We present, on behalf of EuroGentest and the European Society of Human Genetics, guidelines for the evaluation and validation of next-generation sequencing (NGS) applications for the diagnosis of ...genetic disorders. The work was performed by a group of laboratory geneticists and bioinformaticians, and discussed with clinical geneticists, industry and patients' representatives, and other stakeholders in the field of human genetics. The statements that were written during the elaboration of the guidelines are presented here. The background document and full guidelines are available as supplementary material. They include many examples to assist the laboratories in the implementation of NGS and accreditation of this service. The work and ideas presented by others in guidelines that have emerged elsewhere in the course of the past few years were also considered and are acknowledged in the full text. Interestingly, a few new insights that have not been cited before have emerged during the preparation of the guidelines. The most important new feature is the presentation of a 'rating system' for NGS-based diagnostic tests. The guidelines and statements have been applauded by the genetic diagnostic community, and thus seem to be valuable for the harmonization and quality assurance of NGS diagnostics in Europe.
Cerebellar ataxia (CA) and hereditary spastic paraplegia (HSP) are two of the most prevalent motor disorders with extensive locus and allelic heterogeneity. We implemented clinical exome sequencing, ...followed by filtering data for a 'movement disorders' gene panel, as a generic test to increase variant detection in 76 patients with these disorders. Segregation analysis or phenotypic re-evaluation was utilized to substantiate findings. Disease-causing variants were identified in 9 of 28 CA patients, and 8 of 48 HSP patients. In addition, possibly disease-causing variants were identified in 1 and 8 of the remaining CA and HSP patients, respectively. In 10 patients with CA, the total disease-causing or possibly disease-causing variants were detected in 8 different genes, whereas 16 HSP patients had such variants in 12 different genes. In the majority of cases, the identified variants were compatible with the patient phenotype. Interestingly, in some patients variants were identified in genes hitherto related to other movement disorders, such as TH variants in two siblings with HSP. In addition, rare disorders were uncovered, for example, a second case of HSP caused by a VCP variant. For some patients, exome sequencing results had implications for treatment, exemplified by the favorable L-DOPA treatment in a patient with HSP due to ATP13A2 variants (Parkinson type 9). Thus, clinical exome sequencing in this cohort of CA and HSP patients suggests broadening of disease spectra, revealed novel gene-disease associations, and uncovered unanticipated rare disorders. In addition, clinical exome sequencing results have shown their value in guiding practical patient management.
Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with ...autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T p.Thr703Met) and one from the Netherlands (c.563A>C p.Asn188Thr). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.
Hearing impairment (HI) is genetically heterogeneous which hampers genetic counseling and molecular diagnosis. Testing of several single HI-related genes is laborious and expensive. In this study, we ...evaluate the diagnostic utility of whole-exome sequencing (WES) targeting a panel of HI-related genes. Two hundred index patients, mostly of Dutch origin, with presumed hereditary HI underwent WES followed by targeted analysis of an HI gene panel of 120 genes. We found causative variants underlying the HI in 67 of 200 patients (33.5%). Eight of these patients have a large homozygous deletion involving STRC, OTOA or USH2A, which could only be identified by copy number variation detection. Variants of uncertain significance were found in 10 patients (5.0%). In the remaining 123 cases, no potentially causative variants were detected (61.5%). In our patient cohort, causative variants in GJB2, USH2A, MYO15A and STRC, and in MYO6 were the leading causes for autosomal recessive and dominant HI, respectively. Segregation analysis and functional analyses of variants of uncertain significance will probably further increase the diagnostic yield of WES.
Myotonic dystrophy is an autosomal dominant, multisystem disorder that is characterized by myotonic myopathy. The symptoms and severity of myotonic dystrophy type l (DM1) ranges from severe and ...congenital forms, which frequently result in death because of respiratory deficiency, through to late-onset baldness and cataract. In adult patients, cardiac conduction abnormalities may occur and cause a shorter life span. In subsequent generations, the symptoms in DM1 may present at an earlier age and have a more severe course (anticipation). In myotonic dystrophy type 2 (DM2), no anticipation is described, but cardiac conduction abnormalities as in DM1 are observed and patients with DM2 additionally have muscle pain and stiffness. Both DM1 and DM2 are caused by unstable DNA repeats in untranslated regions of different genes: A (CTG)n repeat in the 3'-UTR of the DMPK gene and a (CCTG)n repeat in intron 1 of the CNBP (formerly ZNF9) gene, respectively. The length of the (CTG)n repeat expansion in DM1 correlates with disease severity and age of onset. Nevertheless, these repeat sizes have limited predictive values on individual bases. Because of the disease characteristics in DM1 and DM2, appropriate molecular testing and reporting is very important for the optimal counseling in myotonic dystrophy. Here, we describe best practice guidelines for clinical molecular genetic analysis and reporting in DM1 and DM2, including presymptomatic and prenatal testing.
