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Hi-C sequencing is a DNA-based next-generation sequencing method that preserves the 3D genome conformation and has shown promise in detecting genomic rearrangements in translational research studies. To evaluate Hi-C as a potential clinical diagnostic platform, analytical concordance with routine laboratory testing was assessed using primary pediatric leukemia and sarcoma specimens. Archived viable and non-viable frozen leukemic cells and formalin-fixed paraffin-embedded (FFPE) tumor specimens were analyzed. Pediatric acute myeloid leukemia (AML) and alveolar rhabdomyosarcoma (A-RMS) specimens with known genomic rearrangements were subjected to Hi-C to assess analytical concordance. Subsequently, a discovery cohort consisting of AML and acute lymphoblastic leukemia (ALL) cases without known genomic rearrangements based on prior clinical diagnostic testing was evaluated to determine whether Hi-C could detect rearrangements. Using a standard sequencing depth of 50 million raw read-pairs per sample, or approximately 5X raw genomic coverage, we observed 100% concordance between Hi-C and previous clinical cytogenetic and molecular testing. In the discovery cohort, a clinically relevant gene fusion was detected in 45% of leukemia cases (5/11). This study provides an institutional proof of principle evaluation of Hi-C sequencing to medical diagnostic testing as it identified several clinically relevant rearrangements, including those that were missed by current clinical testing workflows.

Somatic variant detection is an integral part of cancer genomics analysis. While most methods have focused on short-read sequencing, long-read technologies now offer potential advantages in terms of repeat mapping and variant phasing. We present DeepSomatic, a deep learning method for detecting somatic SNVs and insertions and deletions (indels) from both short-read and long-read data, with modes for whole-genome and exome sequencing, and able to run on tumor-normal, tumor-only, and with FFPE-prepared samples. To help address the dearth of publicly available training and benchmarking data for somatic variant detection, we generated and make openly available a dataset of five matched tumor-normal cell line pairs sequenced with Illumina, PacBio HiFi, and Oxford Nanopore Technologies, along with benchmark variant sets. Across samples and technologies (short-read and long-read), DeepSomatic consistently outperforms existing callers, particularly for indels., Competing Interests: Competing interests K.S., D.E.C., P.C., A. Kolesnikov, L.B., J.C.M., and A.C. are employees of Google LLC and own Alphabet stock as part of the standard compensation package. M.S.F. is a part of the speakers bureau for Bayer and PacBio.

