Vandana Shashi

Introduction Congenital heart disease (CHD) occurs in over half of individuals with 22q11.2 deletion syndrome (22q11.2DS), but lesion type varies widely. Methods We analysed basic medical features in 3016 unrelated postnatal individuals with 22q11.2DS from specialised centres in the USA, Canada, Europe, South America, Israel and Australia including 1868 with whole-genome sequencing. Results The typical 3 Mb LCR22 A–D deletion was present in 2788 individuals (92.4%), with smaller A–B (n=172, 5.7%) and A–C (n=56, 1.9%) deletions comprising the remaining cohort. In multivariable mixed-effects logistic regression adjusting for sex, deletion group, genome-wide principal components (PCs) and recruitment site, four non-intercept associations to test relationships met study-wide false discovery rate significance. Compared with the A–B deletion, the A–D deletion was associated with lower odds of persistent truncus arteriosus (OR 0.37, 95% CI 0.18 to 0.75) and higher odds of isolated septal defects (OR 4.68, 95% CI 1.71 to 12.83), although precision was limited by the smaller A–B group. PC2 was associated with lower odds of pulmonary stenosis/atresia with additional lesions (OR 0.73, 95% CI 0.61 to 0.87) and PC4 with higher odds of abnormal origin of the subclavian arteries (ASA; OR 2.59, 95% CI 1.37 to 4.89). The Polygenic Risk Score for proximal pulmonary artery diameter was associated with a lower likelihood of ASA (OR 0.71, 95% CI 0.56 to 0.89, p=3.7E−3). ADMIXTURE-derived continental ancestry proportions did not show independent associations with these two PC-associated outcomes. Conclusion These lesion-specific findings in the data presented indicate that the deletion interval on 22q11.2 and broader genetic background may contribute to CHD variability in 22q11.2DS.

Disorders of GABRA3 , the only epilepsy-associated GABA _A receptor subunit gene on the X chromosome, have eluded clinical clarity due to ambiguous inheritance patterns and variable phenotypes. The long-standing assumption that all pathogenic variants cause loss of function further obscured genotype–phenotype relationships and hindered progress. Here, we curated a cohort of individuals with a GABRA3 variant, integrating deep phenotyping, genotyping, family history, and electrophysiology with a targeted mouse model. Among 43 individuals with 19 GABRA3 variants, functional analyses revealed gain- and loss-of-function effects, each linked to distinct clinical profiles. Gain-of-function variants were associated with severe, treatment-resistant epilepsy and profound intellectual disability, disproportionately affecting males, who were often nonambulant and had cortical visual impairment. Loss-of-function variants produced milder phenotypes, with epilepsy rarely observed; affected males showed behavioral issues and language delay, while females were unaffected carriers. Our gain-of-function ( Gabra3 ^Q242L/+ ) mouse model mirrored these sex-specific differences, showing increased seizure susceptibility, early death, and marked cortical hyperexcitability. These insights resolve longstanding uncertainties surrounding GABRA3 and redefine how X-linked disorders are interpreted. They demonstrate that it is the functional impact of a variant, not its mere presence, that determines whether a condition manifests dominantly or recessively. This distinction carries important implications for genetic counseling, precision medicine, and the broader interpretation of X-linked neurodevelopmental disorders.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is one of the leading causes of sudden cardiac death in children, young adults, and athletes and is characterized by the fibro-fatty replacement of the myocardium, predominantly of the right ventricle. Sixty percent of patients with ACM have a known genetic cause, but for the remainder, the pathogenesis is unknown. This lack of mechanistic understanding has slowed the development of disease-modifying therapies, and children with ACM have a high degree of morbidity and mortality. METHODS: Induced pluripotent stem cells (iPSCs) from 3 family members were differentiated into cardiac myocytes (CMs). Calcium imaging was conducted by labeling calcium with CAL-520 and confocal imaging to capture calcium sparks after iPSC-CMs were electrically paced. A cardiac-specific, inducible knockout mouse ( Tax1bp3 ^−/− ) was made and intracardiac electrophysiology studies conducted to observe arrhythmia inducibility following pacing. RESULTS: We identified a kindred with multiple members affected by ACM cosegregating with biallelic variants in the gene TAX1BP3 , which encodes the protein TAX1BP3 (Tax1-binding protein 3). iPSC-CMs derived from this kindred demonstrated increased intracellular lipid droplets, induction of TRPV4 (transient receptor potential vanilloid type 4) expression, and inducible TRPV4 current. This was associated with depletion of the intracellular sarcoplasmic reticulum Ca ²⁺ store and increased RyR2 (ryanodine receptor 2)-mediated store Ca ²⁺ leak and delayed afterdepolarizations, a known mechanism of Ca ²⁺ -mediated arrhythmogenesis. Similarly, Tax1bp3 cardiac-specific knockout mice had increased Ca ²⁺ leak and were predisposed to ventricular arrhythmias compared with wild-type mice. Ca ²⁺ leak in both the iPSC-CMs and mouse ventricular myocytes was rescued by small molecule TRPV4 inhibition. This strategy also effectively reduced Ca ²⁺ leak in a PKP2 (plakophilin 2) p.His773AlafsX8 iPSC-CM model of ACM. CONCLUSIONS: We conclude that TAX1BP3 is associated with rare autosomal recessive ACM through TRPV4-mediated Ca ²⁺ leak from RyR2. Further, TRPV4 current inhibition has the potential to be a new therapeutic target for ACM.

