A CACNA1D mutation in a patient with persistent hyperinsulinaemic hypoglycaemia, heart defects, and severe hypotonia.

Congenital hyperinsulinaemic hypoglycaemia (HH) can occur in isolation or it may present as part of a wider syndrome. For approximately 40%-50% of individuals with this condition, sequence analysis of the known HH genes identifies a causative mutation. Identifying the underlying genetic aetiology in the remaining cases is important as a genetic diagnosis will inform on recurrence risk, may guide medical management and will provide valuable insights into β-cell physiology. We sequenced the exome of a child with persistent diazoxide-responsive HH, mild aortic insufficiency, severe hypotonia, and developmental delay as well as the unaffected parents. This analysis identified a de novo mutation, p.G403D, in the proband's CACNA1D gene. CACNA1D encodes the main L-type voltage-gated calcium channel in the pancreatic β-cell, a key component of the insulin secretion pathway. The p.G403D mutation had been reported previously as an activating mutation in an individual with primary hyper-aldosteronism, neuromuscular abnormalities, and transient hypoglycaemia. Sequence analysis of the CACNA1D gene in 60 further cases with HH did not identify a pathogenic mutation. Identification of an activating CACNA1D mutation in a second patient with congenital HH confirms the aetiological role of CACNA1D mutations in this disorder. A genetic diagnosis is important as treatment with a calcium channel blocker may be an option for the medical management of this patient

REVEL Is Better at Predicting Pathogenicity of Loss-of-Function than Gain-of-Function Variants

This is the final version. Available on open access from Hindawi via the DOI in this recordData Availability: The list of variants used in this study are included in Supplementary Table 1.In silico predictive tools can help determine the pathogenicity of variants. The 2015 American College of Medical Genetics and Genomics (ACMG) guidelines recommended that scores from these tools can be used as supporting evidence of pathogenicity. A subsequent publication by the ClinGen Sequence Variant Interpretation Working Group suggested that high scores from some tools were sufficiently predictive to be used as moderate or strong evidence of pathogenicity. REVEL is a widely used metapredictor that uses the scores of 13 individual in silico tools to calculate the pathogenicity of missense variants. Its ability to predict missense pathogenicity has been assessed extensively; however, no study has previously tested whether its performance is affected by whether the missense variant acts via a loss-of-function (LoF) or gain-of-function (GoF) mechanism. We used a highly curated dataset of 66 confirmed LoF and 65 confirmed GoF variants to evaluate whether this affected the performance of REVEL. 98% of LoF and 100% of GoF variants met the author-recommended REVEL threshold of 0.5 for pathogenicity, while 89% of LoF and 88% of GoF variants exceeded the 0.75 threshold. However, while 55% of LoF variants met the threshold recommended for a REVEL score to count as strong evidence of pathogenicity from the ACMG guidelines (0.932), only 35% of GoF variants met this threshold (). GoF variants are therefore less likely to receive the highest REVEL scores which would enable the REVEL score to be used as strong evidence of pathogenicity. This has implications for classification with the ACMG guidelines as GoF variants are less likely to meet the criteria for pathogenicity. P = 0.0352 ). GoF variants are therefore less likely to receive the highest REVEL scores which would enable the REVEL score to be used as strong evidence of pathogenicity. This has implications for classification with the ACMG guidelines as GoF variants are less likely to meet the criteria for pathogenicity.Wellcome TrustResearch EnglandNational Institute for Health and Care Research (NIHR

Pitfalls of haplotype phasing from amplicon-based long-read sequencing

This is the final version. Available on open access from Nature Research via the DOI in this recordThe long-read sequencers from Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT) offer the opportunity to phase mutations multiple kilobases apart directly from sequencing reads. In this study, we used long-range PCR with ONT and PacBio sequencing to phase two variants 9 kb apart in the RET gene. We also re-analysed data from a recent paper which had apparently successfully used ONT to phase clinically important haplotypes at the CYP2D6 and HLA loci. From these analyses, we demonstrate PCR-chimera formation during PCR amplification and reference alignment bias are pitfalls that need to be considered when attempting to phase variants using amplicon-based long-read sequencing technologies. These methodological pitfalls need to be avoided if the opportunities provided by long-read sequencers are to be fully exploited.Wellcome Trus

