A systematic assessment of the impact of rare canonical splice site variants on splicing using functional and in silico methods.

BACKGROUND/OBJECTIVES: Canonical splice site variants (CSSVs) are often presumed to cause loss-of-function (LoF) and are assigned very strong evidence of pathogenicity (according to ACMG criterion PVS1). The exact nature and predictability of splicing effects of unselected rare CSSVs in blood-expressed genes is poorly understood. METHODS: 168 rare CSSVs in unselected blood-expressed genes were identified by genome sequencing in 112 individuals, and their impact on splicing was interrogated manually in RNA sequencing (RNA-seq) data. Blind to these RNA-seq data, we attempted to predict the precise impact of CSSVs by applying in silico tools and the ClinGen Sequence Variant Interpretation Working Group 2018 guidelines for applying PVS1 criterion. RESULTS: There was no evidence of a frameshift nor of reduced expression consistent with nonsense-mediated decay for 25.6% of CSSVs: 17.9% had wildtype splicing only and normal junction depths, 3.6% resulted in cryptic splice site usage and in-frame indels, 3.6% resulted in full exon skipping (in-frame), and 0.6% resulted in full intron inclusion (in-frame). The predicted impact on splicing using (i) SpliceAI, (ii) MaxEntScan, and (iii) AutoPVS1, an automatic classification tool for PVS1 interpretation of null variants that utilizes Ensembl Variant Effect Predictor and MaxEntScan, was concordant with RNA-seq analyses for 65%, 63% and 61% of CSSVs, respectively. CONCLUSION: Approximately 1 in 4 rare CSSVs may not cause LoF based on analysis of RNA-seq data. Predictions from in silico methods were often discordant with findings from RNA-seq. More caution may be warranted in applying PVS1-level evidence to CSSVs in the absence of functional data.

Comprehensive whole-genome sequence analyses provide insights into the genomic architecture of cerebral palsy.

We performed whole-genome sequencing (WGS) in 327 children with cerebral palsy (CP) and their biological parents. We classified 37 of 327 (11.3%) children as having pathogenic/likely pathogenic (P/LP) variants and 58 of 327 (17.7%) as having variants of uncertain significance. Multiple classes of P/LP variants included single-nucleotide variants (SNVs)/indels (6.7%), copy number variations (3.4%) and mitochondrial mutations (1.5%). The COL4A1 gene had the most P/LP SNVs. We also analyzed two pediatric control cohorts (n = 203 trios and n = 89 sib-pair families) to provide a baseline for de novo mutation rates and genetic burden analyses, the latter of which demonstrated associations between de novo deleterious variants and genes related to the nervous system. An enrichment analysis revealed previously undescribed plausible candidate CP genes (SMOC1, KDM5B, BCL11A and CYP51A1). A multifactorial CP risk profile and substantial presence of P/LP variants combine to support WGS in the diagnostic work-up across all CP and related phenotypes.

A Study on the Incidence and Prevalence of 5q Spinal Muscular Atrophy in Canada Using Multiple Data Sources.

OBJECTIVES: Spinal muscular atrophy (SMA) is a leading genetic cause of infant death and represents a significant burden of care. An improved understanding of the epidemiology of SMA in Canada may help inform strategies to improve the standard of care for individuals living with SMA. METHODS: We employed a multisource approach to estimate the minimal incidence and prevalence of 5q SMA and to gain greater insight into recent clinical practices and treatment trends for the Canadian SMA population. Data sources included the Canadian Paediatric Surveillance Program (CPSP), Canadian Neuromuscular Disease Registry (CNDR), and molecular genetics laboratories in Canada. RESULTS: The estimated annual minimum incidence of 5q SMA was 4.38, 3.44, and 7.99 cases per 100,000 live births in 2020 and 2021, based on CPSP, CNDR, and molecular genetics laboratories data, respectively, representing approximately 1 in 21,472 births (range 12,516-29,070) in Canada. SMA prevalence was estimated to be 0.85 per 100,000 persons aged 0-79 years. Delay in diagnosis exists across all SMA subtypes. Most common presenting symptoms were delayed milestones, hypotonia, and muscle weakness. Nusinersen was the most common disease-modifying treatment received. Most patients utilized multidisciplinary clinics for management of SMA. CONCLUSION: This study provides data on the annual minimum incidence of pediatric 5q SMA in Canada. Recent therapeutic advances and newborn screening have the potential to drastically alter the natural history of SMA. Findings underline the importance of ongoing surveillance of the epidemiology and long-term health outcomes of SMA in the Canadian population.

