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Abstract Childhood glaucoma (CG) encompasses a heterogeneous group of genetic eye disorders that is responsible for approximately 5% of childhood blindness worldwide. Understanding the molecular aetiology is key to improving diagnosis, prognosis and unlocking the potential for optimising clinical management. In this study, we investigated 86 CG cases from 78 unrelated families of diverse ethnic backgrounds, recruited into the Genomics England 100,000 Genomes Project (GE100KGP) rare disease cohort, to improve the genetic diagnostic yield. Using the Genomics England/Genomic Medicine Centres (GE/GMC) diagnostic pipeline, 13 unrelated families were solved (13/78, 17%). Further interrogation using an expanded gene panel yielded a molecular diagnosis in 7 more unrelated families (7/78, 9%). This analysis effectively raises the total number of solved CG families in the GE100KGP to 26% (20/78 families). Twenty-five percent (5/20) of the solved families had primary congenital glaucoma (PCG), while 75% (15/20) had secondary CG; 53% of this group had non-acquired ocular anomalies (including iris hypoplasia, megalocornea, ectopia pupillae, retinal dystrophy, and refractive errors) and 47% had non-acquired systemic diseases such as cardiac abnormalities, hearing impairment, and developmental delay. CYP1B1 was the most frequently implicated gene, accounting for 55% (11/20) of the solved families. We identified two novel likely pathogenic variants in the TEK gene, in addition to one novel pathogenic copy number variant (CNV) in FOXC1. Variants that passed undetected in the GE100KGP diagnostic pipeline were likely due to limitations of the tiering process, the use of smaller gene panels during analysis, and the prioritisation of coding SNVs and indels over larger structural variants, CNVs, and non-coding variants.

Abstract Cross-species genome comparisons have revealed a substantial number of ultraconserved non-coding elements (UCNEs). Several of these elements have proved to be essential tissue- and cell type-specific cis-regulators of developmental gene expression. Here, we characterize a set of UCNEs as candidate CREs (cCREs) during retinal development and evaluate the contribution of their genomic variation to rare eye diseases, for which pathogenic non-coding variants are emerging. Integration of bulk and single-cell retinal multi-omics data reveals 594 genes under potential cis-regulatory control of UCNEs, of which 45 are implicated in rare eye disease. Mining of candidate cis-regulatory UCNEs in WGS data derived from the rare eye disease cohort of Genomics England reveals 178 ultrarare variants within 84 UCNEs associated with 29 disease genes. Overall, we provide a comprehensive annotation of ultraconserved non-coding regions acting as cCREs during retinal development which can be targets of non-coding variation underlying rare eye diseases.

Abstract Background 5’ untranslated regions (5’UTRs) are essential modulators of protein translation. Predicting the impact of 5’UTR variants is challenging and rarely performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and functional assays to evaluate 5’UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). Methods We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of their 5’UTRs by different whole exome sequencing (WES) kits. The selected 5’UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2397 WGS) and an in-house database (n = 1682 WES), respectively. Identified variants were annotated for 5’UTR-relevant features and classified into seven categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse approaches. Results Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5’UTR compared to that of their canonical isoform. Depending on the probe design, 3–20% of IRD genes have 5’UTRs fully captured by WES. After analyzing these regions in both cohorts, we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 7 were novel. Functional analyses further supported the pathogenicity of three variants. Mis-splicing was demonstrated for the PRPF31:c.-9+1G>T variant. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5’UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. Conclusions This study demonstrates the importance of 5’UTR variants implicated in IRDs and provides a systematic approach for 5’UTR annotation and validation that is applicable to other inherited diseases.

Abstract Background The majority of cases of Dent’s disease are caused by pathogenic variants in the CLCN5 gene, which encodes a voltage-gated chloride ion channel (ClC-5), resulting in proximal tubular dysfunction. We present three members of the same family and one unrelated paediatric patient with the same insertion-deletion CLCN5 variant. The identification of these patients and positive familial segregation led to the re-classification of this variant from one of unknown significance to one of likely pathogenicity. Case presentation A 41 year old male presented with end stage kidney failure, proteinuria and haematuria. Whole genome sequencing identified an insertion-deletion variant in CLCN5, resulting in a missense change (c.1744_1745delinsAA p.(Ala582Lys)). His brother and nephew, who both exhibited renal impairment, haematuria, proteinuria, glycosuria and nephrocalcinosis, were found to have the same variant. In addition, genetic testing of an unrelated paediatric patient who presented with proteinuria and hypercalciuria, demonstrated the same variant. Conclusions The identification of this novel variant in four individuals with features of Dent’s disease, has led to the re-classification of the variant to one of likely pathogenicity. As a result, our patients and any future patients with the same variant can be offered a likely diagnosis, without the need for kidney biopsy, and their family members can be offered genetic screening.

Abstract Background Chronic myeloid leukaemia (CML) is one of the most well characterised human malignancies. Most patients have a cytogenetically visible translocation between chromosomes 9 and 22 which generates the pathognomonic BCR::ABL1 fusion gene. The derivative chromosome 22 (‘Philadelphia’ or Ph chromosome) usually harbours the fusion gene encoding a constitutively active ABL1 kinase domain. A small subset of patients have no visible translocation. Historically, these ‘Philadelphia chromosome negative’ patients caused diagnostic confusion between CML and other myeloproliferative neoplasms; it is now well established that the BCR::ABL1 fusion gene can be generated via submicroscopic intrachromosomal insertion of ABL1 sequence into BCR, or, more rarely, of BCR into ABL1. The fusion genes arising from cryptic insertions are not detectable via G-banded chromosome analysis [karyotype] but can nevertheless always be detected using fluorescence in situ hybridisation (FISH) and/or qualitative reverse transcriptase PCR. Case presentation A 43-year-old female presented with suspected CML in 2007; however, contemporaneous gold standard laboratory investigations, G-banded chromosome analysis and FISH, were both negative. The reverse transcriptase quantitative PCR (RT-qPCR) assay available at the time, which was capable of detecting the common BCR::ABL1 transcripts (e13a2/e14a2), was also negative. Upon review in 2009, the newly recommended reverse transcriptase multiplex PCR (capable of detecting all BCR::ABL1 transcripts including the atypical ones) subsequently detected an e19a2 fusion. The patient then responded to tyrosine kinase inhibitor therapy. In contrast, FISH studies of both samples with three commercially available probes remained consistently negative. Retrospective whole genome sequencing, undertaken as part of the 100,000 Genomes Project, has now revealed that the patient’s BCR::ABL1 fusion gene arose via a uniquely small insertion of 122 kb ABL1 sequences into BCR. Conclusions We present a patient with suspected chronic myeloid leukaemia whose genetic investigations were originally negative at the time of diagnosis despite the use of contemporaneous gold standard methods. This is the first report of a FISH-negative, BCR::ABL1 positive CML which demonstrates that, even after sixty years of research into one of the most well understood human malignancies, whole genome sequencing can yield novel diagnostic findings in CML.

