Publications by authors named "Karine Nguyen"

45 Publications

Heterozygous HTRA1 nonsense or frameshift mutations are pathogenic.

Brain 2021 Oct;144(9):2616-2624

AP-HP, Service de Génétique Moléculaire Neurovasculaire, Hôpital Saint-Louis, France.

Heterozygous missense HTRA1 mutations have been associated with an autosomal dominant cerebral small vessel disease (CSVD) whereas the pathogenicity of heterozygous HTRA1 stop codon variants is unclear. We performed a targeted high throughput sequencing of all known CSVD genes, including HTRA1, in 3853 unrelated consecutive CSVD patients referred for molecular diagnosis. The frequency of heterozygous HTRA1 mutations leading to a premature stop codon in this patient cohort was compared with their frequency in large control databases. An analysis of HTRA1 mRNA was performed in several stop codon carrier patients. Clinical and neuroimaging features were characterized in all probands. Twenty unrelated patients carrying a heterozygous HTRA1 variant leading to a premature stop codon were identified. A highly significant difference was observed when comparing our patient cohort with control databases: gnomAD v3.1.1 [P = 3.12 × 10-17, odds ratio (OR) = 21.9], TOPMed freeze 5 (P = 7.6 × 10-18, OR = 27.1) and 1000 Genomes (P = 1.5 × 10-5). Messenger RNA analysis performed in eight patients showed a degradation of the mutated allele strongly suggesting a haploinsufficiency. Clinical and neuroimaging features are similar to those previously reported in heterozygous missense mutation carriers, except for penetrance, which seems lower. Altogether, our findings strongly suggest that heterozygous HTRA1 stop codons are pathogenic through a haploinsufficiency mechanism. Future work will help to estimate their penetrance, an important information for genetic counselling.
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http://dx.doi.org/10.1093/brain/awab271DOI Listing
October 2021

High rate of hypomorphic variants as the cause of inherited ataxia and related diseases: study of a cohort of 366 families.

Genet Med 2021 11 7;23(11):2160-2170. Epub 2021 Jul 7.

Department of Clinical Genetics, Centre de Référence Maladies Rares Anomalies du Développement, CHU de Rennes, Rennes, France.

Purpose: Diagnosis of inherited ataxia and related diseases represents a real challenge given the tremendous heterogeneity and clinical overlap of the various causes. We evaluated the efficacy of molecular diagnosis of these diseases by sequencing a large cohort of undiagnosed families.

Methods: We analyzed 366 unrelated consecutive patients with undiagnosed ataxia or related disorders by clinical exome-capture sequencing. In silico analysis was performed with an in-house pipeline that combines variant ranking and copy-number variant (CNV) searches. Variants were interpreted according to American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines.

Results: We established the molecular diagnosis in 46% of the cases. We identified 35 mildly affected patients with causative variants in genes that are classically associated with severe presentations. These cases were explained by the occurrence of hypomorphic variants, but also rarely suspected mechanisms such as C-terminal truncations and translation reinitiation.

Conclusion: A significant fraction of the clinical heterogeneity and phenotypic overlap is explained by hypomorphic variants that are difficult to identify and not readily predicted. The hypomorphic C-terminal truncation and translation reinitiation mechanisms that we identified may only apply to few genes, as it relies on specific domain organization and alterations. We identified PEX10 and FASTKD2 as candidates for translation reinitiation accounting for mild disease presentation.
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http://dx.doi.org/10.1038/s41436-021-01250-6DOI Listing
November 2021

AKT Signaling Modifies the Balance between Cell Proliferation and Migration in Neural Crest Cells from Patients Affected with Bosma Arhinia and Microphthalmia Syndrome.

Biomedicines 2021 Jun 29;9(7). Epub 2021 Jun 29.

Aix-Marseille Univ-INSERM, MMG, 13005 Marseille, France.

Over the recent years, the SMCHD1 (Structural Maintenance of Chromosome flexible Hinge Domain Containing 1) chromatin-associated factor has triggered increasing interest after the identification of variants in three rare and unrelated diseases, type 2 Facio Scapulo Humeral Dystrophy (FSHD2), Bosma Arhinia and Microphthalmia Syndrome (BAMS), and the more recently isolated hypogonadotrophic hypogonadism (IHH) combined pituitary hormone deficiency (CPHD) and septo-optic dysplasia (SOD). However, it remains unclear why certain mutations lead to a specific muscle defect in FSHD while other are associated with severe congenital anomalies. To gain further insights into the specificity of SMCHD1 variants and identify pathways associated with the BAMS phenotype and related neural crest defects, we derived induced pluripotent stem cells from patients carrying a mutation in this gene. We differentiated these cells in neural crest stem cells and analyzed their transcriptome by RNA-Seq. Besides classical differential expression analyses, we analyzed our data using MOGAMUN, an algorithm allowing the extraction of active modules by integrating differential expression data with biological networks. We found that in BAMS neural crest cells, all subnetworks that are associated with differentially expressed genes converge toward a predominant role for AKT signaling in the control of the cell proliferation-migration balance. Our findings provide further insights into the distinct mechanism by which defects in neural crest migration might contribute to the craniofacial anomalies in BAMS.
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http://dx.doi.org/10.3390/biomedicines9070751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8301469PMC
June 2021

Phenotype/Genotype Relationship in Left Ventricular Noncompaction: Ion Channel Gene Mutations Are Associated With Preserved Left Ventricular Systolic Function and Biventricular Noncompaction: Phenotype/Genotype of Noncompaction.

J Card Fail 2021 06;27(6):677-681

Cardiology Department, APHM, La Timone Hospital, Marseille, France; Aix Marseille Univ, IRD, APHM, MEPHI, IHU-Méditerranée Infection, Marseille, France. Electronic address:

Background: Few data exist concerning genotype-phenotype relationships in left ventricular noncompaction (LVNC).

Methods And Results: From a multicenter French Registry, we report the genetic and clinical spectrum of 95 patients with LVNC, and their genotype-phenotype relationship. Among the 95 LVNC, 45 had at least 1 mutation, including 14 cases of mutation in ion channel genes. In a complementary analysis including 16 additional patients with ion channel gene mutations, for a total of 30 patients with ion channel gene mutation, we found that those patients had higher median LV ejection fraction (60% vs 40%; P < .001) and more biventricular noncompaction (53.1% vs 18.5%; P < .001) than the 81 other patients with LVNC. Among them, both the 19 patients with an HCN4 mutation and the 11 patients with an RYR2 mutation presented with a higher LV ejection fraction and more frequent biventricular noncompaction than the 81 patients with LVNC but with no mutation in the ion channel gene, but only patients with HCN4 mutation presented with a lower heart rate.

