Publications by authors named "Bernard Grandchamp"

87 Publications

Truncating mutations of TP53AIP1 gene predispose to cutaneous melanoma.

Genes Chromosomes Cancer 2018 Jun 21;57(6):294-303. Epub 2018 Feb 21.

INSERM U976, Centre de Recherche sur la Peau, Hôpital Saint Louis, 75010, Paris, France.

Genetic predisposition to cutaneous malignant melanoma (CMM) involves highly penetrant predisposing genes and low and intermediate penetrant predisposing alleles. However, the missing heritability in (CMM) is still high. For such and in order to identify new genetic factors for CMM, we conducted an exome sequencing study in high-risk CMM patients. Two rounds of exome sequencing were successively performed in 33 and 27 high-risk patients. We focused on genes carrying rare nonsense, frameshift, and splice variants (allelic frequency <1%) that were present in both series of exomes. An extension study was then conducted in a large cohort (1 079 CMM patients and 1 230 Caucasian ethnically matched healthy controls), and the inactivating variants frequency was compared between groups using two-sided Fisher exact test. Two TP53AIP1 truncating mutations were identified in four patients: a frameshift c.63_64insG, p.Q22Afs*81 in two patients from the same family and in the proband of a second family; and a nonsense mutation c.95 C > A, p.Ser32Stop in a patient with multiple CMMs. In all patients, TP53AIP1 truncating variants were strongly associated with CMM risk (two-sided Fisher exact test = 0.004, OR = 3.3[1.3-8.5]). Additionally, we showed that TP53AIP1 mRNA was strongly down-regulated throughout different phases of melanoma progression. TP53AIP1 gene is a TP53 target which plays a key role by inducting apoptosis in response to UV-induced DNA damage. Constitutional mutations of TP53AIP1 had previously been involved in susceptibility to prostate cancer. Our results show that constitutional truncating TP53AIP1 mutations predispose to CMM in the French population. Replication studies in other populations should be performed.
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http://dx.doi.org/10.1002/gcc.22528DOI Listing
June 2018

Prevalence and characteristics of TERT and TERC mutations in suspected genetic pulmonary fibrosis.

Eur Respir J 2016 12 11;48(6):1721-1731. Epub 2016 Nov 11.

APHP, Hôpital Bichat, Service de Pneumologie A, DHU FIRE, Centre de compétence des maladies pulmonaires rares, Paris, France

Telomerase reverse transcriptase (TERT) or telomerase RNA (TERC) gene mutation is a major monogenic cause of pulmonary fibrosis. Sequencing of TERT/TERC genes is proposed to patients with familial pulmonary fibrosis. Little is known about the possible predictors of this mutation and its impact on prognosis.We retrospectively analysed all the genetic diagnoses made between 2007-2014 in patients with pulmonary fibrosis. We evaluated the prevalence of TERT/TERC disease-associated variant (DAV), factors associated with a DAV, and the impact of the DAV on survival.237 patients with pulmonary fibrosis (153 with familial pulmonary fibrosis, 84 with telomere syndrome features without familial pulmonary fibrosis) were tested for TERT/TERC DAV. DAV was diagnosed in 40 patients (16.8%), including five with non-idiopathic interstitial pneumonia. Prevalence of TERT/TERC DAV did not significantly differ between patients with familial pulmonary fibrosis or with only telomere syndrome features (18.2% versus 16.4%). Young age, red blood cell macrocytosis, and low platelet count were associated with the presence of DAV; the probability of DAV was increased for patients 40-60 years. Transplant-free survival was lower with than without TERT/TERC DAV (4.2 versus 7.2 years; p=0.046).TERT/TERC DAV were associated with specific clinical and biological features and reduced transplant-free survival.
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http://dx.doi.org/10.1183/13993003.02115-2015DOI Listing
December 2016

Promoter hypermethylation of HS3ST2, SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscle-invasive bladder cancer.

BMC Cancer 2016 09 1;16:704. Epub 2016 Sep 1.

OncoDiag SAS, Agoranov, Paris, France.

Background: Non-muscle-invasive bladder cancer (NMIBC) is a high incidence form of bladder cancer (BCa), where genetic and epigenetic alterations occur frequently. We assessed the performance of associating a FGFR3 mutation assay and a DNA methylation analysis to improve bladder cancer detection and to predict disease recurrence of NMIBC patients.

Methods: We used allele specific PCR to determine the FGFR3 mutation status for R248C, S249C, G372C, and Y375C. We preselected 18 candidate genes reported in the literature as being hypermethylated in cancer and measured their methylation levels by quantitative multiplex-methylation specific PCR. We selected HS3ST2, SLIT2 and SEPTIN9 as the most discriminative between control and NMIBC patients and we assayed these markers on urine DNA from a diagnostic study consisting of 167 NMIBC and 105 controls and a follow-up study consisting of 158 NMIBC at diagnosis time's and 425 at follow-up time. ROC analysis was performed to evaluate the diagnostic accuracy of each assay alone and in combination.

Results: For Diagnosis: Using a logistic regression analysis with a model consisting of the 3 markers' methylation values, FGFR3 status, age and known smoker status at the diagnosis time we obtained sensitivity/specificity of 97.6 %/84.8 % and an optimism-corrected AUC of 0.96. With an estimated BCa prevalence of 12.1 % in a hematuria cohort, this corresponds to a negative predictive value (NPV) of 99.6 %. For Follow-up: Using a logistic regression with FGFR3 mutation and the CMI at two time points (beginning of the follow-up and current time point), we got sensitivity/specificity/NPV of 90.3 %/65.1 %/97.0 % and a corrected AUC of 0.84. We also tested a thresholding algorithm with FGFR3 mutation and the two time points as described above, obtaining sensitivity/specificity/NPV values of, respectively, 94.5 %/75.9 %/98.5 % and an AUC of 0.82.

