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.

Mucopolysaccharidosis type IVA (MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficient activity of N-acetylgalactosamine-6-sulfatase (GALNS). In this single-center study, we analyzed the clinical and genetic characteristics of 82 pediatric MPS IVA patients, including 68 severe, 12 intermediate, and 2 mild cases. The most common clinical manifestations were pectus carinatum (72%), short stature (56%), and genu valgum (45%). All patients showed markedly reduced GALNS activity, although no significant difference was observed across phenotypic subgroups. Genetic analysis identified 66 distinct GALNS variants, 12 of which were novel. The c.953T>G (p.M318R) variant was the most frequent allele (24.7%). Based on integrated clinical, structural, and in silico assessments, p.M318R and p.R386C were linked to severe disease, whereas p.P499L was associated with mild phenotypes. Protein structure analysis revealed that all homozygous missense variants located in domain 1 correlated with severe presentations. Among patients carrying two missense variants, 85% (34/40) exhibited severe symptoms, and 70.6% (24/34) of these had at least one variant affecting buried residues. Notably, 85.7% (24/28) of patients with biallelic buried-residue missense variants were classified as severe. In summary, this study delineates the phenotypic spectrum of MPS IVA, expands its genotypic landscape, and provides a foundation for further genotype-phenotype investigations.

Schizophrenia (SCZ) and systemic lupus erythematosus (SLE) exhibit epidemiological overlap, suggesting shared genetic underpinnings. Using the largest SLE genome-wide association study (GWAS) to date, we identified a significant genetic correlation between SCZ and SLE (rg = 0.25, p = 9.0 × 10-4). Cross-trait analysis nominated multiple candidate shared loci outside the major histocompatibility complex (MHC), most with aligned effects. The strongest candidate shared association was found at the FURIN-FES locus, previously implicated in SCZ and newly associated with SLE. In addition to FURIN-FES, FCGR2A, DOT1L and an intergenic noncoding RNA locus on chromosome 2 showed additional evidence consistent with shared association. Functional annotation showed that this locus modulated the expression of FURIN, which encodes a proprotein convertase involved in diverse biological processes, and FES, which encodes a tyrosine kinase important in innate immune signalling. The shared risk allele was also associated with altered plasma levels of FURIN, FES, and their functional partners (GDF2, BMP10, NTF4, FER). Tissue- and cell-type-specific eQTL analysis revealed distinct regulatory effects: FURIN expression was perturbed in neuronal and immune cells, while FES expression was altered in immune cells. Mendelian randomisation supported a causal role for FES protein in both disorders. These findings highlight that pleiotropy at the FURIN-FES locus for SCZ and SLE is compatible with disease-specific mechanisms. This underscores that shared genetics across traits does not necessarily mean shared biology.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by progressive motor neuron (MN) loss. Although rare pathogenic variants in the D-amino acid oxidase (DAO) gene have been proposed to cause familial ALS, inconsistent findings across mammalian models have hindered functional validation. Here, we establish and characterise a Drosophila model of DAO loss-of-function by targeting its highly conserved orthologue, Daao1. Constitutive Daao1 knockdown caused early developmental lethality, while surviving adult escapers exhibited profound flight and climbing impairments. Contrary to expectations, Daao1 deficiency did not elevate D-serine levels in larval or adult brains, and simultaneous knockdown of serine racemase (Srr), the enzyme responsible for D-serine synthesis, failed to rescue locomotor phenotypes. Tissue-specific silencing revealed that Daao1 is essential within both MNs and muscles, but not glia, for maintaining motor performance. RNA-seq of Daao1-deficient flies uncovered widespread transcriptional dysregulation affecting metabolism, peptide transport, immune activation and developmental pathways, accompanied by alternative splicing changes in genes required for neuronal morphology and synaptic organisation. Consistent with these molecular alterations, neuromuscular junctions (NMJs) displayed reduced axonal branching and bouton numbers, indicating disrupted neuromuscular connectivity. Remarkably, ubiquitous expression of human DAO rescued lethality and motor defects, demonstrating functional conservation and supporting a role for DAO in maintaining neuromuscular health. Together, our findings reveal a previously unrecognised, D-serine-independent role for DAO in neuromuscular integrity and provide functional evidence connecting DAO dysfunction to ALS-relevant phenotypes. The DAO-ALS Drosophila model provides a powerful platform for mechanistic studies and for evaluating human DAO variants of uncertain significance in ALS.