Patients with undiagnosed and/or rare disorders frequently manifest dysmorphic and neurological features. There is a lack of information on the effectiveness of telehealth in the evaluation of these disorders. We thus compared an unassisted virtual physical examination (PE) with an in‐person PE in undiagnosed individuals and also assessed participant telehealth satisfaction. Twenty‐six individuals enrolled in the Undiagnosed Diseases Network study underwent an in‐home synchronous virtual PE, and a subsequent in‐person PE, by the same clinician. The participants completed surveys on telehealth usability and provider empathy. On PE, general appearance and craniofacial features showed near perfect agreement ( κ = 0.81–1.00) between the telehealth and in‐person evaluations. Specific components of the neurological examination demonstrated substantial agreement (speech, gait, coordination; κ > 0.61), whereas others had moderate agreement (muscle tone, strength; κ = 0.41–0.60), and a few had none to slight agreement (skin; κ < 0.20). Some systems could not be examined in the virtual PE. Importantly, features relevant to diagnosis or management were missed on the virtual PE in only 2/26 individuals. The participants were satisfied with the quality of the telehealth interaction, as well as empathy demonstrated by providers in the virtual interface. Telehealth is effective for PEs in undiagnosed diseases and is acceptable to affected individuals.

The heterotrimeric protein phosphatase 2A (PP2A) complex catalyzes about half of Ser/Thr dephosphorylations in eukaryotic cells. A CAG repeat expansion in the neuron-specific protein PP2A regulatory subunit PPP2R2B gene causes spinocerebellar ataxia type 12 (SCA12). We established five monoallelic missense variants in PPP2R2B (four confirmed as de novo) as a cause of intellectual disability with developmental delay (R149P, T246K, N310K, E37K, I427T). In addition to moderate to severe intellectual disability and developmental delay, affected individuals presented with seizures, microcephaly, aggression, hypotonia, as well as broad-based or stiff gait. We used biochemical and cellular assays, including a novel luciferase complementation assay to interrogate PP2A holoenzyme assembly and activity, as well as deregulated mitochondrial dynamics as possible pathogenic mechanisms. Cell-based assays documented impaired ability of PPP2R2B missense variants to incorporate into the PP2A holoenzyme, localize to mitochondria, induce fission of neuronal mitochondria, and dephosphorylate the mitochondrial fission enzyme dynamin-related protein 1. AlphaMissense-based pathogenicity prediction suggested that an additional seven unreported missense variants may be pathogenic. In conclusion, our studies identify loss-of-function at the PPP2R2B locus as the basis for syndromic intellectual disability with developmental delay. They also extend PPP2R2B-related pathologies from neurodegenerative (SCA12) to neurodevelopmental disorders and suggests that altered mitochondrial dynamics may contribute to mechanisms.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator ( AIRE ) gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic AIRE variants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic AIRE variant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of AIRE -expressing primary patient monocyte-derived dendric cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109–base pair frame-shifting pseudoexon. We also found low-level AIRE expression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic AIRE –expressing cell lines. Through protein modeling and transcriptomic analyses of AIRE -transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal AIRE transcript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing AIRE variant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.

