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.

A framework for an evidence-based gene list relevant to autism spectrum disorder.

Autism spectrum disorder (ASD) is often grouped with other brain-related phenotypes into a broader category of neurodevelopmental disorders (NDDs). In clinical practice, providers need to decide which genes to test in individuals with ASD phenotypes, which requires an understanding of the level of evidence for individual NDD genes that supports an association with ASD. Consensus is currently lacking about which NDD genes have sufficient evidence to support a relationship to ASD. Estimates of the number of genes relevant to ASD differ greatly among research groups and clinical sequencing panels, varying from a few to several hundred. This Roadmap discusses important considerations necessary to provide an evidence-based framework for the curation of NDD genes based on the level of information supporting a clinically relevant relationship between a given gene and ASD.

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.

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.

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.

Correction: Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease.

[This corrects the article DOI: 10.1371/journal.pgen.1009679.].

Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease.

Numerous genetic studies have established a role for rare genomic variants in Congenital Heart Disease (CHD) at the copy number variation (CNV) and de novo variant (DNV) level. To identify novel haploinsufficient CHD disease genes, we performed an integrative analysis of CNVs and DNVs identified in probands with CHD including cases with sporadic thoracic aortic aneurysm. We assembled CNV data from 7,958 cases and 14,082 controls and performed a gene-wise analysis of the burden of rare genomic deletions in cases versus controls. In addition, we performed variation rate testing for DNVs identified in 2,489 parent-offspring trios. Our analysis revealed 21 genes which were significantly affected by rare CNVs and/or DNVs in probands. Fourteen of these genes have previously been associated with CHD while the remaining genes (FEZ1, MYO16, ARID1B, NALCN, WAC, KDM5B and WHSC1) have only been associated in small cases series or show new associations with CHD. In addition, a systems level analysis revealed affected protein-protein interaction networks involved in Notch signaling pathway, heart morphogenesis, DNA repair and cilia/centrosome function. Taken together, this approach highlights the importance of re-analyzing existing datasets to strengthen disease association and identify novel disease genes and pathways.

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.

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.

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.

Data sharing to improve concordance in variant interpretation across laboratories: results from the Canadian Open Genetics Repository.

BACKGROUND: This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation. METHODS: Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin. RESULTS: Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants. CONCLUSIONS: The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.

Hereditary Mucin Deficiency Caused by Biallelic Loss of Function of MUC5B.

Rationale: Mucin homeostasis is fundamental to airway health. Upregulation of airway mucus glycoprotein MUC5B is observed in diverse common lung diseases and represents a potential therapeutic target. In mice, Muc5b is required for mucociliary clearance and for controlling inflammation after microbial exposure. The consequences of its loss in humans are unclear. Objectives: The goal of this study was to identify and characterize a family with congenital absence of MUC5B protein. Methods: We performed whole-genome sequencing in an adult proband with unexplained bronchiectasis, impaired pulmonary function, and repeated Staphylococcus aureus infection. Deep phenotyping over a 12-year period included assessments of pulmonary radioaerosol mucociliary clearance. Genotyping with reverse phenotyping was organized for eight family members. Extensive experiments, including immunofluorescence staining and mass spectrometry for mucins, were performed across accessible sample types. Measurements and Main Results: The proband, and her symptomatic sibling who also had extensive sinus disease with nasal polyps, were homozygous for a novel splicing variant in the MUC5B gene (NM_002458.2: c.1938 + 1G>A). MUC5B was absent from saliva, sputum, and nasal samples. Mucociliary clearance was impaired in the proband, and large numbers of apoptotic macrophages were present in sputum. Three siblings heterozygous for the familial MUC5B variant were asymptomatic but had a shared pattern of mild lung function impairments. Conclusions: Congenital absence of MUC5B defines a new category of genetic respiratory disease. The human phenotype is highly concordant with that of the Muc5b-/- murine model. Further study of individuals with decreased MUC5B production could provide unique mechanistic insights into airway mucus biology.

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.

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.

Within-family influences on dimensional neurobehavioral traits in a high-risk genetic model.

BACKGROUND: Genotype-first and within-family studies can elucidate factors that contribute to psychiatric illness. Combining these approaches, we investigated the patterns of influence of parental scores, a high-impact variant, and schizophrenia on dimensional neurobehavioral phenotypes implicated in major psychiatric disorders. METHODS: We quantitatively assessed cognitive (FSIQ, VIQ, PIQ), social, and motor functioning in 82 adult individuals with a de novo 22q11.2 deletion (22 with schizophrenia), and 148 of their unaffected parents. We calculated within-family correlations and effect sizes of the 22q11.2 deletion and schizophrenia, and used linear regressions to assess contributions to neurobehavioral measures. RESULTS: Proband-parent intra-class correlations (ICC) were significant for cognitive measures (e.g. FSIQ ICC = 0.549, p < 0.0001), but not for social or motor measures. Compared to biparental scores, the 22q11.2 deletion conferred significant impairments for all phenotypes assessed (effect sizes -1.39 to -2.07 s.d.), strongest for PIQ. There were further decrements in those with schizophrenia. Regression models explained up to 37.7% of the variance in IQ and indicated that for proband IQ, parental IQ had larger effects than schizophrenia. CONCLUSIONS: This study, for the first time, disentangles the impact of a high-impact variant from the modifying effects of parental scores and schizophrenia on relevant neurobehavioral phenotypes. The robust proband-parent correlations for cognitive measures, independent of the impact of the 22q11.2 deletion and of schizophrenia, suggest that, for certain phenotypes, shared genetic variation plays a significant role in expression. Molecular genetic and predictor studies are needed to elucidate shared factors and their contribution to psychiatric illness in this and other high-risk groups.

