelamin A. In this article, we present evidence that the mutant lamin A (progerin) accumulates in the nucleus in a cellular age-dependent manner. In human HGPS fibroblast cultures, we observed, concomitantly to nuclear progerin accumulation, severe nuclear envelope deformations and invaginations preventable by farnesyltransferase inhibition. Nuclear alterations affect cell-cycle progression and cell migration and elicit premature senescence. Strikingly, skin biopsy sections from a subject with HGPS showed that the truncated lamin A accumulates primarily in the nuclei of vascular cells. This finding suggests that accumulation of progerin is directly involved in vascular disease in progeria. PMID:16461887

  6. Hutchinson-Gilford progeria mutant lamin A primarily targets human vascular cells as detected by an anti-Lamin A G608G antibody.

    PubMed

    McClintock, Dayle; Gordon, Leslie B; Djabali, Karima

    2006-02-14

    Hutchinson-Gilford progeria syndrome (HGPS; Online Mendelian Inheritance in Man accession no. 176670) is a rare disorder that is characterized by segmental premature aging and death between 7 and 20 years of age from severe premature atherosclerosis. Mutations in the LMNA gene are responsible for this syndrome. Approximately 80% of HGPS cases are caused by a G608 (GGC-->GGT) mutation within exon 11 of LMNA, which elicits a deletion of 50 aa near the C terminus of prelamin A. In this article, we present evidence that the mutant lamin A (progerin) accumulates in the nucleus in a cellular age-dependent manner. In human HGPS fibroblast cultures, we observed, concomitantly to nuclear progerin accumulation, severe nuclear envelope deformations and invaginations preventable by farnesyltransferase inhibition. Nuclear alterations affect cell-cycle progression and cell migration and elicit premature senescence. Strikingly, skin biopsy sections from a subject with HGPS showed that the truncated lamin A accumulates primarily in the nuclei of vascular cells. This finding suggests that accumulation of progerin is directly involved in vascular disease in progeria.

  7. Molecular and functional analysis of the large 5' promoter region of CFTR gene revealed pathogenic mutations in CF and CFTR-related disorders.

    PubMed

    Giordano, Sonia; Amato, Felice; Elce, Ausilia; Monti, Maria; Iannone, Carla; Pucci, Pietro; Seia, Manuela; Angioni, Adriano; Zarrilli, Federica; Castaldo, Giuseppe; Tomaiuolo, Rossella

    2013-05-01

    Patients with cystic fibrosis (CF) manifest a multisystemic disease due to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR); despite extensive testing of coding regions, a proportion of CF alleles remains unidentified. We studied 118 patients with CF and CFTR-related disorders, most with one or both unknown mutations after the scanning of CFTR coding regions, and a non-CF control group (n = 75) by sequencing the 6000-bp region at the 5' of the CFTR gene. We identified 23 mutations, of which 9 were novel. We expressed such mutations in vitro using four cell systems to explore their functional effect, relating the data to the clinical expression of each patient. Some mutations reduced expression of the gene reporter firefly luciferase in various cell lines and may act as disease-causing mutations. Other mutations caused an increase in luciferase expression in some cell lines. One mutation had a different effect in different cells. For other mutations, the expression assay excluded a functional role. Gene variants in the large 5' region may cause altered regulation of CFTR gene expression, acting as disease-causing mutations or modifiers of its clinical phenotype. Studies of in vitro expression in different cell systems may help reveal the effect of such mutations.

  8. Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

    PubMed Central

    Doshi, Urmi; Holliday, Michael J.; Eisenmesser, Elan Z.; Hamelberg, Donald

    2016-01-01

    Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue–residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general. PMID:27071107

  9. Dynamical network of residue-residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation.

    PubMed

    Doshi, Urmi; Holliday, Michael J; Eisenmesser, Elan Z; Hamelberg, Donald

    2016-04-26

    Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue-residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.

  10. Characterization of PTEN mutations in brain cancer reveals that pten mono-ubiquitination promotes protein stability and nuclear localization.

    PubMed

    Yang, Jr-M; Schiapparelli, P; Nguyen, H-N; Igarashi, A; Zhang, Q; Abbadi, S; Amzel, L M; Sesaki, H; Quiñones-Hinojosa, A; Iijima, M

    2017-03-06

    PTEN is a PIP3 phosphatase that antagonizes oncogenic PI3-kinase signalling. Due to its critical role in suppressing the potent signalling pathway, it is one of the most mutated tumour suppressors, especially in brain tumours. It is generally thought that PTEN deficiencies predominantly result from either loss of expression or enzymatic activity. By analysing PTEN in malignant glioblastoma primary cells derived from 16 of our patients, we report mutations that block localization of PTEN at the plasma membrane and nucleus without affecting lipid phosphatase activity. Cellular and biochemical analyses as well as structural modelling revealed that two mutations disrupt intramolecular interaction of PTEN and open its conformation, enhancing polyubiquitination of PTEN and decreasing protein stability. Moreover, promoting mono-ubiquitination increases protein stability and nuclear localization of mutant PTEN. Thus, our findings provide a molecular mechanism for cancer-associated PTEN defects and may lead to a brain cancer treatment that targets PTEN mono-ubiquitination.Oncogene advance online publication, 6 March 2017; doi:10.1038/onc.2016.493.

  11. Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK–STAT pathway in Sézary syndrome

    PubMed Central

    Kiel, Mark J.; Sahasrabuddhe, Anagh A.; Rolland, Delphine C. M.; Velusamy, Thirunavukkarasu; Chung, Fuzon; Schaller, Matthew; Bailey, Nathanael G.; Betz, Bryan L.; Miranda, Roberto N.; Porcu, Pierluigi; Byrd, John C.; Jeffrey Medeiros, L.; Kunkel, Steven L.; Bahler, David W.; Lim, Megan S.; Elenitoba-Johnson, Kojo S. J.

    2015-01-01

    Sézary syndrome (SS) is an aggressive leukaemia of mature T cells with poor prognosis and limited options for targeted therapies. The comprehensive genetic alterations underlying the pathogenesis of SS are unknown. Here we integrate whole-genome sequencing (n=6), whole-exome sequencing (n=66) and array comparative genomic hybridization-based copy-number analysis (n=80) of primary SS samples. We identify previously unknown recurrent loss-of-function aberrations targeting members of the chromatin remodelling/histone modification and trithorax families, including ARID1A in which functional loss from nonsense and frameshift mutations and/or targeted deletions is observed in 40.3% of SS genomes. We also identify recurrent gain-of-function mutations targeting PLCG1 (9%) and JAK1, JAK3, STAT3 and STAT5B (JAK/STAT total ∼11%). Functional studies reveal sensitivity of JAK1-mutated primary SS cells to JAK inhibitor treatment. These results highlight the complex genomic landscape of SS and a role for inhibition of JAK/STAT pathways for the treatment of SS. PMID:26415585

  12. Independent role for presynaptic FMRP revealed by an FMR1 missense mutation associated with intellectual disability and seizures.

    PubMed

    Myrick, Leila K; Deng, Pan-Yue; Hashimoto, Hideharu; Oh, Young Mi; Cho, Yongcheol; Poidevin, Mickael J; Suhl, Joshua A; Visootsak, Jeannie; Cavalli, Valeria; Jin, Peng; Cheng, Xiaodong; Warren, Stephen T; Klyachko, Vitaly A

    2015-01-27

    Fragile X syndrome (FXS) results in intellectual disability (ID) most often caused by silencing of the fragile X mental retardation 1 (FMR1) gene. The resulting absence of fragile X mental retardation protein 1 (FMRP) leads to both pre- and postsynaptic defects, yet whether the pre- and postsynaptic functions of FMRP are independent and have distinct roles in FXS neuropathology remain poorly understood. Here, we demonstrate an independent presynaptic function for FMRP through the study of an ID patient with an FMR1 missense mutation. This mutation, c.413G > A (R138Q), preserves FMRP's canonical functions in RNA binding and translational regulation, which are traditionally associated with postsynaptic compartments. However, neuronally driven expression of the mutant FMRP is unable to rescue structural defects at the neuromuscular junction in fragile x mental retardation 1 (dfmr1)-deficient Drosophila, suggesting a presynaptic-specific impairment. Furthermore, mutant FMRP loses the ability to rescue presynaptic action potential (AP) broadening in Fmr1 KO mice. The R138Q mutation also disrupts FMRP's interaction with the large-conductance calcium-activated potassium (BK) channels that modulate AP width. These results reveal a presynaptic- and translation-independent function of FMRP that is linked to a specific subset of FXS phenotypes.

  13. Independent role for presynaptic FMRP revealed by an FMR1 missense mutation associated with intellectual disability and seizures

    PubMed Central

    Myrick, Leila K.; Deng, Pan-Yue; Hashimoto, Hideharu; Oh, Young Mi; Cho, Yongcheol; Poidevin, Mickael J.; Suhl, Joshua A.; Visootsak, Jeannie; Cavalli, Valeria; Jin, Peng; Cheng, Xiaodong; Warren, Stephen T.; Klyachko, Vitaly A.

    2015-01-01

    Fragile X syndrome (FXS) results in intellectual disability (ID) most often caused by silencing of the fragile X mental retardation 1 (FMR1) gene. The resulting absence of fragile X mental retardation protein 1 (FMRP) leads to both pre- and postsynaptic defects, yet whether the pre- and postsynaptic functions of FMRP are independent and have distinct roles in FXS neuropathology remain poorly understood. Here, we demonstrate an independent presynaptic function for FMRP through the study of an ID patient with an FMR1 missense mutation. This mutation, c.413G > A (R138Q), preserves FMRP’s canonical functions in RNA binding and translational regulation, which are traditionally associated with postsynaptic compartments. However, neuronally driven expression of the mutant FMRP is unable to rescue structural defects at the neuromuscular junction in fragile x mental retardation 1 (dfmr1)-deficient Drosophila, suggesting a presynaptic-specific impairment. Furthermore, mutant FMRP loses the ability to rescue presynaptic action potential (AP) broadening in Fmr1 KO mice. The R138Q mutation also disrupts FMRP’s interaction with the large-conductance calcium-activated potassium (BK) channels that modulate AP width. These results reveal a presynaptic- and translation-independent function of FMRP that is linked to a specific subset of FXS phenotypes. PMID:25561520

  14. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

    SciTech Connect

    Kober, Daniel L.; Alexander-Brett, Jennifer M.; Karch, Celeste M.; Cruchaga, Carlos; Colonna, Marco; Holtzman, Michael J.; Brett, Thomas J.

    2016-12-20

    Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway should be tailored to these genetic and functional differences with patient-specific medicine approaches for neurodegenerative disorders.

  15. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

    PubMed Central

    Kober, Daniel L; Alexander-Brett, Jennifer M; Karch, Celeste M; Cruchaga, Carlos; Colonna, Marco; Holtzman, Michael J; Brett, Thomas J

    2016-01-01

    Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway should be tailored to these genetic and functional differences with patient-specific medicine approaches for neurodegenerative disorders. DOI: http://dx.doi.org/10.7554/eLife.20391.001 PMID:27995897

  16. Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression

    PubMed Central

    Adkisson, Michael; Nava, A. J.; Kirov, Julia V.; Cipollone, Andreanna; Willis, Brandon; Rapp, Jared; de Jong, Pieter J.; Lloyd, Kent C.

    2016-01-01

    The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3’ UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels. PMID:26839965

  17. Fundamental Gating Mechanism of Nicotinic Receptor Channel Revealed by Mutation Causing a Congenital Myasthenic Syndrome

    PubMed Central

    Wang, Hai-Long; Ohno, Kinji; Milone, Margherita; Brengman, Joan M.; Evoli, Amelia; Batocchi, Anna-Paola; Middleton, Lefkos T.; Christodoulou, Kyproula; Engel, Andrew G.; Sine, Steven M.

    2000-01-01

    We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation εA411P in the amphipathic helix of the acetylcholine receptor (AChR) ε subunit. Myasthenic patients from three unrelated families are either homozygous for εA411P or are heterozygous and harbor a null mutation in the second ε allele, indicating that εA411P is recessive. We expressed human AChRs containing wild-type or A411P ε subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by εA411P. Prolines engineered into positions flanking residue 411 of the ε subunit greatly increase the range of activation kinetics similar to εA411P, whereas prolines engineered into positions equivalent to εA411 in β and δ subunits are without effect. Thus, the amphipathic helix of the ε subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well. PMID:10962020

  18. Yeast gain-of-function mutations reveal structure–function relationships conserved among different subfamilies of transient receptor potential channels

    PubMed Central

    Su, Zhenwei; Zhou, Xinliang; Haynes, W. John; Loukin, Stephen H.; Anishkin, Andriy; Saimi, Yoshiro; Kung, Ching

    2007-01-01

    Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across subfamilies. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550I) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of phototaxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure–function principles that can be generalized across different TRP subfamilies. It appears that the conserved aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed. PMID:18042709

  19. In Silico Analysis of Missense Mutations in LPAR6 Reveals Abnormal Phospholipid Signaling Pathway Leading to Hypotrichosis

    PubMed Central

    Raza, Syed Irfan; Muhammad, Dost; Jan, Abid; Ali, Raja Hussain; Hassan, Mubashir; Ahmad, Wasim; Rashid, Sajid

    2014-01-01

    Autosomal recessive hypotrichosis is a rare genetic irreversible hair loss disorder characterized by sparse scalp hair, sparse to absent eyebrows and eyelashes, and sparse axillary and body hair. The study, presented here, established genetic linkage in four families showing similar phenotypes to lysophosphatidic acid receptor 6 (LPAR6) gene on chromosome 13q14.11-q21.32. Subsequently, sequence analysis of the gene revealed two previously reported missense mutations including p.D63V in affected members of one and p.I188F in three other families. Molecular modeling and docking analysis was performed to investigate binding of a ligand oleoyl-L-alpha-lysophosphatidic acid (LPA) to modeled protein structures of normal and mutated (D63V, G146R, I188F, N248Y, S3T, L277P) LPAR6 receptors. The mutant receptors showed a complete shift in orientation of LPA at the binding site. In addition, hydropathy analysis revealed a significant change in the membrane spanning topology of LPAR6 helical segments. The present study further substantiated involvement of LPAR6-LPA signaling in the pathogenesis of hypotrichosis/woolly hair and provided additional insight into the molecular mechanism of hair development. PMID:25119526

  20. Epigenetic involvement in Hutchinson-Gilford progeria syndrome: a mini-review.

    PubMed

    Arancio, Walter; Pizzolanti, Giuseppe; Genovese, Swonild I; Pitrone, Maria; Giordano, Carla

    2014-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare human genetic disease that leads to a severe premature ageing phenotype, caused by mutations in the LMNA gene. The LMNA gene codes for lamin-A and lamin-C proteins, which are structural components of the nuclear lamina. HGPS is usually caused by a de novo C1824T mutation that leads to the accumulation of a dominant negative form of lamin-A called progerin. Progerin also accumulates physiologically in normal ageing cells as a rare splicing form of lamin-A transcripts. From this perspective, HGPS cells seem to be good candidates for the study of the physiological mechanisms of ageing. Progerin accumulation leads to faster cellular senescence, stem cell depletion and the progeroid phenotype. Tissues of mesodermic origin are especially affected by HGPS. HGPS patients usually have a bad quality of life and, with current treatments, their life expectancy does not exceed their second decade at best. Though progerin can be expressed in almost any tissue, when death occurs, it is usually due to cardiovascular complications. In HGPS, severe epigenetic alterations have been reported. Histone-covalent modifications are radically different from control specimens, with the tendency to lose the bipartition into euchromatin and heterochromatin. This is reflected in an altered spatial compartmentalization and conformation of chromatin within the nucleus. Moreover, it seems that microRNAs and microRNA biosynthesis might play a role in HGPS. Exemplary in this connection is the suggested protective effect of miR-9 on the central nervous system of affected individuals. This mini-review will report on the state of the art of HGPS epigenetics, and there will be a discussion of how epigenetic alterations in HGPS cells can alter the cellular metabolism and lead to the systemic syndrome.

  1. Structural analysis of Notch-regulating Rumi reveals basis for pathogenic mutations

    SciTech Connect

    Yu, Hongjun; Takeuchi, Hideyuki; Takeuchi, Megumi; Liu, Qun; Kantharia, Joshua; Haltiwanger, Robert S.; Li, Huilin

    2016-07-18

    We present Rumi O-glucosylates the EGF repeats of a growing list of proteins essential in metazoan development, including Notch. Rumi is essential for Notch signaling, and Rumi dysregulation is linked to several human diseases. Despite Rumi's critical roles, it is unknown how Rumi glucosylates a serine of many but not all EGF repeats. Here we report crystal structures of Drosophila Rumi as binary and ternary complexes with a folded EGF repeat and/or donor substrates. These structures provide insights into the catalytic mechanism and show that Rumi recognizes structural signatures of the EGF motif, the U-shaped consensus sequence, C-X-S-X-(P/A)-C and a conserved hydrophobic region. We found that five Rumi mutations identified in cancers and Dowling–Degos disease are clustered around the enzyme active site and adversely affect its activity. In conclusion, our study suggests that loss of Rumi activity may underlie these diseases, and the mechanistic insights may facilitate the development of modulators of Notch signaling.

  2. Biased clique shuffling reveals stabilizing mutations in cellulase Cel7A.

    PubMed

    Dana, Craig M; Saija, Poonam; Kal, Sarala M; Bryan, Mara B; Blanch, Harvey W; Clark, Douglas S

    2012-11-01

    Renewable fuels produced from biomass-derived sugars are receiving increasing attention. Lignocellulose-degrading enzymes derived from fungi are attractive for saccharification of biomass because they can be produced at higher titers and at significantly less cost than those produced by bacteria or archaea. However, their properties can be suboptimal; for example, they are subject to product inhibition and are sensitive to small changes in pH. Furthermore, increased thermostability would be advantageous for saccharification as increased temperature may reduce the opportunity for microbial contamination. We have developed a mutagenesis platform to improve these properties and applied it to increase the operating temperature and thermostability of the fungal glycosyl hydrolase Cel7A. Secretion of Cel7A at titers of 26 mg/L with limited hyperglycosylation was achieved using a Saccharomyces cerevisiae strain with upregulated protein disulfide isomerase, an engineered α-factor prepro leader, and deletion of a plasma membrane ATPase. Using biased clique shuffling (BCS) of 11 Cel7A genes, we generated a small library (469) rich in activity (86% of the chimeras were active) and identified 51 chimeras with improved thermostability, many of which contained mutations in the loop networks that extend over the enzyme's active site. This BCS library was far superior as a source of active and stable chimeras compared to an equimolar library prepared from the same 11 genes.

  3. Whole Genome Sequencing Reveals a De Novo SHANK3 Mutation in Familial Autism Spectrum Disorder

    PubMed Central

    Nemirovsky, Sergio I.; Córdoba, Marta; Zaiat, Jonathan J.; Completa, Sabrina P.; Vega, Patricia A.; González-Morón, Dolores; Medina, Nancy M.; Fabbro, Mónica; Romero, Soledad; Brun, Bianca; Revale, Santiago; Ogara, María Florencia; Pecci, Adali; Marti, Marcelo; Vazquez, Martin; Turjanski, Adrián; Kauffman, Marcelo A.

    2015-01-01

    Introduction Clinical genomics promise to be especially suitable for the study of etiologically heterogeneous conditions such as Autism Spectrum Disorder (ASD). Here we present three siblings with ASD where we evaluated the usefulness of Whole Genome Sequencing (WGS) for the diagnostic approach to ASD. Methods We identified a family segregating ASD in three siblings with an unidentified cause. We performed WGS in the three probands and used a state-of-the-art comprehensive bioinformatic analysis pipeline and prioritized the identified variants located in genes likely to be related to ASD. We validated the finding by Sanger sequencing in the probands and their parents. Results Three male siblings presented a syndrome characterized by severe intellectual disability, absence of language, autism spectrum symptoms and epilepsy with negative family history for mental retardation, language disorders, ASD or other psychiatric disorders. We found germline mosaicism for a heterozygous deletion of a cytosine in the exon 21 of the SHANK3 gene, resulting in a missense sequence of 5 codons followed by a premature stop codon (NM_033517:c.3259_3259delC, p.Ser1088Profs*6). Conclusions We reported an infrequent form of familial ASD where WGS proved useful in the clinic. We identified a mutation in SHANK3 that underscores its relevance in Autism Spectrum Disorder. PMID:25646853

  4. Genome biogeography reveals the intraspecific spread of adaptive mutations for a complex trait.

    PubMed

    Olofsson, Jill K; Bianconi, Matheus; Besnard, Guillaume; Dunning, Luke T; Lundgren, Marjorie R; Holota, Helene; Vorontsova, Maria S; Hidalgo, Oriane; Leitch, Ilia J; Nosil, Patrik; Osborne, Colin P; Christin, Pascal-Antoine

    2016-12-01

    Physiological novelties are often studied at macro-evolutionary scales such that their micro-evolutionary origins remain poorly understood. Here, we test the hypothesis that key components of a complex trait can evolve in isolation and later be combined by gene flow. We use C4 photosynthesis as a study system, a derived physiology that increases plant productivity in warm, dry conditions. The grass Alloteropsis semialata includes C4 and non-C4 genotypes, with some populations using laterally acquired C4 -adaptive loci, providing an outstanding system to track the spread of novel adaptive mutations. Using genome data from C4 and non-C4 A. semialata individuals spanning the species' range, we infer and date past migrations of different parts of the genome. Our results show that photosynthetic types initially diverged in isolated populations, where key C4 components were acquired. However, rare but recurrent subsequent gene flow allowed the spread of adaptive loci across genetic pools. Indeed, laterally acquired genes for key C4 functions were rapidly passed between populations with otherwise distinct genomic backgrounds. Thus, our intraspecific study of C4 -related genomic variation indicates that components of adaptive traits can evolve separately and later be combined through secondary gene flow, leading to the assembly and optimization of evolutionary innovations.

  5. Arrhythmia Caused by a Drosophila Tropomyosin Mutation Is Revealed Using a Novel Optical Coherence Tomography Instrument

    PubMed Central

    Ma, Lisha; Bradu, Adrian; Podoleanu, Adrian Gh.; Bloor, James W.

    2010-01-01

    Background Dilated cardiomyopathy (DCM) is a severe cardiac condition that causes high mortality. Many genes have been confirmed to be involved in this disease. An ideal system with which to uncover disease mechanisms would be one that can measure the changes in a wide range of cardiac activities associated with mutations in specific, diversely functional cardiac genes. Such a system needs a genetically manipulable model organism that allows in vivo measurement of cardiac phenotypes and a detecting instrument capable of recording multiple phenotype parameters. Methodology and Principal Findings With a simple heart, a transparent body surface at larval stages and available genetic tools we chose Drosophila melanogaster as our model organism and developed for it a dual en-face/Doppler optical coherence tomography (OCT) instrument capable of recording multiple aspects of heart activity, including heart contraction cycle dynamics, ostia dynamics, heartbeat rate and rhythm, speed of heart wall movement and light reflectivity of cardiomyocytes in situ. We applied this OCT instrument to a model of Tropomyosin-associated DCM established in adult Drosophila. We show that DCM pre-exists in the larval stage and is accompanied by an arrhythmia previously unidentified in this model. We also detect reduced mobility and light reflectivity of cardiomyocytes in mutants. Conclusion These results demonstrate the capability of our OCT instrument to characterize in detail cardiac activity in genetic models for heart disease in Drosophila. PMID:21179409

  6. Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

    PubMed

    Akiba, Hiroki; Tsumoto, Kouhei

    2015-07-01

    Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity.

  7. Structural analysis of Notch-regulating Rumi reveals basis for pathogenic mutations

    DOE PAGES

    Yu, Hongjun; Takeuchi, Hideyuki; Takeuchi, Megumi; ...

    2016-07-18

    We present Rumi O-glucosylates the EGF repeats of a growing list of proteins essential in metazoan development, including Notch. Rumi is essential for Notch signaling, and Rumi dysregulation is linked to several human diseases. Despite Rumi's critical roles, it is unknown how Rumi glucosylates a serine of many but not all EGF repeats. Here we report crystal structures of Drosophila Rumi as binary and ternary complexes with a folded EGF repeat and/or donor substrates. These structures provide insights into the catalytic mechanism and show that Rumi recognizes structural signatures of the EGF motif, the U-shaped consensus sequence, C-X-S-X-(P/A)-C and amore » conserved hydrophobic region. We found that five Rumi mutations identified in cancers and Dowling–Degos disease are clustered around the enzyme active site and adversely affect its activity. In conclusion, our study suggests that loss of Rumi activity may underlie these diseases, and the mechanistic insights may facilitate the development of modulators of Notch signaling.« less

  8. Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate

    PubMed Central

    Mangold, Elisabeth; Böhmer, Anne C.; Ishorst, Nina; Hoebel, Ann-Kathrin; Gültepe, Pinar; Schuenke, Hannah; Klamt, Johanna; Hofmann, Andrea; Gölz, Lina; Raff, Ruth; Tessmann, Peter; Nowak, Stefanie; Reutter, Heiko; Hemprich, Alexander; Kreusch, Thomas; Kramer, Franz-Josef; Braumann, Bert; Reich, Rudolf; Schmidt, Gül; Jäger, Andreas; Reiter, Rudolf; Brosch, Sibylle; Stavusis, Janis; Ishida, Miho; Seselgyte, Rimante; Moore, Gudrun E.; Nöthen, Markus M.; Borck, Guntram; Aldhorae, Khalid A.; Lace, Baiba; Stanier, Philip; Knapp, Michael; Ludwig, Kerstin U.

    2016-01-01

    Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10−2). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10−5; ORallelic = 2.46 [95% CI 1.6–3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10−9). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO. PMID:27018475

  9. Craniofacial abnormalities in Hutchinson-Gilford progeria syndrome.

    PubMed

    Ullrich, N J; Silvera, V M; Campbell, S E; Gordon, L B

    2012-09-01

    HGPS is a rare syndrome of segmental premature aging. Our goal was to expand the scope of structural bone and soft-tissue craniofacial abnormalities in HGPS through CT or MR imaging. Using The Progeria Research Foundation Medical and Research Database, 98 imaging studies on 25 patients, birth to 14.1 years of age, were comprehensively reviewed. Eight newly identified abnormalities involving the calvaria, skull base, and soft tissues of the face and orbits were present with prevalences between 43% and 100%. These included J-shaped sellas, a mottled appearance and increased vascular markings of the calvaria, abnormally configured mandibular condyles, hypoplastic articular eminences, small zygomatic arches, prominent parotid glands, and optic nerve kinking. This expanded craniofacial characterization helps link disease features and improves our ability to evaluate how underlying genetic and cellular abnormalities culminate in a disease phenotype.

  10. Next-generation sequencing of a 40 Mb linkage interval reveals TSPAN12 mutations in patients with familial exudative vitreoretinopathy.

    PubMed

    Nikopoulos, Konstantinos; Gilissen, Christian; Hoischen, Alexander; van Nouhuys, C Erik; Boonstra, F Nienke; Blokland, Ellen A W; Arts, Peer; Wieskamp, Nienke; Strom, Tim M; Ayuso, Carmen; Tilanus, Mauk A D; Bouwhuis, Sanne; Mukhopadhyay, Arijit; Scheffer, Hans; Hoefsloot, Lies H; Veltman, Joris A; Cremers, Frans P M; Collin, Rob W J

    2010-02-12

    Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous retinal disorder characterized by abnormal vascularisation of the peripheral retina, often accompanied by retinal detachment. To date, mutations in three genes (FZD4, LRP5, and NDP) have been shown to be causative for FEVR. In two large Dutch pedigrees segregating autosomal-dominant FEVR, genome-wide SNP analysis identified an FEVR locus of approximately 40 Mb on chromosome 7. Microsatellite marker analysis suggested similar at risk haplotypes in patients of both families. To identify the causative gene, we applied next-generation sequencing in the proband of one of the families, by analyzing all exons and intron-exon boundaries of 338 genes, in addition to microRNAs, noncoding RNAs, and other highly conserved genomic regions in the 40 Mb linkage interval. After detailed bioinformatic analysis of the sequence data, prioritization of all detected sequence variants led to three candidates to be considered as the causative genetic defect in this family. One of these variants was an alanine-to-proline substitution in the transmembrane 4 superfamily member 12 protein, encoded by TSPAN12. This protein has very recently been implicated in regulating the development of retinal vasculature, together with the proteins encoded by FZD4, LRP5, and NDP. Sequence analysis of TSPAN12 revealed two mutations segregating in five of 11 FEVR families, indicating that mutations in TSPAN12 are a relatively frequent cause of FEVR. Furthermore, we demonstrate the power of targeted next-generation sequencing technology to identify disease genes in linkage intervals.

  11. Exome sequencing of a colorectal cancer family reveals shared mutation pattern and predisposition circuitry along tumor pathways

    PubMed Central

    Suleiman, Suleiman H.; Koko, Mahmoud E.; Nasir, Wafaa H.; Elfateh, Ommnyiah; Elgizouli, Ubai K.; Abdallah, Mohammed O. E.; Alfarouk, Khalid O.; Hussain, Ayman; Faisal, Shima; Ibrahim, Fathelrahamn M. A.; Romano, Maurizio; Sultan, Ali; Banks, Lawrence; Newport, Melanie; Baralle, Francesco; Elhassan, Ahmed M.; Mohamed, Hiba S.; Ibrahim, Muntaser E.

    2015-01-01

    The molecular basis of cancer and cancer multiple phenotypes are not yet fully understood. Next Generation Sequencing promises new insight into the role of genetic interactions in shaping the complexity of cancer. Aiming to outline the differences in mutation patterns between familial colorectal cancer cases and controls we analyzed whole exomes of cancer tissues and control samples from an extended colorectal cancer pedigree, providing one of the first data sets of exome sequencing of cancer in an African population against a background of large effective size typically with excess of variants. Tumors showed hMSH2 loss of function SNV consistent with Lynch syndrome. Sets of genes harboring insertions–deletions in tumor tissues revealed, however, significant GO enrichment, a feature that was not seen in control samples, suggesting that ordered insertions–deletions are central to tumorigenesis in this type of cancer. Network analysis identified multiple hub genes of centrality. ELAVL1/HuR showed remarkable centrality, interacting specially with genes harboring non-synonymous SNVs thus reinforcing the proposition of targeted mutagenesis in cancer pathways. A likely explanation to such mutation pattern is DNA/RNA editing, suggested here by nucleotide transition-to-transversion ratio that significantly departed from expected values (p-value 5e-6). NFKB1 also showed significant centrality along with ELAVL1, raising the suspicion of viral etiology given the known interaction between oncogenic viruses and these proteins. PMID:26442106

  12. Computational Assay of H7N9 Influenza Neuraminidase Reveals R292K Mutation Reduces Drug Binding Affinity

    NASA Astrophysics Data System (ADS)

    Woods, Christopher J.; Malaisree, Maturos; Long, Ben; McIntosh-Smith, Simon; Mulholland, Adrian J.

    2013-12-01

    The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational `assay.'

  13. Exome sequencing revealed PMM2 gene mutations in a French-Canadian family with congenital atrophy of the cerebellum.

    PubMed

    Noreau, Anne; Beauchemin, Philippe; Dionne-Laporte, Alexandre; Dion, Patrick A; Rouleau, Guy A; Dupré, Nicolas

    2014-01-01

    Two affected and one unaffected siblings from a French-Canadian family were evaluated in our neurogenetic clinic. The oldest brother had intentional and postural hand tremor while his youngest sister presented mild ataxia, a similar hand tremor and global developmental delay. Brain MRIs of the two affected family members further revealed a significant cerebellar atrophy. For this study we conducted a whole exome sequencing (WES) investigation using genomic DNA prepared from the affected brother and sister, alongside DNA prepared from their unaffected mother, and identified two mutations previously reported to cause a rare disorder known as Congenital Disorder of Glycosylation, type Ia (CDG1A) (OMIM #212065). This study emphasizes how the diagnosis of patients presenting a mild tremor phenotype associated with cerebellar atrophy may benefit from WES in establishing genetic defects associated with their conditions.

  14. Mutational analysis of Mycobacterium tuberculosis lysine ɛ-aminotransferase and inhibitor co-crystal structures, reveals distinct binding modes.

    PubMed

    Tripathi, Sarvind Mani; Agarwal, Aparna; Ramachandran, Ravishankar

    Lysine ɛ-aminotransferase (LAT) converts lysine to α-aminoadipate-δ-semialdehyde in a PLP-mediated reaction. We mutated active-site T330, N328 and E243, and structurally rationalized their properties. T330A and T330S mutants cannot bind PLP and are inactive. N328A although inactive, binds to PLP. E243A retains activity, but binds α-ketoglutarate in a different conformation. We had earlier identified 2-aminomethyl piperidine derivative as a LAT inhibitor. The co-crystal structure reveals that it mimics binding of C5 substrates and exhibits two binding modes. E243, that shields R422 in the apo enzyme, exhibits conformational changes to permit the binding of the inhibitor in one of the binding modes. Structure-based analysis of bound water in the active site suggests optimization strategies for synthesis of improved inhibitors.

  15. Blocking farnesylation of the prelamin A variant in Hutchinson-Gilford progeria syndrome alters the distribution of A-type lamins.

    PubMed

    Wang, Yuexia; Ostlund, Cecilia; Choi, Jason C; Swayne, Theresa C; Gundersen, Gregg G; Worman, Howard J

    2012-01-01

    Mutations in the lamin A/C gene that cause Hutchinson-Gilford progeria syndrome lead to expression of a truncated, permanently farnesylated prelamin A variant called progerin. Blocking farnesylation leads to an improvement in the abnormal nuclear morphology observed in cells expressing progerin, which is associated with a re-localization of the variant protein from the nuclear envelope to the nuclear interior. We now show that a progerin construct that cannot be farnesylated is localized primarily in intranuclear foci and that its diffusional mobility is significantly greater than that of farnesylated progerin localized predominantly at the nuclear envelope. Expression of non-farnesylated progerin in transfected cells leads to a redistribution of lamin A and lamin C away from the nuclear envelope into intranuclear foci but does not significantly affect the localization of endogenous lamin B1 at nuclear envelope. There is a similar redistribution of lamin A and lamin C into intranuclear foci in transfected cells expressing progerin in which protein farnesylation is blocked by treatment with a protein farnesyltransferase inhibitor. Blocking farnesylation of progerin can lead to a redistribution of normal A-type lamins away from the inner nuclear envelope. This may have implications for using drugs that block protein prenylation to treat children with Hutchinson-Gilford progeria syndrome. These findings also provide additional evidence that A-type and B-type lamins can form separate microdomains within the nucleus.

  16. Spontaneous mutation reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.

    PubMed

    Horn, Nikki; Wegmann, Udo; Dertli, Enes; Mulholland, Francis; Collins, Samuel R A; Waldron, Keith W; Bongaerts, Roy J; Mayer, Melinda J; Narbad, Arjan

    2013-01-01

    As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

  17. Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK.

    PubMed

    Kim, S I; Andaya, C B; Newman, J W; Goyal, S S; Tai, T H

    2008-11-01

    Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations, which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexakisphosphate). High-performance liquid chromatography and GC-MS analysis of seed extracts from N15-186 indicated that, in addition to phytic acid, inositol monophosphate was significantly reduced whereas inorganic phosphorus and myo-inositol were greatly increased when compared with wild-type. The changes observed in N15-186 resemble those previously described for the maize lpa3 mutant. Analysis of N15-375 revealed changes similar to those observed in previously characterized rice lpa1 mutants (i.e. significant reduction in phytic acid and corresponding increase in inorganic phosphorus with little or no change in inositol phosphate intermediates or myo-inositol). Further genetic analysis of the N15-186 mutant indicated that the mutation, designated lpa N15-186, was located in a region on chromosome 3 between the microsatellite markers RM15875 and RM15907. The rice orthologue of maize lpa3, which encodes a myo-inositol kinase, is in this interval. Sequence analysis of the N15-186 allele of this orthologue (Os03g52760) revealed a single base pair change (C/G to T/A) in the first exon of the gene, which results in a nonsense mutation. Our results indicate that lpa N15-186 is a mutant allele of the rice myo-inositol kinase (OsMIK) gene. Identification and characterization of lpa mutants, such as N15-186, will facilitate studies on the regulation of phytic acid biosynthesis and accumulation and help address questions concerning the contribution of the inositol lipid-dependent and independent biosynthetic pathways to the production of seed phytic acid.

  18. Molecular analysis of genetic mutations among cross-resistant second-line injectable drugs reveals a new resistant mutation in Mycobacterium tuberculosis.

    PubMed

    Malinga, Lesibana; Brand, Jeannette; Olorunju, Steve; Stoltz, Anton; van der Walt, Martie

    2016-08-01

    Mutations causing mono and cross-resistance among amikacin, kanamycin and capreomycin of second-line injectable drugs (SLIDs) namely are not well understood. We investigated 124 isolates of Mycobacterium tuberculosis for mutations within rrs, eis, tlyA and efflux pump (Rv1258c and Rv0194) genes involved in resistance towards SLIDs. The distribution of mutations across these genes were significantly different in strains with mono-resistance or cross-resistance. A new mutation G878A was found in rrs gene, among strains with capreomycin mono-resistant, or in strains with cross-resistance of capreomycin, kanamycin and amikacin. This mutation was associated with the Euro-American X3 lineage (P < 0.0001). Mutations in the two efflux genes Rv1258c and Rv0194 were confined to strains with only capreomycin/amikacin/kanamycin cross-resistance. We further investigated the minimum inhibitory concentration of capreomycin on isolates with new G878A mutation ranging from 8 μg/mL to 64 μg/mL. Inclusion of G878A on new molecular assays could increase the sensitivity of capreomycin resistance detection.

  19. Whole-exome sequencing reveals a novel frameshift mutation in the FAM161A gene causing autosomal recessive retinitis pigmentosa in the Indian population.