Although next‐generation sequencing has enabled diagnoses for many patients with Mendelian disorders, the majority remain undiagnosed. Here, we present a sibling pair who were clinically diagnosed with Escobar syndrome, however targeted gene testing was negative. Exome sequencing (ES), and later genome sequencing (GS), revealed compound heterozygous TTN variants in both siblings, a maternally inherited frameshift variant [(NM_133378.4):c.36812del; p.(Asp12271Valfs*10)], and a paternally inherited missense variant [(NM_133378.4):c.12322G > A; p.(Asp4108Asn)]. This result was considered nondiagnostic due to poor clinical fit and limited pathogenicity evidence for the missense variant of uncertain significance (VUS). Following initial nondiagnostic RNA sequencing (RNAseq) on muscle and further pursuit of other variants detected on the ES/GS, a reanalysis of noncanonical splice sites in the muscle transcriptome identified an out‐of‐frame exon retraction in TTN, near the known VUS. Interim literature included reports of patients with similar TTN variants who had phenotypic concordance with the siblings, and a diagnosis of a congenital titinopathy was given 4 years after the TTN variants had been initially reported. This report highlights the value of reanalysis of RNAseq with a different approach, expands the phenotypic spectrum of congenital titinopathy and also illustrates how a perceived phenotypic mismatch, and failure to consider known variants, can result in a prolongation of the diagnostic journey.

Ultra rare disorders are being diagnosed at an unprecedented rate, due to genomic sequencing. These diagnoses are often a new gene association, for which little is known, and few share the diagnosis. For these diagnoses, we use the term emerging‐ultrarare disorder (E‐URD), defined as <100 diagnosed individuals. We contacted 20 parents of children diagnosed with an E‐URD through the Duke University Research Sequencing Clinic. Seventeen completed semi‐structured interviews exploring parental perspectives (7/17 had children in publications describing the phenotype; 4/17 had children in the first publication establishing a new disorder). Data were analyzed using a directed content approach informed by an empowerment framework. Parents reported a range of responses, including benefits of a diagnosis and challenges of facing the unknown, some described feeling lost and confused, while others expressed empowerment. Empowerment characteristics were hope for the future, positive emotions, engagement, and confidence/self‐efficacy to connect with similar others, partner with healthcare providers, and seek new knowledge. We identified a subset of parents who proactively engaged researchers, supported research and publications, and created patient advocacy and support organizations to connect with and bolster similarly diagnosed families. Other parents reported challenges of low social support, low tolerance for uncertainty, limited knowledge about their child's disorder, as well as difficulty partnering with HCPs and connecting to an E‐URD community. An overarching classification was developed to describe parental actions taken after an E‐URD diagnosis: adjusting, managing, and pioneering. These classifications may help genetic counselors identify and facilitate positive steps with parents of a child with an E‐URD.

Hyperpolarization activated Cyclic Nucleotide (HCN) gated channels are crucial for various neurophysiological functions, including learning and sensory functions, and their dysfunction are responsible for brain disorders, such as epilepsy. To date, HCN2 variants have only been associated with mild epilepsy and recently, one monoallelic missense variant has been linked to developmental and epileptic encephalopathy. Here, we expand the phenotypic spectrum of HCN2- related disorders by describing twenty-one additional individuals from fifteen unrelated families carrying HCN2 variants. Seventeen individuals had developmental delay/intellectual disability (DD/ID), two had borderline DD/ID, and one had borderline DD. Ten individuals had epilepsy with DD/ID, with median age of onset of 10 months, and one had epilepsy with normal development. Molecular diagnosis identified thirteen different pathogenic HCN2 variants, including eleven missense variants affecting highly conserved amino acids, one frameshift variant, and one in-frame deletion. Seven variants were monoallelic of which five occurred de novo, one was not maternally inherited, one was inherited from a father with mild learning disabilities, and one was of unknown inheritance. The remaining six variants were biallelic, with four homozygous and two compound heterozygous variants. Functional studies using two-electrode voltage-clamp recordings in Xenopus laevis oocytes were performed on three monoallelic variants, p.(Arg324His), p.(Ala363Val), and p.(Met374Leu), and three biallelic variants, p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp). The p.(Arg324His) variant induced a strong increase of HCN2 conductance, while p.(Ala363Val) and p.(Met374Leu) displayed dominant negative effects, leading to a partial loss of HCN2 channel function. By confocal imaging, we found that the p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp) pathogenic variants impaired membrane trafficking, resulting in a complete loss of HCN2 elicited currents in Xenopus oocytes. Structural 3D-analysis in depolarized and hyperpolarized states of HCN2 channels, revealed that the pathogenic variants p.(His205Gln), p.(Ser409Leu), p.(Arg324Cys), p.(Asn369Ser) and p.(Gly460Asp) modify molecular interactions altering HCN2 function. Taken together, our data broadens the clinical spectrum associated with HCN2 variants, and disclose that HCN2 is involved in developmental encephalopathy with or without epilepsy.