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Purpose: To evaluate the diagnostic utility of publicly funded clinical exome sequencing (ES) for patients with suspected rare genetic diseases., Methods: We prospectively enrolled 297 probands who met eligibility criteria and received ES across 5 sites in Ontario, Canada, and extracted data from medical records and clinician surveys. Using the Fryback and Thornbury Efficacy Framework, we assessed diagnostic accuracy by examining laboratory interpretation of results and assessed diagnostic thinking by examining the clinical interpretation of results and whether clinical-molecular diagnoses would have been achieved via alternative hypothetical molecular tests., Results: Laboratories reported 105 molecular diagnoses and 165 uncertain results in known and novel genes. Of these, clinicians interpreted 102 of 105 (97%) molecular diagnoses and 6 of 165 (4%) uncertain results as clinical-molecular diagnoses. The 108 clinical-molecular diagnoses were in 104 families (35% diagnostic yield). Each eligibility criteria resulted in diagnostic yields of 30% to 40%, and higher yields were achieved when >2 eligibility criteria were met (up to 45%). Hypothetical tests would have identified 61% of clinical-molecular diagnoses., Conclusion: We demonstrate robustness in eligibility criteria and high clinical validity of laboratory results from ES testing. The importance of ES was highlighted by the potential 40% of patients that would have gone undiagnosed without this test., Competing Interests: Conflict of Interest The authors declare no conflicts of interests., (Copyright © 2023. Published by Elsevier Inc.)

Purpose: We sought to delineate a multisystem disorder caused by recessive cysteine-rich with epidermal growth factor-like domains 1 (CRELD1) gene variants., Methods: The impact of CRELD1 variants was characterized through an international collaboration utilizing next-generation DNA sequencing, gene knockdown, and protein overexpression in Xenopus tropicalis, and in vitro analysis of patient immune cells., Results: Biallelic variants in CRELD1 were found in 18 participants from 14 families. Affected individuals displayed an array of phenotypes involving developmental delay, early-onset epilepsy, and hypotonia, with about half demonstrating cardiac arrhythmias and some experiencing recurrent infections. Most harbored a frameshift in trans with a missense allele, with 1 recurrent variant, p.(Cys192Tyr), identified in 10 families. X tropicalis tadpoles with creld1 knockdown displayed developmental defects along with increased susceptibility to induced seizures compared with controls. Additionally, human CRELD1 harboring missense variants from affected individuals had reduced protein function, indicated by a diminished ability to induce craniofacial defects when overexpressed in X tropicalis. Finally, baseline analyses of peripheral blood mononuclear cells showed similar proportions of immune cell subtypes in patients compared with healthy donors., Conclusion: This patient cohort, combined with experimental data, provide evidence of a multisystem clinical syndrome mediated by recessive variants in CRELD1., Competing Interests: Conflict of Interest Two authors report part ownership of startup companies unrelated to this work: Qiyas Higher Health (Saquib A. Lakhani) and Victory Genomics (Saquib A. Lakhani and Mustafa K. Khokha). Kirsty McWalter is an employee of GeneDx. Kimberly Nugent is currently an employee of Cooper Surgical. Bryan Mak is currently an employee of Genome Medical. All other authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Au-Kline syndrome (AKS) is a neurodevelopmental disorder associated with multiple malformations and a characteristic facial gestalt. The first individuals ascertained carried de novo loss-of-function (LoF) variants in HNRNPK. Here, we report 32 individuals with AKS (26 previously unpublished), including 13 with de novo missense variants. We propose new clinical diagnostic criteria for AKS that differentiate it from the clinically overlapping Kabuki syndrome and describe a significant phenotypic expansion to include individuals with missense variants who present with subtle facial features and few or no malformations. Many gene-specific DNA methylation (DNAm) signatures have been identified for neurodevelopmental syndromes. Because HNRNPK has roles in chromatin and epigenetic regulation, we hypothesized that pathogenic variants in HNRNPK may be associated with a specific DNAm signature. Here, we report a unique DNAm signature for AKS due to LoF HNRNPK variants, distinct from controls and Kabuki syndrome. This DNAm signature is also identified in some individuals with de novo HNRNPK missense variants, confirming their pathogenicity and the phenotypic expansion of AKS to include more subtle phenotypes. Furthermore, we report that some individuals with missense variants have an "intermediate" DNAm signature that parallels their milder clinical presentation, suggesting the presence of an epi-genotype phenotype correlation. In summary, the AKS DNAm signature may help elucidate the underlying pathophysiology of AKS. This DNAm signature also effectively supported clinical syndrome delineation and is a valuable aid for variant interpretation in individuals where a clinical diagnosis of AKS is unclear, particularly for mild presentations., Competing Interests: Declaration of interests H.T.B. is a consultant for Mahzi therapeutics., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: This study investigated the diagnostic utility of nontargeted genomic testing in patients with pediatric heart disease., Methods: We analyzed genome sequencing data of 111 families with cardiac lesions for rare, disease-associated variation., Results: In 14 families (12.6%), we identified causative variants: seven were de novo (ANKRD11, KMT2D, NR2F2, POGZ, PTPN11, PURA, SALL1) and six were inherited from parents with no or subclinical heart phenotypes (FLT4, DNAH9, MYH11, NEXMIF, NIPBL, PTPN11). Outcome of the testing was associated with the presence of extracardiac features (p = 0.02), but not a positive family history for cardiac lesions (p = 0.67). We also report novel plausible gene-disease associations for tetralogy of Fallot/pulmonary stenosis (CDC42BPA, FGD5), hypoplastic left or right heart (SMARCC1, TLN2, TRPM4, VASP), congenitally corrected transposition of the great arteries (UBXN10), and early-onset cardiomyopathy (TPCN1). The identified candidate genes have critical functions in heart development, such as angiogenesis, mechanotransduction, regulation of heart size, chromatin remodeling, or ciliogenesis., Conclusion: This data set demonstrates the diagnostic and scientific value of genome sequencing in pediatric heart disease, anticipating its role as a first-tier diagnostic test. The genetic heterogeneity will necessitate large-scale genomic initiatives for delineating novel gene-disease associations.

