Marcou, Cherisse A.

ABSTRACT Objective Although fetal sex chromosome complement prediction by prenatal cell‐free DNA (cfDNA) screening is highly accurate, discordance between the cfDNA‐inferred chromosomal sex and phenotypic sex observed via ultrasonography or at birth, that is, “fetal sex discordance”, does rarely occur. Such discordance may be caused by differences in sex development (DSD) or technical or biological artifacts. This study aims to provide insights into the proportion of fetal sex discordance cases caused by DSD versus non‐DSD artifacts. Method We retrospectively reviewed 122,748 cases that underwent diagnostic testing at the Mayo Clinic Cytogenetics Laboratory between 2015 and 2023. Thirty‐three singleton‐gestation cases tested for fetal sex discordances were identified. Results Fetal sex discordance was attributed to non‐DSD artifacts in 24 (73%) of the 33 patients. In these cases, the phenotypic sex matched the chromosomal sex revealed by diagnostic cytogenetic testing. The remaining 9 patients included one with mosaic 46,X,psu idic(Y)(q11.21)/45,X, 5 with 46,XY DSD, and 3 with 46,XX DSD. Conclusion The majority of fetal sex discordance cases are caused by non‐DSD artifacts rather than DSD. Accurate clinical counseling of fetal sex discordance requires distinguishing between DSD and non‐DSD causes, emphasizing the need for diagnostic cytogenetic testing.

Abstract Objectives Protein-truncating variants in the TTN gene are a well-established cause of dilated cardiomyopathy (DCM). We report a novel case of DCM caused by a mobile element insertion (MEI) in TTN, through which we highlight the key features of MEIs in next-generation sequencing data. Because of the rarity of MEIs, the next-generation sequencing data features associated with these events may be mistaken as noise, potentially leading to missed diagnoses. Methods Next-generation sequencing gene panel testing for DCM was performed on a 17-year-old male patient presenting with severe left ventricular dilatation and systolic dysfunction. Manta was used for structural variant detection, followed by manual review of NGS data for potential structural variants. Results Manta detected a potential insertion in TTN. Manual review identified hallmark features consistent with a LINE-1 MEI. This finding was orthogonally confirmed by long-range polymerase chain reaction and gel electrophoresis, which indicated an insertion of approximately 4 to 5 kilobase pairs. The insertion disrupted the reading frame of TTN within an A-band exon, resulting in protein truncation that was classified as likely pathogenic. Conclusions This case expands the mutational spectrum of TTN protein-truncating variants. It also underscores the importance of recognizing rarer types of pathogenic variants (eg, MEIs) to produce accurate genetic diagnostics.

AbstractAxenfeld‐Rieger Syndrome (ARS) type 1 is a rare autosomal dominant condition characterized by anterior chamber anomalies, umbilical defects, dental hypoplasia, and craniofacial anomalies, with Meckel's diverticulum in some individuals. Here, we describe a clinically ascertained female of childbearing age with ARS for whom clinical targeted sequencing and deletion/duplication analysis followed by clinical exome and genome sequencing resulted in no pathogenic variants or variants of unknown significance in PITX2 or FOXC1. Advanced bioinformatic analysis of the genome data identified a complex, balanced rearrangement disrupting PITX2. This case is the first reported intrachromosomal rearrangement leading to ARS, illustrating that for patients with compelling clinical phenotypes but negative genomic testing, additional bioinformatic analysis are essential to identify subtle genomic abnormalities in target genes.