Conclusions: Ion channel gene mutations should be searched systematically in patients with LVNC associated with either bradycardia or biventricular noncompaction, particularly when LV systolic function is preserved. Identifying causative mutations is of utmost importance for genetic counselling of at-risk relatives of patients affected by LVNC.
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http://dx.doi.org/10.1016/j.cardfail.2021.01.007DOI Listing
June 2021

Retrospective analysis and reclassification of DYSF variants in a large French series of dysferlinopathy patients.

Genet Med 2021 08 29;23(8):1574-1577. Epub 2021 Apr 29.

Service de Génétique Médicale, Hôpital de la Timone, APHM, Marseille, France.

Purpose: Recent evolution of sequencing technologies and the development of international standards in variant interpretation have profoundly changed the diagnostic approaches in clinical genetics. As a consequence, many variants that were initially claimed to be disease-causing can be now reclassified as benign or uncertain in light of the new data available. Unfortunately, the misclassified variants are still present in the scientific literature and variant databases, greatly interfering with interpretation of diagnostic sequencing results. Despite the urgent need, large-scale efforts to update the classifications of these variants are still not sufficient.

Methods: We retrospectively analyzed 176 DYSF gene variants that were identified in dysferlinopathy patients referred to the Marseille Medical Genetics Department for diagnostic sequencing since 2001.

Results: We reclassified all variants into five-tier American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) pathogenicity classes, revealing changed pathogenicity for 17 variants. We then updated the information for the variants that have been previously published in the variant database and submitted 46 additional DYSF variants.

Conclusion: Besides direct benefit for dysferlinopathy diagnostics, our study contributes to the much needed effort to reanalyze variants from previously published cohorts and to work with curators of variant databases to update the entries for erroneously classified variants.
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http://dx.doi.org/10.1038/s41436-021-01164-3DOI Listing
August 2021

Acute monophasic erythromelalgia pain in five children diagnosed as small-fiber neuropathy.

Eur J Paediatr Neurol 2020 Sep 7;28:198-204. Epub 2020 Jul 7.

Department of Pediatrics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; University of Lausanne, Lausanne, Switzerland.

The small-fiber polyneuropathies (SFN) are a class of diseases in which the small thin myelinated (Aδ) and/or unmyelinated (C) fibers within peripheral nerves malfunction and can degenerate. SFN usually begins in the farthest, most-vulnerable axons, so distal neuropathic pain and symptoms from microvascular dysregulation are common. It is well known in adults, e.g. from diabetes, human immunodeficiency virus, or neurotoxins, but considered extremely rare in children, linked mostly with pathogenic genetic variants in voltage-gated sodium channels. However, increasing evidence suggests that pediatric SFN is not rare, and that dysimmunity is the most common cause. Because most pediatric neurologists are unfamiliar with SFN, we report the diagnosis and management of 5 Swiss children, aged 6-11y, who presented with severe paroxysmal burning pain in the hands and feet temporarily relieved by cooling-the erythromelalgia presentation. Medical evaluations revealed autoimmune diseases in 3 families and 3/5 had preceding or concomitant infections. The standard diagnostic test (PGP9.5-immunolabeled lower-leg skin biopsy) confirmed SFN diagnoses in 3/4, and autonomic function testing (AFT) was abnormal in 2/3. Blood testing for etiology was unrevealing, including genetic testing in 3. Paracetamol and ibuprofen were ineffective. Two children responded to gabapentin plus mexiletine, one to carbamazepine, two to mexiletine plus immunotherapy (methylprednisolone/IVIg). All recovered within 6 months, remaining well for years. These monophasic tempos and therapeutic responses are most consistent with acute post-infectious immune-mediated causality akin to Guillain-Barré large-fiber polyneuropathy. Skin biopsy and AFT for SFN, neuropathic-pain medications and immunotherapy should be considered for acute sporadic pediatric erythromelalgia.
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http://dx.doi.org/10.1016/j.ejpn.2020.06.004DOI Listing
September 2020

Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells.

Cells 2020 06 23;9(6). Epub 2020 Jun 23.

Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France.

Induced pluripotent stem cells (iPSCs) obtained by reprogramming primary somatic cells have revolutionized the fields of cell biology and disease modeling. However, the number protocols for generating mature muscle fibers with sarcolemmal organization using iPSCs remain limited, and partly mimic the complexity of mature skeletal muscle. We used a novel combination of small molecules added in a precise sequence for the simultaneous codifferentiation of human iPSCs into skeletal muscle cells and motor neurons. We show that the presence of both cell types reduces the production time for millimeter-long multinucleated muscle fibers with sarcolemmal organization. Muscle fiber contractions are visible in 19-21 days, and can be maintained over long period thanks to the production of innervated multinucleated mature skeletal muscle fibers with autonomous cell regeneration of PAX7-positive cells and extracellular matrix synthesis. The sequential addition of specific molecules recapitulates key steps of human peripheral neurogenesis and myogenesis. Furthermore, this organoid-like culture can be used for functional evaluation and drug screening. Our protocol, which is applicable to hiPSCs from healthy individuals, was validated in Duchenne Muscular Dystrophy, Myotonic Dystrophy, Facio-Scapulo-Humeral Dystrophy and type 2A Limb-Girdle Muscular Dystrophy, opening new paths for the exploration of muscle differentiation, disease modeling and drug discovery.
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http://dx.doi.org/10.3390/cells9061531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349825PMC
June 2020

Psychosocial Impact of Predictive Genetic Testing in Hereditary Heart Diseases: The PREDICT Study.

J Clin Med 2020 May 6;9(5). Epub 2020 May 6.

APHP, Referral Center for hereditary heart disease, Department of Genetics, Pitié-Salpêtrière University Hospital, 75013 Paris, France.