Long-read genome sequencing reveals a novel intronic retroelement insertion in NR5A1 associated with 46,XY differences of sexual development.

Despite significant advancements in rare genetic disease diagnostics, many patients with rare genetic disease remain without a molecular diagnosis. Novel tools and methods are needed to improve the detection of disease-associated variants and understand the genetic basis of many rare diseases. Long-read genome sequencing provides improved sequencing in highly repetitive, homologous, and low-complexity regions, and improved assessment of structural variation and complex genomic rearrangements compared to short-read genome sequencing. As such, it is a promising method to explore overlooked genetic variants in rare diseases with a high suspicion of a genetic basis. We therefore applied PacBio HiFi sequencing in a large multi-generational family presenting with autosomal dominant 46,XY differences of sexual development (DSD), for whom extensive molecular testing over multiple decades had failed to identify a molecular diagnosis. This revealed a rare SINE-VNTR-Alu retroelement insertion in intron 4 of NR5A1, a gene in which loss-of-function variants are an established cause of 46,XY DSD. The insertion segregated among affected family members and was associated with loss-of-expression of alleles in cis, demonstrating a functional impact on NR5A1. This case highlights the power of long-read genome sequencing to detect genomic variants that have previously been intractable to detection by standard short-read genomic testing.

The landscape of reported VUS in multi-gene panel and genomic testing: Time for a change.

PURPOSE: Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact. METHODS: Rates of inconclusive results due to VUS were collected from over 1.5 million sequencing test results from 19 clinical laboratories in North America from 2020 to 2021. RESULTS: We found a lower rate of inconclusive test results due to VUSs from ES/GS (22.5%) compared with MGPs (32.6%; P < .0001). For MGPs, the rate of inconclusive results correlated with panel size. The use of trios reduced inconclusive rates (18.9% vs 27.6%; P < .0001), whereas the use of GS compared with ES had no impact (22.2% vs 22.6%; P = ns). CONCLUSION: The high rate of VUS observed in diagnostic MGP testing warrants examining current variant reporting practices. We propose several approaches to reduce reported VUS rates, while directing clinician resources toward important VUS follow-up.

Evaluation of the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in infantile epilepsy (Gene-STEPS): an international, multicentre, pilot cohort study.

BACKGROUND: Most neonatal and infantile-onset epilepsies have presumed genetic aetiologies, and early genetic diagnoses have the potential to inform clinical management and improve outcomes. We therefore aimed to determine the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in this population. METHODS: We conducted an international, multicentre, cohort study (Gene-STEPS), which is a pilot study of the International Precision Child Health Partnership (IPCHiP). IPCHiP is a consortium of four paediatric centres with tertiary-level subspecialty services in Australia, Canada, the UK, and the USA. We recruited infants with new-onset epilepsy or complex febrile seizures from IPCHiP centres, who were younger than 12 months at seizure onset. We excluded infants with simple febrile seizures, acute provoked seizures, known acquired cause, or known genetic cause. Blood samples were collected from probands and available biological parents. Clinical data were collected from medical records, treating clinicians, and parents. Trio genome sequencing was done when both parents were available, and duo or singleton genome sequencing was done when one or neither parent was available. Site-specific protocols were used for DNA extraction and library preparation. Rapid genome sequencing and analysis was done at clinically accredited laboratories, and results were returned to families. We analysed summary statistics for cohort demographic and clinical characteristics and the timing, diagnostic yield, and clinical impact of rapid genome sequencing. FINDINGS: Between Sept 1, 2021, and Aug 31, 2022, we enrolled 100 infants with new-onset epilepsy, of whom 41 (41%) were girls and 59 (59%) were boys. Median age of seizure onset was 128 days (IQR 46-192). For 43 (43% [binomial distribution 95% CI 33-53]) of 100 infants, we identified genetic diagnoses, with a median time from seizure onset to rapid genome sequencing result of 37 days (IQR 25-59). Genetic diagnosis was associated with neonatal seizure onset versus infantile seizure onset (14 [74%] of 19 vs 29 [36%] of 81; p=0·0027), referral setting (12 [71%] of 17 for intensive care, 19 [44%] of 43 non-intensive care inpatient, and 12 [28%] of 40 outpatient; p=0·0178), and epilepsy syndrome (13 [87%] of 15 for self-limited epilepsies, 18 [35%] of 51 for developmental and epileptic encephalopathies, 12 [35%] of 34 for other syndromes; p=0·001). Rapid genome sequencing revealed genetic heterogeneity, with 34 unique genes or genomic regions implicated. Genetic diagnoses had immediate clinical utility, informing treatment (24 [56%] of 43), additional evaluation (28 [65%]), prognosis (37 [86%]), and recurrence risk counselling (all cases). INTERPRETATION: Our findings support the feasibility of implementation of rapid genome sequencing in the clinical care of infants with new-onset epilepsy. Longitudinal follow-up is needed to further assess the role of rapid genetic diagnosis in improving clinical, quality-of-life, and economic outcomes. FUNDING: American Academy of Pediatrics, Boston Children's Hospital Children's Rare Disease Cohorts Initiative, Canadian Institutes of Health Research, Epilepsy Canada, Feiga Bresver Academic Foundation, Great Ormond Street Hospital Charity, Medical Research Council, Murdoch Children's Research Institute, National Institute of Child Health and Human Development, National Institute for Health and Care Research Great Ormond Street Hospital Biomedical Research Centre, One8 Foundation, Ontario Brain Institute, Robinson Family Initiative for Transformational Research, The Royal Children's Hospital Foundation, University of Toronto McLaughlin Centre.

