Upstream open reading frame-introducing variants in patients with primary familial brain calcification.

More than 50% of patients with primary familial brain calcification (PFBC), a rare neurological disorder, remain genetically unexplained. While some causative genes are yet to be identified, variants in non-coding regions of known genes may represent a source of missed diagnoses. We hypothesized that 5'-Untranslated Region (UTR) variants introducing an AUG codon may initiate mRNA translation and result in a loss of function in some of the PFBC genes. After reannotation of exome sequencing data of 113 unrelated PFBC probands, we identified two upstream AUG-introducing variants in the 5'UTR of PDGFB. One, NM_002608.4:c.-373C>G, segregated with PFBC in the family. It was predicted to create an upstream open reading frame (ORF). The other one, NM_002608.4:c.-318C>T, was found in a simplex case. It was predicted to result in an ORF overlapping the natural ORF with a frameshift. In a GFP reporter assay, both variants were associated with a dramatic decrease in GFP levels, and, after restoring the reading frame with the GFP sequence, the c.-318C>T variant was associated with a strong initiation of translation as measured by western blotting. Overall, we found upstream AUG-introducing variants in the 5'UTR of PDGFB in 2/113 (1.7%) undiagnosed PFBC cases. Such variants thus represent a source of putative pathogenic variants.

MYH7 p.(Arg1712Gln) is pathogenic founder variant causing hypertrophic cardiomyopathy with overall relatively delayed onset.

INTRODUCTION: The MYH7 c.5135G > A p.(Arg1712Gln) variant has been identified in several patients worldwide and is classified as pathogenic in the ClinVar database. We aimed to delineate its associated phenotype and evaluate a potential founder effect. METHODS: We retrospectively collected clinical and genetic data of 22 probands and 74 family members from an international cohort. RESULTS: In total, 53 individuals carried the MYH7 p.(Arg1712Gln) variant, of whom 38 (72%) were diagnosed with hypertrophic cardiomyopathy (HCM). Mean age at HCM diagnosis was 48.8 years (standard deviation: 18.1; range: 8-74). The clinical presentation ranged from asymptomatic HCM to arrhythmias (atrial fibrillation and malignant ventricular arrhythmias). Aborted sudden cardiac death (SCD) leading to the diagnosis of HCM occurred in one proband at the age of 68 years, and a family history of SCD was reported by 39% (5/13) probands. Neither heart failure deaths nor heart transplants were reported. Women had a generally later-onset disease, with 14% of female carriers diagnosed with HCM at age 50 years compared with 54% of male carriers. In both sexes, the disease was fully penetrant by age 75 years. Haplotypes were reconstructed for 35 patients and showed a founder effect in a subset of patients. CONCLUSION: MYH7 p.(Arg1712Gln) is a pathogenic founder variant with a consistent HCM phenotype that may present with delayed penetrance. This suggested that clinical follow-up should be pursued after the seventh decade in healthy carriers and that longer intervals between screening may be justified in healthy women < 30 years.

Genetic profile and genotype-phenotype correlations in childhood cardiomyopathy.

BACKGROUND: Genetic cardiomyopathy is a rare disease in childhood. AIMS: To analyse clinical and genetic aspects of a paediatric cardiomyopathy population, and to establish genotype-phenotype correlations. METHODS: We performed a retrospective study of all patients with idiopathic cardiomyopathy aged<18years in Southeast France. Secondary causes of cardiomyopathy were excluded. All data (clinical, echocardiography, genetic testing) were collected retrospectively. Patients were classified into six groups: hypertrophic cardiomyopathy; dilated cardiomyopathy; restrictive cardiomyopathy; left ventricular non-compaction; arrhythmogenic right ventricular dysplasia; and mixed cardiomyopathy. Patients who did not have a complete genetic test according to current scientific developments had another deoxyribonucleic acid blood sample during the study time. Genetic tests were considered positive if the variant found was classified as pathogenic, likely pathogenic or a variant of uncertain significance. RESULTS: Eighty-three patients were included between 2005 and 2019. Most patients had hypertrophic cardiomyopathy (39.8%) or dilated cardiomyopathy (27.7%). The median age at diagnosis was 1.28years (interquartile range: 0.27-10.48years). Heart transplantation was performed in 30.1% of patients, and 10.8% died during follow-up. Among 64 patients with a complete genetic analysis, 64.1% had genetic anomalies, mostly in MYH7 (34.2%) and MYBPC3 (12.2%) genes. There were no differences in the whole cohort between genotype-positive and genotype-negative patients. In the hypertrophic cardiomyopathy group, 63.6% had a positive genetic test. Patients with a positive genetic test more often had extracardiac impact (38.1% vs. 8.3%; P=0.009), and more often required an implantable cardiac defibrillator (23.8% vs. 0%; P=0.025) or a heart transplant (19.1% vs. 0%; P=0.047). CONCLUSIONS: In our population, children with cardiomyopathy had a high positive genetic test rate. Hypertrophic cardiomyopathy with a positive genetic test is associated with a worse outcome.