Predictive genetic testing (PGT) is offered to asymptomatic relatives at risk of hereditary heart disease, but the impact of result disclosure has been little studied. We evaluated the psychosocial impacts of PGT in hereditary heart disease, using self-report questionnaires (including the State-Trait Anxiety Inventory) in 517 adults, administered three times to the prospective cohort (PCo: = 264) and once to the retrospective cohort (RCo: = 253). The main motivations for undergoing PGT were "to remove doubt" and "for their children". The level of anxiety increased between pre-test and result appointments ( <0.0001), returned to baseline after the result (PCo), and was moderately elevated at 4.4 years (RCo). Subjects with a history of depression or with high baseline anxiety were more likely to develop anxiety after PGT result ( = 0.004 and <0.0001, respectively), whatever it was. Unfavourable changes in professional and/or family life were observed in 12.4% (PCo) and 18.7% (RCo) of subjects. Few regrets about PGT were expressed (0.8% RCo, 2.3% PCo). Medical benefit was not the main motivation, which emphasises the role of pre/post-test counselling. When PGT was performed by expert teams, the negative impact was modest, but careful management is required in specific categories of subjects, whatever the genetic test result.
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http://dx.doi.org/10.3390/jcm9051365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290753PMC
May 2020

Facioscapulohumeral Muscular Dystrophy-a Tale of Heterogeneity and the Power of Clinical Assessments.

JAMA Netw Open 2020 05 1;3(5):e205004. Epub 2020 May 1.

Aix Marseille University, Marseille Medical Genetics MMG, Marseille, France.

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http://dx.doi.org/10.1001/jamanetworkopen.2020.5004DOI Listing
May 2020

Left-ventricular non-compaction-comparison between different techniques of quantification of trabeculations: Should the diagnostic thresholds be modified?

Arch Cardiovasc Dis 2020 May 2;113(5):321-331. Epub 2020 Apr 2.

Cardiology Department, La Timone Hospital, AP-HM, 13005 Marseille France; Aix Marseille Université, IRD, APHM, MEPHI, IHU - Méditerranée Infection, 13005 Marseille, France. Electronic address:

Background: Diagnosis of left ventricular non-compaction (LVNC) is challenging, and different imaging techniques propose different criteria.

Aim: To compare the value of two-dimensional transthoracic echocardiography (2D-TTE) and cardiac magnetic resonance (CMR) criteria in diagnosing LVNC, and to test a new trabecular quantification method obtained by 2D-TTE, exploring its relationship with CMR non-compacted mass quantification.

Methods: From a multicentre French study, we selected 48 patients with LVNC and 20 with dilated cardiomyopathy (DCM) who underwent 2D-TTE and CMR. Current 2D-TTE (Jenni et al.) and CMR criteria (Petersen et al., Jacquier et al.), were tested. A new 2D-TTE method of trabecular quantification (percentage of trabecular area) was also proposed, and compared with current criteria.

Results: The best cut-off values for the diagnosis of LVNC were a non-compacted/compacted ratio≥2.3 (Petersen et al.), a trabeculated left ventricular mass≥20% (Jacquier et al.) and a non-compacted/compacted ratio≥1.8 (Jenni et al.). Lowering the threshold for the criterion of Jenni et al. from>2 to ≥1.8 improved its sensitivity from 69% to 98%. The 2D-TTE percentage of trabecular area was 25.9±8% in the LVNC group vs. 9.9±4.4% in the DCM group (P<0.05), and was well correlated with CMR non-compacted mass (r=0.65; P<0.05). A 15.8% threshold value for 2D-TTE percentage of trabecular area predicted LVNC diagnosis with a specificity of 95% and a sensitivity of 92%; its sensitivity was better than that for the criteria of Jenni et al. (P<0.01) and Petersen et al. (P=0.03).

Conclusions: Revision of the current threshold for the criterion of Jenni et al. from>2 to ≥1.8 is necessary to improve LVNC diagnosis in patients with left ventricular dysfunction. A new 2D-TTE trabecular quantification method improves TTE diagnosis of LVNC.
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http://dx.doi.org/10.1016/j.acvd.2020.01.004DOI Listing
May 2020

Type 1 FSHD with 6-10 Repeated Units: Factors Underlying Severity in Index Cases and Disease Penetrance in Their Relatives Attention.

Int J Mol Sci 2020 Mar 23;21(6). Epub 2020 Mar 23.

Reference Center of Neuromuscular disorders and ALS, Timone University Hospital, AP-HM, 264 rue Saint-Pierre, Cedex 05 13385 Marseille, France.

Molecular defects in type 1 facioscapulohumeral muscular dystrophy (FSHD) are caused by a heterozygous contraction of the D4Z4 repeat array from 1 to 10 repeat units (RUs) on 4q35. This study compared (1) the phenotype and severity of FSHD1 between patients carrying 6-8 vs. 9-10 RUs, (2) the amount of methylation in different D4Z4 regions between patients with FSHD1 with different clinical severity scores (CSS). This cross-sectional multicenter study was conducted to measure functional scales and for genetic analysis. Patients were classified into two categories according to RUs: Group 1, 6-8; Group 2, 9-10. Methylation analysis was performed in 27 patients. A total of 99 carriers of a contracted D4Z4 array were examined. No significant correlations between RUs and CSS (r = 0.04, = 0.73) and any of the clinical outcome scales were observed between the two groups. Hypomethylation was significantly more pronounced in patients with high CSS (>3.5) than those with low CSS (<1.5) (in DR1 and 5P), indicating that the extent of hypomethylation might modulate disease severity. In Group 1, the disease severity is not strongly correlated with the allele size and is mostly correlated with the methylation of D4Z4 regions.
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http://dx.doi.org/10.3390/ijms21062221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139460PMC
March 2020

Methylation hotspots evidenced by deep sequencing in patients with facioscapulohumeral dystrophy and mosaicism.

Neurol Genet 2019 Dec 14;5(6):e372. Epub 2019 Nov 14.

Aix Marseille University, INSERM, MMG (S.R., C.D., N.B., C.L., M.-C.G., J.D.R., A.L., F.P., E.S.C., S.A., M.B., R.B., K.N., F.M.); Département de Génétique Médicale (A.L., C.V., C.C., R.B., K.N.), AP-HM, Hôpital de la Timone enfants, Marseille; and Centre de référence pour les maladies neuromusculaires et la SLA (E.S.C., S.A.), AP-HM, Hôpital de la Timone, Marseille, France.

Objective: To investigate the distribution of cytosine-guanine dinucleotide (CpG) sites with a variable level of DNA methylation of the D4Z4 macrosatellite element in patients with facioscapulohumeral dystrophy (FSHD).

Methods: By adapting bisulfite modification to deep sequencing, we performed a comprehensive analysis of D4Z4 methylation across D4Z4 repeats and adjacent 4qA sequence in DNA from patients with FSHD1, FSHD2, or mosaicism and controls.

Results: Using hierarchical clustering, we identified clusters with different levels of methylation and separated, thereby the different groups of samples (controls, FSHD1, and FSHD2) based on their respective level of methylation. We further show that deep sequencing-based methylation analysis discriminates mosaic cases for which methylation changes have never been evaluated previously.