Protocol for a Prospective, Observational Cost-effectiveness Analysis of Returning Secondary Findings of Genome Sequencing for Unexplained Suspected Genetic Conditions.

PURPOSE: Although costly, genome-wide sequencing (GWS) detects an extensive range of variants, enhancing our ability to diagnose and assess risk for an increasing number of diseases. In addition to detecting variants related to the indication for testing, GWS can detect secondary variants in BRCA1, BRCA2, and other genes for which early intervention may improve health. As the list of secondary findings grows, there is increased demand for surveillance and management by multiple specialists, adding pressure to constrained health care budgets. Secondary finding testing is actively debated because some consider it opportunistic screening for future health risks that may not manifest. Given the economic implications of secondary finding testing and follow-up and its unproven clinical utility, the objective is to assess the incremental cost-effectiveness of secondary finding ascertainment per case detected and per unit of improved clinical utility in families of children with unexplained suspected genetic conditions undergoing clinical GWS. METHODS: Those undergoing trio genome or exome sequencing are eligible for the study. Positive secondary finding index cases will be matched to negative controls (1:2) based on age group, primary result(s) type, and clinical indication. During the 2-year study, 71 cases and 142 matched controls are expected. Health service use will be collected in patients and 1 adult family member every 6 months. The per-child and per-dyad total cost will be determined by multiplying use of each resource by a corresponding unit price and summing all cost items. Costs will be estimated from the public and societal payer perspectives. The mean cost per child and per dyad for secondary finding-positive and secondary finding-negative groups will be compared statistically. If important demographic differences are observed between groups, ordinary least-squares regression, log transformation, or other nonparametric technique will be used to compare adjusted mean costs. The ratio of the difference in mean cost to the secondary finding yield will be used to estimate incremental cost-effectiveness. In secondary analyses, effectiveness will be estimated using the number of clinical management changes due to secondary findings or the Clinician-Reported Genetic Testing Utility Index (C-GUIDE) score, a validated measure of clinical utility. Sensitivity analysis will be undertaken to assess the robustness of the findings to variation in key parameters. IMPLICATIONS: This study generates key evidence to inform clinical practice and funding allocation related to secondary finding testing. The inclusion of family members and a new measure of clinical utility represent important advancements in economic evaluation in genomics.

Prevalence of Low-Frequency, Antiviral Resistance Variants in SARS-CoV-2 Isolates in Ontario, Canada, 2020-2023.

