Identification of a methylation profile for DNMT1-associated autosomal dominant cerebellar ataxia, deafness, and narcolepsy

Background: DNA methylation is an essential epigenetic mark, controlled by DNA methyltransferase (DNMT) proteins, which regulates chromatin structure and gene expression throughout the genome. In this study, we describe a family with adult-onset autosomal dominant cerebellar ataxia with deafness and narcolepsy (ADCA-DN) caused by mutations in the maintenance methyltransferase DNMT1 and assess the DNA methylation profile of these individuals. Results: We report a family with six individuals affected with ADCA-DN; specifically, patients first developed hearing loss and ataxia, followed by narcolepsy, and cognitive decline. We identified a heterozygous DNMT1 variant, c.1709C\u3eT [p.Ala570Val] by Sanger sequencing, which had been previously reported as pathogenic for ADCA-DN and segregated with disease in the family. DNA methylation analysis by high-resolution genome-wide DNA methylation array identified a decrease in CpGs with 0–10 % methylation and 80–95 % methylation and a concomitant increase in sites with 10–30 % methylation and \u3e95 % methylation. This pattern suggests an increase in methylation of normally unmethylated regions, such as promoters and CpG islands, as well as further methylation of highly methylated gene bodies and intergenic regions. Furthermore, a regional analysis identified 82 hypermethylated loci with consistent robust differences across ≥5 consecutive probes compared to our large reference cohort. Conclusions: This report identifies robust changes in the DNA methylation patterns in ADCA-DN patients, which is an important step towards elucidating disease pathogenesis

Identification of rare-disease genes using blood transcriptome sequencing and large control cohorts.

It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene1. The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches2-5. For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases6-8. This includes muscle biopsies from patients with undiagnosed rare muscle disorders6,9, and cultured fibroblasts from patients with mitochondrial disorders7. However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution

Bi-allelic loss-of-function variants in BCAS3 cause a syndromic neurodevelopmental disorder.

BCAS3 microtubule-associated cell migration factor (BCAS3) is a large, highly conserved cytoskeletal protein previously proposed to be critical in angiogenesis and implicated in human embryogenesis and tumorigenesis. Here, we established BCAS3 loss-of-function variants as causative for a neurodevelopmental disorder. We report 15 individuals from eight unrelated families with germline bi-allelic loss-of-function variants in BCAS3. All probands share a global developmental delay accompanied by pyramidal tract involvement, microcephaly, short stature, strabismus, dysmorphic facial features, and seizures. The human phenotype is less severe compared with the Bcas3 knockout mouse model and cannot be explained by angiogenic defects alone. Consistent with being loss-of-function alleles, we observed absence of BCAS3 in probands' primary fibroblasts. By comparing the transcriptomic and proteomic data based on probands' fibroblasts with those of the knockout mouse model, we identified similar dysregulated pathways resulting from over-representation analysis, while the dysregulation of some proposed key interactors could not be confirmed. Together with the results from a tissue-specific Drosophila loss-of-function model, we demonstrate a vital role for BCAS3 in neural tissue development

PDXK mutations cause polyneuropathy responsive to PLP supplementation

OBJECTIVE: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. METHODS: We performed genome-wide sequencing, homozygosity mapping and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on ATP-binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology and biochemical quantification. RESULTS: We identified bi-allelic mutations in PDXK in five individuals from two unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP-binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in one family, improvement in power, pain and fatigue contributing to patients regaining their ability to ambulate during the first year of PLP normalization. INTERPRETATION: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown aetiology characterised by reduced PLP levels. This article is protected by copyright. All rights reserved

PLPHP deficiency : clinical, genetic, biochemical, and mechanistic insights

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery

Evaluation of exome filtering techniques for the analysis of clinically relevant genes.