Abstract Background Cancer genome sequencing enables accurate classification of tumours and tumour subtypes. However, prediction performance is still limited using exome-only sequencing and for tumour types with low somatic mutation burden such as many paediatric tumours. Moreover, the ability to leverage deep representation learning in discovery of tumour entities remains unknown. Methods We introduce here Mutation-Attention (MuAt), a deep neural network to learn representations of simple and complex somatic alterations for prediction of tumour types and subtypes. In contrast to many previous methods, MuAt utilizes the attention mechanism on individual mutations instead of aggregated mutation counts. Results We trained MuAt models on 2587 whole cancer genomes (24 tumour types) from the Pan-Cancer Analysis of Whole Genomes (PCAWG) and 7352 cancer exomes (20 types) from the Cancer Genome Atlas (TCGA). MuAt achieved prediction accuracy of 89% for whole genomes and 64% for whole exomes, and a top-5 accuracy of 97% and 90%, respectively. MuAt models were found to be well-calibrated and perform well in three independent whole cancer genome cohorts with 10,361 tumours in total. We show MuAt to be able to learn clinically and biologically relevant tumour entities including acral melanoma, SHH-activated medulloblastoma, SPOP-associated prostate cancer, microsatellite instability, POLE proofreading deficiency, and MUTYH-associated pancreatic endocrine tumours without these tumour subtypes and subgroups being provided as training labels. Finally, scrunity of MuAt attention matrices revealed both ubiquitous and tumour-type specific patterns of simple and complex somatic mutations. Conclusions Integrated representations of somatic alterations learnt by MuAt were able to accurately identify histological tumour types and identify tumour entities, with potential to impact precision cancer medicine.

Abstract Background Cardiomyopathies are a leading cause of progressive heart failure and sudden cardiac death; however, their genetic aetiology remains poorly understood. We hypothesised that variants in noncoding regulatory regions and oligogenic inheritance mechanisms may help close the diagnostic gap. Methods We first analysed whole-genome sequencing data of 143 parent–offspring trios from Genomics England 100,000 Genomes Project. We used gene panel testing and a phenotype-based, variant prioritisation framework called Exomiser to identify candidate genes in trios. To assess the contribution of noncoding DNVs to cardiomyopathies, we intersected DNVs with open chromatin sequences from single-cell ATAC-seq data of cardiomyocytes. We also performed a case–control analysis in an exome-negative cohort, including 843 probands and 19,467 controls, to assess the association between noncoding variants in known cardiomyopathy genes and disease. Results In the trio analysis, a definite or probable genetic diagnosis was identified in 21 probands according to the American College of Medical Genetics guidelines. We identified novel DNVs in diagnostic-grade genes (RYR2, TNNT2, PTPN11, MYH7, LZR1, NKX2-5), and five cases harbouring a combination of prioritised variants, suggesting that oligogenic inheritance and genetic modifiers contribute to cardiomyopathies. Phenotype-based ranking of candidate genes identified in noncoding DNV analysis revealed JPH2 as the top candidate. Moreover, a case–control analysis revealed an enrichment of rare noncoding variants in regulatory elements of cardiomyopathy genes (p = .035, OR = 1.43, 95% Cl = 1.095–1.767) versus controls. Of the 25 variants associated with disease (p< 0.5), 23 are novel and nine are predicted to disrupt transcription factor binding motifs. Conclusion Our results highlight complex genetic mechanisms in cardiomyopathies and reveal novel genes for future investigations.

Whole genome sequencing is emerging as a first-line test for rare genetic diseases. In this study, authors maximise diagnoses by supplementing existing semiautomated analyses with clinically driven reevaluation of genomic data by a specialist multidisciplinary team.

Motile and non-motile cilia have distinct functions and protein complexes associated with them. Here, the authors show the conserved protein CFAP20 is important for both motile and non-motile cilia and is distinct from other ciliopathy-associated domains or macromolecular complexes.

Abstract The aim of this study was to investigate coenzyme Q10 (CoQ10) biosynthesis pathway defects in inherited retinal dystrophy. Individuals affected by inherited retinal dystrophy (IRD) underwent exome or genome sequencing for molecular diagnosis of their condition. Following negative IRD gene panel analysis, patients carrying biallelic variants in CoQ10 biosynthesis pathway genes were identified. Clinical data were collected from the medical records. Haplotypes harbouring the same missense variant were characterised from family genome sequencing (GS) data and direct Sanger sequencing. Candidate splice variants were characterised using Oxford Nanopore Technologies single molecule sequencing. The CoQ10 status of the human plasma was determined in some of the study patients. 13 individuals from 12 unrelated families harboured candidate pathogenic genotypes in the genes: PDSS1, COQ2, COQ4 and COQ5. The PDSS1 variant c.589 A > G was identified in three affected individuals from three unrelated families on a possible ancestral haplotype. Three variants (PDSS1 c.468-25 A > G, PDSS1 c.722-2 A > G, COQ5 c.682-7 T > G) were shown to lead to cryptic splicing. 6 affected individuals were diagnosed with non-syndromic retinitis pigmentosa and 7 had additional clinical findings. This study provides evidence of CoQ10 biosynthesis pathway gene defects leading to non-syndromic retinitis pigmentosa in some cases. Intronic variants outside of the canonical splice-sites represent an important cause of disease. RT-PCR nanopore sequencing is effective in characterising these splice defects.

