Background: Heritable thoracic aortic aneurysms and dissections (HTAD) are caused by rare variants in up to 42 genes, but many affected individuals and families remain genetically unsolved. We sought to determine if aortic phenotypes occurred in cases with de novo pathogenic variants in known syndromic genes. Methods: We queried a clinical exome/genome sequencing database for individuals with de novo pathogenic or likely pathogenic variants (PVs) and aortic Human Phenotype Ontology terms. Genes with PVs present in three or more unrelated cases with aortic phenotypes were further evaluated using published evidence, genome-wide rare-variant burden analyses in the UK Biobank and All of Us, and case-level data from independent thoracic aortic disease (TAD) cohorts. Results: Among 250 individuals with de novo PVs and aortic phenotypes, 32 (12.8%) had PVs in genes included on commercial HTAD panels and were excluded from further analyses. The remaining 218 individuals had PVs in 144 genes, including 16 genes identified in three or more unrelated individuals. Genes involved in chromatin regulation and RAS/mitogen-activated protein kinase (MAPK) signaling, as well as several genes associated with overgrowth syndromes, were recurrently observed. Across biobank analyses, independent TAD cohorts, and published reports, the strongest evidence for aortic involvement was observed for DNMT3A, NF1, PTPN11, PPP1CB, NSD1, ANKRD11, ADNP, and ABL1, including significant rare-variant burden associations for three of these genes. Aortic phenotypes also occurred across multiple Coffin-Siris/BAF-complex genes. Conclusions: Aortic phenotypes occur across a diverse range of genetic syndromes beyond established HTAD genes. These findings support broader genomic testing in TAD, expansion of HTAD panels to include additional syndromic genes, and consideration of aortic surveillance in select syndromic disorders.

PURPOSE:Variants of uncertain significance (VUS) represent are clinical challenging. We hypothesize that bioinformatic tools can identify VUS that are "high risk." METHODS:Primary analyses were performed in the Penn Medicine Biobank, which is composed of 43,731 participants, 11,925 of whom carried at least 1 missense VUS in the 11 genes associated with heritable thoracic aortic disease. VUS rare exome variant ensemble learner, AlphaMissense, and minor allele frequency high-risk thresholds were derived using cutpointR. These cutpoints were used to test the association of high-risk VUS with prevalent thoracic aortic disease. RESULTS:In the Penn Medicine Biobank, being heterozygous for a VUS was associated with a modest increased risk of thoracic aortic aneurysm (TAA: OR = 1.14, 95% confidence interval [CI] 1.01 to 1.29, P = .034) but no increased risk of aortic dissection (OR = 1.04, 95% CI 0.55 to 2.00, P = .896). Using cutpointR, we derived are exome variant ensemble learner (>0.649), AlphaMissense (>0.2543), and minor allele frequency (<8.16 × 10-6) cutpoints that identified 435 high-risk VUS that were robustly associated with prevalent dissection (OR = 7.85, 95% CI 4.73 to 13.03, P < .001), although the association with TAA was attenuated (OR = 2.35, 95% CI 1.62 to 3.42, P < .001). Similar results were observed in 2 independent validation cohorts. CONCLUSION:High-risk VUS were strongly associated with aortic dissection, suggesting that high-risk thresholds may be applied clinically.

BACKGROUND:Bicuspid aortic valve (BAV) is a frequent congenital heart defect with a high heritability. Despite this, only a limited number of genes have been associated with the disease, and the molecular mechanisms remain unexplained in most cases. This study aimed to further understand the genetic architecture of BAV. METHODS:A genome-wide association study meta-analysis including 9631 cases among 65 677 participants was performed. Genes were prioritized using transcriptomic analyses based on RNA sequencing in relevant tissues, including human fetal and adult aortic valves. The impact of the knockdown or knockout of 4 candidate genes on cardiac development was verified in zebrafish. A polygenic risk score was developed, its association with BAV was evaluated in an independent cohort, and its association with a wide range of phenotypes (n=976) was evaluated in UK Biobank (n=355 618 individuals). RESULTS:Thirty-six genomic loci were identified, including 32 that were not described previously. Among the prioritized genes, KANK2 and ERBB4 were identified as potentially causal through transcriptomic analyses, colocalization, and Mendelian randomization based on gene expression in human aortic valves (n=484), whereas PRDM6 and STRN were prioritized using similar analyses from aortic (n=326) and left ventricular tissues (n=326), respectively. Targeting 4 candidate genes (WNT4, LEF1, STRN, and KANK2) in zebrafish led to disruption in cardiac development. A polygenic risk score was associated with an odds ratio of 2.07 (95% CI, 1.90-2.25; P=5.43×10-62) per SD for BAV and significantly associated with thoracic aortic aneurysm and atrial fibrillation in UK Biobank. CONCLUSIONS:This study supports a significant polygenic contribution to BAV, where the combination of multiple common variants in genes involved in heart morphogenesis disrupts aortic valve development.

