Neuromuscular disorders (NMDs) encompass conditions that affect the peripheral nervous system. They are often caused by genetic alterations that impair skeletal muscle function. Molecular diagnosis remains challenging because some causal genes have not yet been identified. We therefore used feature-selection approaches to characterize genomic properties that distinguish NMD genes from other protein-coding genes. Curated genes implicated in NMDs (n = 639; GeneTable of NMDs) were merged with 134 genomic features for autosomal protein-coding genes spanning gene complexity, genetic variation, expression patterns, and other gene traits. Machine-learning-based feature selection using Boruta was combined with statistical analyses to compare NMD genes with other protein-coding genes. We also evaluated NMD genes against high-confidence non-NMD disease genes, examined variation across NMD subgroups, and assessed genes added to the NMD catalogue between 2020 and 2024. Compared with other protein-coding genes, NMD genes showed higher expression in disease-relevant tissues, including skeletal muscle and heart; greater transcript complexity and population genetic constraint, and stronger conservation across several evolutionary measures. Of 33 features that distinguished NMD genes from both high-confidence non-NMD disease genes and other autosomal genes, 28 were expression measures. Feature profiles varied across NMD subgroups, while genes added between 2020 and 2024 showed profiles similar to established NMD genes across all Boruta-confirmed features. This study identified genomic features that distinguish NMD genes from high-confidence disease genes and broader autosomal protein-coding genes. These findings provide insight into the gene architecture, expression, constraint, and evolutionary conservation of NMD genes and may inform candidate-gene prioritization in genetically unresolved NMDs.

Intestinal malrotation (IM) is a relatively common congenital anomaly of the gastrointestinal tract resulting from abnormal rotation during embryonic development. While some cases of IM are part of syndromic manifestations, most are isolated, with their genetic basis remaining largely unknown. We present a familial case of apparently autosomal recessive isolated IM in a consanguineous kindred of Arab-Bedouin descent. Nine individuals from two nuclear families were recruited. Homozygosity mapping and logarithm of odds (LOD) score calculations were performed using single nucleotide polymorphism (SNP) array data. Whole-exome and whole-genome sequencing data were analyzed to identify candidate variants. Various analysis and interpretation tools were applied to evaluate and describe potential disease-associated variants. Homozygosity mapping identified a 2 Mb candidate locus on chromosome 21q22.3. Multipoint linkage analysis identified an overlapping locus on chromosome 21 (maximal LOD score: 3.168). A missense variant in PCNT (Gln1951Pro), along with several other segregating candidates, was evaluated. Our findings demonstrate a novel candidate locus at chromosome 21q22.3 associated with isolated IM, with PCNT emerging as a potential candidate gene. Further studies involving additional affected families and functional validation are required to establish the contribution of this locus and the identified variant to the pathogenesis of isolated IM.

Blood lipids are well-established risk factors for cardiovascular and other chronic diseases, yet the underlying functional mechanisms remain unclear. We conducted a large-scale, genome-wide transcriptome analysis of serum lipid levels using whole blood RNA-sequencing data from 6898 individuals of diverse races and ethnicities (17% Black or African American, 15% Hispanic Latino/a, 68% White). We utilized mixed-effects linear regression and meta-analyzed four discovery datasets (n = 4398 participants), identifying significant associations for 27 genes with low-density lipoproteins (LDL), 974 with high-density lipoproteins (HDL), 1229 with triglycerides (TRIG), and 276 with total cholesterol (TCHO). Two replication datasets (n = 2500 participants) confirmed 3, 55, 380, and 31 genes, respectively. Enrichment analysis revealed lipid metabolic processes for LDL, HDL, and TCHO associated genes, including LDLR, NPC2, and SQLE for LDL and TCHO, while TRIG associated genes highlighted immune and allergic response, cancer, and molecular signaling gene enrichment pathways. To infer causality, we performed bidirectional two-sample Mendelian randomization (MR) and colocalization analyses. MR identified the expression of 8 genes as possibly causal for lipid levels; notable EEPD1 expression, linked with cellular cholesterol homeostasis, was inversely linked with TRIG levels. We also identified novel variants shared with LDL levels and gene expression of DDAH2, associated with cardiovascular disease, and TRIG level and expression of FCGRT, linked with maintaining triglyceride homeostasis. Phenome-wide enrichment analyses reported TRIG associated genes were enriched in secondary malignancy of bone suggesting broader systemic health implications of modified lipid levels. These findings highlight novel associations and genetic regulatory mechanisms as potential therapeutic targets for lipid dysregulation.

