The CAV3 p.Ala46Thr mutation is previously associated with skeletal muscle damage only, with its myocardial impact and long-term cardiac risks unclear. This study is aimed at investigating whether it induces subclinical myocardial damage at the cellular level and explore its mechanism using a patient-specific iPSC-CMs model. To achieve this, peripheral blood from a proband carrying the CAV3 c.136G>A (p.Ala46Thr) mutation but only clinical manifestations of skeletal muscle damage was collected. Subsequently, a patient-derived iPSC line was established and directionally differentiated into iPSC-CMs. IF and TEM were then conducted to observe the structure of cardiomyocytes, the arrangement of sarcomeres, and the subcellular localization of CAV3 protein. Additionally, laser confocal calcium imaging technology was used to analyze the basic electrophysiological rhythm of cardiomyocytes and their response to fenoterol. As a result, the mutant iPSC-CMs model was successfully constructed. Compared with controls, mutant iPSC-CMs exhibited a normal cellular structure, but notably, the CAV3 protein exhibited abnormal aggregation around and inside the nucleus, resulting in a loss of membrane localization. Furthermore, functional assays showed that the mutant iPSC-CMs had a significant decrease in basic beating frequency and an irregular rhythm. After stimulation with fenoterol, the increases in beating frequency and rhythm regularity were significantly less pronounced than those in controls. In conclusion, this study is the first to demonstrate that human cardiomyocytes from carriers of the CAV3 p.Ala46Thr mutation without any cardiac symptoms clinically exhibit an abnormal localization of CAV3 protein and definite altered spontaneous activity and calcium-handling behavior. This suggests that the mutation can induce subclinical myocardial damage, and the carriers should be considered as a group with potential risk of heart disease. Overall, this study provides a novel experimental basis for the early warning and accurate treatment of CAV3-related diseases on the site of clinics.

Primary congenital glaucoma (PCG) is an early-onset eye disorder, particularly prevalent in populations with high consanguinity rates. In Pakistan, approximately 63% of marriages are reported to be consanguineous, contributing to an increased burden of autosomal-recessive disorders. The genetic basis of PCG in the highly consanguineous Pakistani population is largely unexplored. This study was conducted to broaden the genetic landscape of PCG in families of Pakistani origin. PCG families identified through clinical characterization were recruited in the study. All probands were initially investigated for CYP1B1 mutations by targeted Sanger sequencing, followed by segregation analysis of the identified variants. Moreover, one family (PCG02) not linked to CYP1B1 was further subjected to a 250K SNP microarray and whole-exome sequencing (WES). The study participants comprised nine large, unrelated consanguineous and three PCG trio families collected from Pakistan. Nine identified variants were recurrent, whereas one previously unreported variant, CYP1B1 (NM_000104.4: c.1390T>C; p.Ser464Pro), was not found in any of the online databases such as gnomAD, ClinVar, and dbSNP, and was predicted to be deleterious by all the pathogenicity predicting tools. Furthermore, a plausible 2-Mb novel locus was identified on Chromosome 7q34 (139,681,371-141,507,822) (GRCh38/hg38) in family PCG02. The current study expands the mutation spectrum of CYP1B1 in the Pakistani population and highlights the genetic heterogeneity of the disease in this population.

Variants in RELN lead to a range of neurodevelopmental phenotypes, from autosomal recessive lissencephaly with cerebellar hypoplasia (LCH) (LIS2) to autosomal dominant focal epilepsies. We carried out exome sequencing (ES) on 10 affected individuals from eight unrelated Iranian families with consanguinity. Nine distinct variants were identified: Four are novel; four had been deposited in ClinVar without a reported affected individual; and the ninth, c.2015C>T, p.(Pro672Leu), had been reported only in heterozygous carriers with dominant epilepsy. In this study, we report the variant for the first time in the homozygous state, in a patient with the full recessive phenotype. The patients were pooled in a systematic review following PRISMA, updated to July 2026, with 28 papers, and all variants were reannotated against NM_005045.4, under a single ACMG/AMP framework. The primary cohort comprised 80 individuals from 48 kindreds (70 previously reported, 10 new) with 48 distinctive variants, of which 41 (85%) were pathogenic or likely pathogenic and five were variants of uncertain significance; two balanced rearrangements were not in either the sequence-variant or copy-number frameworks; and 16 individuals from nine kindreds with monoallelic candidate or susceptibility variants were analyzed separately. The age of onset was not continuous with no reports of onsets between 2.4 and 8 years. Zygosity did not account for this separation because monoallelic individuals occurred in both onset groups; in contrast, malformations of cortical development were documented in all 32 early-onset individuals and in none of the 23 later onset individuals with evaluable neuroimaging. ES was the diagnostic method in 65% of individuals. Thus, zygosity contributes to the phenotypic severity in a broad spectrum of RELNopathies and supports carrier testing and reproductive counseling in consanguineous families.

