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Grybek V.,French Institute of Health and Medical Research | Aubry L.,UEVE UMR 861 | Aubry L.,French Institute of Health and Medical Research | Maupetit-Mehouas S.,French Institute of Health and Medical Research | And 6 more authors.
Stem Cell Reports | Year: 2014

Data from the literature indicate that genomic imprint marks are disturbed in human pluripotent stem cells (PSCs). GNAS is an imprinted locus that produces one biallelic (Gsα) and four monoallelic (NESP55, GNAS-AS1, XLsα, and A/B) transcripts due to differential methylation of their promoters (DMR). To document imprinting at the GNAS locus in PSCs, we studied GNAS locus DMR methylation and transcript (NESP55, XLsα, and A/B) expression in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) derived from two human fibroblasts and their progenies. Results showed that (1) methylation at the GNAS locus DMRs is DMR and cell line specific, (2) changes in allelic transcript expression can be independent of a change in allele-specific DNA methylation, and (3) interestingly, methylation at A/B DMR is correlated with A/B transcript expression. These results indicate that these models are valuable to study the mechanisms controlling GNAS methylation, factors involved in transcript expression, and possibly mechanisms involved in the pathophysiology of pseudohypoparathyroidism type 1B. © 2014 The Authors.


Bricaire L.,University Pierre and Marie Curie | Odou M.-F.,Lille University Hospital Center | Odou M.-F.,Lille 2 University of Health and Law | Cardot-Bauters C.,Lille University Hospital Center | And 20 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2013

Context: Hyperparathyroidism-jaw tumor syndrome (HPT-JT) is an autosomal dominant syndrome with incomplete penetrance that can associate in a single patient parathyroid adenoma or carcinoma, fibro-osseous jaw tumor, cystic kidney lesion, and uterine tumor. Germline mutations of the HRPT2 gene (CDC73) coding for parafibromin are identified in approximately 50%-75% of HPT-JT cases and in approximately 14% of familial isolated hyperparathyroidism. A whole deletion of this gene has recently been reported in 1 sporadic case and in a family presenting with HPT-JT. Objective: The objective of the study was to report molecular abnormalities of the HRPT2 gene in patients with primary hyperparathyroidism in a French National cohort from the Groupe d'Étude des Tumeurs Endocrines. Methods: Patients' genomic DNA was screened by PCR-based sequencing for point mutations affecting HRPT2 and real-time quantitative PCR analysis for gross deletions. Results: We report 20 index patients with a germinal HRPT2 abnormality. Median age at diagnosis of primary hyperparathyroidism was 23 years (range 14-65 years). Median serum total calcium level at diagnosis was 3.19 mmol/L (range 2.8-4.3 mmol/L). Thirteen different mutations were identified by routine sequencing, including 7 mutations never reported. Seven patients (35%) carried a gross deletion of this gene (3 complete and 4 partial deletions). No genotype-phenotype correlation could be identified. Agross deletion of the HRPT2 gene was identified in 7% of patients for whom a routine screening by direct sequencing came up as negative. Conclusion: Gross deletion analysis of the HRPT2 gene is indicated for all patients negative for mutation, presenting with HPT-JT or familial isolated hyperparathyroidism, parathyroid carcinoma, or in patients with apparently sporadic parathyroid adenoma diagnosed at a young age, having a severe hypercalcemia. Copyright © 2013 by The Endocrine Society.


Linglart A.,Institute National Of La Santeet Of La Recherche Megdicale Unite 986 | Fryssira H.,Sophia Genetics | Hiort O.,Jugendmedizin Universitatsklinikum S | Holterhus P.-M.,Universitario Araba | And 22 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2012