Though rare, pediatric high-grade gliomas (pHGG) are a leading cause of cancer-related mortality in children. We wanted to determine whether our currently available clinical laboratory methods could better define diagnosis for pHGG that had been archived at our institution for the past 20 years (1998 to 2017). We investigated 33 formalin-fixed paraffin-embedded pHGG using ThermoFisher Oncoscan SNP microarray with somatic mutation analysis, Sanger sequencing, and whole genome sequencing. These data were correlated with historical histopathological, chromosomal, clinical, and radiological data. Tumors were subsequently classified according to the 2021 WHO Classification of Paediatric CNS Tumours. All 33 tumors were found to have genetic aberrations that placed them within a 2021 WHO subtype and/or provided prognostic information; 6 tumors were upgraded from WHO CNS grade 3 to grade 4. New pHGG genetic features were found including two small cell glioblastomas with H3 G34 mutations not previously described; one tumor with STRN-NTRK2 fusion; and a congenital diffuse leptomeningeal glioneuronal tumor without a chromosomal 1p deletion but with KIAA1549-BRAF fusion. Overall, the combination of laboratory methods yielded key information for tumor classification. Thus, even small studies of these uncommon tumor types may yield new genetic features and possible new subtypes that warrant future investigations., Competing Interests: Declaration of Competing Interest The authors have no competing interests to disclose., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies., Competing Interests: Declaration of interests A.B., M.J.G.S., K. McWalters, R.P., and R.S. are employees of GeneDx., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex, heterogeneous etiology. It is well established that common and rare sequence variants contribute to the formation of CL/P, but the contribution of copy-number variants (CNVs) to cleft formation remains relatively understudied. To fill this knowledge gap, we conducted a large-scale comparative analysis of genome-wide CNV profiles of 869 individuals from the Philippines and 233 individuals of European ancestry with CL/P with three primary goals: first, to evaluate whether differences in CNV number, amount of genomic content, or amount of coding genomic content existed within clefting subtypes; second, to assess whether CNVs in our cohort overlapped with known Mendelian clefting loci; and third, to identify unestablished Mendelian clefting genes. Significant differences in CNVs across cleft types or in individuals with non-syndromic versus syndromic clefts were not observed; however, several CNVs in our cohort overlapped with known syndromic and non-syndromic Mendelian clefting loci. Moreover, employing a filtering strategy relying on population genetics data that rare variants are on the whole more deleterious than common variants, we identify several CNV-associated gene losses likely driving non-syndromic clefting phenotypes. By prioritizing genes deleted at a rare frequency across multiple individuals with clefts yet enriched in our cohort of individuals with clefts compared to control subjects, we identify COBLL1, RIC1, and ARHGEF38 as clefting genes. CRISPR-Cas9 mutagenesis of these genes in Xenopus laevis and Danio rerio yielded craniofacial dysmorphologies, including clefts analogous to those seen in human clefting disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Laboratories utilizing next-generation sequencing align sequence data to a standardized human reference genome (HRG). Several updated versions, or builds, have been released since the original HRG in 2001, including the Genome Reference Consortium Human Build 38 (GRCh38) in 2013. However, most clinical laboratories still use GRCh37, which was released in 2009. We report our laboratory's clinical validation of GRCh38., Methods: Migration to GRCh38 was validated by comparing the coordinates (lifting over) of 9443 internally curated variants from GRCh37 to GRCh38, globally comparing protein coding sequence variants aligned with GRCh37 vs GRCh38 from 917 exomes, assessing genes with known discrepancies, comparing coverage differences, and establishing the analytic sensitivity and specificity of variant detection using Genome in a Bottle data., Results: Eight discrepancies, due to strand swap or reference base, were observed. Three clinically relevant variants had the GRCh37 alternate allele as the reference allele in GRCh38. A comparison of 88 295 calls between builds identified 8 disease-associated genes with sequence differences: ABO, BNC2, KIZ, NEFL, NR2E3, PTPRQ, SHANK2, and SRD5A2. Discrepancies in coding regions in GRCh37 were resolved in GRCh38., Conclusions: There were a small number of clinically significant changes between the 2 genome builds. GRCh38 provided improved detection of nucleotide changes due to the resolution of discrepancies present in GRCh37. Implementation of GRCh38 results in more accurate and consistent reporting., Competing Interests: Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:, (© American Association for Clinical Chemistry 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by oculocutaneous albinism and variable pulmonary fibrosis, granulomatous colitis, or immunodeficiency. The diagnosis relies on clinical findings, platelet transmission electron microscopy studies showing absent dense granules, or the identification of a pathogenic genotype in one of 11 associated genes, including HPS1 We report a 2-wk-old male with significant iris transillumination defects, a pale fundus, and mild corectopia found by clinical exome sequencing to have a previously reported pathogenic variant, c.972dupC p.(Met325HisfsTer128), and a variant of uncertain significance, c.1846G>A p.(Glu616Lys), in HPS1 To determine whether his phenotype was consistent with HPS, follow-up studies of whole blood lumiaggregometry and platelet transmission electron microscopy were performed that revealed absent or markedly reduced platelet ATP secretion and virtually absent platelet dense granules, thus confirming the diagnosis. To the best of our knowledge, our case is the first in which the c.1846G>A p.(Glu616Lys) variant is identified in a patient with HPS. In addition, the case also highlights the importance of leveraging appropriate confirmatory clinical testing and reverse phenotyping, which allowed the care team to establish the clinical diagnosis of HPS and reclassify the previously reported variant of uncertain significance in HPS1 to likely pathogenic., (© 2021 Lansdon et al.; Published by Cold Spring Harbor Laboratory Press.)