Purpose:Vascular Ehlers-Danlos syndrome (vEDS), which is caused by COL3A1 pathogenic variants, is a rare heritable aortic and arterial disorder associated with early mortality, mainly due to spontaneous vascular dissections and ruptures. Improved methods for diagnosing vEDS are needed for guideline-based management to be initiated for preventing deadly complications and differentiating vEDS from overlapping conditions, such as hypermobile EDS (hEDS). Methods:We implemented an artificial intelligence (AI) facial analysis model based on the PhenoScore framework using a support vector machine trained on facial images of 30 individuals, aged 6 to 65 years, with vEDS from the Montalcino Aortic Consortium, control images from the Chicago Face Database, and publicly available images of individuals with hEDS. Cross-validation was used to train the support vector machine, and statistical measures to evaluate the model performance were calculated. Local Interpretable Model-agnostic Explanations was used to generate facial heatmaps highlighting the features driving the model's predictions. Results:The AI classifier showed excellent performance with as few as 13 vEDS training images and distinguished vEDS from both controls and individuals with hEDS with high accuracy, achieving an area under the receiver operating characteristic curve ≥ 0.97. Local Interpretable Model-agnostic Explanations highlighted facial regions already established to characterize the facial features of vEDS patients (eg, prominent eyes). Conclusion:Our results demonstrate the potential of AI-based facial analysis for diagnosing vEDS. This method democratizes the early diagnosis of vEDS by reducing dependence on genetic testing, enabling optimal management and improved outcomes, particularly in resource-limited areas.

Smooth muscle cell-specific myosin heavy chain, encoded by MYH11, is selectively expressed in smooth muscle cells (SMCs). Pathogenic variants in MYH11 predispose to a number of disorders, including heritable thoracic aortic disease associated with patent ductus arteriosus, visceral myopathy, and megacystis-microcolon-intestinal hypoperistalsis syndrome. Rare variants of uncertain significance occur throughout the gene, including MYH11 p.Glu1892Asp, and we sought to determine if this variant causes thoracic aortic disease in mice. Genomic editing was used to generate Myh11E1892D/E1892D mice. Wild-type (WT) and mutant mice underwent cardiovascular phenotyping with and without transverse aortic constriction (TAC). Myh11E1892D/E1892D and WT mice displayed similar growth, blood pressure, root and ascending aortic diameters, and cardiac function up to 13 months of age, along with similar contraction and relaxation on myographic testing. The hypertension induced by TAC was similarly in Myh11E1892D/E1892D and WT mice, but mutant mice showed augmented ascending aortic enlargement and increased elastic fiber fragmentation on histology. Unexpectedly, male Myh11E1892D/E1892D mice undergoing TAC had decreased ejection fraction, stroke volume, fractional shortening, and cardiac output compared to similarly treated male WT mice. Importantly, left ventricular mass increased significantly due to primarily posterior wall thickening, and cardiac histology confirmed cardiomyocyte hypertrophy and increased collagen deposition in the myocardium and surrounding arteries. These results further highlight the phenotypic heterogeneity associated with MYH11 rare variants. Given that MYH11 is selectively expressed in SMCs, these results implicate a role of SMCs in the arteries of the heart contributing to cardiac hypertrophy and failure with pressure overload.

Individuals with heritable thoracic aortic disease (HTAD) face a high risk of deadly aortic dissections, but genetic testing identifies causative variants in only a minority of cases. We explored the contribution of non-canonical splice variants (NCVAS) to thoracic aortic disease (TAD) using SpliceAI and sequencing data from diverse cohorts, including 551 early-onset sporadic dissection cases and 437 HTAD probands with exome sequencing, 57 HTAD pedigrees with whole genome sequencing, and select sporadic cases with clinical panel testing. NCVAS were identified in syndromic HTAD genes such as FBN1, SMAD3, and COL3A1, including intronic variants in FBN1 in two Marfan syndrome (MFS) families. Validation in the Penn Medicine BioBank and UK Biobank showed enrichment of NCVAS in HTAD-associated genes among dissections. These findings suggest NCVAS are an underrecognized contributor to TAD, particularly in sporadic dissection and unsolved MFS cases, highlighting the potential of advanced splice prediction tools in genetic diagnostics.