Importance: Nirmatrelvir-ritonavir is an oral antiviral medication that improves outcomes in SARS-CoV-2 infections. However, there is concern that antiviral resistance will develop and that these viruses could be selected for after treatment. Objective: To determine the prevalence of low-frequency SARS-CoV-2 variants in patient samples that could be selected for by nirmatrelvir-ritonavir. Design, Setting, and Participants: This retrospective cohort study was conducted at 4 laboratories that serve community hospitals, academic tertiary care centers, and COVID-19 assessment centers in Ontario, Canada. Participants included symptomatic or asymptomatic patients who tested positive for SARS-CoV-2 virus and submitted virus samples for diagnostic testing between March 2020 and January 2023. Exposure: SARS-CoV-2 infection. Main Outcomes and Measures: Samples with sufficient viral load underwent next-generation genome sequencing to identify low-frequency antiviral resistance variants that could not be identified through conventional sequencing. Results: This study included 78 866 clinical samples with next-generation whole-genome sequencing data for SARS-CoV-2. Low-frequency variants in the viral nsp5 gene were identified in 128 isolates (0.16%), and no single variant associated with antiviral resistance was predominate. Conclusions and Relevance: This cohort study of low-frequency variants resistant to nirmatrelvir-ritonavir found that these variants were very rare in samples from patients with SARS-CoV-2, suggesting that selection of these variants by nirmatrelvir-ritonavir following the initiation of treatment may also be rare. Surveillance efforts that involve sequencing of viral isolates should continue to monitor for novel resistance variants as nirmatrelvir-ritonavir is used more broadly.

Chromatin regulators in the TBX1 network confer risk for conotruncal heart defects in 22q11.2DS.

Congenital heart disease (CHD) affecting the conotruncal region of the heart, occurs in 40-50% of patients with 22q11.2 deletion syndrome (22q11.2DS). This syndrome is a rare disorder with relative genetic homogeneity that can facilitate identification of genetic modifiers. Haploinsufficiency of TBX1, encoding a T-box transcription factor, is one of the main genes responsible for the etiology of the syndrome. We suggest that genetic modifiers of conotruncal defects in patients with 22q11.2DS may be in the TBX1 gene network. To identify genetic modifiers, we analyzed rare, predicted damaging variants in whole genome sequence of 456 cases with conotruncal defects and 537 controls, with 22q11.2DS. We then performed gene set approaches and identified chromatin regulatory genes as modifiers. Chromatin genes with recurrent damaging variants include EP400, KAT6A, KMT2C, KMT2D, NSD1, CHD7 and PHF21A. In total, we identified 37 chromatin regulatory genes, that may increase risk for conotruncal heart defects in 8.5% of 22q11.2DS cases. Many of these genes were identified as risk factors for sporadic CHD in the general population. These genes are co-expressed in cardiac progenitor cells with TBX1, suggesting that they may be in the same genetic network. The genes KAT6A, KMT2C, CHD7 and EZH2, have been previously shown to genetically interact with TBX1 in mouse models. Our findings indicate that disturbance of chromatin regulatory genes impact the TBX1 gene network serving as genetic modifiers of 22q11.2DS and sporadic CHD, suggesting that there are some shared mechanisms involving the TBX1 gene network in the etiology of CHD.

Gene copy number variation and pediatric mental health/neurodevelopment in a general population.

We assessed the relationship of gene copy number variation (CNV) in mental health/neurodevelopmental traits and diagnoses, physical health and cognition in a community sample of 7100 unrelated children and youth of European or East Asian ancestry (Spit for Science). Clinically significant or susceptibility CNVs were present in 3.9% of participants and were associated with elevated scores on a continuous measure of attention-deficit/hyperactivity disorder (ADHD) traits (P = 5.0 × 10-3), longer response inhibition (a cognitive deficit found in several mental health and neurodevelopmental disorders; P = 1.0 × 10-2) and increased prevalence of mental health diagnoses (P = 1.9 × 10-6, odds ratio: 3.09), specifically ADHD, autism spectrum disorder anxiety and learning problems/learning disorder (P's < 0.01). There was an increased burden of rare deletions in gene-sets related to brain function or expression in brain associated with more ADHD traits. With the current mental health crisis, our data established a baseline for delineating genetic contributors in pediatric-onset conditions.

Trio RNA sequencing in a cohort of medically complex children.

