Use of whole genome sequencing to determine genetic basis of suspected mitochondrial disorders: cohort study

Funder: University of Cambridge; FundRef: http://dx.doi.org/10.13039/501100000735Funder: Alzheimer's Society; FundRef: http://dx.doi.org/10.13039/501100000320Funder: Leverhulme Trust; FundRef: http://dx.doi.org/10.13039/501100000275Funder: National Institute for Health Research; FundRef: http://dx.doi.org/10.13039/501100000272Funder: Department of Health; FundRef: http://dx.doi.org/10.13039/501100000276Funder: Evelyn Trust; FundRef: http://dx.doi.org/10.13039/501100004282Funder: Wellcome Trust; FundRef: http://dx.doi.org/10.13039/100004440Funder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265Abstract: Objective: To determine whether whole genome sequencing can be used to define the molecular basis of suspected mitochondrial disease. Design: Cohort study. Setting: National Health Service, England, including secondary and tertiary care. Participants: 345 patients with suspected mitochondrial disorders recruited to the 100 000 Genomes Project in England between 2015 and 2018. Intervention: Short read whole genome sequencing was performed. Nuclear variants were prioritised on the basis of gene panels chosen according to phenotypes, ClinVar pathogenic/likely pathogenic variants, and the top 10 prioritised variants from Exomiser. Mitochondrial DNA variants were called using an in-house pipeline and compared with a list of pathogenic variants. Copy number variants and short tandem repeats for 13 neurological disorders were also analysed. American College of Medical Genetics guidelines were followed for classification of variants. Main outcome measure: Definite or probable genetic diagnosis. Results: A definite or probable genetic diagnosis was identified in 98/319 (31%) families, with an additional 6 (2%) possible diagnoses. Fourteen of the diagnoses (4% of the 319 families) explained only part of the clinical features. A total of 95 different genes were implicated. Of 104 families given a diagnosis, 39 (38%) had a mitochondrial diagnosis and 65 (63%) had a non-mitochondrial diagnosis. Conclusion: Whole genome sequencing is a useful diagnostic test in patients with suspected mitochondrial disorders, yielding a diagnosis in a further 31% after exclusion of common causes. Most diagnoses were non-mitochondrial disorders and included developmental disorders with intellectual disability, epileptic encephalopathies, other metabolic disorders, cardiomyopathies, and leukodystrophies. These would have been missed if a targeted approach was taken, and some have specific treatments

Widespread genomic influences on phenotype in Dravet syndrome, a ‘monogenic’ condition

Dravet syndrome is an archetypal rare severe epilepsy, considered “monogenic”, typically caused by loss-of-function SCN1A variants. Despite a recognisable core phenotype, its marked phenotypic heterogeneity is incompletely explained by differences in the causal SCN1A variant or clinical factors. In 34 adults with SCN1A-related Dravet syndrome, we show additional genomic variation beyond SCN1A contributes to phenotype and its diversity, with an excess of rare variants in epilepsy-related genes as a set and examples of blended phenotypes, including one individual with an ultra-rare DEPDC5 variant and focal cortical dysplasia. Polygenic risk scores for intelligence are lower, and for longevity, higher, in Dravet syndrome than in epilepsy controls. The causal, major-effect, SCN1A variant may need to act against a broadly compromised genomic background to generate the full Dravet syndrome phenotype, whilst genomic resilience may help to ameliorate the risk of premature mortality in adult Dravet syndrome survivors

Spectrum of mutational signatures in T-cell lymphoma reveals a key role for UV radiation in cutaneous T-cell lymphoma

