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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.

Background Pontocerebellar hypoplasia (PCH) is a heterogeneous group of diseases characterized by lack of development and/or early neurodegeneration of cerebellum and brainstem. According to clinical features, seven subtypes of PCH have been described, PCH type 2 related to TSEN54 mutations being the most frequent. PCH is most often autosomal recessive though de novo anomalies in the X-linked gene CASK have recently been identified in patients, mostly females, presenting with intellectual disability, microcephaly and PCH (MICPCH). Methods Fourteen patients (12 females and two males; aged 16 months-14 years) presenting with PCH at neuroimaging and with clinical characteristics unsuggestive of PCH1 or PCH2 were included. The CASK gene screening was performed using Array-CGH and sequencing. Clinical and neuroradiological features were collected. Results We observed a high frequency of patients with a CASK mutation (13/14). Ten patients (8 girls and 2 boys) had intragenic mutations and three female patients had a Xp11.4 submicroscopic deletion including the CASK gene. All were de novo mutations. Phenotype was variable in severity but highly similar among the 11 girls and was characterized by psychomotor retardation, severe intellectual disability, progressive microcephaly, dystonia, mild dysmorphism, and scoliosis. Other signs were frequently associated, such as growth retardation, ophthalmologic anomalies (glaucoma, megalocornea and optic atrophy), deafness and epilepsy. As expected in an X-linked disease manifesting mainly in females, the boy hemizygous for a splice mutation had a very severe phenotype with nearly no development and refractory epilepsy. We described a mild phenotype in a boy with a mosaic truncating mutation. We found some degree of correlation between severity of the vermis hypoplasia and clinical phenotype. Conclusion This study describes a new series of PCH female patients with CASK inactivating mutations and confirms that these patients have a recognizable although variable phenotype consisting of a specific form of pontocerebellar hypoplasia. In addition, we report the second male patient to present with a severe MICPCH phenotype and a de novo CASK mutation and describe for the first time a mildly affected male patient harboring a mosaic mutation. In our reference centre, CASK related PCH is the second most frequent cause of PCH. The identification of a de novo mutation in these patients enables accurate and reassuring genetic counselling.

IntroductionThis study aims to define the phenotypic and molecular spectrum of the two clinical forms of β-galactosidase (β-GAL) deficiency, GM1-gangliosidosis and mucopolysaccharidosis IVB (Morquio disease type B, MPSIVB).MethodsClinical and genetic data of 52 probands, 47 patients with GM1-gangliosidosis and 5 patients with MPSIVB were analysed.ResultsThe clinical presentations in patients with GM1-gangliosidosis are consistent with a phenotypic continuum ranging from a severe antenatal form with hydrops fetalis to an adult form with an extrapyramidal syndrome. Molecular studies evidenced 47 variants located throughout the sequence of the GLB1 gene, in all exons except 7, 11 and 12. Eighteen novel variants (15 substitutions and 3 deletions) were identified. Several variants were linked specifically to early-onset GM1-gangliosidosis, late-onset GM1-gangliosidosis or MPSIVB phenotypes. This integrative molecular and clinical stratification suggests a variant-driven patient assignment to a given clinical and severity group.ConclusionThis study reports one of the largest series of b-GAL deficiency with an integrative patient stratification combining molecular and clinical features. This work contributes to expand the community knowledge regarding the molecular and clinical landscapes of b-GAL deficiency for a better patient management.

The molecular basis underlying the clinical variability in symptomatic Duchenne muscular dystrophy (DMD) carriers are still to be precised. We report 26 cases of early symptomatic DMD carriers followed in the French neuromuscular network. Clinical presentation, muscular histological analysis and type of gene mutation, as well as X-chromosome inactivation (XCI) patterns using DNA extracted from peripheral blood or muscle are detailed. The initial symptoms were significant weakness (88%) or exercise intolerance (27%). Clinical severity varied from a Duchenne-like progression to a very mild Becker-like phenotype. Cardiac dysfunction was present in 19% of the cases. Cognitive impairment was worthy of notice, as 27% of the carriers are concerned. The muscular analysis was always contributive, revealing muscular dystrophy (83%), mosaic in immunostaining (81%) and dystrophin abnormalities in western blot analysis (84%). In all, 73% had exonic deletions or duplications and 27% had point mutations. XCI pattern was biased in 62% of the cases. In conclusion, we report the largest series of manifesting DMD carriers at pediatric age and show that exercise intolerance and cognitive impairment may reveal symptomatic DMD carriers. The complete histological and immunohistological study of the muscle is the key of the diagnosis leading to the dystrophin gene analysis. Our study shows also that cognitive impairment in symptomatic DMD carriers is associated with mutations in the distal part of the DMD gene. XCI study does not fully explain the mechanisms as well as the wide spectrum of clinical phenotype, though a clear correlation between the severity of the phenotype and inactivation bias was observed.