Congenital microphthalmos (CMIC) is a rare ocular malformation with marked genetic heterogeneity. To investigate its genomic landscape, we performed whole-genome sequencing of 10 proband-parent trios and integrated analyses of inherited variants, candidate germline de novo variants, and tandem-repeat variation. Following quality, population-frequency, and de novo probability filtering, 45 candidate germline de novo loci involving 37 genes were prioritized, including variants in POMT1, MYH11, and NR4A1. Rare homozygous variants in ANXA9 and SCARA5 showed recessive segregation in their respective families. Integration of gene-expression profiles and published functional evidence highlighted potential biological links to ocular development, calcium homeostasis, iron transport, and cellular stress responses. Repeat analysis further identified prominent repeat-length outlier signals in probands 5, 9, and 10, including a candidate AACCCT repeat-length alteration overlapping the SOX9/SOX9-AS1 locus in proband 10. Collectively, these findings broaden the spectrum of candidate genomic alterations in CMIC and nominate genes and regulatory loci that may contribute to its molecular pathogenesis. Their specific pathogenic roles and functional effects require further validation in independent cohorts and experimental models.

Katanins are microtubule-severing ATPases. Dysfunction of katanin subunits has been implicated in impaired neurogenesis, morphogenesis, and neuronal migration. Although heterozygous variants in KATNAL2 (katanin-like 2), a known autism-spectrum disorder (ASD) risk gene, have been reported in patients with ASD or other neurodevelopmental disorders (NDD), the underlying cellular mechanisms remain incompletely defined. Whole-exome sequencing (WES) identified a homozygous missense variant in KATNAL2 (NM_001387690.1:c.1390T>C; p.(Ser464Pro)) in a pediatric patient presenting with severe global developmental delay, intellectual disability, infantile-onset epilepsy, and autistic features, extending the phenotypic spectrum beyond prior autism-focused associations. To elucidate the cellular mechanisms underlying KATNAL2 dysfunction, we established induced pluripotent stem cells (iPSCs) from patient fibroblasts and differentiated them into neural progenitor cells (NPCs) for quantitative immunofluorescence analyses of microtubule organization, microtubule regulatory proteins, and transcriptome profiling. In parallel, we generated CRISPR/Cas9 katnal2 zebrafish crispants and assessed body patterning and axonal projections using the transgenic nbt:dsRed line. Patient iPSC-derived NPCs showed impaired interphase microtubule network organization with increased angular standard deviation in directionality and mitotic/cytokinetic abnormalities, including abnormal positioning of the microtubule-rich cytokinetic midbody region. In vivo, CRISPR/Cas9 katnal2 zebrafish crispants showed abnormal body patterning together with disorganized motor neuron and posterior lateral line nerve axonal projections. Together, these findings support KATNAL2 as a regulator of microtubule network organization and mitotic progression in human NPCs. Our data from patient-derived NPCs and a zebrafish model provide functional evidence linking the p.(Ser464Pro) variant to an expanded syndromic neurodevelopmental phenotype.