Abstract Background The diagnostic rate of Mendelian disorders in sequencing studies continues to increase, along with the pace of novel disease gene discovery. However, variant interpretation in novel genes not currently associated with disease is particularly challenging and strategies combining gene functional evidence with approaches that evaluate the phenotypic similarities between patients and model organisms have proven successful. A full spectrum of intolerance to loss-of-function variation has been previously described, providing evidence that gene essentiality should not be considered as a simple and fixed binary property. Methods Here we further dissected this spectrum by assessing the embryonic stage at which homozygous loss-of-function results in lethality in mice from the International Mouse Phenotyping Consortium, classifying the set of lethal genes into one of three windows of lethality: early, mid, or late gestation lethal. We studied the correlation between these windows of lethality and various gene features including expression across development, paralogy and constraint metrics together with human disease phenotypes. We explored a gene similarity approach for novel gene discovery and investigated unsolved cases from the 100,000 Genomes Project. Results We found that genes in the early gestation lethal category have distinct characteristics and are enriched for genes linked with recessive forms of inherited metabolic disease. We identified several genes sharing multiple features with known biallelic forms of inborn errors of the metabolism and found signs of enrichment of biallelic predicted pathogenic variants among early gestation lethal genes in patients recruited under this disease category. We highlight two novel gene candidates with phenotypic overlap between the patients and the mouse knockouts. Conclusions Information on the developmental period at which embryonic lethality occurs in the knockout mouse may be used for novel disease gene discovery that helps to prioritise variants in unsolved rare disease cases.

Investigation of variation in three cohorts has identified multiple genetic signals associated with the pregnancy-specific liver disorder, intrahepatic cholestasis of pregnancy, giving insight into the disease which can cause preterm birth and stillbirth.

Abstract Background Expansions of short tandem repeats are the cause of many neurogenetic disorders including familial amyotrophic lateral sclerosis, Huntington disease, and many others. Multiple methods have been recently developed that can identify repeat expansions in whole genome or exome sequencing data. Despite the widely recognized need for visual assessment of variant calls in clinical settings, current computational tools lack the ability to produce such visualizations for repeat expansions. Expanded repeats are difficult to visualize because they correspond to large insertions relative to the reference genome and involve many misaligning and ambiguously aligning reads. Results We implemented REViewer, a computational method for visualization of sequencing data in genomic regions containing long repeat expansions and FlipBook, a companion image viewer designed for manual curation of large collections of REViewer images. To generate a read pileup, REViewer reconstructs local haplotype sequences and distributes reads to these haplotypes in a way that is most consistent with the fragment lengths and evenness of read coverage. To create appropriate training materials for onboarding new users, we performed a concordance study involving 12 scientists involved in short tandem repeat research. We used the results of this study to create a user guide that describes the basic principles of using REViewer as well as a guide to the typical features of read pileups that correspond to low confidence repeat genotype calls. Additionally, we demonstrated that REViewer can be used to annotate clinically relevant repeat interruptions by comparing visual assessment results of 44 FMR1 repeat alleles with the results of triplet repeat primed PCR. For 38 of these alleles, the results of visual assessment were consistent with triplet repeat primed PCR. Conclusions Read pileup plots generated by REViewer offer an intuitive way to visualize sequencing data in regions containing long repeat expansions. Laboratories can use REViewer and FlipBook to assess the quality of repeat genotype calls as well as to visually detect interruptions or other imperfections in the repeat sequence and the surrounding flanking regions. REViewer and FlipBook are available under open-source licenses at https://github.com/illumina/REViewer and https://github.com/broadinstitute/flipbook respectively.

Abstract Background Central conventional chondrosarcoma (CS) is the most common subtype of primary malignant bone tumour in adults. Treatment options are usually limited to surgery, and prognosis is challenging. These tumours are characterised by the presence and absence of IDH1 and IDH2 mutations, and recently, TERT promoter alterations have been reported in around 20% of cases. The effect of these mutations on clinical outcome remains unclear. The purpose of this study was to determine if prognostic accuracy can be improved by the addition of genomic data, and specifically by examination of IDH1, IDH2, and TERT mutations. Methods In this study, we combined both archival samples and data sourced from the Genomics England 100,000 Genomes Project (n = 356). Mutations in IDH1, IDH2, and TERT were profiled using digital droplet PCR (n = 346), whole genome sequencing (n=68), or both (n = 64). Complex events and other genetic features were also examined, along with methylation array data (n = 84). We correlated clinical features and patient outcomes with our genetic findings. Results IDH2-mutant tumours occur in older patients and commonly present with high-grade or dedifferentiated disease. Notably, TERT mutations occur most frequently in IDH2-mutant tumours, although have no effect on survival in this group. In contrast, TERT mutations are rarer in IDH1-mutant tumours, yet they are associated with a less favourable outcome in this group. We also found that methylation profiles distinguish IDH1- from IDH2-mutant tumours. IDH wild-type tumours rarely exhibit TERT mutations and tend to be diagnosed in a younger population than those with tumours harbouring IDH1 and IDH2 mutations. A major genetic feature of this group is haploidisation and subsequent genome doubling. These tumours evolve less frequently to dedifferentiated disease and therefore constitute a lower risk group. Conclusions Tumours with IDH1 or IDH2 mutations or those that are IDHwt have significantly different genetic pathways and outcomes in relation to TERT mutation. Diagnostic testing for IDH1, IDH2, and TERT mutations could therefore help to guide clinical monitoring and prognostication.