Facioscapulohumeral dystrophy weakened sarcomeric contractility is mimicked in induced pluripotent stem cells-derived innervated muscle fibres.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is a late-onset autosomal dominant form of muscular dystrophy involving specific groups of muscles with variable weakness that precedes inflammatory response, fat infiltration, and muscle atrophy. As there is currently no cure for this disease, understanding and modelling the typical muscle weakness in FSHD remains a major milestone towards deciphering the disease pathogenesis as it will pave the way to therapeutic strategies aimed at correcting the functional muscular defect in patients. METHODS: To gain further insights into the specificity of the muscle alteration in this disease, we derived induced pluripotent stem cells from patients affected with Types 1 and 2 FSHD but also from patients affected with Bosma arhinia and microphthalmia. We differentiated these cells into contractile innervated muscle fibres and analysed their transcriptome by RNA Seq in comparison with cells derived from healthy donors. To uncover biological pathways altered in the disease, we applied MOGAMUN, a multi-objective genetic algorithm that integrates multiplex complex networks of biological interactions (protein-protein interactions, co-expression, and biological pathways) and RNA Seq expression data to identify active modules. RESULTS: We identified 132 differentially expressed genes that are specific to FSHD cells (false discovery rate < 0.05). In FSHD, the vast majority of active modules retrieved with MOGAMUN converges towards a decreased expression of genes encoding proteins involved in sarcomere organization (P value 2.63e-12 ), actin cytoskeleton (P value 9.4e-5 ), myofibril (P value 2.19e-12 ), actin-myosin sliding, and calcium handling (with P values ranging from 7.9e-35 to 7.9e-21 ). Combined with in vivo validations and functional investigations, our data emphasize a reduction in fibre contraction (P value < 0.0001) indicating that the muscle weakness that is typical of FSHD clinical spectrum might be associated with dysfunction of calcium release (P value < 0.0001), actin-myosin interactions, motor activity, mechano-transduction, and dysfunctional sarcomere contractility. CONCLUSIONS: Identification of biomarkers of FSHD muscle remain critical for understanding the process leading to the pathology but also for the definition of readouts to be used for drug design, outcome measures, and monitoring of therapies. The different pathways identified through a system biology approach have been largely overlooked in the disease. Overall, our work opens new perspectives in the definition of biomarkers able to define the muscle alteration but also in the development of novel strategies to improve muscle function as it provides functional parameters for active molecule screening.

Challenging the traditional approach for interpreting genetic variants: Lessons from Fabry disease.

Fabry disease (FD) is an X-linked genetic disease due to pathogenic variants in GLA. The phenotype varies depending on the GLA variant, alpha-galactosidase residual activity, patient's age and gender and, for females, X chromosome inactivation. Over 1000 variants have been identified, many through screening protocols more susceptible to disclose non-pathogenic variants or variants of unknown significance (VUS). This, together with the non-specificity of some FD symptoms, challenges physicians attempting to interpret GLA variants. The traditional way to interpreting pathogenicity is based on a combined approach using allele frequencies, genomic databases, global and disease-specific clinical databases, and in silico tools proposed by the American College of Medical Genetics and Genomics. Here, a panel of FD specialists convened to study how expertise may compare with the traditional approach. Several GLA VUS, highly controversial in the literature (p.Ser126Gly, p.Ala143Thr, p.Asp313Tyr), were re-analyzed through reviews of patients' charts. The same was done for pathogenic GLA variants with some specificities. Our data suggest that input of geneticists and physicians with wide expertise in disease phenotypes, prevalence, inheritance, biomarkers, alleles frequencies, disease-specific databases, and literature greatly contribute to a more accurate interpretation of the pathogenicity of variants, bringing a significant additional value over the traditional approach.

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

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.

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

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.

Functional classification of ATM variants in ataxia-telangiectasia patients.

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.

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

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 BMPR2 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. BMPR2 was the most frequently mutated gene, followed by TBX4 in both paediatric and adult PAH. BMP9 mutations were identified in 1.2% of adult PAH cases. EIF2AK4 biallelic mutations were restricted to PVOD/PCH. A truncating mutation and a predicted loss-of-function variant were also identified in BMP10 in two severely affected sporadic PAH female patients. CONCLUSION: Our results confirm that mutations are found in genes beyond BMPR2 in heritable PAH, emphasise the role of TBX4 and BMP9, and designate BMP10 as a new PAH gene.

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

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.

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

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.

Cerebral Iron Accumulation Is Not a Major Feature of FA2H/SPG35.

Mutations in the fatty-acid 2-hydroxylase (FA2H) 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 FA2H 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.

Morbidity and mortality from ataxia-telangiectasia are associated with ATM genotype.