Conclusions: Altogether, our approach offers a new high throughput tool for estimation of the D4Z4 methylation level in the different subcategories of patients having FSHD. This methodology allows for a comprehensive and discriminative analysis of different regions along the macrosatellite repeat and identification of focal regions or CpG sites differentially methylated in patients with FSHD1 and FSHD2 but also complex cases such as those presenting mosaicism.
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http://dx.doi.org/10.1212/NXG.0000000000000372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6878839PMC
December 2019

[Marseille welcomes the FSHD Society International Research Conference].

Med Sci (Paris) 2019 Nov 20;35 Hors série n° 2:55-59. Epub 2019 Dec 20.

Aix Marseille Univ, Inserm, MMG, Marseille Medical Genetics, Marseille, France - Centre de référence pour les maladies neuromusculaires et la SLA, Hôpital de la Timone, Marseille, France - Filnemus, Filière Neuromusculaire,.

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http://dx.doi.org/10.1051/medsci/2019184DOI Listing
November 2019

Whole MYBPC3 NGS sequencing as a molecular strategy to improve the efficiency of molecular diagnosis of patients with hypertrophic cardiomyopathy.

Hum Mutat 2020 02 15;41(2):465-475. Epub 2019 Nov 15.

Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Lyon, France.

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiomyopathy, historically believed to affect 1 of 500 people. MYBPC3 pathogenic variations are the most frequent cause of familial HCM and more than 90% of them introduce a premature termination codon. The current study aims to determine the prevalence of deep intronic MYBPC3 pathogenic variations that could lead to splice mutations. To improve molecular diagnosis, a next-generation sequencing (NGS) workflow based on whole MYBPC3 sequencing of a cohort of 93 HCM patients, for whom no putatively causative point mutations were identified after NGS sequencing of a panel of 48 cardiomyopathy-causing genes, was performed. Our approach led us to reconsider the molecular diagnosis of six patients of the cohort (6.5%). These HCM probands were carriers of either a new large MYBPC3 rearrangement or splice intronic variations (five cases). Four pathogenic intronic variations, including three novel ones, were detected. Among them, the prevalence of one of them (NM_000256.3:c.1927+ 600 C>T) was estimated at about 0.35% by the screening of 1,040 unrelated HCM individuals. This study suggests that deep MYBPC3 splice mutations account for a significant proportion of HCM cases (6.5% of this cohort). Consequently, NGS sequencing of MYBPC3 intronic sequences have to be performed systematically.
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http://dx.doi.org/10.1002/humu.23944DOI Listing
February 2020

Analysis of the 4q35 chromatin organization reveals distinct long-range interactions in patients affected with Facio-Scapulo-Humeral Dystrophy.

Sci Rep 2019 07 17;9(1):10327. Epub 2019 Jul 17.

Aix Marseille Univ, INSERM, MMG, U 1251, Marseille, France.

Facio-Scapulo Humeral dystrophy (FSHD) is the third most common myopathy, affecting 1 amongst 10,000 individuals (FSHD1, OMIM #158900). This autosomal dominant pathology is associated in 95% of cases with genetic and epigenetic alterations in the subtelomeric region at the extremity of the long arm of chromosome 4 (q arm). A large proportion of the remaining 5% of cases carry a mutation in the SMCHD1 gene (FSHD2, OMIM #158901). Here, we explored the 3D organization of the 4q35 locus by three-dimensions DNA in situ fluorescent hybridization (3D-FISH) in primary fibroblasts isolated from patients and healthy donors. We found that D4Z4 contractions and/or SMCHD1 mutations impact the spatial organization of the 4q35 region and trigger changes in the expression of different genes. Changes in gene expression were corroborated in muscle biopsies suggesting that the modified chromatin landscape impelled a modulation in the level of expression of a number of genes across the 4q35 locus in FSHD. Using induced pluripotent stem cells (hIPSC), we further examined whether chromatin organization is inherited after reprogramming or acquired during differentiation and showed that folding of the 4q35 region is modified upon differentiation. These results together with previous findings highlight the role of the D4Z4 macrosatellite repeat in the topological organization of chromatin and further indicate that the D4Z4-dependent 3D structure induces transcriptional changes of 4q35 genes expression.
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http://dx.doi.org/10.1038/s41598-019-46861-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637155PMC
July 2019

Functional classification of ATM variants in ataxia-telangiectasia patients.

Hum Mutat 2019 10 17;40(10):1713-1730. Epub 2019 May 17.

CHU de Nancy, Service de Neurologie, Nancy, France.

Ataxia-telangiectasia (A-T) is a recessive disorder caused by biallelic pathogenic variants of ataxia-telangiectasia mutated (ATM). This disease is characterized by progressive ataxia, telangiectasia, immune deficiency, predisposition to malignancies, and radiosensitivity. However, hypomorphic variants may be discovered associated with very atypical phenotypes, raising the importance of evaluating their pathogenic effects. In this study, multiple functional analyses were performed on lymphoblastoid cell lines from 36 patients, comprising 49 ATM variants, 24 being of uncertain significance. Thirteen patients with atypical phenotype and presumably hypomorphic variants were of particular interest to test strength of functional analyses and to highlight discrepancies with typical patients. Western-blot combined with transcript analyses allowed the identification of one missing variant, confirmed suspected splice defects and revealed unsuspected minor transcripts. Subcellular localization analyses confirmed the low level and abnormal cytoplasmic localization of ATM for most A-T cell lines. Interestingly, atypical patients had lower kinase defect and less altered cell-cycle distribution after genotoxic stress than typical patients. In conclusion, this study demonstrated the pathogenic effects of the 49 variants, highlighted the strength of KAP1 phosphorylation test for pathogenicity assessment and allowed the establishment of the Ataxia-TeLangiectasia Atypical Score to predict atypical phenotype. Altogether, we propose strategies for ATM variant detection and classification.
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http://dx.doi.org/10.1002/humu.23778DOI Listing
October 2019

Deciphering the complexity of the 4q and 10q subtelomeres by molecular combing in healthy individuals and patients with facioscapulohumeral dystrophy.

J Med Genet 2019 09 22;56(9):590-601. Epub 2019 Apr 22.

