The new missense G376V-TDP-43 variant induces late-onset distal myopathy but not amyotrophic lateral sclerosis.

TDP-43-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS. Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their 5th-7th decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376 V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies. The G376 V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue. The identification of individuals with TDP-43-related myopathy but not ALS implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype.

Muscular phenotype description of abnormal THOC2 splicing.

Until recently, the disease known to be associated with THOC2 mutations was Intellectual developmental disorder, X-linked 12 (MIM300957). However, recently, fetal arthrogryposis multiplex congenita has been associated with a specific splice site mutation in the THOC2 gene. We report a family with the same splice site mutation in the THOC2 gene involved in fetal arthrogryposis as well. We provide the first description of the muscular phenotype of this disease which reveals the presence of cytoplasmic bodies. Our findings expand the clinical phenotype of THOC2 gene related defects.

Mild limb girdle muscular dystrophy R9 phenotype caused by novel compound heterozygous FKRP gene mutation.

Fukutin-related protein (FKRP) mutations cause a broad spectrum of muscular dystrophies, from a relatively mild limb-girdle muscular dystrophy type 9 (LGMDR9) to severe congenital muscular dystrophy (CMD). This study aims to report two siblings belonging to a non-consanguineous Tunisian family harboring a novel compound heterozygous FKRP variant and presenting a mild LGDMR9 phenotype. For mutation screening, massive parallel sequencing was performed, followed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to validate the existence of the discovered variants. The absence of alpha-dystroglycan was determined by immunohistochemistry. Brain and thigh magnetic resonance imaging (MRI) were performed to detect thigh and brain abnormalities. The two siblings had a late age at onset and clinical examination showed that the pelvic girdles had a predominantly proximal and symmetrical distribution of weakness without cardiac or respiratory involvement. They both had a modified Gardner-Medwin Walton Scale mGMWS grade of 4 and a modified Rankin Scale (mRS) score of 1. The DNA sequencing revealed a novel deletion of exons 2 and 3 in one allele and a missense mutation c.1364C > A, which has been reported to be responsible for congenital muscular dystrophy and mental retardation on the second allele. The simultaneous presence of the two variations in the two cases suggests that the variants segregate with the pathophysiology.

Novel Intronic Mutation in VMA21 Causing Severe Phenotype of X-Linked Myopathy with Excessive Autophagy-Case Report.

X-linked Myopathy with Excessive Autophagy (XMEA) is a rare autophagic vacuolar myopathy caused by mutations in the Vacuolar ATPase assembly factor VMA21 gene; onset usually occurs during childhood and rarely occurs during adulthood. We described a 22-year-old patient with XMEA, whose onset was declared at 11 through gait disorder. He had severe four-limb proximal weakness and amyotrophy, and his proximal muscle MRC score was between 2 and 3/5 in four limbs; creatine kinase levels were elevated (1385 IU/L), and electroneuromyography and muscle MRI were suggestive of myopathy. Muscle biopsy showed abnormalities typical of autophagic vacuolar myopathy. We detected a hemizygous, unreported, intronic, single-nucleotide substitution c.164-20T>A (NM_001017980.4) in intron 2 of the VMA21 gene. Fibroblasts derived from this patient displayed a reduced level of VMA21 transcripts (at 40% of normal) and protein, suggesting a pathogenicity related to an alteration of the splicing efficiency associated with an intron retention. This patient with XMEA displayed a severe phenotype (rapid weakness of upper and lower limbs) due to a new intronic variant of VMA21, related to an alteration in the splicing efficiency associated with intron retention, suggesting that phenotype severity is closely related to the residual expression of the VMA21 protein.

Targeted next-generation sequencing in a large series of fetuses with severe renal diseases.