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder associated with motor neuron degeneration, yet the role of the well-conserved Survival of Motor Neuron (SMN) protein in muscle remains insufficiently understood. Using the Drosophila neuromuscular junction (NMJ) as a model, we combined tissue-specific Smn depletion with transcriptomic profiling and prior genetic modifier screens to identify SMN-dependent genes that contribute to synaptic architecture. Functional screening of candidate genes significantly and selectively effected in muscle identified genes whose knockdown phenocopied NMJ morphometry defects characteristic of Smn loss-of-function (LOF), including FER, CAP, HDAC4, Nhe2, and Raskol. Unlike other genes in this group, analysis of postsynaptic architecture using a subsynaptic reticulum (SSR) biomarker disrupted by Smn LOF singled out the conserved tyrosine kinase FER as an SMN-dependent gene in muscle that influences this prominent feature of the NMJ. Analysis of different FER transcripts suggests that elevation of the FERp100 protein isoform in Smn LOF muscle tissue leads to a dominant-negative effect likely to disrupt signaling via the MAPK-family kinase Basket (Bsk) to regulate the downstream phosphatase Puckered (Puc).

EHMT1 is a lysine methyltransferase that writes and reads histone methylation marks to repress transcription. Deleterious mutations in EHMT1 cause Kleefstra syndrome (KLEFS1; OMIM #610253), a congenital neurodevelopmental disorder. Many of these affect the ankyrin repeat (ANKR) domain, which recognizes histone H3 lysine-9 methylation and mediates interactions with other epigenetic regulators. However, the functional consequences of EHMT1 missense variants within the ANKR domain remain poorly characterized, as nearly 50% are currently classified as variants of unknown significance (VUS). To address this gap, we performed a comprehensive structural genomics analysis to predict how 56 Kleefstra syndrome-associated missense variants alter EHMT1 ANKR domain structure and function. We integrated sequence-based pathogenicity algorithms with protein structural and dynamics metrics to generate variant molecular fitness scores. From these predictions, we selected 16 representative variants for experimental validation. Using differential scanning fluorimetry and circular dichroism, we quantified variant-specific effects on ANKR domain stability and secondary structure. We further assessed methyl-lysine binding using fluorescence polarization assays. By combining computational and biochemical analyses, we re-classified 80% of VUS as tolerated or damaging based on their effects on ANKR domain structural integrity and methyl-lysine binding. The resulting molecular fitness scores reveal mechanisms by which damaging variants disrupt EHMT1 ANKR domain stability, ligand binding, or both. Together, our integrated approach refines EHMT1 variant annotation and provides mechanistic insights that enhance current tools for variant interpretation in Kleefstra syndrome, which can be translated to missense variants in other epigenetic proteins and rare diseases.

Patent ductus arteriosus (PDA) is a common congenital heart disease (CHD) with a complex genetic basis. The transcription factor MEF2C plays a critical role in cardiac development, but the role of promoter region variants in PDA has not been explored. This study aimed to identify MEF2C promoter variants in PDA patients and investigate their functional consequences. We sequenced the MEF2C promoter region in 318 children with PDA and 306 healthy controls. Identified variants were subjected to dual-luciferase reporter assays in A7r5 vascular smooth muscle cells to assess transcriptional activity. Bioinformatics (JASPAR) and electrophoretic mobility shift assays (EMSA) were used to predict and validate changes in transcription factor binding. Seventeen variants were identified, of which seven were present exclusively in PDA patients. Three variants were novel. Dual-luciferase assays showed that six variants significantly altered promoter activity compared to wild-type (range 69.6%-82.3%). JASPAR prediction revealed that these variants affected binding sites for 16 transcription factors, including GATA1, GATA2, GATA3, STAT3, ZEB1 and E2F1. EMSA confirmed altered DNA-protein binding patterns for all six variants, and super-shift assays further validated that the g.24571 A > G variant specifically disrupts GATA3 binding. This is the first study to identify functionally relevant MEF2C promoter variants in PDA. Our findings suggest that dysregulation of MEF2C through promoter variants may contribute to PDA pathogenesis by disrupting critical transcription factor interactions. These results extend the genetic landscape of PDA beyond coding regions and provide new insights into the molecular mechanisms of CHD.

In admixed populations, formed by a mixing of two or more previously isolated populations, genomic segments can be traced to their ancestral populations ('ancestries'). Admixture mapping (AM) associates local ancestry with outcomes in admixed populations, detecting signals when causal variants differ in frequency or effect across ancestral populations. Prior work showed that adjusting for nearby GWAS-identified common variants does not fully explain some AM signals. Here, we assessed two approaches to explain the previously unexplained AM signal: (1) including sets of rare variants; (2) increasing the genomic region considered when searching for common variants. We studied these hypotheses comprehensively using a whole-genome sequencing dataset coupled with metabolomics from the Hispanic Community Health Study/Study of Latinos. From 16 genomic regions of interest, we identified 35 rare-variant sets associated with metabolite levels, of which 4 associations replicated. The locations of identified rare variant sets are either within the same gene or in proximity to adjusted common variants. Overall, our study provides new insight into the genetic architecture underlying AM signals via evaluating the contributions of common and rare variant sets. By integrating rare-variant analysis within the AM framework, our work highlights the potential interplay between variant types and establishes a novel strategy to guide future fine-mapping and causal variant identification in admixed populations. Yet the detected rare variants appear to explain only a small fraction of the AM signal, while inclusion of common variants from a larger genomic region appears to explain most of the AM signals.