Abstract Background Genomic variants which disrupt splicing are a major cause of rare genetic diseases. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. Improving the clinical interpretation of non-canonical splicing variants offers a major opportunity to uplift diagnostic yields from whole genome sequencing data. Methods Here, we examine the landscape of splicing variants in whole-genome sequencing data from 38,688 individuals in the 100,000 Genomes Project and assess the contribution of non-canonical splicing variants to rare genetic diseases. We use a variant-level constraint metric (the mutability-adjusted proportion of singletons) to identify constrained functional variant classes near exon–intron junctions and at putative splicing branchpoints. To identify new diagnoses for individuals with unsolved rare diseases in the 100,000 Genomes Project, we identified individuals with de novo single-nucleotide variants near exon–intron boundaries and at putative splicing branchpoints in known disease genes. We identified candidate diagnostic variants through manual phenotype matching and confirmed new molecular diagnoses through clinical variant interpretation and functional RNA studies. Results We show that near-splice positions and splicing branchpoints are highly constrained by purifying selection and harbour potentially damaging non-coding variants which are amenable to systematic analysis in sequencing data. From 258 de novo splicing variants in known rare disease genes, we identify 35 new likely diagnoses in probands with an unsolved rare disease. To date, we have confirmed a new diagnosis for six individuals, including four in whom RNA studies were performed. Conclusions Overall, we demonstrate the clinical value of examining non-canonical splicing variants in individuals with unsolved rare diseases.

Abstract X-linked Alport syndrome is a genetic kidney disease caused by pathogenic COL4A5 variants, but little is known of the consequences of missense variants affecting the NC1 domain of the corresponding collagen IV α5 chain. This study examined these variants in a normal (gnomAD) and other databases (LOVD, Clin Var and 100,000 Genomes Project) to determine their pathogenicity and clinical significance. Males with Cys substitutions in the collagen IV α5 NC1 domain reported in LOVD (n = 25) were examined for typical Alport features, including age at kidney failure. All NC1 variants in LOVD (n = 86) were then assessed for structural damage using an online computational tool, Missense3D. Variants in the ClinVar, gnomAD and 100,000 Genomes Project databases were also examined for structural effects. Predicted damage associated with NC1 substitutions was then correlated with the level of conservation of the affected residues. Cys substitutions in males were associated with the typical features of X-linked Alport syndrome, with a median age at kidney failure of 31 years. NC1 substitutions predicted to cause structural damage were overrepresented in LOVD (p

Abstract Topoisomerase 3α (TOP3A) is an enzyme that removes torsional strain and interlinks between DNA molecules. TOP3A localises to both the nucleus and mitochondria, with the two isoforms playing specialised roles in DNA recombination and replication respectively. Pathogenic variants in TOP3A can cause a disorder similar to Bloom syndrome, which results from bi‐allelic pathogenic variants in BLM, encoding a nuclear‐binding partner of TOP3A. In this work, we describe 11 individuals from 9 families with an adult‐onset mitochondrial disease resulting from bi‐allelic TOP3A gene variants. The majority of patients have a consistent clinical phenotype characterised by bilateral ptosis, ophthalmoplegia, myopathy and axonal sensory‐motor neuropathy. We present a comprehensive characterisation of the effect of TOP3A variants, from individuals with mitochondrial disease and Bloom‐like syndrome, upon mtDNA maintenance and different aspects of enzyme function. Based on these results, we suggest a model whereby the overall severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants causing adult‐onset mitochondrial disease and more severe variants causing a Bloom‐like syndrome with mitochondrial dysfunction in childhood.

Abstract Background Several common conditions have been widely recognised as risk factors for COVID-19 related death, but risks borne by people with rare diseases are largely unknown. Therefore, we aim to estimate the difference of risk for people with rare diseases comparing to the unaffected. Method To estimate the correlation between rare diseases and COVID-19 related death, we performed a retrospective cohort study in Genomics England 100k Genomes participants, who tested positive for Sars-Cov-2 during the first wave (16-03-2020 until 31-July-2020) of COVID-19 pandemic in the UK (n = 283). COVID-19 related mortality rates were calculated in two groups: rare disease patients (n = 158) and unaffected relatives (n = 125). Fisher’s exact test and logistic regression was used for univariable and multivariable analysis, respectively. Results People with rare diseases had increased risk of COVID19-related deaths compared to the unaffected relatives (OR [95% CI] = 3.47 [1.21– 12.2]). Although, the effect was insignificant after adjusting for age and number of comorbidities (OR [95% CI] = 1.94 [0.65–5.80]). Neurology and neurodevelopmental diseases was significantly associated with COVID19-related death in both univariable (OR [95% CI] = 4.07 [1.61–10.38]) and multivariable analysis (OR [95% CI] = 4.22 [1.60–11.08]). Conclusions Our results showed that rare disease patients, especially ones affected by neurology and neurodevelopmental disorders, in the Genomics England cohort had increased risk of COVID-19 related death during the first wave of the pandemic in UK. The high risk is likely associated with rare diseases themselves, while we cannot rule out possible mediators due to the small sample size. We would like to raise the awareness that rare disease patients may face increased risk for COVID-19 related death. Proper considerations for rare disease patients should be taken when relevant policies (e.g., returning to workplace) are made.

Rare genetic disorders represent some of the most severe and life-limiting conditions that constitute a considerable burden on global healthcare systems and societies. Most individuals affected by rare disorders remain undiagnosed, highlighting the unmet need for improved disease gene discovery and novel variant interpretation. Aberrant (de) phosphorylation can have profound pathological consequences underpinning many disease processes. Numerous phosphatases and associated proteins have been identified as disease genes, with many more likely to have gone undiscovered thus far. To begin to address these issues, we have performed a systematic survey of de novo variants amongst 189 genes encoding phosphatase catalytic subunits found in rare disease patients recruited to the 100,000 Genomes Project (100 kGP), the largest national sequencing project of its kind in the United Kingdom. We found that 49% of phosphatases were found to carry de novo mutation(s) in this cohort. Only 25% of these phosphatases have been previously linked to genetic disorders. A gene-to-patient approach matching variants to phenotypic data identified 9 novel candidate rare-disease genes: PTPRD, PTPRG, PTPRT, PTPRU, PTPRZ1, MTMR3, GAK, TPTE2, PTPN18. As the number of patients undergoing whole genome sequencing increases and information sharing improves, we anticipate that reiterative analysis of genomic and phenotypic data will continue to identify candidate phosphatase disease genes for functional validation. This is the first step towards delineating the aetiology of rare genetic disorders associated with altered phosphatase function, leading to new biological insights and improved clinical outcomes for the affected individuals and their families.