Genome sequencing (GS) is a powerful test for the diagnosis of rare genetic disorders. Although GS can enumerate most non-coding variation, determining which non-coding variants are disease-causing is challenging. RNA sequencing (RNA-seq) has emerged as an important tool to help address this issue, but its diagnostic utility remains understudied, and the added value of a trio design is unknown. We performed GS plus RNA-seq from blood using an automated clinical-grade high-throughput platform on 97 individuals from 39 families where the proband was a child with unexplained medical complexity. RNA-seq was an effective adjunct test when paired with GS. It enabled clarification of putative splice variants in three families, but it did not reveal variants not already identified by GS analysis. Trio RNA-seq decreased the number of candidates requiring manual review when filtering for de novo dominant disease-causing variants, allowing for the exclusion of 16% of gene-expression outliers and 27% of allele-specific-expression outliers. However, clear diagnostic benefit from the trio design was not observed. Blood-based RNA-seq can facilitate genome analysis in children with suspected undiagnosed genetic disease. In contrast to DNA sequencing, the clinical advantages of a trio RNA-seq design may be more limited.

Genome-Wide Sequencing Identified Rare Genetic Variants for Childhood-Onset Monogenic Lupus.

OBJECTIVE: Genetics play an important role in systemic lupus erythematosus (SLE) pathogenesis. We calculated the prevalence of rare variants in known monogenic lupus genes among children suspected of monogenic lupus. METHODS: We completed paired-end genome-wide sequencing (whole genome sequencing [WGS] or whole exome sequencing) in patients suspected of monogenic lupus, and focused on 36 monogenic lupus genes. We prioritized rare (minor allele frequency < 1%) exonic, nonsynonymous, and splice variants with predicted pathogenicity classified as deleterious variants (Combined Annotation Dependent Depletion [CADD], PolyPhen2, and Sorting Intolerant From Tolerant [SIFT] scores). Additional filtering restricted to predicted damaging variants by considering reported zygosity. In those with WGS (n = 69), we examined copy number variants (CNVs) > 1 kb in size. We created additive non-HLA and HLA SLE genetic risk scores (GRSs) using common SLE-risk single-nucleotide polymorphisms. We tested the relationship between SLE GRSs and the number of rare variants with multivariate logistic models, adjusted for sex, ancestry, and age of diagnosis. RESULTS: The cohort included 71 patients, 80% female, with a mean age at diagnosis of 8.9 (SD 3.2) years. We identified predicted damaging variants in 9 (13%) patients who were significantly younger at diagnosis compared to those without a predicted damaging variant (6.8 [SD 2.1] years vs 9.2 [SD 3.2] years, P = 0.01). We did not identify damaging CNVs. There was no significant association between non-HLA or HLA SLE GRSs and the odds of carrying ≥ 1 rare variant in multivariate analyses. CONCLUSION: In a cohort of patients with suspected monogenic lupus who underwent genome-wide sequencing, 13% carried rare predicted damaging variants for monogenic lupus. Additional studies are needed to validate our findings.

Pharmacogenetic profiling via genome sequencing in children with medical complexity.

BACKGROUND: Children with medical complexity (CMC) are a priority pediatric population, with high resource use and associated costs. Genome-wide sequencing is increasingly organized for CMC early in life as a diagnostic test. Polypharmacy becomes common as CMC age. Clinically relevant pharmacogenetic (PGx) information can be extracted from existing genome sequencing (GS) data via GS-PGx profiling. The role of GS-PGx profiling in the CMC population is unclear. METHODS: Prescribed medications were extracted from care plans of 802 eligible CMC enrolled in a structured Complex Care Program over a 10-year period. Drug-gene associations were annotated using curated Clinical Pharmacogenetics Implementation Consortium data. GS-PGx profiling was then performed for a subset of 50 CMC. RESULTS: Overall, 546 CMC (68%) were prescribed at least one medication with an established PGx association. In the GS-PGx subgroup, 24 (48%) carried variants in pharmacogenes with drug-gene guidelines for one or more of their current medications. All had findings of potential relevance to some medications, including 32 (64%) with variants in CYP2C19 that could affect their metabolism of proton-pump inhibitors. CONCLUSION: GS-PGx profiling at the time of diagnostics-focused genetic testing could be an efficient way to incorporate precision prescribing practices into the lifelong care of CMC. IMPACT: Polypharmacy and genetic test utilization are both common in children with medical complexity. The role of repurposing genome sequencing data for pharmacogenetic profiling in children with medical complexity was previously unclear. We identified a high rate of medication use with clinically relevant drug-gene associations in this priority pediatric population and demonstrated that relevant pharmacogenetic information can be extracted from their existing genome sequencing data. Pharmacogenetic profiling at the time of diagnostics-focused genetic testing could be an efficient way to incorporate precision prescribing practices into the lifelong care of children with medical complexity.

