Bi-allelic genetic variants in the translational GTPases GTPBP1 and GTPBP2 cause a distinct identical neurodevelopmental syndrome.

The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species.

Use of genome sequencing to hunt for cryptic second-hit variants: analysis of 31 cases recruited to the 100 000 Genomes Project.

BACKGROUND: Current clinical testing methods used to uncover the genetic basis of rare disease have inherent limitations, which can lead to causative pathogenic variants being missed. Within the rare disease arm of the 100 000 Genomes Project (100kGP), families were recruited under the clinical indication 'single autosomal recessive mutation in rare disease'. These participants presented with strong clinical suspicion for a specific autosomal recessive disorder, but only one suspected pathogenic variant had been identified through standard-of-care testing. Whole genome sequencing (WGS) aimed to identify cryptic 'second-hit' variants. METHODS: To investigate the 31 families with available data that remained unsolved following formal review within the 100kGP, SVRare was used to aggregate structural variants present in <1% of 100kGP participants. Small variants were assessed using population allele frequency data and SpliceAI. Literature searches and publicly available online tools were used for further annotation of pathogenicity. RESULTS: Using these strategies, 8/31 cases were solved, increasing the overall diagnostic yield of this cohort from 10/41 (24.4%) to 18/41 (43.9%). Exemplar cases include a patient with cystic fibrosis harbouring a novel exonic LINE1 insertion in CFTR and a patient with generalised arterial calcification of infancy with complex interlinked duplications involving exons 2-6 of ENPP1. Although ambiguous by short-read WGS, the ENPP1 variant structure was resolved using optical genome mapping and RNA analysis. CONCLUSION: Systematic examination of cryptic variants across a multi-disease cohort successfully identifies additional pathogenic variants. WGS data analysis in autosomal recessive rare disease should consider complex structural and small intronic variants as potentially pathogenic second hits.

Genome sequencing identifies KMT2E-disrupting cryptic structural variant in a female with O'Donnell-Luria-Rodan syndrome.

We describe a patient from the 100,000 Genomes Project with a complex de novo structural variant within KMT2E leading to O'Donnell-Luria-Rodan syndrome. This case expands the mutational spectrum for this syndrome and highlights the importance of revisiting unsolved cases using better SV prioritisation tools and updated gene panels.

The prevalence and phenotypic range associated with biallelic PKDCC variants.

PKDCC encodes a component of Hedgehog signalling required for normal chondrogenesis and skeletal development. Although biallelic PKDCC variants have been implicated in rhizomelic shortening of limbs with variable dysmorphic features, this association was based on just two patients. In this study, data from the 100 000 Genomes Project was used in conjunction with exome sequencing and panel-testing results accessed via international collaboration to assemble a cohort of eight individuals from seven independent families with biallelic PKDCC variants. The allelic series included six frameshifts, a previously described splice-donor site variant and a likely pathogenic missense variant observed in two families that was supported by in silico structural modelling. Database queries suggested that the prevalence of this condition is between 1 of 127 and 1 of 721 in clinical cohorts with skeletal dysplasia of unknown aetiology. Clinical assessments, combined with data from previously published cases, indicate a predominantly upper limb involvement. Micrognathia, hypertelorism and hearing loss appear to be commonly co-occurring features. In conclusion, this study strengthens the link between biallelic inactivation of PKDCC and rhizomelic limb-shortening and will enable clinical testing laboratories to better interpret variants in this gene.

Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 macrodomain of SARS-CoV-2.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.

Conclusion of diagnostic odysseys due to inversions disrupting GLI3 and FBN1.

