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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. [ABSTRACT FROM AUTHOR]

Activating variants in the PIK3CA gene cause a heterogeneous spectrum of disorders that involve congenital or early‐onset segmental/focal overgrowth, now referred to as PIK3CA‐related overgrowth spectrum (PROS). Historically, the clinical diagnoses of patients with PROS included a range of distinct syndromes, including CLOVES syndrome, dysplastic megalencephaly, hemimegalencephaly, focal cortical dysplasia, Klippel–Trenaunay syndrome, CLAPO syndrome, fibroadipose hyperplasia or overgrowth, hemihyperplasia multiple lipomatosis, and megalencephaly capillary malformation‐polymicrogyria (MCAP) syndrome. MCAP is a sporadic overgrowth disorder that exhibits core features of progressive megalencephaly, vascular malformations, distal limb malformations, cortical brain malformations, and connective tissue dysplasia. In 2012, our research group contributed to the identification of predominantly mosaic, gain‐of‐function variants in PIK3CA as an underlying genetic cause of the syndrome. Mosaic variants are technically more difficult to detect and require implementation of more sensitive sequencing technologies and less stringent variant calling algorithms. In this study, we demonstrated the utility of deep sequencing using the Illumina TruSight Oncology 500 (TSO500) sequencing panel in identifying variants with low allele fractions in a series of patients with PROS and suspected mosaicism: pathogenic, mosaic PIK3CA variants were identified in all 13 individuals, including 6 positive controls. This study highlights the importance of screening for low‐level mosaic variants in PROS patients. The use of targeted panels with deep sequencing in clinical genetic testing laboratories would improve diagnostic yield and accuracy within this patient population. [ABSTRACT FROM AUTHOR]

Background: Intronic variants outside the canonical splice site are challenging to interpret and therefore likely represent an underreported cause of human disease. Autosomal recessive variants in DYNC2H1 are associated with short‐rib thoracic dysplasia 3 with or without polydactyly (SRTD3), a clinically heterogeneous disease generally presenting with short ribs, shortened tubular bones, narrow thorax and acetabular roof anomalies. We describe a case of SRTD3 with compound heterozygous frameshift and intronic variants and highlight the essential role of RNA sequencing (RNA‐Seq) in variant interpretation. Methods: Following inconclusive clinical genetic testing identifying a likely pathogenic frameshift variant and an intronic variant of uncertain significance (VUS) in DYNC2H1 in trans, the family enrolled in the Care4Rare Canada research program, where RNA‐Seq studies were performed. Results: The proband presented with post‐axial polydactyly of all four limbs, a significantly small chest with a pectus excavatum and anterior flaring of the ribs. RNA‐Seq investigations revealed a novel splice junction as a result of the intronic VUS and significantly decreased DYNC2H1 gene expression in the proband. Conclusion: This case demonstrates the diagnostic utility of RNA‐Seq for variant interpretation following inconclusive clinical testing, which can ultimately lead to diagnosis for patients with rare disease. [ABSTRACT FROM AUTHOR]

We examined the utility of clinical and research processes in the reanalysis of publicly‐funded clinical exome sequencing data in Ontario, Canada. In partnership with eight sites, we recruited 287 families with suspected rare genetic diseases tested between 2014 and 2020. Data from seven laboratories was reanalyzed with the referring clinicians. Reanalysis of clinically relevant genes identified diagnoses in 4% (13/287); four were missed by clinical testing. Translational research methods, including analysis of novel candidate genes, identified candidates in 21% (61/287). Of these, 24 families have additional evidence through data sharing to support likely diagnoses (8% of cohort). This study indicates few diagnoses are missed by clinical laboratories, the incremental gain from reanalysis of clinically‐relevant genes is modest, and the highest yield comes from validation of novel disease‐gene associations. Future implementation of translational research methods, including continued reporting of compelling genes of uncertain significance by clinical laboratories, should be considered to maximize diagnoses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We thank Compute Canada for the Research Allocation Competitions allocation, which facilitated our analysis of the IMAG INE and EGA genomes, and Julia Handra for coordinating the STR molecular testing of the clinical samples. Genome-wide sequencing as a first-tier screening test for short tandem repeat expansions. [Extracted from the article]

Partial duplications of genes can be challenging to detect and interpret and, therefore, likely represent an underreported cause of human disease. X-linked dominant variants in ATRX are associated with Alpha-thalassemia/impaired intellectual development syndrome, X-linked (ATR-X syndrome), a clinically heterogeneous disease generally presenting with intellectual disability, hypotonia, characteristic facies, genital anomalies, and alpha-thalassemia. We describe an affected male with a de novo hemizygous intragenic duplication of ~43.6 kb in ATRX, detected by research genome sequencing following non-diagnostic clinical testing. RNA sequencing and DNA methylation episignature analyses were central in variant interpretation, and this duplication was subsequently interpreted as disease-causing. This represents the smallest reported tandem duplication within ATRX associated with disease. This case demonstrates the diagnostic utility of integrating multiple omics technologies, which can ultimately lead to a definitive diagnosis for rare disease patients., (© 2023. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