Uncovering the Association Between Complete AZFc Microduplications and Spermatogenic Ability: The First Reported Series.

Purpose This article aims to report the first series of men with complete AZFc microduplications and their clinical and reproductive characteristics. Methods We sampled 3000 men who presented for reproductive urology evaluation from 2012-2020, of which 104 men underwent high-resolution Y-chromosome microarray testing, and five men were identified to have complete AZFc microduplications. Medical, surgical, and reproductive histories were obtained. Semen and hormonal parameters as well as response to fertility therapies were recorded. Results Five men were identified as having complete AZFc microduplications. The mean age was 33.75 years, representing 0.2% (5/3000) of men presenting for fertility investigation, 4.8% (5/104) of men undergoing microarray testing, and 21% (5/24) of men with AZFc abnormalities. Two of the men had prior undescended testicles and one had several autoimmune processes. The mean follicle-stimulating hormone (FSH) was 5.5 IU/L, luteinizing hormone (LH) 3.6 IU/L, and testosterone 14.56 nmol/L. One man was azoospermic, one man alternated between severe oligospermia and rare non-motile sperm, one had variable parameters, with one semen analysis demonstrating azoospermia and a second demonstrating a total motile sperm count (TMSC) of 4 ×106, one man was persistently oligospermic with TMSCs ranging 3.96-12.6 ×106, and one man initially had severe oligospermia, with a mean TMSC of 1.5 ×106, which increased to 21.7 ×106 after intervention (varicocele embolization, clomiphene citrate). This last man then fathered a spontaneous pregnancy. Conclusion AZFc complete microduplications are a rare cause of spermatogenic failure but not an uncommon form of AZFc abnormality. Clinically, they represent a heterogeneous group, having a variable reproductive potential. Cases should be managed on an individual basis.

Deep intronic variant in MVK as a cause for mevalonic aciduria initially presenting as non-syndromic retinitis pigmentosa.

Non-syndromic retinitis pigmentosa (NSRP) is a clinically and genetically heterogeneous group of disorders characterized by progressive degeneration of the rod and cone photoreceptors, often leading to blindness. The evolving association of syndromic genes to cause NSRP and the increasing role of intronic variants in explaining missing heritability in genetic disorders present challenges in establishing conclusive clinical and genetic diagnoses. This study sought to identify and validate the causative genetic variant(s) in a 13-year-old male initially diagnosed with NSRP. Genome sequencing identified a pathogenic missense variant in MVK [NM_000431.3:c.803T>C (p.Ile268Thr)], in trans with a novel intronic variant predicted to create a new donor splice site (c.768+71C>A). Proband cDNA analysis confirmed the inclusion of the first 68 base pairs of intron 8 that resulted in a frameshift in MVK (r.768_769ins[768+1_768+68]) and significantly reduced the expression of reference transcript (17.6%). Patient re-phenotyping revealed ataxia, cerebellar atrophy, elevated urinary mevalonate and LTE4 , in keeping with mild mevalonic aciduria and associated syndromic retinitis pigmentosa. Leakage of reference transcript likely explains the milder phenotype observed in our patient. This is the first association of a deep intronic splice variant to cause MVK-related disorder. This report highlights the importance of variant validation and patient re-phenotyping in establishing accurate diagnosis in the era of genome sequencing.

Genome-wide tandem repeat expansions contribute to schizophrenia risk.

Tandem repeat expansions (TREs) can cause neurological diseases but their impact in schizophrenia is unclear. Here we analyzed genome sequences of adults with schizophrenia and found that they have a higher burden of TREs that are near exons and rare in the general population, compared with non-psychiatric controls. These TREs are disproportionately found at loci known to be associated with schizophrenia from genome-wide association studies, in individuals with clinically-relevant genetic variants at other schizophrenia loci, and in families where multiple individuals have schizophrenia. We showed that rare TREs in schizophrenia may impact synaptic functions by disrupting the splicing process of their associated genes in a loss-of-function manner. Our findings support the involvement of genome-wide rare TREs in the polygenic nature of schizophrenia.