    PubMed

    Zhou, Yu; Saikia, Bibhuti B; Jiang, Zhilin; Zhu, Xiong; Liu, Yuqing; Huang, Lulin; Kim, Ramasamy; Yang, Yin; Qu, Chao; Hao, Fang; Gong, Bo; Tai, Zhengfu; Niu, Lihong; Yang, Zhenglin; Sundaresan, Periasamy; Zhu, Xianjun

    2015-10-01

    Retinitis pigmentosa (RP) is a heterogenous group of inherited retinal degenerations caused by mutations in at least 50 genes. To identify genetic mutations underlying autosomal recessive RP (arRP), we performed whole-exome sequencing study on two consanguineous marriage Indian families (RP-252 and RP-182) and 100 sporadic RP patients. Here we reported novel mutation in FAM161A in RP-252 and RP-182 with two patients affected with RP in each family. The FAM161A gene was identified as the causative gene for RP28, an autosomal recessive form of RP. By whole-exome sequencing we identified several homozygous genomic regions, one of which included the recently identified FAM161A gene mutated in RP28-linked arRP. Sequencing analysis revealed the presence of a novel homozygous frameshift mutation p.R592FsX2 in both patients of family RP-252 and family RP-182. In 100 sporadic Indian RP patients, this novel homozygous frameshift mutation p.R592FsX2 was identified in one sporadic patient ARRP-S-I-46 by whole-exome sequencing and validated by Sanger sequencing. Meanwhile, this homozygous frameshift mutation was absent in 1000 ethnicity-matched control samples screened by direct Sanger sequencing. In conclusion, we identified a novel homozygous frameshift mutations of RP28-linked RP gene FAM161A in Indian population.

  20. Exomic Sequencing of Medullary Thyroid Cancer Reveals Dominant and Mutually Exclusive Oncogenic Mutations in RET and RAS

    PubMed Central

    Jiao, Yuchen; Sausen, Mark; Leary, Rebecca; Bettegowda, Chetan; Roberts, Nicholas J.; Bhan, Sheetal; Ho, Allen S.; Khan, Zubair; Bishop, Justin; Westra, William H.; Wood, Laura D.; Hruban, Ralph H.; Tufano, Ralph P.; Robinson, Bruce; Dralle, Henning; Toledo, Sergio P. A.; Toledo, Rodrigo A.; Morris, Luc G. T.; Ghossein, Ronald A.; Fagin, James A.; Chan, Timothy A.; Velculescu, Victor E.; Vogelstein, Bert; Kinzler, Kenneth W.; Papadopoulos, Nickolas; Nelkin, Barry D.; Ball, Douglas W.

    2013-01-01

    Context: Medullary thyroid cancer (MTC) is a rare thyroid cancer that can occur sporadically or as part of a hereditary syndrome. Objective: To explore the genetic origin of MTC, we sequenced the protein coding exons of approximately 21,000 genes in 17 sporadic MTCs. Patients and Design: We sequenced the exomes of 17 sporadic MTCs and validated the frequency of all recurrently mutated genes and other genes of interest in an independent cohort of 40 MTCs comprised of both sporadic and hereditary MTC. Results: We discovered 305 high-confidence mutations in the 17 sporadic MTCs in the discovery phase, or approximately 17.9 somatic mutations per tumor. Mutations in RET, HRAS, and KRAS genes were identified as the principal driver mutations in MTC. All of the other additional somatic mutations, including mutations in spliceosome and DNA repair pathways, were not recurrent in additional tumors. Tumors without RET, HRAS, or KRAS mutations appeared to have significantly fewer mutations overall in protein coding exons. Conclusions: Approximately 90% of MTCs had mutually exclusive mutations in RET, HRAS, and KRAS, suggesting that RET and RAS are the predominant driver pathways in MTC. Relatively few mutations overall and no commonly recurrent driver mutations other than RET, HRAS, and KRAS were seen in the MTC exome. PMID:23264394

  1. Progerin impairs chromosome maintenance by depleting CENP-F from metaphase kinetochores in Hutchinson-Gilford progeria fibroblasts.

    PubMed

    Eisch, Veronika; Lu, Xiang; Gabriel, Diana; Djabali, Karima

    2016-04-26

    Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare premature aging disorder that leads to death at an average age of 14.7 years due to myocardial infarction or stroke. The most common mutation in HGPS is at position G608G (GGC>GGT) within exon 11 of the LMNA gene. This mutation results in the deletion of 50 amino acids at the carboxyl-terminal tail of prelamin A, producing a truncated farnesylated protein called progerin. Lamins play important roles in the organization and structure of the nucleus. The nuclear build-up of progerin causes severe morphological and functional changes in interphase HGPS cells. In this study, we investigated whether progerin elicits spatiotemporal deviations in mitotic processes in HGPS fibroblasts. We analyzed the nuclear distribution of endogenous progerin during mitosis in relation to components of the nuclear lamina, nuclear envelope (NE) and nuclear pores. We found that progerin caused defects in chromosome segregation as early as metaphase, delayed NE reformation and trapped lamina components and inner NE proteins in the endoplasmic reticulum at the end of mitosis. Progerin displaced the centromere protein F (CENP-F) from metaphase chromosome kinetochores, which caused increased chromatin lagging, binucleated cells and genomic instability. This accumulation of progerin-dependent defects with each round of mitosis predisposes cells to premature senescence.

  2. Progerin impairs chromosome maintenance by depleting CENP-F from metaphase kinetochores in Hutchinson-Gilford progeria fibroblasts

    PubMed Central

    Eisch, Veronika; Lu, Xiang; Gabriel, Diana; Djabali, Karima

    2016-01-01

    Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare premature aging disorder that leads to death at an average age of 14.7 years due to myocardial infarction or stroke. The most common mutation in HGPS is at position G608G (GGC>GGT) within exon 11 of the LMNA gene. This mutation results in the deletion of 50 amino acids at the carboxyl-terminal tail of prelamin A, producing a truncated farnesylated protein called progerin. Lamins play important roles in the organization and structure of the nucleus. The nuclear build-up of progerin causes severe morphological and functional changes in interphase HGPS cells. In this study, we investigated whether progerin elicits spatiotemporal deviations in mitotic processes in HGPS fibroblasts. We analyzed the nuclear distribution of endogenous progerin during mitosis in relation to components of the nuclear lamina, nuclear envelope (NE) and nuclear pores. We found that progerin caused defects in chromosome segregation as early as metaphase, delayed NE reformation and trapped lamina components and inner NE proteins in the endoplasmic reticulum at the end of mitosis. Progerin displaced the centromere protein F (CENP-F) from metaphase chromosome kinetochores, which caused increased chromatin lagging, binucleated cells and genomic instability. This accumulation of progerin-dependent defects with each round of mitosis predisposes cells to premature senescence. PMID:27015553

  3. Complex mutations & subpopulations of deletions at exon 19 of EGFR in NSCLC revealed by next generation sequencing: potential clinical implications.

    PubMed

    Marchetti, Antonio; Del Grammastro, Maela; Filice, Giampaolo; Felicioni, Lara; Rossi, Giulio; Graziano, Paolo; Sartori, Giuliana; Leone, Alvaro; Malatesta, Sara; Iacono, Michele; Guetti, Luigi; Viola, Patrizia; Mucilli, Felice; Cuccurullo, Franco; Buttitta, Fiamma

    2012-01-01

    Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.

  4. Mutational analysis of the pumpkin (Cucurbita maxima) phloem exudate lectin, PP2 reveals Ser-104 is crucial for carbohydrate binding.

    PubMed

    Bobbili, Kishore Babu; Bandari, Shyam; Grobe, Kay; Swamy, Musti J

    2014-07-18

    The pumpkin phloem lectin (PP2) is an RNA-binding, defense-related, chitooligosaccharide-specific, homodimeric lectin of Mr 48 kDa expressed at high concentrations in the sieve elements and companion cells of pumpkin (Cucurbita maxima). In the present study, PP2 was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway as a prerequisite for unimpaired folding and posttranslational glycosylation of recombinant PP2. Previous computational modeling and ligand docking studies predicted a putative chitooligosaccharide-binding site on the PP2 surface, which was divided into three subsites, with two amino acid residues in each subsite identified as possible candidates for interaction with chitooligosaccharides (CHOs). In this work, mutational analysis and hemagglutination assays were employed to verify the role of the predicted residues in the carbohydrate binding activity of the protein. The results obtained revealed that mutation of Ser-104 to Ala (S104A) at subsite-2 resulted in about 90% loss of agglutination activity of the protein, indicating that Ser-104 is crucial for the binding of CHOs to PP2. Also, L100A (at subsite-1) and K200A (at subsite-3) independently decreased the lectin activity by about 40%, indicating that these two residues also contribute significantly to sugar binding by PP2. Together, these findings confirm that all the three subsites contribute to varying degrees toward PP2-carbohydrate interaction, and confirm the validity of the computational model, as proposed earlier.

  5. Functional analysis reveals splicing mutations of the CASQ2 gene in patients with CPVT: implication for genetic counselling and clinical management.

    PubMed

    Roux-Buisson, Nathalie; Rendu, John; Denjoy, Isabelle; Guicheney, Pascale; Goldenberg, Alice; David, Nadine; Faivre, Laurence; Barthez, Olivier; Danieli, Gian Antonio; Marty, Isabelle; Lunardi, Joel; Fauré, Julien

    2011-09-01

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare and severe arrhythmogenic disorder. Although usually transmitted in a recessive form, few cases of dominant mutations have been reported. Thirteen mutations in the CASQ2 gene have been reported so far in association with CPVT. We performed molecular analysis of the CASQ2 gene in 43 probands with CPVT and identified eight mutations in five patients. Six mutations were novel: one was a single nucleotide deletion, three affected consensus splice sites, and two had unknown consequences: the c.939 + 5G>C and the synonymous c.381C>T variations. We demonstrated that these two variations affected CASQ2 splicing using a splicing minigene assay. These data increased significantly the number of CASQ2 mutations described in association with CPVT, revealed the high prevalence of splicing and truncating mutations in this gene and brought new insight regarding the dominant inheritance of the disease. Moreover, our report of the first splicing abnormalities in CASQ2 caused by intronic mutation or synonymous change underlines the absolute necessity to perform extensive molecular analysis for genetic diagnosis and counseling of CPVT.

  6. Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage.

    PubMed

    Kennedy, Scott R; Salk, Jesse J; Schmitt, Michael W; Loeb, Lawrence A

    2013-01-01

    Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >10(7) wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ~5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G → T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G → A and T → C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.

  7. Multiregion sequencing reveals the intratumor heterogeneity of driver mutations in TP53-driven non-small cell lung cancer.

    PubMed

    Zhang, Le-Le; Kan, Mengyuan; Zhang, Man-Man; Yu, Sha-Sha; Xie, Hui-Jun; Gu, Zhao-Hui; Wang, Hai-Ning; Zhao, Shuang-Xia; Zhou, Guang-Biao; Song, Huai-Dong; Zheng, Cui-Xia

    2017-01-01

    Intratumor heterogeneity (ITH) in non-small cell lung cancer (NSCLC) may account for resistance after a period of targeted therapies because drugs destroy only a portion of tumor cells. The recognition of ITH helps identify high-risk patients to make effective treatment decisions. However, ITH studies are confounded by interpatient heterogeneity in NSCLC and a large amount of passenger mutations. To address these issues, we recruited NSCLC patients carrying TP53 mutations and selected driver mutations within recurrently mutated genes in NSCLC. A total of 12-paired normal-tumor tissues were subjected to whole-genome/whole-exome sequencing. From these, 367 non-silent mutations were selected as driver mutations and deeply sequenced in 61 intratumoral microdissections. We identified a universal prevalence of heterogeneity in all 12 tumors, indicating branched evolution. Although TP53 mutations were observed in single biopsy of all 12 tumors, most tumors consist of both TP53 mutated and non-mutated cells in separate regions within the same tumor. This suggests the late molecular timing of the acquisition of TP53 mutations; therefore, the detection of TP53 mutations in a single biopsy may simply not reflect the early malignant potential. In addition, we identified regions of loss of heterozygosity surrounding TP53 and CDKN2A mutations in tumor 711, which also exhibited heterogeneity in different regional samples. Because the ITH of driver mutations likely has clinical consequences, further efforts are needed to limit the impact of ITH and to improve therapeutic efficiency, which will benefit NSCLC patients receiving targeted treatments.

  8. Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors

    PubMed Central

    Goriely, Anne; Hansen, Ruth M. S.; Taylor, Indira B.; Olesen, Inge A.; Jacobsen, Grete Krag; McGowan, Simon J.; Pfeifer, Susanne P.; McVean, Gilean A. T.; Meyts, Ewa Rajpert-De; Wilkie, Andrew O.M.

    2010-01-01

    Genes mutated in congenital malformation syndromes are frequently implicated in oncogenesis1,2, but the causative germline and somatic mutations occur in separate cells at different times of an organism’s life. Here we unify these processes for mutations arising in male germ cells that show a paternal age effect3. Screening of 30 spermatocytic seminomas4,5 for oncogenic mutations in 17 genes identified 2 mutations in FGFR3 (both 1948A>G encoding K650E, which causes thanatophoric dysplasia in the germline)6 and 5 mutations in HRAS. Massively parallel sequencing of sperm DNA showed that the FGFR3 mutation increases with paternal age, with a similar mutation spectrum at the K650 codon to that in bladder cancer7,8. Most spermatocytic seminomas show increased immunoreactivity for FGFR3 and/or HRAS. We propose that paternal age effect mutations activate a common “selfish” pathway supporting proliferation in the testis, leading to diverse phenotypes in the next generation including fetal lethality, congenital syndromes and cancer. PMID:19855393

  9. Coronary artery disease in a Werner syndrome-like form of progeria characterized by low levels of progerin, a splice variant of lamin A.

    PubMed

    Hisama, Fuki M; Lessel, Davor; Leistritz, Dru; Friedrich, Katrin; McBride, Kim L; Pastore, Matthew T; Gottesman, Gary S; Saha, Bidisha; Martin, George M; Kubisch, Christian; Oshima, Junko

    2011-12-01

    Classical Hutchinson-Gilford progeria syndrome (HGPS) is caused by LMNA mutations that generate an alternatively spliced form of lamin A, termed progerin. HGPS patients present in early childhood with atherosclerosis and striking features of accelerated aging. We report on two pedigrees of adult-onset coronary artery disease with progeroid features, who were referred to our International Registry of Werner Syndrome (WS) because of clinical features consistent with the diagnosis. No mutations were identified in the WRN gene that is responsible for WS, among these patients. Instead, we found two novel heterozygous mutations at the junction of exon 10 and intron 11 of the LMNA gene. These mutations resulted in the production of progerin at a level substantially lower than that of HGPS. Our findings indicate that LMNA mutations may result in coronary artery disease presenting in the fourth to sixth decades along with short stature and a progeroid appearance resembling WS. The absence of early-onset cataracts in this setting should suggest the diagnosis of progeroid laminopathy. This study illustrates the evolving genotype-phenotype relationship between the amount of progerin produced and the age of onset among the spectrum of restrictive dermopathy, HGPS, and atypical forms of WS.

  10. Enhanced SRSF5 Protein Expression Reinforces Lamin A mRNA Production in HeLa Cells and Fibroblasts of Progeria Patients.

    PubMed

    Vautrot, Valentin; Aigueperse, Christelle; Oillo-Blanloeil, Florence; Hupont, Sébastien; Stevenin, James; Branlant, Christiane; Behm-Ansmant, Isabelle

    2016-03-01

    The Hutchinson Gilford Progeria Syndrome (HGPS) is a rare genetic disease leading to accelerated aging. Three mutations of the LMNA gene leading to HGPS were identified. The more frequent ones, c.1824C>T and c.1822G>A, enhance the use of the intron 11 progerin 5'splice site (5'SS) instead of the LMNA 5'SS, leading to the production of the truncated dominant negative progerin. The less frequent c.1868C>G mutation creates a novel 5'SS (LAΔ35 5'SS), inducing the production of another truncated LMNA protein (LAΔ35). Our data show that the progerin 5'SS is used at low yield in the absence of HGPS mutation, whereas utilization of the LAΔ35 5'SS is dependent upon the presence of the c.1868C>G mutation. In the perspective to correct HGPS splicing defects, we investigated whether SR proteins can modify the relative yields of utilization of intron 11 5'SSs. By in cellulo and in vitro assays, we identified SRSF5 as a direct key regulator increasing the utilization of the LMNA 5'SS in the presence of the HGPS mutations. Enhanced SRSF5 expression in dermal fibroblasts of HGPS patients as well as PDGF-BB stimulation of these cells decreased the utilization of the progerin 5'SS, and improves nuclear morphology, opening new therapeutic perspectives for premature aging.

  11. Coronary Artery Disease in a Werner Syndrome-Like Form of Progeria Characterized by Low Levels of Progerin, a Splice Variant of Lamin A

    PubMed Central

    Hisama, Fuki M.; Lessel, Davor; Leistritz, Dru; Friedrich, Katrin; McBride, Kim L.; Pastore, Matthew T.; Gottesman, Gary S.; Saha, Bidisha; Martin, George M.; Kubisch, Christian; Oshima, Junko

    2015-01-01

    Classical Hutchinson–Gilford progeria syndrome (HGPS) is caused by LMNA mutations that generate an alternatively spliced form of lamin A, termed progerin. HGPS patients present in early childhood with atherosclerosis and striking features of accelerated aging. We report on two pedigrees of adult-onset coronary artery disease with progeroid features, who were referred to our International Registry of Werner Syndrome (WS) because of clinical features consistent with the diagnosis. No mutations were identified in the WRN gene that is responsible for WS, among these patients. Instead, we found two novel heterozygous mutations at the junction of exon 10 and intron 11 of the LMNA gene. These mutations resulted in the production of progerin at a level substantially lower than that of HGPS. Our findings indicate that LMNA mutations may result in coronary artery disease presenting in the fourth to sixth decades along with short stature and a progeroid appearance resembling WS. The absence of early-onset cataracts in this setting should suggest the diagnosis of progeroid laminopathy. This study illustrates the evolving genotype–phenotype relationship between the amount of progerin produced and the age of onset among the spectrum of restrictive dermopathy, HGPS, and atypical forms of WS. PMID:22065502

  12. The pugilistDominant Mutation of Drosophila melanogaster: A Simple-Sequence Repeat Disorder Reveals Localized Transport in the Eye.

    PubMed

    Rong, Yikang S; Golic, Mary M; Golic, Kent G

    2016-01-01

    The pugilist-Dominant mutation results from fusion of a portion of the gene encoding the tri-functional Methylene Tetrahydrofolate Dehydrogenase (E.C.1.5.1.5, E.C.3.5.4.9, E.C.6.3.4.3) to approximately one kb of a heterochromatic satellite repeat. Expression of this fusion gene results in an unusual ring pattern of pigmentation around the eye. We carried out experiments to determine the mechanism for this pattern. By using FLP-mediated DNA mobilization to place different pugD transgenes at pre-selected sites we found that variation in repeat length makes a strong contribution to variability of the pug phenotype. This variation is manifest primarily as differences in the thickness of the pigmented ring. We show that similar phenotypic variation can also be achieved by changing gene copy number. We found that the pugD pattern is not controlled by wingless, which is normally expressed in a similar ring pattern. Finally, we found that physical injury to a pugD eye can lead to pigment deposition in parts of the eye that would not have been pigmented in the absence of injury. Our results are consistent with a model in which a metabolite vital for pigment formation is imported from the periphery of the eye, and pugD limits the extent of its transport towards the center of the eye, thus revealing the existence of a hitherto unknown mechanism of localized transport in the eye.

  13. Hutchinson-Gilford progeria syndrome, cardiovascular disease and oxidative stress.

    PubMed

    Trigueros-Motos, Laia; Gonzalez, Jose M; Rivera, Jose; Andres, Vicente

    2011-06-01

    Hutchinson-Gilford Progeria Syndrome (HGPS), a rare human disease characterized by premature aging, is mainly caused by the abnormal accumulation of progerin, a mutant form of the mammalian nuclear envelope component lamin A. HGPS patients exhibit vascular alterations and die at an average age of 13 years, predominantly from myocardial infarction or stroke. Animal models of HGPS have been a valuable tool in the study of the pathological processes implicated in the origin of this disease and its associated cardiovascular alterations. Some of the molecular mechanisms of HGPS might be relevant to the process of normal aging, since progerin is detected in cells from normal elderly humans. Conversely, processes linked to normal aging, such as the increase in oxidative stress, might be relevant to the pathogenic mechanisms of HGPS. In this review, we discuss recent advances in the understanding of the molecular mechanisms underlying the cardiovascular alterations associated with HGPS, the potential role of oxidative stress, and therapeutic approaches for the treatment of this devastating disease.

  14. A BLOC-1 Mutation Screen Reveals a Novel BLOC1S3 Mutation in Hermansky-Pudlak Syndrome Type 8 (HPS-8)

    PubMed Central

    Cullinane, Andrew R; Curry, James A; Golas, Gretchen; Pan, James; Carmona-Rivera, Carmelo; Hess, Richard A; White, James G; Huizing, Marjan; Gahl, William A

    2012-01-01

    Summary Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder of lysosome-related organelle biogenesis and is characterized by oculocutaneous albinism and a bleeding diathesis. Over the past decade, we screened 250 patients with HPS-like symptoms for mutations in the genes responsible for HPS subtypes 1–6. We identified 38 individuals with no functional mutations, and therefore, we analyzed all 8 genes encoding the Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1) proteins in these individuals. Here we describe the identification of a novel nonsense mutation in BLOC1S3 (HPS-8) in a 6 year-old Iranian boy. This mutation caused nonsense mediated decay of BLOC1S3 mRNA and destabilized the BLOC-1 complex. Our patient’s melanocytes showed aberrant localization of TYRP1, with increased plasma-membrane trafficking. These findings confirm a common cellular defect for HPS patients with defects in BLOC-1 subunits. We identified only 2 patients with BLOC-1 defects in our cohort, suggesting that other HPS genes remain to be identified. PMID:22709368

  15. Tissue-specific signaling networks rewired by major somatic mutations in human cancer revealed by proteome-wide discovery.

    PubMed

    Zhao, Junfei; Cheng, Feixiong; Zhao, Zhongming

    2017-03-31

    Massive somatic mutations discovered by large cancer genome sequencing projects provide unprecedented opportunities in the development of precision oncology. However, deep understanding of functional consequences of somatic mutations and identifying actionable mutations and the related drug responses currently remain formidable challenges. Dysfunction of protein post-translational modification plays critical roles in tumorigenesis and drug responses. In this study, we proposed a novel computational oncoproteomics approach, named kinome-wide network module for cancer pharmacogenomics (KNMPx), for identifying actionable mutations that rewired signaling networks and further characterized tumorigenesis and anticancer drug responses. Specifically, we integrated 746,631 missense mutations in 4,997 tumor samples across 16 major cancer types/subtypes from The Cancer Genome Atlas into over 170,000 carefully curated non-redundant phosphorylation sites covering 18,610 proteins. We found 47 mutated proteins (e.g., ERBB2, TP53, and CTNNB1) that had enriched missense mutations at their phosphorylation sites in pan-cancer analysis. In addition, tissue-specific kinase-substrate interaction modules altered by somatic mutations identified by KNMPx were significantly associated with patient survival. We further reported a kinome-wide landscape of pharmacogenomic interactions by incorporating somatic mutation-rewired signaling networks in 1,001 cancer cell lines via KNMPx. Interestingly, we found that cell lines could highly reproduce oncogenic phosphorylation site mutations identified in primary tumors, supporting the confidence in their associations with sensitivity/resistance of inhibitors targeting EGF, MAPK, PI3K, mTOR, and Wnt signaling pathways. In summary, this systematic oncoproteomics analysis of kinome phosphorylation site mutations illustrates new capabilities to speed the development of precision oncology.

  16. Exome sequencing reveals RAG1 mutations in a child with autoimmunity and sterile chronic multifocal osteomyelitis evolving into disseminated granulomatous disease

    PubMed Central

    Reiff, Andreas; Bassuk, Alexander G; Church, Joseph A; Campbell, Elizabeth; Bing, Xinyu; Ferguson, Polly J

    2013-01-01

    We describe a boy who developed autoinflammatory (chronic sterile multifocal osteomyelitis) and autoimmune (autoimmune cytopenias; vitiligo) phenotypes who subsequently developed disseminated granulomatous disease. Whole exome sequencing revealed homozygous RAG1 mutations thus expanding the spectrum of combined immunodeficiency with autoimmunity and granuloma that can occur with RAG deficiency. PMID:24122031

  17. A multi-species comparative structural bioinformatics analysis of inherited mutations in α-D-Mannosidase reveals strong genotype-phenotype correlation

    PubMed Central

    2009-01-01

    Background Lysosomal α-mannosidase is an enzyme that acts to degrade N-linked oligosaccharides and hence plays an important role in mannose metabolism in humans and other mammalian species, especially livestock. Mutations in the gene (MAN2B1) encoding lysosomal α-D-mannosidase cause improper coding, resulting in dysfunctional or non-functional protein, causing the disease α-mannosidosis. Mapping disease mutations to the structure of the protein can help in understanding the functional consequences of these mutations and thus indirectly, the finer aspects of the pathology and clinical manifestations of the disease, including phenotypic severity as a function of the genotype. Results A comprehensive homology modeling study of all the wild-type and inherited mutations of lysosomal α-mannosidase in four different species, human, cow, cat and guinea pig, reveals a significant correlation between the severity of the genotype and the phenotype in α-mannosidosis. We used the X-ray crystallographic structure of bovine lysosomal α-mannosidase as template, containing only two disulphide bonds and some ligands, to build structural models of wild-type structures with four disulfide linkages and all bound ligands. These wild-type models were then used as templates for disease mutations. All the truncations and substitutions involving the residues in and around the active site and those that destabilize the fold led to severe genotypes resulting in lethal phenotypes, whereas the mutations lying away from the active site were milder in both their genotypic and phenotypic expression. Conclusion Based on the co-location of mutations from different organisms and their proximity to the enzyme active site, we have extrapolated observed mutations from one species to homologous positions in other organisms, as a predictive approach for detecting likely α-mannosidosis. Besides predicting new disease mutations, this approach also provides a way for detecting mutation hotspots in the

  18. Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

    PubMed Central

    Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Razafsky, David S.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

    2012-01-01

    Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies. PMID:22649085

  19. X chromosome exome sequencing reveals a novel ALG13 mutation in a nonsyndromic intellectual disability family with multiple affected male siblings.

    PubMed

    Bissar-Tadmouri, Nesrine; Donahue, Whithey L; Al-Gazali, Lihadh; Nelson, Stanley F; Bayrak-Toydemir, Pinar; Kantarci, Sibel

    2014-01-01

    X-linked intellectual disability (XLID) is a heterogeneous condition associated with mutations in >100 genes, accounting for over 10% of all cases of intellectual impairment. The majority of XLID cases show nonsyndromic forms (NSXLID), in which intellectual disability is the sole clinically consistent manifestation. Here we performed X chromosome exome (X-exome) sequencing to identify the causative mutation in an NSXLID family with four affected male siblings and five unaffected female siblings. The X-exome sequencing at 88× coverage in one affected male sibling revealed a novel missense mutation (p.Tyr1074Cys) in the asparagine-linked glycosylation 13 homolog (ALG13) gene. Segregation analysis by Sanger sequencing showed that the all affected siblings were hemizygous and the mother was heterozygous for the mutation. Recently, a de novo missense mutation in ALG13 has been reported in a patient with X-linked congenital disorders of glycosylation type I. Our study reports the first case of NSXLID caused by a mutation in ALG13 involved in protein N-glycosylation.

  20. A postnatal role for embryonic myosin revealed by MYH3 mutations that alter TGFβ signaling and cause autosomal dominant spondylocarpotarsal synostosis

    PubMed Central

    Zieba, Jennifer; Zhang, Wenjuan; Chong, Jessica X.; Forlenza, Kimberly N.; Martin, Jorge H.; Heard, Kelly; Grange, Dorothy K.; Butler, Merlin G.; Kleefstra, Tjitske; Lachman, Ralph S.; Nickerson, Deborah; Regnier, Michael; Cohn, Daniel H.; Bamshad, Michael; Krakow, Deborah

    2017-01-01

    Spondylocarpotarsal synostosis (SCT) is a skeletal disorder characterized by progressive vertebral, carpal and tarsal fusions, and mild short stature. The majority of affected individuals have an autosomal recessive form of SCT and are homozygous or compound heterozygous for nonsense mutations in the gene that encodes the cytoskeletal protein filamin B (FLNB), but a subset do not have FLNB mutations. Exome sequence analysis of three SCT patients negative for FLNB mutations identified an autosomal dominant form of the disease due to heterozygosity for missense or nonsense mutations in MYH3, which encodes embryonic myosin. Cells transfected with the MYH3 missense mutations had reduced TGFβ signaling, revealing a regulatory role for embryonic myosin in the TGFβ signaling pathway. In wild-type mice, there was persistent postnatal expression of embryonic myosin in the small muscles joining the neural arches of the spine suggesting that loss of myosin function in these muscles contribute to the disease. Our findings demonstrate that dominant mutations in MYH3 underlie autosomal dominant SCT, identify a postnatal role for embryonic myosin and suggest that altered regulation of signal transduction in the muscles within the spine may lead to the development of vertebral fusions. PMID:28205584

  1. A postnatal role for embryonic myosin revealed by MYH3 mutations that alter TGFβ signaling and cause autosomal dominant spondylocarpotarsal synostosis.

    PubMed

    Zieba, Jennifer; Zhang, Wenjuan; Chong, Jessica X; Forlenza, Kimberly N; Martin, Jorge H; Heard, Kelly; Grange, Dorothy K; Butler, Merlin G; Kleefstra, Tjitske; Lachman, Ralph S; Nickerson, Deborah; Regnier, Michael; Cohn, Daniel H; Bamshad, Michael; Krakow, Deborah

    2017-02-16

    Spondylocarpotarsal synostosis (SCT) is a skeletal disorder characterized by progressive vertebral, carpal and tarsal fusions, and mild short stature. The majority of affected individuals have an autosomal recessive form of SCT and are homozygous or compound heterozygous for nonsense mutations in the gene that encodes the cytoskeletal protein filamin B (FLNB), but a subset do not have FLNB mutations. Exome sequence analysis of three SCT patients negative for FLNB mutations identified an autosomal dominant form of the disease due to heterozygosity for missense or nonsense mutations in MYH3, which encodes embryonic myosin. Cells transfected with the MYH3 missense mutations had reduced TGFβ signaling, revealing a regulatory role for embryonic myosin in the TGFβ signaling pathway. In wild-type mice, there was persistent postnatal expression of embryonic myosin in the small muscles joining the neural arches of the spine suggesting that loss of myosin function in these muscles contribute to the disease. Our findings demonstrate that dominant mutations in MYH3 underlie autosomal dominant SCT, identify a postnatal role for embryonic myosin and suggest that altered regulation of signal transduction in the muscles within the spine may lead to the development of vertebral fusions.

  2. Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues.

    PubMed

    Krimmel, Jeffrey D; Schmitt, Michael W; Harrell, Maria I; Agnew, Kathy J; Kennedy, Scott R; Emond, Mary J; Loeb, Lawrence A; Swisher, Elizabeth M; Risques, Rosa Ana

    2016-05-24

    Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue.

  3. Integrative proteomics, genomics, and translational immunology approaches reveal mutated forms of Proteolipid Protein 1 (PLP1) and mutant-specific immune response in multiple sclerosis.

    PubMed

    Qendro, Veneta; Bugos, Grace A; Lundgren, Debbie H; Glynn, John; Han, May H; Han, David K

    2017-03-01

    In order to gain mechanistic insights into multiple sclerosis (MS) pathogenesis, we utilized a multi-dimensional approach to test the hypothesis that mutations in myelin proteins lead to immune activation and central nervous system autoimmunity in MS. Mass spectrometry-based proteomic analysis of human MS brain lesions revealed seven unique mutations of PLP1; a key myelin protein that is known to be destroyed in MS. Surprisingly, in-depth genomic analysis of two MS patients at the genomic DNA and mRNA confirmed mutated PLP1 in RNA, but not in the genomic DNA. Quantification of wild type and mutant PLP RNA levels by qPCR further validated the presence of mutant PLP RNA in the MS patients. To seek evidence linking mutations in abundant myelin proteins and immune-mediated destruction of myelin, specific immune response against mutant PLP1 in MS patients was examined. Thus, we have designed paired, wild type and mutant peptide microarrays, and examined antibody response to multiple mutated PLP1 in sera from MS patients. Consistent with the idea of different patients exhibiting unique mutation profiles, we found that 13 out of 20 MS patients showed antibody responses against specific but not against all the mutant-PLP1 peptides. Interestingly, we found mutant PLP-directed antibody response against specific mutant peptides in the sera of pre-MS controls. The results from integrative proteomic, genomic, and immune analyses reveal a possible mechanism of mutation-driven pathogenesis in human MS. The study also highlights the need for integrative genomic and proteomic analyses for uncovering pathogenic mechanisms of human diseases.

  4. Mechanisms of premature vascular aging in children with Hutchinson-Gilford progeria syndrome.

    PubMed

    Gerhard-Herman, Marie; Smoot, Leslie B; Wake, Nicole; Kieran, Mark W; Kleinman, Monica E; Miller, David T; Schwartzman, Armin; Giobbie-Hurder, Anita; Neuberg, Donna; Gordon, Leslie B

    2012-01-01

    Hutchinson-Gilford progeria syndrome is a rare, segmental premature aging syndrome of accelerated atherosclerosis and early death from myocardial infarction or stroke. This study sought to establish comprehensive characterization of the fatal vasculopathy in Hutchinson-Gilford progeria syndrome and its relevance to normal aging. We performed cardiovascular assessments at a single clinical site on the largest prospectively studied cohort to date. Carotid-femoral pulse wave velocity was dramatically elevated (mean: 13.00±3.83 m/s). Carotid duplex ultrasound echobrightness, assessed in predefined tissue sites as a measure of arterial wall density, was significantly greater than age- and sex-matched controls in the intima-media (P<0.02), near adventitia (P<0.003), and deep adventitia (P<0.01), as was internal carotid artery mean flow velocity (P<0.0001). Ankle-brachial indices were abnormal in 78% of patients. Effective disease treatments may be heralded by normalizing trends of these noninvasive cardiovascular measures. The data demonstrate that, along with peripheral vascular occlusive disease, accelerated vascular stiffening is an early and pervasive mechanism of vascular disease in Hutchinson-Gilford progeria syndrome. There is considerable overlap with cardiovascular changes of normal aging, which reinforces the view that defining mechanisms of cardiovascular disease in Hutchinson-Gilford progeria syndrome provides a unique opportunity to isolate a subset of factors influencing cardiovascular disease in the general aging population.

  5. Speeding up the clock: The past, present and future of progeria.

    PubMed

    Swahari, Vijay; Nakamura, Ayumi

    2016-01-01

    Progeria is a devastating disorder in which patients exhibit signs of premature aging. The most well-known progeroid syndromes include Hutchinson-Gilford Progeria Syndrome (HGPS) and Werner Syndrome (WS). While HGPS and WS are rare, they often result in severe age-associated complications starting in the early developmental period or after the pubertal growth spurt during adolescence, respectively. In addition, patients with HGPS ultimately die of diseases normally seen in the elderly population, with stroke and myocardial infarction as the leading causes of death. Many WS patients develop similar cardiovascular complications but also have an increased predisposition to developing multiple rare malignancies. These premature aging disorders, as well as animal and cell culture models that recapitulate these diseases, have provided insight into the genetics and cellular pathways that underlie these human conditions and have also uncovered possible mechanisms behind normal aging. Here we discuss the history, the types of progeria, and the various pathophysiological mechanisms that drive these diseases. We also address recent medical advances and treatment modalities for patients with progeria.

  6. Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing*#

    PubMed Central

    Li, Shan; Zheng, Yun-chao; Cui, Hai-rui; Fu, Hao-wei; Shu, Qing-yao; Huang, Jian-zhong

    2016-01-01

    Mutation breeding is based on the induction of genetic variations; hence knowledge of the frequency and type of induced mutations is of paramount importance for the design and implementation of a mutation breeding program. Although γ ray irradiation has been widely used since the 1960s in the breeding of about 200 economically important plant species, molecular elucidation of its genetic effects has so far been achieved largely by analysis of target genes or genomic regions. In the present study, the whole genomes of six γ-irradiated M2 rice plants were sequenced; a total of 144–188 million high-quality (Q>20) reads were generated for each M2 plant, resulting in genome coverage of >45 times for each plant. Single base substitution (SBS) and short insertion/deletion (Indel) mutations were detected at the average frequency of 7.5×10−6–9.8×10−6 in the six M2 rice plants (SBS being about 4 times more frequent than Indels). Structural and copy number variations, though less frequent than SBS and Indel, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots. The present study is the first genome-wide single-nucleotide resolution study on the feature and frequency of γ irradiation-induced mutations in a seed propagated crop; the findings are of practical importance for mutation breeding of rice and other crop species. PMID:27921396

  7. Comprehensive Mutational Analysis of Sucrose-Metabolizing Pathways in Streptococcus mutans Reveals Novel Roles for the Sucrose Phosphotransferase System Permease

    PubMed Central

    Zeng, Lin

    2013-01-01

    Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO4 hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIALev) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA. PMID:23222725

  8. Comprehensive mutational analysis of sucrose-metabolizing pathways in Streptococcus mutans reveals novel roles for the sucrose phosphotransferase system permease.

    PubMed

    Zeng, Lin; Burne, Robert A

    2013-02-01

    Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO(4) hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIA(Lev)) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA.

  9. The E646D-ATP13A4 mutation associated with autism reveals a defect in calcium regulation.

    PubMed

    Vallipuram, Janaki; Grenville, Jeffrey; Crawford, Dorota A

    2010-03-01

    ATP13A4 is a member of the subfamily of P5-type ATPases. P5-type ATPases are the least studied of the P-type ATPase subfamilies with no ion specificities assigned to them. In order to elucidate ATP13A4 function, we studied the protein's subcellular localization and tested whether it is involved in calcium regulation. The intracellular calcium concentration was measured in COS-7 cells over-expressing mouse ATP13A4 using ratiometric calcium imaging with fura-2 AM as a calcium indicator. The results of this study show that ATP13A4 is localized to the endoplasmic reticulum (ER). Furthermore, we demonstrate that over-expression of ATP13A4 in COS-7 cells caused a significant increase in the intracellular calcium level. Interestingly, over-expression of the sequence variant containing a substitution of aspartic acid for a glutamic acid (E646D), previously found in patients with autism spectrum disorder (ASD), did not increase the free cellular calcium likely due to the mutation. In this study, we also describe the expression of ATP13A4 during mouse embryonic development. Quantitative real-time PCR revealed that ATP13A4 was highly expressed at embryonic days 15-17, when neurogenesis takes place. The present study is the first to provide further insights into the biological role of a P5-type ATPase. Our results demonstrate that ATP13A4 may be involved in calcium regulation and that its expression is developmentally regulated. Overall, this study provides support for the hypothesis that ATP13A4 may play a vital role in the developing nervous system and its impairment can contribute to the symptoms seen in ASD.