Abstract Cardiomyopathy (CMP) is a heritable disorder. Over 50% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 × 10−7 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7–58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP.

Abstract Alport syndrome is the commonest inherited kidney disease and nearly half the pathogenic variants in the COL4A3–COL4A5 genes that cause Alport syndrome result in Gly substitutions. This study examined the molecular characteristics of Gly substitutions that determine the severity of clinical features. Pathogenic COL4A5 variants affecting Gly in the Leiden Open Variation Database in males with X-linked Alport syndrome were correlated with age at kidney failure (n = 157) and hearing loss diagnosis (n = 80). Heterozygous pathogenic COL4A3 and COL4A4 variants affecting Gly (n = 304) in autosomal dominant Alport syndrome were correlated with the risk of haematuria in the UK 100,000 Genomes Project. Gly substitutions were stratified by exon location (1 to 20 or 21 to carboxyl terminus), being adjacent to a non-collagenous region (interruption or terminus), and the degree of instability caused by the replacement residue. Pathogenic COL4A5 variants that resulted in a Gly substitution with a highly destabilising residue reduced the median age at kidney failure by 7 years (p = 0.002), and age at hearing loss diagnosis by 21 years (p = 0.004). Substitutions adjacent to a non-collagenous region delayed kidney failure by 19 years (p = 0.014). Heterozygous pathogenic COL4A3 and COL4A4 variants that resulted in a Gly substitution with a highly destabilising residue (Arg, Val, Glu, Asp, Trp) were associated with an increased risk of haematuria (p = 0.018), and those adjacent to a non-collagenous region were associated with a reduced risk (p = 0.046). Exon location had no effect. In addition, COL4A5 variants adjacent to non-collagenous regions were over-represented in the normal population in gnomAD (p

Abstract We report new genetic diagnoses of Dravet syndrome in a group of adults with complex epilepsy of unknown cause, under follow‐up at a tertiary epilepsy center. Individuals with epilepsy and other features of unknown cause from our unit underwent whole‐genome sequencing through the 100 000 Genomes Project. Virtual gene panels were applied to frequency‐filtered variants based on phenotype summary. Of 1078 individuals recruited, 8 (0.74%) were identified to have a pathogenic or likely pathogenic variant in SCN1A. Variant types were as follows: nonsense (stopgain) in five (62.5%) and missense in three (37.5%). Detailed review of childhood history confirmed a phenotype compatible with Dravet syndrome. Median age at genetic diagnosis was 44.5 years (range 28‐52 years). Tonic‐clonic seizures were ongoing in all despite polytherapy including valproate. All had a history of fever sensitivity and myoclonic seizures, which were ongoing in two (25%) and three (37.5%) individuals, respectively. Salient features of Dravet syndrome may be less apparent in adulthood, making clinical diagnosis difficult. Regardless of age, benefits of a genetic diagnosis include access to syndrome‐specific treatment options, avoidance of harmful drugs, and monitoring for common complications.

Chondrosarcoma (CS) is a rare tumour type and the most common primary malignant bone cancer in adults. The prognosis, currently based on tumour grade, imaging and anatomical location, is not reliable, and more objective biomarkers are required. We aimed to determine whether the level of circulating tumour DNA (ctDNA) in the blood of CS patients could be used to predict outcome. In this multi‐institutional study, we recruited 145 patients with cartilaginous tumours, of which 41 were excluded. ctDNA levels were assessed in 83 of the remaining 104 patients, whose tumours harboured a hotspot mutation in IDH1/2 or GNAS. ctDNA was detected pre‐operatively in 31/83 (37%) and in 12/31 (39%) patients postoperatively. We found that detection of ctDNA was more accurate than pathology for identification of high‐grade tumours and was associated with a poor prognosis; ctDNA was never associated with CS grade 1/atypical cartilaginous tumours (ACT) in the long bones, in neoplasms sited in the small bones of the hands and feet or in tumours measuring less than 80 mm. Although the results are promising, they are based on a small number of patients, and therefore, introduction of this blood test into clinical practice as a complementary assay to current standard‐of‐care protocols would allow the assay to be assessed more stringently and developed for a more personalised approach for the treatment of patients with CS.

Abstract Multiple myeloma is characterized by chromosomal abnormalities and genetic variation, which may inform prognosis and guide treatment. This pilot study sought to examine the feasibility of incorporating Whole Genome Sequencing (WGS) alongside the routine laboratory evaluation of 14 patients with newly diagnosed multiple myeloma who had enrolled in the 100,000 Genomes Project. In all 14 cases, WGS data could be obtained in a timely fashion within existing clinical frameworks in a tertiary hospital setting. The data not only replicated standard‐of‐care FISH analysis of chromosomal abnormalities but also provided further chromosomal and molecular genetic insights that may influence patient management.

Abstract Objective The objective of this study was to employ ensemble clustering and tree-based risk model approaches to identify interactions between clinicogenomic features for colorectal cancer using the 100,000 Genomes Project. Results Among the 2211 patients with colorectal cancer (mean age of diagnosis: 67.7; 59.7% male), 16.3%, 36.3%, 39.0% and 8.4% had stage 1, 2, 3 and 4 cancers, respectively. Almost every patient had surgery (99.7%), 47.4% had chemotherapy, 7.6% had radiotherapy and 1.4% had immunotherapy. On average, tumour mutational burden (TMB) was 18 mutations/Mb and 34.4%, 31.3% and 25.7% of patients had structural or copy number mutations in KRAS, BRAF and NRAS, respectively. In the fully adjusted Cox model, patients with advanced cancer [stage 3 hazard ratio (HR) = 3.2; p < 0.001; stage 4 HR = 10.2; p < 0.001] and those who had immunotherapy (HR = 1.8; p < 0.04) or radiotherapy (HR = 1.5; p < 0.02) treatment had a higher risk of dying. The ensemble clustering approach generated four distinct clusters where patients in cluster 2 had the best survival outcomes (1-year: 98.7%; 2-year: 96.7%; 3-year: 93.0%) while patients in cluster 3 (1-year: 87.9; 2-year: 70.0%; 3-year: 53.1%) had the worst outcomes. Kaplan–Meier analysis and log rank test revealed that the clusters were separated into distinct prognostic groups (p