Funder: Galderma; doi: http://dx.doi.org/10.13039/501100009754Funder: NIHR-BRC Cambridge core grantFunder: National Institute for Health Research; doi: http://dx.doi.org/10.13039/501100000272Funder: NHS EnglandAbstract: T-cell non-Hodgkin’s lymphomas develop following transformation of tissue resident T-cells. We performed a meta-analysis of whole exome sequencing data from 403 patients with eight subtypes of T-cell non-Hodgkin’s lymphoma to identify mutational signatures and associated recurrent gene mutations. Signature 1, indicative of age-related deamination, was prevalent across all T-cell lymphomas, reflecting the derivation of these malignancies from memory T-cells. Adult T-cell leukemia-lymphoma was specifically associated with signature 17, which was found to correlate with the IRF4 K59R mutation that is exclusive to Adult T-cell leukemia-lymphoma. Signature 7, implicating UV exposure was uniquely identified in cutaneous T-cell lymphoma (CTCL), contributing 52% of the mutational burden in mycosis fungoides and 23% in Sezary syndrome. Importantly this UV signature was observed in CD4 + T-cells isolated from the blood of Sezary syndrome patients suggesting extensive re-circulation of these T-cells through skin and blood. Analysis of non-Hodgkin’s T-cell lymphoma cases submitted to the national 100,000 WGS project confirmed that signature 7 was only identified in CTCL strongly implicating UV radiation in the pathogenesis of cutaneous T-cell lymphoma

Functional Analysis of a Carotid Intima-Media Thickness Locus Implicates <i>BCAR1</i> and Suggests a Causal Variant

Background— Carotid intima-media thickness (IMT) is a marker of subclinical atherosclerosis that can predict cardiovascular disease events over traditional risk factors. This study examined the BCAR1-CFDP1-TMEM170A locus on chromosome 16, associated with carotid IMT and coronary artery disease in the IMT and IMT-Progression as Predictors of Vascular Events (IMPROVE) cohort, to identify the functional variant. Methods and Results— In analysis of the locus lead single nucleotide polymorphism (SNP; rs4888378, intronic in CFDP1 ) in Progressione della Lesione Intimale Carotidea (PLIC), the protective AA genotype was associated with slower IMT progression in women ( P =0.04) but not in men. Meta-analysis of 5 cohort studies also supported a protective effect of the A allele on common carotid IMT in women only (women: β=−0.0047, P =1.63×10 –4 ; men: β=−0.0029, P =0.0678). Two hundred fourteen noncoding variants in strong linkage disequilibrium ( r 2 ≥0.8) with rs4888378 were identified from 1000 Genome Project. ENCODE regulatory chromatin marks were used to create a shortlist of 6 possible regulatory variants. Electrophoretic mobility shift assays on the shortlist detected allele-specific protein binding to the lead SNP rs4888378; multiplexed competitor electrophoretic mobility shift assays implicated FOXA as the protein. Luciferase reporter assays on rs4888378 showed a significant 35% to 92% ( P =0.0057; P =4.0×10 –22 ) decrease in gene expression with the A allele. Expression quantitative trait loci analysis confirmed previously reported associations of rs4888378 with BCAR1 in vascular tissues. Conclusions— Molecular studies suggest the lead SNP as a potentially causal SNP at the BCAR1 - CFDP1 - TMEM170A locus, and expression quantitative trait loci studies implicate BCAR1 as the causal gene. This variant showed stronger effects on common carotid IMT in women, raising questions about the mechanism of the causal SNP on atherosclerosis. </jats:sec

Evaluating the performance of a clinical genome sequencing program for diagnosis of rare genetic disease, seen through the lens of craniosynostosis

PurposeGenome sequencing (GS) for diagnosis of rare genetic disease is being introduced into the clinic, but the complexity of the data poses challenges for developing pipelines with high diagnostic sensitivity. We evaluated the performance of the Genomics England 100,000 Genomes Project (100kGP) panel-based pipelines, using craniosynostosis as a test disease.MethodsGS data from 114 probands with craniosynostosis and their relatives (314 samples), negative on routine genetic testing, were scrutinized by a specialized research team, and diagnoses compared with those made by 100kGP.ResultsSixteen likely pathogenic/pathogenic variants were identified by 100kGP. Eighteen additional likely pathogenic/pathogenic variants were identified by the research team, indicating that for craniosynostosis, 100kGP panels had a diagnostic sensitivity of only 47%. Measures that could have augmented diagnoses were improved calling of existing panel genes (+18% sensitivity), review of updated panels (+12%), comprehensive analysis of de novo small variants (+29%), and copy-number/structural variants (+9%). Recent NHS England recommendations that partially incorporate these measures should achieve 85% overall sensitivity (+38%).ConclusionGS identified likely pathogenic/pathogenic variants in 29.8% of previously undiagnosed patients with craniosynostosis. This demonstrates the value of research analysis and the importance of continually improving algorithms to maximize the potential of clinical GS.</p