We report the screening of a large panel of genes in a series of 100 fetuses (98 families) affected with severe renal defects. Causative variants were identified in 22% of cases, greatly improving genetic counseling. The percentage of variants explaining the phenotype was different according to the type of phenotype. The highest diagnostic yield was found in cases affected with the ciliopathy-like phenotype (11/15 families and, in addition, a single heterozygous or a homozygous Class 3 variant in PKHD1 in three unrelated cases with autosomal recessive polycystic kidney disease). The lowest diagnostic yield was observed in cases with congenital anomalies of the kidney and urinary tract (9/78 families and, in addition, Class 3 variants in GREB1L in three unrelated cases with bilateral renal agenesis). Inheritance was autosomal recessive in nine genes (PKHD1, NPHP3, CEP290, TMEM67, DNAJB11, FRAS1, ACE, AGT, and AGTR1), and autosomal dominant in six genes (PKD1, PKD2, PAX2, EYA1, BICC1, and MYOCD). Finally, we developed an original approach of next-generation sequencing targeted RNA sequencing using the custom capture panel used for the sequencing of DNA, to validate one MYOCD heterozygous splicing variant identified in two male siblings with megabladder and inherited from their healthy mother.

School level of children carrying a HNF1B variant or a deletion.

The prevalence of neurological involvement in patients with a deletion of or a variant in the HNF1B gene remains discussed. The aim of this study was to investigate the neuropsychological outcomes in a large cohort of children carrying either a HNF1B whole-gene deletion or a disease-associated variant, revealed by the presence of kidney anomalies. The neuropsychological development-based on school level-of 223 children included in this prospective cohort was studied. Data from 180 children were available for analysis. Patients mean age was 9.6 years, with 39.9% of girls. Among these patients, 119 carried a HNF1B deletion and 61 a disease-associated variant. In the school-aged population, 12.7 and 3.6% of patients carrying a HNF1B deletion and a disease-associated variant had special educational needs, respectively. Therefore, the presence of a HNF1B deletion increases the risk to present with a neuropsychiatric involvement when compared with the general population. On the other hand, almost 90% of patients carrying a HNF1B disease-associated variant or deletion have a normal schooling in a general educational environment. Even if these findings do not predict the risk of neuropsychiatric disease at adulthood, most patients diagnosed secondary to kidney anomalies do not show a neurological outcome severe enough to impede standard schooling at elementary school. These results should be taken into account in prenatal counseling.

A novel disease-causing mutation in the Renin gene in a Tunisian family with autosomal dominant tubulointerstitial kidney disease.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a rare group of disease that affect the tubules of the kidney. There are 4 known subtypes of ADTKD classified based on causative genes and clinical features. In our study, we aimed to identify the causative subtypes of ADTKD in a Tunisian ADTKD family (3 affected members), in whom standard nephrological diagnosis did not provide clear subtype of ADTKD, until genetic testing was performed. Sanger sequencing was performed for UMOD, HNF1ß and REN genes. Mutational analysis allowed us to detect a heterozygous mutation in the REN gene: c.1172C > G, (p.T391R) in exon 10. In silico analyses predicted that the novel likely pathogenic mutation affect protein stability and 3D structure. Our study highlights the importance of establishing a genetic diagnosis to identify the subtype of ADTKD for better patient care. To the best of our knowledge, we report here a first Tunisian ADTKD-REN family.

Clinical courses and complications of young adults with Autosomal Recessive Polycystic Kidney Disease (ARPKD).

Autosomal recessive polycystic kidney disease (ARPKD) is a severe pediatric hepatorenal disorder with pronounced phenotypic variability. A substantial number of patients with early diagnosis reaches adulthood and some patients are not diagnosed until adulthood. Yet, clinical knowledge about adult ARPKD patients is scarce. Here, we describe forty-nine patients with longitudinal follow-up into young adulthood that were identified in the international ARPKD cohort study ARegPKD. Forty-five patients were evaluated in a cross-sectional analysis at a mean age of 21.4 (±3.3) years describing hepatorenal findings. Renal function of native kidneys was within CKD stages 1 to 3 in more than 50% of the patients. Symptoms of hepatic involvement were frequently detected. Fourteen (31%) patients had undergone kidney transplantation and six patients (13%) had undergone liver transplantation or combined liver and kidney transplantation prior to the visit revealing a wide variability of clinical courses. Hepatorenal involvement and preceding complications in other organs were also evaluated in a time-to-event analysis. In summary, we characterize the broad clinical spectrum of young adult ARPKD patients. Importantly, many patients have a stable renal and hepatic situation in young adulthood. ARPKD should also be considered as a differential diagnosis in young adults with fibrocystic hepatorenal disease.