Abstract Background Despite clinically demonstrated accuracy in next generation sequencing (NGS) data, many clinical laboratories continue to confirm variants with Sanger sequencing, which increases cost of testing and turnaround time. Several studies have assessed the accuracy of NGS in detecting single nucleotide variants; however, less has been reported about insertion, deletion, and deletion-insertion variants (indels). Methods We performed a retrospective analysis from 2015–2022 of indel results from a subset of NGS targeted gene panel tests offered through the Mayo Clinic Genomics Laboratories. We compared results from NGS and Sanger sequencing of indels observed in clinical runs and during the intra-assay validation of the tests. Results Results demonstrated 100% concordance between NGS and Sanger sequencing for over 490 indels (217 unique), ranging in size from 1 to 68 basepairs (bp). The majority of indels were deletions (77%) and 1 to 5 bp in length (90%). Variant frequencies ranged from 11.4% to 67.4% and 85.1% to 100% for heterozygous and homozygous variants, respectively, with a median depth of coverage of 2562×. A subset of indels (7%) were located in complex regions of the genome, and these were accurately detected by NGS. We also demonstrated 100% reproducibility of indel detection (n = 179) during intra-assay validation. Conclusions Together this data demonstrates that reportable indel variants up to 68 bp can be accurately assessed using NGS, even when they occur in complex regions. Depending on the complexity of the region or variant, Sanger sequence confirmation of indels is usually not necessary if the variants meet appropriate coverage and allele frequency thresholds.

Background: The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. Methods: In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell–derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. Results: WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio . Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell–derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. Conclusions: We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.

ABSTRACTBackgroundCurrently available structural variant (SV) detection methods do not span the complete spectrum of disease‐causing SVs. Optical genome mapping (OGM), an emerging technology with the potential to resolve diagnostic dilemmas, was performed to investigate clinically‐relevant SVs in a 4‐year‐old male with an epileptic encephalopathy of undiagnosed molecular origin.MethodsOGM was utilized to image long, megabase‐size DNA molecules, fluorescently labeled at specific sequence motifs throughout the genome with high sensitivity for detection of SVs greater than 500 bp in size. OGM results were confirmed in a CLIA‐certified laboratory via mate‐pair sequencing.ResultsOGM identified a mosaic, de novo 90 kb deletion and inversion on the X chromosome disrupting the CDKL5 gene. Detection of the mosaic deletion, which had been previously undetected by chromosomal microarray, an infantile epilepsy panel including exon‐level microarray for CDKL5, exome sequencing as well as genome sequencing, resulted in a diagnosis of X‐linked dominant early infantile epileptic encephalopathy‐2.ConclusionOGM affords an effective technology for the detection of SVs, especially those that are mosaic, since these remain difficult to detect with current NGS technologies and with conventional chromosomal microarrays. Further research in undiagnosed populations with OGM is warranted.

AbstractPrader–Willi syndrome (PWS) is a prototypic genetic condition related to imprinting. Causative mechanisms include paternal 15q11‐q13 deletion, maternal chromosome 15 uniparental disomy (UPD15), Prader–Willi Syndrome/Angelman Syndrome (PWS/AS) critical region imprinting defects, and complex chromosomal rearrangements. Maternal UPD15‐related PWS poses risks of concomitant autosomal recessive (AR) disorders when the mother carries a pathogenic variant in one of the genes on chromosome 15 associated with autosomal recessive inherited disease. Co‐occurrence of autosomal recessive conditions in the setting of UPD leads to increased complexity of the clinical phenotype, and may delay the diagnosis of PWS. We report a patient with PWS and associated congenital ichthyosis due to maternal UPD15, and a homozygous novel pathogenic variant in ceramide synthase 3 (CERS3). We also review the literature of associated disorders reported in the setting of maternal UPD15‐related PWS and provide a summary of the previously described CERS3 variants. This represents the second case of autosomal recessive congenital ichthyosis (ARCI) in the setting of PWS and UPD15. There needs to be a high index of suspicion of this genetic mechanism when there is unexpected phenotype or evolution of the clinical course in a patient with PWS.

The gene encoding SAP97 ( DLG1) joins a growing list of genes encoding ion channel interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. In this study we provide the first data supporting DLG1-encoded SAP97’s candidacy as a minor Brugada syndrome susceptibility gene.