Ciliopathies represent a diverse group of inherited disorders resulting from dysfunctional cilia. A rare subset is associated with biallelic variants in WDR19, with fewer than 100 affected individuals reported worldwide. This study is aimed at reviewing the clinical and molecular spectrum of WDR19-associated ciliopathies, with a particular focus on genotype-phenotype correlations. We report a 32-year-old woman with renal, ocular and hepatic manifestations attributed to compound heterozygous variants in WDR19, and reviewed available data from 93 previously published cases. Missense, truncating, splice-site and copy number variants have all been reported, with renal, ocular, skeletal and hepatic involvement most frequently observed. Genotype-phenotype analyses indicated that the presence of a truncating variant was associated with increased likelihood of skeletal involvement and reduced likelihood of hepatobiliary disease compared with individuals harbouring only missense variants. Amongst individuals with only missense variants, variants located outside repeat protein domains were associated with early symptom onset (before 16 years of age) and with ocular manifestations. Notably, recurrent variants demonstrated considerable phenotypic variability. This study represents the largest review of WDR19 variants to date and contributes to current understanding of WDR19-related disease.

Familial isolated pituitary adenoma (FIPA) accounts for approximately 2%-5% of all pituitary adenomas, with inactivating variants of the aryl hydrocarbon receptor-interacting protein (AIP) gene representing the most frequent known genetic cause. Clinically, patients with AIP variants often have young-onset macroadenomas with growth hormone hypersecretion, although disease severity and penetrance are variable. Most reported AIP variants are point mutations, whereas large deletions are rare and potentially underdiagnosed. Accurate detection of AIP copy-number variants requires methods such as multiplex ligation-dependent probe amplification or validated copy-number analysis of next-generation sequencing data, as Sanger sequencing alone may fail to identify these alterations. Due to the rarity of the disease, it is unknown whether large deletions in the ubiquitously expressed AIP gene are associated with potentially more severe phenotype. Available data suggest that large deletions may occur in 8%-10% of AIP mutation-positive pedigrees, highlighting the importance of incorporating copy-number variant detection into AIP testing workflows. We analysed data from all published patients with large AIP deletions (n = 25) and report here two novel large AIP deletions (Exons 3-4 and Exons 2-6 deletions) and three additional three families, including an Albanian kindred associated with metastatic Hürthle cell thyroid carcinoma. No major differences compared with other AIP variants were found in age at diagnosis, tumour size, hormonal profile, sex distribution or presence of other tumours. A role for AIP variants in thyroid carcinogenesis is unlikely.

Inherited diseases have a strong genetic basis, with copy number variants (CNVs) playing a crucial role in their pathogenesis. However, the detection and validation of CNVs remain challenging. In this study, we introduce target enrichment polymerase chain reaction (tecPCR), a novel technique that integrates multiplex polymerase chain reaction (PCR) with next-generation sequencing (NGS) for enhanced CNV validation, and applied it in familial exudative vitreoretinopathy (FEVR). Using NGS, 97 variants linked to FEVR were identified, including 91 SNVs and 6 CNVs. Of the CNVs, 66.67% (4/6) were validated both by quantitative PCR and tecPCR, yielding a positive predictive value of 100% (95% confidence intervals: 39.8%-100%) and negative predictive value of 100% (95% confidence intervals: 15.8%-100%) for tecPCR in this pilot validation. The validated FEVR genetic test showed a positivity rate of 53.07% (95/179). Moreover, the demographic and ocular features of the FEVR patients with CNVs were comparable to those with SNVs. This study broadens the mutation spectrum of FEVR and demonstrates that tecPCR provides exceptional specificity and sensitivity, making it a promising tool for genetic diagnostics.