Genome sequencing among children with medical complexity: What constitutes value from parents' perspective?

Genome sequencing (GS) has demonstrated high diagnostic yield in pediatric patients with complex, clinically heterogeneous presentations. Emerging evidence shows generally favorable experiences for patients and families receiving GS. As a result, implementation of GS in pediatrics is gaining momentum. To inform implementation, we conducted a qualitative study to explore the personal utility of GS for parents of children with medical complexity (CMC). GS was performed at an academic tertiary-care center for CMC for whom a genetic etiology was suspected. Following the return of GS results, semi-structured interviews were conducted with 14 parents about their child's diagnostic journey. Of the children whose parents were interviewed, six children received a diagnosis, two received a possible diagnosis, and six did not receive a diagnosis. A predominantly deductive thematic analysis approach to the interview data was used by applying Kohler's personal utility framework to understand affective, cognitive, behavioral and social impacts of GS. Both the diagnosed and undiagnosed groups experienced enhanced emotion-focused coping (affective). The diagnosed group experienced favorable utility related to knowledge of condition (cognitive) and communication with relatives (behavioral). A domain beyond Kohler's framework related to the presence or absence of GS impact on medical management was also described by parents. The deployment of GS late in the diagnostic odyssey and the limited knowledge available for the rare genetic disorders diagnosed in this cohort appeared to diminish the perceived utility of GS. As GS capabilities continue to evolve at a rapid pace and become available earlier in the diagnostic journey, it is important to consider the impact and timing of testing on parents of CMC.

Bi-allelic mutations of LONP1 encoding the mitochondrial LonP1 protease cause pyruvate dehydrogenase deficiency and profound neurodegeneration with progressive cerebellar atrophy.

LonP1 is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. We identified a novel homozygous missense LONP1 variant, c.2282 C > T, (p.Pro761Leu), by whole-exome and Sanger sequencing in two siblings born to healthy consanguineous parents. Both siblings presented with stepwise regression during infancy, profound hypotonia and muscle weakness, severe intellectual disability and progressive cerebellar atrophy on brain imaging. Muscle biopsy revealed the absence of ragged-red fibers, however, scattered cytochrome c oxidase-negative staining and electron dense mitochondrial inclusions were observed. Primary cultured fibroblasts from the siblings showed normal levels of mtDNA and mitochondrial transcripts, and normal activities of oxidative phosphorylation complexes I through V. Interestingly, fibroblasts of both siblings showed glucose-repressed oxygen consumption compared to their mother, whereas galactose and palmitic acid utilization were similar. Notably, the siblings' fibroblasts had reduced pyruvate dehydrogenase (PDH) activity and elevated intracellular lactate:pyruvate ratios, whereas plasma ratios were normal. We demonstrated that in the siblings' fibroblasts, PDH dysfunction was caused by increased levels of the phosphorylated E1α subunit of PDH, which inhibits enzyme activity. Blocking E1α phosphorylation activated PDH and reduced intracellular lactate concentrations. In addition, overexpressing wild-type LonP1 in the siblings' fibroblasts down-regulated phosphoE1α. Furthermore, in vitro studies demonstrated that purified LonP1-P761L failed to degrade phosphorylated E1α, in contrast to wild-type LonP1. We propose a novel mechanism whereby homozygous expression of the LonP1-P761L variant leads to PDH deficiency and energy metabolism dysfunction, which promotes severe neurologic impairment and neurodegeneration.

A Comprehensive Workflow for Read Depth-Based Identification of Copy-Number Variation from Whole-Genome Sequence Data.

A remaining hurdle to whole-genome sequencing (WGS) becoming a first-tier genetic test has been accurate detection of copy-number variations (CNVs). Here, we used several datasets to empirically develop a detailed workflow for identifying germline CNVs >1 kb from short-read WGS data using read depth-based algorithms. Our workflow is comprehensive in that it addresses all stages of the CNV-detection process, including DNA library preparation, sequencing, quality control, reference mapping, and computational CNV identification. We used our workflow to detect rare, genic CNVs in individuals with autism spectrum disorder (ASD), and 120/120 such CNVs tested using orthogonal methods were successfully confirmed. We also identified 71 putative genic de novo CNVs in this cohort, which had a confirmation rate of 70%; the remainder were incorrectly identified as de novo due to false positives in the proband (7%) or parental false negatives (23%). In individuals with an ASD diagnosis in which both microarray and WGS experiments were performed, our workflow detected all clinically relevant CNVs identified by microarrays, as well as additional potentially pathogenic CNVs < 20 kb. Thus, CNVs of clinical relevance can be discovered from WGS with a detection rate exceeding microarrays, positioning WGS as a single assay for genetic variation detection.

Genome sequencing for detection of pathogenic deep intronic variation: A clinical case report illustrating opportunities and challenges.

Variants in JAM3 have been reported in four families manifesting a severe autosomal recessive disorder characterized by hemorrhagic destruction of the brain, subependymal calcification, and cataracts. We describe a 7-year-old male with a similar presentation found by research-based quad genome sequencing to have two novel splicing variants in trans in JAM3, including one deep intronic variant (NM_032801.4: c.256+1260G>C) not detectable by standard exome sequencing. Targeted sequencing of RNA isolated from transformed lymphoblastoid cell lines confirmed that each of the two variants has a deleterious effect on JAM3 mRNA splicing. The role for genome sequencing as a clinical diagnostic test extends to those patients with phenotypes strongly suggestive of a specific Mendelian disorder, especially when the causal genetic variant(s) are not found by a more targeted approach. Barriers to diagnosis via identification of pathogenic deep intronic variation include lack of laboratory consensus regarding in silico splicing prediction tools and limited access to clinically validated confirmatory RNA experiments.