  10. A bioinformatics analysis of Lamin-A regulatory network: a perspective on epigenetic involvement in Hutchinson-Gilford progeria syndrome.

    PubMed

    Arancio, Walter

    2012-04-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare human genetic disease that leads to premature aging. HGPS is caused by mutation in the Lamin-A (LMNA) gene that leads, in affected young individuals, to the accumulation of the progerin protein, usually present only in aging differentiated cells. Bioinformatics analyses of the network of interactions of the LMNA gene and transcripts are presented. The LMNA gene network has been analyzed using the BioGRID database (http://thebiogrid.org/) and related analysis tools such as Osprey (http://biodata.mshri.on.ca/osprey/servlet/Index) and GeneMANIA ( http://genemania.org/). The network of interaction of LMNA transcripts has been further analyzed following the competing endogenous (ceRNA) hypotheses (RNA cross-talk via microRNAs [miRNAs]) and using the miRWalk database and tools (www.ma.uni-heidelberg.de/apps/zmf/mirwalk/). These analyses suggest particular relevance of epigenetic modifiers (via acetylase complexes and specifically HTATIP histone acetylase) and adenosine triphosphate (ATP)-dependent chromatin remodelers (via pBAF, BAF, and SWI/SNF complexes).

  11. A farnesyltransferase inhibitor prevents both the onset and late progression of cardiovascular disease in a progeria mouse model.

    PubMed

    Capell, Brian C; Olive, Michelle; Erdos, Michael R; Cao, Kan; Faddah, Dina A; Tavarez, Urraca L; Conneely, Karen N; Qu, Xuan; San, Hong; Ganesh, Santhi K; Chen, Xiaoyan; Avallone, Hedwig; Kolodgie, Frank D; Virmani, Renu; Nabel, Elizabeth G; Collins, Francis S

    2008-10-14

    Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic form of human premature aging. Death occurs at a mean age of 13 years, usually from heart attack or stroke. Almost all cases of HGPS are caused by a de novo point mutation in the lamin A (LMNA) gene that results in production of a mutant lamin A protein termed progerin. This protein is permanently modified by a lipid farnesyl group, and acts as a dominant negative, disrupting nuclear structure. Treatment with farnesyltransferase inhibitors (FTIs) has been shown to prevent and even reverse this nuclear abnormality in cultured HGPS fibroblasts. We have previously created a mouse model of HGPS that shows progressive loss of vascular smooth muscle cells in the media of the large arteries, in a pattern that is strikingly similar to the cardiovascular disease seen in patients with HGPS. Here we show that the dose-dependent administration of the FTI tipifarnib (R115777, Zarnestra) to this HGPS mouse model can significantly prevent both the onset of the cardiovascular phenotype as well as the late progression of existing cardiovascular disease. These observations provide encouraging evidence for the current clinical trial of FTIs for this rare and devastating disease.

  12. Activating the synthesis of progerin, the mutant prelamin A in Hutchinson-Gilford progeria syndrome, with antisense oligonucleotides.

    PubMed

    Fong, Loren G; Vickers, Timothy A; Farber, Emily A; Choi, Christine; Yun, Ui Jeong; Hu, Yan; Yang, Shao H; Coffinier, Catherine; Lee, Roger; Yin, Liya; Davies, Brandon S J; Andres, Douglas A; Spielmann, H Peter; Bennett, C Frank; Young, Stephen G

    2009-07-01

    Hutchinson-Gilford progeria syndrome (HGPS) is caused by point mutations that increase utilization of an alternate splice donor site in exon 11 of LMNA (the gene encoding lamin C and prelamin A). The alternate splicing reduces transcripts for wild-type prelamin A and increases transcripts for a truncated prelamin A (progerin). Here, we show that antisense oligonucleotides (ASOs) against exon 11 sequences downstream from the exon 11 splice donor site promote alternate splicing in both wild-type and HGPS fibroblasts, increasing the synthesis of progerin. Indeed, wild-type fibroblasts transfected with these ASOs exhibit progerin levels similar to (or greater than) those in fibroblasts from HGPS patients. This progerin was farnesylated, as judged by metabolic labeling studies. The synthesis of progerin in wild-type fibroblasts was accompanied by the same nuclear shape and gene-expression perturbations observed in HGPS fibroblasts. An ASO corresponding to the 5' portion of intron 11 also promoted alternate splicing. In contrast, an ASO against exon 11 sequences 5' to the alternate splice site reduced alternate splicing in HGPS cells and modestly lowered progerin levels. Thus, different ASOs can be used to increase or decrease 'HGPS splicing'. ASOs represent a new and powerful tool for recreating HGPS pathophysiology in wild-type cells.

  13. Heterogeneous evolution of microsatellites revealed by reconstruction of recent mutation history in an invasive apomictic snail, Potamopyrgus antipodarum.

    PubMed

    Weetman, David; Hauser, Lorenz; Carvalho, Gary R

    2006-05-01

    Heterogeneous patterns of microsatellite evolution present a major challenge for the development of mutation models, and an improved understanding of the determinants of variation in mutation rates and patterns among loci, alleles and taxa is required. A 19th Century bottleneck associated with the introduction of clones of the snail Potamopyrgus antipodarum to Britain presented an opportunity to reconstruct recent microsatellite evolution within the most common apomictic lineage. There was significant variation in both the number and step size of mutations among the seven loci studied. Patterns of mutability were consistent with higher mutation rates for di- than trinucleotides and for longer alleles at a locus. Mutation size was influenced in a more complex way, decreasing with relative allele length much more strongly for tri-, than dinucleotides. We found support for this latter, highly novel result in the literature via reanalysis of data in a recent genome-scan study of human microsatellites, which showed a similarly disparate pattern of length-dependence between di- and trinucleotides. In spite of the apomictic form of reproduction and an unusually strong excess of microsatellite contractions in P. antipodarum, there were notable similarities with mutation processes of human microsatellites, supporting the wider taxonomic generality of such evolutionary mechanisms.

  14. Structural Analysis Reveals the Deleterious Effects of Telomerase Mutations in Telomerase-Associated Bone Marrow Failure Syndromes.

    PubMed

    Hoffman, Hunter; Rice, Cory; Skordalakes, Emmanuel

    2017-02-01

    Naturally occurring mutations in the ribonucleoprotein reverse transcriptase, telomerase, are associated with the bone marrow failure syndromes dyskeratosis congenita (DKC), aplastic anemia (AA), and idiopathic pulmonary fibrosis (IPF). However, the mechanism by which these mutations impact telomerase function remains unknown. Here we present the structure of the human telomerase c-terminal extension (CTE or thumb domain) determined by the method of single-wavelength anomalous diffraction (SAD) to 2.31 A resolution. We also used direct telomerase activity and nucleic acid binding assays to explain how naturally occurring mutations within this portion of telomerase contribute to human disease. The single mutations localize within three highly conserved regions of the telomerase thumb domain referred to as motifs E-I, (thumb loop and helix) E-II and E-III (the FVYL pocket, comprising the hydrophobic residues F1012, V1025, Y1089 and L1092). Biochemical data shows that the mutations associated with DKC, AA and IFP disrupt the binding between telomerases protein subunit reverse transcriptase (TERT) and its nucleic acid substrates leading to loss of telomerase activity and processivity. Collectively our data shows that although these mutations do not alter the overall stability or expression of TERT, these rare genetic disorders are associated with an impaired telomerase holoenzyme that is unable to correctly assemble with its nucleic acid substrates, leading to incomplete telomere extension and telomere attrition, which are hallmarks of these diseases.

  15. Mutation-Structure-Function Relationship Based Integrated Strategy Reveals the Potential Impact of Deleterious Missense Mutations in Autophagy Related Proteins on Hepatocellular Carcinoma (HCC): A Comprehensive Informatics Approach

    PubMed Central

    Awan, Faryal Mehwish; Obaid, Ayesha; Ikram, Aqsa; Janjua, Hussnain Ahmed

    2017-01-01

    Autophagy, an evolutionary conserved multifaceted lysosome-mediated bulk degradation system, plays a vital role in liver pathologies including hepatocellular carcinoma (HCC). Post-translational modifications (PTMs) and genetic variations in autophagy components have emerged as significant determinants of autophagy related proteins. Identification of a comprehensive spectrum of genetic variations and PTMs of autophagy related proteins and their impact at molecular level will greatly expand our understanding of autophagy based regulation. In this study, we attempted to identify high risk missense mutations that are highly damaging to the structure as well as function of autophagy related proteins including LC3A, LC3B, BECN1 and SCD1. Number of putative structural and functional residues, including several sites that undergo PTMs were also identified. In total, 16 high-risk SNPs in LC3A, 18 in LC3B, 40 in BECN1 and 43 in SCD1 were prioritized. Out of these, 2 in LC3A (K49A, K51A), 1 in LC3B (S92C), 6 in BECN1 (S113R, R292C, R292H, Y338C, S346Y, Y352H) and 6 in SCD1 (Y41C, Y55D, R131W, R135Q, R135W, Y151C) coincide with potential PTM sites. Our integrated analysis found LC3B Y113C, BECN1 I403T, SCD1 R126S and SCD1 Y218C as highly deleterious HCC-associated mutations. This study is the first extensive in silico mutational analysis of the LC3A, LC3B, BECN1 and SCD1 proteins. We hope that the observed results will be a valuable resource for in-depth mechanistic insight into future investigations of pathological missense SNPs using an integrated computational platform. PMID:28085066

  16. Mutation-Structure-Function Relationship Based Integrated Strategy Reveals the Potential Impact of Deleterious Missense Mutations in Autophagy Related Proteins on Hepatocellular Carcinoma (HCC): A Comprehensive Informatics Approach.

    PubMed

    Awan, Faryal Mehwish; Obaid, Ayesha; Ikram, Aqsa; Janjua, Hussnain Ahmed

    2017-01-11

    Autophagy, an evolutionary conserved multifaceted lysosome-mediated bulk degradation system, plays a vital role in liver pathologies including hepatocellular carcinoma (HCC). Post-translational modifications (PTMs) and genetic variations in autophagy components have emerged as significant determinants of autophagy related proteins. Identification of a comprehensive spectrum of genetic variations and PTMs of autophagy related proteins and their impact at molecular level will greatly expand our understanding of autophagy based regulation. In this study, we attempted to identify high risk missense mutations that are highly damaging to the structure as well as function of autophagy related proteins including LC3A, LC3B, BECN1 and SCD1. Number of putative structural and functional residues, including several sites that undergo PTMs were also identified. In total, 16 high-risk SNPs in LC3A, 18 in LC3B, 40 in BECN1 and 43 in SCD1 were prioritized. Out of these, 2 in LC3A (K49A, K51A), 1 in LC3B (S92C), 6 in BECN1 (S113R, R292C, R292H, Y338C, S346Y, Y352H) and 6 in SCD1 (Y41C, Y55D, R131W, R135Q, R135W, Y151C) coincide with potential PTM sites. Our integrated analysis found LC3B Y113C, BECN1 I403T, SCD1 R126S and SCD1 Y218C as highly deleterious HCC-associated mutations. This study is the first extensive in silico mutational analysis of the LC3A, LC3B, BECN1 and SCD1 proteins. We hope that the observed results will be a valuable resource for in-depth mechanistic insight into future investigations of pathological missense SNPs using an integrated computational platform.

  17. Exome sequencing reveals a heterozygous DLX5 mutation in a Chinese family with autosomal-dominant split-hand/foot malformation

    PubMed Central

    Wang, Xue; Xin, Qian; Li, Lin; Li, Jiangxia; Zhang, Changwu; Qiu, Rongfang; Qian, Chenmin; Zhao, Hailing; Liu, Yongchao; Shan, Shan; Dang, Jie; Bian, Xianli; Shao, Changshun; Gong, Yaoqin; Liu, Qiji

    2014-01-01

    Split-hand/foot malformation (SHFM) is a congenital limb deformity due to the absence or dysplasia of central rays of the autopod. Six SHFM loci have already been identified. Here we describe a Chinese family with autosomal-dominant SHFM1 that has previously been mapped to 7q21.2-21.3. The two affected family members, mother and son, showed deep median clefts between toes, ectrodactyly and syndactyly; the mother also showed triphalangeal thumbs. Exome sequencing and variant screening of candidate genes in the six loci known to be responsible for SHFM revealed a novel heterozygous mutation, c.558G>T (p.(Gln186His)), in distal-less homeobox 5 (DLX5). As DLX5 encodes a transcription factor capable of transactivating MYC, we also tested whether the mutation could affect DLX5 transcription acitivity. Results from luciferase reporter assay revealed that a mutation in DLX5 compromised its transcriptional activity. This is the first report of a mutation in DLX5 leading to autosomal-dominant SHFM1. PMID:24496061

  18. Whole Exome Sequencing Reveals Compound Heterozygosity for Ethnically Distinct PEX7 Mutations Responsible for Rhizomelic Chondrodysplasia Punctata, Type 1

    PubMed Central

    Jacobsen, Jessie C.; Glamuzina, Emma; Taylor, Juliet; Swan, Brendan; Handisides, Shona; Wilson, Callum; Fietz, Michael; van Dijk, Tessa; Appelhof, Bart; Hill, Rosamund; Marks, Rosemary; Love, Donald R.; Robertson, Stephen P.; Snell, Russell G.; Lehnert, Klaus

    2015-01-01

    We describe two brothers who presented at birth with bone growth abnormalities, followed by development of increasingly severe intellectual and physical disability, growth restriction, epilepsy, and cerebellar and brain stem atrophy, but normal ocular phenotypes. Case 1 died at 19 years of age due to chronic respiratory illnesses without a unifying diagnosis. The brother remains alive but severely disabled at 19 years of age. Whole exome sequencing identified compound heterozygous stop mutations in the peroxisome biogenesis factor 7 gene in both individuals. Mutations in this gene cause rhizomelic chondrodysplasia punctata, type 1 (RCDP1). One mutation, p.Arg232∗, has only been documented once before in a Japanese family, which is of interest given these two boys are of European descent. The other mutation, p.Leu292∗, is found in approximately 50% of RCDP1 patients. These are the first cases of RCDP1 that describe the coinheritance of the p.Arg232∗ and p.Leu292∗ mutations and demonstrate the utility of WES in cases with unclear diagnoses. PMID:26587300

  19. Excess of mutational jackpot events in expanding populations revealed by spatial Luria–Delbrück experiments

    PubMed Central

    Fusco, Diana; Gralka, Matti; Kayser, Jona; Anderson, Alex; Hallatschek, Oskar

    2016-01-01

    The genetic diversity of growing cellular populations, such as biofilms, solid tumours or developing embryos, is thought to be dominated by rare, exceptionally large mutant clones. Yet, the emergence of these mutational jackpot events is only understood in well-mixed populations, where they stem from mutations that arise during the first few cell divisions. To study jackpot events in spatially structured populations, we track mutant clones in microbial populations using fluorescence microscopy and population sequencing. High-frequency mutations are found to be massively enriched in microbial colonies compared with well-shaken liquid cultures, as a result of late-occurring mutations surfing at the edge of range expansions. Thus, jackpot events can be generated not only when mutations arise early but also when they occur at favourable locations, which exacerbates their role in adaptation and disease. In particular, because spatial competition with the wild type keeps most mutant clones in a quiescent state, strong selection pressures that kill the wild type promote drug resistance. PMID:27694797

  20. Whole Exome Sequencing Reveals a Mutation in CRYBB2 in a Large Mexican Family with Autosomal Dominant Pulverulent Cataract

    PubMed Central

    Messina-Baas, Olga; Gonzalez-Garay, Manuel L.; González-Huerta, Luz M.; Toral-López, Jaime; Cuevas-Covarrubias, Sergio A.

    2016-01-01

    Congenital cataract, an important cause of reversible blindness, is due to several causes including Mendelian inheritance. Thirty percent of cataracts are hereditary with participation of the gamma crystallin genes. Clinical and genetic heterogeneity is observed in patients with gene mutations and congenital cataract; about 40 genetic loci have been associated with hereditary cataract. In this study, we identified the underlying genetic cause of an autosomal dominant pulverulent cataract (ADPC) in a large Mexican family. Twenty-one affected patients and 20 healthy members of a family with ADPC were included. Genomic DNA was analyzed by whole exome sequencing in the proband, a normal daughter, and in an affected son, whereas DNA Sanger sequencing was performed in all members of the family. After the bioinformatics analysis, all samples were genotyped using Sanger sequencing to eliminate variants that do not cosegregate with the cataract. We observed a perfect cosegregation of a nonsense mutation c.475C>T (p.Q155*) in exon 6 of the CRYBB2 gene with ADPC. We calculated a logarithm of the odds score of 5.5. This mutation was not detected in healthy members of the family and in 100 normal controls. This is the first Mexican family with ADPC associated with a p.Q155* mutation. Interestingly, this specific mutation in the CRYBB2 gene seems to be exclusively associated with pulverulent/cerulean cataract (with some clinical variability) independent of the population's genetic background. PMID:27385965

  1. Targeted sequencing of refractory myeloma reveals a high incidence of mutations in CRBN and Ras pathway genes.

    PubMed

    Kortüm, K Martin; Mai, Elias K; Hanafiah, Nur H; Shi, Chang-Xi; Zhu, Yuan-Xiao; Bruins, Laura; Barrio, Santiago; Jedlowski, Patrick; Merz, Maximilian; Xu, Jing; Stewart, Robert A; Andrulis, Mindaugas; Jauch, Anna; Hillengass, Jens; Goldschmidt, Hartmut; Bergsagel, P Leif; Braggio, Esteban; Stewart, A Keith; Raab, Marc S

    2016-09-01

    In this study, targeted sequencing to screen 50 multidrug refractory multiple myeloma (rMM) patients was performed by using the Multiple Myeloma Mutation Panel. Patients were pretreated with both immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs), and 88%, 78%, and 68% were refractory to an IMiD, a PI, or both, respectively. The majority of patients had progressive (82%) or refractory (78%) disease immediately before sampling, with 43% being IMiD refractory and 46% being PI refractory in the most recent line of therapy. Compared with newly diagnosed MM, an increased prevalence of mutations in the Ras pathway genes KRAS, NRAS, and/or BRAF (72%), as well as TP53 (26%), CRBN (12%), and CRBN pathway genes (10%) was observed. Longitudinal analyses performed in 3 patients with CRBN mutations at time of IMiD resistance confirmed that these mutations were undetectable at earlier, IMiD-sensitive time points. Furthermore, the functional introduction of these mutations in MM cells conferred lenalidomide resistance in vitro. These data indicate a differential genetic landscape in rMM associated with drug response.

  2. Revealing the Effects of Missense Mutations Causing Snyder-Robinson Syndrome on the Stability and Dimerization of Spermine Synthase.

    PubMed

    Peng, Yunhui; Norris, Joy; Schwartz, Charles; Alexov, Emil

    2016-01-08

    Missense mutations in spermine synthase (SpmSyn) protein have been shown to cause the Snyder-Robinson syndrome (SRS). Depending on the location within the structure of SpmSyn and type of amino acid substitution, different mechanisms resulting in SRS were proposed. Here we focus on naturally occurring amino acid substitutions causing SRS, which are situated away from the active center of SpmSyn and thus are not directly involved in the catalysis. Two of the mutations, M35R and P112L, are reported for the first time in this study. It is demonstrated, both experimentally and computationally, that for such mutations the major effect resulting in dysfunctional SpmSyn is the destabilization of the protein. In vitro experiments indicated either no presence or very little amount of the mutant SpmSyn in patient cells. In silico modeling predicted that all studied mutations in this work destabilize SpmSyn and some of them abolish homo-dimer formation. Since dimerization and structural stability are equally important for the wild type function of SpmSyn, it is proposed that the SRS caused by mutations occurring in the N-domain of SpmSyn is a result of dysfunctional mutant proteins being partially unfolded and degraded by the proteomic machinery of the cell or being unable to form a homo-dimer.

  3. Large-scale mutational analysis of Kv11.1 reveals molecular insights into type 2 long QT syndrome

    NASA Astrophysics Data System (ADS)

    Anderson, Corey L.; Kuzmicki, Catherine E.; Childs, Ryan R.; Hintz, Caleb J.; Delisle, Brian P.; January, Craig T.

    2014-11-01

    It has been suggested that deficient protein trafficking to the cell membrane is the dominant mechanism associated with type 2 Long QT syndrome (LQT2) caused by Kv11.1 potassium channel missense mutations, and that for many mutations the trafficking defect can be corrected pharmacologically. However, this inference was based on expression of a small number of Kv11.1 mutations. We performed a comprehensive analysis of 167 LQT2-linked missense mutations in four Kv11.1 structural domains and found that deficient protein trafficking is the dominant mechanism for all domains except for the distal carboxy-terminus. Also, most pore mutations—in contrast to intracellular domain mutations—were found to have severe dominant-negative effects when co-expressed with wild-type subunits. Finally, pharmacological correction of the trafficking defect in homomeric mutant channels was possible for mutations within all structural domains. However, pharmacological correction is dramatically improved for pore mutants when co-expressed with wild-type subunits to form heteromeric channels.

  4. The FasFADD death domain complex structure reveals the basis of DISC assembly and disease mutations

    SciTech Connect

    Wang, Liwei; Yang, Jin Kuk; Kabaleeswaran, Venkataraman; Rice, Amanda J.; Cruz, Anthony C.; Park, Ah Young; Yin, Qian; Damko, Ermelinda; Jang, Se Bok; Raunser, Stefan; Robinson, Carol V.; Siegel, Richard M.; Walz, Thomas; Wu, Hao

    2010-10-10

    The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.

  5. Excess of mutational jackpot events in expanding populations revealed by spatial Luria-Delbrück experiments.

    PubMed

    Fusco, Diana; Gralka, Matti; Kayser, Jona; Anderson, Alex; Hallatschek, Oskar

    2016-10-03

    The genetic diversity of growing cellular populations, such as biofilms, solid tumours or developing embryos, is thought to be dominated by rare, exceptionally large mutant clones. Yet, the emergence of these mutational jackpot events is only understood in well-mixed populations, where they stem from mutations that arise during the first few cell divisions. To study jackpot events in spatially structured populations, we track mutant clones in microbial populations using fluorescence microscopy and population sequencing. High-frequency mutations are found to be massively enriched in microbial colonies compared with well-shaken liquid cultures, as a result of late-occurring mutations surfing at the edge of range expansions. Thus, jackpot events can be generated not only when mutations arise early but also when they occur at favourable locations, which exacerbates their role in adaptation and disease. In particular, because spatial competition with the wild type keeps most mutant clones in a quiescent state, strong selection pressures that kill the wild type promote drug resistance.

  6. In-vitro characterization of androgen receptor mutations associated with complete androgen insensitivity syndrome reveals distinct functional deficits.

    PubMed

    Werner, R; Zhan, J; Gesing, J; Struve, D; Hiort, O

    2008-01-01

    Adequate androgen receptor (AR) function is crucial for male sex development and maintenance of secondary male characteristics. Mutations in the AR lead to androgen insensitivity syndrome (AIS) characterized by an end-organ resistance to androgens. The clinical appearance of individuals with 46,XY karyotype and an AR mutation varies widely from normal male to the ultimate completely female phenotype of complete androgen insensitivity syndrome (CAIS). We have analyzed the androgen receptor missense mutations P723S, P904S, and H917R, clinically associated with CAIS, which were described to have a normal maximum androgen binding (Bmax) but elevated equilibrium dissociation constants (Kd's) and compared their properties with the F916X deletion mutant, leading to the loss of the last four amino acids of the AR. Functional analysis allowed a quantitative and qualitative discrimination of these mutants in transactivation, amino-terminal/carboxy-terminal (N/C)-interaction, and coactivation capacity, varying widely with each distinct mutation. We conclude that mutations in the AR have to be characterized meticulously, not only to prove any quantitative functional deficit as a proof of consequence, but also to gain knowledge on qualitative functional properties. This is necessary as a possible link to genotype-phenotype correlation in AIS, but also with respect to medical decision making in CAIS.

  7. Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.

    PubMed

    Vermeer, Sascha; Hoischen, Alexander; Meijer, Rowdy P P; Gilissen, Christian; Neveling, Kornelia; Wieskamp, Nienke; de Brouwer, Arjan; Koenig, Michel; Anheim, Mathieu; Assoum, Mirna; Drouot, Nathalie; Todorovic, Slobodanka; Milic-Rasic, Vedrana; Lochmüller, Hanns; Stevanin, Giovanni; Goizet, Cyril; David, Albert; Durr, Alexandra; Brice, Alexis; Kremer, Berry; van de Warrenburg, Bart P C; Schijvenaars, Mascha M V A P; Heister, Angelien; Kwint, Michael; Arts, Peer; van der Wijst, Jenny; Veltman, Joris; Kamsteeg, Erik-Jan; Scheffer, Hans; Knoers, Nine

    2010-12-10

    Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation sequencing technology, we identified a mutation in a gene and have shown its association with autosomal-recessive cerebellar ataxia. In a Dutch consanguineous family with three affected siblings a homozygous 12.5 Mb region on chromosome 3 was targeted by array-based sequence capture. Prioritization of all detected sequence variants led to four candidate genes, one of which contained a variant with a high base pair conservation score (phyloP score: 5.26). This variant was a leucine-to-arginine substitution in the DUF 590 domain of a 16K transmembrane protein, a putative calcium-activated chloride channel encoded by anoctamin 10 (ANO10). The analysis of ANO10 by Sanger sequencing revealed three additional mutations: a homozygous mutation (c.1150_1151del [p.Leu384fs]) in a Serbian family and a compound-heterozygous splice-site mutation (c.1476+1G>T) and a frameshift mutation (c.1604del [p.Leu535X]) in a French family. This illustrates the power of using initial homozygosity mapping with next-generation sequencing technology to identify genes involved in autosomal-recessive diseases. Moreover, identifying a putative calcium-dependent chloride channel involved in cerebellar ataxia adds another pathway to the list of pathophysiological mechanisms that may cause cerebellar ataxia.

  8. Whole Exome Sequencing Reveals Homozygous Mutations in RAI1, OTOF, and SLC26A4 Genes Associated with Nonsyndromic Hearing Loss in Altaian Families (South Siberia)

    PubMed Central

    Karafet, Tatiana M.; Morozov, Igor V.; Mikhalskaia, Valeriia Yu.; Zytsar, Marina V.; Bondar, Alexander A.

    2016-01-01

    Hearing loss (HL) is one of the most common sensorineural disorders and several dozen genes contribute to its pathogenesis. Establishing a genetic diagnosis of HL is of great importance for clinical evaluation of deaf patients and for estimating recurrence risks for their families. Efforts to identify genes responsible for HL have been challenged by high genetic heterogeneity and different ethnic-specific prevalence of inherited deafness. Here we present the utility of whole exome sequencing (WES) for identifying candidate causal variants for previously unexplained nonsyndromic HL of seven patients from four unrelated Altaian families (the Altai Republic, South Siberia). The WES analysis revealed homozygous missense mutations in three genes associated with HL. Mutation c.2168A>G (SLC26A4) was found in one family, a novel mutation c.1111G>C (OTOF) was revealed in another family, and mutation c.5254G>A (RAI1) was found in two families. Sanger sequencing was applied for screening of identified variants in an ethnically diverse cohort of other patients with HL (n = 116) and in Altaian controls (n = 120). Identified variants were found only in patients of Altaian ethnicity (n = 93). Several lines of evidences support the association of homozygosity for discovered variants c.5254G>A (RAI1), c.1111C>G (OTOF), and c.2168A>G (SLC26A4) with HL in Altaian patients. Local prevalence of identified variants implies possible founder effect in significant number of HL cases in indigenous population of the Altai region. Notably, this is the first reported instance of patients with RAI1 missense mutation whose HL is not accompanied by specific traits typical for Smith-Magenis syndrome. Presumed association of RAI1 gene variant c.5254G>A with isolated HL needs to be proved by further experimental studies. PMID:27082237

  9. Mutational Studies on Resurrected Ancestral Proteins Reveal Conservation of Site-Specific Amino Acid Preferences throughout Evolutionary History

    PubMed Central

    Risso, Valeria A.; Manssour-Triedo, Fadia; Delgado-Delgado, Asunción; Arco, Rocio; Barroso-delJesus, Alicia; Ingles-Prieto, Alvaro; Godoy-Ruiz, Raquel; Gavira, Jose A.; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2015-01-01

    Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. PMID:25392342

  10. Genetic characterization of T-PLL reveals two major biologic subgroups and JAK3 mutations as prognostic marker.

    PubMed

    Stengel, Anna; Kern, Wolfgang; Zenger, Melanie; Perglerová, Karolína; Schnittger, Susanne; Haferlach, Torsten; Haferlach, Claudia

    2016-01-01

    T-cell prolymphocytic leukemia (T-PLL) is a rare post-thymic T-cell neoplasm with aggressive clinical course and short overall survival. So far, due to the rareness of this disease, genetic data are available only from individual cases or small cohorts. In our study, we aimed at performing a comprehensive cytogenetic and molecular genetic characterization of T-PLL comprising the largest cohort of patients with T-PLL analyzed so far, including correlations between the respective markers and their impact on prognosis. Genetic abnormalities were found in all 51 cases with T-PLL, most frequently involving the TCRA/D locus (86%). Deletions were detected for ATM (69%) and TP53 (31%), whereas i(8)(q10) was observed in 61% of cases. Mutations in ATM, TP53, JAK1, and JAK3 were detected in 73, 14, 6, and 21% of patients, respectively. Additionally, BCOR mutations were observed for the first time in a lymphoid malignancy (8%). Two distinct genetic subgroups of T-PLL were identified: A large subset (86% of patients) showed abnormalities involving the TCRA/D locus activating the proto-oncogenes TCL1 or MTCP1, while the second group was characterized by a high frequency of TP53 mutations (4/7 cases). Further, analyses of overall survival identified JAK3 mutations as important prognostic marker, showing a significant negative impact.

  11. Exome sequencing reveals recurrent REV3L mutations in cisplatin-resistant squamous cell carcinoma of head and neck

    PubMed Central

    Huang, Kie Kyon; Jang, Kang Won; Kim, Sangwoo; Kim, Han Sang; Kim, Sung-Moo; Kwon, Hyeong Ju; Kim, Hye Ryun; Yun, Hwan Jung; Ahn, Myung Ju; Park, Keon Uk; Ramnarayanan, Kalpana; McPherson, John R.; Zhang, Shenli; Rhee, Je-Keun; Vettore, André L.; Das, Kakoli; Ishimoto, Takatsugu; Kim, Joo Hang; Koh, Yoon Woo; Kim, Se Hun; Choi, Eun Chang; Teh, Bin Tean; Rozen, Steven G.; Kim, Tae-Min; Tan, Patrick; Cho, Byoung Chul

    2016-01-01

    Dacomitinib, an irreversible pan-HER inhibitor, had shown modest clinical activity in squamous cell carcinoma of head and neck (SCCHN) patients. Therefore, validated predictive biomarkers are required to identify patients most likely to benefit from this therapeutic option. To characterize the genetic landscape of cisplatin-treated SCCHN genomes and identify potential predictive biomarkers for dacomitinib sensitivity, we performed whole exome sequencing on 18 cisplatin-resistant metastatic SCCHN tumors and their matched germline DNA. Platinum-based chemotherapy elevated the mutation rates of SCCHN compared to chemotherapy-naïve SCCHNs. Cisplatin-treated SCCHN genomes uniquely exhibited a novel mutational signature characterized by C:G to A:T transversions at CCR sequence contexts that may have arisen due to error-prone translesional synthesis. Somatic mutations in REV3L, the gene encoding the catalytic subunit of DNA polymerase ζ involved in translesional synthesis, are significantly enriched in a subset of patients who derived extended clinical benefit to dacomitinib (P = 0.04). Functional assays showed that loss-of-function of REV3L dramatically enhanced the sensitivity of SCCHN cells to dacomitinib by the loss of both translesion synthesis and homologous recombination pathways. Our data suggest that the ‘platinum’ mutational signature and inactivation of REV3L may inform treatment options in patients of recurrent SCCHN. PMID:26790612

  12. Reconstruction of microsatellite mutation history reveals a strong and consistent deletion bias in invasive clonal snails, Potamopyrgus antipodarum.

    PubMed

    Weetman, David; Hauser, Lorenz; Carvalho, Gary R

    2002-10-01

    Direct observations of mutations and comparative analyses suggest that nuclear microsatellites show a tendency to expand, with reports of deletion biases limited to very long alleles or a few loci in multilocus studies. Here we investigate microsatellite evolution in clonal snails, Potamopyrgus antipodarum, since their introduction to Britain in the 19th century, using an analysis based on minimum spanning networks of multilocus microsatellite genotypes. British populations consist of a small number of highly distinct genotype groups with very few outlying genotypes, suggesting clonal lineages containing minor variation generated by mutation. Network patterns suggest that a single introduced genotype was the ancestor of all extant variation and also provide support for wholly apomictic reproduction within the most common clonal lineage (group A). Microsatellites within group A showed a strong tendency to delete repeats, with an overall bias exceeding 88%, irrespective of the exact method used to infer mutations. This highly unusual pattern of deletion bias is consistent across populations and loci and is unrelated to allele size. We suggest that for persistence of microsatellites in this clone, some change in the mutation mechanism must have occurred in relatively recent evolutionary time. Possible causes of such a change in mechanism are discussed.

  13. Structural Instability of the Prion Protein upon M205S/R Mutations Revealed by Molecular Dynamics Simulations

    PubMed Central

    Hirschberger, Thomas; Stork, Martina; Schropp, Bernhard; Winklhofer, Konstanze F.; Tatzelt, Jörg; Tavan, Paul

    2006-01-01

    The point mutations M205S and M205R have been demonstrated to severely disturb the folding and maturation process of the cellular prion protein (PrPC). These disturbances have been interpreted as consequences of mutation-induced structural changes in PrP, which are suggested to involve helix 1 and its attachment to helix 3, because the mutated residue M205 of helix 3 is located at the interface of these two helices. Furthermore, current models of the prion protein scrapie (PrPSc), which is the pathogenic isoform of PrPC in prion diseases, imply that helix 1 disappears during refolding of PrPC into PrPSc. Based on molecular-dynamics simulations of wild-type and mutant PrPC in aqueous solution, we show here that the native PrPC structure becomes strongly distorted within a few nanoseconds, once the point mutations M205S and M205R have been applied. In the case of M205R, this distortion is characterized by a motion of helix 1 away from the hydrophobic core into the aqueous environment and a subsequent structural decay. Together with experimental evidence on model peptides, this decay suggests that the hydrophobic attachment of helix 1 to helix 3 at M205 is required for its correct folding into its stable native structure. PMID:16513786

  14. Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize mik.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexaki...

  15. Correlation of phenotype/genotype in a cohort of 23 xeroderma pigmentosum-variant patients reveals 12 new disease-causing POLH mutations.

    PubMed

    Opletalova, Kristina; Bourillon, Agnès; Yang, Wei; Pouvelle, Caroline; Armier, Jacques; Despras, Emmanuelle; Ludovic, Martin; Mateus, Christine; Robert, Caroline; Kannouche, Patricia; Soufir, Nadem; Sarasin, Alain

    2014-01-01

    Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XPV patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV-hypersensitivity in the presence of caffeine, a signature of the XP-V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal-cell carcinoma was 13 per patient, six for squamous-cell carcinoma, and five for melanoma. XP-V is due to defects in the translesion-synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life-cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP-Vs from sun exposure.

  16. Exome sequencing reveals a novel mutation for autosomal recessive non-syndromic mental retardation in the TECR gene on chromosome 19p13.

    PubMed

    Çalışkan, Minal; Chong, Jessica X; Uricchio, Lawrence; Anderson, Rebecca; Chen, Peixian; Sougnez, Carrie; Garimella, Kiran; Gabriel, Stacey B; dePristo, Mark A; Shakir, Khalid; Matern, Dietrich; Das, Soma; Waggoner, Darrel; Nicolae, Dan L; Ober, Carole

    2011-04-01

    Exome sequencing is a powerful tool for discovery of the Mendelian disease genes. Previously, we reported a novel locus for autosomal recessive non-syndromic mental retardation (NSMR) in a consanguineous family [Nolan, D.K., Chen, P., Das, S., Ober, C. and Waggoner, D. (2008) Fine mapping of a locus for nonsyndromic mental retardation on chromosome 19p13. Am. J. Med. Genet. A, 146A, 1414-1422]. Using linkage and homozygosity mapping, we previously localized the gene to chromosome 19p13. The parents of this sibship were recently included in an exome sequencing project. Using a series of filters, we narrowed the putative causal mutation to a single variant site that segregated with NSMR: the mutation was homozygous in five affected siblings but in none of eight unaffected siblings. This mutation causes a substitution of a leucine for a highly conserved proline at amino acid 182 in TECR (trans-2,3-enoyl-CoA reductase), a synaptic glycoprotein. Our results reveal the value of massively parallel sequencing for identification of novel disease genes that could not be found using traditional approaches and identifies only the seventh causal mutation for autosomal recessive NSMR.

  17. Differences in potential for amino acid change after mutation reveals distinct strategies for kappa and lambda light-chain variation.