Aix Marseille Univ, INSERM, MMG, Marseille Medical Genetics U1251, Marseille, France

Background: Subtelomeres are variable regions between telomeres and chromosomal-specific regions. One of the most studied pathologies linked to subtelomeric imbalance is facioscapulohumeral dystrophy (FSHD). In most cases, this disease involves shortening of an array of D4Z4 macrosatellite elements at the 4q35 locus. The disease also segregates with a specific A-type haplotype containing a degenerated polyadenylation signal distal to the last repeat followed by a repetitive array of β-satellite elements. This classification applies to most patients with FSHD. A subset of patients called FSHD2 escapes this definition and carries a mutation in the gene. We also recently described patients carrying a complex rearrangement consisting of a -duplication of the distal 4q35 locus identified by molecular combing.

Methods: Using this high-resolution technology, we further investigated the organisation of the 4q35 region linked to the disease and the 10q26 locus presenting with 98% of homology in controls and patients.

Results: Our analyses reveal a broad variability in size of the different elements composing these loci highlighting the complexity of these subtelomeres and the difficulty for genomic assembly. Out of the 1029 DNA samples analysed in our centre in the last 7 years, we also identified 54 cases clinically diagnosed with FSHD carrying complex genotypes. This includes mosaic patients, patients with deletions of the proximal 4q region and 23 cases with an atypical chromosome 10 pattern, infrequently found in the control population and never reported before.

Conclusion: Overall, this work underlines the complexity of these loci challenging the diagnosis and genetic counselling for this disease.
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http://dx.doi.org/10.1136/jmedgenet-2018-105949DOI Listing
September 2019

SMCHD1 is involved in de novo methylation of the DUX4-encoding D4Z4 macrosatellite.

Nucleic Acids Res 2019 04;47(6):2822-2839

Aix Marseille Univ, INSERM MMG, Nerve and Muscle Department, Marseille, France.

The DNA methylation epigenetic signature is a key determinant during development. Rules governing its establishment and maintenance remain elusive especially at repetitive sequences, which account for the majority of methylated CGs. DNA methylation is altered in a number of diseases including those linked to mutations in factors that modify chromatin. Among them, SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain Containing 1) has been of major interest following identification of germline mutations in Facio-Scapulo-Humeral Dystrophy (FSHD) and in an unrelated developmental disorder, Bosma Arhinia Microphthalmia Syndrome (BAMS). By investigating why germline SMCHD1 mutations lead to these two different diseases, we uncovered a role for this factor in de novo methylation at the pluripotent stage. SMCHD1 is required for the dynamic methylation of the D4Z4 macrosatellite upon reprogramming but seems dispensable for methylation maintenance. We find that FSHD and BAMS patient's cells carrying SMCHD1 mutations are both permissive for DUX4 expression, a transcription factor whose regulation has been proposed as the main trigger for FSHD. These findings open new questions as to what is the true aetiology for FSHD, the epigenetic events associated with the disease thus calling the current model into question and opening new perspectives for understanding repetitive DNA sequences regulation.
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http://dx.doi.org/10.1093/nar/gkz005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451109PMC
April 2019

Widening the landscape of heritable pulmonary hypertension mutations in paediatric and adult cases.

Eur Respir J 2019 03 14;53(3). Epub 2019 Mar 14.

Département de Génétique, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.

Background: Heritable forms of pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease/pulmonary capillary haemangiomatosis (PVOD/PCH) diverge by lung histopathological lesions, clinical and para-clinical presentation, their responsible genes, and mode of transmission. Since the identification of the gene in families affected by PAH, mutations in several other genes have been discovered for both forms. The mutation landscape in these new genes is not yet well known.

Methods: We set up a next-generation sequencing-based targeted sequencing gene panel allowing known genes for PAH and PVOD/PCH to be analysed simultaneously Genetic analysis was prospectively performed on 263 PAH and PVOD/PCH patients (adult and paediatric cases).

Results: Pathogenic mutations were identified in 19.5% of sporadic PAH patients (n=180), 54.5% of familial PAH patients and 13.3% of PVOD/PCH patients. was the most frequently mutated gene, followed by in both paediatric and adult PAH. mutations were identified in 1.2% of adult PAH cases. biallelic mutations were restricted to PVOD/PCH. A truncating mutation and a predicted loss-of-function variant were also identified in in two severely affected sporadic PAH female patients.

Conclusion: Our results confirm that mutations are found in genes beyond in heritable PAH, emphasise the role of and , and designate as a new PAH gene.
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http://dx.doi.org/10.1183/13993003.01371-2018DOI Listing
March 2019

Targeted panel sequencing in adult patients with left ventricular non-compaction reveals a large genetic heterogeneity.

Clin Genet 2019 03 27;95(3):356-367. Epub 2018 Dec 27.

Sorbonne Universités, UPMC Univ. Paris 06, INSERM, UMR_S 1166 and ICAN Institute for Cardiometabolism and Nutrition, Paris, France.

Left ventricular non-compaction (LVNC) is a cardiomyopathy that may be of genetic origin; however, few data are available about the yield of mutation, the spectrum of genes and allelic variations. The aim of this study was to better characterize the genetic spectrum of isolated LVNC in a prospective cohort of 95 unrelated adult patients through the molecular investigation of 107 genes involved in cardiomyopathies and arrhythmias. Fifty-two pathogenic or probably pathogenic variants were identified in 40 patients (42%) including 31 patients (32.5%) with single variant and 9 patients with complex genotypes (9.5%). Mutated patients tended to have younger age at diagnosis than patients with no identified mutation. The most prevalent genes were TTN, then HCN4, MYH7, and RYR2. The distribution includes 13 genes previously reported in LVNC and 10 additional candidate genes. Our results show that LVNC is basically a genetic disease and support genetic counseling and cardiac screening in relatives. There is a large genetic heterogeneity, with predominant TTN null mutations and frequent complex genotypes. The gene spectrum is close to the one observed in dilated cardiomyopathy but with specific genes such as HCN4. We also identified new candidate genes that could be involved in this sub-phenotype of cardiomyopathy.
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http://dx.doi.org/10.1111/cge.13484DOI Listing
March 2019

Typical features of Parkinson disease and diagnostic challenges with microdeletion 22q11.2.

Neurology 2018 06 11;90(23):e2059-e2067. Epub 2018 May 11.