Abstract The development of computational methods to assess pathogenicity of pre-messenger RNA splicing variants is critical for diagnosis of human disease. We assessed the capability of eight algorithms, and a consensus approach, to prioritize 249 variants of uncertain significance (VUSs) that underwent splicing functional analyses. The capability of algorithms to differentiate VUSs away from the immediate splice site as being ‘pathogenic’ or ‘benign’ is likely to have substantial impact on diagnostic testing. We show that SpliceAI is the best single strategy in this regard, but that combined usage of tools using a weighted approach can increase accuracy further. We incorporated prioritization strategies alongside diagnostic testing for rare disorders. We show that 15% of 2783 referred individuals carry rare variants expected to impact splicing that were not initially identified as ‘pathogenic’ or ‘likely pathogenic’; one in five of these cases could lead to new or refined diagnoses.

Abstract Background It is estimated that 1–13% of cases of bronchiectasis in adults globally are attributable to primary ciliary dyskinesia (PCD) but many adult patients with bronchiectasis have not been investigated for PCD. PCD is a disorder caused by mutations in genes required for motile cilium structure or function, resulting in impaired mucociliary clearance. Symptoms appear in infancy but diagnosis is often late or missed, often due to the lack of a “gold standard” diagnostic tool and non-specific symptoms. Mutations in > 50 genes account for around 70% of cases, with additional genes, and non-coding, synonymous, missense changes or structural variants (SVs) in known genes presumed to account for the missing heritability. Methods UK patients with no identified genetic confirmation for the cause of their PCD or bronchiectasis were eligible for whole genome sequencing (WGS) in the Genomics England Ltd 100,000 Genomes Project. 21 PCD probands and 52 non-cystic fibrosis (CF) bronchiectasis probands were recruited in Wessex Genome Medicine Centre (GMC). We carried out analysis of single nucleotide variants (SNVs) and SVs in all families recruited in Wessex GMC. Results 16/21 probands in the PCD cohort received confirmed (n = 9), probable (n = 4) or possible (n = 3) diagnosis from WGS, although 13/16 of these could have been picked up by current standard of care gene panel testing. In the other cases, SVs were identified which were missed by panel testing. We identified variants in novel PCD candidate genes (IFT140 and PLK4) in 2 probands in the PCD cohort. 3/52 probands in the non-CF bronchiectasis cohort received a confirmed (n = 2) or possible (n = 1) diagnosis of PCD. We identified variants in novel PCD candidate genes (CFAP53 and CEP164) in 2 further probands in the non-CF bronchiectasis cohort. Conclusions Genetic testing is an important component of diagnosing PCD, especially in cases of atypical disease history. WGS is effective in cases where prior gene panel testing has found no variants or only heterozygous variants. In these cases it may detect SVs and is a powerful tool for novel gene discovery.

Posterior urethral valves (PUV) are the commonest cause of end-stage renal disease in children, but the genetic architecture of this rare disorder remains unknown. We performed a sequencing-based genome-wide association study (seqGWAS) in 132 unrelated male PUV cases and 23,727 controls of diverse ancestry, identifying statistically significant associations with common variants at 12q24.21 (p=7.8 × 10−12; OR 0.4) and rare variants at 6p21.1 (p=2.0 × 10-8; OR 7.2), that were replicated in an independent European cohort of 395 cases and 4151 controls. Fine mapping and functional genomic data mapped these loci to the transcription factor TBX5 and planar cell polarity gene PTK7, respectively, the encoded proteins of which were detected in the developing urinary tract of human embryos. We also observed enrichment of rare structural variation intersecting with candidate cis-regulatory elements, particularly inversions predicted to affect chromatin looping (p=3.1 × 10-5). These findings represent the first robust genetic associations of PUV, providing novel insights into the underlying biology of this poorly understood disorder and demonstrate how a diverse ancestry seqGWAS can be used for disease locus discovery in a rare disease.

There is considerable variability in the susceptibility and progression for COVID-19 and it appears to be strongly correlated with age, gender, ethnicity and pre-existing health conditions. However, to our knowledge, cohort studies of COVID-19 in clinically vulnerable groups are lacking. Host genetics has also emerged as a major risk factor for COVID-19, and variation in the ACE2 receptor, which facilitates entry of the SARS-CoV-2 virus into the cell, has become a major focus of attention. Thus, we interrogated an ethnically diverse cohort of National Health Service (NHS) patients in the United Kingdom (United Kingdom) to assess the association between variants in the ACE2 locus and COVID-19 risk. We analysed whole-genome sequencing (WGS) data of 1,837 cases who were tested positive for SARS-CoV-2, and 37,207 controls who were not tested, from the UK’s 100,000 Genomes Project (100KGP) for the presence of ACE2 coding variants and extract expression quantitative trait loci (eQTLs). We identified a splice site variant (rs2285666) associated with increased ACE2 expression with an overrepresentation in SARS-CoV-2 positive patients relative to 100KGP controls (p = 0.015), and in hospitalised European patients relative to outpatients in intra-ethnic comparisons (p = 0.029). We also compared the prevalence of 288 eQTLs, of which 23 were enriched in SARS-CoV-2 positive patients. The eQTL rs12006793 had the largest effect size (d = 0.91), which decreases ACE2 expression and is more prevalent in controls, thus potentially reducing the risk of COVID-19. We identified three novel nonsynonymous variants predicted to alter ACE2 function, and showed that three variants (p.K26R, p. H378R, p. Y515N) alter receptor affinity for the viral Spike (S) protein. Variant p. N720D, more prevalent in the European population (p < 0.001), potentially increases viral entry by affecting the ACE2-TMPRSS2 complex. The spectrum of genetic variants in ACE2 may inform risk stratification of COVID-19 patients and could partially explain the differences in disease susceptibility and severity among different ethnic groups.

Abstract 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.