    PubMed

    Hershberg, Uri; Shlomchik, Mark J

    2006-10-24

    B cells generate varied yet functional clones under high rates of mutation of their V genes. It has been proposed that as a result of the opposing demands of diversification and preservation of integrity, the V genes of heavy and light chains have evolved to overexpress codons prone to amino acid change in their complementarity determining regions (CDR) compared with the framework (FW) regions. We have analyzed the germ-line V genes of heavy and light chains (both kappa and lambda), comparing codons of CDR and FW of the germ-line V regions both to each other and to control regions. We found that in both germ-line heavy chains and lambda chains, CDR codons are prone to replacement mutations, whereas in the FW, the opposite is true. Furthermore, the difference between CDR and FW in heavy chains and lambda chains is based on codons that are prone to nonconservative changes of amino acid. In contrast, in germ-line kappa chains, the codons in both CDR and FW are more prone to replacement mutations. We also demonstrated that negative selection during immune responses is more sensitive to nonconservative amino acid substitutions than overall amino acid change, demonstrating the applicability of our analysis to real-time process of selection in the immune system. The differences in germ-line kappa and lambda light chains' potential reaction to mutation suggests that via these two differently evolved light-chain types, the B cell repertoire encompasses two different strategies to balance diversity and stability in an immune response.

  18. Whole Exome Sequencing of Rapid Autopsy Tumors and Xenograft Models Reveals Possible Driver Mutations Underlying Tumor Progression

    PubMed Central

    Xie, Tao; Musteanu, Monica; Lopez-Casas, Pedro P.; Shields, David J.; Olson, Peter; Rejto, Paul A.; Hidalgo, Manuel

    2015-01-01

    Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal malignancy due to its propensity to invade and rapidly metastasize and remains very difficult to manage clinically. One major hindrance towards a better understanding of PDAC is the lack of molecular data sets and models representative of end stage disease. Moreover, it remains unclear how molecularly similar patient-derived xenograft (PDX) models are to the primary tumor from which they were derived. To identify potential molecular drivers in metastatic pancreatic cancer progression, we obtained matched primary tumor, metastases and normal (peripheral blood) samples under a rapid autopsy program and performed whole exome sequencing (WES) on tumor as well as normal samples. PDX models were also generated, sequenced and compared to tumors. Across the matched data sets generated for three patients, there were on average approximately 160 single-nucleotide mutations in each sample. The majority of mutations in each patient were shared among the primary and metastatic samples and, importantly, were largely retained in the xenograft models. Based on the mutation prevalence in the primary and metastatic sites, we proposed possible clonal evolution patterns marked by functional mutations affecting cancer genes such as KRAS, TP53 and SMAD4 that may play an important role in tumor initiation, progression and metastasis. These results add to our understanding of pancreatic tumor biology, and demonstrate that PDX models derived from advanced or end-stage likely closely approximate the genetics of the disease in the clinic and thus represent a biologically and clinically relevant pre-clinical platform that may enable the development of effective targeted therapies for PDAC. PMID:26555578

  19. Molecular Analysis of Collagen XVIII Reveals Novel Mutations, Presence of a Third Isoform, and Possible Genetic Heterogeneity in Knobloch Syndrome

    PubMed Central

    Suzuki, O. T.; Sertié, A. L.; Der Kaloustian, V. M.; Kok, F.; Carpenter, M.; Murray, J.; Czeizel, A. E.; Kliemann, S. E.; Rosemberg, S.; Monteiro, M.; Olsen, B. R.; Passos-Bueno, M. R.

    2002-01-01

    Knobloch syndrome (KS) is a rare disease characterized by severe ocular alterations, including vitreoretinal degeneration associated with retinal detachment and occipital scalp defect. The responsible gene, COL18A1, has been mapped to 21q22.3, and, on the basis of the analysis of one family, we have demonstrated that a mutation affecting only one of the three COL18A1 isoforms causes this phenotype. We report here the results of the screening of both the entire coding region and the exon-intron boundaries of the COL18A1 gene (which includes 43 exons), in eight unrelated patients with KS. Besides 20 polymorphic changes, we identified 6 different pathogenic changes in both alleles of five unrelated patients with KS (three compound heterozygotes and two homozygotes). All are truncating mutations leading to deficiency of one or all collagen XVIII isoforms and endostatin. We have verified that, in exon 41, the deletion c3514-3515delCT, found in three unrelated alleles, is embedded in different haplotypes, suggesting that this mutation has occurred more than once. In addition, our results provide evidence of nonallelic genetic heterogeneity in KS. We also show that the longest human isoform (NC11-728) is expressed in several tissues (including the human eye) and that lack of either the short variant or all of the collagen XVIII isoforms causes similar phenotypes but that those patients who lack all forms present more-severe ocular alterations. Despite the small sample size, we found low endostatin plasma levels in those patients with mutations leading to deficiency of all isoforms; in addition, it seems that absence of all collagen XVIII isoforms causes predisposition to epilepsy. PMID:12415512

  20. Somatic MED12 Nonsense Mutation Escapes mRNA Decay and Reveals a Motif Required for Nuclear Entry.

    PubMed

    Heikkinen, Tuomas; Kämpjärvi, Kati; Keskitalo, Salla; von Nandelstadh, Pernilla; Liu, Xiaonan; Rantanen, Ville; Pitkänen, Esa; Kinnunen, Matias; Kuusanmäki, Heikki; Kontro, Mika; Turunen, Mikko; Mäkinen, Netta; Taipale, Jussi; Heckman, Caroline; Lehti, Kaisa; Mustjoki, Satu; Varjosalo, Markku; Vahteristo, Pia

    2017-03-01

    MED12 is a key component of the transcription-regulating Mediator complex. Specific missense and in-frame insertion/deletion mutations in exons 1 and 2 have been identified in uterine leiomyomas, breast tumors, and chronic lymphocytic leukemia. Here, we characterize the first MED12 5' end nonsense mutation (c.97G>T, p.E33X) identified in acute lymphoblastic leukemia and show that it escapes nonsense-mediated mRNA decay (NMD) by using an alternative translation initiation site. The resulting N-terminally truncated protein is unable to enter the nucleus due to the lack of identified nuclear localization signal (NLS). The absence of NLS prevents the mutant MED12 protein to be recognized by importin-α and subsequent loading into the nuclear pore complex. Due to this mislocalization, all interactions between the MED12 mutant and other Mediator components are lost. Our findings provide new mechanistic insights into the MED12 functions and indicate that somatic nonsense mutations in early exons may avoid NMD.

  1. Whole Exome Sequencing Reveals Mutations in Known Retinal Disease Genes in 33 out of 68 Israeli Families with Inherited Retinopathies

    PubMed Central

    Beryozkin, Avigail; Shevah, Elia; Kimchi, Adva; Mizrahi-Meissonnier, Liliana; Khateb, Samer; Ratnapriya, Rinki; Lazar, Csilla H.; Blumenfeld, Anat; Ben-Yosef, Tamar; Hemo, Yitzhak; Pe’er, Jacob; Averbuch, Eduard; Sagi, Michal; Boleda, Alexis; Gieser, Linn; Zlotogorski, Abraham; Falik-Zaccai, Tzipora; Alimi-Kasem, Ola; Jacobson, Samuel G.; Chowers, Itay; Swaroop, Anand; Banin, Eyal; Sharon, Dror

    2015-01-01

    Whole exome sequencing (WES) is a powerful technique for identifying sequence changes in the human genome. The goal of this study was to delineate the genetic defects in patients with inherited retinal diseases (IRDs) using WES. WES was performed on 90 patient DNA samples from 68 families and 226 known genes for IRDs were analyzed. Sanger sequencing was used to validate potential pathogenic variants that were also subjected to segregation analysis in families. Thirty-three causative mutations (19 novel and 14 known) in 25 genes were identified in 33 of the 68 families. The vast majority of mutations (30 out of 33) have not been reported in the Israeli and the Palestinian populations. Nine out of the 33 mutations were detected in additional families from the same ethnic population, suggesting a founder effect. In two families, identified phenotypes were different from the previously reported clinical findings associated with the causative gene. This is the largest genetic analysis of IRDs in the Israeli and Palestinian populations to date. We also demonstrate that WES is a powerful tool for rapid analysis of known disease genes in large patient cohorts. PMID:26306921

  2. Targeted deletion of the tub mouse obesity gene reveals that tubby is a loss-of-function mutation.

    PubMed

    Stubdal, H; Lynch, C A; Moriarty, A; Fang, Q; Chickering, T; Deeds, J D; Fairchild-Huntress, V; Charlat, O; Dunmore, J H; Kleyn, P; Huszar, D; Kapeller, R

    2000-02-01

    The mouse tubby phenotype is characterized by maturity-onset obesity accompanied by retinal and cochlear degeneration. A positional cloning effort to find the gene responsible for this phenotype led to the identification of tub, a member of a novel gene family of unknown function. A splice defect mutation in the 3' end of the tub gene, predicted to disrupt the C terminus of the Tub protein, has been implicated in the genesis of the tubby phenotype. It is not clear, however, whether the Tub mutant protein retains any biological activity, or perhaps has some dominant function, nor is it established that the tubby mutation is itself responsible for all of the observed tubby phenotypes. To address these questions, we generated tub-deficient mice and compared their phenotype to that of tubby mice. Our results demonstrate that tubby is a loss-of-function mutation of the tub gene and that loss of the tub gene is sufficient to give rise to the full spectrum of tubby phenotypes. We also demonstrate that loss of photoreceptors in the retina of tubby and tub-deficient mice occurs by apoptosis. In addition, we show that Tub protein expression is not significantly altered in the ob, db, or melanocortin 4 receptor-deficient mouse model of obesity.

  3. Assessing the efficacy of protein farnesyltransferase inhibitors in mouse models of progeria.

    PubMed

    Yang, Shao H; Chang, Sandy Y; Andres, Douglas A; Spielmann, H Peter; Young, Stephen G; Fong, Loren G

    2010-02-01

    Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of a farnesylated form of prelamin A (progerin). Previously, we showed that blocking protein farnesylation with a farnesyltransferase inhibitor (FTI) ameliorates the disease phenotypes in mouse model of HGPS (Lmna(HG/+)). However, the interpretation of the FTI treatment studies is open to question in light of recent studies showing that mice expressing a nonfarnesylated version of progerin (Lmna(nHG/+)) develop progeria-like disease phenotypes. The fact that Lmna(nHG/+) mice manifest disease raised the possibility that the beneficial effects of an FTI in Lmna(HG/+) mice were not due to the effects of the drug on the farnesylation of progerin, but may have been due to unanticipated secondary effects of the drug on other farnesylated proteins. To address this issue, we compared the ability of an FTI to improve progeria-like disease phenotypes in both Lmna(HG/+) and Lmna(nHG/+) mice. In Lmna(HG/+) mice, the FTI reduced disease phenotypes in a highly significant manner, but the drug had no effect in Lmna(nHG/+) mice. The failure of the FTI to ameliorate disease in Lmna(nHG/+) mice supports the idea that the beneficial effects of an FTI in Lmna(HG/+) mice are due to the effect of drug on the farnesylation of progerin.

  4. Treatment considerations in hutchinson-gilford progeria syndrome: a case report.

    PubMed

    Hazan-Molina, H; Aizenbud, D; Dror, Aizenbud D

    2015-01-01

    Hutchinson-Guilford progeria syndrome is an extremely rare condition classified as one of the premature ageing syndromes. This case presents a 16-year-old Israeli female patient, suffering from a variant of Hutchinson-Guilford progeria with a history of treatment with oral biphosphnates. The patient presented with typical cranial and facial features of the syndrome including delayed teeth eruption and root development probably due to insufficient jaw growth and severs retrognatic position of the maxilla and mandible. Orthodontic treatment considerations are described along with those required in light of the previous treatment by oral biphosphonates.All primary teeth were extracted in three appointments while creating as minimal trauma as possible to the surrounding tissue and alveolar bone. For now, the patient refuses to begin the orthodontic treatment course. There are no limitations to conduct any dental procedures in progeria patients, however, extreme caution must be exercised during oral surgery due to the inelasticity of tissues and dermal atrophy. Orthodontic procedure commencement should be early enough to manage the delayed development and eruption of teeth. Patients taking oral biphosphonates should be advised of this potential complication. If orthodontic treatment is considered appropriate, plans should be assessed and modified to include compromises.

  5. Biomechanical Strain Exacerbates Inflammation on a Progeria-on-a-Chip Model.

    PubMed

    Ribas, João; Zhang, Yu Shrike; Pitrez, Patrícia R; Leijten, Jeroen; Miscuglio, Mario; Rouwkema, Jeroen; Dokmeci, Mehmet Remzi; Nissan, Xavier; Ferreira, Lino; Khademhosseini, Ali

    2017-02-17

    Organ-on-a-chip platforms seek to recapitulate the complex microenvironment of human organs using miniaturized microfluidic devices. Besides modeling healthy organs, these devices have been used to model diseases, yielding new insights into pathophysiology. Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease showing accelerated vascular aging, leading to the death of patients due to cardiovascular diseases. HGPS targets primarily vascular cells, which reside in mechanically active tissues. Here, a progeria-on-a-chip model is developed and the effects of biomechanical strain are examined in the context of vascular aging and disease. Physiological strain induces a contractile phenotype in primary smooth muscle cells (SMCs), while a pathological strain induces a hypertensive phenotype similar to that of angiotensin II treatment. Interestingly, SMCs derived from human induced pluripotent stem cells of HGPS donors (HGPS iPS-SMCs), but not from healthy donors, show an exacerbated inflammatory response to strain. In particular, increased levels of inflammation markers as well as DNA damage are observed. Pharmacological intervention reverses the strain-induced damage by shifting gene expression profile away from inflammation. The progeria-on-a-chip is a relevant platform to study biomechanics in vascular biology, particularly in the setting of vascular disease and aging, while simultaneously facilitating the discovery of new drugs and/or therapeutic targets.

  6. Chemical screening identifies ROCK as a target for recovering mitochondrial function in Hutchinson-Gilford progeria syndrome.

    PubMed

    Kang, Hyun Tae; Park, Joon Tae; Choi, Kobong; Choi, Hyo Jei Claudia; Jung, Chul Won; Kim, Gyu Ree; Lee, Young-Sam; Park, Sang Chul

    2017-03-19

    Hutchinson-Gilford progeria syndrome (HGPS) constitutes a genetic disease wherein an aging phenotype manifests in childhood. Recent studies indicate that reactive oxygen species (ROS) play important roles in HGPS phenotype progression. Thus, pharmacological reduction in ROS levels has been proposed as a potentially effective treatment for patient with this disorder. In this study, we performed high-throughput screening to find compounds that could reduce ROS levels in HGPS fibroblasts and identified rho-associated protein kinase (ROCK) inhibitor (Y-27632) as an effective agent. To elucidate the underlying mechanism of ROCK in regulating ROS levels, we performed a yeast two-hybrid screen and discovered that ROCK1 interacts with Rac1b. ROCK activation phosphorylated Rac1b at Ser71 and increased ROS levels by facilitating the interaction between Rac1b and cytochrome c. Conversely, ROCK inactivation with Y-27632 abolished their interaction, concomitant with ROS reduction. Additionally, ROCK activation resulted in mitochondrial dysfunction, whereas ROCK inactivation with Y-27632 induced the recovery of mitochondrial function. Furthermore, a reduction in the frequency of abnormal nuclear morphology and DNA double-strand breaks was observed along with decreased ROS levels. Thus, our study reveals a novel mechanism through which alleviation of the HGPS phenotype is mediated by the recovery of mitochondrial function upon ROCK inactivation.

  7. Lifespan extension by dietary intervention in a mouse model of Cockayne syndrome uncouples early postnatal development from segmental progeria.

    PubMed

    Brace, Lear E; Vose, Sarah C; Vargas, Dorathy F; Zhao, Shuangyun; Wang, Xiu-Ping; Mitchell, James R

    2013-12-01

    Cockayne syndrome (CS) is a rare autosomal recessive segmental progeria characterized by growth failure, lipodystrophy, neurological abnormalities, and photosensitivity, but without skin cancer predisposition. Cockayne syndrome life expectancy ranges from 5 to 16 years for the two most severe forms (types II and I, respectively). Mouse models of CS have thus far been of limited value due to either very mild phenotypes, or premature death during postnatal development prior to weaning. The cause of death in severe CS models is unknown, but has been attributed to extremely rapid aging. Here, we found that providing mutant pups with soft food from as late as postnatal day 14 allowed survival past weaning with high penetrance independent of dietary macronutrient balance in a novel CS model (Csa(-/-) | Xpa(-/-)). Survival past weaning revealed a number of CS-like symptoms including small size, progressive loss of adiposity, and neurological symptoms, with a maximum lifespan of 19 weeks. Our results caution against interpretation of death before weaning as premature aging, and at the same time provide a valuable new tool for understanding mechanisms of progressive CS-related progeroid symptoms including lipodystrophy and neurodysfunction.

  8. Cardiac electrical defects in progeroid mice and Hutchinson-Gilford progeria syndrome patients with nuclear lamina alterations.

    PubMed

    Rivera-Torres, José; Calvo, Conrado J; Llach, Anna; Guzmán-Martínez, Gabriela; Caballero, Ricardo; González-Gómez, Cristina; Jiménez-Borreguero, Luis J; Guadix, Juan A; Osorio, Fernando G; López-Otín, Carlos; Herraiz-Martínez, Adela; Cabello, Nuria; Vallmitjana, Alex; Benítez, Raul; Gordon, Leslie B; Jalife, José; Pérez-Pomares, José M; Tamargo, Juan; Delpón, Eva; Hove-Madsen, Leif; Filgueiras-Rama, David; Andrés, Vicente

    2016-11-15

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24(-/-) mouse model of HGPS. Challenge of Zmpste24(-/-) mice with the β-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24(-/-) cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24(-/-) progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death.

  9. Cardiac electrical defects in progeroid mice and Hutchinson–Gilford progeria syndrome patients with nuclear lamina alterations

    PubMed Central

    Rivera-Torres, José; Calvo, Conrado J.; Llach, Anna; Guzmán-Martínez, Gabriela; Caballero, Ricardo; González-Gómez, Cristina; Jiménez-Borreguero, Luis J.; Guadix, Juan A.; Osorio, Fernando G.; López-Otín, Carlos; Herraiz-Martínez, Adela; Cabello, Nuria; Vallmitjana, Alex; Benítez, Raul; Gordon, Leslie B.; Pérez-Pomares, José M.; Tamargo, Juan; Delpón, Eva; Hove-Madsen, Leif; Filgueiras-Rama, David; Andrés, Vicente

    2016-01-01

    Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24−/− mouse model of HGPS. Challenge of Zmpste24−/− mice with the β-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24−/− cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24−/− progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death. PMID:27799555

  10. Comprehensive Mutational Analysis Reveals p6Gag Phosphorylation To Be Dispensable for HIV-1 Morphogenesis and Replication

    PubMed Central

    Radestock, Benjamin; Morales, Ivonne; Rahman, Sheikh Abdul; Radau, Sonja; Glass, Bärbel; Zahedi, René Peiman; Müller, Barbara

    2013-01-01

    The structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture. PMID:23115284

  11. Comprehensive mutational analysis reveals p6Gag phosphorylation to be dispensable for HIV-1 morphogenesis and replication.

    PubMed

    Radestock, Benjamin; Morales, Ivonne; Rahman, Sheikh Abdul; Radau, Sonja; Glass, Bärbel; Zahedi, René Peiman; Müller, Barbara; Kräusslich, Hans-Georg

    2013-01-01

    The structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture.

  12. Structural modeling and patch-clamp analysis of pain-related mutation TRPA1-N855S reveal inter-subunit salt bridges stabilizing the channel open state.

    PubMed

    Zíma, Vlastimil; Witschas, Katja; Hynkova, Anna; Zímová, Lucie; Barvík, Ivan; Vlachova, Viktorie

    2015-06-01

    The ankyrin transient receptor potential channel TRPA1 is a polymodal sensor for noxious stimuli, and hence a promising target for treating chronic pain. This tetrameric six-transmembrane segment (S1-S6) channel can be activated by various pungent chemicals, such as allyl isothiocyanate or cinnamaldehyde, but also by intracellular Ca(2+) or depolarizing voltages. Within the S4-S5 linker of human TRPA1, a gain-of-function mutation, N855S, was recently found to underlie familial episodic pain syndrome, manifested by bouts of severe upper body pain, triggered by physical stress, fasting, or cold. To clarify the structural basis for this channelopathy, we derive a structural model of TRPA1 by combining homology modeling, molecular dynamics simulations, point mutagenesis and electrophysiology. In the vicinity of N855, the model reveals inter-subunit salt bridges between E854 and K868. Using the heterologous expression of recombinant wild-type and mutant TRPA1 channels in HEK293T cells, we indeed found that the charge-reversal mutants E854R and K868E exhibited dramatically reduced responses to chemical and voltage stimuli, whereas the charge-swapping mutation E854R/K868E substantially rescued their functionalities. Moreover, mutation analysis of highly conserved charged residues within the S4-S5 region revealed a gain-of-function phenotype for R852E with an increased basal channel activity, a loss of Ca(2+)-induced potentiation and an accelerated Ca(2+)-dependent inactivation. Based on the model and on a comparison with the recently revealed atomic-level structure of the related channel TRPV1, we propose that inter-subunit salt bridges between adjacent S4-S5 regions are crucial for stabilizing the conformations associated with chemically and voltage-induced gating of the TRPA1 ion channel.

  13. Inbreeding reveals stronger net selection on Drosophila melanogaster males: implications for mutation load and the fitness of sexual females.

    PubMed

    Mallet, M A; Chippindale, A K

    2011-06-01

    Stronger selection on males has the potential to lower the deleterious mutation load of females, reducing the cost of sex. However, few studies have directly quantified the strength of selection for both sexes. As the magnitude of inbreeding depression (ID) is related to the strength of selection, we measured the cost of inbreeding for both males and females in a laboratory population of Drosophila melanogaster. Using a novel technique for inbreeding, we found significant ID for both juvenile viability and adult fitness in both sexes. The genetic variation responsible for this depression in fitness appeared to be recessive for adult fitness (h=0.11) and partially additive for juvenile viability (h=0.29). ID was identical across the sexes in terms of juvenile viability but was significantly more deleterious for males than females as adults, even though female X-chromosome homogamety should predispose them to a higher inbreeding load. We estimated the strength of selection on adult males to be 1.24 greater than on adult females, and this appears to be a consequence of selection arising from competition for mates. Combined with the generally positive intersexual genetic correlation for inbred lines, our results suggest that the mutation load of sexual females could be meaningfully reduced by stronger selection acting on males.

  14. Mechanism of Drug Resistance Revealed by the Crystal Structure of the Unliganded HIV-1 Protease with F53L Mutation

    SciTech Connect

    Liu, Fengling; Kovalevsky, Andrey Y.; Louis, John M.; Boross, Peter I.; Wang, Yuan-Fang; Harrison, Robert W.; Weber, Irene T.

    2010-12-03

    Mutations in HIV-1 protease (PR) that produce resistance to antiviral PR inhibitors are a major problem in AIDS therapy. The mutation F53L arising from antiretroviral therapy was introduced into the flexible flap region of the wild-type PR to study its effect and potential role in developing drug resistance. Compared to wild-type PR, PR{sub F53L} showed lower (15%) catalytic efficiency, 20-fold weaker inhibition by the clinical drug indinavir, and reduced dimer stability, while the inhibition constants of two peptide analog inhibitors were slightly lower than those for PR. The crystal structure of PR{sub F53L} was determined in the unliganded form at 1.35 {angstrom} resolution in space group P4{sub 1}2{sub 1}2. The tips of the flaps in PR{sub F53L} had a wider separation than in unliganded wild-type PR, probably due to the absence of hydrophobic interactions of the side-chains of Phe53 and Ile50{prime}. The changes in interactions between the flaps agreed with the reduced stability of PR{sub F53L} relative to wild-type PR. The altered flap interactions in the unliganded form of PR{sub F53L} suggest a distinct mechanism for drug resistance, which has not been observed in other common drug-resistant mutants.

  15. NMR structure of the human prion protein with the pathological Q212P mutation reveals unique structural features.

    PubMed

    Ilc, Gregor; Giachin, Gabriele; Jaremko, Mariusz; Jaremko, Łukasz; Benetti, Federico; Plavec, Janez; Zhukov, Igor; Legname, Giuseppe

    2010-07-22

    Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrP(C)) conformer, denoted as infectious scrapie isoform or PrP(Sc). In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrP(Sc) in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90-124) and a globular domain (residues 125-231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the beta(2)-alpha(2) loop region. This structure might provide new insights into the early events of conformational transition of PrP(C) into PrP(Sc). Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of beta(2)-alpha(2) loop and alpha(3) helix.

  16. Kinetic network models of tryptophan mutations in β-hairpins reveal the importance of non-native interactions.

    PubMed

    Razavi, Asghar M; Voelz, Vincent A

    2015-06-09

    We present an analysis of the most extensive explicit-solvent simulations of β-hairpins to date (9.4 ms in aggregate), with the aim of probing the effects of tryptophan mutations on folding. From molecular simulations of GB1 hairpin, trpzip4, trpzip5, and trpzip6 performed on Folding@home, Markov State Models (MSMs) were constructed using a unified set of metastable states, enabling objective comparison of folding mechanisms. MSM models display quantitative agreement with experimental structural observables and folding kinetics, and predict multimodal kinetics due to specific non-native kinetic traps, which be identified as on- or off-pathway from the network topology. We quantify kinetic frustration by several means, including the s-ensemble method to evaluate glasslike behavior. Free-energy profiles and transition state movement clearly show stabilization of non-native states as Trp mutations are introduced. Remarkably, we find that "β-capped" sequences (trpzip4 and trpzip5) are able to overcome this frustration and remain cooperative two-state folders with a large time-scale gap. These results suggest that, while β-capping motifs are robust, fold stabilization by tryptophan generally may require overcoming significant non-native kinetic traps, perhaps explaining their under-representation in natural proteins.

  17. Whole Exome Sequencing Reveals a BSCL2 Mutation Causing Progressive Encephalopathy with Lipodystrophy (PELD) in an Iranian Pediatric Patient

    PubMed Central

    Alaei, Mohammad Reza; Talebi, Saeed; Ghofrani, Mohammad; Taghizadeh, Mohsen; Keramatipour, Mohammad

    2016-01-01

    Background: Progressive encephalopathy with or without lipodystrophy is a rare autosomal recessive childhood-onset seipin-associated neurodegenerative syndrome, leading to developmental regression of motor and cognitive skills. In this study, we introduce a patient with developmental regression and autism. The causative mutation was found by exome sequencing. Methods: The proband showed a generalized hypertonia and regression of all developmental milestones. Based on the advantages of next-generation sequencing (NGS), whole exome sequencing (WES) was requested. The functional significance of variants was evaluated by NGS-specific prediction servers. Sanger sequencing was used for segregation analysis in the family. Results: There was no specific sign in the clinical and paraclinical investigations of the patient to establish a conclusive clinical diagnosis. WES detected a known homozygous nonsense mutation in BSCL2 (NM_001122955.3:c. 985C>T; p.Arg329*). The variant is segregating in the pedigree with an autosomal recessive pattern. Conclusion: Exome sequencing is a robust method for identifying the candidate gene variants in Mendelian traits. PMID:27452399

  18. Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator).

    PubMed

    Ramjeesingh, M; Li, C; Garami, E; Huan, L J; Galley, K; Wang, Y; Bear, C E

    1999-02-02

    The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATPase and as a chloride channel. It has been hypothesized, on the basis of electrophysiological findings, that the catalytic activity of CFTR is tightly coupled to the opening and closing of the channel gate. In the present study, to determine the structural basis for the ATPase activity of CFTR, we assessed the effect of mutations within the "Walker A" consensus motifs on ATP hydrolysis by the purified, intact protein. Mutation of the lysine residue in the "Walker A" motif of either the first nucleotide binding fold (CFTRK464A) or the second nucleotide binding fold (CFTRK1250A) inhibited the ATPase activity of the purified intact CFTR protein significantly, by greater than 50%. This finding suggests that the two nucleotide binding folds of CFTR are functioning cooperatively in catalysis. However, the rate of channel gating was only significantly inhibited in one of these purified mutants, CFTRK1250A, suggesting that ATPase activity may not be tightly coupled to channel gating as previously hypothesized.

  19. New Lmna knock-in mice provide a molecular mechanism for the 'segmental aging' in Hutchinson-Gilford progeria syndrome.

    PubMed

    Jung, Hea-Jin; Tu, Yiping; Yang, Shao H; Tatar, Angelica; Nobumori, Chika; Wu, Daniel; Young, Stephen G; Fong, Loren G

    2014-03-15

    Lamins A and C (products of the LMNA gene) are found in roughly equal amounts in peripheral tissues, but the brain produces mainly lamin C and little lamin A. In HeLa cells and fibroblasts, the expression of prelamin A (the precursor to lamin A) can be reduced by miR-9, but the relevance of those cell culture studies to lamin A regulation in the brain was unclear. To address this issue, we created two new Lmna knock-in alleles, one (Lmna(PLAO-5NT)) with a 5-bp mutation in a predicted miR-9 binding site in prelamin A's 3' UTR, and a second (Lmna(PLAO-UTR)) in which prelamin A's 3' UTR was replaced with lamin C's 3' UTR. Neither allele had significant effects on lamin A levels in peripheral tissues; however, both substantially increased prelamin A transcript levels and lamin A protein levels in the cerebral cortex and the cerebellum. The increase in lamin A expression in the brain was more pronounced with the Lmna(PLAO-UTR) allele than with the Lmna(PLAO-5NT) allele. With both alleles, the increased expression of prelamin A transcripts and lamin A protein was greater in the cerebral cortex than in the cerebellum. Our studies demonstrate the in vivo importance of prelamin A's 3' UTR and its miR-9 binding site in regulating lamin A expression in the brain. The reduced expression of prelamin A in the brain likely explains why children with Hutchinson-Gilford progeria syndrome (a progeroid syndrome caused by a mutant form of prelamin A) are spared from neurodegenerative disease.

  20. Mutations in CSTA, encoding Cystatin A, underlie exfoliative ichthyosis and reveal a role for this protease inhibitor in cell-cell adhesion.

    PubMed

    Blaydon, Diana C; Nitoiu, Daniela; Eckl, Katja-Martina; Cabral, Rita M; Bland, Philip; Hausser, Ingrid; van Heel, David A; Rajpopat, Shefali; Fischer, Judith; Oji, Vinzenz; Zvulunov, Alex; Traupe, Heiko; Hennies, Hans Christian; Kelsell, David P

    2011-10-07

    Autosomal-recessive exfoliative ichthyosis presents shortly after birth as dry, scaly skin over most of the body with coarse peeling of nonerythematous skin on the palms and soles, which is exacerbated by excessive moisture and minor trauma. Using whole-genome homozygosity mapping, candidate-gene analysis and deep sequencing, we have identified loss-of-function mutations in the gene for protease inhibitor cystatin A (CSTA) as the underlying genetic cause of exfoliative ichthyosis. We found two homozygous mutations, a splice-site and a nonsense mutation, in two consanguineous families of Bedouin and Turkish origin. Electron microscopy of skin biopsies from affected individuals revealed that the level of detachment occurs in the basal and lower suprabasal layers. In addition, in vitro modeling suggests that in the absence of cystatin A protein, there is a cell-cell adhesion defect in human keratinocytes that is particularly prominent when cells are subject to mechanical stress. We show here evidence of a key role for a protease inhibitor in epidermal adhesion within the lower layers of the human epidermis.

  1. Epidermolysis Bullosa Simplex-Type Mutations Alter the Dynamics of the Keratin Cytoskeleton and Reveal a Contribution of Actin to the Transport of Keratin SubunitsD⃞V⃞

    PubMed Central

    Werner, Nicola Susann; Windoffer, Reinhard; Strnad, Pavel; Grund, Christine; Leube, Rudolf Eberhard; Magin, Thomas Michael

    2004-01-01

    Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs. PMID:14668478

  2. A BAFF-R mutation associated with non-Hodgkin lymphoma alters TRAF recruitment and reveals new insights into BAFF-R signaling.

    PubMed

    Hildebrand, Joanne M; Luo, Zhenghua; Manske, Michelle K; Price-Troska, Tammy; Ziesmer, Steven C; Lin, Wai; Hostager, Bruce S; Slager, Susan L; Witzig, Thomas E; Ansell, Stephen M; Cerhan, James R; Bishop, Gail A; Novak, Anne J

    2010-11-22

    The cytokine B cell activating factor (BAFF) and its receptor, BAFF receptor (BAFF-R), modulate signaling cascades critical for B cell development and survival. We identified a novel mutation in TNFRSF13C, the gene encoding human BAFF-R, that is present in both tumor and germline tissue from a subset of patients with non-Hodgkin lymphoma. This mutation encodes a His159Tyr substitution in the cytoplasmic tail of BAFF-R adjacent to the TRAF3 binding motif. Signaling through this mutant BAFF-R results in increased NF-κB1 and NF-κB2 activity and increased immunoglobulin production compared with the wild-type (WT) BAFF-R. This correlates with increased TRAF2, TRAF3, and TRAF6 recruitment to His159Tyr BAFF-R. In addition, we document a requirement for TRAF6 in WT BAFF-R signaling. Together, these data identify a novel lymphoma-associated mutation in human BAFF-R that results in NF-κB activation and reveals TRAF6 as a necessary component of normal BAFF-R signaling.

  3. Correlated alterations in genome organization, histone methylation, and DNA-lamin A/C interactions in Hutchinson-Gilford progeria syndrome.

    PubMed

    McCord, Rachel Patton; Nazario-Toole, Ashley; Zhang, Haoyue; Chines, Peter S; Zhan, Ye; Erdos, Michael R; Collins, Francis S; Dekker, Job; Cao, Kan

    2013-02-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease that is frequently caused by a de novo point mutation at position 1824 in LMNA. This mutation activates a cryptic splice donor site in exon 11, and leads to an in-frame deletion within the prelamin A mRNA and the production of a dominant-negative lamin A protein, known as progerin. Here we show that primary HGPS skin fibroblasts experience genome-wide correlated alterations in patterns of H3K27me3 deposition, DNA-lamin A/C associations, and, at late passages, genome-wide loss of spatial compartmentalization of active and inactive chromatin domains. We further demonstrate that the H3K27me3 changes associate with gene expression alterations in HGPS cells. Our results support a model that the accumulation of progerin in the nuclear lamina leads to altered H3K27me3 marks in heterochromatin, possibly through the down-regulation of EZH2, and disrupts heterochromatin-lamina interactions. These changes may result in transcriptional misregulation and eventually trigger the global loss of spatial chromatin compartmentalization in late passage HGPS fibroblasts.

  4. Proliferation of progeria cells is enhanced by lamina-associated polypeptide 2α (LAP2α) through expression of extracellular matrix proteins.

    PubMed

    Vidak, Sandra; Kubben, Nard; Dechat, Thomas; Foisner, Roland

    2015-10-01

    Lamina-associated polypeptide 2α (LAP2α) localizes throughout the nucleoplasm and interacts with the fraction of lamins A/C that is not associated with the peripheral nuclear lamina. The LAP2α-lamin A/C complex negatively affects cell proliferation. Lamins A/C are encoded by LMNA, a single heterozygous mutation of which causes Hutchinson-Gilford progeria syndrome (HGPS). This mutation generates the lamin A variant progerin, which we show here leads to loss of LAP2α and nucleoplasmic lamins A/C, impaired proliferation, and down-regulation of extracellular matrix components. Surprisingly, contrary to wild-type cells, ectopic expression of LAP2α in cells expressing progerin restores proliferation and extracellular matrix expression but not the levels of nucleoplasmic lamins A/C. We conclude that, in addition to its cell cycle-inhibiting function with lamins A/C, LAP2α can also regulate extracellular matrix components independently of lamins A/C, which may help explain the proliferation-promoting function of LAP2α in cells expressing progerin.

  5. Molecular analysis of the beta-globin gene cluster in the Niokholo Mandenka population reveals a recent origin of the beta(S) Senegal mutation.

    PubMed

    Currat, Mathias; Trabuchet, Guy; Rees, David; Perrin, Pascale; Harding, Rosalind M; Clegg, John B; Langaney, André; Excoffier, Laurent

    2002-01-01

    A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.

  6. Murine viable motheaten mutation reveals a gene critical to the development of both B and T lymphocytes

    SciTech Connect

    Sidman, C.L.; Marshall, J.D.; Allen, R.D. )

    1989-08-01

    In lethally irradiated normal mice reconstituted with both normal and autoimmune mutant viable moth-eaten (me{sup v}) bone marrow, the me{sup v}-derived B and T cells display aberrant behavior, while those derived from the normal bone marrow develop and function normally. The observed developmental abnormalities of me{sup v} B and T lymphocytes are therefore intrinsic to these cell types, rather than being determined by defective influences from the cells' environment. These data bring into question the in vivo significance of reported intracellular regulatory defects in motheaten (me) and me{sup v} mice and suggest that these mutations affect a gene whose product acts cell autonomously in the development of several hematopoietic cell lineages including B and T lymphocytes.