Comparing genome sequencing technologies to improve rare disease diagnostics: a protocol for the evaluation of a pilot project, Genome-wide Sequencing Ontario.

BACKGROUND: Genome-wide sequencing has emerged as a promising strategy for the timely diagnosis of rare diseases, but it is not yet available as a clinical test performed in Canadian diagnostic laboratories. We describe the protocol for evaluating a 2-year pilot project, Genome-wide Sequencing Ontario, to offer high-quality clinical genome-wide sequencing in Ontario, Canada. METHODS: The Genome-wide Sequencing Ontario protocol was codesigned by the Ontario Ministry of Health, the Hospital for Sick Children in Toronto and the Children's Hospital of Eastern Ontario in Ottawa. Enrolment of a prospective cohort of patients began on Apr. 1, 2021. Eligible cases with blood samples available for the index case and both parents (i.e., trios) are randomized to receive exome sequencing or genome sequencing. We will collect patient-level data and ascertain costs associated with the laboratory workflow for exome sequencing and genome sequencing. We will compare point estimates for the diagnostic utility and timeliness of exome sequencing and genome sequencing, and we will determine an incremental cost-effectiveness ratio (expressed as the incremental cost of genome sequencing versus exome sequencing per additional patient with a causal variant detected). INTERPRETATION: Findings from this work will provide robust evidence for the diagnostic utility, cost-effectiveness and timeliness of exome sequencing and genome sequencing, and will be disseminated via academic publications and policy briefs. Findings will inform provincial and cross-provincial policy related to the long-term organization, delivery and reimbursement of clinical-grade genome diagnostics for rare disease.

Genomics4RD: An integrated platform to share Canadian deep-phenotype and multiomic data for international rare disease gene discovery.

Despite recent progress in the understanding of the genetic etiologies of rare diseases (RDs), a significant number remain intractable to diagnostic and discovery efforts. Broad data collection and sharing of information among RD researchers is therefore critical. In 2018, the Care4Rare Canada Consortium launched the project C4R-SOLVE, a subaim of which was to collect, harmonize, and share both retrospective and prospective Canadian clinical and multiomic data. Here, we introduce Genomics4RD, an integrated web-accessible platform to share Canadian phenotypic and multiomic data between researchers, both within Canada and internationally, for the purpose of discovering the mechanisms that cause RDs. Genomics4RD has been designed to standardize data collection and processing, and to help users systematically collect, prioritize, and visualize participant information. Data storage, authorization, and access procedures have been developed in collaboration with policy experts and stakeholders to ensure the trusted and secure access of data by external researchers. The breadth and standardization of data offered by Genomics4RD allows researchers to compare candidate disease genes and variants between participants (i.e., matchmaking) for discovery purposes, while facilitating the development of computational approaches for multiomic data analyses and enabling clinical translation efforts for new genetic technologies in the future.

Trio genome sequencing for developmental delay and pediatric heart conditions: A comparative microcost analysis.

PURPOSE: Genome sequencing (GS) can aid clinical management of multiple pediatric conditions. Insurers require accurate cost information to inform funding and implementation decisions. The objective was to compare the laboratory workflows and microcosts of trio GS testing in children with developmental delay (DD) and in children with cardiac conditions. METHODS: Cost items related to each step in trio GS (child and 2 parents) for both populations were identified and measured. Program costs over 5 years were estimated. Probabilistic and deterministic analyses were conducted. RESULTS: The mean cost per trio GS was CAD$6634.11 (95% CI = 6352.29-6913.40) for DD and CAD$8053.10 (95% CI = 7699.30-8558.10) for cardiac conditions. The 5-year program cost was CAD$28.11 million (95% CI = 26.91-29.29) for DD and CAD$5.63 million (95% CI = 5.38-5.98) for cardiac conditions. Supplies constituted the largest cost component for both populations. The higher cost per sample for the population with cardiac conditions was due to the inclusion of pharmacogenomics, higher bioinformatics labor costs, and a more labor intensive case review. CONCLUSION: This analysis indicated important variation in trio GS workflow and costs between pediatric populations in a single institution. Enhanced understanding of the clinical utility and costs of GS can inform harmonization and implementation decision-making.