The human dopaminergic system is vital for a broad range of neurological processes, including the control of voluntary movement. Here we report a proband presenting with clinical features of dopamine deficiency: severe infantile parkinsonism-dystonia, characterised by frequent oculogyric crises, dysautonomia and global neurodevelopmental impairment. CSF neurotransmitter analysis was unexpectedly normal. Triome whole-genome sequencing revealed a homozygous variant (c.110C>A, (p.T37K)) in DRD1, encoding the most abundant dopamine receptor (D1) in the central nervous system, most highly expressed in the striatum. This variant was absent from gnomAD, with a CADD score of 27.5. Using an in vitro heterologous expression system, we determined that DRD1-T37K results in loss of protein function. Structure-function modelling studies predicted reduced substrate binding, which was confirmed in vitro. Exposure of mutant protein to the selective D1 agonist Chloro APB resulted in significantly reduced cyclic AMP levels. Numerous D1 agonists failed to rescue the cellular defect, reflected clinically in the patient, who had no benefit from dopaminergic therapy. Our study identifies DRD1 as a new disease-associated gene, suggesting a crucial role for the D1 receptor in motor control.

Abstract The largest whole genome sequencing (WGS) endeavour involving cancer and rare diseases was initiated in the UK in 2015 and ran for 5 years. Despite its rarity, sarcoma ranked third overall among the number of patients' samples sent for sequencing. Herein, we recount the lessons learned by a specialist sarcoma centre that recruited close to 1000 patients to the project, so that we and others may learn from our experience. WGS data was generated from 597 patients, but samples from the remaining approximately 400 patients were not sequenced. This was largely accounted for by unsuitability due to extensive necrosis, secondary to neoadjuvant radiotherapy or chemotherapy, or being placed in formalin. The number of informative genomes produced was reduced further by a PCR amplification step. We showed that this loss of genomic data could be mitigated by sequencing whole genomes from needle core biopsies. Storage of resection specimens at 4 °C for up to 96 h overcame the challenge of freezing tissue out of hours including weekends. Removing access to formalin increased compliance to these storage arrangements. With over 70 different sarcoma subtypes described, WGS was a useful tool for refining diagnoses and identifying novel alterations. Genomes from 350 of the cohort of 597 patients were analysed in this study. Overall, diagnoses were modified for 3% of patients following review of the WGS findings. Continued refinement of the variant‐calling bioinformatic pipelines is required as not all alterations were identified when validated against histology and standard of care diagnostic tests. Further research is necessary to evaluate the impact of germline mutations in patients with sarcoma, and sarcomas with evidence of hypermutation. Despite 50% of the WGS exhibiting domain 1 alterations, the number of patients with sarcoma who were eligible for clinical trials remains small, highlighting the need to revaluate clinical trial design.

Lipoedema is a chronic adipose tissue disorder mainly affecting women, causing excess subcutaneous fat deposition on the lower limbs with pain and tenderness. There is often a family history of lipoedema, suggesting a genetic origin, but the contribution of genetics is currently unclear. A tightly phenotyped cohort of 200 lipoedema patients was recruited from two UK specialist clinics. Objective clinical characteristics and measures of quality of life data were obtained. In an attempt to understand the genetic architecture of the disease better, genome-wide single nucleotide polymorphism (SNP) genotype data were obtained, and a genome wide association study (GWAS) was performed on 130 of the recruits. The analysis revealed genetic loci suggestively associated with the lipoedema phenotype, with further support provided by an independent cohort taken from the 100,000 Genomes Project. The top SNP rs1409440 (ORmeta ≈ 2.01, Pmeta ≈ 4 x 10-6) is located upstream of LHFPL6, which is thought to be involved with lipoma formation. Exactly how this relates to lipoedema is not yet understood. This first GWAS of a UK lipoedema cohort has identified genetic regions of suggestive association with the disease. Further replication of these findings in different populations is warranted.

Summary: Robinow syndrome (RS) is a genetically heterogeneous disorder with six genes that converge on the WNT/planar cell polarity (PCP) signaling pathway implicated (DVL1, DVL3, FZD2, NXN, ROR2, and WNT5A). RS is characterized by skeletal dysplasia and distinctive facial and physical characteristics. To further explore the genetic heterogeneity, paralog contribution, and phenotypic variability of RS, we investigated a cohort of 22 individuals clinically diagnosed with RS from 18 unrelated families. Pathogenic or likely pathogenic variants in genes associated with RS or RS phenocopies were identified in all 22 individuals, including the first variant to be reported in DVL2. We retrospectively collected medical records of 16 individuals from this cohort and extracted clinical descriptions from 52 previously published cases. We performed Human Phenotype Ontology (HPO) based quantitative phenotypic analyses to dissect allele-specific phenotypic differences. Individuals with FZD2 variants clustered into two groups with demonstrable phenotypic differences between those with missense and truncating alleles. Probands with biallelic NXN variants clustered together with the majority of probands carrying DVL1, DVL2, and DVL3 variants, demonstrating no phenotypic distinction between the NXN-autosomal recessive and dominant forms of RS. While phenotypically similar diseases on the RS differential matched through HPO analysis, clustering using phenotype similarity score placed RS-associated phenotypes in a unique cluster containing WNT5A, FZD2, and ROR2 apart from non-RS-associated paralogs. Through human phenotype analyses of this RS cohort and OMIM clinical synopses of Mendelian disease, this study begins to tease apart specific biologic roles for non-canonical WNT-pathway proteins.

Recent evidence has questioned the dogma of strict maternal transmission of mitochondrial DNA (mtDNA) in humans. Wei et al. saw no evidence of paternal transmission of mtDNA in 11,035 human trios, and show that nuclear-mitochondrial segments (NUMTs) can give the impression of paternal mtDNA transmission, but are actually inherited through the nuclear genome.