Immune-related DNA methylation patterns may provide interpretable markers of biological ageing and disease relevance. This exploratory study integrated four Illumina HumanMethylation450 and MethylationEPIC datasets comprising paediatric peripheral-blood leukocytes (GSE36054; 192 retained after quality control), infant buccal cells (n = 542), neuronal nuclei (n = 58), and peripheral blood from a polybrominated-biphenyl exposure cohort (n = 659). The paediatric peripheral-blood dataset served as the primary modelling cohort, with infant buccal cells providing an independent early-life cross-tissue reference. Processing included cohort-specific quality control and normalisation, batch adjustment, empirical-Bayes differential methylation analysis, pathway enrichment, repeated 10-fold cross-validation, biologically informed neural-network modelling, feature attribution, cross-tissue assessment, gene-ablation analysis, disease mapping, and two-sample Mendelian randomisation. IL6, CXCR4, CDK11 and CREBBP showed the strongest coherent methylation signals and stable predictive contributions, whereas BCLAF1 contributed less consistently. These loci mapped to cytokine-receptor interaction, chemokine signalling, G2/M checkpoint regulation, histone acetylation, and apoptosis-related pathways. The neural network achieved a cross-validated mean absolute error of 1.87 years and R2 of 0.781; removing the polybrominated-biphenyl-associated feature filter yielded comparable performance (MAE 1.91 years; R2 = 0.772). Prioritised features also showed cross-tissue consistency in infant buccal cells. Mendelian randomisation identified genetically supported associations of IL6 methylation with preterm birth and lower mean birth weight and of CXCR4 methylation with preterm birth after multiplicity correction. These associations do not establish that methylation itself causes the outcomes. The findings prioritise immune-related methylation features across paediatric blood and infant buccal contexts within an interpretable framework for age-related methylation and outcome-oriented molecular investigation.

Targeted precision therapies are increasingly used in the treatment of individuals with vascular anomalies (VAs). This increases the need for rapid, accurate and inexpensive genetic diagnosis. Droplet digital polymerase chain reaction (ddPCR) is an alternative to next-generation sequencing (NGS), permitting rapid, highly sensitive interrogation of recurrent pathogenic mosaic variants. We examined the feasibility of ddPCR as a primary diagnostic tool in a large cohort of individuals with VAs. Lesional tissue was collected for ddPCR of up to 46 recurrent pathogenic variants across 16 genes associated with VAs. Specimens were assessed on a subset of assays for each individual based on clinical phenotype. Most individuals who had negative ddPCR results went on to high-depth gene panel or deep exome NGS, or Sanger sequencing. Here we report the phenotypic and molecular findings for 78 newly recruited and tested individuals in addition to the 60 individuals already reported from our cohort. The overall diagnostic yield for our cohort when combined with individuals previously reported was 104/138 (75%). Of 138 individuals tested, recurrent pathogenic variants were detected in 71 (51%) on ddPCR. Variants were most frequently identified in PIK3CA (n = 28), TEK (n = 18), GNAQ (n = 12), or MAP2K1 (n = 7). In a further 33 individuals, pathogenic variants were identified on NGS or Sanger sequencing. Our findings indicate that ddPCR is an efficient method achieving a high diagnostic yield in our cohort when used prior to sequencing.

Variants in the transcription factor CRX cause a spectrum of retinal degenerative disorders including Leber congenital amaurosis 7 (LCA7). LCA7-associated variants exert a dominant effect in developing photoreceptor cells, leading to cellular dysfunction and poor to absent visual acuity in patients. Here, we dissect the allelic heterogeneity of LCA7 using multimodal single-cell gene expression and chromatin accessibility analysis of retinal organoids. We generated mature (240 day) retinal organoids from patient-derived iPSCs carrying missense (K88Q) and nonsense (T155fs) variants, as well as from CRISPR-corrected control lines. We show that these variants, while both causing LCA7, produce unique transcriptional changes in human photoreceptors. Furthermore, the K88Q variant causes aberrant chromatin accessibility at sites typically bound by non-photoreceptor retinal transcription factors, indicating a partial derangement of normal retinal cell fate programming. This work demonstrates the gene regulatory mechanisms of pathogenicity of two disease-causing CRX variants in human retinal cells.