Conclusions: We showed that combined analysis of FGFR3 mutation and DNA methylation markers on urine can be a useful strategy in diagnosis, surveillance and for risk stratification of patients with NMIBC. These results provide the basis for a highly accurate noninvasive test for population screening and allowing to decrease the frequency of cystoscopy, an important feature for both patient quality of life improvement and care cost reduction.
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http://dx.doi.org/10.1186/s12885-016-2748-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007990PMC
September 2016

PARKIN Inactivation Links Parkinson's Disease to Melanoma.

J Natl Cancer Inst 2016 Mar 17;108(3). Epub 2015 Dec 17.

Affiliations of authors: INSERM, U976, Centre de Recherche sur la Peau, Hôpital Saint Louis , Paris , France (HHH, JA, SM, LM, VD, NBS, MB, AB, CL, ND, NS); AP-HP, Hôpital Bichat Claude Bernard, Département de Génétique , Paris , France (HHH, CK, AT, BG, NS); Université Paris Diderot, Sorbonne Paris Cité , UMRS976, Paris , France (HHH, CK, JA, LM, NBS, MB, AB, LD, AT, BG, ND, NS); Université Paris 6, INSERM UMRS975, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epinière, Hôpital Pitié-Salpêtrière, AP-HP , Paris , France (SL, AB); INSERM, U940, Laboratoire de Pharmacologie, Hôpital Saint Louis Paris , France (SM); AP-HP, Hôpital Bichat Claude Bernard, Service de Dermatologie , Paris , France (VD); AP-HP, Hôpital Saint Louis, Service de Dermatologie , Paris , France (NBS, MB, CL, ND); INSERM, CRB3, Département de Pathologie, Hôpital Bichat, AP-HP , Paris , France (LD); AP-HP, Hôpital Ambroise Paré, Service de Dermatologie , Boulogne Billancourt , France (PS); CHU Grenoble, Service de Dermatologie , Grenoble , France (MTL); Gustave Roussy, Service de Génétique, Département de Biopathologie , Villejuif , France (BBdP); Division of Molecular Genetic Epidemiology, German Cancer Research Center, Im Neuenheimer Feld 580 , Heidelberg , Germany (RK); Department of Neurology, University Hospital of Würzburg , Würzburg , Germany (SK); Inserm UMR1037, Centre de Recherche en Cancérologie de Toulouse , Toulouse , France (NAA); Hôpital de l'Hôtel-Dieu, Service de Dermatologie , Lyon , France (LT); AP-HP, Groupe Pitié-Salpêtrière, Département de Génétique, Cytogénétique et Embryologie , Paris , France (AB).

Background: Melanoma incidence is higher in patients affected by Parkinson's disease (PD) and vice versa, but the genetic link shared by both diseases is unknown. As PARK2 is both a tumor suppressor gene and frequently mutated in young onset PD, we evaluated the role of PARK2 in melanoma predisposition and progression.

Methods: An in-depth PARK2 gene dosage analysis and sequencing was performed on 512 French case patients and 562 healthy control patients, as well as sporadic tumors and melanoma cell lines. The frequency of genetic alterations was compared between case patients and control patients using two-sided Fisher's exact tests and odds ratio (OR) calculations. We used western blotting to determine PARKIN expression in melanocytes and melanoma cell lines and transfection followed by clonogenic assays to evaluate the effect of PARKIN expression on cellular proliferation. All statistical tests were two-sided.

Results: Germline PARK2 mutations (including copy number variations, splicing, and putative deleterious missense mutations) were present in 25 case patients but only four control patients (OR = 3.95, 95% confidence interval = 1.34 to 15.75). Copy number variations (CNVs) and loss of heterozygosity were present in 60% and 74%, respectively, of primary tumors. PARKIN protein was expressed in melanocytes but not in most melanoma cell lines, and its expression decreased following melanocyte transformation by oncogenic NRAS. Re-expression of PARKIN in melanoma cell lines resulted in a drastic reduction of cell proliferation and inhibition of PARKIN in melanocytes stimulated their proliferation.

Conclusion: Our results show an important role for PARK2 as a tumor suppressor both in melanoma predisposition and progression, which could explain the epidemiological association of these diseases.
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http://dx.doi.org/10.1093/jnci/djv340DOI Listing
March 2016

The Diagnostic and Prognostic Performance of Urinary FGFR3 Mutation Analysis in Bladder Cancer Surveillance: A Prospective Multicenter Study.

Urology 2015 Dec 11;86(6):1185-90. Epub 2015 Sep 11.

AP-HP, Department of Genetics, Bichat Hospital, Paris, France. Electronic address:

Objective: To assess the diagnostic and prognostic performance of a noninvasive FGFR3 mutation analysis. After transurethral resection (TUR) of noninvasive bladder transitional cell carcinoma (B-TCC), recurrence occurs in 70% of patients, thus justifying cystoscopic surveillance.

Materials And Methods: A prospective multicenter study was carried out with a 2-year follow-up of patients with superficial B-TCC. Urine samples were collected before TUR and then before each cystoscopy during follow-up. Screening for the most prevalent FGFR3 mutations was done using urinary cells. The prognostic significance of an FGFR3 mutation at the time of the initial diagnosis was determined. The performance of the test in diagnosing and/or predicting recurrence during follow-up was assessed by calculating sensitivity and specificity.

Results: Of 191 patients studied, 74 (39%) had a positive analysis before TUR (FGFR3 mutation group). The presence of an FGFR3 mutation at the time of diagnosis was associated with a shorter time to recurrence (P = .02). During follow-up, 68 patients from the FGFR3 mutation group were evaluated. FGFR3 mutation analysis showed a sensitivity of 0.73 and a specificity of 0.87 when compared with the results of cystoscopy. A positive urine test was predictive of recurrence either at the time of the positive result or later during the 2-year follow-up, with a sensitivity of 0.70 and a specificity of 0.87.

Conclusion: Among patients with an FGFR3 mutation in the initial tumor, a noninvasive urine test during follow-up can be valuable in diagnosing or predicting subsequent recurrence.
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http://dx.doi.org/10.1016/j.urology.2015.07.036DOI Listing
December 2015

Heterozygous RTEL1 mutations are associated with familial pulmonary fibrosis.