Many genetic testing methodologies are biased towards picking up structural variants (SVs) that alter copy number. Copy-neutral rearrangements such as inversions are therefore likely to suffer from underascertainment. In this study, manual review prompted by a virtual multidisciplinary team meeting and subsequent bioinformatic prioritisation of data from the 100K Genomes Project was performed across 43 genes linked to well-characterised skeletal disorders. Ten individuals from three independent families were found to harbour diagnostic inversions. In two families, inverted segments of 1.2/14.8 Mb unequivocally disrupted GLI3 and segregated with skeletal features consistent with Greig cephalopolysyndactyly syndrome. For one family, phenotypic blending was due to the opposing breakpoint lying ~45 kb from HOXA13 In the third family, long suspected to have Marfan syndrome, a 2.0 Mb inversion disrupting FBN1 was identified. These findings resolved lengthy diagnostic odysseys of 9-20 years and highlight the importance of direct interaction between clinicians and data-analysts. These exemplars of a rare mutational class inform future SV prioritisation strategies within the NHS Genomic Medicine Service and similar genome sequencing initiatives. In over 30 years since these two disease-gene associations were identified, large inversions have yet to be described and so our results extend the mutational spectra linked to these conditions.

Biallelic PRMT7 pathogenic variants are associated with a recognizable syndromic neurodevelopmental disorder with short stature, obesity, and craniofacial and digital abnormalities.

PURPOSE: Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyzes the methylation of arginine residues on several protein substrates. Biallelic pathogenic PRMT7 variants have previously been associated with a syndromic neurodevelopmental disorder characterized by short stature, brachydactyly, intellectual developmental disability, and seizures. To our knowledge, no comprehensive study describes the detailed clinical characteristics of this syndrome. Thus, we aim to delineate the phenotypic spectrum of PRMT7-related disorder. METHODS: We assembled a cohort of 51 affected individuals from 39 different families, gathering clinical information from 36 newly described affected individuals and reviewing data of 15 individuals from the literature. RESULTS: The main clinical characteristics of the PRMT7-related syndrome are short stature, mild to severe developmental delay/intellectual disability, hypotonia, brachydactyly, and distinct facial morphology, including bifrontal narrowing, prominent supraorbital ridges, sparse eyebrows, short nose with full/broad nasal tip, thin upper lip, full and everted lower lip, and a prominent or squared-off jaw. Additional variable findings include seizures, obesity, nonspecific magnetic resonance imaging abnormalities, eye abnormalities (i.e., strabismus or nystagmus), and hearing loss. CONCLUSION: This study further delineates and expands the molecular, phenotypic spectrum and natural history of PRMT7-related syndrome characterized by a neurodevelopmental disorder with skeletal, growth, and endocrine abnormalities.

Biallelic variants in PIGN cause Fryns syndrome, multiple congenital anomalies-hypotonia-seizures syndrome, and neurologic phenotypes: A genotype-phenotype correlation study.

PURPOSE: Biallelic PIGN variants have been described in Fryns syndrome, multiple congenital anomalies-hypotonia-seizure syndrome (MCAHS), and neurologic phenotypes. The full spectrum of clinical manifestations in relation to the genotypes is yet to be reported. METHODS: Genotype and phenotype data were collated and analyzed for 61 biallelic PIGN cases: 21 new and 40 previously published cases. Functional analysis was performed for 2 recurrent variants (c.2679C>G p.Ser893Arg and c.932T>G p.Leu311Trp). RESULTS: Biallelic-truncating variants were detected in 16 patients-10 with Fryns syndrome, 1 with MCAHS1, 2 with Fryns syndrome/MCAHS1, and 3 with neurologic phenotype. There was an increased risk of prenatal or neonatal death within this group (6 deaths were in utero or within 2 months of life; 6 pregnancies were terminated). Incidence of polyhydramnios, congenital anomalies (eg, diaphragmatic hernia), and dysmorphism was significantly increased. Biallelic missense or mixed genotype were reported in the remaining 45 cases-32 showed a neurologic phenotype and 12 had MCAHS1. No cases of diaphragmatic hernia or abdominal wall defects were seen in this group except patient 1 in which we found the missense variant p.Ser893Arg to result in functionally null alleles, suggesting the possibility of an undescribed functionally important region in the final exon. For all genotypes, there was complete penetrance for developmental delay and near-complete penetrance for seizures and hypotonia in patients surviving the neonatal period. CONCLUSION: We have expanded the described spectrum of phenotypes and natural history associated with biallelic PIGN variants. Our study shows that biallelic-truncating variants usually result in the more severe Fryns syndrome phenotype, but neurologic problems, such as developmental delay, seizures, and hypotonia, present across all genotypes. Functional analysis should be considered when the genotypes do not correlate with the predicted phenotype because there may be other functionally important regions in PIGN that are yet to be discovered.