Recurrent pregnancy loss affects 1%-2% of reproductive-aged couples, with chromosomal abnormalities accounting for 40%-60% of cases. There is a close link between adverse pregnancy history (APH) and recurrent pregnancy loss (RPL). However, cryptic balanced translocations with translocated fragments smaller than the resolution of karyotyping or with similar banding patterns are often missed. This study, based on the testing results of 586 families who underwent preimplantation genetic testing for aneuploidy (PGT-A) due to RPL or APH, identified cryptic balanced translocations that were not detectable by conventional karyotyping, and validated them using Optical Genome Mapping (OGM). The prevalence of cryptic balanced translocations in the overall RPL/APH cohort was 1.19% (7/586, 95% CI: 0.58%-2.45%, Wilson score). It determined the incidence of cryptic balanced translocations in RPL or APH and changed the clinical management of these patients. Additionally, OGM resolved a 4-chromosome complex chromosomal rearrangement (CCR) involving 6 breakpoints and elucidated the genetic basis of two whole-exome sequencing-negative monogenic disorders by demonstrating pathogenic disruptions of EXT1 and PITX2 genes. OGM can be used as a second-line diagnostic tool, in addition to karyotype analysis and CNV-seq, for etiological screening of couples with recurrent pregnancy loss and adverse pregnancy history. The main conclusion of this study is that OGM can identify cryptic structural rearrangements, thereby enabling timely conversion of clinical management from PGT-A to preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR), and facilitating the transfer of euploid, non-carrier embryos for couples with RPL or APH.

Angelman syndrome is a neurodevelopmental disorder caused by the loss of the maternal allele of the ubiquitin-protein ligase E3A (UBE3A) gene. UBE3A is imprinted with maternal-allelic expression in neurons of the central nervous system (CNS) and biallelic expression in other cell types. Consequently, in Angelman syndrome, UBE3A is substantially reduced in CNS neurons and reduced by half in other cells. It is unclear how cell-type-specific gene expression in the brain is dysregulated in Angelman syndrome, as previous studies have lacked cell type resolution. Using single nuclei RNA-sequencing, we show that gene expression is dysregulated in neuronal subtypes in the frontal cortex of neonatal pigs with a UBE3A maternal deletion. A total of 3812 unique genes were dysregulated across ten cell type clusters, with most of the dysregulated genes (3154 genes) in excitatory neurons. Pathway analysis revealed alterations in oxidative phosphorylation, proteasome function, and synaptic function. Additionally, somatostatin (SST)-a secreted neuropeptide involved in GABAergic inhibition and synaptic plasticity- was reduced in inhibitory neurons in the cortex and hypothalamus, which correlated with lower circulating SST protein in neonates but not adolescent pigs. Overall, these findings provide greater clarity on the cell-type-specific dysregulation of gene expression and cellular pathways caused by the loss of UBE3A in CNS neurons, expanding our understanding of the molecular pathology in Angelman syndrome.

APOL1 variants G1 and G2 are major genetic risk factors for chronic kidney disease in people with recent sub-Saharan African ancestry, yet the mechanisms linking genotype to disease remain poorly defined. Circulating APOL1 is likely to play a critical role, although how specific genotypes and regulatory variants influence circulating APOL1 levels is incompletely understood. We analysed Olink-measured APOL1 NPX (Normalized Protein eXpression) levels from 43 587 UK Biobank participants. Associations between APOL1 NPX and known genotypes were assessed in individuals with recent sub-Saharan African ancestry, including analyses of G1, G2, E150K (rs2239785) and N264K (rs73885316). We also investigated protein quantitative trait loci and cis-acting variants across the APOL1 locus, including predicted microRNA binding sites. Results indicated that APOL1 NPX varied in an APOL1 genotype-dependent manner. E150K, G1, and G2 demonstrate a clear dose-dependent relationship with APOL1 NPX, with the largest effect for G2. After accounting for APOL1 genotype, APOL1 NPX remained higher in individuals with self-reported Black or Black British ethnicity, consistent with additional regulatory elements. Multiple cis-regulatory variants near APOL1 were independently associated with APOL1 NPX, and are likely to contribute to ancestry-related differences. Together these findings demonstrate that APOL1 genotype is a major determinant of APOL1 NPX, and that G1 and G2 have distinct biological effects. Regulatory variation adds another layer of control over APOL1 NPX and may help explain population differences. These findings provide a biological framework for understanding genotype-specific molecular effects of APOL1 variation.