Eur Respir J 2015 Aug 28;46(2):474-85. Epub 2015 May 28.

Université Paris Diderot, Sorbonne Paris Cité, Paris, France APHP, Hôpital Bichat, Service de Pneumologie A, DHU FIRE Centre de compétence des maladies pulmonaires rares, Paris, France.

Pulmonary fibrosis is a fatal disease with progressive loss of respiratory function. Defective telomere maintenance leading to telomere shortening is a cause of pulmonary fibrosis, as mutations in the telomerase component genes TERT (reverse transcriptase) and TERC (RNA component) are found in 15% of familial pulmonary fibrosis (FPF) cases. However, so far, about 85% of FPF remain genetically uncharacterised.Here, in order to identify new genetic causes of FPF, we performed whole-exome sequencing, with a candidate-gene approach, of 47 affected subjects from 35 families with FPF without TERT and TERC mutations.We identified heterozygous mutations in regulator of telomere elongation helicase 1 (RTEL1) in four families. RTEL1 is a DNA helicase with roles in DNA replication, genome stability, DNA repair and telomere maintenance. The heterozygous RTEL1 mutations segregated as an autosomal dominant trait in FPF, and were predicted by structural analyses to severely affect the function and/or stability of RTEL1. In agreement with this, RTEL1-mutated patients exhibited short telomeres in comparison with age-matched controls.Our results provide evidence that heterozygous RTEL1 mutations are responsible for FPF and, thereby, extend the clinical spectrum of RTEL1 deficiency. Thus, RTEL1 enlarges the number of telomere-associated genes implicated in FPF.
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http://dx.doi.org/10.1183/09031936.00040115DOI Listing
August 2015

Genes involved in the WNT and vesicular trafficking pathways are associated with melanoma predisposition.

Int J Cancer 2015 May 24;136(9):2109-19. Epub 2014 Oct 24.

Department of Haematology and Medical Oncology, Biomedical Research Institute INCLIVA, Valencia, 46010, Spain.

Multifactorial predisposition to melanoma includes genes involved in pigmentation, immunity and DNA repair. Nonetheless, missing heritability in melanoma is still important. We studied the role of 335 candidate SNPs in melanoma susceptibility by using a dedicated chip and investigating 110 genes involved in different pathways. A discovery set was comprised of 1069 melanoma patients and 925 controls from France. Data were replicated using validation phases II (1085 cases and 801 controls from Spain) and III (1808 cases and 1894 controls from Germany and a second set of Spanish samples). In addition, an exome sequencing study was performed in three high-risk French melanoma families. Nineteen SNPs in 17 genes were initially associated with melanoma in the French population. Six SNPs were replicated in phase II, including two new SNPs in the WNT3 (rs199524) and VPS41 (rs11773094) genes. The role of VPS41 and WNT3 was confirmed in a meta-analysis (3940 melanoma cases and 3620 controls) with two-side p values of 0.002, (OR = 0.86) and 4.07 × 10(-10) (OR = 0.80), respectively. Exome sequencing revealed a non-synonymous VPS41 variant in one family that was shown to be strongly associated with familial melanoma (OR = 4.46, p = 0.001) in an independent sample of 178 melanoma families. WNT3 belongs to WNT pathway known to play a crucial role in melanoma, whereas VPS41 regulates vesicular trafficking and is thought to play a role in pigmentation. Our work identified two new pathways involved in melanoma predisposition. These results may be useful in the future for identifying individuals highly predisposed to melanoma.
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http://dx.doi.org/10.1002/ijc.29257DOI Listing
May 2015

Functional and clinical impact of novel TMPRSS6 variants in iron-refractory iron-deficiency anemia patients and genotype-phenotype studies.

Hum Mutat 2014 Nov 10;35(11):1321-9. Epub 2014 Sep 10.

CEINGE, Biotecnologie Avanzate, Naples, Italy.

Iron-refractory iron-deficiency anemia (IRIDA) is a rare autosomal-recessive disorder characterized by hypochromic microcytic anemia, low transferrin saturation, and inappropriate high levels of the iron hormone hepcidin. The disease is caused by variants in the transmembrane protease serine 6 (TMPRSS6) gene that encodes the type II serine protease matriptase-2, a negative regulator of hepcidin transcription. Sequencing analysis of the TMPRSS6 gene in 21 new IRIDA patients from 16 families with different ethnic origin reveal 17 novel mutations, including the most frequent mutation in Southern Italy (p.W590R). Eight missense mutations were analyzed in vitro. All but the p.T287N variant impair matriptase-2 autoproteotylic activation, decrease the ability to cleave membrane HJV and inhibit the HJV-dependent hepcidin activation. Genotype-phenotype studies in IRIDA patients have been so far limited due to the relatively low number of described patients. Our genotype-phenotype correlation analysis demonstrates that patients carrying two nonsense mutations present a more severe anemia and microcytosis and higher hepcidin levels than the other patients. We confirm that TMPRSS6 mutations are spread along the gene and that mechanistically they fully or partially abrogate hepcidin inhibition. Genotyping IRIDA patients help in predicting IRIDA severity and may be useful for predicting response to iron treatment.
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http://dx.doi.org/10.1002/humu.22632DOI Listing
November 2014

A large French case-control study emphasizes the role of rare Mc1R variants in melanoma risk.

Biomed Res Int 2014 10;2014:925716. Epub 2014 Apr 10.

INSERM U976, Centre de Recherche sur la Peau, Hôpital Saint Louis, 1 Avenue Claude Vellefaux, 75010 Paris, France ; Laboratoire de Génétique, Hôpital Bichat Claude Bernard, APHP, IFR02, Université Paris 7, 46 rue Henri Hucahrd, 75018 Paris, France.

Background: The MC1R gene implicated in melanogenesis and skin pigmentation is highly polymorphic. Several alleles are associated with red hair and fair skin phenotypes and contribute to melanoma risk.

Objective: This work aims to assess the effect of different classes of MC1R variants, notably rare variants, on melanoma risk. Methods. MC1R coding region was sequenced in 1131 melanoma patients and 869 healthy controls. MC1R variants were classified as RHC (R) and non-RHC (r). Rare variants (frequency < 1%) were subdivided into two subgroups, predicted to be damaging (D) or not (nD).