A significant challenge facing clinical translation of exome sequencing is meaningful and efficient variant interpretation. Each exome contains ∼500 rare coding variants; laboratories must systematically and efficiently identify which variant(s) contribute to the patient\u27s phenotype. In silico filtering is an approach that reduces analysis time while decreasing the chances of incidental findings. We retrospectively assessed 55 solved exomes using available datasets as in silico filters: Online Mendelian Inheritance in Man (OMIM), Orphanet, Human Phenotype Ontology (HPO), and Radboudumc University Medical Center curated panels. We found that personalized panels produced using HPO terms for each patient had the highest success rate (100%), while producing considerably less variants to assess. HPO panels also captured multiple diagnoses in the same individual. We conclude that custom HPO-derived panels are an efficient and effective way to identify clinically relevant exome variants. Hum Mutat 2018 Feb; 39(2):197-201

RNA sequencing resolves novel DYNC2H1 variants causing short‐rib thoracic dysplasia type 3: Case report

Abstract Background Intronic variants outside the canonical splice site are challenging to interpret and therefore likely represent an underreported cause of human disease. Autosomal recessive variants in DYNC2H1 are associated with short‐rib thoracic dysplasia 3 with or without polydactyly (SRTD3), a clinically heterogeneous disease generally presenting with short ribs, shortened tubular bones, narrow thorax and acetabular roof anomalies. We describe a case of SRTD3 with compound heterozygous frameshift and intronic variants and highlight the essential role of RNA sequencing (RNA‐Seq) in variant interpretation. Methods Following inconclusive clinical genetic testing identifying a likely pathogenic frameshift variant and an intronic variant of uncertain significance (VUS) in DYNC2H1 in trans, the family enrolled in the Care4Rare Canada research program, where RNA‐Seq studies were performed. Results The proband presented with post‐axial polydactyly of all four limbs, a significantly small chest with a pectus excavatum and anterior flaring of the ribs. RNA‐Seq investigations revealed a novel splice junction as a result of the intronic VUS and significantly decreased DYNC2H1 gene expression in the proband. Conclusion This case demonstrates the diagnostic utility of RNA‐Seq for variant interpretation following inconclusive clinical testing, which can ultimately lead to diagnosis for patients with rare disease

Specific heterozygous variants in MGP lead to endoplasmic reticulum stress and cause spondyloepiphyseal dysplasia

Abstract Matrix Gla protein (MGP) is a vitamin K-dependent post-translationally modified protein, highly expressed in vascular and cartilaginous tissues. It is a potent inhibitor of extracellular matrix mineralization. Biallelic loss-of-function variants in the MGP gene cause Keutel syndrome, an autosomal recessive disorder characterized by widespread calcification of various cartilaginous tissues and skeletal and vascular anomalies. In this study, we report four individuals from two unrelated families with two heterozygous variants in MGP, both altering the cysteine 19 residue to phenylalanine or tyrosine. These individuals present with a spondyloepiphyseal skeletal dysplasia characterized by short stature with a short trunk, diffuse platyspondyly, midface retrusion, progressive epiphyseal anomalies and brachytelephalangism. We investigated the cellular and molecular effects of one of the heterozygous deleterious variants (C19F) using both cell and genetically modified mouse models. Heterozygous ‘knock-in’ mice expressing C19F MGP recapitulate most of the skeletal anomalies observed in the affected individuals. Our results suggest that the main underlying mechanism leading to the observed skeletal dysplasia is endoplasmic reticulum stress-induced apoptosis of the growth plate chondrocytes. Overall, our findings support that heterozygous variants in MGP altering the Cys19 residue cause autosomal dominant spondyloepiphyseal dysplasia, a condition distinct from Keutel syndrome both clinically and molecularly

Recessive mutations in NDUFA2 cause mitochondrial leukoencephalopathy

Deficiencies of mitochondrial respiratory chain complex I frequently result in leukoencephalopathy in young patients, and different mutations in the genes encoding its subunits are still being uncovered. We report 2 patients with cystic leukoencephalopathy and complex I deficiency with recessive mutations in NDUFA2, an accessory subunit of complex I. The first patient was initially diagnosed with a primary systemic carnitine deficiency associated with a homozygous variant in SLC22A5, but also exhibited developmental regression and cystic leukoencephalopathy, and an additional diagnosis of complex I deficiency was suspected. Biochemical analysis confirmed a complex I deficiency, and whole-exome sequencing revealed a homozygous mutation in NDUFA2 (c.134A>C, p.Lys45Thr). Review of a biorepository of patients with unsolved genetic leukoencephalopathies who underwent whole-exome or genome sequencing allowed us to identify a second patient with compound heterozygous mutations in NDUFA2 (c.134A>C, p.Lys45Thr; c.225del, p.Asn76Metfs*4). Only 1 other patient with mutations in NDUFA2 and a different phenotype (Leigh syndrome) has previously been reported. This is the first report of cystic leukoencephalopathy caused by mutations in NDUFA2