  7. The allergy gene: how a mutation in a skin protein revealed a link between eczema and asthma

    PubMed Central

    2011-01-01

    Ichthyosis vulgaris is a common genetic skin disorder characterized by dry, scaly skin. About 1% of the European population have the full presentation of ichthyosis vulgaris; up to 10% have a milder, subclinical form. Atopic eczema is the most common, inflammatory skin condition, affecting 20% of children. It is often accompanied by a number of other allergies, including atopic asthma. Atopic eczema is a complex trait, where predisposing genes in combination with environmental stimuli produce the disease. Recently, we reported the first loss-of-function genetic mutations in the filaggrin gene as the cause of ichthyosis vulgaris. We noted people with ichthyosis vulgaris also have atopic eczema (and vice versa) and that the filaggrin gene sits in a known atopic eczema susceptibility locus. We went on to confirm that filaggrin mutations, carried by up to 10% of the population, are the major genetic predisposing factor for atopic eczema and the various allergies associated with atopic eczema. Filaggrin is a highly abundant protein expressed in the uppermost part of the epidermis that is critical to the formation and hydration of the stratum corneum—the outermost dead cell layers responsible for the barrier function of the skin. Filaggrin deficiency leads to a “leaky” skin barrier that allows higher than normal water loss (explaining the dry, scaly skin), as well as allowing entry of allergens through the epidermis where they trigger inflammatory and allergic immune responses (atopic eczema and allergies). This work has placed the skin barrier at the center stage of eczema and allergy research and has kick-started new therapy development programs aimed at repairing or enhancing skin-barrier function as a means of treating or preventing these very common diseases. PMID:21399759

  8. Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends

    PubMed Central

    Chan, K. C. Allen; Jiang, Peiyong; Sun, Kun; Cheng, Yvonne K. Y.; Tong, Yu K.; Cheng, Suk Hang; Wong, Ada I. C.; Hudecova, Irena; Leung, Tak Y.; Chiu, Rossa W. K.; Lo, Yuk Ming Dennis

    2016-01-01

    Plasma DNA obtained from a pregnant woman was sequenced to a depth of 270× haploid genome coverage. Comparing the maternal plasma DNA sequencing data with the parental genomic DNA data and using a series of bioinformatics filters, fetal de novo mutations were detected at a sensitivity of 85% and a positive predictive value of 74%. These results represent a 169-fold improvement in the positive predictive value over previous attempts. Improvements in the interpretation of the sequence information of every base position in the genome allowed us to interrogate the maternal inheritance of the fetus for 618,271 of 656,676 (94.2%) heterozygous SNPs within the maternal genome. The fetal genotype at each of these sites was deduced individually, unlike previously, where the inheritance was determined for a collection of sites within a haplotype. These results represent a 90-fold enhancement in the resolution in determining the fetus’s maternal inheritance. Selected genomic locations were more likely to be found at the ends of plasma DNA molecules. We found that a subset of such preferred ends exhibited selectivity for fetal- or maternal-derived DNA in maternal plasma. The ratio of the number of maternal plasma DNA molecules with fetal preferred ends to those with maternal preferred ends showed a correlation with the fetal DNA fraction. Finally, this second generation approach for noninvasive fetal whole-genome analysis was validated in a pregnancy diagnosed with cardiofaciocutaneous syndrome with maternal plasma DNA sequenced to 195× coverage. The causative de novo BRAF mutation was successfully detected through the maternal plasma DNA analysis. PMID:27799561

  9. Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends.

    PubMed

    Chan, K C Allen; Jiang, Peiyong; Sun, Kun; Cheng, Yvonne K Y; Tong, Yu K; Cheng, Suk Hang; Wong, Ada I C; Hudecova, Irena; Leung, Tak Y; Chiu, Rossa W K; Lo, Yuk Ming Dennis

    2016-12-13

    Plasma DNA obtained from a pregnant woman was sequenced to a depth of 270× haploid genome coverage. Comparing the maternal plasma DNA sequencing data with the parental genomic DNA data and using a series of bioinformatics filters, fetal de novo mutations were detected at a sensitivity of 85% and a positive predictive value of 74%. These results represent a 169-fold improvement in the positive predictive value over previous attempts. Improvements in the interpretation of the sequence information of every base position in the genome allowed us to interrogate the maternal inheritance of the fetus for 618,271 of 656,676 (94.2%) heterozygous SNPs within the maternal genome. The fetal genotype at each of these sites was deduced individually, unlike previously, where the inheritance was determined for a collection of sites within a haplotype. These results represent a 90-fold enhancement in the resolution in determining the fetus's maternal inheritance. Selected genomic locations were more likely to be found at the ends of plasma DNA molecules. We found that a subset of such preferred ends exhibited selectivity for fetal- or maternal-derived DNA in maternal plasma. The ratio of the number of maternal plasma DNA molecules with fetal preferred ends to those with maternal preferred ends showed a correlation with the fetal DNA fraction. Finally, this second generation approach for noninvasive fetal whole-genome analysis was validated in a pregnancy diagnosed with cardiofaciocutaneous syndrome with maternal plasma DNA sequenced to 195× coverage. The causative de novo BRAF mutation was successfully detected through the maternal plasma DNA analysis.

  10. A single point mutation reveals gating of the human ClC-5 Cl-/H+ antiporter.

    PubMed

    De Stefano, Silvia; Pusch, Michael; Zifarelli, Giovanni

    2013-12-01

    ClC-5 is a 2Cl(-)/1H(+) antiporter highly expressed in endosomes of proximal tubule cells. It is essential for endocytosis and mutations in ClC-5 cause Dent's disease, potentially leading to renal failure. However, the physiological role of ClC-5 is still unclear. One of the main issues is whether the strong rectification of ClC-5 currents observed in heterologous systems, with currents elicited only at positive voltages, is preserved in vivo and what is the origin of this rectification. In this work we identified a ClC-5 mutation, D76H, which, besides the typical outward currents of the wild-type (WT), shows inward tail currents at negative potentials that allow the estimation of the reversal of ClC-5 currents for the first time. A detailed analysis of the dependence of these inward tail currents on internal and external pH and [Cl(-)] shows that they are generated by a coupled transport of Cl(-) and H(+) with a 2 : 1 stoichiometry. From this result we conclude that the inward tail currents are caused by a gating mechanism that regulates ClC-5 transport activity and not by a major alteration of the transport mechanism itself. This implies that the strong rectification of the currents of WT ClC-5 is at least in part caused by a gating mechanism that activates the transporter at positive potentials. These results elucidate the biophysical properties of ClC-5 and contribute to the understanding of its physiological role.

  11. Whole-exome sequencing in a single proband reveals a mutation in the CHST8 gene in autosomal recessive peeling skin syndrome.

    PubMed

    Cabral, Rita M; Kurban, Mazen; Wajid, Muhammad; Shimomura, Yutaka; Petukhova, Lynn; Christiano, Angela M

    2012-04-01

    Generalized peeling skin syndrome (PSS) is an autosomal recessive genodermatosis characterized by lifelong, continuous shedding of the upper epidermis. Using whole-genome homozygozity mapping and whole-exome sequencing, we identified a novel homozygous missense mutation (c.229C>T, R77W) within the CHST8 gene, in a large consanguineous family with non-inflammatory PSS type A. CHST8 encodes a Golgi transmembrane N-acetylgalactosamine-4-O-sulfotransferase (GalNAc4-ST1), which we show by immunofluorescence staining to be expressed throughout normal epidermis. A colorimetric assay for total sulfated glycosaminoglycan (GAG) quantification, comparing human keratinocytes (CCD1106 KERTr) expressing wild type and mutant recombinant GalNAc4-ST1, revealed decreased levels of total sulfated GAGs in cells expressing mutant GalNAc4-ST1, suggesting loss of function. Western blotting revealed lower expression levels of mutant recombinant GalNAc4-ST1 compared to wild type, suggesting that accelerated degradation may result in loss of function, leading to PSS type A. This is the first report describing a mutation as the cause of PSS type A.

  12. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    SciTech Connect

    Carmen Herranz, Ma; Mingarro, Ismael; Pallas, Vicente . E-mail: vpallas@ibmcp.upv.es

    2005-08-15

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed.

  13. Simultaneous shoulder and hip dislocation in a 12-year-old girl with Hutchinson-Gilford progeria syndrome.

    PubMed

    Espandar, Ramin; Eraghi, Amir Sobhani; Mardookhpour, Shirin

    2012-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature ageing disorder that is characterized by accelerated degenerative changes of the cutaneous, musculoskeletal and cardiovascular systems. Mean age at diagnosis is 2.9 years and generally leading to death at approximately 13 years of age due to myocardial infarction or stroke. Orthopedic manifestations of HGPS are multiple and shoulder dislocation is a rare skeletal trauma in progeria syndrome. Our patient had simultaneous shoulder and hip dislocation associated with a low energy trauma. This subject has not been reported. Treatment accomplished as close reduction under general anesthesia and immobilization.

  14. Discordant gene expression signatures and related phenotypic differences in lamin A- and A/C-related Hutchinson-Gilford progeria syndrome (HGPS).

    PubMed

    Plasilova, Martina; Chattopadhyay, Chandon; Ghosh, Apurba; Wenzel, Friedel; Demougin, Philippe; Noppen, Christoph; Schaub, Nathalie; Szinnai, Gabor; Terracciano, Luigi; Heinimann, Karl

    2011-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disorder displaying features reminiscent of premature senescence caused by germline mutations in the LMNA gene encoding lamin A and C, essential components of the nuclear lamina. By studying a family with homozygous LMNA mutation (K542N), we showed that HGPS can also be caused by mutations affecting both isoforms, lamin A and C. Here, we aimed to elucidate the molecular mechanisms underlying the pathogenesis in both, lamin A- (sporadic) and lamin A and C-related (hereditary) HGPS. For this, we performed detailed molecular studies on primary fibroblasts of hetero- and homozygous LMNA K542N mutation carriers, accompanied with clinical examinations related to the molecular findings. By assessing global gene expression we found substantial overlap in altered transcription profiles (13.7%; 90/657) in sporadic and hereditary HGPS, with 83.3% (75/90) concordant and 16.7% (15/90) discordant transcriptional changes. Among the concordant ones we observed down-regulation of TWIST2, whose inactivation in mice and humans leads to loss of subcutaneous fat and dermal appendages, and loss of expression in dermal fibroblasts and periadnexial cells from a LMNA(K542N/K542N) patient further confirming its pivotal role in skin development. Among the discordant transcriptional profiles we identified two key mediators of vascular calcification and bone metabolism, ENPP1 and OPG, which offer a molecular explanation for the major phenotypic differences in vascular and bone disease in sporadic and hereditary HGPS. Finally, this study correlates reduced TWIST2 and OPG expression with increased osteocalcin levels, thereby linking altered bone remodeling to energy homeostasis in hereditary HGPS.

  15. A connexin30 mutation rescues hearing and reveals roles for gap junctions in cochlear amplification and micromechanics

    PubMed Central

    Lukashkina, Victoria A.; Levic, Snezana; Lukashkin, Andrei N.; Strenzke, Nicola; Russell, Ian J.

    2017-01-01

    Accelerated age-related hearing loss disrupts high-frequency hearing in inbred CD-1 mice. The p.Ala88Val (A88V) mutation in the gene coding for the gap-junction protein connexin30 (Cx30) protects the cochlear basal turn of adult CD-1Cx30A88V/A88V mice from degeneration and rescues hearing. Here we report that the passive compliance of the cochlear partition and active frequency tuning of the basilar membrane are enhanced in the cochleae of CD-1Cx30A88V/A88V compared to CBA/J mice with sensitive high-frequency hearing, suggesting that gap junctions contribute to passive cochlear mechanics and energy distribution in the active cochlea. Surprisingly, the endocochlear potential that drives mechanoelectrical transduction currents in outer hair cells and hence cochlear amplification is greatly reduced in CD-1Cx30A88V/A88V mice. Yet, the saturating amplitudes of cochlear microphonic potentials in CD-1Cx30A88V/A88V and CBA/J mice are comparable. Although not conclusive, these results are compatible with the proposal that transmembrane potentials, determined mainly by extracellular potentials, drive somatic electromotility of outer hair cells. PMID:28220769

  16. Mutational Analysis Reveals a Noncontractile but Interactive Role of Actin and Profilin in Viral RNA-Dependent RNA Synthesis▿

    PubMed Central

    Harpen, Mary; Barik, Tiasha; Musiyenko, Alla; Barik, Sailen

    2009-01-01

    As obligatory parasites, viruses co-opt a variety of cellular functions for robust replication. The expression of the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV), a significant pediatric pathogen, absolutely requires actin and is stimulated by the actin-regulatory protein profilin. As actin is a major contractile protein, it was important to determine whether the known functional domains of actin and profilin were important for their ability to activate RSV transcription. Analyses of recombinant mutants in a reconstituted RSV transcription system suggested that the divalent-cation-binding domain of actin is critically needed for binding to the RSV genome template and for the activation of viral RNA synthesis. In contrast, the nucleotide-binding domain and the N-terminal acidic domain were needed neither for template binding nor for transcription. Specific surface residues of actin, required for actin-actin contact during filamentation, were also nonessential for viral transcription. Unlike actin, profilin did not directly bind to the viral template but was recruited by actin. Mutation of the interactive residues of actin or profilin, resulting in the loss of actin-profilin binding, also abolished profilin's ability to stimulate viral transcription. Together, these results suggest that actin acts as a classical transcription factor for the virus by divalent-cation-dependent binding to the viral template and that profilin acts as a transcriptional cofactor, in part by associating with actin. This essential viral role of actin is independent of its contractile cellular role. PMID:19710142

  17. Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies

    PubMed Central

    Lee, Wei-Hua; Higuchi, Hitoshi; Ikeda, Sakae; Macke, Erica L; Takimoto, Tetsuya; Pattnaik, Bikash R; Liu, Che; Chu, Li-Fang; Siepka, Sandra M; Krentz, Kathleen J; Rubinstein, C Dustin; Kalejta, Robert F; Thomson, James A; Mullins, Robert F; Takahashi, Joseph S; Pinto, Lawrence H; Ikeda, Akihiro

    2016-01-01

    While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases. DOI: http://dx.doi.org/10.7554/eLife.19264.001 PMID:27863209

  18. Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling

    PubMed Central

    Chaki, Moumita; Airik, Rannar; Ghosh, Amiya K.; Giles, Rachel H.; Chen, Rui; Slaats, Gisela G.; Wang, Hui; Hurd, Toby W.; Zhou, Weibin; Cluckey, Andrew; Gee, Heon-Yung; Ramaswami, Gokul; Hong, Chen-Jei; Hamilton, Bruce A.; Červenka, Igor; Ganji, Ranjani Sri; Bryja, Vitezslav; Arts, Heleen H.; van Reeuwijk, Jeroen; Oud, Machteld M.; Letteboer, Stef J.F.; Roepman, Ronald; Husson, Hervé; Ibraghimov-Beskrovnaya, Oxana; Ysunaga, Takayuki; Walz, Gerd; Eley, Lorraine; Sayer, John A.; Schermer, Bernhard; Liebau, Max C.; Benzing, Thomas; Le Corre, Stephanie; Drummond, Iain; Joles, Jaap A.; Janssen, Sabine; Allen, Susan J.; Natarajan, Sivakumar; O Toole, John F.; Attanasio, Massimo; Saunier, Sophie; Antignac, Corinne; Koenekoop, Robert K.; Ren, Huanan; Lopez, Irma; Nayir, Ahmet; Stoetzel, Corinne; Dollfus, Helene; Massoudi, Rustin; Gleeson, Joseph G.; Andreoli, Sharon P.; Doherty, Dan G.; Lindstrad, Anna; Golzio, Christelle; Katsanis, Nicholas; Pape, Lars; Abboud, Emad B.; Al-Rajhi, Ali A.; Lewis, Richard A.; Lupski, James R.; Omran, Heymut; Lee, Eva; Wang, Shaohui; Sekiguchi, JoAnn M.; Saunders, Rudel; Johnson, Colin A.; Garner, Elizabeth; Vanselow, Katja; Andersen, Jens S.; Shlomai, Joseph; Nurnberg, Gudrun; Nurnberg, Peter; Levy, Shawn; Smogorzewska, Agata; Otto, Edgar A.; Hildebrandt, Friedhelm

    2012-01-01

    SUMMARY Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as ‘ciliopathies’. However, disease mechanisms remain poorly understood. Here we identify by whole exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway, hitherto not implicated in ciliopathies. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164 and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents, and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. We identify TTBK2, CCDC92, NPHP3 and DVL3 as novel CEP164 interaction partners. Our findings link degenerative diseases of kidney and retina, disorders of increasing prevalence, to mechanisms of DDR. PMID:22863007

  19. Using RNA-Seq SNP data to reveal potential causal mutations related to pig production traits and RNA editing.

    PubMed

    Martínez-Montes, A M; Fernández, A; Pérez-Montarelo, D; Alves, E; Benítez, R M; Nuñez, Y; Óvilo, C; Ibañez-Escriche, N; Folch, J M; Fernández, A I

    2017-04-01

    RNA-Seq technology is widely used in quantitative gene expression studies and identification of non-annotated transcripts. However this technology also can be used for polymorphism detection and RNA editing in transcribed regions in an efficient and cost-effective way. This study used SNP data from an RNA-Seq assay to identify genes and mutations underlying production trait variations in an experimental pig population. The hypothalamic and hepatic transcriptomes of nine extreme animals for growth and fatness from an (Iberian × Landrace) × Landrace backcross were analyzed by RNA-Seq methodology, and SNP calling was conducted. More than 125 000 single nucleotide variants (SNVs) were identified in each tissue, and 78% were considered to be potential SNPs, those SNVs segregating in the context of this study. Potential informative SNPs were detected by considering those showing a homozygous or heterozygous genotype in one extreme group and the alternative genotype in the other group. In this way, 4396 and 1862 informative SNPs were detected in hypothalamus and liver respectively. Out of the 32 SNPs selected for validation, 25 (80%) were confirmed as actual SNPs. Association analyses for growth, fatness and premium cut yields with 19 selected SNPs were carried out, and four potential causal genes (RETSAT, COPA, RNMT and PALMD) were identified. Interestingly, new RNA editing modifications were detected and validated for the NR3C1:g.102797 (ss1985401074) and ACSM2B:g.13374 (ss1985401075) positions and for the COG3:g3.4525 (ss1985401087) modification previously identified across vertebrates, which could lead to phenotypic variation and should be further investigated.

  20. Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation

    PubMed Central

    Martínez-Barricarte, Rubén; Heurich, Meike; Valdes-Cañedo, Francisco; Vazquez-Martul, Eduardo; Torreira, Eva; Montes, Tamara; Tortajada, Agustín; Pinto, Sheila; Lopez-Trascasa, Margarita; Morgan, B. Paul; Llorca, Oscar; Harris, Claire L.; Rodríguez de Córdoba, Santiago

    2010-01-01

    Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associates disease with a mutation in the C3 gene. Mutant C3923ΔDG, which lacks 2 amino acids, could not be cleaved to C3b by the AP C3-convertase and was therefore the predominant circulating C3 protein in the patients. However, upon activation to C3b by proteases, or to C3(H2O) by spontaneous thioester hydrolysis, C3923ΔDG generated an active AP C3-convertase that was regulated normally by decay accelerating factor (DAF) but was resistant to decay by fH. Moreover, activated C3b923ΔDG and C3(H2O)923ΔDG were resistant to proteolysis by factor I (fI) in the presence of fH, but were efficiently inactivated in the presence of membrane cofactor protein (MCP). These characteristics cause a fluid phase–restricted AP dysregulation in the patients that continuously activated and consumed C3 produced by the normal C3 allele. These findings expose structural requirements in C3 that are critical for recognition of the substrate C3 by the AP C3-convertase and for the regulatory activities of fH, DAF, and MCP, all of which have implications for therapeutic developments. PMID:20852386

  1. Analysis of Leigh syndrome mutations in the yeast SURF1 homolog reveals a new member of the cytochrome oxidase assembly factor family.

    PubMed

    Bestwick, Megan; Jeong, Mi-Young; Khalimonchuk, Oleh; Kim, Hyung; Winge, Dennis R

    2010-09-01

    Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F(249)T and Y(344)D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G(137)E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G(137)E Shy1 mutant phenocopied shy1Delta cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G(137)E Shy1 mutant revealed Coa2, Cox10, and a novel factor designated Coa4. Coa2 and Cox10 are previously characterized CcO assembly factors. Coa4 is a twin CX(9)C motif mitochondrial protein localized in the intermembrane space and associated with the inner membrane. Cells lacking Coa4 are depressed in CcO activity but show no impairment in Cox1 maturation or formation of the Shy1-stabilized Cox1 assembly intermediate. To glean insights into the functional role of Coa4 in CcO biogenesis, an unbiased suppressor screen of coa4Delta cells was conducted. Respiratory function of coa4Delta cells was restored by the overexpression of CYC1 encoding cytochrome c. Cyc1 is known to be important at an ill-defined step in the assembly and/or stability of CcO. This new link to Coa4 may begin to further elucidate the role of Cyc1 in CcO biogenesis.

  2. A Novel Mutation of LAMB2 in a Multi-Generational Mennonite Family Reveals a New Phenotypic Variant of Pierson Syndrome

    PubMed Central

    Mohney, Brian G.; Pulido, Jose S.; Lindor, Noralane M.; Hogan, Marie C.; Consugar, Mark B.; Peters, Justin; Pankratz, V. Shane; Nasr, Samih H.; Smith, Stephen J.; Gloor, James; Kubly, Vickie; Spencer, Dorothy; Nielson, Rebecca; Puffenberger, Erik G.; Strauss, Kevin A.; Morton, D. Holmes; Eldahdah, Lama; Harris, Peter C.

    2011-01-01

    Purpose To describe a novel laminin beta-2 (LAMB2) mutation associated with nephritic syndrome and severe retinal disease without microcoria in a large, multi-generational family with Pierson syndrome. Design Retrospective chart review and prospective family examination. Participants An extended consanguineous family of 52 members. Methods The eyes, urine, and serum DNA were evaluated in all family members after discovering 2 patients, both less than 10 years of age, with bilateral retinal detachments and concurrent renal dysfunction. Linkage analysis was performed in the 9 living affected individuals, 7 using the Illumina Human Hap370 Duo Bead Array and 2 using GeneChip 10K mapping arrays. Main Outcome Measures The prevalence and severity of ocular and kidney involvement and genetic findings. Results Eleven affected family members were identified (9 living), all manifesting chronic kidney disease and bilateral chrioretinal pigmentary changes, with or without retinal detachments, but without microcoria or neurodevelopmental deficits, segregating in an autosomal recessive pattern. The causative gene was localized to a 9Mb region on chromosome 3. Comprehensive gene sequencing revealed a novel LAMB2 variant (c. 440A>G; His147R) that was homozygous in the 9 living, affected family members, observed at a frequency of 2.1% in the Old Order Mennonite population, and absent in 91 non-Mennonite controls. The mutation is located in a highly conserved site in the N-terminal domain VI of LAMB2. Conclusions This study describes a novel mutation of LAMB2 and further expands the spectrum of eye and renal manifestations associated with defects in the laminin beta-2 chain. PMID:21236492

  3. Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation.

    PubMed

    Yuan, Feng-Jie; Zhu, Dan-Hua; Tan, Yuan-Yuan; Dong, De-Kun; Fu, Xu-Jun; Zhu, Shen-Long; Li, Bai-Quan; Shu, Qing-Yao

    2012-11-01

    Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is an important constituent of soybean meal. Since phytic acid and its mineral salts (phytates) are almost indigestible for monogastrics, their abundance in grain food/feed causes nutritional and environmental problems; interest in breeding low phytic acid has therefore increased considerably. Based on gene mapping and the characteristics of inositol polyphosphates profile in the seeds of a soybean mutant line Gm-lpa-ZC-2, the soybean ortholog of inositol 1,3,4,5,6 pentakisphosphate (InsP(5)) 2-kinase (IPK1), which transforms InsP(5) into phytic acid, was first hypothesized as the candidate gene responsible for the low phytic acid alteration in Gm-lpa-ZC-2. One IPK1 ortholog (Glyma14g07880, GmIPK1) was then identified in the mapped region on chromosome 14. Sequencing revealed a G → A point mutation in the genomic DNA sequence and the exclusion of the entire fifth exon in the cDNA sequence of GmIPK1 in Gm-lpa-ZC-2 compared with its wild-type progenitor Zhechun No. 3. The excluded exon encodes 37 amino acids that spread across two conserved IPK1 motifs. Furthermore, complete co-segregation of low phytic acid phenotype with the G → A mutation was observed in the F(2) population of ZC-lpa x Zhexiandou No. 4 (a wild-type cultivar). Put together, the G → A point mutation affected the pre-mRNA splicing and resulted in the exclusion of the fifth exon of GmIPK1 which is expected to disrupt the GmIPK1 functionality, leading to low phytic acid level in Gm-lpa-ZC-2. Gm-lpa-ZC-2, would be a good germplasm source in low phytic acid soybean breeding.

  4. Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics.

    PubMed

    Matsumoto, Ken'ichiro; Tanaka, Yoshikazu; Watanabe, Tsuyoshi; Motohashi, Ren; Ikeda, Koji; Tobitani, Kota; Yao, Min; Tanaka, Isao; Taguchi, Seiichi

    2013-10-01

    NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.

  5. Yeast DNA ligase IV mutations reveal a nonhomologous end joining function of BRCT1 distinct from XRCC4/Lif1 binding.

    PubMed

    Chiruvella, Kishore K; Renard, Brian M; Birkeland, Shanda R; Sunder, Sham; Liang, Zhuobin; Wilson, Thomas E

    2014-12-01

    LIG4/Dnl4 is the DNA ligase that (re)joins DNA double-strand breaks (DSBs) via nonhomologous end joining (NHEJ), an activity supported by binding of its tandem BRCT domains to the ligase accessory protein XRCC4/Lif1. We screened a panel of 88 distinct ligase mutants to explore the structure–function relationships of the yeast Dnl4 BRCT domains and inter-BRCT linker in NHEJ. Screen results suggested two distinct classes of BRCT mutations with differential effects on Lif1 interaction as compared to NHEJ completion. Validated constructs confirmed that D800K and GG(868:869)AA mutations, which target the Lif1 binding interface, showed a severely defective Dnl4–Lif1 interaction but a less consistent and often small decrease in NHEJ activity in some assays, as well as nearly normal levels of Dnl4 accumulation at DSBs. In contrast, mutants K742A and KTT(742:744)ATA, which target the β3-α2 region of the first BRCT domain, substantially decreased NHEJ function commensurate with a large defect in Dnl4 recruitment to DSBs, despite a comparatively greater preservation of the Lif1 interaction. Together, these separation-of-function mutants indicate that Dnl4 BRCT1 supports DSB recruitment and NHEJ in a manner distinct from Lif1 binding and reveal a complexity of Dnl4 BRCT domain functions in support of stable DSB association.

  6. Impaired Acid Catalysis by Mutation of a Protein Loop Hinge Residue in a YopH Mutant Revealed by Crystal Structures

    SciTech Connect

    Brandao, T.; Robinson, H; Johnson, S; Hengge, A

    2009-01-01

    Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presence of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.

  7. Cloning of wrinkle-free, a previously uncharacterized mouse mutation, reveals crucial roles for fatty acid transport protein 4 in skin and hair development.

    PubMed

    Moulson, Casey L; Martin, Daniel R; Lugus, Jesse J; Schaffer, Jean E; Lind, Anne C; Miner, Jeffrey H

    2003-04-29

    Wrinkle-free (wrfr) is a previously uncharacterized, spontaneous, autosomal recessive mouse mutation resulting in very tight, thick skin. wrfr mutant mice exhibit severe breathing difficulties secondary to their tight skin and die shortly after birth. This phenotype is strikingly similar to a very rare human genetic disorder, restrictive dermopathy. wrfr mutant mice display a defective skin barrier, which is normally imparted by the cornified envelope, a composite of protein and lipid that prevents loss of water from within and entry of potentially harmful substances from without. In addition, hair growth from grafted wrfr skin is impaired. Positional cloning of the wrfr mutation revealed a retrotransposon insertion into a coding exon of Slc27a4, the gene encoding fatty acid transport protein (FATP)4. FATP4 is the primary intestinal FATP and is thought to play a major role in dietary fatty acid uptake; it therefore is viewed as a target to prevent or reverse obesity. However, its function in vivo had not been determined. Our results demonstrate an unexpected yet critical role for FATP4 in skin and hair development and suggest Slc27a4 to be a candidate gene for restrictive dermopathy.

  8. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    PubMed

    Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  9. Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

    PubMed Central

    Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I.; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity. PMID:24278284

  10. Whole-exome sequencing of a patient with severe and complex hemostatic abnormalities reveals a possible contributing frameshift mutation in C3AR1

    PubMed Central

    Leinøe, Eva; Nielsen, Ove Juul; Jønson, Lars; Rossing, Maria

    2016-01-01

    The increasing availability of genome-wide analysis has made it possible to rapidly sequence the exome of patients with undiagnosed or unresolved medical conditions. Here, we present the case of a 64-yr-old male patient with schistocytes in the peripheral blood smear and a complex and life-threatening coagulation disorder causing recurrent venous thromboembolic events, severe thrombocytopenia, and subdural hematomas. Whole-exome sequencing revealed a frameshift mutation (C3AR1 c.355-356dup, p.Asp119Alafs*19) resulting in a premature stop codon in C3AR1 (Complement Component 3a Receptor 1). Based on this finding, atypical hemolytic uremic syndrome was suspected because of a genetic predisposition, and a targeted treatment regime with eculizumab was initiated. Life-threatening hemostatic abnormalities would most likely have persisted had it not been for the implementation of whole-exome sequencing in this particular clinical setting. PMID:27551680

  11. Temsirolimus Partially Rescues the Hutchinson-Gilford Progeria Cellular Phenotype.

    PubMed

    Gabriel, Diana; Gordon, Leslie B; Djabali, Karima

    2016-01-01

    Hutchinson-Gilford syndrome (HGPS, OMIM 176670, a rare premature aging disorder that leads to death at an average age of 14.7 years due to myocardial infarction or stroke, is caused by mutations in the LMNA gene. Lamins help maintain the shape and stability of the nuclear envelope in addition to regulating DNA replication, DNA transcription, proliferation and differentiation. The LMNA mutation results in the deletion of 50 amino acids from the carboxy-terminal region of prelamin A, producing the truncated, farnesylated protein progerin. The accumulation of progerin in HGPS nuclei causes numerous morphological and functional changes that lead to premature cellular senescence. Attempts to reverse this HGPS phenotype have identified rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), as a drug that is able to rescue the HGPS cellular phenotype by promoting autophagy and reducing progerin accumulation. Rapamycin is an obvious candidate for the treatment of HGPS disease but is difficult to utilize clinically. To further assess rapamycin's efficacy with regard to proteostasis, mitochondrial function and the degree of DNA damage, we tested temsirolimus, a rapamycin analog with a more favorable pharmacokinetic profile than rapamycin. We report that temsirolimus decreases progerin levels, increases proliferation, reduces misshapen nuclei, and partially ameliorates DNA damage, but does not improve proteasome activity or mitochondrial dysfunction. Our findings suggest that future therapeutic strategies should identify new drug combinations and treatment regimens that target all the dysfunctional hallmarks that characterize HGPS cells.

  12. Temsirolimus Partially Rescues the Hutchinson-Gilford Progeria Cellular Phenotype

    PubMed Central

    Gabriel, Diana; Gordon, Leslie B.

    2016-01-01

    Hutchinson-Gilford syndrome (HGPS, OMIM 176670, a rare premature aging disorder that leads to death at an average age of 14.7 years due to myocardial infarction or stroke, is caused by mutations in the LMNA gene. Lamins help maintain the shape and stability of the nuclear envelope in addition to regulating DNA replication, DNA transcription, proliferation and differentiation. The LMNA mutation results in the deletion of 50 amino acids from the carboxy-terminal region of prelamin A, producing the truncated, farnesylated protein progerin. The accumulation of progerin in HGPS nuclei causes numerous morphological and functional changes that lead to premature cellular senescence. Attempts to reverse this HGPS phenotype have identified rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), as a drug that is able to rescue the HGPS cellular phenotype by promoting autophagy and reducing progerin accumulation. Rapamycin is an obvious candidate for the treatment of HGPS disease but is difficult to utilize clinically. To further assess rapamycin’s efficacy with regard to proteostasis, mitochondrial function and the degree of DNA damage, we tested temsirolimus, a rapamycin analog with a more favorable pharmacokinetic profile than rapamycin. We report that temsirolimus decreases progerin levels, increases proliferation, reduces misshapen nuclei, and partially ameliorates DNA damage, but does not improve proteasome activity or mitochondrial dysfunction. Our findings suggest that future therapeutic strategies should identify new drug combinations and treatment regimens that target all the dysfunctional hallmarks that characterize HGPS cells. PMID:28033363

  13. Structure and Mutational Analyses of Escherichia coli ZapD Reveal Charged Residues Involved in FtsZ Filament Bundling

    PubMed Central

    Roach, Elyse J.; Wroblewski, Charles; Seidel, Laura; Berezuk, Alison M.; Brewer, Dyanne; Kimber, Matthew S.

    2016-01-01

    ABSTRACT Bacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associated proteins (Zap) for stability throughout the process of division. In Escherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure of Escherichia coli ZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Using in vitro FtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments. IMPORTANCE Z-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins in E. coli that associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure of E. coli

  14. Blocking protein farnesylation improves nuclear shape abnormalities in keratinocytes of mice expressing the prelamin A variant in Hutchinson-Gilford progeria syndrome.

    PubMed

    Wang, Yuexia; Ostlund, Cecilia; Worman, Howard J

    2010-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by mutations in LMNA leading to expression of a truncated prelamin A variant termed progerin. Whereas a farnesylated polypeptide is normally removed from the carboxyl-terminus of prelamin A during endoproteolytic processing to lamin A, progerin lacks the cleavage site and remains farnesylated. Cultured cells from human subjects with HGPS and genetically modified mice expressing progerin have nuclear morphological abnormalities, which are reversed by inhibitors of protein farnesylation. In addition, treatment with protein farnesyltransferase inhibitors improves whole animal phenotypes in mouse models of HGPS. However, improvement in nuclear morphology in tissues after treatment of animals has not been demonstrated. We therefore treated transgenic mice that express progerin in epidermis with the protein farnesyltransferase inhibitor FTI-276 or a combination of pravastatin and zoledronate to determine if they reversed nuclear morphological abnormalities in tissue. Immunofluorescence microscopy and "blinded" electron microscopic analysis demonstrated that systemic administration of FTI-276 or pravastatin plus zoledronate significantly improved nuclear morphological abnormalities in keratinocytes of transgenic mice. These results show that pharmacological blockade of protein prenylation reverses nuclear morphological abnormalities that occur in HGPS in vivo. They further suggest that skin biopsy may be useful to determine if protein farnesylation inhibitors are exerting effects in subjects with HGPS in clinical trials.

  15. Naïve adult stem cells from patients with Hutchinson-Gilford progeria syndrome express low levels of progerin in vivo.

    PubMed

    Wenzel, Vera; Roedl, Daniela; Gabriel, Diana; Gordon, Leslie B; Herlyn, Meenhard; Schneider, Reinhard; Ring, Johannes; Djabali, Karima

    2012-06-15

    Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare disorder characterized by segmental accelerated aging and early death from coronary artery disease or stroke. Nearly 90% of HGPS sufferers carry a G608G mutation within exon 11 of LMNA, producing a truncated form of prelamin A, referred to as "progerin". Here, we report the isolation of naïve multipotent skin-derived precursor (SKP) cells from dermal fibroblast cultures from HGPS donors. These cells form spheres and express the neural crest marker, nestin, in addition to the multipotent markers, OCT4, Sox2, Nanog and TG30; these cells can self-renew and differentiate into smooth muscle cells (SMCs) and fibroblasts. The SMCs derived from the HGPS-SKPs accumulate nuclear progerin with increasing passages. A subset of the HGPS-naïve SKPs express progerin in vitro and in situ in HGPS skin sections. This is the first in vivo evidence that progerin is produced in adult stem cells, and implies that this protein could induce stem cells exhaustion as a mechanism contributing to aging. Our study provides a basis on which to explore therapeutic applications for HGPS stem cells and opens avenues for investigating the pathogenesis of other genetic diseases.

  16. Analysis of Ankyrin Repeats Reveals How a Single Point Mutation in RFXANK Results in Bare Lymphocyte Syndrome

    PubMed Central

    Nekrep, Nada; Geyer, Matthias; Jabrane-Ferrat, Nabila; Peterlin, B. Matija

    2001-01-01

    Ankyrin repeats are well-known structural modules that mediate interactions between a wide spectrum of proteins. The regulatory factor X with ankyrin repeats (RFXANK) is a subunit of a tripartite RFX complex that assembles on promoters of major histocompatibility complex class II (MHC II) genes. Although it is known that RFXANK plays a central role in the nucleation of RFX, it was not clear how its ankyrin repeats mediate the interactions within the complex and with other proteins. To answer this question, we modeled the RFXANK protein and determined the variable residues of the ankyrin repeats that should contact other proteins. Site-directed alanine mutagenesis of these residues together with in vitro and in vivo binding studies elucidated how RFXAP and CIITA, which simultaneously interact with RFXANK in vivo, bind to two opposite faces of its ankyrin repeats. Moreover, the binding of RFXAP requires two separate surfaces on RFXANK. One of them, which is located in the ankyrin groove, is severely affected in the FZA patient with the bare lymphocyte syndrome. This genetic disease blocks the expression of MHC II molecules on the surface of B cells. By pinpointing the interacting residues of the ankyrin repeats of RFXANK, the mechanism of this subtype of severe combined immunodeficiency was revealed. PMID:11463838

  17. Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

    PubMed Central

    Sakamoto, Yasumitsu; Suzuki, Yoshiyuki; Iizuka, Ippei; Tateoka, Chika; Roppongi, Saori; Fujimoto, Mayu; Inaka, Koji; Tanaka, Hiroaki; Yamada, Mitsugu; Ohta, Kazunori; Gouda, Hiroaki; Nonaka, Takamasa; Ogasawara, Wataru; Tanaka, Nobutada

    2015-01-01

    The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double β-barrel fold and a recently identified regulatory α-helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms. PMID:26057589

  18. Shear stress-induced mechanotransduction protein deregulation and vasculopathy in a mouse model of progeria

    PubMed Central

    2014-01-01

    Introduction A mouse model of progeria derived by insertion of the human mutant LMNA gene (mLMNA), producing mutant lamin A, shows loss of smooth muscle cells in the media of the ascending aorta. We hypothesized that high shear stress, in the presence of mutant lamin A, induces this vasculopathy and tried to define the molecular and cellular basis for aortic vasculopathy. Methods Ascending and descending aortas from wild type (WT) and mLMNA+ mice were compared using proteomics, Western blots, PCR and immunostaining. To determine whether high fluidic shear stress, known to occur in the ascending aorta, contributed to the vasculopathy, we exposed descending aortas of mLMNA+ mice, with no apparent vasculopathy, to 75 dynes/cm2 shear stress for 30 minutes using a microfluidic system. Results When the mice were one year of age, expression of several mechanotransduction proteins in the ascending aorta, including vimentin, decreased in mLMNA+ mice but no decrease occurred in the descending aorta. High fluidic shear stress produced a significant reduction in vimentin of mLMNA+ mice but not in similarly treated WT mice. Conclusions The occurrence of mutant lamin A and high shear stress correlate with a reduction in the level of mechanotransduction proteins in smooth muscle cells of the media. Reduction of these proteins may contribute over time to development of vasculopathy in the ascending aorta in progeria syndrome. PMID:24661531

  19. Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes

    PubMed Central

    Kreienkamp, Ray; Croke, Monica; Neumann, Martin A.; Bedia-Diaz, Gonzalo; Graziano, Simona; Dusso, Adriana; Dorsett, Dale; Carlberg, Carsten; Gonzalo, Susana

    2016-01-01

    Hutchinson-Gilford Progeria Syndrome (HGPS) is a devastating incurable premature aging disease caused by accumulation of progerin, a toxic lamin A mutant protein. HGPS patient-derived cells exhibit nuclear morphological abnormalities, altered signaling pathways, genomic instability, and premature senescence. Here we uncover new molecular mechanisms contributing to cellular decline in progeria. We demonstrate that HGPS cells reduce expression of vitamin D receptor (VDR) and DNA repair factors BRCA1 and 53BP1 with progerin accumulation, and that reconstituting VDR signaling via 1α,25-dihydroxyvitamin D3 (1,25D) treatment improves HGPS phenotypes, including nuclear morphological abnormalities, DNA repair defects, and premature senescence. Importantly, we discovered that the 1,25D/VDR axis regulates LMNA gene expression, as well as expression of DNA repair factors. 1,25D dramatically reduces progerin production in HGPS cells, while stabilizing BRCA1 and 53BP1, two key factors for genome integrity. Vitamin D/VDR axis emerges as a new target for treatment of HGPS and potentially other lamin-related diseases exhibiting VDR deficiency and genomic instability. Because progerin expression increases with age, maintaining vitamin D/VDR signaling could keep the levels of progerin in check during physiological aging. PMID:27145372

  20. Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes.