Rare disease gene association discovery from burden analysis of the 100,000 Genomes Project data.

To discover rare disease-gene associations, we developed a gene burden analytical framework and applied it to rare, protein-coding variants from whole genome sequencing of 35,008 cases with rare diseases and their family members recruited to the 100,000 Genomes Project (100KGP). Following in silico triaging of the results, 88 novel associations were identified including 38 with existing experimental evidence. We have published the confirmation of one of these associations, hereditary ataxia with UCHL1 , and independent confirmatory evidence has recently been published for four more. We highlight a further seven compelling associations: hypertrophic cardiomyopathy with DYSF and SLC4A3 where both genes show high/specific heart expression and existing associations to skeletal dystrophies or short QT syndrome respectively; monogenic diabetes with UNC13A with a known role in the regulation of ß cells and a mouse model with impaired glucose tolerance; epilepsy with KCNQ1 where a mouse model shows seizures and the existing long QT syndrome association may be linked; early onset Parkinson's disease with RYR1 with existing links to tremor pathophysiology and a mouse model with neurological phenotypes; anterior segment ocular abnormalities associated with POMK showing expression in corneal cells and with a zebrafish model with developmental ocular abnormalities; and cystic kidney disease with COL4A3 showing high renal expression and prior evidence for a digenic or modifying role in renal disease. Confirmation of all 88 associations would lead to potential diagnoses in 456 molecularly undiagnosed cases within the 100KGP, as well as other rare disease patients worldwide, highlighting the clinical impact of a large-scale statistical approach to rare disease gene discovery.

Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 Macrodomain of SARS-CoV-2.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 152 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated protein dynamics within the active site, and key inhibitor motifs that will template future drug development against Mac1.

Recommendations for clinical interpretation of variants found in non-coding regions of the genome.

BACKGROUND: The majority of clinical genetic testing focuses almost exclusively on regions of the genome that directly encode proteins. The important role of variants in non-coding regions in penetrant disease is, however, increasingly being demonstrated, and the use of whole genome sequencing in clinical diagnostic settings is rising across a large range of genetic disorders. Despite this, there is no existing guidance on how current guidelines designed primarily for variants in protein-coding regions should be adapted for variants identified in other genomic contexts. METHODS: We convened a panel of nine clinical and research scientists with wide-ranging expertise in clinical variant interpretation, with specific experience in variants within non-coding regions. This panel discussed and refined an initial draft of the guidelines which were then extensively tested and reviewed by external groups. RESULTS: We discuss considerations specifically for variants in non-coding regions of the genome. We outline how to define candidate regulatory elements, highlight examples of mechanisms through which non-coding region variants can lead to penetrant monogenic disease, and outline how existing guidelines can be adapted for the interpretation of these variants. CONCLUSIONS: These recommendations aim to increase the number and range of non-coding region variants that can be clinically interpreted, which, together with a compatible phenotype, can lead to new diagnoses and catalyse the discovery of novel disease mechanisms.

Venus: Elucidating the Impact of Amino Acid Variants on Protein Function Beyond Structure Destabilisation.

Exploring the functional effect of a non-synonymous coding variant at the protein level requires multiple pieces of information to be interpreted appropriately. This is particularly important when embarking on the study of a potentially pathogenic variant linked to a rare or monogenic disease. Whereas accurate protein stability predictions alone are generally informative, other effects, such as disruption of post-translational modifications or weakened ligand binding, may also contribute to the disease phenotype. Furthermore, consideration of nearby variants that are found in the healthy population may strengthen or refute a given mechanistic hypothesis. Whilst there are several bioinformatics tools available that score a genetic variant in terms of deleteriousness, there is no single tool that assembles multiple effects of a variant on the encoded protein, beyond structural stability, and presents them on the structure for inspection. Venus is a web application which, given a protein substitution, rapidly estimates the predicted effect on protein stability of the variant, flags if the variant affects a post-translational modification site, a predicted linear motif or known annotation, and determines the effect on protein stability of variants which affect nearby residues and have been identified in healthy populations. Venus is built upon Michelanglo and the results can be exported to it, allowing them to be annotated and shared with other researchers. Venus is freely accessible at https://venus.cmd.ox.ac.uk and its source code is openly available at https://github.com/CMD-Oxford/Michelanglo-and-Venus.