G-proteins are ubiquitously expressed heterotrimeric proteins consisting of α, β and γ subunits and mediate G-protein coupled receptor signalling cascades. The β subunit is encoded by one of five highly similar paralogs (GNB1-GNB5, accordingly). The developmental importance of G-proteins is highlighted by the clinical relevance of variants in genes such as GNB1, which cause severe neurodevelopmental disease (NDD). Recently the candidacy of GNB2 was raised in association with NDD in an individual with a de novo variant affecting a codon conserved across paralogs and recurrently mutated in GNB1-related disease, c.229G>A p.(Gly77Arg), in association with global developmental delay, intellectual disability and dysmorphic features. Here, we report a patient with strikingly similar facial features and NDD in association with a de novo GNB2 variant affecting the same codon, c.229G>T p.(Gly77Trp). In addition, this individual has epilepsy and overgrowth. Our report is the second to implicate a de novo GNB2 variant with a severe yet variable NDD., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The most recent build of the human reference genome, GRCh38, was released in 2013. However, many laboratories performing next-generation sequencing (NGS) continue to align to GRCh37. Our aim was to assess the number of clinical diagnostic laboratories that have migrated to GRCh38 and discern factors impeding migration for those still using GRCh37. A brief, five-question survey was electronically administered to 71 clinical laboratories offering constitutional NGS-based testing and analyzed categorically. Twenty-eight responses meeting inclusion criteria were collected from 24 academic and four commercial diagnostic laboratories. Most of these (14; 50%) reported volumes of <500 NGS-based tests in 2019. Only two respondents (7%) had already migrated entirely to GRCh38; most laboratories (15; 54%) had no plans to migrate. The two prevailing reasons for not yet migrating were as follows: laboratories did not feel the benefits outweighed the time and monetary costs (14; 50%); and laboratories had insufficient staff to facilitate the migration (12; 43%). These data, although limited, suggest most clinical molecular laboratories are reluctant to migrate to GRCh38, and there appear to be multiple obstacles to overcome before GRCh38 is widely adopted., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The chromodomain family member chromodomain 1 (CHD1) has been shown to have numerous critical molecular functions including transcriptional regulation, splicing, and DNA repair. Complete loss of function of this gene is not compatible with life. On the other hand, missense and copy number variants of CHD1 can result in intellectual disabilities and craniofacial malformations in human patients including cleft palate and Pilarowski-Bjornsson Syndrome. We have used the aquatic developmental model organism Xenopus laevis, to determine a specific role for Chd1 in such cranioafcial disorders. Protein and gene knockdown techniques in Xenopus, including antisense oligos and mosaic Crispr/Cas9-mediated mutagenesis, recapitulated the craniofacial defects observed in humans. Further analysis indicated that embryos deficient in Chd1 had defects in cranial neural crest development and jaw cartilage morphology. Additionally, flow cytometry and immunohistochemistry revealed that decreased Chd1 resulted in increased in apoptosis in the developing head. Together, these experiments demonstrate that Chd1 is critical for fundamental processes and cell survival in craniofacial development. We also presented evidence that Chd1 is regulated by retinoic acid signaling during craniofacial development. Expression levels of chd1 mRNA, specifically in the head, were increased by RAR agonist exposure and decreased upon antagonist treatment. Subphenotypic levels of an RAR antagonist and Chd1 morpholinos synergized to result in orofacial defects. Further, RAR DNA binding sequences (RAREs) were detected in chd1 regulatory regions by bioinformatic analysis. In summary, by combining human genetics and experiments in an aquatic model we now have a better understanding of the role of CHD1 in craniofacial disorders., (© 2020 Wiley Periodicals LLC.)

Orofacial clefts are one of the most common birth defects, affecting 1-2 per 1000 births, and have a complex etiology. High-resolution array-based comparative genomic hybridization has increased the ability to detect copy number variants (CNVs) that can be causative for complex diseases such as cleft lip and/or palate. Utilizing this technique on 97 nonsyndromic cleft lip and palate cases and 43 cases with cleft palate only, we identified a heterozygous deletion of Isthmin 1 in one affected case, as well as a deletion in a second case that removes putative 3' regulatory information. Isthmin 1 is a strong candidate for clefting, as it is expressed in orofacial structures derived from the first branchial arch and is also in the same "synexpression group" as fibroblast growth factor 8 and sprouty RTK signaling antagonist 1a and 2 , all of which have been associated with clefting. CNVs affecting Isthmin 1 are exceedingly rare in control populations, and Isthmin 1 scores as a likely haploinsufficiency locus. Confirming its role in craniofacial development, knockdown or clustered randomly interspaced short palindromic repeats/Cas9-generated mutation of isthmin 1 in Xenopus laevis resulted in mild to severe craniofacial dysmorphologies, with several individuals presenting with median clefts. Moreover, knockdown of isthmin 1 produced decreased expression of LIM homeobox 8 , itself a gene associated with clefting, in regions of the face that pattern the maxilla. Our study demonstrates a successful pipeline from CNV identification of a candidate gene to functional validation in a vertebrate model system, and reveals Isthmin 1 as both a new human clefting locus as well as a key craniofacial patterning gene., (Copyright © 2018 by the Genetics Society of America.)

Here we describe the genotype-phenotype correlations of diseases caused by variants in Fibroblast Growth Factor Receptor 1 ( FGFR1) and report a novel, de novo variant in FGFR1 in an individual with multiple congenital anomalies. The proband presented with bilateral cleft lip and palate, malformed auricles, and bilateral ectrodactyly of his hands and feet at birth. He was later diagnosed with diabetes insipidus, spastic quadriplegia, developmental delay, agenesis of the corpus callosum, and enlargement of the third cerebral ventricle. We noted the substantial phenotypic overlap with individuals with Hartsfield syndrome, the rare combination of holoprosencephaly and ectrodactyly. Sequencing of FGFR1 identified a previously unreported de novo variant in exon 11 (p.Gly487Cys), which we modeled to determine its predicted effect on the protein structure. Although it was not predicted to significantly alter protein folding stability, it is possible this variant leads to the formation of nonnative intra- or intermolecular disulfide bonds. We then mapped this and other disease-associated variants to a 3-dimensional model of FGFR1 to assess which protein domains harbored the highest number of pathogenic changes. We observed the greatest number of variants within the domains involved in FGF binding and FGFR activation. To further explore the contribution of each variant to disease, we recorded the phenotype resulting from each FGFR1 variant to generate a series of phenotype-specific protein maps and compared our results to benign variants appearing in control databases. It is our hope that the use of phenotypic maps such as these will further the understanding of genetic disease in general and diseases caused by variation in FGFR1 specifically.