    PubMed

    Kreienkamp, Ray; Croke, Monica; Neumann, Martin A; Bedia-Diaz, Gonzalo; Graziano, Simona; Dusso, Adriana; Dorsett, Dale; Carlberg, Carsten; Gonzalo, Susana

    2016-05-24

    Hutchinson-Gilford Progeria Syndrome (HGPS) is a devastating incurable premature aging disease caused by accumulation of progerin, a toxic lamin A mutant protein. HGPS patient-derived cells exhibit nuclear morphological abnormalities, altered signaling pathways, genomic instability, and premature senescence. Here we uncover new molecular mechanisms contributing to cellular decline in progeria. We demonstrate that HGPS cells reduce expression of vitamin D receptor (VDR) and DNA repair factors BRCA1 and 53BP1 with progerin accumulation, and that reconstituting VDR signaling via 1α,25-dihydroxyvitamin D3 (1,25D) treatment improves HGPS phenotypes, including nuclear morphological abnormalities, DNA repair defects, and premature senescence. Importantly, we discovered that the 1,25D/VDR axis regulates LMNA gene expression, as well as expression of DNA repair factors. 1,25D dramatically reduces progerin production in HGPS cells, while stabilizing BRCA1 and 53BP1, two key factors for genome integrity. Vitamin D/VDR axis emerges as a new target for treatment of HGPS and potentially other lamin-related diseases exhibiting VDR deficiency and genomic instability. Because progerin expression increases with age, maintaining vitamin D/VDR signaling could keep the levels of progerin in check during physiological aging.

  1. Mutational and Structural Analysis of l-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

    PubMed Central

    García-Pino, Abel; Las Heras-Vázquez, Francisco Javier; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; García-Ruiz, Juan M.; Loris, Remy; Gavira, Jose Antonio

    2012-01-01

    N-Carbamoyl-l-amino acid amidohydrolases (l-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first l-carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg234 in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a “small-molecule binding domain,” allowing further evolutionary considerations for this enzyme family. PMID:22904279

  2. Differential screening of mutated SOD1 transgenic mice reveals early up-regulation of a fast axonal transport component in spinal cord motor neurons.

    PubMed

    Dupuis, L; de Tapia, M; René, F; Lutz-Bucher, B; Gordon, J W; Mercken, L; Pradier, L; Loeffler, J P

    2000-08-01

    In the present study we analyze the molecular mechanisms underlying motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS). For this, we used a transgenic mouse model expressing the Cu/Zn superoxide dismutase (SOD1) gene with a Gly(86) to Arg (G86R) mutation equivalent to that found in a subset of human FALS. Using an optimized suppression subtractive hybridization method, a cDNA specifically up-regulated during the asymptomatic phase in the lumbar spinal cord of G86R mice was identified by sequence analysis as the KIF3-associated protein (KAP3), a regulator of fast axonal transport. RT-PCR analysis revealed that KAP3 induction was an early event arising long before axonal degeneration. Immunohistochemical studies further revealed that KAP3 protein predominantly accumulates in large motor neurons of the ventral spinal cord. We further demonstrated that KAP3 up-regulation occurs independent of any change in the other components of the kinesin II complex. However, since the ubiquitous KIF1A motor is up-regulated, our results show an early and complex rearrangement of the fast axonal transport machinery in the course of FALS pathology.

  3. Genome sequencing reveals unique mutations in characteristic metabolic pathways and the transfer of virulence genes between V. mimicus and V. cholerae.

    PubMed

    Wang, Duochun; Wang, Haiyin; Zhou, Yanyan; Zhang, Qiuxiang; Zhang, Fanfei; Du, Pengcheng; Wang, Shujing; Chen, Chen; Kan, Biao

    2011-01-01

    Vibrio mimicus, the species most similar to V. cholerae, is a microbe present in the natural environmental and sometimes causes diarrhea and internal infections in humans. It shows similar phenotypes to V. cholerae but differs in some biochemical characteristics. The molecular mechanisms underlying the differences in biochemical metabolism between V. mimicus and V. cholerae are currently unclear. Several V. mimicus isolates have been found that carry cholera toxin genes (ctxAB) and cause cholera-like diarrhea in humans. Here, the genome of the V. mimicus isolate SX-4, which carries an intact CTX element, was sequenced and annotated. Analysis of its genome, together with those of other Vibrio species, revealed extensive differences within the Vibrionaceae. Common mutations in gene clusters involved in three biochemical metabolism pathways that are used for discrimination between V. mimicus and V. cholerae were found in V. mimicus strains. We also constructed detailed genomic structures and evolution maps for the general types of genomic drift associated with pathogenic characters in polysaccharides, CTX elements and toxin co-regulated pilus (TCP) gene clusters. Overall, the whole-genome sequencing of the V. mimicus strain carrying the cholera toxin gene provides detailed information for understanding genomic differences among Vibrio spp. V. mimicus has a large number of diverse gene and nucleotide differences from its nearest neighbor, V. cholerae. The observed mutations in the characteristic metabolism pathways may indicate different adaptations to different niches for these species and may be caused by ancient events in evolution before the divergence of V. cholerae and V. mimicus. Horizontal transfers of virulence-related genes from an uncommon clone of V. cholerae, rather than the seventh pandemic strains, have generated the pathogenic V. mimicus strain carrying cholera toxin genes.

  4. Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

    PubMed Central

    Wu, Hong; Zeng, Hong; Dong, Aiping; Li, Fengling; He, Hao; Senisterra, Guillermo; Seitova, Alma; Duan, Shili; Brown, Peter J.; Vedadi, Masoud; Arrowsmith, Cheryl H.; Schapira, Matthieu

    2013-01-01

    Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation. PMID:24367611

  5. A multi-omic analysis of an Enterococcus faecium mutant reveals specific genetic mutations and dramatic changes in mRNA and protein expression

    PubMed Central

    2013-01-01

    Background For a long time, Enterococcus faecium was considered a harmless commensal of the mammalian gastrointestinal (GI) tract and was used as a probiotic in fermented foods. In recent decades, E. faecium has been recognised as an opportunistic pathogen that causes diseases such as neonatal meningitis, urinary tract infections, bacteremia, bacterial endocarditis and diverticulitis. E. faecium could be taken into space with astronauts and exposed to the space environment. Thus, it is necessary to observe the phenotypic and molecular changes of E. faecium after spaceflight. Results An E. faecium mutant with biochemical features that are different from those of the wild-type strain was obtained from subculture after flight on the SHENZHOU-8 spacecraft. To understand the underlying mechanism causing these changes, the whole genomes of both the mutant and the WT strains were sequenced using Illumina technology. The genomic comparison revealed that dprA, a recombination-mediator gene, and arpU, a gene associated with cell wall growth, were mutated. Comparative transcriptomic and proteomic analyses showed that differentially expressed genes or proteins were involved with replication, recombination, repair, cell wall biogenesis, glycometabolism, lipid metabolism, amino acid metabolism, predicted general function and energy production/conversion. Conclusion This study analysed the comprehensive genomic, transcriptomic and proteomic changes of an E. faecium mutant from subcultures that were loaded on the SHENZHOU-8 spacecraft. The implications of these gene mutations and expression changes and their underlying mechanisms should be investigated in the future. We hope that the current exploration of multiple “-omics” analyses of this E. faecium mutant will provide clues for future studies on this opportunistic pathogen. PMID:24373636

  6. A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals novel NS5 mutation

    PubMed Central

    Schmidt, DJ; Pickett, BE; Camacho, D; Comach, G; Xhaja, K; Lennon, NJ; Rizzolo, K; de Bosch, N; Becerra, A; Nogueira, ML; Mondini, A; da Silva, EV; Vasconcelos, PF; Muñoz-Jordán, JL; Santiago, GA; Ocazionez, R; Gehrke, L; Lefkowitz, EJ; Birren, BW; Henn, MR; Bosch, I

    2013-01-01

    Dengue virus currently causes 50-100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3(DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008 respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of 7 amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may

  7. Whole Genome Sequencing Reveals Novel Non-Synonymous Mutation in Ectodysplasin A (EDA) Associated with Non-Syndromic X-Linked Dominant Congenital Tooth Agenesis

    PubMed Central

    Sarkar, Tanmoy; Bansal, Rajesh; Das, Parimal

    2014-01-01

    Congenital tooth agenesis in human is characterized by failure of tooth development during tooth organogenesis. 300 genes in mouse and 30 genes in human so far have been known to regulate tooth development. However, candidature of only 5 genes viz. PAX9, MSX1, AXIN2, WNT10A and EDA have been experimentally established for congenitally missing teeth like hypodontia and oligodontia. In this study an Indian family with multiple congenital tooth agenesis was identified. Pattern of inheritance was apparently autosomal dominant type with a rare possibility to be X-linked. Whole genome sequencing of two affected individuals was carried out which revealed 119 novel non-synonymous single nucleotide variations (SNVs) distributed among 117 genes. Out of these only one variation (c.956G>T) located at exon 9 of X-linked EDA gene was considered as pathogenic and validated among all the affected and unaffected family members and unrelated controls. This variation leads to p.Ser319Ile change in the TNF homology domain of EDA (transcript variant 1) protein. In silico analysis predicts that this Ser319 is well conserved across different vertebrate species and a part of putative receptor binding site. Structure based homology modeling predicts that this amino acid residue along with four other amino acid residues nearby, those when mutated known to cause selective tooth agenesis, form a cluster that may have functional significance. Taken together these results suggest that c.956G>T (p.Ser319Ile) mutation plausibly reduces the receptor binding activity of EDA leading to distinct tooth agenesis in this family. PMID:25203534

  8. Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/Deletion (δβ)0 Mutation in Antalya

    PubMed Central

    Bilgen, Türker; Altıok Clark, Özden; Öztürk, Zeynep; Yeşilipek, M. Akif; Keser, İbrahim

    2016-01-01

    Objective: Although the calculated carrier frequency for point mutations of the β-globin gene is around 10% for Antalya Province, nothing is known about the profile of large deletional mutations involving the β-globin gene. In this study, we aimed to screen common deletional mutations in the β-globin gene cluster in patients for whom direct DNA sequencing was not able to demonstrate the mutation(s) responsible for the disease phenotype. Materials and Methods: Thirty-one index cases selected with a series of selection events among 60 cases without detected β-globin gene mutation from 580 thalassemia-related cases tested by direct sequencing over the last 4 years in our diagnostic center were screened for the most common 8 different large deletional mutations of the β-globin gene cluster by gap-PCR. Results: We detected 1 homozygous and 9 heterozygous novel unrelated cases for the Turkish inversion/deletion (δβ)0 mutation in our series of 31 cases. Our study showed that the Turkish inversion/deletion (δβ)0 mutation per se accounts for 16.6% of the unidentified causative alleles and also accounts for 1.5% of all detected mutations over the last 4 years in our laboratory. Conclusion: Since molecular diagnosis of deletional mutations in the β-globin gene cluster warrants different approaches, it deserves special attention in order to provide prenatal diagnosis and prevention opportunities to the families involved. We conclude that the Turkish inversion/deletion (δβ)0, as the most prevalent deletional mutation detected so far, has to be routinely tested for in Antalya, and the gap-PCR approach has valuable diagnostic potential in the patients at risk. PMID:26377447

  9. Application of an NGS-based 28-gene panel in myeloproliferative neoplasms reveals distinct mutation patterns in essential thrombocythaemia, primary myelofibrosis and polycythaemia vera.

    PubMed

    Delic, Sabit; Rose, Dominic; Kern, Wolfgang; Nadarajah, Niroshan; Haferlach, Claudia; Haferlach, Torsten; Meggendorfer, Manja

    2016-11-01

    Molecular routine diagnostics for BCR-ABL1-negative myeloproliferative neoplasms (MPN) currently focusses on mutations in JAK2, CALR and MPL. In recent years, recurrent mutations in MPNs have been identified in several other genes. We here present the validation of a next generation sequencing (NGS)-based 28-gene panel and its use in MPN. We analysed the mutation status of 28 genes in 100 MPN patients [40 essential thrombocythaemia (ET), 30 primary myelofibrosis (PMF), 30 polycythaemia vera (PV)] and found two or more mutated genes in 53 patients. Moreover, significantly more mutated splicing genes (SF3B1, SRSF2 and U2AF1) were present in PMF (0·60 mutated genes/patient) compared to ET (0·15) while no mutations in splicing genes were found in PV. Additionally, chromatin modification genes (ASXL1 and EZH2) were frequently mutated in PMF patients (0·50) and, to a significantly lesser extent, in ET (0·13) and PV (0·07). Contrarily, DNA methylation genes (DNMT3A, IDH1, IDH2 and TET2) were mutated most often in PV (0·5) and less frequently in ET (0·23) and PMF (0·20), but without reaching statistical significance. Our results demonstrate the feasibility and utility of NGS-based panel diagnostics for MPN. With 53% of the patients bearing two or more mutated genes, their prognostic relevance needs further studies.

  10. Incomplete processing of mutant lamin A in Hutchinson-Gilford progeria leads to nuclear abnormalities, which are reversed by farnesyltransferase inhibition.

    PubMed

    Glynn, Michael W; Glover, Thomas W

    2005-10-15

    Hutchinson-Gilford progeria syndrome (HGPS) is typically caused by mutations in codon 608 (G608G) of the LMNA gene, which activates a cryptic splice site resulting in the in-frame loss of 150 nucleotides from the lamin A message. The deleted region includes a protein cleavage site that normally removes 15 amino acids, including a CAAX box farnesylation site, from the lamin A protein. We investigated the processing of the C-terminus of the mutant protein, 'progerin', and found that it does not undergo cleavage and, indeed, remains farnesylated. The retention of the farnesyl group may have numerous consequences, as farnesyl groups increase lipophilicity and are involved in membrane association and in protein interactions, and is likely to be an important factor in the HGPS phenotype. To further investigate this, we studied the effects of farnesylation inhibition on nuclear phenotypes in cells expressing normal and mutant lamin A. Expression of a GFP-progerin fusion protein in normal fibroblasts caused a high incidence of nuclear abnormalities, as was also seen in HGPS fibroblasts, and resulted in abnormal nuclear localization of GFP-progerin in comparison with the localization pattern of GFP-lamin A. Expression of a GFP-lamin A fusion containing a mutation preventing the final cleavage step, causing the protein to remain farnesylated, displayed identical localization patterns and nuclear abnormalities as in HGPS cells and in cells expressing GFP-progerin. Exposure to a farnesyltransferase inhibitor (FTI), PD169541, caused a significant improvement in the nuclear morphology of cells expressing GFP-progerin and in HGPS cells. These results implicate the abnormal farnesylation of progerin in the cellular phenotype in HGPS cells and suggest that FTIs may represent a therapeutic option for patients with HGPS.

  11. Multi-country Survey Revealed Prevalent and Novel F1534S Mutation in Voltage-Gated Sodium Channel (VGSC) Gene in Aedes albopictus

    PubMed Central

    Xu, Jiabao; Bonizzoni, Mariangela; Zhong, Daibin; Zhou, Guofa; Cai, Songwu; Li, Yiji; Wang, Xiaoming; Lo, Eugenia; Lee, Rebecca; Sheen, Roger; Duan, Jinhua; Yan, Guiyun; Chen, Xiao-Guang

    2016-01-01

    Background Aedes albopictus is an important dengue vector because of its aggressive biting behavior and rapid spread out of its native home range in Southeast Asia. Pyrethroids are widely used for adult mosquito control, and resistance to pyrethroids should be carefully monitored because vector control is the only effective method currently available to prevent dengue transmission. The voltage-gated sodium channel gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). Previous studies reported various mutations in the voltage-gated sodium channel (VGSC) gene, but the spatial distribution of kdr mutations in Ae. albopictus has not been systematically examined, and the association between kdr mutation and phenotypic resistance has not been established. Methods A total of 597 Ae. albopictus individuals from 12 populations across Asia, Africa, America and Europe were examined for mutations in the voltage-gated sodium channel gene. Three domains for a total of 1,107 bp were sequenced for every individual. Two populations from southern China were examined for pyrethroid resistance using the World Health Organization standard tube bioassay, and the association between kdr mutations and phenotypic resistance was tested. Results A total of 29 synonymous mutations were found across domain II, III and IV of the VGSC gene. Non-synonymous mutations in two codons of the VGSC gene were detected in 5 populations from 4 countries. A novel mutation at 1532 codon (I1532T) was found in Rome, Italy with a frequency of 19.7%. The second novel mutation at codon 1534 (F1534S) was detected in southern China and Florida, USA with a frequency ranging from 9.5–22.6%. The WHO insecticide susceptibility bioassay found 90.1% and 96.1% mortality in the two populations from southern China, suggesting resistance and probable resistance. Positive association between kdr mutations with deltamethrin resistance was established in these two populations

  12. DHPLC analysis of patients with Nevoid Basal Cell Carcinoma Syndrome reveals novel PTCH missense mutations in the sterol-sensing domain.

    PubMed

    Marsh, A; Wicking, C; Wainwright, B; Chenevix-Trench, G

    2005-09-01

    Nevoid Basal Cell Carcinoma Syndrome (NBCCS) is an autosomal dominant disorder characterised by multiple basal cell carcinomas, palmar and plantar pitting, odontogenic keratocysts of the jaws and bilamellar calcification of the falx. Mutations in the PTCH gene are responsible for NBCCS but most studies have found mutations in less than half of the cases tested. We used denaturing high performance liquid chromatography (DHPLC) to screen for PTCH mutations in 28 NBCCS cases, most of whom had been previously evaluated by single stranded conformation polymorphism analysis but found to be negative. Protein truncating (n = 10) and missense or indel (n = 4) mutations were found in 14/28 (50%) cases and one additional case carried an unclassified variant, c.2777G>C. Thirteen of the variants were novel. The mutation frequency was similar in inherited and de novo cases. Three of the missense and indel mutations were in the sterol-sensing domain, and one was in the sixth transmembrane domain.

  13. VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

    USGS Publications Warehouse

    Vogler, Amy J.; Nottingham, Roxanne; Busch, Joseph D.; Sahl, Jason W.; Shuey, Megan M.; Foster, Jeffrey T.; Schupp, James M.; Smith, Susan; Rocke, Tonie E.; Klein, Paul; Wagner, David M.

    2016-01-01

    Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used.

  14. A novel phosphorylation site mutation in profilin 1 revealed in a large screen of US, Nordic, and German amyotrophic lateral sclerosis/frontotemporal dementia cohorts.

    PubMed

    Ingre, Caroline; Landers, John E; Rizik, Naji; Volk, Alexander E; Akimoto, Chizuru; Birve, Anna; Hübers, Annemarie; Keagle, Pamela J; Piotrowska, Katarzyna; Press, Rayomand; Andersen, Peter Munch; Ludolph, Albert C; Weishaupt, Jochen H

    2013-06-01

    Profilin 1 is a central regulator of actin dynamics. Mutations in the gene profilin 1 (PFN1) have very recently been shown to be the cause of a subgroup of amyotrophic lateral sclerosis (ALS). Here, we performed a large screen of US, Nordic, and German familial and sporadic ALS and frontotemporal dementia (FTLD) patients for PFN1 mutations to get further insight into the spectrum and pathogenic relevance of this gene for the complete ALS/FTLD continuum. Four hundred twelve familial and 260 sporadic ALS cases and 16 ALS/FTLD cases from Germany, the Nordic countries, and the United States were screened for PFN1 mutations. Phenotypes of patients carrying PFN1 mutations were studied. In a German ALS family we identified the novel heterozygous PFN1 mutation p.Thr109Met, which was absent in controls. This novel mutation abrogates a phosphorylation site in profilin 1. The recently described p.Gln117Gly sequence variant was found in another familial ALS patient from the United States. The ALS patients with mutations in PFN1 displayed spinal onset motor neuron disease without overt cognitive involvement. PFN1 mutations were absent in patients with motor neuron disease and dementia, and in patients with only FTLD. We provide further evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. Patients carrying PFN1 mutations reported so far represent the "classic" ALS end of the ALS-FTLD spectrum. The novel p.Thr109Met mutation provides additional proof-of-principle that mutant proteins involved in the regulation of cytoskeletal dynamics can cause motor neuron degeneration. Moreover, this new mutation suggests that fine-tuning of actin polymerization by phosphorylation of profilin 1 might be necessary for motor neuron survival.

  15. Clinical and Molecular Studies Reveal a PSEN1 Mutation (L153V) in a Peruvian Family with Early-Onset Alzheimer's Disease

    PubMed Central

    Cornejo-Olivas, Mario R.; Yu, Chang-En; Mazzetti, Pilar; Mata, Ignacio F.; Meza, Maria; Lindo-Samanamud, Saul; Leverenz, James B.; Bird, Thomas D.

    2014-01-01

    Presenilin 1 (PSEN1) gene mutations are found in 30 to 70% of familial early onset Alzheimer disease (EOAD) cases (onset <60 years). Prevalence of these mutations is highly variable including ethnic differences worldwide. No Peruvian kindred with familial AD (FAD) have been described. Standardized clinical evaluation and cognitive assessment was completed in a Peruvian family with severe EOAD. Clinical course was characterized by very early onset (before age 35 years), progressive cognitive impairment with early memory loss, spatial disorientation and executive dysfunction. We sequenced all exons of PSEN1 in the proband and identified a c.475C>G DNA change resulting in a p.L153V missense mutation in the transmembrane domain 2 of the gene. This mutation is also present in the three additional affected siblings but not in a non-affected family member consistent with segregation of this mutation with the disease. This is the first report of a Peruvian family affected with EOAD associated with a PSEN1 mutation. This same mutation has been reported previously in English and French families, but a novel variants very close to the mutation and ancestry informative markers analysis suggests the mutation might be of Amerindian or African origin in this Peruvian family. PMID:24495933

  16. Clinical and molecular studies reveal a PSEN1 mutation (L153V) in a Peruvian family with early-onset Alzheimer's disease.

    PubMed

    Cornejo-Olivas, Mario R; Yu, Chang-En; Mazzetti, Pilar; Mata, Ignacio F; Meza, Maria; Lindo-Samanamud, Saul; Leverenz, James B; Bird, Thomas D

    2014-03-20

    Presenilin 1 (PSEN1) gene mutations are found in 30-70% of familial early-onset Alzheimer disease (EOAD) cases (onset <60 years). Prevalence of these mutations is highly variable including ethnic differences worldwide. No Peruvian kindred with familial AD (FAD) have been described. Standardized clinical evaluation and cognitive assessment were completed in a Peruvian family with severe EOAD. Clinical course was characterized by very early onset (before age 35 years), progressive cognitive impairment with early memory loss, spatial disorientation and executive dysfunction. We sequenced all exons of PSEN1 in the proband and identified a c.475C>G DNA change resulting in a p.L153V missense mutation in the transmembrane domain 2 of the gene. This mutation is also present in the three additional affected siblings but not in a non-affected family member consistent with segregation of this mutation with the disease. This is the first report of a Peruvian family affected with EOAD associated with a PSEN1 mutation. This same mutation has been reported previously in English and French families, but a novel variants very close to the mutation and ancestry informative markers analysis suggests the mutation might be of Amerindian or African origin in this Peruvian family.

  17. Rapid growth of a hepatocellular carcinoma and the driving mutations revealed by cell-population genetic analysis of whole-genome data.

    PubMed

    Tao, Yong; Ruan, Jue; Yeh, Shiou-Hwei; Lu, Xuemei; Wang, Yu; Zhai, Weiwei; Cai, Jun; Ling, Shaoping; Gong, Qiang; Chong, Zecheng; Qu, Zhengzhong; Li, Qianqian; Liu, Jiang; Yang, Jin; Zheng, Caihong; Zeng, Changqing; Wang, Hurng-Yi; Zhang, Jing; Wang, Sheng-Han; Hao, Lingtong; Dong, Lili; Li, Wenjie; Sun, Min; Zou, Wei; Yu, Caixia; Li, Chaohua; Liu, Guojing; Jiang, Lan; Xu, Jin; Huang, Huanwei; Li, Chunyan; Mi, Shuangli; Zhang, Bing; Chen, Baoxian; Zhao, Wenming; Hu, Songnian; Zhuang, Shi-Mei; Shen, Yang; Shi, Suhua; Brown, Christopher; White, Kevin P; Chen, Ding-Shinn; Chen, Pei-Jer; Wu, Chung-I

    2011-07-19

    We present the analysis of the evolution of tumors in a case of hepatocellular carcinoma. This case is particularly informative about cancer growth dynamics and the underlying driving mutations. We sampled nine different sections from three tumors and seven more sections from the adjacent nontumor tissues. Selected sections were subjected to exon as well as whole-genome sequencing. Putative somatic mutations were then individually validated across all 9 tumor and 7 nontumor sections. Among the mutations validated, 24 were amino acid changes; in addition, 22 large indels/copy number variants (>1 Mb) were detected. These somatic mutations define four evolutionary lineages among tumor cells. Separate evolution and expansion of these lineages were recent and rapid, each apparently having only one lineage-specific protein-coding mutation. Hence, by using a cell-population genetic definition, this approach identified three coding changes (CCNG1, P62, and an indel/fusion gene) as tumor driver mutations. These three mutations, affecting cell cycle control and apoptosis, are functionally distinct from mutations that accumulated earlier, many of which are involved in inflammation/immunity or cell anchoring. These distinct functions of mutations at different stages may reflect the genetic interactions underlying tumor growth.

  18. Agonist-dependent endocytosis of γ-aminobutyric acid type A (GABAA) receptors revealed by a γ2(R43Q) epilepsy mutation.

    PubMed

    Chaumont, Severine; André, Caroline; Perrais, David; Boué-Grabot, Eric; Taly, Antoine; Garret, Maurice

    2013-09-27

    GABA-gated chloride channels (GABAARs) trafficking is involved in the regulation of fast inhibitory transmission. Here, we took advantage of a γ2(R43Q) subunit mutation linked to epilepsy in humans that considerably reduces the number of GABAARs on the cell surface to better understand the trafficking of GABAARs. Using recombinant expression in cultured rat hippocampal neurons and COS-7 cells, we showed that receptors containing γ2(R43Q) were addressed to the cell membrane but underwent clathrin-mediated dynamin-dependent endocytosis. The γ2(R43Q)-dependent endocytosis was reduced by GABAAR antagonists. These data, in addition to a new homology model, suggested that a conformational change in the extracellular domain of γ2(R43Q)-containing GABAARs increased their internalization. This led us to show that endogenous and recombinant wild-type GABAAR endocytosis in both cultured neurons and COS-7 cells can be amplified by their agonists. These findings revealed not only a direct relationship between endocytosis of GABAARs and a genetic neurological disorder but also that trafficking of these receptors can be modulated by their agonist.

  19. Agonist-dependent Endocytosis of γ-Aminobutyric Acid Type A (GABAA) Receptors Revealed by a γ2(R43Q) Epilepsy Mutation*

    PubMed Central

    Chaumont, Severine; André, Caroline; Perrais, David; Boué-Grabot, Eric; Taly, Antoine; Garret, Maurice

    2013-01-01

    GABA-gated chloride channels (GABAARs) trafficking is involved in the regulation of fast inhibitory transmission. Here, we took advantage of a γ2(R43Q) subunit mutation linked to epilepsy in humans that considerably reduces the number of GABAARs on the cell surface to better understand the trafficking of GABAARs. Using recombinant expression in cultured rat hippocampal neurons and COS-7 cells, we showed that receptors containing γ2(R43Q) were addressed to the cell membrane but underwent clathrin-mediated dynamin-dependent endocytosis. The γ2(R43Q)-dependent endocytosis was reduced by GABAAR antagonists. These data, in addition to a new homology model, suggested that a conformational change in the extracellular domain of γ2(R43Q)-containing GABAARs increased their internalization. This led us to show that endogenous and recombinant wild-type GABAAR endocytosis in both cultured neurons and COS-7 cells can be amplified by their agonists. These findings revealed not only a direct relationship between endocytosis of GABAARs and a genetic neurological disorder but also that trafficking of these receptors can be modulated by their agonist. PMID:23935098

  20. A RelA(p65) Thr505 phospho-site mutation reveals an important mechanism regulating NF-κB-dependent liver regeneration and cancer

    PubMed Central

    Moles, A; Butterworth, J A; Sanchez, A; Hunter, J E; Leslie, J; Sellier, H; Tiniakos, D; Cockell, S J; Mann, D A; Oakley, F; Perkins, N D

    2016-01-01

    Post-translational modifications of nuclear factor (NF)-κB subunits provide a mechanism to differentially regulate their activity in response to the many stimuli that induce this pathway. However, the physiological significance of these modifications is largely unknown, and it remains unclear if these have a critical role in the normal and pathological functions of NF-κB in vivo. Among these, phosphorylation of the RelA(p65) Thr505 residue has been described as an important regulator of NF-κB activity in cell lines, but its physiological significance was not known. Therefore, to learn more about the role of this pathway in vivo, we generated a knockin mouse with a RelA T505A mutation. Unlike RelA knockout mice, the RelA T505A mice develop normally but exhibit aberrant hepatocyte proliferation following liver partial hepatectomy or damage resulting from carbon tetrachloride (CCl4) treatment. Consistent with these effects, RelA T505A mice exhibit earlier onset of cancer in the N-nitrosodiethylamine model of hepatocellular carcinoma. These data reveal a critical pathway controlling NF-κB function in the liver that acts to suppress the tumour-promoting activities of RelA. PMID:26853469

  1. Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis

    PubMed Central

    Yang, Li; Wu, Gang; Poethig, R. Scott

    2012-01-01

    Plant microRNAs (miRNAs) typically mediate RNA cleavage, but examples of miRNA-mediated translational repression have also been reported. However, the functional significance of this latter process is unknown. We identified SUO in a screen for Arabidopsis mutations that increase the accumulation of the miR156-regulated gene SPL3. suo has a loss-of-function phenotype characteristic of plants with reduced Argonaute (AGO)1 activity. An analysis of RNA and protein levels in suo mutants demonstrated that this phenotype is a consequence of a defect in miRNA-mediated translational repression; the effect of suo on vegetative phase change is attributable to a reduction in miR156/miR157 activity. SUO encodes a large protein with N-terminal bromo-adjacent homology (BAH) and transcription elongation factor S-II (TFS2N) domains and two C-terminal GW (glycine and tryptophan) repeats. SUO is present in the nucleus, and colocalizes with the processing-body component DCP1 in the cytoplasm. Our results reveal that SOU is a component of the miRNA pathway in Arabidopsis and demonstrate that translational repression is a functionally important aspect of miRNA activity in plants. PMID:22184231

  2. Linkage disequilibrium analysis reveals an albuminuria risk haplotype containing three missense mutations in the cubilin gene with striking differences among European and African ancestry populations

    PubMed Central

    2012-01-01

    Background A recent meta-analysis described a variant (p.Ile2984Val) in the cubilin gene (CUBN) that is associated with levels of albuminuria in the general population and in diabetics. Methods We implemented a Linkage Disequilibrium (LD) search with data from the 1000 Genomes Project, on African and European population genomic sequences. Results We found that the p.Ile2984Val variation is part of a larger haplotype in European populations and it is almost absent in west Africans. This haplotype contains 19 single nucleotide polymorphisms (SNPs) in very high LD, three of which are missense mutations (p.Leu2153Phe, p.Ile2984Val, p.Glu3002Gly), and two have not been previously reported. Notably, this European haplotype is absent in west African populations, and the frequency of each individual polymorphism differs significantly in Africans. Conclusions Genotyping of these variants in existing African origin sample sets coupled to measurements of urine albumin excretion levels should reveal which is the most likely functional candidate for albuminuria risk. The unique haplotypic structure of CUBN in different populations may leverage the effort to identify the functional variant and to shed light on evolution of the CUBN gene locus. PMID:23114252

  3. Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis.

    PubMed

    Yang, Li; Wu, Gang; Poethig, R Scott

    2012-01-03

    Plant microRNAs (miRNAs) typically mediate RNA cleavage, but examples of miRNA-mediated translational repression have also been reported. However, the functional significance of this latter process is unknown. We identified SUO in a screen for Arabidopsis mutations that increase the accumulation of the miR156-regulated gene SPL3. suo has a loss-of-function phenotype characteristic of plants with reduced Argonaute (AGO)1 activity. An analysis of RNA and protein levels in suo mutants demonstrated that this phenotype is a consequence of a defect in miRNA-mediated translational repression; the effect of suo on vegetative phase change is attributable to a reduction in miR156/miR157 activity. SUO encodes a large protein with N-terminal bromo-adjacent homology (BAH) and transcription elongation factor S-II (TFS2N) domains and two C-terminal GW (glycine and tryptophan) repeats. SUO is present in the nucleus, and colocalizes with the processing-body component DCP1 in the cytoplasm. Our results reveal that SOU is a component of the miRNA pathway in Arabidopsis and demonstrate that translational repression is a functionally important aspect of miRNA activity in plants.

  4. Molecular dynamics simulation reveals insights into the mechanism of unfolding by the A130T/V mutations within the MID1 zinc-binding Bbox1 domain.

    PubMed

    Zhao, Yunjie; Zeng, Chen; Massiah, Michael A

    2015-01-01

    The zinc-binding Bbox1 domain in protein MID1, a member of the TRIM family of proteins, facilitates the ubiquitination of the catalytic subunit of protein phosphatase 2A and alpha4, a protein regulator of PP2A. The natural mutation of residue A130 to a valine or threonine disrupts substrate recognition and catalysis. While NMR data revealed the A130T mutant Bbox1 domain failed to coordinate both structurally essential zinc ions and resulted in an unfolded structure, the unfolding mechanism is unknown. Principle component analysis revealed that residue A130 served as a hinge point between the structured β-strand-turn-β-strand (β-turn-β) and the lasso-like loop sub-structures that constitute loop1 of the ββα-RING fold that the Bbox1 domain adopts. Backbone RMSD data indicate significant flexibility and departure from the native structure within the first 5 ns of the molecular dynamics (MD) simulation for the A130V mutant (>6 Å) and after 30 ns for A130T mutant (>6 Å). Overall RMSF values were higher for the mutant structures and showed increased flexibility around residues 125 and 155, regions with zinc-coordinating residues. Simulated pKa values of the sulfhydryl group of C142 located near A130 suggested an increased in value to ~9.0, paralleling the increase in the apparent dielectric constants for the small cavity near residue A130. Protonation of the sulfhydryl group would disrupt zinc-coordination, directly contributing to unfolding of the Bbox1. Together, the increased motion of residues of loop 1, which contains four of the six zinc-binding cysteine residues, and the increased pKa of C142 could destabilize the structure of the zinc-coordinating residues and contribute to the unfolding.

  5. Functional coupling between the extracellular matrix and nuclear lamina by Wnt signaling in progeria.

    PubMed

    Hernandez, Lidia; Roux, Kyle J; Wong, Esther Sook Miin; Mounkes, Leslie C; Mutalif, Rafidah; Navasankari, Raju; Rai, Bina; Cool, Simon; Jeong, Jae-Wook; Wang, Honghe; Lee, Hyun-Shik; Kozlov, Serguei; Grunert, Martin; Keeble, Thomas; Jones, C Michael; Meta, Margarita D; Young, Stephen G; Daar, Ira O; Burke, Brian; Perantoni, Alan O; Stewart, Colin L

    2010-09-14

    The segmental premature aging disease Hutchinson-Gilford Progeria (HGPS) is caused by a truncated and farnesylated form of Lamin A. In a mouse model for HGPS, a similar Lamin A variant causes the proliferative arrest and death of postnatal, but not embryonic, fibroblasts. Arrest is due to an inability to produce a functional extracellular matrix (ECM), because growth on normal ECM rescues proliferation. The defects are associated with inhibition of canonical Wnt signaling, due to reduced nuclear localization and transcriptional activity of Lef1, but not Tcf4, in both mouse and human progeric cells. Defective Wnt signaling, affecting ECM synthesis, may be critical to the etiology of HGPS because mice exhibit skeletal defects and apoptosis in major blood vessels proximal to the heart. These results establish a functional link between the nuclear envelope/lamina and the cell surface/ECM and may provide insights into the role of Wnt signaling and the ECM in aging.