Results: Both R and r alleles were associated with melanoma (OR = 2.66 [2.20-3.23] and 1.51 [1.32-1.73]) and had similar population attributable risks (15.8% and 16.6%). We also identified 69 rare variants, of which 25 were novel. D variants were strongly associated with melanoma (OR = 2.38 [1.38-4.15]) and clustered in the same MC1R domains as R alleles (intracellular 2, transmembrane 2 and 7).

Conclusion: This work confirms the role of R and r alleles in melanoma risk in the French population and proposes a novel class of rare D variants as important melanoma risk factors. These findings may improve the definition of high-risk subjects that could be targeted for melanoma prevention and screening.
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http://dx.doi.org/10.1155/2014/925716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4003837PMC
February 2015

Early-onset osteoarthritis, Charcot-Marie-Tooth like neuropathy, autoimmune features, multiple arterial aneurysms and dissections: an unrecognized and life threatening condition.

PLoS One 2014 7;9(5):e96387. Epub 2014 May 7.

INSERM U698, Hôpital Bichat, Paris, France; AP-HP, Hôpital Bichat, Centre de référence pour les syndromes de Marfan et apparentés, Service de Cardiologie, Paris, France; Université Denis Diderot, Paris 7, UFR de Médecine, Paris, France.

Background: Severe osteoarthritis and thoracic aortic aneurysms have recently been associated with mutations in the SMAD3 gene, but the full clinical spectrum is incompletely defined.

Methods: All SMAD3 gene mutation carriers coming to our centre and their families were investigated prospectively with a structured panel including standardized clinical workup, blood tests, total body computed tomography, joint X-rays. Electroneuromyography was performed in selected cases.

Results: Thirty-four SMAD3 gene mutation carriers coming to our centre were identified and 16 relatives were considered affected because of aortic surgery or sudden death (total 50 subjects). Aortic disease was present in 72%, complicated with aortic dissection, surgery or sudden death in 56% at a mean age of 45 years. Aneurysm or tortuosity of the neck arteries was present in 78%, other arteries were affected in 44%, including dissection of coronary artery. Overall, 95% of mutation carriers displayed either aortic or extra-aortic arterial disease. Acrocyanosis was also present in the majority of patients. Osteoarticular manifestations were recorded in all patients. Joint involvement could be severe requiring surgery in young patients, of unusual localization such as tarsus or shoulder, or mimicking crystalline arthropathy with fibrocartilage calcifications. Sixty eight percent of patients displayed neurological symptoms, and 9 suffered peripheral neuropathy. Electroneuromyography revealed an axonal motor and sensory neuropathy in 3 different families, very evocative of type II Charcot-Marie-Tooth (CMT2) disease, although none had mutations in the known CMT2 genes. Autoimmune features including Sjogren's disease, rheumatoid arthritis, Hashimoto's disease, or isolated autoantibodies- were found in 36% of patients.

Interpretation: SMAD3 gene mutations are associated with aortic dilatation and osteoarthritis, but also autoimmunity and peripheral neuropathy which mimics type II Charcot-Marie-Tooth.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096387PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4012990PMC
October 2015

Comprehensive functional annotation of 18 missense mutations found in suspected hemochromatosis type 4 patients.

Hum Mol Genet 2014 Sep 8;23(17):4479-90. Epub 2014 Apr 8.

Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Inserm U1078, Université de Brest, SFR SnInBioS, CHRU de Brest, Etablissement Français du Sang - Bretagne, Brest, France CHRU de Brest, Inserm CIC0502, Brest, France

Hemochromatosis type 4 is a rare form of primary iron overload transmitted as an autosomal dominant trait caused by mutations in the gene encoding the iron transport protein ferroportin 1 (SLC40A1). SLC40A1 mutations fall into two functional categories (loss- versus gain-of-function) underlying two distinct clinical entities (hemochromatosis type 4A versus type 4B). However, the vast majority of SLC40A1 mutations are rare missense variations, with only a few showing strong evidence of causality. The present study reports the results of an integrated approach collecting genetic and phenotypic data from 44 suspected hemochromatosis type 4 patients, with comprehensive structural and functional annotations. Causality was demonstrated for 10 missense variants, showing a clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups of loss-of-function mutations were distinguished: one impairing cell-surface expression and one altering only iron egress. Additionally, a new gain-of-function mutation was identified, and the degradation of ferroportin on hepcidin binding was shown to probably depend on the integrity of a large extracellular loop outside of the hepcidin-binding domain. Eight further missense variations, on the other hand, were shown to have no discernible effects at either protein or RNA level; these were found in apparently isolated patients and were associated with a less severe phenotype. The present findings illustrate the importance of combining in silico and biochemical approaches to fully distinguish pathogenic SLC40A1 mutations from benign variants. This has profound implications for patient management.
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http://dx.doi.org/10.1093/hmg/ddu160DOI Listing
September 2014

Antisense oligonucleotide-based therapy in human erythropoietic protoporphyria.

Am J Hum Genet 2014 Apr 27;94(4):611-7. Epub 2014 Mar 27.

Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France; Assistance Publique-Hôpitaux de Paris, Centre Français des Porphyries, Hôpital Louis Mourier, 178 Rue des Renouillers, F-92701 Colombes, France; Université de Versailles Saint Quentin en Yvelines, F-78035 Versailles, France; Assistance Publique-Hôpitaux de Paris, Laboratoire de Biochimie Hormonale et Génétique, Hôpital Ambroise Paré, F-92100 Boulogne Billancourt, France; Laboratory of Excellence GR-Ex, 75000 Paris, France.

In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.
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http://dx.doi.org/10.1016/j.ajhg.2014.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3980518PMC
April 2014

RHOXF2 gene, a new candidate gene for spermatogenesis failure.

Basic Clin Androl 2014 10;24. Epub 2014 Feb 10.