A National French consensus on gene lists for the diagnosis of myopathies using next-generation sequencing.

Next-generation sequencing (NGS) gene-panel-based analyses constitute diagnosis strategies which are adapted to the genetic heterogeneity within the field of myopathies, including more than 200 implicated genes to date. Nonetheless, important inter-laboratory diversity of gene panels exists at national and international levels, complicating the exchange of data and the visibility of the diagnostic offers available for referring neurologists. To address this issue, we here describe the initiative of the genetic diagnosis section of the French National Network for Rare Neuromuscular Diseases (Filière Nationale des Maladies Rares Neuromusculaires, FILNEMUS), which led to set up a consensual nationwide diagnostic strategy among the nine French genetic diagnosis laboratories using NGS for myopathies. The strategy is based on the determination of 13 clinical and/or histological entry-diagnosis groups, and consists for each group either in a successive NGS analysis of a "core gene list" followed in case of a negative result by the analysis of an "exhaustive gene list", or in the NGS analysis of a "unique exhaustive gene list".

[PKHD1 mutations in autosomal recessive polycystic kidney disease (ARPKD): Genotype-phenotype correlations from a series of 308 cases to improve prenatal counselling]. / Mutations PKHD1 dans la polykystose autosomique récessive : corrélations génotype­phénotype dans une série de 308 cas pour guider le diagnostic anténatal.

OBJECTIVES: ARPKD is a recessive rare disease due to PKHD1 mutation. The main objective of the study was to characterize the phenotypic variability according to the different types of PKHD1 mutations. METHODS: This study was performed in 308 ARPKD patients with a genetic diagnosis from our genetic center. Related physicians provided minimal clinical and biological data. RESULTS: Patients were divided into three genotypic groups: the first group (G1; n=65) consisted of patients with two truncating mutations, the second group (G2; n=117) of patients with one truncating and one non-truncating mutation, and the third group (G3; n=126) of patients with two non-truncating mutations. In the entire cohort, the outcomes consisted of 31% of pregnancy termination, 18% of neonatal deaths and 51% of patient survival after the neonatal period. The proportion of severe ARPKD (pregnancy termination or neonatal death) was significantly greater in G1: 94% versus 47% in G2 and 27% in G3 (P<0.001). CONCLUSION: The presence of two truncating mutations in PKHD1 is associated with the most severe perinatal phenotype. However, the phenotypic variability observed in the other genotypic groups requires caution for prenatal counseling.

Fetal anomalies associated with HNF1B mutations: report of 20 autopsy cases.

OBJECTIVES: To describe macroscopic and microscopic anomalies present in fetuses carrying hepatocyte nuclear factor-1 ß mutation, their frequency, and genotype/phenotype correlations. METHODS: Clinical data, ultrasound findings, genetic studies, and autopsy reports of 20 fetal autopsies were analyzed. Histology was reviewed by two pathologists. RESULTS: Macroscopic findings were typically unilateral or bilateral renal enlargement and cortical cysts. Renal lesions were associated with congenital anomalies of the kidney and urinary tract in 25% of cases. Microscopic renal anomalies were dominated by glomerulocystic kidney and renal dysplasia. Extra-renal manifestations such as pancreatic hypoplasia (75%) and genital anomalies (68%) were only detected at autopsy. In 40% of cases, there was heterozygous deletion of the whole gene. There were de novo mutations in 40%. CONCLUSION: This study underlines the importance of considering hepatocyte nuclear factor-1 ß mutations in fetuses with congenital anomalies of the kidney and urinary tract, especially when associated with pancreatic hypoplasia. No correlation between phenotype and genotype was found, highlighting high intra-familial variability in cases with inherited mutations. © 2016 John Wiley & Sons, Ltd.

Compound heterozygous PKHD1 variants cause a wide spectrum of ductal plate malformations.