Genetic prion disease accounts for 10–15% of prion disease. While insertion of four or more octapeptide repeats are clearly pathogenic, smaller repeat insertions have an unclear pathogenicity. The goal of this case series was to provide an insight into the characteristics of the 2-octapeptide repeat genetic variant and to provide insight into the risk for Creutzfeldt–Jakob disease in asymptomatic carriers. 2-octapeptide repeat insertion prion disease cases were collected from the National Prion Disease Pathology Surveillance Center (US), the National Prion Clinic (UK), and the National Creutzfeldt–Jakob Disease Registry (Australia). Three largescale population genetic databases were queried for the 2-octapeptide repeat insertion allele. Eight cases of 2-octapeptide repeat insertion were identified. The cases were indistinguishable from the sporadic Creutzfeldt–Jakob cases of the same molecular subtype. Western blot characterization of the prion protein in the absence of enzymatic digestion with proteinase K revealed that 2-octapeptide repeat insertion and sporadic Creutzfeldt–Jakob disease have distinct prion protein profiles. Interrogation of large-scale population datasets suggested the variant is of very low penetrance. The 2-octapeptide repeat insertion is at most a low-risk genetic variant. Predictive genetic testing for asymptomatic blood relatives is not likely to be justified given the low risk.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PurposeThe purpose of this study was to report retinal dystrophy as a novel clinical feature and expand the ocular phenotype in patients harboring biallelic candidate FDXR variants.MethodsPatients carrying biallelic candidate FDXR variants were identified by whole genome sequencing (WGS) as part of the National Institute for Health Research BioResource rare-disease and the UK's 100,000 Genomes Project (100KGP) with an additional case identified by exome sequencing. Retrospective clinical data were collected from the medical records. Haplotype reconstruction was performed in families harboring the same missense variant.ResultsTen individuals from 8 unrelated families with biallelic candidate variants in FDXR were identified. In addition to bilateral optic atrophy and variable extra-ocular findings, 7 of 10 individuals manifested retinal dystrophy comprising dysfunction and degeneration of both rod and cone photoreceptors. Five of 10 subjects had sensorineural hearing loss. The previously unreported missense variant (c.1115C > A, p.(Pro372His)) was found in 5 of 8 (62.5%) study families. Haplotype reconstruction using WGS data demonstrated a likely ancestral haplotype.ConclusionsFDXR-associated disease is a phenotypically heterogeneous disorder with retinal dystrophy being a major clinical feature observed in this cohort. In addition, we hypothesize that a number of factors are likely to drive the pathogenesis of optic atrophy, retinal degeneration, and perhaps the associated systemic manifestations.

Cross-species genome comparisons have revealed a substantial number of ultraconserved non-coding elements (UCNEs). Several of these elements have proved to be essential tissue- and cell type-specific cis-regulators of developmental gene expression. Here, we characterize a set of UCNEs as candidate CREs (cCREs) during retinal development and evaluate the contribution of their genomic variation to rare eye diseases, for which pathogenic non-coding variants are emerging. Integration of bulk and single-cell retinal multi-omics data reveals 594 genes under potential cis-regulatory control of UCNEs, of which 45 are implicated in rare eye disease. Mining of candidate cis-regulatory UCNEs in WGS data derived from the rare eye disease cohort of Genomics England reveals 178 ultrarare variants within 84 UCNEs associated with 29 disease genes. Overall, we provide a comprehensive annotation of ultraconserved non-coding regions acting as cCREs during retinal development which can be targets of non-coding variation underlying rare eye diseases. Ultraconserved non-coding elements (UCNEs) can regulate developmental gene expression. Retinal multi-omics data integration revealed UCNEs to be candidate cis-regulatory elements during retinal development, which may be implicated in rare eye diseases. [ABSTRACT FROM AUTHOR]

Cardiomyopathy has variable penetrance. We analyzed age and sex-related genetic differences in 1,397 cardiomyopathy patients (Ontario, UK) with whole genome sequencing. Pediatric cases (n = 471) harbored more deleterious protein-coding variants in Tier 1 cardiomyopathy genes compared to adults (n = 926) (34.6% vs 25.9% respectively, p = 0.0015), with variant enrichment in constrained coding regions. Pediatric patients had a higher burden of sarcomere and lower burden of channelopathy gene variants compared to adults. Specifically, pediatric patients had more MYH7 and MYL3 variants in hypertrophic cardiomyopathy, and fewer TTN truncating variants in dilated cardiomyopathy. MYH7 variants clustered in the myosin head and neck domains in children. OBSCN was a top mutated gene in adults, enriched for protein-truncating variants. In dilated cardiomyopathy, female patients had a higher burden of z-disc gene variants compared to males. Genetic differences may explain age and sex-related variability in cardiomyopathy penetrance. Genotype-guided predictions of age of onset can inform pre-test genetic counseling. Pediatric cardiomyopathy patients were more likely to be genotype-positive than adults with a higher burden of variants in MYH7, MYL3, TNNT2, VCL. Adults had a higher burden of OBSCN and TTN variants. Females with dilated cardiomyopathy (DCM) had a higher burden of z-disc gene variants compared to males. [ABSTRACT FROM AUTHOR]

Background: The majority of cases of Dent's disease are caused by pathogenic variants in the CLCN5 gene, which encodes a voltage-gated chloride ion channel (ClC-5), resulting in proximal tubular dysfunction. We present three members of the same family and one unrelated paediatric patient with the same insertion-deletion CLCN5 variant. The identification of these patients and positive familial segregation led to the re-classification of this variant from one of unknown significance to one of likely pathogenicity. Case presentation: A 41 year old male presented with end stage kidney failure, proteinuria and haematuria. Whole genome sequencing identified an insertion-deletion variant in CLCN5, resulting in a missense change (c.1744_1745delinsAA p.(Ala582Lys)). His brother and nephew, who both exhibited renal impairment, haematuria, proteinuria, glycosuria and nephrocalcinosis, were found to have the same variant. In addition, genetic testing of an unrelated paediatric patient who presented with proteinuria and hypercalciuria, demonstrated the same variant. Conclusions: The identification of this novel variant in four individuals with features of Dent's disease, has led to the re-classification of the variant to one of likely pathogenicity. As a result, our patients and any future patients with the same variant can be offered a likely diagnosis, without the need for kidney biopsy, and their family members can be offered genetic screening. [ABSTRACT FROM AUTHOR]