Pyridine Nucleotide-Disulfide Oxidoreductase Domain 2 (PYROXD2; previously called YueF) is a mitochondrial inner membrane/matrix-residing protein and is reported to regulate mitochondrial function. The clinical importance of PYROXD2 has been unclear, and little is known of the protein's precise biological function. In the present paper, we report biallelic variants in PYROXD2 identified by genome sequencing in a patient with suspected mitochondrial disease. The child presented with acute neurological deterioration, unresponsive episodes, and extreme metabolic acidosis, and received rapid genomic testing. He died shortly after. Magnetic resonance imaging (MRI) brain imaging showed changes resembling Leigh syndrome, one of the more common childhood mitochondrial neurological diseases. Functional studies in patient fibroblasts showed a heightened sensitivity to mitochondrial metabolic stress and increased mitochondrial superoxide levels. Quantitative proteomic analysis demonstrated decreased levels of subunits of the mitochondrial respiratory chain complex I, and both the small and large subunits of the mitochondrial ribosome, suggesting a mitoribosomal defect. Our findings support the critical role of PYROXD2 in human cells, and suggest that the biallelic PYROXD2 variants are associated with mitochondrial dysfunction, and can plausibly explain the child's clinical presentation.
Abstract Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes 1 . Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome 2 . We identify an 18 base pair region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals in whom it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologues. Using RNA sequencing, we show how 5′ splice-site use is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 base pair region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.
Abstract Internationally, the practice of offering additional findings (AFs) when undertaking a clinically indicated genomic test differs. In the USA, the recommendation is to include analysis for AFs alongside diagnostic analysis, unless a patient opts‐out, whereas European and Canadian guidelines recommend opt‐in models. These guidelines all consider the offer of AFs as an activity concurrent with the offer of diagnostic testing. This paper describes a novel two‐step model for managing AFs within the healthcare system in Victoria, Australia and presents the study protocol for its evaluation. Adults who have received results of diagnostic whole exome sequencing undertaken within the healthcare system are invited to attend a genetic counseling appointment to consider reanalysis of their stored genomic data for AFs. The evaluation protocol addresses uptake, decision‐making, understanding, counseling challenges, and explores preferences for future models of care. Recruitment commenced in November 2017 and will cease when 200 participants have been approached. When the study is concluded, the evaluation results will contribute to the evidence base guiding approaches to counseling and models of care for AFs.
Tuberous sclerosis complex (TSC) is a multi-system genetic disorder. Most patients have germline mutations in TSC1 or TSC2 but, 10%–15% patients do not have TSC1/TSC2 mutations detected on routine clinical genetic testing. We investigated the contribution of low-level mosaic TSC1/TSC2 mutations in unsolved sporadic patients and families with TSC. Thirty-one sporadic TSC patients negative on routine testing and eight families with suspected parental mosaicism were sequenced using deep panel sequencing followed by droplet digital polymerase chain reaction. Pathogenic variants were found in 22/31 (71%) unsolved sporadic patients, 16 were mosaic (median variant allele fraction [VAF] 6.8% in blood) and 6 had missed germline mutations. Parental mosaicism was detected in 5/8 families (median VAF 1% in blood). Clinical testing laboratories typically only report pathogenic variants with allele fractions above 10%. Our findings highlight the critical need to change laboratory practice by implementing higher sensitivity assays to improve diagnostic yield, inform patient management and guide reproductive counseling.
The ability of cytotoxic lymphocytes (CL) to eliminate virus-infected or cancerous target cells through the granule exocytosis death pathway is critical to immune homoeostasis. Congenital loss of CL function due to bi-allelic mutations in PRF1, UNC13D, STX11 or STXBP2 leads to a potentially fatal immune dysregulation, familial haemophagocytic lymphohistiocytosis (FHL). This occurs due to the failure of CLs to release functional pore-forming protein perforin and, therefore, inability to kill the target cell. Bi-allelic mutations in partner proteins STXBP2 or STX11 impair CL cytotoxicity due to failed docking/fusion of cytotoxic secretory granules to the synaptic cleft between the lymphocyte and a target cell. One unique feature of STXBP2- and STX11- deficient patient cytotoxic lymphocytes is that their short-term in vitro treatment with a low concentration of IL-2 partially or completely restores natural killer cell degranulation and cytotoxicity, suggesting the existence of a secondary, yet unknown, pathway for secretory granule exocytosis. In the current report, we studied natural killer and T cell function in an individual with late presentation of FHL due to hypomorphic bi-allelic mutations in STXBP2. Intriguingly, in addition to the expected alterations in the STXBP2 and STX11 proteins, we also observed a concomitant significant reduction in the expression of homologous STXBP1 protein, which had never been implicated in CL function. Further analysis of human NK and T cells demonstrated a functional role for STXBP1 in natural killer and CD8+ T-cell cytotoxicity, where it appears to be responsible for as much as 50% of their cytotoxic activity. This discovery provides an explanation for a paradoxical recovery of natural killer cell function in patients with STXBP2 (and, potentially, STX11) mutations, and suggests a unique and previously unappreciated interplay between STXBP/Munc proteins regulating the same essential granule exocytosis pathway.
Abstract Canavan disease (CD) is a neurodegenerative disorder caused by biallelic disease-causing variants in the ASPA gene. Here, we utilized long-read sequencing (LRS) to investigate eight individuals clinically diagnosed with Canavan disease but without definitive genetic diagnoses. Our analyses identified a recurring previously unreported intronic SVA_E retrotransposon insertion within ASPA in all eight individuals. Surprisingly, the frequency of this variant in population databases suggests it is the most common pathogenic variant in ASPA and should be evaluated in diagnostic testing and carrier screening for CD. Additionally, this finding has implications for the broader rare disease community, as it highlights a substantial blind spot in standard short-read diagnostic pipelines, which historically have missed or overlooked these types of insertions. This discovery highlights the power of emerging technologies, such as LRS and RNA-sequencing (RNA-seq), to bring new classes of variants into diagnostic utility for genetic disorders like CD.
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