From The Dalglish Family 22q Clinic for Adults and Department of Psychiatry (E.B., A.M.F., A.S.B.), Toronto General Research Institute (A.S.B.), and Division of Cardiology, Department of Medicine (A.S.B.), University Health Network, Toronto, Canada; De Hartekamp Groep (E.B.), Centre for People with Intellectual Disability, Haarlem; Department of Nuclear Medicine (E.B., J.B.), Academic Medical Center, Amsterdam, the Netherlands; Clinical Genetics Research Program and Campbell Family Mental Health Research Institute (N.J.B., A.M.F., A.S.B.), Centre for Addiction and Mental Health, Toronto; Institute of Medical Science (N.J.B., M.M., A.E.L., A.S.B.), Division of Neurology, Department of Medicine (C.M., M.M., A.E.L.), and Department of Psychiatry (A.S.B.), University of Toronto; Deer Lodge Movement Disorders Centre (S.U.); Section of Neurology (S.U.), Division of Internal Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg; Morton and Gloria Shulman Movement Disorders Centre and the Edmond J. Safra Program in Parkinson's Disease Research (C.M., A.E.L.), Toronto Western Hospital and University of Toronto, Canada; Department of Molecular Neuroscience (K.Y.M., N.W.W.), UCL Institute of Neurology, London, UK; Department of Neurology (S.K.), Kansai Medical University, Osaka, Japan; Department of Neurology (M.J.B.), University of Virginia School of Medicine, Charlottesville; Medical Genetics Unit (P.P.), Perugia University Hospital, Italy; Department of Neurology (B.D.B.), University of Colorado Anschutz Medical Campus, Aurora; Neurology Section (B.D.B.), VA Eastern Colorado Health Care System, Denver; Cognitive & Movement Disorders Clinic and Hurvitz Brain Sciences Research Program (M.M.), Sunnybrook Health Sciences Centre, Toronto, Canada; Departments of Clinical Neurosciences (Movement Disorders) (B.D.) and Genetics (Neurogenetics) (K.N.), Timone University Hospital (AP-HM), Provence-Alpes-Côte d'Azur; Aix-Marseille University (B.D., K.N.), Marseille; Department of Genetics (Neurogenetics) (P.C., A.J.), Pitié-Salpêtrière University Hospital; Sorbonne University (P.C., A.J.), Paris; Department of Neurosciences (Movement Disorders) (E.M.), Lille University Hospital; Lille University (E.M.); Department of Neurology (Movement Disorders) (T.D.), Pierre Wertheimer University Hospital, Lyon; Marc Jeannerod Center for Cognitive Neurosciences (T.D.), Lyon-1 University; Department of Neurology (Movement Disorders) and Clinical Investigation Center (Clinical and Experimental Neurosciences) (O.C.), Poitiers University Hospital; Department of Neurology (Movement Disorders) (S.D.), Rennes University Hospital; Rennes-1 University (S.D.); Department of Clinical Neurosciences (Movement Disorders) (M.B.), Nice University Hospital, France; Department of Psychiatry (A.M.F.), Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, the Netherlands; Center for Human Genetics (E.V., A.S., A.V.), University Hospital Leuven; Department of Human Genetics (A.S.), KU Leuven, Belgium; Department of Neurology (A.P.), University of Munich, Germany; Scientific and Technological Coordination Unit of the ANLIS Directorate (C.P.), National Administration of Laboratories and Institutes of Health, Argentina; Department of Neurodegenerative Diseases (T.G.), Center of Neurology and Hertie-Institute for Clinical Brain Research, University of Tübingen; German Center for Neurodegenerative Diseases (DZNE) (T.G.); Department of Neurology (K.C.), AZ Turnhout, Antwerp, Belgium; Neurology Unit and Stroke Center (F.B.), Hôpital Foch, Suresnes, France; Movement Disorder Division (K.M.), Johns Hopkins University, Baltimore, MD; and Psychological Medicine and Clinical Neurosciences (N.M.W.), MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University School of Medicine, Cardiff University, UK.

Objective: To delineate the natural history, diagnosis, and treatment response of Parkinson disease (PD) in individuals with 22q11.2 deletion syndrome (22q11.2DS), and to determine if these patients differ from those with idiopathic PD.

Methods: In this international observational study, we characterized the clinical and neuroimaging features of 45 individuals with 22q11.2DS and PD (mean follow-up 7.5 ± 4.1 years).

Results: 22q11.2DS PD had a typical male excess (32 male, 71.1%), presentation and progression of hallmark motor symptoms, reduced striatal dopamine transporter binding with molecular imaging, and initial positive response to levodopa (93.3%). Mean age at motor symptom onset was relatively young (39.5 ± 8.5 years); 71.4% of cases had early-onset PD (<45 years). Despite having a similar age at onset, the diagnosis of PD was delayed in patients with a history of antipsychotic treatment compared with antipsychotic-naive patients (median 5 vs 1 year, = 0.001). Preexisting psychotic disorders (24.5%) and mood or anxiety disorders (31.1%) were common, as were early dystonia (19.4%) and a history of seizures (33.3%).

Conclusions: Major clinical characteristics and response to standard treatments appear comparable in 22q11.2DS-associated PD to those in idiopathic PD, although the average age at onset is earlier. Importantly, treatment of preexisting psychotic illness may delay diagnosis of PD in 22q11.DS patients. An index of suspicion and vigilance for complex comorbidity may assist in identifying patients to prioritize for genetic testing.
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http://dx.doi.org/10.1212/WNL.0000000000005660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993183PMC
June 2018

Molecular combing reveals complex 4q35 rearrangements in Facioscapulohumeral dystrophy.

Hum Mutat 2017 10 6;38(10):1432-1441. Epub 2017 Aug 6.

Aix Marseille Université, INSERM GMGF UMR S_910, Marseille, 13385, France.

Facioscapulohumeral dystrophy (FSHD), one of the most common hereditary neuromuscular disorders, is associated with a complex combination of genetic variations at the subtelomeric 4q35 locus. As molecular diagnosis relying on Southern blot (SB) might be challenging in some cases, molecular combing (MC) was recently developed as an additional technique for FSHD diagnosis and exploration of the genomic organization of the 4q35 and 10q26 regions. In complement to the usual SB, we applied MC in a large cohort of 586 individuals with clinical FSHD. In 332 subjects, the two 4q alleles were normal in size, allowing exclusion of FSHD1 while we confirmed FSHD1 in 230 patients. In 14 patients from 10 families, we identified a recurrent complex heterozygous rearrangement at 4q35 consisting of a duplication of the D4Z4 array and a 4qA haplotype, irresolvable by the SB technique. In five families, we further identified variations in the SMCHD1 gene. Impact of the different mutations was tested using a minigene assay and we analyzed DNA methylation after sodium bisulfite modification and NGS sequencing. We discuss the involvement of this rearrangement in FSHD since all mutations in SMCHD1 are not associated with D4Z4 hypomethylation and do not always segregate with the disease.
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http://dx.doi.org/10.1002/humu.23304DOI Listing
October 2017

Affected female carriers of MTM1 mutations display a wide spectrum of clinical and pathological involvement: delineating diagnostic clues.

