Scaling national and international improvement in virtual gene panel curation via a collaborative approach to discordance resolution.

Clinical validity assessments of gene-disease associations underpin analysis and reporting in diagnostic genomics, and yet wide variability exists in practice, particularly in use of these assessments for virtual gene panel design and maintenance. Harmonization efforts are hampered by the lack of agreed terminology, agreed gene curation standards, and platforms that can be used to identify and resolve discrepancies at scale. We undertook a systematic comparison of the content of 80 virtual gene panels used in two healthcare systems by multiple diagnostic providers in the United Kingdom and Australia. The process was enabled by a shared curation platform, PanelApp, and resulted in the identification and review of 2,144 discordant gene ratings, demonstrating the utility of sharing structured gene-disease validity assessments and collaborative discordance resolution in establishing national and international consensus.

100,000 Genomes Pilot on Rare-Disease Diagnosis in Health Care - Preliminary Report.

BACKGROUND: The U.K. 100,000 Genomes Project is in the process of investigating the role of genome sequencing in patients with undiagnosed rare diseases after usual care and the alignment of this research with health care implementation in the U.K. National Health Service. Other parts of this project focus on patients with cancer and infection. METHODS: We conducted a pilot study involving 4660 participants from 2183 families, among whom 161 disorders covering a broad spectrum of rare diseases were present. We collected data on clinical features with the use of Human Phenotype Ontology terms, undertook genome sequencing, applied automated variant prioritization on the basis of applied virtual gene panels and phenotypes, and identified novel pathogenic variants through research analysis. RESULTS: Diagnostic yields varied among family structures and were highest in family trios (both parents and a proband) and families with larger pedigrees. Diagnostic yields were much higher for disorders likely to have a monogenic cause (35%) than for disorders likely to have a complex cause (11%). Diagnostic yields for intellectual disability, hearing disorders, and vision disorders ranged from 40 to 55%. We made genetic diagnoses in 25% of the probands. A total of 14% of the diagnoses were made by means of the combination of research and automated approaches, which was critical for cases in which we found etiologic noncoding, structural, and mitochondrial genome variants and coding variants poorly covered by exome sequencing. Cohortwide burden testing across 57,000 genomes enabled the discovery of three new disease genes and 19 new associations. Of the genetic diagnoses that we made, 25% had immediate ramifications for clinical decision making for the patients or their relatives. CONCLUSIONS: Our pilot study of genome sequencing in a national health care system showed an increase in diagnostic yield across a range of rare diseases. (Funded by the National Institute for Health Research and others.).

Targeting de novo loss-of-function variants in constrained disease genes improves diagnostic rates in the 100,000 Genomes Project.

BACKGROUND: Genome sequencing was first offered clinically in the UK through the 100,000 Genomes Project (100KGP). Analysis was restricted to predefined gene panels associated with the patient's phenotype. However, panels rely on clearly characterised phenotypes and risk missing diagnoses outside of the panel(s) applied. We propose a complementary method to rapidly identify pathogenic variants, including those missed by 100KGP methods. METHODS: The Loss-of-function Observed/Expected Upper-bound Fraction (LOEUF) score quantifies gene constraint, with low scores correlated with haploinsufficiency. We applied DeNovoLOEUF, a filtering strategy to sequencing data from 13,949 rare disease trios in the 100KGP, by filtering for rare, de novo, loss-of-function variants in disease genes with a LOEUF score < 0.2. We compared our findings with the corresponding patient's diagnostic reports. RESULTS: 324/332 (98%) of the variants identified using DeNovoLOEUF were diagnostic or partially diagnostic (whereby the variant was responsible for some of the phenotype). We identified 39 diagnoses that were "missed" by 100KGP standard analyses, which are now being returned to patients. CONCLUSION: We have demonstrated a highly specific and rapid method with a 98% positive predictive value that has good concordance with standard analysis, low false-positive rate, and can identify additional diagnoses. Globally, as more patients are being offered genome sequencing, we anticipate that DeNovoLOEUF will rapidly identify new diagnoses and facilitate iterative analyses when new disease genes are discovered.

Molecular diagnoses in the congenital malformations caused by ciliopathies cohort of the 100,000 Genomes Project.