SavvyCNV: Genome-wide CNV calling from off-target reads

This is the uncorrected proof. The final version is available on open access from Public Library of Science via the DOI in this recordData Availability: The SavvyCNV tool and the code used to run the benchmarking comparisons are freely available on github. The tool is available at https://github.com/rdemolgen/SavvySuite. The code used to run the benchmarking comparisons is available at: https://github.com/exeter-matthew-wakeling/SavvyCNV_benchmarking. Our study uses the ICR96 data set for benchmarking, which is publicly available and can be accessed through the European-Genome phenome Archive (EGA) under the accession number EGAS00001002428. The dataset of 2591 samples referred to the molecular genetics department at the Royal Devon and Exeter Hospital for genetic testing cannot be shared due to patient confidentiality issues, as the genotype data could be used to identify individuals and so cannot be made openly available. Requests for access to the anonymised data by researchers will be considered following an application to the Genetic Beta Cell Research Bank (https://www.diabetesgenes.org/current-research/genetic-beta-cell-research-bank/) with proposals reviewed by the Genetic Data Access Committee.Identifying copy number variants (CNVs) can provide diagnoses to patients and provide important biological insights into human health and disease. Current exome and targeted sequencing approaches cannot detect clinically and biologically-relevant CNVs outside their target area. We present SavvyCNV, a tool which uses off-target read data from exome and targeted sequencing data to call germline CNVs genome-wide. Up to 70% of sequencing reads from exome and targeted sequencing fall outside the targeted regions. We have developed a new tool, SavvyCNV, to exploit this 'free data' to call CNVs across the genome. We benchmarked SavvyCNV against five state-of-the-art CNV callers using truth sets generated from genome sequencing data and Multiplex Ligation-dependent Probe Amplification assays. SavvyCNV called CNVs with high precision and recall, outperforming the five other tools at calling CNVs genome-wide, using off-target or on-target reads from targeted panel and exome sequencing. We then applied SavvyCNV to clinical samples sequenced using a targeted panel and were able to call previously undetected clinically-relevant CNVs, highlighting the utility of this tool within the diagnostic setting. SavvyCNV outperforms existing tools for calling CNVs from off-target reads. It can call CNVs genome-wide from targeted panel and exome data, increasing the utility and diagnostic yield of these tests. SavvyCNV is freely available at https://github.com/rdemolgen/SavvySuite.Medical Research Council (MRC)Research EnglandDiabetes U

The Role of ONECUT1 Variants in Monogenic and Type 2 Diabetes Mellitus

This is the author accepted manuscript. The final version is available from the American Diabetes Association via the DOI in this recordData and Resource Availability: The monogenic diabetes patient data used in this study are available from the corresponding author upon reasonable request. The type 2 diabetes data used in this study are available via UK Biobank (https://www.ukbiobank.ac.uk/), subject to necessary approvals.ONECUT1 (also known as HNF6) is a transcription factor involved in pancreatic development and beta-cell function. Recently, biallelic variants in ONECUT1 were reported as a cause of neonatal diabetes mellitus (NDM) in 2 subjects and missense monoallelic variants were associated with type 2 diabetes and possibly maturity-onset diabetes of the young (MODY). Here we examine the role of ONECUT1 variants in NDM, MODY and Type 2 diabetes in large international cohorts of subjects with monogenic diabetes and >400,000 subjects from UK Biobank. We identified a biallelic frameshift ONECUT1 variant as the cause of NDM in one individual. However, we found no enrichment of missense or null ONECUT1 variants among 484 individuals clinically suspected of MODY, in whom all known genes had been excluded. Finally, using a rare variant burden test in the UK Biobank European cohort, we identified a significant association between heterozygous ONECUT1 null variants and type 2 diabetes (P=0.006) but did not find association between missense variants and type 2 diabetes. Our results confirm biallelic ONECUT1 variants as a cause of NDM and highlight monoallelic null variants as a risk factor for type 2 diabetes. These findings confirm the critical role of ONECUT1 in human beta-cell function.Diabetes UKMedical Research Council (MRC)Wellcome TrustNational Institute for Health Research (NIHR

Uncovering treatment burden as a key concept for stroke care: a systematic review of qualitative research