  6. Targeting isoprenylcysteine methylation ameliorates disease in a mouse model of progeria.

    PubMed

    Ibrahim, Mohamed X; Sayin, Volkan I; Akula, Murali K; Liu, Meng; Fong, Loren G; Young, Stephen G; Bergo, Martin O

    2013-06-14

    Several progeroid disorders, including Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (ZMPSTE24 deficiency), arise when a farnesylated and methylated form of prelamin A accumulates at the nuclear envelope. Here, we found that a hypomorphic allele of isoprenylcysteine carboxyl methyltransferase (ICMT) increased body weight, normalized grip strength, and prevented bone fractures and death in Zmpste24-deficient mice. The reduced ICMT activity caused prelamin A mislocalization within the nucleus and triggered prelamin A-dependent activation of AKT-mammalian target of rapamycin (mTOR) signaling, which abolished the premature senescence of Zmpste24-deficient fibroblasts. ICMT inhibition increased AKT-mTOR signaling and proliferation and delayed senescence in human HGPS fibroblasts but did not reduce the levels of misshapen nuclei in mouse and human cells. Thus, targeting ICMT might be useful for treating prelamin A-associated progeroid disorders.

  7. Hip pathology in Hutchinson-Gilford progeria syndrome: a report of two children.

    PubMed

    Akhbari, Pouya; Jha, Shilpa; James, Kyle D; Hinves, Barry L; Buchanan, Jamie A F

    2012-11-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder. The estimated incidence is one in 4 million births. Orthopaedic manifestations include abnormality of the hips occurring early in the disease process. Severe coxa valga can be apparent by the age of 2 years. We report two cases of HGPS, one in a 7-year-old girl with avascular necrosis of the left hip and the second in a 13-year-old girl with recurrent traumatic hip dislocations. We demonstrate the pathoanatomical changes in the hip with HGPS using a combination of imaging modalities including radiographic, computed tomographic and MRI scans. These include coxa magna, coxa valga and acetabular dysplasia. We also comment on how these would affect the surgical management of this high-risk group of patients.

  8. Unraveling the mysteries of aging through a Hutchinson-Gilford progeria syndrome model.

    PubMed

    Serio, Ryan N

    2011-04-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare, deadly laminopathy. Research into its nature has provided valuable insights into understanding molecular mechanisms underlying cell senescence. Phenotypic changes in nuclear structure and heterochromatin, resulting from increased progerin production following overuse of a cryptic splice site in the LMNA gene have profound effects on cell cycle progression and DNA repair mechanisms. A high degree of genomic instability occurs as DNA double-strand breaks are introduced but not repaired, despite appropriate machinery. Damage-response mechanisms become deregulated as a result of failed recruitment of elongation factors and possibly ancillary to disruption of chromatin organization. Key regulatory regions on mRNA transcripts governing splicing activity have been exposed using antisense technology, and identification of novel molecular targets, such as xeroderma pigmentosum group A, have generated optimism for the possibility of finally yielding an effective therapy for slowing the aging process in HGPS patients while concomitantly providing valuable insights into physiological aging in general.

  9. Impact of Farnesylation Inhibitors on Survival in Hutchinson-Gilford Progeria Syndrome

    PubMed Central

    Gordon, Leslie B.; Massaro, Joe; D'Agostino, Ralph B.; Campbell, Susan E.; Brazier, Joan; Brown, W. Ted; Kleinman, Monica E; Kieran, Mark W.

    2014-01-01

    Background Hutchinson-Gilford progeria syndrome is an ultra-rare segmental premature aging disease resulting in early death from heart attack or stroke. There is no approved treatment, but starting in 2007, several recent single arm clinical trials have administered inhibitors of protein farnesylation aimed at reducing toxicity of the disease-producing protein progerin. No study has assessed whether treatments influence patient survival. The key elements necessary for this analysis are a robust natural history of survival and comparison with a sufficiently large patient population that has been treated for a sufficient time period with disease-targeting medications. Methods and Results We generated survival Kaplan-Meier survival analyses for the largest untreated Hutchinson-Gilford progeria syndrome cohort to date. Mean survival was 14.6 years. Comparing survival for treated versus age-and-gender-matched untreated cohorts, hazard ratio was 0.13 (95% CI 0.04-0.37; P<0.001) with median follow-up of 5.3 years from time of treatment initiation. There were 21/43 deaths in untreated versus 5/43 deaths among treated subjects. Treatment increased mean survival by 1.6 years. Conclusions This study provides a robust untreated disease survival profile, which can be utilized for comparisons now and in the future to assess changes in survival with treatments for HGPS. The current comparisons estimating increased survival with protein farnesylation inhibitors provide the first evidence of treatments influencing survival for this fatal disease. Clinical Trial Registration Information www.clinicaltrials.gov. Indentifiers: NCT00425607, NCT00879034 and NCT00916747. PMID:24795390

  10. Hyaluronan is not elevated in urine or serum in Hutchinson-Gilford Progeria Syndrome.

    PubMed

    Gordon, Leslie B; Harten, Ingrid A; Calabro, Anthony; Sugumaran, Geetha; Csoka, Antonei B; Brown, W Ted; Hascall, Vincent; Toole, Bryan P

    2003-07-01

    Elevations in urinary hyaluronan have been used as the principal laboratory indicator for diagnosis of Hutchinson-Gilford Progeria Syndrome (HGPS). Previous reports have provided evidence suggesting that children with HGPS have altered hyaluronan metabolism as indicated by a mean 17-fold increase in urinary hyaluronan over normal values. In addition, adults with Werner's syndrome have elevated urinary hyaluronan and even more prominent elevations in serum hyaluronan over age-matched controls. It is not known whether serum hyaluronan is elevated or whether serum hyaluronan levels correlate with urinary hyaluronan levels in children with HGPS. In a large cohort of 19 HGPS patients, we sought to confirm elevations in urinary hyaluronan concentration, to establish whether serum hyaluronan is elevated, to measure the size of urinary hyaluronan, and to determine whether serum or urine hyaluronidase levels are altered. We have analyzed urinary and serum hyaluronan levels in patients with HGPS and control patients (1) by using an enzyme-linked immunosorbent assay (ELISA)-like method in which sample hyaluronan in solution and hyaluronan in solid phase compete for a solution of biotinylated hyaluronan-binding protein, and (2) by fluorophore-assisted carbohydrate electrophoresis. The size of urinary hyaluronan was measured by using Sepharose CL-6B size exclusion chromatography. Serum and urinary hyaluronidases were evaluated quantitatively, by using ELISA, and qualitatively, by using a gel detection method. HGPS patients did not show a significant elevation in either urinary or serum hyaluronan. We detected no difference in the size of urinary hyaluronan between HGPS children and age-matched controls. Serum and urinary hyaluronidase levels were not significantly different in normal and HGPS patients. These studies indicate that neither serum nor urinary hyaluronan concentration is a reliable diagnostic or prognostic marker for HGPS and underscore a difference between adult

  11. Resveratrol rescues SIRT1-dependent adult stem cell decline and alleviates progeroid features in laminopathy-based progeria.

    PubMed

    Liu, Baohua; Ghosh, Shrestha; Yang, Xi; Zheng, Huiling; Liu, Xinguang; Wang, Zimei; Jin, Guoxiang; Zheng, Bojian; Kennedy, Brian K; Suh, Yousin; Kaeberlein, Matt; Tryggvason, Karl; Zhou, Zhongjun

    2012-12-05

    Abnormal splicing of LMNA gene or aberrant processing of prelamin A results in progeroid syndrome. Here we show that lamin A interacts with and activates SIRT1. SIRT1 exhibits reduced association with nuclear matrix (NM) and decreased deacetylase activity in the presence of progerin or prelamin A, leading to rapid depletion of adult stem cells (ASCs) in Zmpste24(-/-) mice. Resveratrol enhances the binding between SIRT1 and A-type lamins to increases its deacetylase activity. Resveratrol treatment rescues ASC decline, slows down body weight loss, improves trabecular bone structure and mineral density, and significantly extends the life span in Zmpste24(-/-) mice. Our data demonstrate lamin A as an activator of SIRT1 and provide a mechanistic explanation for the activation of SIRT1 by resveratrol. The link between conserved SIRT1 longevity pathway and progeria suggests a stem cell-based and SIRT1 pathway-dependent therapeutic strategy for progeria.

  12. Hutchinson-Gilford progeria: faithful DNA maintenance, inheritance and allelic transcription of beta(1-4) galactosyltransferase.

    PubMed

    O'Brien, M E; Jensen, S; Weiss, A S

    1998-03-16

    Hutchinson-Gilford progeria syndrome (HGPS) is a fatal segmental aging disorder affecting children. There is a paucity of prior data at the nucleotide level on DNA maintenance in HGPS. We have examined the specific nucleotide sequences and production of allelic transcripts from the locus GGTB2 encoding beta(1-4) galactosyltransferase. Quantitative Northern blots of mRNA from HGPS and control fibroblasts indicated identical mature beta(1-4) galactosyltransferase transcript sizes and amounts, regardless of their altered glycosylation status. DNA sequencing of cDNA derived from HGPS beta(1-4) galactosyltransferase mRNA populations confirmed the encoded amino acid sequence was unaffected. Population studies of 41 unrelated individuals provided allelic frequency estimates for a novel FokI polymorphism, which was identified in two of six progeria cell strains. The polymorphism was faithfully inherited in a progeria pedigree in a Mendelian manner. Furthermore, the polymorphism provided direct evidence through sequencing of reverse transcription polymerase chain reaction products that both alleles were transcribed and generated mature mRNA. Any defects in transcripts were below detectable levels over the lengths of coding sequences examined, despite multiple replication events from conception leading to the production and maintenance of patient-derived cells. These results indicate faithful transcription in HGPS.

  13. Screening in ALS and FTD patients reveals 3 novel UBQLN2 mutations outside the PXX domain and a pure FTD phenotype.

    PubMed

    Synofzik, Matthis; Maetzler, Walter; Grehl, Torsten; Prudlo, Johannes; Vom Hagen, Jennifer Müller; Haack, Tobias; Rebassoo, Piret; Munz, Marita; Schöls, Ludger; Biskup, Saskia

    2012-12-01

    Mutations in UBQLN2 have recently been shown to cause dominant X-linked amyotrophic lateral sclerosis (ALS) and ALS plus frontotemporal dementia (FTD). Information on their frequency in different populations is still rare, and a pure FTD phenotype has not yet been reported. Moreover, the mutational spectrum of known UBQLN2 mutations is still limited to its PXX repeat region. Based on a screening of 206 ALS and FTD patients, we here report 3 novel UBQLN2 mutations, accounting for 1.2% (2/161) ALS and 2.2% (1/45) FTD patients, including a patient with pure FTD. All mutations were located in highly conserved domains outside the PXX repeat region and not observed in 1450 ethnically matched control X-chromosomes. All affected patients presented with apparently sporadic disease. UBQLN2 mutations are rare in Central European ALS and FTD patients, but contribute significantly to patients with seemingly sporadic disease. UBQLN2 is able to cause any disease on the ALS-FTD continuum, including pure FTD. Because the pathogenic mechanism of UBQLN2 mutations is not limited to its PXX region, UBQLN2 screening in neurodegenerative patients should not be limited to this region.

  14. Signaling pathway activation drift during aging: Hutchinson-Gilford Progeria Syndrome fibroblasts are comparable to normal middle-age and old-age cells.

    PubMed

    Aliper, Alexander M; Csoka, Antonei Benjamin; Buzdin, Anton; Jetka, Tomasz; Roumiantsev, Sergey; Moskalev, Alexy; Zhavoronkov, Alex

    2015-01-01

    For the past several decades, research in understanding the molecular basis of human aging has progressed significantly with the analysis of premature aging syndromes. Progerin, an altered form of lamin A, has been identified as the cause of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), and may be a contributing causative factor in normal aging. However, the question of whether HGPS actually recapitulates the normal aging process at the cellular and organismal level, or simply mimics the aging phenotype is widely debated. In the present study we analyzed publicly available microarray datasets for fibroblasts undergoing cellular aging in culture, as well as fibroblasts derived from young, middle-age, and old-age individuals, and patients with HGPS. Using GeroScope pathway analysis and drug discovery platform we analyzed the activation states of 65 major cellular signaling pathways. Our analysis reveals that signaling pathway activation states in cells derived from chronologically young patients with HGPS strongly resemble cells taken from normal middle-aged and old individuals. This clearly indicates that HGPS may truly represent accelerated aging, rather than being just a simulacrum. Our data also points to potential pathways that could be targeted to develop drugs and drug combinations for both HGPS and normal aging.

  15. Signaling pathway activation drift during aging: Hutchinson-Gilford Progeria Syndrome fibroblasts are comparable to normal middle-age and old-age cells

    PubMed Central

    Aliper, Alexander M.; Csoka, Antonei Benjamin; Buzdin, Anton; Jetka, Tomasz; Roumiantsev, Sergey; Moskalev, Alexey; Zhavoronkov, Alex

    2015-01-01

    For the past several decades, research in understanding the molecular basis of human aging has progressed significantly with the analysis of premature aging syndromes. Progerin, an altered form of lamin A, has been identified as the cause of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), and may be a contributing causative factor in normal aging. However, the question of whether HGPS actually recapitulates the normal aging process at the cellular and organismal level, or simply mimics the aging phenotype is widely debated. In the present study we analyzed publicly available microarray datasets for fibroblasts undergoing cellular aging in culture, as well as fibroblasts derived from young, middle-age, and old-age individuals, and patients with HGPS. Using GeroScope pathway analysis and drug discovery platform we analyzed the activation states of 65 major cellular signaling pathways. Our analysis reveals that signaling pathway activation states in cells derived from chronologically young patients with HGPS strongly resemble cells taken from normal middle-aged and old individuals. This clearly indicates that HGPS may truly represent accelerated aging, rather than being just a simulacrum. Our data also points to potential pathways that could be targeted to develop drugs and drug combinations for both HGPS and normal aging. PMID:25587796

  16. Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant.

    PubMed

    Li, Jun; Woods, Susan L; Healey, Sue; Beesley, Jonathan; Chen, Xiaoqing; Lee, Jason S; Sivakumaran, Haran; Wayte, Nicci; Nones, Katia; Waterfall, Joshua J; Pearson, John; Patch, Anne-Marie; Senz, Janine; Ferreira, Manuel A; Kaurah, Pardeep; Mackenzie, Robertson; Heravi-Moussavi, Alireza; Hansford, Samantha; Lannagan, Tamsin R M; Spurdle, Amanda B; Simpson, Peter T; da Silva, Leonard; Lakhani, Sunil R; Clouston, Andrew D; Bettington, Mark; Grimpen, Florian; Busuttil, Rita A; Di Costanzo, Natasha; Boussioutas, Alex; Jeanjean, Marie; Chong, George; Fabre, Aurélie; Olschwang, Sylviane; Faulkner, Geoffrey J; Bellos, Evangelos; Coin, Lachlan; Rioux, Kevin; Bathe, Oliver F; Wen, Xiaogang; Martin, Hilary C; Neklason, Deborah W; Davis, Sean R; Walker, Robert L; Calzone, Kathleen A; Avital, Itzhak; Heller, Theo; Koh, Christopher; Pineda, Marbin; Rudloff, Udo; Quezado, Martha; Pichurin, Pavel N; Hulick, Peter J; Weissman, Scott M; Newlin, Anna; Rubinstein, Wendy S; Sampson, Jone E; Hamman, Kelly; Goldgar, David; Poplawski, Nicola; Phillips, Kerry; Schofield, Lyn; Armstrong, Jacqueline; Kiraly-Borri, Cathy; Suthers, Graeme K; Huntsman, David G; Foulkes, William D; Carneiro, Fatima; Lindor, Noralane M; Edwards, Stacey L; French, Juliet D; Waddell, Nicola; Meltzer, Paul S; Worthley, Daniel L; Schrader, Kasmintan A; Chenevix-Trench, Georgia

    2016-05-05

    Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

  17. Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant

    PubMed Central

    Li, Jun; Woods, Susan L.; Healey, Sue; Beesley, Jonathan; Chen, Xiaoqing; Lee, Jason S.; Sivakumaran, Haran; Wayte, Nicci; Nones, Katia; Waterfall, Joshua J.; Pearson, John; Patch, Anne-Marie; Senz, Janine; Ferreira, Manuel A.; Kaurah, Pardeep; Mackenzie, Robertson; Heravi-Moussavi, Alireza; Hansford, Samantha; Lannagan, Tamsin R.M.; Spurdle, Amanda B.; Simpson, Peter T.; da Silva, Leonard; Lakhani, Sunil R.; Clouston, Andrew D.; Bettington, Mark; Grimpen, Florian; Busuttil, Rita A.; Di Costanzo, Natasha; Boussioutas, Alex; Jeanjean, Marie; Chong, George; Fabre, Aurélie; Olschwang, Sylviane; Faulkner, Geoffrey J.; Bellos, Evangelos; Coin, Lachlan; Rioux, Kevin; Bathe, Oliver F.; Wen, Xiaogang; Martin, Hilary C.; Neklason, Deborah W.; Davis, Sean R.; Walker, Robert L.; Calzone, Kathleen A.; Avital, Itzhak; Heller, Theo; Koh, Christopher; Pineda, Marbin; Rudloff, Udo; Quezado, Martha; Pichurin, Pavel N.; Hulick, Peter J.; Weissman, Scott M.; Newlin, Anna; Rubinstein, Wendy S.; Sampson, Jone E.; Hamman, Kelly; Goldgar, David; Poplawski, Nicola; Phillips, Kerry; Schofield, Lyn; Armstrong, Jacqueline; Kiraly-Borri, Cathy; Suthers, Graeme K.; Huntsman, David G.; Foulkes, William D.; Carneiro, Fatima; Lindor, Noralane M.; Edwards, Stacey L.; French, Juliet D.; Waddell, Nicola; Meltzer, Paul S.; Worthley, Daniel L.; Schrader, Kasmintan A.; Chenevix-Trench, Georgia

    2016-01-01

    Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present. PMID:27087319

  18. Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation.

    PubMed

    Sato, Wataru; Hitaoka, Seiji; Inoue, Kaoru; Imai, Mizue; Saio, Tomohide; Uchida, Takeshi; Shinzawa-Itoh, Kyoko; Yoshikawa, Shinya; Yoshizawa, Kazunari; Ishimori, Koichiro

    2016-07-15

    Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271-12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions that were almost cancelled out by large favorable (Columbic) electrostatic interactions but resulted in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, whereas the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO.

  19. Mutational and structural analysis of KIR3DL1 reveals a lineage-defining allotypic dimorphism that impacts both HLA and peptide sensitivity.

    PubMed

    O'Connor, Geraldine M; Vivian, Julian P; Widjaja, Jacqueline M; Bridgeman, John S; Gostick, Emma; Lafont, Bernard A P; Anderson, Stephen K; Price, David A; Brooks, Andrew G; Rossjohn, Jamie; McVicar, Daniel W

    2014-03-15

    Killer Ig-like receptors (KIRs) control the activation of human NK cells via interactions with peptide-laden HLAs. KIR3DL1 is a highly polymorphic inhibitory receptor that recognizes a diverse array of HLA molecules expressing the Bw4 epitope, a group with multiple polymorphisms incorporating variants within the Bw4 motif. Genetic studies suggest that KIR3DL1 variation has functional significance in several disease states, including HIV infection. However, owing to differences across KIR3DL1 allotypes, HLA-Bw4, and associated peptides, the mechanistic link with biological outcome remains unclear. In this study, we elucidated the impact of KIR3DL1 polymorphism on peptide-laden HLA recognition. Mutational analysis revealed that KIR residues involved in water-mediated contacts with the HLA-presented peptide influence peptide binding specificity. In particular, residue 282 (glutamate) in the D2 domain underpins the lack of tolerance of negatively charged C-terminal peptide residues. Allotypic KIR3DL1 variants, defined by neighboring residue 283, displayed differential sensitivities to HLA-bound peptide, including the variable HLA-B*57:01-restricted HIV-1 Gag-derived epitope TW10. Residue 283, which has undergone positive selection during the evolution of human KIRs, also played a central role in Bw4 subtype recognition by KIR3DL1. Collectively, our findings uncover a common molecular regulator that controls HLA and peptide discrimination without participating directly in peptide-laden HLA interactions. Furthermore, they provide insight into the mechanics of interaction and generate simple, easily assessed criteria for the definition of KIR3DL1 functional groupings that will be relevant in many clinical applications, including bone marrow transplantation.

  20. Structural Studies of a Rationally Selected Multi-Drug Resistant HIV-1 Protease Reveal Synergistic Effect of Distal Mutations on Flap Dynamics.

    PubMed

    Agniswamy, Johnson; Louis, John M; Roche, Julien; Harrison, Robert W; Weber, Irene T

    2016-01-01

    We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker than PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir.

  1. Structural Studies of a Rationally Selected Multi-Drug Resistant HIV-1 Protease Reveal Synergistic Effect of Distal Mutations on Flap Dynamics

    SciTech Connect

    Agniswamy, Johnson; Louis, John M.; Roche, Julien; Harrison, Robert W.; Weber, Irene T.; Sluis-Cremer, Nicolas

    2016-12-16

    We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker than PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir.

  2. Structural Studies of a Rationally Selected Multi-Drug Resistant HIV-1 Protease Reveal Synergistic Effect of Distal Mutations on Flap Dynamics

    PubMed Central

    Agniswamy, Johnson; Louis, John M.; Roche, Julien; Harrison, Robert W.

    2016-01-01

    We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker than PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir. PMID:27992544

  3. Older paternal age and fresh gene mutation: data on additional disorders.

    PubMed

    Jones, K L; Smith, D W; Harvey, M A; Hall, B D; Quan, L

    1975-01-01

    Older paternal age has previously been documented as a factor in sporadic fresh mutational cases of several autosomal dominant disorders. In this collaborative study, an older mean paternal age has been documented in sporadic cases of at least five additional dominantly inheritable disorders; the basal cell nevus syndrome, the Waardenburg syndrome, the Crouzon syndrome, the oculo-dental-digital sysdrome, and the Treacher-Collins syndrome. It was also found to be a factor in acrodysostosis and progeria, suggesting a fresh mutant gene etiology for these two conditions in which virtually all cases have been sporadic and the mode of genetic etiology has been unknown.

  4. Genomic profiling of stage II and III colon cancers reveals APC mutations to be associated with survival in stage III colon cancer patients

    PubMed Central

    van den Broek, Evert; Krijgsman, Oscar; Sie, Daoud; Tijssen, Marianne; Mongera, Sandra; van de Wiel, Mark A.; Th. Belt, Eric J.; den Uil, Sjoerd H.; Bril, Herman; Stockmann, Hein B.A.C.; Ylstra, Bauke; Carvalho, Beatriz; Meijer, Gerrit A.; Fijneman, Remond J.A.

    2016-01-01

    Tumor profiling of DNA alterations, i.e. gene point mutations, somatic copy number aberrations (CNAs) and structural variants (SVs), improves insight into the molecular pathology of cancer and clinical outcome. Here, associations between genomic aberrations and disease recurrence in stage II and III colon cancers were investigated. A series of 114 stage II and III microsatellite stable colon cancer samples were analyzed by high-resolution array-comparative genomic hybridization (array-CGH) to detect CNAs and CNA-associated chromosomal breakpoints (SVs). For 60 of these samples mutation status of APC, TP53, KRAS, PIK3CA, FBXW7, SMAD4, BRAF and NRAS was determined using targeted massive parallel sequencing. Loss of chromosome 18q12.1-18q12.2 occurred more frequently in tumors that relapsed than in relapse-free tumors (p < 0.001; FDR = 0.13). In total, 267 genes were recurrently affected by SVs (FDR < 0.1). CNAs and SVs were not associated with disease-free survival (DFS). Mutations in APC and TP53 were associated with increased CNAs. APC mutations were associated with poor prognosis in (5-fluorouracil treated) stage III colon cancers (p = 0.005; HR = 4.1), an effect that was further enhanced by mutations in MAPK pathway (KRAS, NRAS, BRAF) genes. We conclude that among multiple genomic alterations in CRC, strongest associations with clinical outcome were observed for common mutations in APC. PMID:27729614

  5. Pancreatic endocrine tumours: mutational and immunohistochemical survey of protein kinases reveals alterations in targetable kinases in cancer cell lines and rare primaries

    PubMed Central

    Corbo, V.; Beghelli, S.; Bersani, S.; Antonello, D.; Talamini, G.; Brunelli, M.; Capelli, P.; Falconi, M.; Scarpa, A.

    2012-01-01

    Background: Kinases represent potential therapeutic targets in pancreatic endocrine tumours (PETs). Patients and methods: Thirty-five kinase genes were sequenced in 36 primary PETs and three PET cell lines: (i) 4 receptor tyrosine kinases (RTK), epithelial growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tyrosine-protein kinase KIT (KIT), platelet-derived growth factor receptor alpha (PDGFRalpha); (ii) 6 belonging to the Akt/mTOR pathway; and (iii) 25 frequently mutated in cancers. The immunohistochemical expression of the four RTKs and the copy number of EGFR and HER2 were assessed in 140 PETs. Results: Somatic mutations were found in KIT in one and ATM in two primary neoplasms. Among 140 PETs, EGFR was immunopositive in 18 (13%), HER2 in 3 (2%), KIT in 16 (11%), and PDGFRalpha in 135 (96%). HER2 amplification was found in 2/130 (1.5%) PETs. KIT membrane immunostaining was significantly associated with tumour aggressiveness and shorter patient survival. PET cell lines QGP1, CM and BON harboured mutations in FGFR3, FLT1/VEGFR1 and PIK3CA, respectively. Conclusions: Only rare PET cases, harbouring either HER2 amplification or KIT mutation, might benefit from targeted drugs. KIT membrane expression deserves further attention as a prognostic marker. ATM mutation is involved in a proportion of PET. The finding of specific mutations in PET cell lines renders these models useful for preclinical studies involving pathway-specific therapies. PMID:21447618

  6. Loss of H3K9me3 Correlates with ATM Activation and Histone H2AX Phosphorylation Deficiencies in Hutchinson-Gilford Progeria Syndrome

    PubMed Central

    Zhang, Haoyue; Sun, Linlin; Wang, Kun; Wu, Di; Trappio, Mason; Witting, Celeste; Cao, Kan

    2016-01-01

    Compelling evidence suggests that defective DNA damage response (DDR) plays a key role in the premature aging phenotypes in Hutchinson-Gilford progeria syndrome (HGPS). Studies document widespread alterations in histone modifications in HGPS cells, especially, the global loss of histone H3 trimethylated on lysine 9 (H3K9me3). In this study, we explore the potential connection(s) between H3K9me3 loss and the impaired DDR in HGPS. When cells are exposed to a DNA-damaging agent Doxorubicin (Dox), double strand breaks (DSBs) are generated that result in the phosphorylation of histone H2A variant H2AX (gammaH2AX) within an hour. We find that the intensities of gammaH2AX foci appear significantly weaker in the G0/G1 phase HGPS cells compared to control cells. This reduction is associated with a delay in the recruitment of essential DDR factors. We further demonstrate that ataxia-telangiectasia mutated (ATM) is responsible for the amplification of gammaH2AX signals at DSBs during G0/G1 phase, and its activation is inhibited in the HGPS cells that display significant loss of H3K9me3. Moreover, methylene (MB) blue treatment, which is known to save heterochromatin loss in HGPS, restores H3K9me3, stimulates ATM activity, increases gammaH2AX signals and rescues deficient DDR. In summary, this study demonstrates an early DDR defect of attenuated gammaH2AX signals in G0/G1 phase HGPS cells and provides a plausible connection between H3K9me3 loss and DDR deficiency. PMID:27907109

  7. Loss of H3K9me3 Correlates with ATM Activation and Histone H2AX Phosphorylation Deficiencies in Hutchinson-Gilford Progeria Syndrome.

    PubMed

    Zhang, Haoyue; Sun, Linlin; Wang, Kun; Wu, Di; Trappio, Mason; Witting, Celeste; Cao, Kan

    2016-01-01

    Compelling evidence suggests that defective DNA damage response (DDR) plays a key role in the premature aging phenotypes in Hutchinson-Gilford progeria syndrome (HGPS). Studies document widespread alterations in histone modifications in HGPS cells, especially, the global loss of histone H3 trimethylated on lysine 9 (H3K9me3). In this study, we explore the potential connection(s) between H3K9me3 loss and the impaired DDR in HGPS. When cells are exposed to a DNA-damaging agent Doxorubicin (Dox), double strand breaks (DSBs) are generated that result in the phosphorylation of histone H2A variant H2AX (gammaH2AX) within an hour. We find that the intensities of gammaH2AX foci appear significantly weaker in the G0/G1 phase HGPS cells compared to control cells. This reduction is associated with a delay in the recruitment of essential DDR factors. We further demonstrate that ataxia-telangiectasia mutated (ATM) is responsible for the amplification of gammaH2AX signals at DSBs during G0/G1 phase, and its activation is inhibited in the HGPS cells that display significant loss of H3K9me3. Moreover, methylene (MB) blue treatment, which is known to save heterochromatin loss in HGPS, restores H3K9me3, stimulates ATM activity, increases gammaH2AX signals and rescues deficient DDR. In summary, this study demonstrates an early DDR defect of attenuated gammaH2AX signals in G0/G1 phase HGPS cells and provides a plausible connection between H3K9me3 loss and DDR deficiency.

  8. Exome sequencing reveals a novel Moroccan founder mutation in SLC19A3 as a new cause of early-childhood fatal Leigh syndrome.

    PubMed

    Gerards, Mike; Kamps, Rick; van Oevelen, Jo; Boesten, Iris; Jongen, Eveline; de Koning, Bart; Scholte, Hans R; de Angst, Isabel; Schoonderwoerd, Kees; Sefiani, Abdelaziz; Ratbi, Ilham; Coppieters, Wouter; Karim, Latifa; de Coo, René; van den Bosch, Bianca; Smeets, Hubert

    2013-03-01

    Leigh syndrome is an early onset, often fatal progressive neurodegenerative disorder caused by mutations in the mitochondrial or nuclear DNA. Until now, mutations in more than 35 genes have been reported to cause Leigh syndrome, indicating an extreme genetic heterogeneity for this disorder, but still only explaining part of the cases. The possibility of whole exome sequencing enables not only mutation detection in known candidate genes, but also the identification of new genes associated with Leigh syndrome in small families and isolated cases. Exome sequencing was combined with homozygosity mapping to identify the genetic defect in a Moroccan family with fatal Leigh syndrome in early childhood and specific magnetic resonance imaging abnormalities in the brain. We detected a homozygous nonsense mutation (c.20C>A; p.Ser7Ter) in the thiamine transporter SLC19A3. In vivo overexpression of wild-type SLC19A3 showed an increased thiamine uptake, whereas overexpression of mutant SLC19A3 did not, confirming that the mutation results in an absent or non-functional protein. Seventeen additional patients with Leigh syndrome were screened for mutations in SLC19A3 using conventional Sanger sequencing. Two unrelated patients, both from Moroccan origin and one from consanguineous parents, were homozygous for the same p.Ser7Ter mutation. One of these patients showed the same MRI abnormalities as the patients from the first family. Strikingly, patients receiving thiamine had an improved life-expectancy. One patient in the third family deteriorated upon interruption of the thiamine treatment and recovered after reinitiating. Although unrelated, all patients came from the province Al Hoceima in Northern Morocco. Based on the recombination events the mutation was estimated to have occurred 1250-1750 years ago. Our data shows that SLC19A3 is a new candidate for mutation screening in patients with Leigh syndrome, who might benefit from high doses of thiamine and/or biotin. Especially

  9. B-cell receptor configuration and mutational analysis of patients with chronic lymphocytic leukaemia and trisomy 12 reveal recurrent molecular abnormalities.

    PubMed

    Falisi, Erika; Novella, Elisabetta; Visco, Carlo; Guercini, Nicola; Maura, Francesco; Giaretta, Ilaria; Pomponi, Fabrizio; Nichele, Ilaria; Finotto, Silvia; Montaldi, Annamaria; Neri, Antonino; Rodeghiero, Francesco

    2014-03-01

    Trisomy 12 (+12) is the third most frequent cytogenetic aberration in chronic lymphocytic leukaemia (CLL) retrievable both as the sole chromosomal abnormality or in association with additional alterations. NOTCH1 mutations are known to be more prevalent among +12 patients, whereas mutations of FBXW7, a gene involved in NOTCH1 degradation, that lead to the constitutional activation of NOTCH1 have not been investigated in this setting. We analyzed a unicentric cohort of 44 +12 patients with CLL for mutations of TP53, NOTCH1 and FBXW7 genes, and we correlated them with B-cell receptor (BCR) configurations. FBXW7, TP53 and NOTCH1 mutations were identified in 4.5%, 6.8% and 18.2% of patients, respectively. FBXW7 and NOTCH1 mutations appeared in a mutually exclusive fashion, suggesting that both aberrations might affect the same biological pathway. We found that 44.1% of +12 CLL patients had stereotyped B-cell receptors, which is significantly higher than that observed in patients with CLL and no +12 (27%, p = 0.01). Subsets #1, #8, #10, #28 and #59 were the most represented stereotyped patterns, and IGHV4-39*01 was the gene configuration most commonly used. There was a significantly higher risk for Richter's syndrome (RS) transformation in patients with NOTCH1 or FBXW7 mutations, with four of the seven (57%) patients developing RS and characterized at least by one of the two abnormalities. These observations suggest that, similarly to the aberrations of NOTCH1, FBXW7 gene mutations may also result in cell proliferation and evasion from apoptosis in patients with +12 CLL. Together with the extremely high frequency of stereotyped BCRs and RS transformation, these abnormalities appear to cluster in these CLL patients with additional chromosome 12, suggesting a connection with the prognosis of the disease.

  10. Next Generation Sequencing of Cytokeratin 20-Negative Merkel Cell Carcinoma Reveals Ultraviolet Signature Mutations and Recurrent TP53 and RB1 Inactivation

    PubMed Central

    Harms, Paul W.; Collie, Angela M. B.; Hovelson, Daniel H.; Cani, Andi K.; Verhaegen, Monique E.; Patel, Rajiv M.; Fullen, Douglas R.; Omata, Kei; Dlugosz, Andrzej A.; Tomlins, Scott A.; Billings, Steven D.

    2016-01-01

    Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin-20 (CK20) is expressed in approximately 95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (ten Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%)) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping

  11. Embryonic mutation as a possible cause of in utero carcinogenesis in mice revealed by postnatal treatment with 12-O-tetradecanoylphorbol-13-acetate

    SciTech Connect

    Nomura, T.; Nakajima, H.; Hatanaka, T.; Kinuta, M.; Hongyo, T. )

    1990-04-01

    Although in utero irradiation at early stages induced a high incidence of somatic mutations at coat color genes in the embryos of a specified tester strain (PT x HT F1) of mice, it was not carcinogenic by itself. However, in utero-irradiated animals did develop skin tumors and hepatomas (but not leukemias) by the postnatal administration of 12-O-tetradecanoylphorbol-13-acetate. The incidence of both tumors and embryonic mutations increased with in utero doses of X-rays. Furthermore, a large reduction of tumor incidence, about 80%, was observed by low-dose-rate irradiation, similar to the 75% reduction in spot size found for embryonic mutations. The tumor nodule size was also dramatically reduced by low-dose-rate irradiation. Consequently, the induced incidence and size of tumors produced by 12-O-tetradecanoylphorbol-13-acetate treatment parallel those which are observed for coat color mutations as expected, because somatic mutations observed in the pigment cells must similarly occur in embryonic cells of other organs. The larger the clone of mutant cells, the greater their chance of becoming tumorigenic by 12-O-tetradecanoylphorbol-13-acetate posttreatment. These results strongly support the recent epidemiological survey showing that adult types of cancers, but not leukemias, are increasing in the atomic bomb survivors exposed in utero, since humans are continuously exposed to a variety of cancer-promoting agents in contrast to experimental animals reared without such exposures.

  12. Direct resequencing of the complete ERBB2 coding sequence reveals an absence of activating mutations in ERBB2 amplified breast cancer.

    PubMed

    Zito, Christina I; Riches, David; Kolmakova, Julia; Simons, Jan; Egholm, Michael; Stern, David F

    2008-07-01

    Gene amplification is among the most common genetic abnormalities that cause cancer. One of the most clinically important gene amplifications in human cancer causes extensive reduplication of ERBB2. A variety of cancers also occasionally harbor somatic mutations in ERBB2. Gene amplification and activating mutations both have predictive value for clinical response to targeted inhibitors. Since the number of gene copies in an amplicon may exceed 100, and since amplicons may encompass multiple genes, high-resolution analysis of gene amplifications poses considerable technical challenges. We have overcome this obstacle by using emulsion-based resequencing to determine the sequence of many independently-amplified individual DNA molecules in parallel. We used this high throughput sequencing technology to analyze ERBB2 mutational status in five ERBB2 amplified cell lines (four breast, one ovarian) and two breast tumors. Genomic DNA was isolated and the 28 exons of ERBB2 were independently amplified. Amplicons were then pooled at equimolar ratios, subjected to emulsion PCR (emPCR) and finally to picotiter plate pyrosequencing. High-quality sequence data were obtained for all amplicons analyzed and no activating mutations within ERBB2 were identified. Although we did not find activating mutations within the multiple copies of ERBB2 in these samples, the results establish the utility of this technology as a feasible and cost-effective approach for high resolution analysis of amplified genes.

  13. A cis-regulatory mutation in troponin-I of Drosophila reveals the importance of proper stoichiometry of structural proteins during muscle assembly.

    PubMed

    Firdaus, Hena; Mohan, Jayaram; Naz, Sarwat; Arathi, Prabhashankar; Ramesh, Saraf R; Nongthomba, Upendra

    2015-05-01

    Rapid and high wing-beat frequencies achieved during insect flight are powered by the indirect flight muscles, the largest group of muscles present in the thorax. Any anomaly during the assembly and/or structural impairment of the indirect flight muscles gives rise to a flightless phenotype. Multiple mutagenesis screens in Drosophila melanogaster for defective flight behavior have led to the isolation and characterization of mutations that have been instrumental in the identification of many proteins and residues that are important for muscle assembly, function, and disease. In this article, we present a molecular-genetic characterization of a flightless mutation, flightless-H (fliH), originally designated as heldup-a (hdp-a). We show that fliH is a cis-regulatory mutation of the wings up A (wupA) gene, which codes for the troponin-I protein, one of the troponin complex proteins, involved in regulation of muscle contraction. The mutation leads to reduced levels of troponin-I transcript and protein. In addition to this, there is also coordinated reduction in transcript and protein levels of other structural protein isoforms that are part of the troponin complex. The altered transcript and protein stoichiometry ultimately culminates in unregulated acto-myosin interactions and a hypercontraction muscle phenotype. Our results shed new insights into the importance of maintaining the stoichiometry of structural proteins during muscle assembly for proper function with implications for the identification of mutations and disease phenotypes in other species, including humans.