Context: Acrodysostosis is a rare skeletal dysplasia that is associated with multiple resistance to G protein-coupled receptor (GPCR) signaling hormones in a subset of patients. Acrodysostosis is genetically heterogeneous because it results from heterozygous mutations in PRKAR1A or PDE4D, two key actors in the GPCR-cAMP-protein kinase A pathway. Objective: Our objective was to identify the phenotypic features that distinguish the two genotypes causing acrodysostosis. Patients and Methods: Sixteen unrelated patients with acrodysostosis underwent a candidategene approach and were investigated for phenotypic features. Results: All patients had heterozygous de novo mutations. Fourteen patients carried a PRKAR1A mutation (PRKAR1A patients), five each a novel PRKAR1A mutation (p.Q285R, p.G289E, p.A328V, p.R335L, or p.Q372X), nine the reported PRKAR1A p.R368X mutation; two patients harbored a mutation in PDE4D (PDE4D patients) (one novel mutation, p.A227S; one reported, p.E590A). All PRKAR1A, but none of the PDE4D mutated patients were resistant to PTH and TSH. Two PRKAR1A patients each with a novel mutation presented a specific pattern of brachydactyly. One PDE4D patient presented with acroskyphodysplasia. Additional phenotypic differences included mental retardation in PDE4D patients. In addition, we report the presence of pigmented skin lesions in PRKAR1A and PDE4D patients, a feature not yet described in the acrodysostosis entity. Conclusions: All PRKAR1A and PDE4D patients present similar bone dysplasia characterizing acrodysostosis. Phenotypic differences, including the presence of resistance to GPCR-cAMP signaling hormones in PRKAR1A but not PDE4D patients, indicate phenotype-genotype correlations and highlight the specific contributions of PRKAR1A and PDE4D in cAMP signaling in different tissues. Copyright © 2012 by The Endocrine Society.


Garin I.,Hospital Universitario Araba Txagorritxu | Mantovani G.,University of Milan | Barlier A.,Aix - Marseille University | Brix B.,University of Lübeck | And 10 more authors.
European Journal of Human Genetics | Year: 2015

Pseudohypoparathyroidism is a rare endocrine disorder that can be caused by genetic (mainly maternally inherited inactivating point mutations, although intragenic and gross deletions have rarely been reported) or epigenetic alterations at GNAS locus. Clinical and molecular characterization of this disease is not that easy because of phenotypic, biochemical and molecular overlapping features between both subtypes of the disease. The European Consortium for the study of PHP (EuroPHP) designed the present work with the intention of generating the standards of diagnostic clinical molecular (epi)genetic testing in PHP patients. With this aim, DNA samples of eight independent PHP patients carrying GNAS genetic and/or epigenetic defects (three patients with GNAS deletions, two with 20q uniparental disomy and three with a methylation defect of unknown origin) without GNAS point mutations were anonymized and sent to the five participant laboratories for their routine genetic analysis (methylation-specific (MS)-MLPA, pyrosequencing and EpiTYPER) and interpretations. All laboratories were able to detect methylation defects and, after the data analysis, the Consortium compared the results to define technical advantages and disadvantages of different techniques. To conclude, we propose as first-level investigation in PHP patients copy number and methylation analysis by MS-MLPA. Then, in patients with partial methylation defect, the result should be confirmed by single CpG bisulphite-based methods (ie pyrosequencing), whereas in case of a complete methylation defect without detectable deletion, microsatellites or SNP genotyping should be performed to exclude uniparental disomy 20. © 2015 Macmillan Publishers Limited All rights reserved.


Reis A.F.,Federal University of São Paulo | Kannengiesser C.,Laboratoire Of Biochimie Hormonale Et Genetique | Kannengiesser C.,University Paris Diderot | Jennane F.,CHU Ibn Rochd | And 9 more authors.
Pediatric Diabetes | Year: 2011

Wolcott-Rallison syndrome (WRS, OMIM 226980) is a rare autosomal recessive disorder characterized by permanent neonatal diabetes mellitus, epiphyseal dysplasia, and other multisystemic clinical manifestations. We described two novel mutations in the EIF2AK3 gene in two consanguineous families with WRS from Brazil and Morocco. We have observed in case 1 a homozygous C > T replacement at base pair c.1192 at exon 7, generating a stop codon at position 398 (Gln398Stop). Both of his parents were found to be heterozygous for the mutation. We detected in both parents of case 2, a deceased Moroccan girl, a duplication of base pair c.851A at exon 5 (c.851dupA) leading to a frameshift and a stop codon at position 285 (p.Pro285AlafsX3). Both cases 1 and 2 had neonatal diabetes mellitus, multiple epiphyseal dysplasia, and growth delay, and presented episodes of acute hepatic dysfunction. Case 1 presented central hypothyroidism, developmental delay, and mild mental retardation. Case 2 presented a fatal episode of acute renal failure. The clinical phenotype associated with the syndrome can be variable, but a combination of infancy-onset diabetes mellitus, multiple epiphyseal dysplasia, and hepatic and/or renal dysfunction is the mainstay of diagnosis. © 2010 John Wiley & Sons A/S.