Next-generation sequencing (NGS) allows the detection of plentiful mutations increasing the rate of patients getting a positive diagnosis. However, while single-nucleotide variants (SNVs) or small indels can be easily detected, structural variations (SVs) such as copy number variants (CNVs) are often not researched. In Charcot–Marie–Tooth disease (CMT), the most common hereditary peripheral neuropathy, the PMP22-duplication was the first variation detected. Since then, more than 90 other genes have been associated with CMT, with point mutations or small indels mostly described. Herein, we present a personalized approach we performed to obtain a positive diagnosis of a patient suffering from demyelinating CMT. His NGS data were aligned to the human reference sequence but also studied using the CovCopCan software, designed to detect large CNVs. This approach allowed the detection of only one mutation in SH3TC2, the frequent p.Arg954*, while SH3TC2 is known to be responsible for autosomal recessive demyelinating CMT forms. Interestingly, by modifying the standard CovCopCan use, we detected the second mutation of this patient corresponding to a 922 bp deletion in SH3TC2 (Chr5:148,390,609-Chr5:148,389,687), including only one exon (exon 14). This highlights that SVs, different from PMP22 duplication, can be responsible for peripheral neuropathy and should be searched systematically. This approach could also be employed to improve the diagnosis of all inherited diseases.

Treatment with the chimeric anti-CD20 monoclonal antibody rituximab induces rapid and long-lasting depletion of circulating B cells. We report the occurrence of enteroviral meningoencephalitis following rituximab therapy in 1 child with immune thrombocytopenia and in 1 adult patient with relapsed B cell lymphoma.

Holoprosencephaly (HPE) is the most common severe brain anomaly in humans, which results from incomplete cleavage of the forebrain during early embryogenesis. The aetiology of HPE is very heterogeneous. Among the genetic factors, TGIF ( TG-interacting factor), which codes for a transcription factor modulating the signalling pathway of TGF-beta, was previously implicated. We investigated 127 HPE probands by sequencing their TGIF gene and identified the first nonsense mutation reported so far and also a novel missense mutation, in two families that presented a large range of disease severity. The low number of mutations in TGIF suggests that this gene has no major contribution to the aetiology of HPE and our study confirms the wide clinical heterogeneity of the disease.

Holoprosencephaly (HPE; 1 out of 16,000 live births; 1 out of 250 conceptuses) is a complex brain malformation resulting from incomplete cleavage of the prosencephalon, affecting both the forebrain and the face. Clinical expressivity is variable, ranging from a single cerebral ventricle and cyclopia to clinically unaffected carriers in familial dominant autosomic HPE. The disease is genetically heterogeneous, but additional environmental agents also contribute to the etiology of HPE. In our cohort of 200 patients, 34 heterozygous mutations were identified, 24 of them being novel ones: 13 out of 17 in the Sonic hedgehog gene (SHH); 4 out of 7 in ZIC2; and 7 out of 8 in SIX3. The two mutations identified in TGIF have already been reported. Novel phenotypes associated with a mutation have been described, such as abnormalities of the pituitary gland and corpus callosum, colobomatous microphthalmia, choanal aperture stenosis, and isolated cleft lip. This study confirms the great genetic heterogeneity of the disease, the important phenotypic variability in HPE families, and the difficulty to establish genotype‐phenotype correlations. Hum Mutat 24:43–51, 2004. © 2004 Wiley‐Liss, Inc.