BACKGROUND: Primary ciliopathies represent a group of inherited disorders due to defects in the primary cilium, the 'cell's antenna'. The 100,000 Genomes Project was launched in 2012 by Genomics England (GEL), recruiting National Health Service (NHS) patients with eligible rare diseases and cancer. Sequence data were linked to Human Phenotype Ontology (HPO) terms entered by recruiting clinicians. METHODS: Eighty-three prescreened probands were recruited to the 100,000 Genomes Project suspected to have congenital malformations caused by ciliopathies in the following disease categories: Bardet-Biedl syndrome (n=45), Joubert syndrome (n=14) and 'Rare Multisystem Ciliopathy Disorders' (n=24). We implemented a bespoke variant filtering and analysis strategy to improve molecular diagnostic rates for these participants. RESULTS: We determined a research molecular diagnosis for n=43/83 (51.8%) probands. This is 19.3% higher than previously reported by GEL (n=27/83 (32.5%)). A high proportion of diagnoses are due to variants in non-ciliopathy disease genes (n=19/43, 44.2%) which may reflect difficulties in clinical recognition of ciliopathies. n=11/83 probands (13.3%) had at least one causative variant outside the tiers 1 and 2 variant prioritisation categories (GEL's automated triaging procedure), which would not be reviewed in standard 100,000 Genomes Project diagnostic strategies. These include four structural variants and three predicted to cause non-canonical splicing defects. Two unrelated participants have biallelic likely pathogenic variants in LRRC45, a putative novel ciliopathy disease gene. CONCLUSION: These data illustrate the power of linking large-scale genome sequence to phenotype information. They demonstrate the value of research collaborations in order to maximise interpretation of genomic data.

ERF-related craniosynostosis and surgical management in the paediatric cohort.

INTRODUCTION: ERF mutation is one of the most recently identified genetic aberrations associated with syndromic craniosynostosis. Data on the pattern of craniosynostosis, surgical management of ERF-related craniosynostosis and outcomes is limited. We report on our single-centre experience in paediatric cohort of patients with syndromic craniosynostosis secondary to ERF mutation. METHODS: A retrospective review of all paediatric craniofacial cases was performed over an 8-year period (2014-2022). All patients with genetically confirm ERF-related craniosynostosis were identified, and clinical parameters including, age, sex, pattern of craniosynostosis, associated tonsillar herniation and follow-up period were further analysed from electronic clinical and imaging systems. All patients were selected and discussed in multidisciplinary craniofacial meeting (composed of neurosurgical, maxillofacial, plastics and genetics teams) prior to any surgical intervention. RESULTS: Overall, 10 patients with ERF-related craniosynostosis were identified with a male-to-female ratio of 4:1 with mean age at the time of surgery of 21.6 months with a mean follow-up period of 5.2 years. ERF-confirmed cases led to variable craniosynostosis pattern with multi-sutural synostosis with concurrent sagittal and bilateral lambdoid involvement as the most common pattern (7/10). No patient pre-operatively had evidence of papilloedema on ophthalmological assessment. Eight out of 10 patients had associated low-lying tonsils/hind brain hernia pre-operatively. Eight out of 10 patients required surgery which included 2 fronto-orbital advancement, 3 calvarial remodelling, 2 posterior calvarial remodelling/release and 1 insertion of ventriculoperitoneal shunt. CONCLUSION: Involvement of sagittal and lambdoid sutures is the most common pattern of craniosynostosis. ERF-related craniosynostosis can have variable pattern of suture fusion, and management of each patient requires unique surgical planning and execution based on clinical needs for the optimal outcomes.

A gene-to-patient approach uplifts novel disease gene discovery and identifies 18 putative novel disease genes.

PURPOSE: Exome and genome sequencing have drastically accelerated novel disease gene discoveries. However, discovery is still hindered by myriad variants of uncertain significance found in genes of undetermined biological function. This necessitates intensive functional experiments on genes of equal predicted causality, leading to a major bottleneck. METHODS: We apply the loss-of-function observed/expected upper-bound fraction metric of intolerance to gene inactivation to curate a list of predicted haploinsufficient disease genes. Using data from the 100,000 Genomes Project, we adopt a gene-to-patient approach that matches de novo loss-of-function variants in constrained genes to patients with rare disease. Through large-scale aggregation of data, we reduce excess analytical noise currently hindering novel discoveries. RESULTS: Results from 13,949 trios revealed 643 rare, de novo predicted loss-of-function events filtered from 1044 loss-of-function observed/expected upper-bound fraction-constrained genes. A total of 168 variants occurred within 126 genes without a known disease-gene relationship. Of these, 27 genes had >1 kindred affected, and for 18 of these genes, multiple kindreds had overlapping phenotypes. Two years after initial analysis, 11 of 18 (61%) of these genes have been independently published as novel disease gene discoveries. CONCLUSION: Using large cohorts and adopting gene-based approaches can rapidly and objectively accelerate dominantly inherited novel gene discovery by targeting the most appropriate genes for functional validation.

The p190 RhoGAPs, ARHGAP35, and ARHGAP5 are implicated in GnRH neuronal development: Evidence from patients with idiopathic hypogonadotropic hypogonadism, zebrafish, and in vitro GAP activity assay.