Krabbe disease is an autosomal recessive demyelinating lysosomal storage disorder caused by a deficiency of galactocerebrosidase. The adult-onset variant is very rare. Hematopoietic stem cell transplantation (HSCT) is reported to be successful in treating infants with Krabbe disease prior to the onset of symptoms, but there are no reported cases of its use for adult-onset disease. We report the first follow-up data for a patient with adult-onset Krabbe disease who underwent HSCT at age 41, 16 years after the onset of symptoms. HSCT resulted in a sustained normalization of peripheral GALC enzyme activity, halted the progression of symptoms at 24 months post-allograft, and led to improvements in gait and balance. Serial imaging also confirmed that no significant progression of demyelination has occurred. Although long-term follow-up is needed to confirm the effects of HSCT, our 24-month results suggest that HSCT is a viable therapeutic option for symptomatic patients with adult-onset Krabbe disease.

This study arose from our finding that SubH2Bv, a histone H2B variant residing in the subacrosomal compartment of mammalian spermatozoa, contains a bipartite nuclear localization signal (bNLS) but in spite of this did not enter the spermatid nucleus. Instead, it associated with proacrosomic and acrosomic vesicles, which were targeted to the nuclear surface to form the acrosome. On this basis we proposed that SubH2Bv targets proacrosomic/acrosomic vesicles from the Golgi apparatus to the nuclear envelope by utilizing the classical bipartite/karyopherin alpha (KPNA) nuclear import pathway. To test the protein's nuclear targeting ability, SubH2Bv, with and without targeted mutations of the basic residues of bNLS, as well as bNLS alone, were transfected into mammalian cells as GFP-fusion proteins. Only the intact bNLS conferred nuclear entry. Subsequently, we showed that a KPNA, most likely KPNA6, occupies the same sperm head compartment and follows the same pattern of acrosomal association during spermiogenesis as SubH2Bv. Sperm head fractionation combined with Western blotting located this KPNA to the subacrosomal layer of the perinuclear theca, while immunocytochemistry of testicular sections showed that it associates with the surface of proacrosomic/acrosomic vesicles during acrosomal biogenesis. The identical sperm-localization and testicular-expression patterns between KPNA and SubH2Bv suggested a potential binding interaction between these proteins. This was supported by recombinant SubH2Bv affinity pull-down assays on germ cell extracts. The results of this study provide a compelling argument that these two nuclear homing proteins work in concert to direct the acrosomic vesicle to the nucleus. Their final residence in the subacrosomal layer of the perinuclear theca of spermatozoa indicates a role for SubH2Bv and KPNA in acrosomal-nuclear docking.

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking., (Copyright 2001 Academic Press.)

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking., (Copyright 2001 Academic Press.)