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly characterized by impairment of both diaphragm and lung development in utero. CDH presents as a spectrum of forms and severities, with diaphragm defects arising in the dorsal/posterior region typically correlating with more severe pulmonary disease and higher risk of mortality than those appearing in ventral/anterior regions. The genetic etiology underlying CDH is complex, with many implicated genes showing variable expressivity and incomplete penetrance in both human patients and mouse models. Here we present in vivo validation of two genes previously unassociated with CDH: the CDC42-interacting kinase CDC42BPB; and CNOT1, a scaffolding protein of the CCR4-NOT protein complex, critical for mRNA regulation through modifications such as deadenylation. Each gene was found to have a damaging, de novo missense variant in a recent large-scale CDH patient sequencing screen. Loss of Cdc42bpb leads to ventral diaphragmatic hernias, heart septal defects, and minor lung epithelial differentiation defects in mouse embryos. Installation of the orthologous patient-specific missense variant through CRISPR/Cas9 editing leads to less severe ventral diaphragm defects. Mouse embryos with either one or two copies of the orthologous Cnot1 variant, c.1867C > T (p.R623W), develop dorsal diaphragmatic hernias with low (<50%) penetrance, and mutants showed alterations in mRNA isoform expression consistent with the molecular role of Cnot1 in RNA splicing. These results underscore the power of in vivo functional modeling to validate genes and patient-specific variants uncovered by patient sequencing, reveal two previously unrecognized genetic causes of CDH, and highlight the heterogeneity of different patient anatomic presentations.

More than 500 genes associated with hearing loss have been identified, yet the pathogenic mechanisms underlying most deafness-causing mutations remain to be clarified, hindering the development of viable clinical therapeutic strategies. Clinical studies indicated that patients with DUOXA2 mutations present mucosal immune dysregulation and hypothyroidism. In addition, genetics screening in mouse models linked Duoxa2 deficiency to severe deafness, yet its pathogenic mechanism remains elusive. In this study, we demonstrated that Duoxa2 is essential for normal auditory function and clarified its underlying mechanism by establishing the Duoxa2 conditional knockout (cKO) mouse model. We found that Duoxa2 deficiency impairs the structure and function of outer hair cells (OHCs) and stria vascularis (SV), core auditory components, via disrupted thyroid hormone synthesis. Duoxa2 cKO mice exhibited disorganized stereociliary bundles in OHCs, altered distribution patterns of the critical KCNQ4, aberrant ribbon synapse localization, and capillary dilation within the intermediate cell layer of SV. Notably, Atoh1-Cre drivers not only express Cre recombinase in hair cells but also show high expression in thyroid. This finding elucidates the reduced thyroid hormone levels in Atoh1-Duoxa2-/- mice and reveals an indirect mechanism affecting cochlear function. While postnatal thyroid hormone supplementation completely reversed these pathological changes, the effect was minimal in adulthood. Our findings provided insights into the intrinsic link between distinct thyroid disorders and hearing impairment and offered evidence for clinical intervention in patients with DUOXA2 variants.

Mutations in the LRRK2 gene are the most frequent cause of familial Parkinson's disease (PD) whereas common variants are associated with an increased risk for sporadic PD. LRRK2 encodes a multi-domain protein displaying two functional enzymatic activities: GTPase and kinase. In addition to familial mutations, several common LRRK2 coding variants have been associated with PD risk in different ethnic populations. Little is known about how these coding variants modulate the risk of developing PD. In this study, we aimed to evaluate the properties of a collection of LRRK2 coding risk variants (A419V, N551K, R1398H, R1628P, M1646T, S1647T, G2385R). With the exception of G2385R, we find that coding risk variants have minimal impact on LRRK2 protein levels, GTP-binding, LRRK2 phosphorylation, and subcellular localization in human cells and primary neurons. G2385R LRRK2 exhibits reduced phosphorylation at Ser910/Ser935 and Ser1292, consistent with diminished kinase activity. Notably, however, LRRK2 variants associated with increased PD risk (R1628P, M1646T, and G2385R) significantly elevate the levels of LRRK2-mediated Rab10 phosphorylation by >two-fold in cells. In contrast, LRRK2 variants associated with reduced PD risk (N551K, R1398H, and N551K-R1398H) do not alter pRab10 levels. The G2385R LRRK2 variant significantly inhibits neurite outgrowth in primary cortical neurons compared to wild-type LRRK2. Our study indicates that LRRK2-dependent Rab phosphorylation represents a relevant readout of PD risk induced by LRRK2 variants and demonstrates that the G2385R variant creates a hyperactive kinase that can impair neuronal integrity at comparable levels to the effects of G2019S LRRK2.