Acta Neuropathol 2017 Dec 6;134(6):889-904. Epub 2017 Jul 6.

Laboratoire Diagnostic Génétique, Faculté de Médecine, CHRU, Nouvel Hôpital Civil, 1 place de l'Hôpital, 67091, Strasbourg, France.

X-linked myotubular myopathy (XLMTM), a severe congenital myopathy, is caused by mutations in the MTM1 gene located on the X chromosome. A majority of affected males die in the early postnatal period, whereas female carriers are believed to be usually asymptomatic. Nevertheless, several affected females have been reported. To assess the phenotypic and pathological spectra of carrier females and to delineate diagnostic clues, we characterized 17 new unrelated affected females and performed a detailed comparison with previously reported cases at the clinical, muscle imaging, histological, ultrastructural and molecular levels. Taken together, the analysis of this large cohort of 43 cases highlights a wide spectrum of clinical severity ranging from severe neonatal and generalized weakness, similar to XLMTM male, to milder adult forms. Several females show a decline in respiratory function. Asymmetric weakness is a noteworthy frequent specific feature potentially correlated to an increased prevalence of highly skewed X inactivation. Asymmetry of growth was also noted. Other diagnostic clues include facial weakness, ptosis and ophthalmoplegia, skeletal and joint abnormalities, and histopathological signs that are hallmarks of centronuclear myopathy such as centralized nuclei and necklace fibers. The histopathological findings also demonstrate a general disorganization of muscle structure in addition to these specific hallmarks. Thus, MTM1 mutations in carrier females define a specific myopathy, which may be independent of the presence of an XLMTM male in the family. As several of the reported affected females carry large heterozygous MTM1 deletions not detectable by Sanger sequencing, and as milder phenotypes present as adult-onset limb-girdle myopathy, the prevalence of this myopathy is likely to be greatly underestimated. This report should aid diagnosis and thus the clinical management and genetic counseling of MTM1 carrier females. Furthermore, the clinical and pathological history of this cohort may be useful for therapeutic projects in males with XLMTM, as it illustrates the spectrum of possible evolution of the disease in patients surviving long term.
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http://dx.doi.org/10.1007/s00401-017-1748-0DOI Listing
December 2017

Segregation between SMCHD1 mutation, D4Z4 hypomethylation and Facio-Scapulo-Humeral Dystrophy: a case report.

BMC Med Genet 2016 Sep 15;17(1):66. Epub 2016 Sep 15.

Aix Marseille Univ, INSERM, GMGF, Marseille, France.

Background: The main form of Facio-Scapulo-Humeral muscular Dystrophy is linked to copy number reduction of the 4q D4Z4 macrosatellite (FSHD1). In 5 % of cases, FSHD phenotype appears in the absence of D4Z4 reduction (FSHD2). In 70-80 % of these patients, variants of the SMCHD1 gene segregate with 4qA haplotypes and D4Z4 hypomethylation.

Case Presentation: We report a family presenting with neuromuscular symptoms reminiscent of FSHD but without D4Z4 copy reduction. We characterized the 4q35 region using molecular combing, searched for mutation in the SMCHD1 gene and determined D4Z4 methylation level by sodium bisulfite sequencing. We further investigated the impact of the SMCHD1 mutation at the protein level and on the NMD-dependent degradation of transcript. In muscle, we observe moderate but significant reduction in D4Z4 methylation, not correlated with DUX4-fl expression. Exome sequencing revealed a heterozygous insertion of 7 bp in exon 37 of the SMCHD1 gene producing a loss of frame with premature stop codon 4 amino acids after the insertion (c.4614-4615insTATAATA). Both wild-type and mutated transcripts are detected.

Conclusion: The truncated protein is absent and the full-length protein level is similar in patients and controls indicating that in this family, FSHD is not associated with SMCHD1 haploinsufficiency.
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http://dx.doi.org/10.1186/s12881-016-0328-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025538PMC
September 2016

The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype.

Eur J Hum Genet 2016 07 2;24(7):992-1000. Epub 2015 Dec 2.

Département de Génétique et INSERM U781, Université Paris Descartes-Sorbonne Paris Cité, Fondation Imagine, Hôpital Necker-Enfants malades, AP-HP, Paris, France.

Heterozygous COL2A1 variants cause a wide spectrum of skeletal dysplasia termed type II collagenopathies. We assessed the impact of this gene in our French series. A decision tree was applied to select 136 probands (71 Stickler cases, 21 Spondyloepiphyseal dysplasia congenita cases, 11 Kniest dysplasia cases, and 34 other dysplasia cases) before molecular diagnosis by Sanger sequencing. We identified 66 different variants among the 71 positive patients. Among those patients, 18 belonged to multiplex families and 53 were sporadic. Most variants (38/44, 86%) were located in the triple helical domain of the collagen chain and glycine substitutions were mainly observed in severe phenotypes, whereas arginine to cysteine changes were more often encountered in moderate phenotypes. This series of skeletal dysplasia is one of the largest reported so far, adding 44 novel variants (15%) to published data. We have confirmed that about half of our Stickler patients (46%) carried a COL2A1 variant, and that the molecular spectrum was different across the phenotypes. To further address the question of genotype-phenotype correlation, we plan to screen our patients for other candidate genes using a targeted next-generation sequencing approach.
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http://dx.doi.org/10.1038/ejhg.2015.250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070901PMC
July 2016

Cerebral Iron Accumulation Is Not a Major Feature of /SPG35.

Mov Disord Clin Pract 2015 Mar 18;2(1):56-60. Epub 2015 Feb 18.

Institute of Genetics and Cellular and Molecular Biology INSERM U964 CNRS UMR7104 University of Strasbourg Illkirch France.

Mutations in the fatty-acid 2-hydroxylase () gene cause an autosomal recessive spastic paraplegia (SPG35), often associating with cerebellar ataxia; cerebral MRI may show iron accumulation in the basal ganglia, leading to the inclusion of SPG35 among the causes of neurodegeneration with brain iron accumulation. This finding was initially considered strongly relevant for diagnosis, although its frequency is not yet established. We found 5 novel patients (from two families) with mutations in the gene: none of them showed cerebral iron accumulation (T2-weighted images performed in all; T2 gradient-echo in 2); notably, in 1 case, iron accumulation was absent even after 18 years from disease onset on both T2 gradient-echo and susceptibility-weight MRI sequences. Cerebral iron accumulation is not a prominent feature in SPG35 and is not always dependent on disease duration; its absence should not discourage from evoking this diagnosis.
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http://dx.doi.org/10.1002/mdc3.12118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353525PMC
March 2015

Low penetrance in facioscapulohumeral muscular dystrophy type 1 with large pathological D4Z4 alleles: a cross-sectional multicenter study.