EA 2493, University of Versailles Saint-Quentin, Versailles, F-78035 France ; Department of Reproductive Biology, Cytogenetics, Gynecology and Obstetrics, Poissy Saint Germain Hospital, Poissy, F-78303 France.

Introduction: Genes involved in testicular differentiation, spermatogenesis, proliferation and apoptosis of germ cells have been shown to evolve rapidly and display rapid DNA changes. These genes are therefore good candidates for explaining impairments in spermatogenesis. Initial studies of some of these genes appear to confirm this hypothesis. The RHOXF2 candidate gene belongs to the RHOX family clustered in Xq24 and is specifically expressed in the testis. It contains four exons and codes for a 288 amino acid (aa) transcription factor. It has a high degree of homology (>99.9%) with its paralogue RHOXF2B, which is also preferentially expressed in the testis.

Objectives: To sequence RHOXF2 and RHOXF2B in intracytoplasmic sperm injection (ICSI) patients and identify any single-nucleotide polymorphisms (SNPs) associated with impaired spermatogenesis.

Materials: A cohort of 327 patients in ICSI programmes at Poissy and Bichat hospitals. All patients gave their written, informed consent to participation. One hundred patients had unaffected spermatogenesis and 227 displayed impaired spermatogenesis.

Methods: The four exons in each of RHOXF2 and RHOXF2B were sequenced in 47 patients with oligospermia or non-obstructive azoospermia. Given that exons 2 and 3 were found to harbour most of the SNPs, only these two exons were sequenced in the remaining 280 subjects.

Results: Due to the extremely high degree of sequence identity between RHOXF2 and RHOXF2B, we were not able to distinguish between the sequences of these two genes. Although 9 SNPs were identified, there were no significant frequency differences between ICSI patients with normal vs. impaired spermatogenesis. Two insertions were identified: a 21-nucleotide insertion was retrieved in both groups and a guanine insertion (inducing a premature stop codon) only found in two patients with impaired spermatogenesis.

Conclusion/outlook: RHOXF2 is a good candidate for rapid evolution by positive selection. Analysis of the polymorphism frequency in exons 2 and 3 did not allow us to correlate the identified SNPs with male infertility. However, a single nucleotide insertion was identified only in men with impaired spermatogenesis. Further work will be needed to establish whether genetic changes in RHOXF2 can give rise to defects in spermatogenesis.
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http://dx.doi.org/10.1186/2051-4190-24-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4349744PMC
March 2015

X-linked sideroblastic anemia due to ALAS2 intron 1 enhancer element GATA-binding site mutations.

Am J Hematol 2014 Mar 20;89(3):315-9. Epub 2013 Nov 20.

Department of Pathology, Boston Children's Hospital, Boston, Massachusetts.

X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia. In affected males, it is uniformly associated with partial loss-of-function missense mutations in the erythroid-specific heme biosynthesis protein 5-aminolevulinate synthase 2 (ALAS2). Here, we report five families with XLSA owing to mutations in a GATA transcription factor binding site located in a transcriptional enhancer element in intron 1 of the ALAS2 gene. As such, this study defines a new class of mutations that should be evaluated in patients undergoing genetic testing for a suspected diagnosis of XLSA.
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http://dx.doi.org/10.1002/ajh.23616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943703PMC
March 2014

Epistasis in iron metabolism: complex interactions between Cp, Mon1a, and Slc40a1 loci and tissue iron in mice.

Mamm Genome 2013 Dec;24(11-12):427-38

Disorders of iron metabolism are among the most common acquired and constitutive diseases. Hemochromatosis has a solid genetic basis and in Northern European populations it is usually associated with homozygosity for the C282Y mutation in the HFE protein. However, the penetrance of this mutation is incomplete and the clinical presentation is highly variable. The rare and common variants identified so far as genetic modifiers of HFE-related hemochromatosis are unable to account for the phenotypic heterogeneity of this disorder. There are wide variations in the basal iron status of common inbred mouse strains, and this diversity may reflect the genetic background of the phenotypic diversity under pathological conditions. We therefore examined the genetic basis of iron homeostasis using quantitative trait loci mapping applied to the HcB-15 recombinant congenic strains for tissue and serum iron indices. Two highly significant QTL containing either the N374S Mon1a mutation or the Ferroportin locus were found to be major determinants in spleen and liver iron loading. Interestingly, when considering possible epistatic interactions, the effects of Mon1a on macrophage iron export are conditioned by the genotype at the Slc40a1 locus. Only mice that are C57BL/10ScSnA homozygous at both loci display a lower spleen iron burden. Furthermore, the liver-iron lowering effect of the N374S Mon1a mutation is observed only in mice that display a nonsense mutation in the Ceruloplasmin (Cp) gene. This study highlights the existence of genetic interactions between Cp, Mon1a, and the Slc40a1 locus in iron metabolism, suggesting that epistasis may be a crucial determinant of the variable biological and clinical presentations in iron disorders.
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http://dx.doi.org/10.1007/s00335-013-9479-6DOI Listing
December 2013

The MUC5B variant is associated with idiopathic pulmonary fibrosis but not with systemic sclerosis interstitial lung disease in the European Caucasian population.

PLoS One 2013 5;8(8):e70621. Epub 2013 Aug 5.

Service de Pneumologie A, APHP, Hopital Bichat, Paris, France.

A polymorphism on the MUC5B promoter (rs35705950) has been associated with idiopathic pulmonary fibrosis (IPF) but not with systemic sclerosis (SSc) with interstitial lung disease (ILD). We genotyped the MUC5B promoter in the first 142 patients of the French national prospective cohort of IPF, in 981 French patients with SSc (346 ILD), 598 Italian patients with SSc (207 ILD), 1383 French controls and 494 Italian controls. A meta-analysis was performed including all American data available. The T risk allele was present in 41.9% of the IPF patients, 10.8% of the controls (P = 2 × 10(-44)), OR 6.3 [4.6-8.7] for heterozygous patients and OR 21.7 [10.4-45.3] for homozygous patients. Prevalence of the T allele was not modified according to age, gender, smoking in IPF patients. However, none of the black patients with IPF presented the T allele. The prevalence of the T risk allele was similar between French (10%) and Italian (12%) cohorts of SSc whatever the presence of an ILD (11.1% and 13.5%, respectively). Meta-analysis confirmed the similarity between French, Italian and American cohorts of IPF or SSc-ILD. This study confirms 1) an association between the T allele risk and IPF, 2) an absence of association with SSc-ILD, suggesting different pathophysiology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070621PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734256PMC
March 2014

Identification of mutations in TMEM5 and ISPD as a cause of severe cobblestone lissencephaly.