Clinical and epidemiological evidence demonstrates a robust bidirectional link between inflammatory bowel disease (IBD) and periodontitis, with IBD patients having a significantly elevated risk of periodontitis. Although this association is well established, the shared molecular mechanisms remain unclear. Both IBD, including Crohn's disease (CD) and ulcerative colitis (UC), and periodontitis feature aberrant ferroptosis activation, which drives oxidative damage and tissue breakdown. We therefore hypothesized that ferroptosis is a core pathogenic mechanism common to all three conditions. Using GEO transcriptomic data, we identified 116, 134, and 255 exploratory candidate ferroptosis-related differentially expressed genes in CD, UC, and periodontitis, respectively, with 55 candidate genes overlapping across all three conditions. Four machine learning-based feature selection algorithms consistently ranked COPZ1 as the top hub gene. In animal models of colitis and periodontitis, COPZ1 showed markedly elevated protein levels in inflamed intestinal and gingival tissues. In vitro experiments demonstrated that COPZ1 knockdown upregulated the expression of ferroptosis-inhibitory genes (Gpx4, Slc7a11, and Fth1) in RAW264.7-ASC macrophages under both basal and LPS-induced inflammatory conditions, and reversed LPS-mediated downregulation of Gpx4, suggesting that COPZ1 is involved in the regulation of ferroptosis in macrophages. Immune infiltration analysis revealed correlations between COPZ1 expression and the abundance of macrophages, neutrophils, B cell subsets, and T cell subsets, suggesting its association with differences in estimated immune cell composition. Single-cell RNA sequencing from CD patient biopsies further demonstrated heterogeneous COPZ1 expression across diverse intestinal immune cell types (including neutrophils, CD8[+] T cells, CD4[+] T cells, and macrophages) and linked to ferroptosis-associated intercellular signaling. In silico drug prediction identified nine small-molecule compounds that stably bound to COPZ1 through hydrogen bonds. Molecular dynamics simulations subsequently ranked BRD-K66453893 as the compound exhibiting the greatest structural stability among the tested candidates. Collectively, our findings identify COPZ1 as a shared candidate ferroptosis-related hub gene for CD, UC, and periodontitis, and highlight its association with the inflammatory immune microenvironment, thereby providing a promising therapeutic target for these interrelated chronic inflammatory disorders.

Charcot-Marie-Tooth (CMT) neuropathy is a clinically and genetically heterogeneous group of diseases characterized by the length-dependent axonal degeneration of peripheral nerves. We previously mapped a rare form of X-linked CMT, CMTX3, to a 5.7-Mb interval on chromosome Xq26.3-q27.1 and excluded the coding region of all known genes in the linkage interval for mutations. Whole genome sequencing subsequently identified a 78-kb region of chromosome 8q24.3 that had been duplicated and inserted into the CMTX3 locus between the genes HAPSTR2 and SOX3. The 78-kb insertion, which contains a partial transcript of ARHGAP39, fully segregated in families with CMTX3 and was absent in neurologically normal controls. To retain the CMTX3 insertion and investigate its consequences in appropriate neuronal tissue, we generated induced pluripotent stem cells (iPSCs) from CMTX3 fibroblasts. Using bulk RNA sequencing of patient-derived spinal motor neurons, ARHGAP39 was deemed nonpathogenic by excluding both the formation of novel fusion transcripts and dosage effects from the partial duplication. Subsequent NanoString expression analyses of candidate genes within the CMTX3 locus, across different stages of neuronal differentiation, identified spatiotemporal dysregulation of SOX3. NanoString showed reduced SOX3 expression in patient iPSCs. RNA sequencing detected SOX3 downregulation in CMTX3 neuroepithelial progenitor cells, which was further confirmed by quantitative proteomics. Given the early onset and relatively rapid progression of CMTX3, these data prioritise SOX3 as a leading candidate gene, consistent with its role as one of the earliest transcription factors expressed in the developing nervous system and a key regulator of neuronal fate.

