Expanding SPTAN1 monoallelic variant associated disorders: From epileptic encephalopathy to pure spastic paraplegia and ataxia.

PURPOSE: Nonerythrocytic αII-spectrin (SPTAN1) variants have been previously associated with intellectual disability and epilepsy. We conducted this study to delineate the phenotypic spectrum of SPTAN1 variants. METHODS: We carried out SPTAN1 gene enrichment analysis in the rare disease component of the 100,000 Genomes Project and screened 100,000 Genomes Project, DECIPHER database, and GeneMatcher to identify individuals with SPTAN1 variants. Functional studies were performed on fibroblasts from 2 patients. RESULTS: Statistically significant enrichment of rare (minor allele frequency < 1 × 10-5) probably damaging SPTAN1 variants was identified in families with hereditary ataxia (HA) or hereditary spastic paraplegia (HSP) (12/1142 cases vs 52/23,847 controls, p = 2.8 × 10-5). We identified 31 individuals carrying SPTAN1 heterozygous variants or deletions. A total of 10 patients presented with pure or complex HSP/HA. The remaining 21 patients had developmental delay and seizures. Irregular αII-spectrin aggregation was noted in fibroblasts derived from 2 patients with p.(Arg19Trp) and p.(Glu2207del) variants. CONCLUSION: We found that SPTAN1 is a genetic cause of neurodevelopmental disorder, which we classified into 3 distinct subgroups. The first comprises developmental epileptic encephalopathy. The second group exhibits milder phenotypes of developmental delay with or without seizures. The final group accounts for patients with pure or complex HSP/HA.

GM2 gangliosidosis AB variant: first case of late onset and review of the literature.

AB variant is the rarest form of GM2 gangliosidosis, neurodegenerative diseases caused by lysosomal accumulation of GM2 gangliosides. Less than thirty cases are referenced in the literature, and to date, no late-onset form has been described. Our proband is a 22-year-old male with spinocerebellar ataxia and lower limbs motor deficiency. His symptoms started at the age of 10. A genetic analysis revealed two mutations in the GM2A gene encoding the GM2 activator protein (GM2-AP), an essential co-factor of hexosaminidase A. Both mutations, GM2A:c.79A > T:p.Lys27* and GM2A:c.415C > T:p.Pro139Ser, were inherited respectively from his father and his mother. The nonsense mutation was predicted to be likely pathogenic, but the missense mutation was of unknown significance. To establish the pathogenicity of this variant, we studied GM2 accumulation and GM2A gene expression. Electron microscopy and immunofluorescence performed on patient's fibroblasts did not reveal any lysosomal accumulation of GM2. There was also no difference in GM2A gene expression using RT-qPCR, and both mutations were found on cDNA Sanger sequencing. Measurement of plasma gangliosides by liquid-phase chromatography-tandem mass spectrometry showed an accumulation of GM2 in our patient's plasma at 83.5 nmol/L, and a GM2/GM3 ratio at 0.066 (median of negative control at 30.2 nmol/L [19.7-46.8] and 0.019 respectively). Therefore, the association of both p.Lys27* and p.Pro169Ser mutations leads to a GM2-AP functional deficiency. Whereas the first mutation is more likely to be linked with infantile form of GM2 gangliosidosis, the hypomorphic p.Pro169Ser variant may be the first associated with a late-onset form of AB variant.

RFC1 nonsense and frameshift variants cause CANVAS: clues for an unsolved pathophysiology.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is an inherited late-onset neurological disease caused by bi-allelic AAGGG pentanucleotide expansions within intron 2 of RFC1. Despite extensive studies, the pathophysiological mechanism of these intronic expansions remains elusive. We screened by clinical exome sequencing two unrelated patients presenting with late-onset ataxia. A repeat-primer polymerase chain reaction was used for RFC1 AAGGG intronic expansion identification. RFC1 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction. We identified the first two CANVAS affected patients who are compound heterozygous for RFC1 truncating variants (p.Arg388* and c.575delA, respectively) and a pathological AAGGG expansion. RFC1 expression studies in whole blood showed a significant reduction of RFC1 mRNA for both patients compared to three patients with bi-allelic RFC1 expansions. In conclusion, this observation provides clues that suggest bi-allelic RFC1 conditional loss-of-function as the cause of the disease.

Biallelic PI4KA variants cause a novel neurodevelopmental syndrome with hypomyelinating leukodystrophy.

Phosphoinositides are lipids that play a critical role in processes such as cellular signalling, ion channel activity and membrane trafficking. When mutated, several genes that encode proteins that participate in the metabolism of these lipids give rise to neurological or developmental phenotypes. PI4KA is a phosphoinositide kinase that is highly expressed in the brain and is essential for life. Here we used whole exome or genome sequencing to identify 10 unrelated patients harbouring biallelic variants in PI4KA that caused a spectrum of conditions ranging from severe global neurodevelopmental delay with hypomyelination and developmental brain abnormalities to pure spastic paraplegia. Some patients presented immunological deficits or genito-urinary abnormalities. Functional analyses by western blotting and immunofluorescence showed decreased PI4KA levels in the patients' fibroblasts. Immunofluorescence and targeted lipidomics indicated that PI4KA activity was diminished in fibroblasts and peripheral blood mononuclear cells. In conclusion, we report a novel severe metabolic disorder caused by PI4KA malfunction, highlighting the importance of phosphoinositide signalling in human brain development and the myelin sheath.