Grandchamp B.,Laboratoire Of Biochimie Hormonale Et Genetique | Grandchamp B.,French Institute of Health and Medical Research | Hetet G.,Laboratoire Of Biochimie Hormonale Et Genetique | Kannengiesser C.,Laboratoire Of Biochimie Hormonale Et Genetique | And 11 more authors.
Blood | Year: 2011

STEAP3/TSAP6 encodes a ferrireductase that is involved in the acquisition of iron by developing erythroblasts and steap3/tsap6 null-mice display severe microcytic anemia. We report a family in which 3 siblings born to healthy parents display transfusion-dependent hypochromic anemia. A nonsense STEAP3/TSAP6 was identified in the siblings at the heterozygous state. This mutation was inherited from their father while no mutation was found in their mother. A large variability of expression was found between normal alleles in a control population, confirming a previous report that STEAP3/TSAPS6 is an expressed quantitative trait locus (e-QTL). Determination of the relative allele expression showed that the "normal" allele was expressed at a significantly higher level in the father than in the affected siblings relative to the shared mutated allele. The blood level of STEAP3/TSAP6 mRNA was severely reduced in the siblings, while both parents were in the lower range of normal controls. The STEAP3/TSAP6 protein was also reduced in lymphocytic cell lines from the patients. Collectively, our data support the hypothesis that STEAP3/TSAP6 deficiency leads to severe anemia in the affected siblings and results from the combination of a mutated allele inherited from their father and a weakly expressed allele inherited from their mother. © 2011 by The American Society of Hematology.


Verine J.,Laboratoire Of Pathologie | Verine J.,French Institute of Health and Medical Research | Verine J.,University Paris Diderot | Pluvinage A.,University Paris Diderot | And 10 more authors.
European Urology | Year: 2010

Context: Hereditary renal cancers (HRCs) comprise approximately 3-5% of renal cell carcinomas (RCCs). Objective: Our aim was to provide an overview of the currently known HRC syndromes in adults. Evidence acquisition: Data on HRC syndromes were analysed using PubMed and Online Mendelian Inheritance in Man with an emphasis on kidney cancer, clinical criteria, management, treatment, and genetic counselling and screening. Evidence synthesis: Ten HRC syndromes have been described that are inherited with an autosomal dominant trait. Eight genes have already been identified (VHL, MET, FH, FLCN, TSC1, TSC2, CDC73, and SDHB). These HRC syndromes involve one or more RCC histologic subtypes and are generally bilateral and multiple. Computed tomography and magnetic resonance imaging are the best imaging techniques for surveillance and assessment of renal lesions, but there are no established guidelines for follow-up after imaging. Except for hereditary leiomyomatosis RCC tumours, conservative treatments favour both an oncologically effective therapeutic procedure and a better preservation of renal function. Conclusions: HRC involves multiple clinical manifestations, histologic subtypes, genetic alterations, and molecular pathways. Urologists should know about HRC syndromes in the interest of their patients and families. © 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved.