Am J Hum Genet 2012 Dec;91(6):1135-43

Assistance Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, Biochimie, Paris 75877, France.

Cobblestone lissencephaly is a peculiar brain malformation with characteristic radiological anomalies. It is defined as cortical dysplasia that results when neuroglial overmigration into the arachnoid space forms an extracortical layer that produces agyria and/or a "cobblestone" brain surface and ventricular enlargement. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal-recessive diseases characterized by cerebral, ocular, and muscular deficits. These include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN, and FKRP identified these diseases as alpha-dystroglycanopathies. Our exhaustive screening of these six genes, in a cohort of 90 fetal cases, led to the identification of a mutation in only 53% of the families, suggesting that other genes might also be involved. We therefore decided to perform a genome-wide study in two multiplex families. This allowed us to identify two additional genes: TMEM5 and ISPD. Because TMEM has a glycosyltransferase domain and ISPD has an isoprenoid synthase domain characteristic of nucleotide diP-sugar transferases, these two proteins are thought to be involved in the glycosylation of dystroglycan. Further screening of 40 families with cobblestone lissencephaly identified nonsense and frameshift mutations in another four unrelated cases for each gene, increasing the mutational rate to 64% in our cohort. All these cases displayed a severe phenotype of cobblestone lissencephaly A. TMEM5 mutations were frequently associated with gonadal dysgenesis and neural tube defects, and ISPD mutations were frequently associated with brain vascular anomalies.
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http://dx.doi.org/10.1016/j.ajhg.2012.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3516603PMC
December 2012

Genetic variation at KIT locus may predispose to melanoma.

Pigment Cell Melanoma Res 2013 Jan 23;26(1):88-96. Epub 2012 Nov 23.

Département de Génétique, Hôpital Bichat-Claude Bernard, APHP, Paris, France.

As loss of KIT frequently occurs in melanoma progression, we hypothesized that KIT is implicated in predisposition to melanoma (MM). Thus, we sequenced the KIT coding region in 112 familial MM cases and 143 matched controls and genotyped tag single-nucleotide polymorphisms (SNPs) in two cohorts of melanoma patients and matched controls. Five rare KIT substitutions, all predicted possibly or probably deleterious, were identified in five patients, but none in controls [RR = 2.26 (1.26-2.26)]. Expressed in melanocyte lines, three substitutions inhibited KIT signaling. Comparison with exomes database (7020 alleles) confirmed a significant excess of rare deleterious KIT substitutions in patients. Additionally, a common SNP, rs2237028, was associated with MM risk, and 6 KIT variants were associated with nevus count. Our data strongly suggest that rare KIT substitutions predispose to melanoma and that common variants at KIT locus may also impact nevus count and melanoma risk.
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http://dx.doi.org/10.1111/pcmr.12032DOI Listing
January 2013

TGFB2 mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of Marfan syndrome.

Nat Genet 2012 Jul 8;44(8):916-21. Epub 2012 Jul 8.

Institut National de la Santé et de la Recherche Médicale (INSERM) U698, Hôpital Bichat, Paris, France.

A predisposition for thoracic aortic aneurysms leading to acute aortic dissections can be inherited in families in an autosomal dominant manner. Genome-wide linkage analysis of two large unrelated families with thoracic aortic disease followed by whole-exome sequencing of affected relatives identified causative mutations in TGFB2. These mutations-a frameshift mutation in exon 6 and a nonsense mutation in exon 4-segregated with disease with a combined logarithm of odds (LOD) score of 7.7. Sanger sequencing of 276 probands from families with inherited thoracic aortic disease identified 2 additional TGFB2 mutations. TGFB2 encodes transforming growth factor (TGF)-β2, and the mutations are predicted to cause haploinsufficiency for TGFB2; however, aortic tissue from cases paradoxically shows increased TGF-β2 expression and immunostaining. Thus, haploinsufficiency for TGFB2 predisposes to thoracic aortic disease, suggesting that the initial pathway driving disease is decreased cellular TGF-β2 levels leading to a secondary increase in TGF-β2 production in the diseased aorta.
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http://dx.doi.org/10.1038/ng.2348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4033668PMC
July 2012

Inactive matriptase-2 mutants found in IRIDA patients still repress hepcidin in a transfection assay despite having lost their serine protease activity.

Hum Mutat 2012 Sep 30;33(9):1388-96. Epub 2012 May 30.

Université Paris Diderot, Paris, France.

Mutations of the TMPRSS6 gene, which encodes Matriptase-2, are responsible for iron-refractory iron-deficiency anemia. Matriptase-2 is a transmembrane protease that downregulates hepcidin expression. We report one frameshift (p.Ala605ProfsX8) and four novel missense mutations (p.Glu114Lys, p.Leu235Pro, p.Tyr418Cys, p.Pro765Ala) found in IRIDA patients. These mutations lead to changes in both the catalytic and noncatalytic domains of Matriptase-2. Analyses of the mutant proteins revealed a reduction of autoactivating cleavage and the loss of N-Boc-Gln-Ala-Arg-p-nitroanilide hydrolysis. This resulted either from a direct modification of the active site or from the lack of the autocatalytic cleavage that transforms the zymogen into an active protease. In a previously described transfection assay measuring the ability of Matriptase-2 to repress the hepcidin gene (HAMP) promoter, all mutants retained some, if not all, of their transcriptional repression activity. This suggests that caution is called for in interpreting the repression assay in assessing the functional relevance of Matriptase-2 substitutions. We propose that Matriptase-2 activity should be measured directly in the cell medium of transfected cells using the chromogenic substrate. This simple test can be used to determine whether a sequence variation leading to an amino acid substitution is functionally relevant or not.
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http://dx.doi.org/10.1002/humu.22116DOI Listing
September 2012

[Molecular diagnosis of HFE mutations in routine laboratories. Results of a survey from reference laboratories in France].