Correction: SMAD6 variants in craniosynostosis: genotype and phenotype evaluation (Genetics in Medicine, (2020), 10.1038/s41436-020-0817-2)

The version of the Article previously published did not acknowledge Freya Boardman-Pretty17,18 and the Genomics England Research Consortium in the author list. This has now been corrected in both the PDF and HTML versions of the Article

Certain heterozygous variants in the kinase domain of the serine/threonine kinase NEK8 can cause an autosomal dominant form of polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.</p

Whole genome sequencing for the diagnosis of neurological repeat expansion disorders in the UK: a retrospective diagnostic accuracy and prospective clinical validation study

Background: repeat expansion disorders affect about 1 in 3000 individuals and are clinically heterogeneous diseases caused by expansions of short tandem DNA repeats. Genetic testing is often locus-specific, resulting in underdiagnosis of people who have atypical clinical presentations, especially in paediatric patients without a previous positive family history. Whole genome sequencing is increasingly used as a first-line test for other rare genetic disorders, and we aimed to assess its performance in the diagnosis of patients with neurological repeat expansion disorders. Methods: we retrospectively assessed the diagnostic accuracy of whole genome sequencing to detect the most common repeat expansion loci associated with neurological outcomes (AR, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, C9orf72, CACNA1A, DMPK, FMR1, FXN, HTT, and TBP) using samples obtained within the National Health Service in England from patients who were suspected of having neurological disorders; previous PCR test results were used as the reference standard. The clinical accuracy of whole genome sequencing to detect repeat expansions was prospectively examined in previously genetically tested and undiagnosed patients recruited in 2013–17 to the 100 000 Genomes Project in the UK, who were suspected of having a genetic neurological disorder (familial or early-onset forms of ataxia, neuropathy, spastic paraplegia, dementia, motor neuron disease, parkinsonian movement disorders, intellectual disability, or neuromuscular disorders). If a repeat expansion call was made using whole genome sequencing, PCR was used to confirm the result. Findings: the diagnostic accuracy of whole genome sequencing to detect repeat expansions was evaluated against 793 PCR tests previously performed within the NHS from 404 patients. Whole genome sequencing correctly classified 215 of 221 expanded alleles and 1316 of 1321 non-expanded alleles, showing 97·3% sensitivity (95% CI 94·2–99·0) and 99·6% specificity (99·1–99·9) across the 13 disease-associated loci when compared with PCR test results. In samples from 11 631 patients in the 100 000 Genomes Project, whole genome sequencing identified 81 repeat expansions, which were also tested by PCR: 68 were confirmed as repeat expansions in the full pathogenic range, 11 were non-pathogenic intermediate expansions or premutations, and two were non-expanded repeats (16% false discovery rate). Interpretation: In our study, whole genome sequencing for the detection of repeat expansions showed high sensitivity and specificity, and it led to identification of neurological repeat expansion disorders in previously undiagnosed patients. These findings support implementation of whole genome sequencing in clinical laboratories for diagnosis of patients who have a neurological presentation consistent with a repeat expansion disorder. Funding: Medical Research Council, Department of Health and Social Care, National Health Service England, National Institute for Health Research, and Illumina.</p

Sex, Genotype, and Liver Volume Progression as Risk of Hospitalization Determinants in Autosomal Dominant Polycystic Liver Disease

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