Lalou C.,University Paris Diderot | Scamuffa N.,University Paris Diderot | Mourah S.,University Paris Diderot | Plassa F.,Laboratoire Of Biochimie | And 6 more authors.
PLoS ONE | Year: 2010

Background: Altered tumor suppressor p53 and/or CDKN2A as well as Ras genes are frequently found in primary and metastatic melanomas. These alterations were found to be responsible for acquisition of invasive and metastatic potential through their defective regulatory control of metalloproteinases and urokinase genes. Methodology/Principal Findings: Using primary human melanoma M10 cells with altered p53, CDKN2A and N-Ras genes, we found that inhibition of the proprotein convertases (PCs), enzymes involved in the proteolytic activation of various cancer-related protein precursors resulted in significantly reduced invasiveness. Analysis of M10 cells and their gastric and lymph node derived metastatic cells revealed the presence of all the PCs found in the secretory pathway. Expression of the general PCs inhibitor α1-PDX in these cells in a stable manner (M10/PDX) had no effect on the mRNA expression levels of these PCs. Whereas, in vitro digestion assays and cell transfection experiments, revealed that M10/PDX cells display reduced PCs activity and are unable to process the PCs substrates proIGF-1R and proPDGF-A. These cells showed reduced migration and invasion that paralleled decreased gelatinase MMP-2 activity and increased expression and secretion of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Furthermore, these cells showed decreased levels of urokinase-type plasminogen activator receptor (uPAR) and increased levels of plasminogen activator inhibitor-1 (PAI-1). Conclusions: Taken together, these data suggest that inhibition of PCs activity results in decreased invasiveness of primary human melanoma cells despite their altered p53, CDKN2A and N-Ras genes, suggesting that PCs may serve as novel therapeutic targets in melanoma. © 2010 Lalou et al.


Guillem F.,French Institute of Health and Medical Research | Grandchamp B.,French Institute of Health and Medical Research | Grandchamp B.,Laboratoire Of Biochimie Hormonale Et Genetique
Hematologie | Year: 2011

The transmembrane serine protease matriptase-2, encoded by the TMPRSS6 gene, has a major role in the regulation of iron homeostasis by repressing hepcidin. Mutations of TMPRSS6 gene are responsible for hypochromic microcytic anemia Iron Refractory Iron Deficiency Anemia (IRIDA) characterized by impaired iron absorption in the intestine resulting from a defect in hepcidin repression. Furthermore, genome wide association studies show that frequent variants of TMPRSS6 gene are associated with hematological parameters like hemoglobin concentration and mean globular volume. Here, we will review recent informations about MT2 biology and TMPRSS6 mutations in patients with IRIDA, and we will summarize the results of genome wide association studies showing the association of common TMPRSS6 gene polyporphisms with hematological biological parameter.


Aguilar-Martinez P.,Montpellier University Hospital Center | Grandchamp B.,Laboratoire Of Biochimie Hormonale Et Genetique | Cunat S.,Montpellier University Hospital Center | Cadet E.,University of Picardie Jules Verne | And 5 more authors.
Haematologica | Year: 2011

Background: Heterozygotes for the p.Cys282Tyr (C282Y) mutation of the HFE gene do not usually express a hemochromatosis phenotype. Apart from the compound heterozygous state for C282Y and the widespread p.His63Asp (H63D) variant allele, other rare HFE mutations can be found in trans on chromosome 6. Design and Methods: We performed molecular investigation of the genes implicated in hereditary hemochromatosis in six patients who presented with iron overload but were simple heterozygotes for the HFE C282Y mutation at first genetic testing. Functional impairment of new variants was deduced from computational methods including molecular modeling studies. Results: We identified four rare HFE mutant alleles, three of which have not been previously described. One mutation is a 13-nucleotide deletion in exon 6 (c.1022_1034del13, p.His341_Ala345>LeufsX119), which is predicted to lead to an elongated and unstable protein. The second one is a substitution of the last nucleotide of exon 2 (c.340G>A, p.Glu114Lys) which modifies the relative solvent accessibility in a loop interface. The third mutation, p.Arg67Cys, also lies in exon 2 and introduces a destabilization of the secondary structure within a loop of the α1 domain. We also found the previously reported c.548T>C (p.Leu183Pro) missense mutation in exon 3. No other known iron genes were mutated. We present an algorithm at the clinical and genetic levels for identifying patients deserving further investigation. Conclusions: Our results suggest that additional mutations in HFE may have a clinical impact in C282Y carriers. In conjunction with results from previously described cases we conclude that an elevated transferrin saturation level and elevated hepatic iron index should indicate the utility of searching for further HFE mutations in C282Y heterozygotes prior to other iron gene studies. © 2011 Ferrata Storti Foundation.

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