The Bartter-Gitelman Spectrum: 50-Year Follow-up With Revision of Diagnosis After Whole-Genome Sequencing.

Bartter syndrome (BS) and Gitelman syndrome (GS) are renal tubular disorders affecting sodium, potassium, and chloride reabsorption. Clinical features include muscle cramps and weakness, in association with hypokalemia, hypochloremic metabolic alkalosis, and hyperreninemic hyperaldosteronism. Hypomagnesemia and hypocalciuria are typical of GS, while juxtaglomerular hyperplasia is characteristic of BS. GS is due to SLC12A3 variants, whereas BS is due to variants in SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND, MAGED2, or CASR. We had the opportunity to follow up one of the first reported cases of a salt-wasting tubulopathy, who based on clinical features was diagnosed with GS. The patient had presented at age 10 years with tetany precipitated by vomiting or diarrhea. She had hypokalemia, a hypochloremic metabolic alkalosis, hyponatremia, mild hypercalcemia, and normomagnesemia, and subsequently developed hypocalciuria and hypomagnesemia. A renal biopsy showed no evidence for juxtaglomerular hyperplasia. She developed chronic kidney failure at age 55 years, and ocular sclerochoroidal calcification, associated with BS and GS, at older than 65 years. Our aim was therefore to establish the genetic diagnosis in this patient using whole-genome sequencing (WGS). Leukocyte DNA was used for WGS analysis, and this revealed a homozygous c.226C > T (p.Arg76Ter) nonsense CLCNKB mutation, thereby establishing a diagnosis of BS type-3. WGS also identified 2 greater than 5-Mb regions of homozygosity that suggested likely mutational heterozygosity in her parents, who originated from a Greek island with fewer than 1500 inhabitants and may therefore have shared a common ancestor. Our results demonstrate the utility of WGS in establishing the correct diagnosis in renal tubular disorders with overlapping phenotypes.

Can polygenic risk scores contribute to cost-effective cancer screening? A systematic review.

PURPOSE: Polygenic risk influences susceptibility to cancer. We assessed whether polygenic risk scores could be used in conjunction with other predictors of future disease status in cost-effective risk-stratified screening for cancer. METHODS: We undertook a systematic review of papers that evaluated the cost-effectiveness of screening interventions informed by polygenic risk scores compared with more conventional screening modalities. We included papers reporting cost-effectiveness outcomes with no restriction on type of cancer or form of polygenic risk modeled. We evaluated studies using the Quality of Health Economic Studies checklist. RESULTS: A total of 10 studies were included in the review, which investigated 3 cancers: prostate (n = 5), colorectal (n = 3), and breast (n = 2). Of the 10 papers, 9 scored highly (score >75 on a 0-100 scale) when assessed using the quality checklist. Of the 10 studies, 8 concluded that polygenic risk-informed cancer screening was likely to be more cost-effective than alternatives. CONCLUSION: Despite the positive conclusions of the included studies, it is unclear if polygenic risk stratification will contribute to cost-effective cancer screening given the absence of robust evidence on the costs of polygenic risk stratification, the effects of differential ancestry, potential downstream economic sequalae, and how large volumes of polygenic risk data would be collected and used.

Germline MBD4 deficiency causes a multi-tumor predisposition syndrome.

We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.

GREEN-DB: a framework for the annotation and prioritization of non-coding regulatory variants from whole-genome sequencing data.