Variants of uncertain significance (VUS) in mismatch repair (MMR) genes represent a persistent bottleneck in germline interpretation for Lynch syndrome, creating a critical opportunity to leverage tumor biology to refine pathogenicity assessment. Although tumor features such as microsatellite instability (MSI) and immunohistochemistry (IHC) are routinely evaluated, they are typically interpreted separately from germline classification, and their quantitative contribution within ACMG/AMP frameworks remains poorly defined. We therefore analyzed paired germline and tumor sequencing data from 1110 tumors across 1073 patients with colorectal or endometrial cancer to determine whether mismatch repair-deficient (MMR-d) mutational signatures can be quantitatively integrated into Bayesian germline variant interpretation. Using COSMIC single-base substitution signatures, tumors were classified as MMR-d or MMR proficient, and an empirically derived likelihood ratio (LR) quantified the association between MMR-d signatures and pathogenic germline MMR variants. The presence of an MMR-d signature increased the likelihood of an underlying pathogenic germline MMR variant approximately eightfold (LR ≈ 8; log10 LR ≈ 0.90), whereas its absence provided moderate-to-strong benign evidence (LR ≈ 0.156; log10 LR ≈ -0.81). Applying this integrative framework to 45 germline MMR VUS, joint modeling of tumor mutational signatures with additional somatic and variant-level evidence resulted in clinically significant reclassification of 38 (84.4%) variants, including three reclassified as pathogenic or likely pathogenic and 35 as likely benign. A total of 16 downgraded variants were independently downgraded by Invitae. These findings demonstrate that tumor mutational signatures can be formally incorporated into Bayesian germline interpretation, transforming tumor data into quantitative pathogenicity evidence and offering a principled strategy to reduce VUS burden in hereditary cancer genetics.

Nonsmall cell lung cancer (NSCLC) is characterized by substantial heterogeneity in driver mutation backgrounds and tumor microenvironment (TME) phenotypes, yet the transcriptional regulatory states linking genotype context to immune-stromal variation remain incompletely defined. In this study, we integrated mutation, transcriptomic, and clinical data from TCGA-LUAD/LUSC to construct a mutation-expression-matched NSCLC cohort and evaluated RUNX1 as a mutation context-associated transcriptional/regulatory state rather than as a recurrently mutated driver. Driver contexts were assessed using a prespecified six-gene panel comprising EGFR, KRAS, TP53, STK11, KEAP1, and BRAF in the overall NSCLC cohort, the primary LUAD analysis, and a descriptive LUSC sensitivity analysis. The prespecified driver-context-negative operational group was defined as tumors without mutations in any of these six genes. RUNX1 mutation status was reported separately and did not contribute to this definition. Analyses included RUNX1 distributions, six-gene overlap, the RUNX1 cutoff, comutation burden, model sample-size audits, and adjusted context models. RUNX1 expression and inferred RUNX1 target-gene-set activity were evaluated across mutation contexts, whereas TME features were characterized using MCP-counter, xCell, ssGSEA, and ESTIMATE. Single-cell RNA sequencing and Visium spatial transcriptomic datasets were further used to localize RUNX1 expression, inferred target projections, and candidate TME-supporting genes. RUNX1 was prioritized within a prespecified candidate-screening framework and was significantly associated with the inferred RUNX1 target-gene-set readout. RUNX1 expression differences required interpretation in relation to histology and comutation structure. In the primary LUAD analysis, the KRAS-mutant context was associated with higher RUNX1 expression, whereas the KEAP1-mutant context was associated with lower expression. LUSC findings were retained as a descriptive sensitivity analysis and were not given the same inferential weight. Six-gene overlap, comutation burden, the RUNX1 cutoff, and model sample-size audits further showed that driver labels were nonmutually exclusive and that the prespecified driver-context-negative group was an operational reference rather than a biologically homogeneous subtype. RUNX1-high and RUNX1-low tumors displayed mutation context-dependent immune and stromal differences, involving stromal/cancer-associated fibroblast, myeloid/dendritic cell, endothelial, immune-score, and tumor-purity axes. Single-cell analysis localized RUNX1-associated signals and target-gene projections across epithelial-like and myeloid compartments, and spatial transcriptomics supported concordance between RUNX1 and selected TME-supporting genes in NSCLC tissue sections. Public immune checkpoint inhibitor cohorts were limited by small sample size and incomplete annotations and did not support RUNX1 as a stable immunotherapy-response predictor. Overall, these findings propose a mutation-context-aware RUNX1-TME stratification framework and generate testable hypotheses for mechanistic validation and mutation-annotated immunotherapy studies.