Ann Biol Clin (Paris) 2012 May-Jun;70(3):305-13

Laboratoire de génétique moléculaire, Centre de référence des surcharges en fer rares d'origine génétique, CHU Pontchaillou, Rennes.

HFE-related hemochromatosis (HFE hemochromatosis) or type 1 hemochromatosis is an autosomal recessive disease characterized by progressive iron overload usually expressed in adulthood. The HFE gene, located on the short arm of chromosome 6 (6p21.3), encodes a protein that plays a crucial role in iron metabolism by modulating hepcidin synthesis in the liver. Homozygosity for the p.Cys282Tyr mutation accounts for nearly 80% of cases of hemochromatosis in France. Genetic testing is the key investigation to confirm the diagnosis of HFE hemochromatosis. A survey on routine practices was carried out among the eight reference laboratories of the French national network on genetic iron disorders. The main findings from this survey are as follows: 1) the p.Cys282Tyr mutation must be searched for as an initial step to establish the diagnosis of HFE hemochromatosis. This is in agreement with the recommendations of the French Health Authority (HAS) published in 2005. In these recommendations, homozygosity for the p.Cys282Tyr mutation with at least elevated transferrin saturation, is considered the only genotype that confirms of the diagnosis of HFE hemochromatosis; 2) in combination with the p.Cys282Tyr mutation (compound heterozygous genotypes), the p.Ser65Cys and the p.His63Asp variants may contribute to the occurrence of mild iron overload; 3) family screening is mandatory following the detection of homozygous individuals for the p.Cys282Tyr mutation.
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http://dx.doi.org/10.1684/abc.2012.0704DOI Listing
August 2012

Two novel mutations in the L ferritin coding sequence associated with benign hyperferritinaemia unmasked by glycosylated ferritin assay.

Ann Clin Biochem 2012 May 25;49(Pt 3):302-5. Epub 2012 Apr 25.

Department of Pathology, Princess Royal University Hospital, Farnborough, Orpington, Kent BR6 8ND, UK.

Investigating persistent hyperferritinaemia without apparent iron overload is challenging. Even when inflammation, cirrhosis, Still's disease, fatty liver and malignancy are excluded, there remains a group of patients with unexplained hyperferritinaemia for whom rare forms of haemochromatosis (ferroportin disease) are a consideration. Preliminary results suggest that abnormal percentage glycosylation of serum ferritin is seen in some cases of genetically determined hyperferritinaemia. Serum ferritin is normally 50-81% glycosylated, but low glycosylation (20-42%) prevails in hereditary hyperferritinaemia cataract syndrome. This contrasts with hyperglycosylation (>90%) associated with the benign hyperferritinaemia related to missense L ferritin (p.Thr30Ile) mutation. Here, we describe two novel missense L ferritin variants also associated with hyperglycosylation, p.Gln26Ile and p.Ala27Val. Ferritin glycosylation, a comparatively simple measurement, can identify patients for DNA sequencing as hyperglycosylation (>90%) is associated with benign hyperferritinaemia and mutant L ferritin chain.
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http://dx.doi.org/10.1258/acb.2011.011229DOI Listing
May 2012

A new human NHERF1 mutation decreases renal phosphate transporter NPT2a expression by a PTH-independent mechanism.

PLoS One 2012 10;7(4):e34764. Epub 2012 Apr 10.

Faculté de Médecine, Université Paris Descartes, Paris, France.

Background: The sodium-hydrogen exchanger regulatory factor 1 (NHERF1) binds to the main renal phosphate transporter NPT2a and to the parathyroid hormone (PTH) receptor. We have recently identified mutations in NHERF1 that decrease renal phosphate reabsorption by increasing PTH-induced cAMP production in the renal proximal tubule.

Methods: We compared relevant parameters of phosphate homeostasis in a patient with a previously undescribed mutation in NHERF1 and in control subjects. We expressed the mutant NHERF1 protein in Xenopus Oocytes and in cultured cells to study its effects on phosphate transport and PTH-induced cAMP production.

Results: We identified in a patient with inappropriate renal phosphate reabsorption a previously unidentified mutation (E68A) located in the PDZ1 domain of NHERF1.We report the consequences of this mutation on NHERF1 function. E68A mutation did not modify cAMP production in the patient. PTH-induced cAMP synthesis and PKC activity were not altered by E68A mutation in renal cells in culture. In contrast to wild-type NHERF1, expression of the E68A mutant in Xenopus oocytes and in human cells failed to increase phosphate transport. Pull down experiments showed that E68A mutant did not interact with NPT2a, which robustly interacted with wild type NHERF1 and previously identified mutants. Biotinylation studies revealed that E68A mutant was unable to increase cell surface expression of NPT2a.

Conclusions: Our results indicate that the PDZ1 domain is critical for NHERF1-NPT2a interaction in humans and for the control of NPT2a expression at the plasma membrane. Thus we have identified a new mechanism of renal phosphate loss and shown that different mutations in NHERF1 can alter renal phosphate reabsorption via distinct mechanisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034764PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323571PMC
October 2012

A novel type of congenital hypochromic anemia associated with a nonsense mutation in the STEAP3/TSAP6 gene.

Blood 2011 Dec 26;118(25):6660-6. Epub 2011 Oct 26.

Assistance Publique des Hôpitaux de Paris, Hôpital Bichat, Laboratoire de Biochimie Hormonale et Génétique, Paris, France.

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.
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http://dx.doi.org/10.1182/blood-2011-01-329011DOI Listing
December 2011

Assessment of tyrosinase variants and skin cancer risk in a large cohort of French subjects.

J Dermatol Sci 2011 Nov 22;64(2):127-33. Epub 2011 Aug 22.