PURPOSE: The study aimed to identify novel genes for idiopathic hypogonadotropic hypogonadism (IHH). METHODS: A cohort of 1387 probands with IHH underwent exome sequencing and de novo, familial, and cohort-wide investigations. Functional studies were performed on 2 p190 Rho GTPase-activating proteins (p190 RhoGAP), ARHGAP35 and ARHGAP5, which involved in vivo modeling in larval zebrafish and an in vitro p190A-GAP activity assay. RESULTS: Rare protein-truncating variants (PTVs; n = 5) and missense variants in the RhoGAP domain (n = 7) in ARHGAP35 were identified in IHH cases (rare variant enrichment: PTV [unadjusted P = 3.1E-06] and missense [adjusted P = 4.9E-03] vs controls). Zebrafish modeling using gnrh3:egfp phenotype assessment showed that mutant larvae with deficient arhgap35a, the predominant ARHGAP35 paralog in the zebrafish brain, display decreased GnRH3-GFP+ neuronal area, a readout for IHH. In vitro GAP activity studies showed that 1 rare missense variant [ARHGAP35 p.(Arg1284Trp)] had decreased GAP activity. Rare PTVs (n = 2) also were discovered in ARHGAP5, a paralog of ARHGAP35; however, arhgap5 zebrafish mutants did not display significant GnRH3-GFP+ abnormalities. CONCLUSION: This study identified ARHGAP35 as a new autosomal dominant genetic driver for IHH and ARHGAP5 as a candidate gene for IHH. These observations suggest a novel role for the p190 RhoGAP proteins in GnRH neuronal development and integrity.

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

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

High Rate of Recurrent De Novo Mutations in Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

Biallelic human ITCH variants causing a multisystem disease with dysmorphic features: A second report.

We report a 23 year old female with biallelic truncating variants in the ITCH (Itchy E3 Ubiquitin protein ligase, mouse homolog of; OMIM60649) gene associated with marked short stature, severe early onset chronic lung disease resembling asthma, dysmorphic facial features, and symmetrical camptodactyly of the fingers but normal intellect. The condition has only been reported once previously (Lohr et al., American Journal of Human Genetics, 2010, 86, 447-453) in 10 children from an Old Order Amish family found to have a homozygous frameshift truncating variant in association with failure to thrive, chronic lung disease, motor and cognitive delay, and variable autoimmune diseases including autoimmune hepatitis, enteropathy, hypothyroidism, and diabetes. The condition is listed in OMIM as Autoimmune disease, Multisystem with Facial Dysmorphism (OMIM613385). The clinical course as well as the dysmorphic facial and limb features overlap closely with our patient. We believe the triad of marked syndromic short stature, chronic lung disease, and dysmorphism (with or without cognitive impairment and wider autoimmune involvement) is distinctive.

Cascade Fumarate Hydratase mutation screening allows early detection of kidney tumour: a case report.

BACKGROUND: Fumarate hydratase (FH) deficiency is a rare autosomal recessive disorder which results in a major defect in cellular metabolism. It presents in infancy with progressive encephalopathy, hypotonia, seizures and failure to thrive and is often fatal in childhood. It is caused by mutations in the FH gene (1q42.1) that result in deficiency of the citric acid cycle enzyme fumarate hydratase, resulting in accumulation of fumaric acid. Heterozygous germline mutations in the FH gene predispose to an aggressive autosomal dominant inherited early-onset kidney cancer syndrome: hereditary leiomyomatosis and renal cell cancer (HLRCC). CASE PRESENTATION: Cascade FH mutation screening enabled the early diagnosis of a renal tumour in an asymptomatic parent of a child with fumarate hydratase deficiency, resulting in timely and possibly life-saving treatment. CONCLUSION: While the theoretical risk of kidney cancer in parents of children with recessive fumarate hydratase deficiency is well recognized, to our knowledge this is the first report of a kidney tumour being detected in a parent by screening performed for this indication. This underscores the importance of offering lifelong kidney surveillance to such parents and other heterozygous relatives of children born with fumarate hydratase deficiency.

Improving diagnosis and broadening the phenotypes in early-onset seizure and severe developmental delay disorders through gene panel analysis.