Dyslipidemia is a leading modifiable risk factor for cardiovascular disease (CVD), yet the shared genetic architecture of lipid traits remains poorly characterized, particularly in African-ancestry populations who bear a disproportionate CVD burden but are underrepresented in genomic research. We conducted multi-trait meta-analyses of genome-wide association summary statistics for HDL cholesterol, LDL cholesterol, triglycerides, and total cholesterol in 34 039 individuals of African ancestry, comprising 27 289 discovery participants from three continental African cohorts (ACCME, AWI-Gen, and UGR) and 6750 replication participants from the UK Biobank. Multi-trait analysis and eQTL colocalization across metabolic and cardiovascular tissues were then performed to identify shared genetic signals and characterize their tissue-specific regulatory mechanisms. Multi-trait analysis identified 90 genome-wide significant variants. Of these, 43 were novel variants with no prior exact-variant lipid association records. Eight variants across six genomic regions reached genome-wide significance in the multi-trait analysis and 70 of the 90 primary signals remained significant after total cholesterol was excluded. The strongest associations localized to established lipid loci including APOE/NECTIN2/TOMM40 (P = 8.09 × 10-133), CETP (P = 9.44 × 10-108), LIPC (P = 9.42 × 10-51), and LPL (P = 5.79 × 10-22), with 92% of signals replicating in the UK Biobank cohort. eQTL colocalization linked seven lead variants to tissue-specific gene expression and prioritized candidate regulatory genes. Together, these findings characterize shared lipid-associated signals with heterogeneous, trait-dependent effects in continental African populations and provide population-relevant evidence for candidate regulatory mechanisms.

Next-generation sequencing (NGS) has markedly expanded the mutational spectrum of monogenic congenital hypothyroidism (CH), but has also led to the identification of numerous variants of uncertain significance, limiting the clinical utility of genetic testing. We previously developed an SLC5A5-specific machine learning-based predictor that improved the interpretation of missense variants associated with CH. Here, we systematically evaluated the pathogenicity of all CH-associated SLC5A5 variants reported in the literature using an integrated multidisciplinary framework based on the American College of Medical Genetics and Genomics (ACMG) guidelines. Among the 63 identified SLC5A5 variants, 5% were classified as benign, 16% as likely benign, 15% as of uncertain significance, 37% as likely pathogenic, and 27% as pathogenic. The computational variant effect predictor AlphaMissense was the most appropriate tool for predicting the functional impact of SLC5A5 missense variants. Experimental functional assays assessing the functional impact of SLC5A5 missense variants on radioiodide accumulation were instrumental in reclassifying variants of uncertain significance into clinically actionable categories. Moreover, patients' phenotype revealing reduced to absent thyroid radioiodide accumulation provided valuable phenotypic evidence supporting variant classification, which highlights the diagnostic value of thyroid scintigraphy in CH-associated SLC5A5 variants. Overall, our findings highlight the value of a multidisciplinary pathogenicity evaluation based on the ACMG guidelines, integrating computational predictions, functional assays, and phenotypic data into a standardized and evidence-based framework for the accurate interpretation of SLC5A5 variants, thereby representing a significant advance in the molecular diagnosis of congenital hypothyroidism.