Laboratoire de Biochimie Hormonale et Génétique, Hôpital Bichat Claude Bernard, APHP, IFR02, 75018, Paris, France.

Background: Tyrosinase (TYR) is a key pigmentation gene that is highly polymorphic and responsible for the most common form of autosomal recessive albinism, OCA1.

Objective: To assess the role of frequent and rare TYR variants in predisposition to skin cancer (SK) in the French population.

Methods: We genotyped a frequent TYR variant (p.R402Q) in 1273 patients {1047 cutaneous melanoma (CM) and 226 basal cell carcinoma (BCC)} and 925 controls, and the full coding region of TYR was sequenced in 287 patients suspected of genetic predisposition to SK (familial and/or multiple SK and/or onset before 40 years) and 187 controls.

Results: The homozygous p.R402Q variant was significantly associated with SK risk (P value=0.008; OR=1.57), and was mostly associated with multiple CM risk (P value=0.021; OR=2.50) and familial CM risk (P value=0.022; OR=2.16). In addition, 19 rare TYR variants, mainly albinism mutations, were identified in 15 patients and 8 controls. Among these, 3 clearly deleterious mutations (1 non-sense and 2 affecting mRNA splicing) were identified in 3 patients, one of which was homozygous.

Conclusion: Our data confirmed the association of TYR p.R402Q with SK risk in the French population, and support that rare deleterious TYR variants may also play a role in multi-factorial genetic predisposition to SK. These results should be confirmed by replications studies.
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http://dx.doi.org/10.1016/j.jdermsci.2011.07.003DOI Listing
November 2011

Two novel mutations in the EIF2AK3 gene in children with Wolcott-Rallison syndrome.

Pediatr Diabetes 2011 May 7;12(3 Pt 1):187-91. Epub 2010 Sep 7.

Endocrinology Unit, Federal University of São Paulo (UNIFESP), São Paulo, Brazil.

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.
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http://dx.doi.org/10.1111/j.1399-5448.2010.00679.xDOI Listing
May 2011

Missense SLC25A38 variations play an important role in autosomal recessive inherited sideroblastic anemia.

Haematologica 2011 Jun 10;96(6):808-13. Epub 2011 Mar 10.

AP-HP, Service de Biochimie Génétique et Hormonale, Hopital Bichat, Paris, INSERM, Centre de Recherche Biomédicale Bichat Beaujon, Université Paris Diderot, France.

Background: Congenital sideroblastic anemias are rare disorders with several genetic causes; they are characterized by erythroblast mitochondrial iron overload, differ greatly in severity and some occur within a syndrome. The most common cause of non-syndromic, microcytic sideroblastic anemia is a defect in the X-linked 5-aminolevulinate synthase 2 gene but this is not always present. Recently, variations in the gene for the mitochondrial carrier SLC25A38 were reported to cause a non-syndromic, severe type of autosomal-recessive sideroblastic anemia. Further evaluation of the importance of this gene was required to estimate the proportion of patients affected and to gain further insight into the range and types of variations involved.

Design And Methods: In three European diagnostic laboratories sequence analysis of SLC25A38 was performed on DNA from patients affected by congenital sideroblastic anemia of a non-syndromic nature not caused by variations in the 5-aminolevulinate synthase 2 gene.

Results: Eleven patients whose ancestral origins spread across several continents were homozygous or compound heterozygous for ten different SLC25A38 variations causing premature termination of translation (p.Arg117X, p.Tyr109LeufsX43), predicted splicing alteration (c.625G>C; p.Asp209His) or missense substitution (p.Gln56Lys, p.Arg134Cys, p.Ile147Asn, p.Arg187Gln, p.Pro190Arg, p.Gly228Val, p.Arg278Gly). Only three of these variations have been described previously (p.Arg117X, p.Tyr109LeufsX43 and p.Asp209His). All new variants reported here are missense and affect conserved amino acids. Structure modeling suggests that these variants may influence different aspects of transport as described for mutations in other mitochondrial carrier disorders.

Conclusions: Mutations in the SLC25A38 gene cause severe, non-syndromic, microcytic/hypochromic sideroblastic anemia in many populations. Missense mutations are shown to be of importance as are mutations that affect protein production. Further investigation of these mutations should shed light on structure-function relationships in this protein.
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http://dx.doi.org/10.3324/haematol.2010.039164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105641PMC
June 2011

Sideroblastic anemia: molecular analysis of the ALAS2 gene in a series of 29 probands and functional studies of 10 missense mutations.

Hum Mutat 2011 Jun 24;32(6):590-7. Epub 2011 Feb 24.

INSERM, Centre de Recherche Biomédicale Bichat-Beaujon, Paris, France.

X-linked Sideroblastic Anemia (XLSA) is the most common genetic form of sideroblastic anemia, a heterogeneous group of disorders characterized by iron deposits in the mitochondria of erythroid precursors. XLSA is due to mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Thirteen different ALAS2 mutations were identified in 16 out of 29 probands with sideroblastic anemia. One third of the patients were females with a highly skewed X-chromosome inactivation. The identification of seven novel mutations in the ALAS2 gene, six missense mutations, and one deletion in the proximal promoter extends the allelic heterogeneity of XSLA. Most of the missense mutations were predicted to be deleterious, and 10 of them, without any published functional characterization, were expressed in Escherichia coli. ALAS2 activities were assayed in vitro. Five missense mutations resulted in decreased enzymatic activity under standard conditions, and two other mutated proteins had decreased activity when assayed in the absence of exogenous pyridoxal phosphate and increased thermosensitivity. Although most amino acid substitutions result in a clearly decreased enzymatic activity in vitro, a few mutations have a more subtle effect on the protein that is only revealed by in vitro tests under specific conditions.
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http://dx.doi.org/10.1002/humu.21455DOI Listing
June 2011

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

Haematologica 2011 Apr 12;96(4):507-14. Epub 2011 Jan 12.

Laboratoire d'Hématologie, CHU de Montpellier, Montpellier, France.

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.
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http://dx.doi.org/10.3324/haematol.2010.029751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3069226PMC
April 2011
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