Orphanet J Rare Dis 2015 Jan 21;10. Epub 2015 Jan 21.

AP-HM, Reference Center of Neuromuscular Disorders and ALS, Timone University Hospital, Aix-Marseille University, 264 rue Saint-Pierre, Marseille, Cedex 05, 13385, France.

Background: Facioscapulohumeral muscular dystrophy type 1(FSHD1) is an autosomal dominant disorder associated with the contraction of D4Z4 less than 11 repeat units (RUs) on chromosome 4q35. Penetrance in the range of the largest alleles is poorly known. Our objective was to study the penetrance of FSHD1 in patients carrying alleles ranging between 6 to10 RUs and to evaluate the influence of sex, age, and several environmental factors on clinical expression of the disease.

Methods: A cross-sectional multicenter study was conducted in six French and one Swiss neuromuscular centers. 65 FSHD1 affected patients carrying a 4qA allele of 6-10 RUs were identified as index cases (IC) and their 119 at-risk relatives were included. The age of onset was recorded for IC only. Medical history, neurological examination and manual muscle testing were performed for each subject. Genetic testing determined the allele size (number of RUs) and the 4qA/4qB allelic variant. The clinical status of relatives was established blindly to their genetic testing results. The main outcome was the penetrance defined as the ratio between the number of clinically affected carriers and the total number of carriers.

Results: Among the relatives, 59 carried the D4Z4 contraction. At the clinical level, 34 relatives carriers were clinically affected and 25 unaffected. Therefore, the calculated penetrance was 57% in the range of 6-10 RUs. Penetrance was estimated at 62% in the range of 6-8 RUs, and at 47% in the range of 9-10 RUs. Moreover, penetrance was lower in women than men. There was no effect of drugs, anesthesia, surgery or traumatisms on the penetrance.

Conclusions: Penetrance of FSHD1 is low for largest alleles in the range of 9-10 RUs, and lower in women than men. This is of crucial importance for genetic counseling and clinical management of patients and families.
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http://dx.doi.org/10.1186/s13023-014-0218-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320820PMC
January 2015

Differential DNA methylation of the D4Z4 repeat in patients with FSHD and asymptomatic carriers.

Neurology 2014 Aug 16;83(8):733-42. Epub 2014 Jul 16.

From Aix Marseille Universite (M.-C.G., S.R., C.D., A.T., G.B., E.S.-C., N.B., J.M., F.P., M.B., N.L., R.B., S.A., K.N., F.M.), INSERM GMGF UMR S_910, Marseille; and APHM, Centre de Référence des Maladies Neuromusculaires et de la SLA (E.S.-C., S.A.), and APHM, Laboratoire de Génétique Médicale (C.V., C.C., N.L., R.B., K.N.), Hôpital de la Timone, Marseille, France.

Objective: We investigated the link between DNA hypomethylation and clinical penetrance in facioscapulohumeral dystrophy (FSHD) because hypomethylation is moderate and heterogeneous in patients and could not thus far be correlated with disease presence or severity.

Methods: To investigate the link between clinical signs of FSHD and DNA methylation, we explored 95 cases (37 FSHD1, 29 asymptomatic individuals carrying a shortened D4Z4 array, 9 patients with FSHD2, and 20 controls) by implementing 2 approaches: methylated DNA immunoprecipitation and sodium bisulfite sequencing.

Results: Both methods revealed statistically significant differences between asymptomatic carriers or controls and individuals with clinical FSHD, especially in the proximal region of the repeat. Absence of clinical expression in asymptomatic carriers is associated with a level of methylation similar to controls.

Conclusions: We provide a proof of concept that the targeted approaches that we describe could be applied systematically to patient samples in routine diagnosis and suggest that local hypomethylation within D4Z4 might serve as a modifier for clinical expression of FSHD phenotype.

Classification Of Evidence: This study provides Class III evidence that assays for hypomethylation within the D4Z4 region accurately distinguish patients with FSHD from individuals with D4Z4 contraction without FSHD.
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http://dx.doi.org/10.1212/WNL.0000000000000708DOI Listing
August 2014

Dysregulation of 4q35- and muscle-specific genes in fetuses with a short D4Z4 array linked to facio-scapulo-humeral dystrophy.

Hum Mol Genet 2013 Oct 17;22(20):4206-14. Epub 2013 Jun 17.

The authors wish it to be known that in their opinion the first two and the last two authors should be regarded as joint authors.

Facio-scapulo-humeral dystrophy (FSHD) results from deletions in the subtelomeric macrosatellite D4Z4 array on the 4q35 region. Upregulation of the DUX4 retrogene from the last D4Z4 repeated unit is thought to underlie FSHD pathophysiology. However, no one knows what triggers muscle defect and when alteration arises. To gain further insights into the molecular mechanisms of the disease, we evaluated at the molecular level, the perturbation linked to the FSHD genotype with no a priori on disease onset, severity or penetrance and prior to any infiltration by fibrotic or adipose tissue in biopsies from fetuses carrying a short pathogenic D4Z4 array (n = 6) compared with fetuses with a non-pathogenic D4Z4 array (n = 21). By measuring expression of several muscle-specific markers and 4q35 genes including the DUX4 retrogene by an RT-PCR and western blotting, we observed a global dysregulation of genes involved in myogenesis including MYOD1 in samples with <11 D4Z4. The DUX4-fl pathogenic transcript was detected in FSHD biopsies but also in controls. Importantly, in FSHD fetuses, we mainly detected the non-spliced DUX4-fl isoform. In addition, several other genes clustered at the 4q35 locus are upregulated in FSHD fetuses. Our study is the first to examine fetuses carrying an FSHD-linked genotype and reveals an extensive dysregulation of several muscle-specific and 4q35 genes at early development stage at a distance from any muscle defect. Overall, our work suggests that even if FSHD is an adult-onset muscular dystrophy, the disease might also involve early molecular defects arising during myogenesis or early differentiation.
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http://dx.doi.org/10.1093/hmg/ddt272DOI Listing
October 2013
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