We show that haploinsufficiency of KANSL1 is sufficient to cause the 17q21.31 microdeletion syndrome, a multisystem disorder characterized by intellectual disability, hypotonia and distinctive facial ...features. The KANSL1 protein is an evolutionarily conserved regulator of the chromatin modifier KAT8, which influences gene expression through histone H4 lysine 16 (H4K16) acetylation. RNA sequencing studies in cell lines derived from affected individuals and the presence of learning deficits in Drosophila melanogaster mutants suggest a role for KANSL1 in neuronal processes.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
In view of the population-specific heterogeneity in reported genetic risk factors for Parkinson's disease (PD), we conducted a genome-wide association study (GWAS) in a large sample of PD cases and ...controls from the Netherlands. After quality control (QC), a total of 514,799 SNPs genotyped in 772 PD cases and 2024 controls were included in our analyses. Direct replication of SNPs within SNCA and BST1 confirmed these two genes to be associated with PD in the Netherlands (SNCA, rs2736990: P = 1.63 × 10(-5), OR = 1.325 and BST1, rs12502586: P = 1.63 × 10(-3), OR = 1.337). Within SNCA, two independent signals in two different linkage disequilibrium (LD) blocks in the 3' and 5' ends of the gene were detected. Besides, post-hoc analysis confirmed GAK/DGKQ, HLA and MAPT as PD risk loci among the Dutch (GAK/DGKQ, rs2242235: P = 1.22 × 10(-4), OR = 1.51; HLA, rs4248166: P = 4.39 × 10(-5), OR = 1.36; and MAPT, rs3785880: P = 1.9 × 10(-3), OR = 1.19).
Spastic paraplegia type 7 is an autosomal recessive neurodegenerative disorder mainly characterized by progressive bilateral lower limb spasticity and referred to as a form of hereditary spastic ...paraplegia. Additional disease features may also be observed as part of a more complex phenotype. Many different mutations have already been identified, but no genotype-phenotype correlations have been found so far. From a total of almost 800 patients referred for testing, we identified 60 patients with mutations in the SPG7 gene. We identified 14 previously unreported mutations and detected a high recurrence rate of several earlier reported mutations. We were able to collect detailed clinical data for 49 patients, who were ranked based on a pure versus complex phenotype, ataxia versus no ataxia and missense versus null mutations. A generally complex phenotype occurred in 69% of all patients and was associated with a younger age at onset (trend with P = 0.07). Ataxia was observed in 57% of all patients. We found that null mutations were associated with the co-occurrence of cerebellar ataxia (trend with P = 0.06). The c.1409 G > A (p.Arg470Gln) mutation, which was found homozygously in two sibs, was associated with a specific complex phenotype that included predominant visual loss due to optical nerve atrophy. Neuropathology in one of these cases showed severe degeneration of the optic system, with less severe degeneration of the ascending tracts of the spinal cord and cerebellum. Other disease features encountered in this cohort included cervical dystonia, vertical gaze palsy, ptosis and severe intellectual disability. In this large Dutch cohort, we seem to have identified the first genotype-phenotype correlation in spastic paraplegia type 7 by observing an association between the cerebellar phenotype of spastic paraplegia type 7 and SPG7 null alleles. An overlapping phenotypic presentation with its biological counterpart AFG3L2, which when mutated causes spinocerebellar ataxia type 28, is apparent and possibly suggests that abnormal levels of the SPG7 protein impact the function of the mitochondrial ATPases associated with diverse cellular activities-protease complex (formed by SPG7 and AFG3L2) in the cerebellum. In addition, a missense mutation in exon 10 resulted in predominant optical nerve atrophy, which might suggest deleterious interactions of this SPG7 variant with its substrate OPA1, the mutated gene product in optic atrophy type 1. Functional studies are required to further investigate these interactions.
Revertant mosaicism has been reported in several inherited diseases, including the genetic skin fragility disorder epidermolysis bullosa (EB). Here, we describe the largest cohort of seven patients ...with revertant mosaicism and dystrophic EB (DEB), associated with mutations in the COL7A1 gene, and determine the underlying molecular mechanisms. We show that revertant mosaicism occurs both in autosomal dominantly and recessively inherited DEB. We found that null mutations resulting in complete loss of collagen VII and severe disease, as well as missense or splice-site mutations associated with some preserved collagen VII function and a milder phenotype, were corrected by revertant mosaicism. The mutation, subtype, and severity of the disease are thus not decisive for the presence of revertant mosaicism. Although collagen VII is synthesized and secreted by both keratinocytes and fibroblasts, evidence for reversion was only found in keratinocytes. The reversion mechanisms included back mutations/mitotic recombinations in 70% of the cases and second-site mutations affecting splicing in 30%. We conclude that revertant mosaicism is more common than previously assumed in patients with DEB, and our findings will have implications for future therapeutic strategies using the patient’s naturally corrected cells as a source for cell-based therapies.
Dystrophic epidermolysis bullosa (DEB) is a devastating blistering disease affecting skin and mucous membranes. It is caused by pathogenic variants in the COL7A1 gene encoding type VII collagen, and ...can be inherited dominantly or recessively. Recently, promising proof-of-principle has been shown for antisense oligonucleotide (AON)-mediated exon skipping as a therapeutic approach for DEB. However, the precise phenotypic effect to be anticipated from exon skipping, and which patient groups could benefit, is not yet clear. To answer these questions, we studied new clinical and molecular data on seven patients from the Dutch EB registry and reviewed the literature on COL7A1 exon skipping variants. We found that phenotypes associated with dominant exon skipping cannot be distinguished from phenotypes caused by other dominant DEB variants. Recessive exon skipping phenotypes are generally relatively mild in the spectrum of recessive DEB. Therefore, for dominant DEB, AON-mediated exon skipping is unlikely to ameliorate the phenotype. In contrast, the overall severity of phenotypes associated with recessive natural exon skipping pivots toward the milder end of the spectrum. Consequently, we anticipate AON-mediated exon skipping for recessive DEB caused by bi-allelic null variants should lead to a clinically relevant improvement of this devastating phenotype.
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