BACKGROUND: Ataxia-telangiectasia (A-T) is a rare genetic disease caused by germline biallelic mutations in the ataxia-telangiectasia mutated gene (ATM) that result in partial or complete loss of ATM expression or activity. The course of the disease is characterized by neurologic manifestations, infections, and cancers. OBJECTIVE: We studied A-T progression and investigated whether manifestations were associated with the ATM genotype. METHODS: We performed a retrospective cohort study in France of 240 patients with A-T born from 1954 to 2005 and analyzed ATM mutations in 184 patients, along with neurologic manifestations, infections, and cancers. RESULTS: Among patients with A-T, the Kaplan-Meier 20-year survival rate was 53.4%; the prognosis for these patients has not changed since 1954. Life expectancy was lower among patients with mutations in ATM that caused total loss of expression or function of the gene product (null mutations) compared with that seen in patients with hypomorphic mutations because of earlier onset of cancer (mainly hematologic malignancies). Cancer (hazard ratio, 2.7; 95% CI, 1.6-4.5) and respiratory tract infections (hazard ratio, 2.3; 95% CI, 1.4-3.8) were independently associated with mortality. Cancer (hazard ratio, 5.8; 95% CI, 2.9-11.6) was a major risk factor for mortality among patients with null mutations, whereas respiratory tract infections (hazard ratio, 4.1; 95% CI, 1.8-9.1) were the leading cause of death among patients with hypomorphic mutations. CONCLUSION: Morbidity and mortality among patients with A-T are associated with ATM genotype. This information could improve our prognostic ability and lead to adapted therapeutic strategies.

A naturally occurring human minidysferlin protein repairs sarcolemmal lesions in a mouse model of dysferlinopathy.

Dysferlinopathies are autosomal recessive, progressive muscle dystrophies caused by mutations in DYSF, leading to a loss or a severe reduction of dysferlin, a key protein in sarcolemmal repair. Currently, no etiological treatment is available for patients affected with dysferlinopathy. As for other muscular dystrophies, gene therapy approaches based on recombinant adeno-associated virus (rAAV) vectors are promising options. However, because dysferlin messenger RNA is far above the natural packaging size of rAAV, full-length dysferlin gene transfer would be problematic. In a patient presenting with a late-onset moderate dysferlinopathy, we identified a large homozygous deletion, leading to the production of a natural "minidysferlin" protein. Using rAAV-mediated gene transfer into muscle, we demonstrated targeting of the minidysferlin to the muscle membrane and efficient repair of sarcolemmal lesions in a mouse model of dysferlinopathy. Thus, as previously demonstrated in the case of dystrophin, a deletion mutant of the dysferlin gene is also functional, suggesting that dysferlin's structure is modular. This minidysferlin protein could be used as part of a therapeutic strategy for patients affected with dysferlinopathies.

Analysis of the DYSF mutational spectrum in a large cohort of patients.

Dysferlinopathies belong to the heterogeneous group of autosomal recessive muscular dystrophies. Mutations in the gene encoding dysferlin (DYSF) lead to distinct phenotypes, mainly Limb Girdle Muscular Dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). Here, we analysed the mutational data from the largest cohort described to date, a cohort of 134 patients, included based on clinical suspicion of primary dysferlinopathy and/or dysferlin protein deficiency identified on muscle biopsy samples. Data were compiled from 38 patients previously screened for mutations in our laboratory (Nguyen, et al., 2005; Nguyen, et al., 2007), and 96 supplementary patients screened for DYSF mutations using genomic DHPLC analysis, and subsequent sequencing of detected variants, in a routine diagnostic setting. In 89 (66%) out of 134 patients, molecular analysis identified two disease causing mutations, confirming the diagnosis of primary Dysferlinopathy on a genetic basis. Furthermore, one mutation was identified in 30 patients, without identification of a second deleterious allele. We are currently developing complementary analysis for patients in whom only one or no disease-causing allele could be identified using the genomic screening procedure. Altogether, 64 novel mutations have been identified in this cohort, which corresponds to approximately 25% of all DYSF mutations reported to date. The mutational spectrum of this cohort significantly shows a higher proportion of nonsense mutations, but a lower proportion of deleterious missense changes as compared to previous series. (c) 2008 Wiley-Liss, Inc.

Exon deletions of SPG4 are a frequent cause of hereditary spastic paraplegia.

BACKGROUND: Point mutations in SPG4, the gene encoding spastin, are a frequent cause of autosomal dominant hereditary spastic paraplegia (AD-HSP). However, standard methods for genetic analyses fail to detect exonic microdeletions. METHODS: 121 mutation-negative probands were screened for rearrangements in SPG4 by multiplex ligation-dependent probe amplification. RESULTS: 24 patients with 16 different heterozygotic exon deletions in SPG4 (20%) were identified, ranging from one exon to the whole coding sequence. Comparison with 78 patients with point mutations showed a similar clinical picture but an earlier age at onset. CONCLUSIONS: Exon deletions in SPG4 are as frequent as point mutations, and SPG4 is responsible for 40% of AD-HSP.