BACKGROUND: We sought to investigate the diagnostic yield and mutation spectrum in previously reported genes for early-onset epilepsy and disorders of severe developmental delay. METHODS: In 400 patients with these disorders with no known underlying aetiology and no major structural brain anomaly, we analysed 46 genes using a combination of targeted sequencing on an Illumina MiSeq platform and targeted, exon-level microarray copy number analysis. RESULTS: We identified causative mutations in 71/400 patients (18%). The diagnostic rate was highest among those with seizure onset within the first two months of life (39%), although overall it was similar in those with and without seizures. The most frequently mutated gene was SCN2A (11 patients, 3%). Other recurrently mutated genes included CDKL5, KCNQ2, SCN8A (six patients each), FOXG1, MECP2, SCN1A, STXBP1 (five patients each), KCNT1, PCDH19, TCF4 (three patients each) and ATP1A3, PRRT2 and SLC9A6 (two patients each). Mutations in EHMT1, GABRB3, LGI1, MBD5, PIGA, UBE3A and ZEB2 were each found in single patients. We found mutations in a number of genes in patients where either the electroclinical features or dysmorphic phenotypes were atypical for the identified gene. In only 11 cases (15%) had the clinician sufficient certainty to specify the mutated gene as the likely cause before testing. CONCLUSIONS: Our data demonstrate the considerable utility of a gene panel approach in the diagnosis of patients with early-onset epilepsy and severe developmental delay disorders., They provide further insights into the phenotypic spectrum and genotype-phenotype correlations for a number of the causative genes and emphasise the value of exon-level copy number testing in their analysis.

Achondroplasia: Really rhizomelic?

Achondroplasia is the most common form of short limb dwarfism in humans. The shortening of the limb lengths in achondroplasia is widely described as "rhizomelic." While this appearance may be convincing clinically, the description is not necessarily true or helpful radiologically. The aims of this study, were therefore, to determine whether rhizomelic shortening is a true feature of achondroplasia at diagnosis in infancy. Humeral, radial, femoral, and tibial diaphyseal lengths were recorded by two independent observers from 22 skeletal surveys of infants with achondroplasia and compared with 150 normal age-matched control subjects. Upper and lower limb bone length ratios (radial/humeral and tibial/femoral lengths, respectively) in both groups were compared using an unpaired t-test. Mean upper limb length ratios were statistically higher within the achondroplasia group at 0.87 ± 0.04 (n = 22, mean age 70 ± 94 days) compared to normal controls at 0.79 ± 0.02 (n = 150, mean age 113 days ± 88 days; P < 0.0001). Lower limb length ratios were not significantly different between groups (0.84 ± 0.04 vs. 0.83 ± 0.02, P = 0.46). There was good inter-observer agreement of limb length measurements, with an average measurement difference of 0.1 ± 1.4 mm. In conclusion, infants with achondroplasia demonstrate statistically significant rhizomelic shortening within the upper limbs, but not lower limbs at diagnosis, compared to normal controls. The term "rhizomelic shortening" in relation to achondroplasia should be reserved when describing upper limb proportions. © 2016 Wiley Periodicals, Inc.

The rise of the genome and personalised medicine.

Virtually all medical specialties are impacted by genetic disease. Enhanced understanding of the role of genetics in human disease, coupled with rapid advancement in sequencing technology, is transforming the speed of diagnosis for patients and providing increasing opportunities to tailor management. As set out in the Annual report of the Chief Medical Officer 2016: Generation Genome1 and the recent NHS England board paper Creating a genomic medicine service to lay the foundations to deliver personalised interventions and treatments,2 the increasing 'mainstreaming' of genetic testing into routine practice and plans to embed whole genome sequencing in the NHS mean that the profile and importance of genomics is on the rise for many clinicians. This article provides a brief overview of genomics and its current clinical applications, including its contribution to personalised medicine. Physicians will be signposted to key issues that will allow the successful implementation of genomics for rare disease diagnosis and cancer management.

Epileptic spasms and early-onset photosensitive epilepsy in Patau syndrome: An EEG study.

INTRODUCTION: Patau syndrome, trisomy 13, is the third commonest autosomal trisomy. It is associated with a 25-50% prevalence of epilepsy, but detailed electroclinical descriptions are rare. The occurrence of early-onset photosensitivity has recently been reported in single patients. MATERIALS/PATIENTS: We collected electroclinical data on 8 infants (age range from 2 months to 3 years and 9 months, median: 17 months) with Patau syndrome referred for an EEG in our Clinical Neurophysiology Department between 1991 and 2011. METHODS: All EEGs, case-notes, cytogenetic diagnosis and neuroimaging when available were reviewed; data on the occurrence of seizures, epileptiform discharges, photoparoxysmal response and their characteristics in terms of positive frequencies, latencies, grade and duration were noted and analysed. RESULTS: Two patients had been previously diagnosed with epilepsy (one with tonic spasms and one with multiple seizure types). We found 3 patients with photosensitive myoclonic epilepsy (37.5%), and one with non-photosensitive myoclonic epilepsy. We also recorded non-epileptic myoclonic jerks in one patient known to suffer from epileptic spasms. Among photosensitive patients we found self-limited, Waltz's grade 2-4, spike-wave/polyspike-wave discharges in low, medium and high frequency ranges in two patients and in the high frequency range in the third patient, with latencies and duration from less than 1s to a maximum of 9s. CONCLUSIONS: In our cohort of Patau syndrome patients, we found a high prevalence of spasms and photic-induced myoclonic jerks. Photosensitivity shows an unusual early age of onset.