Purpose: Polycomb group proteins are key epigenetic transcriptional regulators. Multiple neurodevelopmental disorders are associated with pathogenic variants of the genes encoding Polycomb group proteins. RYBP is a core component of the noncanonical Polycomb Repressor Complex 1; however, its role in disease is unclear. Methods: Functional consequences of RYBP variants were assessed using in vitro cellular and in vivo Drosophila melanogaster studies. Results: We described 7 individuals with heterozygous de novo variants of RYBP and their clinical findings, including severe developmental delay, dysmorphisms, and multiple congenital anomalies. We showed that all single-nucleotide variants in RYBP localize to the N-terminal domain of the gene, which encodes the zinc-finger domain and ubiquitin-binding moiety. In vitro studies have demonstrated that the RYBP c.132C>G p.(Cys44Trp) variant causes reduced protein expression but does not affect the binding of YY1, RING1B, or ubiquitin. In vivo overexpression studies in Drosophila melanogaster showed a dramatic functional difference between human RYBP and its variant forms, affecting the C44 amino acid residue. DNA methylation studies suggested a possible episignature associated with RYBP-related disorder. Conclusion: Heterozygous de novo variants in RYBP are associated with an identifiable syndromic neurodevelopmental disorder with multiple congenital anomalies. (c) 2025 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

PURPOSE:Advancements in sequencing technologies have significantly improved clinical genetic testing; yet, the diagnostic yield remains around 30% to 40%. Emerging technologies are now being deployed to address the remaining diagnostic gap. METHODS:We tested whether short-read genome sequencing could increase the diagnostic yield in individuals enrolled into the UCI-GREGoR research study, who had suspected Mendelian conditions and prior inconclusive testing. Two other collaborative research cohorts, focused on aortopathy and dilated cardiomyopathy, consisted of individuals who were undiagnosed but had not undergone harmonized prior testing. RESULTS:We sequenced 353 families (754 participants) and found a molecular diagnosis in 54 (15.3%) of them. Of these diagnoses, 55.5% were previously missed because the causative variants were in regions not originally interrogated. In 5 cases, they were deep intronic variants, all of which led to abnormal splicing and pseudoexons, as directly shown by RNA sequencing. All 5 of these variants had inconclusive spliceAI scores. In 26% of newly diagnosed cases, the causal variant could have been detected by exome sequencing reanalysis. CONCLUSION:Genome sequencing can overcome limitations of clinical genetic testing, such as the inability to call intronic variants. Our findings highlight pseudoexons as a common mechanism via which deep intronic variants cause Mendelian disease.

CHASERR encodes a human long noncoding RNA (lncRNA) adjacent to CHD2, a coding gene in which de novo loss-of-function variants cause developmental and epileptic encephalopathy. Here, we report our findings in three unrelated children with a syndromic, early-onset neurodevelopmental disorder, each of whom had a de novo deletion in the CHASERR locus. The children had severe encephalopathy, shared facial dysmorphisms, cortical atrophy, and cerebral hypomyelination - a phenotype that is distinct from the phenotypes of patients with CHD2 haploinsufficiency. We found that the CHASERR deletion results in increased CHD2 protein abundance in patient-derived cell lines and increased expression of the CHD2 transcript in cis. These findings indicate that CHD2 has bidirectional dosage sensitivity in human disease, and we recommend that other lncRNA-encoding genes be evaluated, particularly those upstream of genes associated with mendelian disorders. (Funded by the National Human Genome Research Institute and others.) Deletions in a gene (CHASERR) that encodes a long noncoding RNA cause severe developmental delay and an increase in expression of a neighboring gene, CHD2.

Objective Data from DNA genotyping via a 96-SNP panel in a study of 25,015 clinical samples were utilized for quality control and tracking of sample identity in a clinical sequencing network. The study aimed to demonstrate the value of both the precise SNP tracking and the utility of the panel for predicting the sex-by-genotype of the participants, to identify possible sample mix-ups. Results Precise SNP tracking showed no sample swap errors within the clinical testing laboratories. In contrast, when comparing predicted sex-by-genotype to the provided sex on the test requisition, we identified 110 inconsistencies from 25,015 clinical samples (0.44%), that had occurred during sample collection or accessioning. The genetic sex predictions were confirmed using additional SNP sites in the sequencing data or high-density genotyping arrays. It was determined that discrepancies resulted from clerical errors (49.09%), samples from transgender participants (3.64%) and stem cell or bone marrow transplant patients (7.27%) along with undetermined sample mix-ups (40%) for which sample swaps occurred prior to arrival at genome centers, however the exact cause of the events at the sampling sites resulting in the mix-ups were not able to be determined.