Ductal plate malformations (DPM) present with a wide phenotypic spectrum comprising Von Meyenburg complexes (VMC), Caroli disease (CD), Caroli syndrome (CS), and autosomal recessive polycystic kidney disease (ARPKD). Variants in PKHD1 are responsible for ARPKD and CS with a high inter- and intra-familial phenotypic variability. Rare familial cases of CD had been reported and exceptional cases of CD are associated with PKHD1 variants. In a family of three siblings presenting with a wide spectrum of severity of DPM, we performed whole exome sequencing and identified two PKHD1 compound heterozygous variants (c.10444G>A; p.Arg3482Cys and c.5521C>T; p.Glu1841Lys), segregating with the symptoms. Two compound heterozygous PKHD1 variants, including one hypomorphic variant, were identified in two other familial cases of DPM with at least one patient presenting with CD. This report widens the phenotypic variability of PKHD1 variants to VMC, and others hepatic bile ducts malformations with inconstant renal phenotype in adults and highlights the important intra-familial phenotypic variability. It also showed that PKHD1 might be a major gene for CD. This work adds an example of the contribution of exome sequencing, not only in the discovery of new genes but also in expanding the phenotypic spectrum of well-known disease-associated genes, using reverse phenotyping.

A splicing mutation in the DMD gene detected by next-generation sequencing and confirmed by mRNA and protein analysis.

BACKGROUND: Dystrophinopathies, either the severe Duchenne Muscular Dystrophy (DMD) or the milder Becker Muscular Dystrophy (BMD), are X-linked recessive disorders caused by mutations in the DMD gene. DMD is one of the longest human genes. Large deletions or duplications account for 60-80% of the mutations. Remaining anomalies consist in point mutations or small rearrangements. Routinely, the molecular diagnosis is done by a Multiplex Ligation-dependent Probe Amplification (MLPA) or array Comparative Genome Hybridization (aCGH), followed, if negative, by Sanger sequencing of all exons. METHODS: In this study, massive parallel sequencing (MPS) or next generation sequencing (NGS) was used to make a rapid and costless molecular diagnosis in a young boy suspected of DMD. RESULTS: A small deletion: NM_004006.2:c.2803+5_2803+8del was identified. The diagnosis was performed in one single manipulation and within a week. The consequence of this intronic mutation is a skipping of exon 21 confirmed by mRNA and protein analysis. CONCLUSIONS: NGS appears to be an efficient new strategy in DMD molecular diagnosis. It highlights the major evolution of the diagnostic strategy towards high throughput technologies, where bioinformatics analysis becomes the real challenge for variations detection. This is the first study reporting in vivo impact of this intronic mutation.

Genotype-phenotype correlations in fetuses and neonates with autosomal recessive polycystic kidney disease.

The prognosis of autosomal recessive polycystic kidney disease is known to correlate with genotype. The presence of two truncating mutations in the PKHD1 gene encoding the fibrocystin protein is associated with neonatal death while patients who survive have at least one missense mutation. To determine relationships between genotype and renal and hepatic abnormalities we correlated the severity of renal and hepatic histological lesions to the type of PKHD1 mutations in 54 fetuses (medical pregnancy termination) and 20 neonates who died shortly after birth. Within this cohort, 55.5% of the mutations truncated fibrocystin. The severity of cortical collecting duct dilatations, cortical tubule and glomerular lesions, and renal cortical and hepatic portal fibrosis increased with gestational age. Severe genotypes, defined by two truncating mutations, were more frequent in patients of less than 30 weeks gestation compared to older fetuses and neonates. When adjusted to gestational age, the extension of collecting duct dilatation into the cortex and cortical tubule lesions, but not portal fibrosis, was more prevalent in patients with severe than in those with a non-severe genotype. Our results show the presence of two truncating mutations of the PKHD1 gene is associated with the most severe renal forms of prenatally detected autosomal recessive polycystic kidney disease. Their absence, however, does not guarantee survival to the neonatal period.

Familial acampomelic form of campomelic dysplasia caused by a 960 kb deletion upstream of SOX9.

