Functional Assessment of Variants Associated with Wolfram Syndrome

Wolfram Syndrome (WS) is a heterogeneous multisystem neurodegenerative disorder with two allelic variations in addition to a separate subtype known as WS type 2. The wide phenotypic spectrum of WS includes diabetes mellitus and optic atrophy which is often accompanied by diabetes insipidus, deafness, urological and neurological complications in combination or in isolation. To date, the understanding of the genotype phenotype relationship in this complex syndrome remains poorly understood. In this study we identified and explored the functionality of rare and novel variants in the two causative WS genes WFS1 and CISD2 by assessing the effects of the mutations on the encoded proteins Wolframin and ERIS, in a cohort of 12 patients with autosomal recessive WS, dominant WS and WS type 2. The identified pathogenic variants included missense changes, frameshift deletions and insertions in WFS1 and an exonic deletion in CISD2 which all altered the respective encoded protein in a manner that did not correlate to the phenome previously described. These observations suggest the lack of genotype phenotype correlation in this complex syndrome and the need to explore other molecular genetic mechanisms. Additionally, our findings highlight the importance of functionally assessing variants for their pathogenicity to tackle the problem of increasing variants of unknown significance (VUS) in the public genetic databases

Biochemical analysis of novel NAA10 variants suggests distinct pathogenic mechanisms involving impaired protein N-terminal acetylation

NAA10 is the catalytic subunit of the N-terminal acetyltransferase complex, NatA, which is responsible for N-terminal acetylation of nearly half the human proteome. Since 2011, at least 21 different NAA10 missense variants have been reported as pathogenic in humans. The clinical features associated with this X-linked condition vary, but commonly described features include developmental delay, intellectual disability, cardiac anomalies, brain abnormalities, facial dysmorphism and/or visual impairment. Here, we present eight individuals from five families with five different de novo or inherited NAA10 variants. In order to determine their pathogenicity, we have performed biochemical characterisation of the four novel variants c.16G>C p.(A6P), c.235C>T p.(R79C), c.386A>C p.(Q129P) and c.469G>A p.(E157K). Additionally, we clinically describe one new case with a previously identified pathogenic variant, c.384T>G p.(F128L). Our study provides important insight into how different NAA10 missense variants impact distinct biochemical functions of NAA10 involving the ability of NAA10 to perform N-terminal acetylation. These investigations may partially explain the phenotypic variability in affected individuals and emphasise the complexity of the cellular pathways downstream of NAA10.publishedVersio

DOORS syndrome and a recurrent truncating ATP6V1B2 variant

PURPOSE: Biallelic variants in TBC1D24, which encodes a protein that regulates vesicular transport, are frequently identified in patients with DOORS (deafness, onychodystrophy, osteodystrophy, intellectual disability [previously referred to as mental retardation], and seizures) syndrome. The aim of the study was to identify a genetic cause in families with DOORS syndrome and without a TBC1D24 variant. METHODS: Exome or Sanger sequencing was performed in individuals with a clinical diagnosis of DOORS syndrome without TBC1D24 variants. RESULTS: We identified the same truncating variant in ATP6V1B2 (NM_001693.4:c.1516C>T; p.Arg506*) in nine individuals from eight unrelated families with DOORS syndrome. This variant was already reported in individuals with dominant deafness onychodystrophy (DDOD) syndrome. Deafness was present in all individuals, along with onychodystrophy and abnormal fingers and/or toes. All families but one had developmental delay or intellectual disability and five individuals had epilepsy. We also describe two additional families with DDOD syndrome in whom the same variant was found. CONCLUSION: We expand the phenotype associated with ATP6V1B2 and propose another causal gene for DOORS syndrome. This finding suggests that DDOD and DOORS syndromes might lie on a spectrum of clinically and molecularly related conditions

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.</p

Biochemical analysis of novel NAA10 variants suggests distinct pathogenic mechanisms involving impaired protein N‑terminal acetylation

NAA10 is the catalytic subunit of the N-terminal acetyltransferase complex, NatA, which is responsible for N-terminal acetylation of nearly half the human proteome. Since 2011, at least 21 different NAA10 missense variants have been reported as pathogenic in humans. The clinical features associated with this X-linked condition vary, but commonly described features include developmental delay, intellectual disability, cardiac anomalies, brain abnormalities, facial dysmorphism and/or visual impairment. Here, we present eight individuals from five families with five different de novo or inherited NAA10 variants. In order to determine their pathogenicity, we have performed biochemical characterisation of the four novel variants c.16G>C p.(A6P), c.235C>T p.(R79C), c.386A>C p.(Q129P) and c.469G>A p.(E157K). Additionally, we clinically describe one new case with a previously identified pathogenic variant, c.384T>G p.(F128L). Our study provides important insight into how different NAA10 missense variants impact distinct biochemical functions of NAA10 involving the ability of NAA10 to perform N-terminal acetylation. These investigations may partially explain the phenotypic variability in affected individuals and emphasise the complexity of the cellular pathways downstream of NAA10

Deciphering the premature mortality in PIGA-CDG – An untold story

Objective: Congenital disorder of glycosylation (CDG) due to a defective phosphatidylinositol glycan anchor biosynthesis class A protein (PIGA) is a severe X-linked developmental and epileptic encephalopathy. Seizures are often treatment refractory, and patients have intellectual disability and global developmental delay. Previous reports have suggested that patients with PIGA-CDG have a high risk of premature mortality. This study aimed to evaluate the observed high mortality and the causes of death in PIGA-CDG patients. Methods: We reviewed the literature and collected additional unpublished patients through an international network. Results: In total, we reviewed the data of 88 patients of whom 30 patients born alive were deceased, and the overall mortality before the age of 20 years was 30 % (26/88). Age at death ranged from 15 days to 48 years of life. The median age at death was two years and more than half of the patients deceased in early childhood. The PIGA-specific mortality rate/1000 person-years was 44.9/1000 person-years (95 %, CI 31.4–64.3). There were no cases of definite or probable sudden unexpected death in epilepsy (SUDEP) and half of the patients died due to respiratory failure (15/30, 50 %) or possible SUDEP (3/30, 10 %). Three patients (10 %) died from severe cardiomyopathy, liver failure and gastrointestinal bleeding, respectively. The cause of death was unclassified in nine patients (30 %). Autopsies were rarely performed and the true cause of death remains unknown for the majority of patients. Significance: Our data indicate an increased risk of premature death in patients with PIGA-CDG when compared to most monogenic developmental and epileptic encephalopathies