<b>Background</b> Patients with chronic disease may experience complicated management plans requiring significant personal investment. This has been termed ‘treatment burden’ and has been associated with unfavourable outcomes. The aim of this systematic review is to examine the qualitative literature on treatment burden in stroke from the patient perspective.<p></p> <b>Methods and findings</b> The search strategy centred on: stroke, treatment burden, patient experience, and qualitative methods. We searched: Scopus, CINAHL, Embase, Medline, and PsycINFO. We tracked references, footnotes, and citations. Restrictions included: English language, date of publication January 2000 until February 2013. Two reviewers independently carried out the following: paper screening, data extraction, and data analysis. Data were analysed using framework synthesis, as informed by Normalization Process Theory. Sixty-nine papers were included. Treatment burden includes: (1) making sense of stroke management and planning care, (2) interacting with others, (3) enacting management strategies, and (4) reflecting on management. Health care is fragmented, with poor communication between patient and health care providers. Patients report inadequate information provision. Inpatient care is unsatisfactory, with a perceived lack of empathy from professionals and a shortage of stimulating activities on the ward. Discharge services are poorly coordinated, and accessing health and social care in the community is difficult. The study has potential limitations because it was restricted to studies published in English only and data from low-income countries were scarce.<p></p> <b>Conclusions</b> Stroke management is extremely demanding for patients, and treatment burden is influenced by micro and macro organisation of health services. Knowledge deficits mean patients are ill equipped to organise their care and develop coping strategies, making adherence less likely. There is a need to transform the approach to care provision so that services are configured to prioritise patient needs rather than those of health care systems

Non-coding variants disrupting a tissue-specific regulatory element in HK1 cause congenital hyperinsulinism.

This is the author accepted manuscript. The final version is available from Nature Research via the DOI in this recordData availability statement: All non‐clinical data analysed during this study are included in this published article (and its supplementary information files). Clinical and genotype data is available only through collaboration as this can be used to identify individuals and so cannot be made openly available. Requests for collaboration will be considered following an application to the Genetic Beta Cell Research Bank (https://www.diabetesgenes.org/current‐research/genetic‐ beta‐cell‐research‐bank/). Contact by email should be directed to the Corresponding author.Code availability statement: All code and software versions used specified in Methods.Gene expression is tightly regulated, with many genes exhibiting cell-specific silencing when their protein product would disrupt normal cellular function1. This silencing is largely controlled by non-coding elements, and their disruption might cause human disease2. We performed gene-agnostic screening of the non-coding regions to discover new molecular causes of congenital hyperinsulinism. This identified 14 non-coding de novo variants affecting a 42-bp conserved region encompassed by a regulatory element in intron 2 of the hexokinase 1 gene (HK1). HK1 is widely expressed across all tissues except in the liver and pancreatic beta cells and is thus termed a 'disallowed gene' in these specific tissues. We demonstrated that the variants result in a loss of repression of HK1 in pancreatic beta cells, thereby causing insulin secretion and congenital hyperinsulinism. Using epigenomic data accessed from public repositories, we demonstrated that these variants reside within a regulatory region that we determine to be critical for cell-specific silencing. Importantly, this has revealed a disease mechanism for non-coding variants that cause inappropriate expression of a disallowed gene.Wellcome Trus

Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes

Spatio-temporal dynamics of intracellular calcium, [Ca2+]i, regulate the contractile function of cardiac muscle cells. Measuring [Ca2+]i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca2+]i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca2+ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils.We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca2+]i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca2+]i transient and of the simulated fluorescence intensity signal, F/F0, reached values similar to that found in the literature ([Ca2+]i 1 μM; F/F0 5.5). However, our model predicted the variation in [Ca2+]i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F0 signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca2+]i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structureinduced heterogeneities in [Ca2+]i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca2+]i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes

Transverse tubule remodelling: a cellular pathology driven by both sides of the plasmalemma?

Transverse (t)-tubules are invaginations of the plasma membrane that form a complex network of ducts, 200–400 nm in diameter depending on the animal species, that penetrates deep within the cardiac myocyte, where they facilitate a fast and synchronous contraction across the entire cell volume. There is now a large body of evidence in animal models and humans demonstrating that pathological distortion of the t-tubule structure has a causative role in the loss of myocyte contractility that underpins many forms of heart failure. Investigations into the molecular mechanisms of pathological t-tubule remodelling to date have focused on proteins residing in the intracellular aspect of t-tubule membrane that form linkages between the membrane and myocyte cytoskeleton. In this review, we shed light on the mechanisms of t-tubule remodelling which are not limited to the intracellular side. Our recent data have demonstrated that collagen is an integral part of the t-tubule network and that it increases within the tubules in heart failure, suggesting that a fibrotic mechanism could drive cardiac junctional remodelling. We examine the evidence that the linkages between the extracellular matrix, t-tubule membrane and cellular cytoskeleton should be considered as a whole when investigating the mechanisms of t-tubule pathology in the failing heart