Campomelic dysplasia (CD) is a rare autosomal dominant osteochondrodysplasia with or without XY disorders of sexual development (DSD). Campomelia is absent in about 10% of the cases, referred to as the acampomelic form of CD (ACD). Most CDs are caused by mutations within the SOX9 coding region. Several CD patients with balanced chromosome rearrangements involving the 17q24 region have been reported suggesting the presence of cis-regulatory elements upstream and/or downstream of the gene. Deletions upstream of SOX9 represent a third mechanism of mutation. To date, a 1.5 Mb de novo deletion in the SOX9 upstream region has been identified in a single 46,XY patient with ACD and DSD. We report here for the first time on a familial ACD caused by an inherited deletion mapping upstream of the SOX9 gene. Using high-density oligoarray comparative genomic hybridization (CGH), we showed that the size of the deletion was 960 kb in the XY-DSD child and her mother, both affected. The deletion lying from 517 kb to 1.477 Mb upstream of SOX9 remove several highly conserved elements and reduce the minimum critical size and therefore the number of highly conserved sequence elements responsible for ACD.

Genotype-phenotype analysis in 2,405 patients with a dystrophinopathy using the UMD-DMD database: a model of nationwide knowledgebase.

UMD-DMD France is a knowledgebase developed through a multicenter academic effort to provide an up-to-date resource of curated information covering all identified mutations in patients with a dystrophinopathy. The current release includes 2,411 entries consisting in 2,084 independent mutational events identified in 2,046 male patients and 38 expressing females, which corresponds to an estimated number of 39 people per million with a genetic diagnosis of dystrophinopathy in France. Mutations consist in 1,404 large deletions, 215 large duplications, and 465 small rearrangements, of which 39.8% are nonsense mutations. The reading frame rule holds true for 96% of the DMD patients and 93% of the BMD patients. Quality control relies on the curation by four experts for the DMD gene and related diseases. Data on dystrophin and RNA analysis, phenotypic groups, and transmission are also available. About 24% of the mutations are de novo events. This national centralized resource will contribute to a greater understanding of prevalence of dystrophinopathies in France, and in particular, of the true frequency of BMD, which was found to be almost half (43%) that of DMD. UMD-DMD is a searchable anonymous database that includes numerous newly developed tools, which can benefit to all the scientific community interested in dystrophinopathies. Dedicated functions for genotype-based therapies allowed the prediction of a new multiexon skipping (del 45-53) potentially applicable to 53% of the deleted DMD patients. Finally, such a national database will prove to be useful to implement the international global DMD patients' registries under development.

Identification of two novel mutations and long-term follow-up in abetalipoproteinemia: a report of four cases.

Abetalipoproteinemia (ABL; OMIM 200100) is an inherited disorder resulting from mutations in the microsomal triglyceride transfer protein gene and characterized by a major lipid malabsorption leading to extremely low plasma cholesterol and triglyceride levels and fat-soluble vitamins deficiencies. We report two novel mutations (c.59del17 and c.582C>A) and the long-term follow-up of four ABL subjects treated with vitamin E. The good outcome of the early-treated patients contrasts with severe ataxia and retinopathy observed in the patient with delayed treatment. In conclusion, early diagnosis and early management are essential to prevent the manifestations following the fat-soluble vitamin deficiencies.

Gonadal dysgenesis without adrenal insufficiency in a 46, XY patient heterozygous for the nonsense C16X mutation: a case of SF1 haploinsufficiency.

Targeted disruption of the orphan nuclear receptor SF1 results in the absence of adrenals and gonads, establishing that this transcription factor is implicated in gonadal determination and adrenal development. Four human SF1 gene mutations have been described to date: three (G35E, R92Q, R255L) were responsible for adrenal insufficiency associated with a gonadal dysgenesis in two 46, XY individuals, one (8 bp deletion in exon 6) resulted in gonadal dysgenesis without adrenal insufficiency. We identified a new heterozygous SF1 gene mutation, C16X, in a 46, XY patient showing gonadal dysgenesis with normal adrenal function: low basal levels of AMH and testosterone (T), weak T response to hCG, hypoplastic testes with abundant seminiferous tubules but rare germ cells. This mutation causes premature termination of translation and should abolish all SF1 activity. Therefore haploinsufficiency could explain the deleterious effect of this mutation in our patient suggesting that testis development is more SF1 dose-dependent than adrenal development. Although the same mechanism explains the deleterious effects of SF1 missense mutations, recent studies have demonstrated an additional dominant negative effect. These data suggest that heterozygous mutation impaired adrenal development only if the two mechanisms, gene dosage and dominant negative effects occur.