Introduction: Pathogenic (P) or likely pathogenic (LP) aortopathy gene variants cause hereditary thoracic aortic aneurysm and dissection, but P/LP variants only explain approximately 20% of cases of thoracic aortic disease. Variants of unknown significance (VUS) are frequently identified among individuals undergoing genetic testing for thoracic aortic disease, however managing patients with VUSs remains clinically challenging. Hypothesis: Bioinformatic tools can be used to determine thresholds above which VUSs may be considered “high-risk.” Methods: Primary analyses were performed in the Penn Medicine Biobank (PMBB) which is composed of 43,731 participants who volunteered to have clinical information linked to biospecimen data including DNA which has undergone whole exome sequencing. PMBB participants were screened for VUSs in one of 11 aortopathy genes. VUS REVEL, AlphaMissense and minor allele frequency (MAF) high-risk thresholds were derived using cutpointR, a statistical package to optimize continuous variable thresholds based on binary outcomes. These thresholds were applied to VUS carriers in the PMBB and two independent validation cohorts. Logistic regression analysis was performed to test the association of high-risk VUSs with prevalent thoracic aortic disease. Results: There were 11,925 individuals in PMBB who carried at least one missense VUS. Carrying a VUS was associated with a modest increased risk of thoracic aortic aneurysm (TAA: OR=1.14, 95% confidence interval [CI] 1.01 to 1.29, P =0.034) but no increased risk of thoracic aortic dissection (OR=1.04, 95%CI 0.55 to 2.00, P =0.896). As VUS REVEL or AlphaMissense increased, or MAF decreased, the association between VUSs and thoracic aortic disease became statistically significant. Using cutpointR, we derived REVEL (>0.649), AlphaMissense (>0.2543), and MAF (<8.16x10 -6 ) thresholds that together identified 435 high-risk VUSs robustly associated with prevalent dissection (OR=7.85, 95%CI 4.73 to 13.03, P <0.001), though the association with TAA was attenuated (OR=2.35, 95%CI 1.62 to 3.42, P <0.001). Similar results were observed in the UK Biobank (UKB) and Early Onset Sporadic Thoracic Aortic Dissection (ESTAD) cohorts. Conclusions: VUSs that met high-risk bioinformatic thresholds were strongly associated with prevalent aortic dissection in the PMBB, UKB and ESTAD. Future investigation is warranted to determine if these thresholds can instruct clinical care of individuals carrying aortopathy gene VUSs.

Genes encoding long non-coding RNAs (lncRNAs) comprise a large fraction of the human genome, yet haploinsufficiency of a lncRNA has not been shown to cause a Mendelian disease. CHASERR is a highly conserved human lncRNA adjacent to CHD2-a coding gene in which de novo loss-of-function variants cause developmental and epileptic encephalopathy. Here we report three unrelated individuals each harboring an ultra-rare heterozygous de novo deletion in the CHASERR locus. We report similarities in severe developmental delay, facial dysmorphisms, and cerebral dysmyelination in these individuals, distinguishing them from the phenotypic spectrum of CHD2 haploinsufficiency. We demonstrate reduced CHASERR mRNA expression and corresponding increased CHD2 mRNA and protein in whole blood and patient-derived cell lines-specifically increased expression of the CHD2 allele in cis with the CHASERR deletion, as predicted from a prior mouse model of Chaserr haploinsufficiency. We show for the first time that de novo structural variants facilitated by Alu-mediated non-allelic homologous recombination led to deletion of a non-coding element (the lncRNA CHASERR) to cause a rare syndromic neurodevelopmental disorder. We also demonstrate that CHD2 has bidirectional dosage sensitivity in human disease. This work highlights the need to carefully evaluate other lncRNAs, particularly those upstream of genes associated with Mendelian disorders.

BackgroundKinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown.MethodsTo understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy.ResultsC. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells.ConclusionWe report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.