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

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.

Optic neuropathy linked to ACAD9 pathogenic variants: A potentially riboflavin-responsive disorder?

Mitochondrial complex I (CI) deficiencies (OMIM 252010) are the commonest inherited mitochondrial disorders in children. Acyl-CoA dehydrogenase 9 (ACAD9) is a flavoenzyme involved chiefly in CI assembly and possibly in fatty acid oxidation. Biallelic pathogenic variants result in CI dysfunction, with a phenotype ranging from early onset and sometimes fatal mitochondrial encephalopathy with lactic acidosis to late-onset exercise intolerance. Cardiomyopathy is often associated. We report a patient with childhood-onset optic and peripheral neuropathy without cardiac involvement, related to CI deficiency. Genetic analysis revealed compound heterozygous pathogenic variants in ACAD9, expanding the clinical spectrum associated to ACAD9 mutations. Importantly, riboflavin treatment (15 mg/kg/day) improved long-distance visual acuity and demonstrated significant rescue of CI activity in vitro.

Biallelic RFC1-expansion in a French multicentric sporadic ataxia cohort.

OBJECTIVE: Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a recessively inherited multisystem ataxia compromising cerebellar, vestibular, and sensory nerves, which has been associated to a pathogenic AAGGG(n) biallelic expansion repeat in the RFC1 gene. Our objective was to assess its prevalence in a French cohort of patients with idiopathic sporadic late-onset ataxia (ILOA), idiopathic early-onset ataxia (IEOA), or Multiple System Atrophy of Cerebellar type (MSA-C). METHODS: 163 patients were recruited in 3 French tertiary centers: 100 ILOA, 21 IEOA, and 42 patients with possible or probable MSA-C. RESULTS: A pathogenic biallelic RFC1 AAGGG(n) repeat expansion was found in 15 patients: 15/100 in the ILOA group, but none in the IEOA and MSA-C subgroups. 14/15 patients had a CANVAS phenotype. Only 1/15 had isolated cerebellar ataxia, but also shorter biallelic expansions. Two RFC1 AAGGG(n) alleles were found in 78% of patients with a CANVAS phenotype. In one post-mortem case, the pathophysiological involvement of cerebellum and medullar posterior columns was found. CONCLUSION: Our study confirms the genetic heterogeneity of the CANVAS and that RFC1 repeat expansions should be searched for preferentially in case of unexplained ILOA associated with a sensory neuronopathy, but not particularly in patients classified as MSA-C.

Expanding the clinical spectrum of STIP1 homology and U-box containing protein 1-associated ataxia.

BACKGROUND: STUB1 has been first associated with autosomal recessive (SCAR16, MIM# 615768) and later with dominant forms of ataxia (SCA48, MIM# 618093). Pathogenic variations in STUB1 are now considered a frequent cause of cerebellar ataxia. OBJECTIVE: We aimed to improve the clinical, radiological, and molecular delineation of SCAR16 and SCA48. METHODS: Retrospective collection of patients with SCAR16 or SCA48 diagnosed in three French genetic centers (Montpellier, Strasbourg and Nancy). RESULTS: Here, we report four SCAR16 and nine SCA48 patients from two SCAR16 and five SCA48 unrelated French families. All presented with slowly progressive cerebellar ataxia. Additional findings included cognitive decline, dystonia, parkinsonism and swallowing difficulties. The age at onset was highly variable, ranging from 14 to 76 years. Brain MRI showed marked cerebellar atrophy in all patients. Phenotypic findings associated with STUB1 pathogenic variations cover a broad spectrum, ranging from isolated slowly progressive ataxia to severe encephalopathy, and include extrapyramidal features. We described five new pathogenic variations, two previously reported pathogenic variations, and two rare variants of unknown significance in association with STUB1-related disorders. We also report the first pathogenic variation associated with both dominant and recessive forms of inheritance (SCAR16 and SCA48). CONCLUSION: Even though differences are observed between the recessive and dominant forms, it appears that a continuum exists between these two entities. While adding new symptoms associated with STUB1 pathogenic variations, we insist on the difficulty of genetic counselling in STUB1-related pathologies. Finally, we underscore the usefulness of DAT-scan as an additional clue for diagnosis.

ATP8A2-related disorders as recessive cerebellar ataxia.

ATP8A2-related disorders are autosomal recessive conditions that associate encephalopathy with or without hypotonia, psychomotor delay, abnormal movements, chorea, tremor, optic atrophy and cerebellar atrophy (CARMQ4). Through a multi-centric collaboration, we identified six point mutations (one splice site and five missense mutations) involving ATP8A2 in six individuals from five families. Two patients from one family with the homozygous p.Gly585Val mutation had a milder presentation without encephalopathy. Expression and functional studies of the missense mutations demonstrated that protein levels of four of the five missense variants were very low and lacked phosphatidylserine-activated ATPase activity. One variant p.Ile215Leu, however, expressed at normal levels and displayed phospholipid-activated ATPase activity similar to the non-mutated protein. We therefore expand for the first time the phenotype related to ATP8A2 mutations to less severe forms characterized by cerebellar ataxia without encephalopathy and suggest that ATP8A2 should be analyzed for all cases of syndromic or non-syndromic recessive or sporadic ataxia.