Non-coding variants have long been recognized as important contributors to common disease risks, but with the expansion of clinical whole genome sequencing, examples of rare, high-impact non-coding variants are also accumulating. Despite recent advances in the study of regulatory elements and the availability of specialized data collections, the systematic annotation of non-coding variants from genome sequencing remains challenging. Here, we propose a new framework for the prioritization of non-coding regulatory variants that integrates information about regulatory regions with prediction scores and HPO-based prioritization. Firstly, we created a comprehensive collection of annotations for regulatory regions including a database of 2.4 million regulatory elements (GREEN-DB) annotated with controlled gene(s), tissue(s) and associated phenotype(s) where available. Secondly, we calculated a variation constraint metric and showed that constrained regulatory regions associate with disease-associated genes and essential genes from mouse knock-outs. Thirdly, we compared 19 non-coding impact prediction scores providing suggestions for variant prioritization. Finally, we developed a VCF annotation tool (GREEN-VARAN) that can integrate all these elements to annotate variants for their potential regulatory impact. In our evaluation, we show that GREEN-DB can capture previously published disease-associated non-coding variants as well as identify additional candidate disease genes in trio analyses.

Provocation Testing and Therapeutic Response in a Newly Described Channelopathy: RyR2 Calcium Release Deficiency Syndrome.

BACKGROUND: A novel familial arrhythmia syndrome, cardiac ryanodine receptor (RyR2) calcium release deficiency syndrome (CRDS), has recently been described. We evaluated a large and well characterized family to assess provocation testing, risk factor stratification and response to therapy in CRDS. METHODS: We present a family with multiple unheralded sudden cardiac deaths and aborted cardiac arrests, primarily in children and young adults, with no clear phenotype on standard clinical testing. RESULTS: Genetic analysis, including whole genome sequencing, firmly established that a missense mutation in RYR2, Ala4142Thr, was the underlying cause of disease in the family. Functional study of the variant in a cell model showed RyR2 loss-of-function, indicating that the family was affected by CRDS. EPS (Electrophysiological Study) was undertaken in 9 subjects known to carry the mutation, including a survivor of aborted sudden cardiac death, and the effects of flecainide alone and in combination with metoprolol were tested. There was a clear gradation in inducibility of nonsustained and sustained ventricular arrhythmia between subjects at EPS, with the survivor of aborted sudden cardiac death being the most inducible subject. Administration of flecainide substantially reduced arrhythmia inducibility in this subject and abolished arrhythmia in all others. Finally, the effects of additional metoprolol were tested; it increased inducibility in 4/9 subjects. CONCLUSIONS: The Ala4142Thr mutation of RYR2 causes the novel heritable arrhythmia syndrome CRDS, which is characterized by familial sudden death in the absence of prior symptoms or a recognizable phenotype on ambulatory monitoring or exercise stress testing. We increase the experience of a specific EPS protocol in human subjects and show that it is helpful in establishing the clinical status of gene carriers, with potential utility for risk stratification. Our data provide evidence that flecainide is protective in human subjects with CRDS, consistent with the effect previously shown in a mouse model.

Biallelic TMEM260 variants cause truncus arteriosus, with or without renal defects.

Only two families have been reported with biallelic TMEM260 variants segregating with structural heart defects and renal anomalies syndrome (SHDRA). With a combination of genome, exome sequencing and RNA studies, we identified eight individuals from five families with biallelic TMEM260 variants. Variants included one multi-exon deletion, four nonsense/frameshifts, two splicing changes and one missense change. Together with the published cases, analysis of clinical data revealed ventricular septal defects (12/12), mostly secondary to truncus arteriosus (10/12), elevated creatinine levels (6/12), horse-shoe kidneys (1/12) and renal cysts (1/12) in patients. Three pregnancies were terminated on detection of severe congenital anomalies. Six patients died between the ages of 6 weeks and 5 years. Using a range of stringencies, carrier frequency for SHDRA was estimated at 0.0007-0.007 across ancestries. In conclusion, this study confirms the genetic basis of SHDRA, expands its known mutational spectrum and clarifies its clinical features. We demonstrate that SHDRA is a severe condition associated with substantial mortality in early childhood and characterised by congenital cardiac malformations with a variable renal phenotype.