BACKGROUND:Disabling pansclerotic morphea (DPM) is a rare systemic inflammatory disorder, characterized by poor wound healing, fibrosis, cytopenias, hypogammaglobulinemia, and squamous-cell carcinoma. The cause is unknown, and mortality is high. METHODS:We evaluated four patients from three unrelated families with an autosomal dominant pattern of inheritance of DPM. Genomic sequencing independently identified three heterozygous variants in a specific region of the gene that encodes signal transducer and activator of transcription 4 (STAT4). Primary skin fibroblast and cell-line assays were used to define the functional nature of the genetic defect. We also assayed gene expression using single-cell RNA sequencing of peripheral-blood mononuclear cells to identify inflammatory pathways that may be affected in DPM and that may respond to therapy. RESULTS:Genome sequencing revealed three novel heterozygous missense gain-of-function variants in STAT4. In vitro, primary skin fibroblasts showed enhanced interleukin-6 secretion, with impaired wound healing, contraction of the collagen matrix, and matrix secretion. Inhibition of Janus kinase (JAK)-STAT signaling with ruxolitinib led to improvement in the hyperinflammatory fibroblast phenotype in vitro and resolution of inflammatory markers and clinical symptoms in treated patients, without adverse effects. Single-cell RNA sequencing revealed expression patterns consistent with an immunodysregulatory phenotype that were appropriately modified through JAK inhibition. CONCLUSIONS:Gain-of-function variants in STAT4 caused DPM in the families that we studied. The JAK inhibitor ruxolitinib attenuated the dermatologic and inflammatory phenotype in vitro and in the affected family members. (Funded by the American Academy of Allergy, Asthma, and Immunology Foundation and others.).

Objective:Data from DNA genotyping via a 96-SNP panel in a study of 25,015 clinical samples were utilized for quality control and tracking of sample identity in a clinical sequencing network. The study aimed to demonstrate the value of both the precise SNP tracking and the utility of the panel for predicting the sex-by-genotype of the participants, to identify possible sample mix-ups.Results:Precise SNP tracking showed no sample swap errors within the clinical testing laboratories. In contrast, when comparing predicted sex-by-genotype to the provided sex on the test requisition, we identified 110 inconsistencies from 25,015 clinical samples (0.44%), that had occurred during sample collection or accessioning. The genetic sex predictions were confirmed using additional SNP sites in the sequencing data or high-density genotyping arrays. It was determined that discrepancies resulted from clerical errors, samples from transgender participants and stem cell or bone marrow transplant patients along with undetermined sample mix-ups.

Pre-mRNA splicing factors are crucial in regulating transcript diversity, by removing introns from eukaryotic transcripts, an essential step in gene expression. Splicing of pre-mRNA is catalyzed by spliceosomes. CWC27 is a cyclophilin associated with spliceosome, in which genetic defects of its components have been linked to spliceosomopathies with clinical phenotypes including skeletal developmental defects, retinitis pigmentosa (RP), short stature, skeletal anomalies, and neurological disorders. We report two siblings (male and female) of Mexican descent with a novel homozygous frameshift variant in CWC27 and aim to highlight the cardinal features among the previously described 12 cases as well as expand the currently recognized phenotypic spectrum. Both siblings presented with a range of ocular and extraocular manifestations including novel features such as solitary kidney and tarsal coalition in the male sibling, together with gait abnormalities, and Hashimoto's thyroiditis in the female sibling. Finally, we highlight ectodermal involvement including sparse scalp hair, eyebrows and lashes, pigmentary differences, nail dysplasia, and dental anomalies as a core phenotype associated with the CWC27 spliceosomopathy.

Exome sequencing of genes associated with heritable thoracic aortic disease (HTAD) failed to identify a pathogenic variant in a large family with Marfan syndrome (MFS). A genome-wide linkage analysis for thoracic aortic disease identified a peak at 15q21.1, and genome sequencing identified a novel deep intronic FBN1 variant that segregated with thoracic aortic disease in the family (LOD score 2.7) and was predicted to alter splicing. RT-PCR and bulk RNA sequencing of RNA harvested from fibroblasts explanted from the affected proband revealed an insertion of a pseudoexon between exons 13 and 14 of the FBN1 transcript, predicted to lead to nonsense mediated decay (NMD). Treating the fibroblasts with an NMD inhibitor, cycloheximide, greatly improved the detection of the pseudoexon-containing transcript. Family members with the FBN1 variant had later onset aortic events and fewer MFS systemic features than typical for individuals with haploinsufficiency of FBN1. Variable penetrance of the phenotype and negative genetic testing in MFS families should raise the possibility of deep intronic FBN1 variants and the need for additional molecular studies.