Background and Objectives: The human genome contains ∼20,000 genes, each of which has its own set of complex regulatory systems to govern precise expression in each developmental stage and cell type. Here, we report a female patient with congenital weakness, respiratory failure, skeletal dysplasia, contractures, short stature, intellectual delay, respiratory failure, and amenorrhea who presented to Medical Genetics service with no known cause for her condition., Methods: Whole-exome and whole-genome sequencing were conducted, as well as investigational functional studies to assess the effect of SOX8 variant., Results: The patient was found to have biallelic SOX8 variants (NM_014587.3:c.422+5G>C; c.583dup p.(His195ProfsTer11)). SOX8 is a transcriptional regulator, which is predicted to be imprinted (expressed from only one parental allele), but this has not yet been confirmed. We provide evidence that while SOX8 was maternally expressed in adult-derived fibroblasts and lymphoblasts, it was biallelically expressed in other cell types and therefore suggest that biallelic variants are associated with this recessive condition. Functionally, we showed that the paternal variant had the capacity to affect mRNA splicing while the maternal variant resulted in low levels of a truncated protein, which showed decreased binding at and altered expression of SOX8 targets., Discussion: Our findings associate SOX8 variants with this novel condition, highlight how complex genome regulation can complicate novel disease-gene identification, and provide insight into the molecular pathogenesis of this disease., Competing Interests: The authors report no relevant disclosures. Go to Neurology.org/NG for full disclosures, (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Genome-wide sequencing (GWS) is a standard of care for diagnosis of suspected genetic disorders, but the proportion of patients found to have pathogenic or likely pathogenic variants ranges from less than 30% to more than 60% in reported studies. It has been suggested that the diagnostic rate can be improved by interpreting genomic variants in the context of each affected individual's full clinical picture and by regular follow-up and reinterpretation of GWS laboratory results. Trio exome sequencing was performed in 415 families and trio genome sequencing in 85 families in the CAUSES study. The variants observed were interpreted by a multidisciplinary team including laboratory geneticists, bioinformaticians, clinical geneticists, genetic counselors, pediatric subspecialists, and the referring physician, and independently by a clinical laboratory using standard American College of Medical Genetics and Genomics (ACMG) criteria. Individuals were followed for an average of 5.1 years after testing, with clinical reassessment and reinterpretation of the GWS results as necessary. The multidisciplinary team established a diagnosis of genetic disease in 43.0% of the families at the time of initial GWS interpretation, and longitudinal follow-up and reinterpretation of GWS results produced new diagnoses in 17.2% of families whose initial GWS interpretation was uninformative or uncertain. Reinterpretation also resulted in rescinding a diagnosis in four families (1.9%). Of the families studied, 33.6% had ACMG pathogenic or likely pathogenic variants related to the clinical indication. Close collaboration among clinical geneticists, genetic counselors, laboratory geneticists, bioinformaticians, and individuals' primary physicians, with ongoing follow-up, reanalysis, and reinterpretation over time, can improve the clinical value of GWS., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Autism spectrum disorder (ASD) is a constellation of neurodevelopmental disorders with high phenotypic and genetic heterogeneity, complicating the discovery of causative genes. Through a forward genetics approach selecting for defective vocalization in mice, we identified Kdm5a as a candidate ASD gene. To validate our discovery, we generated a Kdm5a knockout mouse model ( Kdm5a -/- ) and confirmed that inactivating Kdm5a disrupts vocalization. In addition, Kdm5a -/- mice displayed repetitive behaviors, sociability deficits, cognitive dysfunction, and abnormal dendritic morphogenesis. Loss of KDM5A also resulted in dysregulation of the hippocampal transcriptome. To determine if KDM5A mutations cause ASD in humans, we screened whole exome sequencing and microarray data from a clinical cohort. We identified pathogenic KDM5A variants in nine patients with ASD and lack of speech. Our findings illustrate the power and efficacy of forward genetics in identifying ASD genes and highlight the importance of KDM5A in normal brain development and function., Competing Interests: LE, IT, NN, JR, SL, KK, XL, PA, MT, AG, AH, PZ, HA, AR, KH, MA, CW, BD, RL, MC, MF, RR, RA, EE, RM, HH, AL, BB, MC No competing interests declared, (© 2020, El Hayek et al.)