  14. Hybridisation-based resequencing of 17 X-linked intellectual disability genes in 135 patients reveals novel mutations in ATRX, SLC6A8 and PQBP1

    PubMed Central

    Jensen, Lars R; Chen, Wei; Moser, Bettina; Lipkowitz, Bettina; Schroeder, Christopher; Musante, Luciana; Tzschach, Andreas; Kalscheuer, Vera M; Meloni, Ilaria; Raynaud, Martine; van Esch, Hilde; Chelly, Jamel; de Brouwer, Arjan P M; Hackett, Anna; van der Haar, Sigrun; Henn, Wolfram; Gecz, Jozef; Riess, Olaf; Bonin, Michael; Reinhardt, Richard; Ropers, Hans-Hilger; Kuss, Andreas W

    2011-01-01

    X-linked intellectual disability (XLID), also known as X-linked mental retardation, is a highly genetically heterogeneous condition for which mutations in >90 different genes have been identified. In this study, we used a custom-made sequencing array based on the Affymetrix 50k platform for mutation screening in 17 known XLID genes in patients from 135 families and found eight single-nucleotide changes that were absent in controls. For four mutations affecting ATRX (p.1761M>T), PQBP1 (p.155R>X) and SLC6A8 (p.390P>L and p.477S>L), we provide evidence for a functional involvement of these changes in the aetiology of intellectual disability. PMID:21267006

  15. Mutational Analysis of the Myxococcus xanthus Ω4499 Promoter Region Reveals Shared and Unique Properties in Comparison with Other C-Signal-Dependent Promoters

    PubMed Central

    Yoder, Deborah R.; Kroos, Lee

    2004-01-01

    The bacterium Myxococcus xanthus undergoes multicellular development during times of nutritional stress and uses extracellular signals to coordinate cell behavior. C-signal affects gene expression late in development, including that of Ω4499, an operon identified by insertion of Tn5 lac into the M. xanthus chromosome. The Ω4499 promoter region has several sequences in common with those found previously to be important for expression of other C-signal-dependent promoters. To determine if these sequences are important for Ω4499 promoter activity, the effects of mutations on expression of a downstream reporter gene were tested in M. xanthus. Although the promoter resembles those recognized by Escherichia coli σ54, mutational analysis implied that a σ70-type σ factor likely recognizes the promoter. A 7-bp sequence known as a C box and a 5-bp element located 6 bp upstream of the C box have been shown to be important for expression of other C-signal-dependent promoters. The Ω4499 promoter region has C boxes centered at −33 and −55 bp, with 5-bp elements located 7 and 8 bp upstream, respectively. A multiple-base-pair mutation in any of these sequences reduced Ω4499 promoter activity more than twofold. Single base-pair mutations in the C box centered at −33 bp yielded a different pattern of effects on expression than similar mutations in other C boxes, indicating that each functions somewhat differently. An element from about −81 to −77 bp exerted a twofold positive effect on expression but did not appear to be responsible for the C-signal dependence of the Ω4499 promoter. Mutations in sigD and sigE, which are genes that encode σ factors, reduced expression from the Ω4499 promoter. The results provide further insight into the regulation of C-signal-dependent genes, demonstrating both shared and unique properties among the promoter regions so far examined. PMID:15175290

  16. Novel Candidate Genes and a Wide Spectrum of Structural and Point Mutations Responsible for Inherited Retinal Dystrophies Revealed by Exome Sequencing

    PubMed Central

    de Castro-Miró, Marta; Tonda, Raul; Escudero-Ferruz, Paula; Andrés, Rosa; Mayor-Lorenzo, Andrés; Castro, Joaquín; Ciccioli, Marcela; Hidalgo, Daniel A.; Rodríguez-Ezcurra, Juan José; Farrando, Jorge; Pérez-Santonja, Juan J.; Cormand, Bru; Marfany, Gemma

    2016-01-01

    Background NGS-based genetic diagnosis has completely revolutionized the human genetics field. In this study, we have aimed to identify new genes and mutations by Whole Exome Sequencing (WES) responsible for inherited retinal dystrophies (IRD). Methods A cohort of 33 pedigrees affected with a variety of retinal disorders was analysed by WES. Initial prioritization analysis included around 300 IRD-associated genes. In non-diagnosed families a search for pathogenic mutations in novel genes was undertaken. Results Genetic diagnosis was attained in 18 families. Moreover, a plausible candidate is proposed for 10 more cases. Two thirds of the mutations were novel, including 4 chromosomal rearrangements, which expand the IRD allelic heterogeneity and highlight the contribution of private mutations. Our results prompted clinical re-evaluation of some patients resulting in assignment to a syndromic instead of non-syndromic IRD. Notably, WES unveiled four new candidates for non-syndromic IRD: SEMA6B, CEP78, CEP250, SCLT1, the two latter previously associated to syndromic disorders. We provide functional data supporting that missense mutations in CEP250 alter cilia formation. Conclusion The diagnostic efficiency of WES, and strictly following the ACMG/AMP criteria is 55% in reported causative genes or functionally supported new candidates, plus 30% families in which likely pathogenic or VGUS/VUS variants were identified in plausible candidates. Our results highlight the clinical utility of WES for molecular diagnosis of IRD, provide a wider spectrum of mutations and concomitant genetic variants, and challenge our view on syndromic vs non-syndromic, and causative vs modifier genes. PMID:28005958

  17. Increased progerin expression associated with unusual LMNA mutations causes severe progeroid syndromes.

    PubMed

    Moulson, Casey L; Fong, Loren G; Gardner, Jennifer M; Farber, Emily A; Go, Gloriosa; Passariello, Annalisa; Grange, Dorothy K; Young, Stephen G; Miner, Jeffrey H

    2007-09-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare precocious aging syndrome caused by mutations in LMNA that lead to synthesis of a mutant form of prelamin A, generally called progerin, that cannot be processed to mature lamin A. Most HGPS patients have a recurrent heterozygous de novo mutation in exon 11 of LMNA, c.1824C>T/p.G608G; this synonymous mutation activates a nearby cryptic splice donor site, resulting in synthesis of the mutant prelamin A, progerin, which lacks 50 amino acids within the carboxyl-terminal domain. Abnormal splicing is incomplete, so the mutant allele produces some normally-spliced transcripts. Nevertheless, the synthesis of progerin is sufficient to cause misshapen nuclei in cultured cells and severe disease phenotypes in affected patients. Here we present two patients with extraordinarily severe forms of progeria caused by unusual mutations in LMNA. One had a splice site mutation (c.1968+1G>A; or IVS11+1G>A), and the other had a novel synonymous coding region mutation (c.1821G>A/p.V607V). Both mutations caused very frequent use of the same exon 11 splice donor site that is activated in typical HGPS patients. As a consequence, the ratios of progerin mRNA and protein to wild-type were higher than in typical HGPS patients. Fibroblasts from both patients exhibited nuclear shape abnormalities typical of HGPS, and cells treated with a protein farnesyltransferase inhibitor exhibited fewer misshapen nuclei. Thus, farnesyltransferase inhibitors may prove to be useful even when progerin expression levels are higher than those in typical HGPS patients.

  18. An exome sequencing study of Moebius syndrome including atypical cases reveals an individual with CFEOM3A and a TUBB3 mutation

    PubMed Central

    Liu, David; Gonzaga-Jauregui, Claudia; Jhangiani, Shalini; Lu, James T.; Sutton, V. Reid; Fernbach, Susan D.; Azamian, Mahshid; White, Lisa; Edmond, Jane C.; Paysse, Evelyn A.; Belmont, John W.; Muzny, Donna; Lupski, James R.; Gibbs, Richard A.; Lewis, Richard Alan; Lee, Brendan H.; Lalani, Seema R.

    2017-01-01

    Moebius syndrome is characterized by congenital unilateral or bilateral facial and abducens nerve palsies (sixth and seventh cranial nerves) causing facial weakness, feeding difficulties, and restricted ocular movements. Abnormalities of the chest wall such as Poland anomaly and variable limb defects are frequently associated with this syndrome. Most cases are isolated; however, rare families with autosomal dominant transmission with incomplete penetrance and variable expressivity have been described. The genetic basis of this condition remains unknown. In a cohort study of nine individuals suspected to have Moebius syndrome (six typical, three atypical), we performed whole-exome sequencing to try to identify a commonly mutated gene. Although no such gene was identified and we did not find mutations in PLXND1 and REV3L, we found a de novo heterozygous mutation, p.E410K, in the gene encoding tubulin beta 3 class III (TUBB3), in an individual with atypical Moebius syndrome. This individual was diagnosed with near-complete ophthalmoplegia, agenesis of the corpus callosum, and absence of the septum pellucidum. No substantial limb abnormalities were noted. Mutations in TUBB3 have been associated with complex cortical dysplasia and other brain malformations and congenital fibrosis of extraocular muscles type 3A (CFEOM3A). Our report highlights the overlap of genetic etiology and clinical differences between CFEOM and Moebius syndrome and describes our approach to identifying candidate genes for typical and atypical Moebius syndrome. PMID:28299356

  19. Rett-causing mutations reveal two domains critical for MeCP2 function and for toxicity in MECP2 duplication syndrome mice.

    PubMed

    Heckman, Laura Dean; Chahrour, Maria H; Zoghbi, Huda Y

    2014-06-26

    Loss of function of the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2) causes the progressive neurological disorder Rett syndrome (RTT). Conversely, duplication or triplication of Xq28 causes an equally wide-ranging progressive neurological disorder, MECP2 duplication syndrome, whose features overlap somewhat with RTT. To understand which MeCP2 functions cause toxicity in the duplication syndrome, we generated mouse models expressing endogenous Mecp2 along with a RTT-causing mutation in either the methyl-CpG binding domain (MBD) or the transcriptional repression domain (TRD). We determined that both the MBD and TRD must function for doubling MeCP2 to be toxic. Mutating the MBD reproduces the null phenotype and expressing the TRD mutant produces milder RTT phenotypes, yet both mutations are harmless when expressed with endogenous Mecp2. Surprisingly, mutating the TRD is more detrimental than deleting the entire C-terminus, indicating a dominant-negative effect on MeCP2 function, likely due to the disruption of a basic cluster.

  20. Global expression profiling reveals gain-of-function onco-genic activity of a mutated thyroid hormone receptor in thyroid carcinogenesis

    PubMed Central

    Lu, Changxue; Mishra, Alok; Zhu, Yuelin J; Meltzer, Paul; Cheng, Sheue-yann

    2011-01-01

    Thyroid hormone receptors (TRs) are critical in regulating gene expression in normal physiological processes. Decreased expression and/or somatic mutations of TRs have been shown to be associated several types of human cancers including liver, breast, lung, and thyroid. To understand the molecular mechanisms by which mutated TRs promote carcinogenesis, an animal model of follicular thyroid carcinoma (FTC) (Thrbpv/pv mice) was used in the present study. The Thrbpv/pv mouse harbors a knockin dominant negative PV mutation, identified in a patient with resistance to thyroid hormone. To understand whether oncogenic actions of PV involve not only the loss of normal TR functions but also gain-of-function activities, we compared the gene expression profiles of thyroid lesions in Thrbpv/pv mice and Thra1-/- Thrb-/- mice that also spontaneously develop FTC, but with less severe malignancy. Analysis of the cDNA microarray data derived from microdissected thyroid tumor cells of these two mice showed contrasting global gene expression profiles. With stringent selection using 2.5-fold change (p<0.01) in cDNA microarray analysis, 241 genes with altered gene expression were identified. Nearly half of the genes (n=103: 42.7% of total) with altered gene expression in thyroid tumor cells of Thrbpv/pv mice were associated with tumorigenesis and metastasis; some of these genes function as oncogenes in human thyroid cancers. The remaining genes were found to function in transcriptional regulation, RNA processing, cell proliferation, apoptosis, angiogenesis, and cytoskeleton modification. These results indicate that the more aggressive thyroid tumor progression in Thrbpv/pv mice was not due simply to the loss of tumor suppressor functions of TR via mutation but also, importantly, to gain-of-function in the oncogenic activities of PV to drive thyroid carcinogenesis. Thus, the present study identifies a novel mechanism by which a mutated TRβ evolves with an oncogenic advantage to promote

  1. Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

    PubMed

    Agarwal, A; MacKenzie, R J; Eide, C A; Davare, M A; Watanabe-Smith, K; Tognon, C E; Mongoue-Tchokote, S; Park, B; Braziel, R M; Tyner, J W; Druker, B J

    2015-06-04

    To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2Rγ) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2Rγ abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2Rγ in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2Rγ completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2Rγ interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2Rγ, indicating IL2Rγ and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2Rγ in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth.

  2. Crystal structure of type I ryanodine receptor amino-terminal [beta]-trefoil domain reveals a disease-associated mutation 'hot spot' loop

    SciTech Connect

    Amador, Fernando J.; Liu, Shuang; Ishiyama, Noboru; Plevin, Michael J.; Wilson, Aaron; MacLennan, David H.; Ikura, Mitsuhiko

    2009-12-01

    Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca{sup 2+}. Ca{sup 2+} is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca{sup 2+} transient by activation of a class of Ca{sup 2+} release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca{sup 2+}-ATPases (SERCAs) to terminate the Ca{sup 2+} transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyR{sub NTD}) at 2.5 {angstrom}. The RyR{sub NTD} structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP3Rsup), but lacks most of the long helix-turn-helix segment of the 'arm' domain in IP3Rsup. The N-terminal {beta}-trefoil fold, found in both RyR and IP{sub 3}R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation 'hot spot' loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyRNTD, supporting previously described mechanisms whereby mutations perturb protein-protein interactions.

  3. cDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark.

    PubMed

    Duno, Morten; Sveen, Marie-Louise; Schwartz, Marianne; Vissing, John

    2008-08-01

    Calpainopathy or limb-girdle muscular dystrophy type 2A (LGMD2A) is generally recognized as the most prevalent form of recessive LGMD and is caused by mutations in the CAPN3 gene. Out of a cohort of 119 patients fulfilling clinical criteria for LGMD2, referred to our neuromuscular clinic, 46 were suspected to have LGMD2A, based on western blot results. Four of these patients were shown to have LGMD2I upon molecular analysis, whereas 16 of the remaining 42 patients harbored mutations in CAPN3 by both direct genomic sequencing and cDNA analyses. In 10 patients, we identified both mutant alleles. In three other, only one heterozygous mutation could be identified on the genomic level; however, CAPN3 cDNA analyses demonstrated homozygosity for the mutant allele, indicating the presence of an unidentified allele that somehow compromise correct CAPN3 RNA processing. In the three remaining patients, only a single heterozygous mutation could be identified both at the genomic level and on full-length CAPN3 cDNA. All three patients exhibited a highly abnormal western blot for calpain-3 and clinical characteristics of LGMD2A. Only three of the genetically confirmed LGMD2A patients were of Danish origin, indicating a five- to sixfold lower prevalence in Denmark compared to other European countries. A total of 16 different CAPN3 mutations were identified, of which 5 were novel. The present study demonstrates the value of cDNA analysis for CAPN3 in LGMD2A patients and indicates that calpainopathy is an uncommon cause of LGMD in the Denmark.

  4. Analysis of Mutations in Streptomycin-Resistant Strains Reveals a Simple and Reliable Genetic Marker for Identification of the Mycobacterium tuberculosis Beijing Genotype

    PubMed Central

    Villellas, Cristina; Aristimuño, Liselotte; Vitoria, María-Asunción; Prat, Cristina; Blanco, Silvia; García de Viedma, Darío; Domínguez, José; Samper, Sofía

    2013-01-01

    The Mycobacterium tuberculosis pandemic is a major health problem, further complicated by an increasing incidence of drug-resistant isolates and the existence of highly transmissible strains, such as those in the Beijing family. Streptomycin (STR)-resistant M. tuberculosis clinical isolates have been analyzed to look for mutations in the rpsL, rrs, and gidB genes. In addition, the Rv1258c gene, which encodes Tap, an efflux pump that transports STR, has been sequenced. Mutations affecting codons 43 and 88 of the rpsL gene were found in 44.4% of the strains, and 16.7% of the strains carried mutations in the rrs gene, both of which probably contribute to STR resistance. Many strains presented with mutations in the gidB gene, but the implication of those mutations in STR resistance remains unclear. Interestingly, a cytosine nucleotide insertion between positions 580 and 581 (denominated Tap580) in the Rv1258c gene has been found in all Beijing isolates included in this study, suggesting that it might be a novel polymorphism specific to the Beijing family of M. tuberculosis. A simple and fast restriction fragment length polymorphism (RFLP)-PCR method for detecting the Tap580 insertion has been developed and used to screen a collection of 220 DNA samples obtained from cultures of M. tuberculosis isolates and 30 respiratory specimens. In all cases, the Beijing and non-Beijing representative samples were identified correctly. Tap580 is a novel polymorphism specific to the highly transmissible Beijing family, which allows for fast detection of these strains even at the very early stages of infection. PMID:23616454

  5. Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components.

    PubMed

    Şahin, Aslı; Held, Aaron; Bredvik, Kirsten; Major, Paxton; Achilli, Toni-Marie; Kerson, Abigail G; Wharton, Kristi; Stilwell, Geoff; Reenan, Robert

    2017-02-01

    Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc superoxide dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, we have genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y, and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical, and behavioral phenotypes. Contrary to previous transgenic models, we have recapitulated ALS-like phenotypes without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R, and H71Y) exhibited neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) was phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes were recessive, a gain-of-function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which failed to show severe locomotor defects or nerve degeneration. We conclude that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies.

  6. Cardiovascular Pathology in Hutchinson-Gilford Progeria: Correlation with the Vascular Pathology of Aging

    PubMed Central

    Olive, Michelle; Harten, Ingrid; Mitchell, Richard; Beers, Jeanette; Djabali, Karima; Cao, Kan; Erdos, Michael R.; Blair, Cecilia; Funke, Birgit; Smoot, Leslie; Gerhard-Herman, Marie; Machan, Jason T.; Kutys, Robert; Virmani, Renu; Collins, Francis S.; Wight, Thomas N.; Nabel, Elizabeth G.; Gordon, Leslie B.

    2010-01-01

    Objective Children with Hutchinson–Gilford progeria syndrome (HGPS) exhibit dramatically accelerated cardiovascular disease (CVD) causing death from myocardial infarction or stroke between ages 7 and 20 years. We undertook the first histological comparative evaluation between genetically confirmed HGPS and the CVD of aging. Methods and Results We present structural and immunohistological analysis of cardiovascular tissues from two children with HGPS, who died of myocardial infarction. Both had features classically associated with the atherosclerosis of aging, as well as arteriolosclerosis of small vessels. Additionally, vessels exhibited prominent adventitial fibrosis, a previously undescribed feature of HGPS. Importantly, though progerin was detected at higher rates in the HGPS coronary arteries, it was also present in non-HGPS individuals. Between ages one month and 97 years, progerin staining increased an average of 3.34% per year (P<0.0001) in coronary arteries. Conclusion We find concordance between many aspects of cardiovascular pathology in both HGPS and geriatric patients. HGPS generates a more prominent adventitial fibrosis than typical CVD. Vascular progerin generation in young non-HGPS individuals, which significantly increases throughout life, strongly suggests that progerin has a role in the CV aging of the general population. PMID:20798379

  7. A pathway linking oxidative stress and the Ran GTPase system in progeria.

    PubMed

    Datta, Sutirtha; Snow, Chelsi J; Paschal, Bryce M

    2014-04-01

    Maintaining the Ran GTPase at a proper concentration in the nucleus is important for nucleocytoplasmic transport. Previously we found that nuclear levels of Ran are reduced in cells from patients with Hutchinson-Gilford progeria syndrome (HGPS), a disease caused by constitutive attachment of a mutant form of lamin A (termed progerin) to the nuclear membrane. Here we explore the relationship between progerin, the Ran GTPase, and oxidative stress. Stable attachment of progerin to the nuclear membrane disrupts the Ran gradient and results in cytoplasmic localization of Ubc9, a Ran-dependent import cargo. Ran and Ubc9 disruption can be induced reversibly with H2O2. CHO cells preadapted to oxidative stress resist the effects of progerin on Ran and Ubc9. Given that HGPS-patient fibroblasts display elevated ROS, these data suggest that progerin inhibits nuclear transport via oxidative stress. A drug that inhibits pre-lamin A cleavage mimics the effects of progerin by disrupting the Ran gradient, but the effects on Ran are observed before a substantial ROS increase. Moreover, reducing the nuclear concentration of Ran is sufficient to induce ROS irrespective of progerin. We speculate that oxidative stress caused by progerin may occur upstream or downstream of Ran, depending on the cell type and physiological setting.

  8. Metformin Alleviates Aging Cellular Phenotypes in Hutchinson-Gilford Progeria Syndrome Dermal Fibroblasts.

    PubMed

    Park, Seul-Ki; Shin, Ok Sarah

    2017-02-13

    Metformin is a popular antidiabetic biguanide, which has been considered as a candidate drug for cancer treatment and aging prevention. Hutchinson-Gilford progeria syndrome (HGPS) is a devastating disease characterized by premature aging and severe age-associated complications leading to death. The effects of metformin on HGPS dermal fibroblasts remain largely undefined. In this study, we investigated whether metformin could exert a beneficial effect on nuclear abnormalities and delay senescence in fibroblasts derived from HGPS patients. Metformin treatment partially restored normal nuclear phenotypes, delayed senescence, activated the phosphorylation of AMP-activated protein kinase, and decreased reactive oxygen species formation in HGPS dermal fibroblasts. Interestingly, metformin reduced the number of phosphorylated histone variant H2AX-positive DNA damage foci and suppressed progerin protein expression, compared to the control. Furthermore, metformin-supplemented aged mice showed higher splenocyte proliferation and mRNA expression of the antioxidant enzyme, superoxide dismutase 2 than the control mice. Collectively, our results show that metformin treatment alleviates the nuclear defects and premature aging phenotypes in HGPS fibroblasts. Thus, metformin can be considered a promising therapeutic approach for life extension in HGPS. This article is protected by copyright. All rights reserved.

  9. Age-dependent loss of MMP-3 in Hutchinson-Gilford progeria syndrome.

    PubMed

    Harten, Ingrid A; Zahr, Rima S; Lemire, Joan M; Machan, Jason T; Moses, Marsha A; Doiron, Robert J; Curatolo, Adam S; Rothman, Frank G; Wight, Thomas N; Toole, Bryan P; Gordon, Leslie B

    2011-11-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare, progressive segmental premature aging disease that includes scleroderma-like skin, progressive joint contracture, and atherosclerosis. Affected individuals die prematurely of heart attacks or strokes. Extracellular matrix dysregulation is implicated as a factor in disease progression. We analyzed messenger RNA and protein levels for matrix metalloproteinases (MMPs)-2,-3, and -9 in HGPS primary human dermal fibroblasts using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and gelatin zymography. MMP-3 messenger RNA and protein levels decreased significantly with increasing donor age in HGPS fibroblasts but not in controls. MMP-2 messenger RNA also showed a donor age-dependent decrease in HGPS fibroblasts, but levels of secreted protein were unchanged. MMP-9 was similar in HGPS and control cultures. The decreased MMP-3 may represent a shift in the inherent extracellular matrix-degrading proteolytic balance in favor of matrix deposition in HGPS. This metalloproteinase has the potential to serve as a biomarker of therapeutic efficacy when assessing treatments for HGPS.

  10. A-type lamins and Hutchinson-Gilford progeria syndrome: pathogenesis and therapy.

    PubMed

    Gonzalez, Jose M; Pla, Davinia; Perez-Sala, Dolores; Andres, Vicente

    2011-06-01

    Lamin A and lamin C (A-type lamins, both encoded by the LMNA gene) are major components of the mammalian nuclear lamina, a complex proteinaceous structure that acts as a scaffold for protein complexes that regulate nuclear structure and function. Abnormal accumulation of farnesylated-progerin, a mutant form of prelamin A, plays a key role in the pathogenesis of the Hutchinson-Gilford progeria syndrome (HGPS), a devastating disorder that causes the death of affected children at an average age of 13.5 years, predominantly from premature atherosclerosis and myocardial infarction or stroke. Remarkably, progerin is also present in normal cells and appears to progressively accumulate during aging of non-HGPS cells. Therefore, understanding how this mutant form of lamin A provokes HGPS may shed significant insight into physiological aging. In this review, we discuss recent advances into the pathogenic mechanisms underlying HGPS, the main murine models of the disease, and the therapeutic strategies developed in cellular and animal models with the aim of reducing the accumulation of farnesylated-progerin, as well as their use in clinical trials of HGPS.

  11. Rapamycin reverses cellular phenotypes and enhances mutant protein clearance in Hutchinson-Gilford progeria syndrome cells.

    PubMed

    Cao, Kan; Graziotto, John J; Blair, Cecilia D; Mazzulli, Joseph R; Erdos, Michael R; Krainc, Dimitri; Collins, Francis S

    2011-06-29

    Hutchinson-Gilford progeria syndrome (HGPS) is a lethal genetic disorder characterized by premature aging. HGPS is most commonly caused by a de novo single-nucleotide substitution in the lamin A/C gene (LMNA) that partially activates a cryptic splice donor site in exon 11, producing an abnormal lamin A protein termed progerin. Accumulation of progerin in dividing cells adversely affects the integrity of the nuclear scaffold and leads to nuclear blebbing in cultured cells. Progerin is also produced in normal cells, increasing in abundance as senescence approaches. Here, we report the effect of rapamycin, a macrolide antibiotic that has been implicated in slowing cellular and organismal aging, on the cellular phenotypes of HGPS fibroblasts. Treatment with rapamycin abolished nuclear blebbing, delayed the onset of cellular senescence, and enhanced the degradation of progerin in HGPS cells. Rapamycin also decreased the formation of insoluble progerin aggregates and induced clearance through autophagic mechanisms in normal fibroblasts. Our findings suggest an additional mechanism for the beneficial effects of rapamycin on longevity and encourage the hypothesis that rapamycin treatment could provide clinical benefit for children with HGPS.

  12. Skeletal muscle contractile function and neuromuscular performance in Zmpste24 -/- mice, a murine model of human progeria.

    PubMed

    Greising, Sarah M; Call, Jarrod A; Lund, Troy C; Blazar, Bruce R; Tolar, Jakub; Lowe, Dawn A

    2012-08-01

    Human progeroid syndromes and premature aging mouse models present as segmental, accelerated aging because some tissues and not others are affected. Skeletal muscle is detrimentally changed by normal aging but whether it is an affected tissue in progeria has not been resolved. We hypothesized that mice which mimic Hutchinson-Gilford progeria syndrome would exhibit age-related alterations of skeletal muscle. Zmpste24 (-/-) and Zmpste24 (+/+) littermates were assessed for skeletal muscle functions, histo-morphological characteristics, and ankle joint mechanics. Twenty-four-hour active time, ambulation, grip strength, and whole body tension were evaluated as markers of neuromuscular performance, each of which was at least 33% lower in Zmpste24 (-/-) mice compared with littermates (p < 0.06). Contractile capacity of the posterior leg muscles were not affected in Zmpste24 (-/-) mice, but muscles of the anterior leg were 30-90% weaker than those of Zmpste24 (+/+) mice (p < 0.01). Leg muscles were 32-47% smaller in the Zmpste24 (-/-) mice and contained ~60% greater collagen relative to littermates (p < 0.01). Soleus and extensor digitorum longus muscles of Zmpste24 (-/-) mice had excessive myonuclei and altered fiber size distributions but, otherwise, appeared normal. Ankle range of motion was 70% lower and plantar- and dorsiflexion passive torques were nearly 3-fold greater in Zmpste24 (-/-) than Zmpste24 (+/+) mice (p ≤ 0.01). The combined factors of muscle atrophy, collagen accumulation, and perturbed joint mechanics likely contributed to poor neuromuscular performance and selective muscle weakness displayed by Zmpste24 (-/-)mice. In summary, these characteristics are similar to those of aged mice indicating accelerated aging of skeletal muscle in progeria.

  13. Defective ATM-Kap-1-mediated chromatin remodeling impairs DNA repair and accelerates senescence in progeria mouse model.

    PubMed

    Liu, Baohua; Wang, Zimei; Ghosh, Shrestha; Zhou, Zhongjun

    2013-04-01

    ATM-mediated phosphorylation of KAP-1 triggers chromatin remodeling and facilitates the loading and retention of repair proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs) derived from Zmpste24(-/-) mice undergo early senescence, attributable to delayed recruitment of DNA repair proteins. Here, we show that ATM-Kap-1 signaling is compromised in Zmpste24(-/-) MEFs, leading to defective DNA damage-induced chromatin remodeling. Knocking down Kap-1 rescues impaired chromatin remodeling, defective DNA repair and early senescence in Zmpste24(-/-) MEFs. Thus, ATM-Kap-1-mediated chromatin remodeling plays a critical role in premature aging, carrying significant implications for progeria therapy.

  14. HIV-2 Surveillance with Next-Generation Sequencing Reveals Mutations in a Cytotoxic Lymphocyte-Restricted Epitope Involved in Long-Term Nonprogression.

    PubMed

    Yamaguchi, Julie; Brennan, Catherine A; Alessandri-Gradt, Elodie; Plantier, Jean-Christophe; Cloherty, Gavin A; Berg, Michael G

    2017-04-01

    HIV-2 exhibits a natural history of infection distinct from HIV-1. Primarily found in West Africa and in only 10%-20% of HIV infections in this region, patients with HIV-2 typically exhibit a slower progression to AIDS, lower viral loads, and decreased rates of transmission. Here, we used next-generation sequencing to determine the sequence and phylogenetic classification of nine HIV-2 genomes. We identified a patient with a series of mutations in an invariant cytotoxic lymphocyte (CTL)-restricted gag epitope required for retroviral structure and replication and implicated in long-term nonprogression to AIDS. The presence of wild-type sequence argues these mutations are involved in immune escape, whereas its reversion to a sequence seen only in the sooty mangabey reservoir suggests an alternate means of controlling infection. Surveillance and molecular characterization of circulating strains are essential for continued development of monitoring tools and may provide greater insight into the reduced pathogenicity of HIV-2.

  15. A Library of Infectious Hepatitis C Viruses with Engineered Mutations in the E2 Gene Reveals Growth-Adaptive Mutations That Modulate Interactions with Scavenger Receptor Class B Type I.

    PubMed

    Zuiani, Adam; Chen, Kevin; Schwarz, Megan C; White, James P; Luca, Vincent C; Fremont, Daved H; Wang, David; Evans, Matthew J; Diamond, Michael S

    2016-12-01

    While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties.

  16. In-Depth Mutational Analysis of the Promyelocytic Leukemia Zinc Finger BTB/POZ Domain Reveals Motifs and Residues Required for Biological and Transcriptional Functions

    PubMed Central

    Melnick, Ari; Ahmad, K. Farid; Arai, Sally; Polinger, Adam; Ball, Helen; Borden, Katherine L.; Carlile, Graeme W.; Prive, Gilbert G.; Licht, Jonathan D.

    2000-01-01

    The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression. PMID:10938130

  17. An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli.

    PubMed Central

    Fisher, K E; Eisenstein, E

    1993-01-01

    High-level expression of the regulatory enzyme threonine deaminase in Escherichia coli strains grown on minimal medium that are deficient in the activities of enzymes needed for branched-chain amino acid biosynthesis result in growth inhibition, possibly because of the accumulation of toxic levels of alpha-ketobutyrate, the product of the committed step in isoleucine biosynthesis. This condition affords a means for selecting genetic variants of threonine deaminase that are deficient in catalysis by suppression of growth inhibition. Strains harboring mutations in ilvA that decreased the catalytic activity of threonine deaminase were found to grow more rapidly than isogenic strains containing wild-type ilvA. Modification of the ilvA gene to introduce additional unique, evenly spaced restriction enzyme sites facilitated the identification of suppressor mutations by enabling small DNA fragments to be subcloned for sequencing. The 10 mutations identified in ilvA code for enzymes with significantly reduced activity relative to that of wild-type threonine deaminase. Values for their specific activities range from 40% of that displayed by wild-type enzyme to complete inactivation as evidenced by failure to complement an ilvA deletion strain to isoleucine prototrophy. Moreover, some mutant enzymes showed altered allosteric properties with respect to valine activation and isoleucine inhibition. The location of the 10 mutations in the 5' two-thirds of the ilvA gene is consistent with suggestions that threonine deaminase is organized functionally with an amino-terminal domain that is involved in catalysis and a carboxy-terminal domain that is important for regulation. Images PMID:8407838

  18. Sequencing of the coding exons of the LRP1 and LDLR genes on individual DNA samples reveals novel mutations in both genes.

    PubMed

    Van Leuven, F; Thiry, E; Lambrechts, M; Stas, L; Boon, T; Bruynseels, K; Muls, E; Descamps, O

    2001-02-15

    Five coding polymorphisms in de LRP1 gene, i.e. A217V, A775P, D2080N, D2632E and G4379S were discovered by sequencing its 89 exons in three test-groups of 22 healthy individuals, 29 Alzheimer patients and 18 individuals with different clinical and molecularly uncharacterized lipid metabolism problems. No genetic defect was evident in the LRP1 gene of any of the Alzheimer's disease (AD) patients, further excluding LRP1 as a major genetic problem in AD. Lipoprotein receptor related protein (LRP) A217V (exon 6) was clearly present in all groups as a polymorphism, while D2632E was observed only once in a healthy volunteer. On the other hand, LRP1 alleles A775P, D2080N, and G4379 were encountered only in patients with FH or with undefined problems of lipid metabolism. This finding forced one to also analyze the LDL receptor (LDLR) gene, for which a method was devised to sequence the entire region comprising LDLR exons 2-18. The resulting sequence contig of 33567 nucleotides yielded finally an exact physical map that corrects published and listed LDLR gene maps in many positions. In addition, next to known mutations in LDLR that cause FH, four novel LDLR defects were defined, i.e. del e7-10, exon 9 mutation N407T, a 20 bp insertion in exon 4, and a double mutation C292W/K290R in exon 6. No evidence for pathology connected to the LRP1 'mutations' was obtained by subsequent screening for the five LRP1 variants in larger groups of 110 FH patients and 118 patients with molecularly undefined, clinical problems of cholesterol and/or lipid metabolism. In three individuals with a mutant LDLR gene a variant LRP1 allele was also present, but without direct, obvious clinical compound effects, indicating that the variant LRP1 alleles must, for the present, be considered polymorphisms.

  19. Mutation analysis of the gene encoding Bruton`s tyrosine kinase in a family with a sporadic case of X-linked agammaglobulinemia reveals three female carriers

    SciTech Connect

    Hagemann, T.L.; Kwan, Sau-Ping; Assa`ad, A.H.

    1995-11-06

    Bruton`s tyrosine kinase (Btk) has been identified as the protein responsible for the primary immunodeficiency X-linked agammaglobulinemia (XLA). We and others have cloned the gene for Btk and recently reported the genomic organization. Nineteen exons were positioned within the 37 kb gene. With the sequence data derived from our genomic map, we have designed a PCR based assay to directly identify mutations of the Btk gene in germline DNA of patients with XLA. In this report, the assay was used to analyze a family with a sporadic case of XLA to determine if other female relatives carry the disease. A four base-pair deletion was found in the DNA of the affected boy and was further traced through three generations. With the direct identification of the mutations responsible for XLA, we can now diagnose conclusively the disease and identify the immunologically normal female carriers. This same technique can easily be applied to prenatal diagnosis in families where the mutation can be identified. 34 refs., 3 figs.

  20. 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

  1. A DFT study of the unusual substrate-assisted mechanism of Serratia marcescens chitinase B reveals the role of solvent and mutational effect on catalysis.

    PubMed

    Jitonnom, Jitrayut; Sattayanon, Chanchai; Kungwan, Nawee; Hannongbua, Supa

    2015-03-01

    Serratia marcescens chitinase B (SmChiB) catalyzes the hydrolysis of β-1,4-glycosidic bond, via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile. In this paper, the catalytic mechanism of SmChiB has been investigated by using density functional theory. The details of two consecutive steps (glycosylation and deglycosylation), the structures and energetics along the whole catalytic reaction, and the roles of solvent molecules as well as some conserved SmChiB residues (Asp142, Tyr214, Asp215, and Arg294) during catalysis are highlighted. Our calculations show that the formation of the oxazolinium cation intermediate in the glycosylation step was found to be a rate-determining step (with a barrier of 23 kcal/mol), in line with our previous computational studies (Jitonnom et al., 2011, 2014). The solvent water molecules have a significant effect on a catalytic efficiency in the degycosylation step: the catalytic water is essentially placed in a perfect position for nucleophic attack by hydrogen bond network, lowering the barrier height of this step from 11.3 kcal/mol to 2.9 kcal/mol when more water molecules were introduced. Upon the in silico mutations of the four conserved residues, their mutational effects on the relative stability of the reaction intermediates and the computed energetics can be obtained by comparing with the wild-type results. Mutations of Tyr214 to Phe or Ala have shown a profound effect on the relative stability of the oxazolinium intermediate, emphasizing a direct role of this residue in destabilizing the intermediate. In line with the experiment that the D142A mutation leads to almost complete loss of SmChiB activity, this mutation greatly decreases the stability of the intermediate, resulting in a very large increase in the activation barrier up to 50 kcal/mol. The salt-bridges residues (Asp215 and Arg294) were also found to play a role in stabilizing the oxazolinium intermediate.

  2. Prospective mutation screening of three common deafness genes in a large Taiwanese Cohort with idiopathic bilateral sensorineural hearing impairment reveals a difference in the results between families from hospitals and those from rehabilitation facilities.

    PubMed

    Wu, Chen-Chi; Chen, Pei-Jer; Chiu, Yu-Hsun; Lu, Ying-Chang; Wu, Ming-Chueh; Hsu, Chuan-Jen

    2008-01-01

    Accurate epidemiological data on common deafness genes are essential to improve the efficiency and to reduce the cost of molecular diagnosis. They m