Human Molecular Genetics

BackgroundHeterozygous c.283+1G>A and c.283G>A variants in the THRB gene, encoding for thyroid hormone receptor (TR){beta}1 and {beta}2, lead to autosomal dominant macular dystrophy (ADMD). We report the detailed clinical characterization of two first-degree relatives with ADMD, heterozygous for THRB c.283+1G>A, and an unrelated ADMD patient with a novel variant, c.283G>C. The genomic and molecular consequences of both variants were studied. MethodsgDNA and mRNA were obtained from leukocytes. Clinical characterization included biochemistry, bone density and body composition, ECG, echocardiography, ultrasound, audiometry and color-vision. In vitro assays investigated TR function and DNA binding. ResultsThe patients manifested no resistance to thyroid hormone beta (RTH{beta}) and had normal FT4 and TSH. Detailed studies in two patients showed no goiter, tachycardia, hypercholesterinemia or hepatic steatosis. Hearing was not impaired. Both had impaired color vision and reduced bone density. RT-PCR from all three patients revealed skipping of exon 4 exclusive to TR{beta}1, producing a deletion of 87 amino acids in the N-terminal domain (TR{beta}1{Delta}NTD). In vitro, DNA-binding affinity of TR{beta}1{Delta}NTD to DR4-TRE with or without RXR was comparable to TR{beta}1WT. Surprisingly, TR{beta}1{Delta}NTD was transcriptionally twice more active than TR{beta}1WT with a similar EC50 for T3, demonstrating gain-of-function of TR{beta}1{Delta}NTD. THRA expression in leukocytes was increased by 3-fold compared to unrelated controls and different from RTH{beta} patients. ConclusionThese THRB splice site variants produce TR{beta}1 exon 4 skipping, resulting in a gain-of-function mutant, TR{beta}1{Delta}NTD. This explains the dominant ADMD phenotype devoid of RTH{beta} and suggests a TR{beta}1 gain-of-function syndrome.

Pancreatic cancer disproportionately affects Black individuals in the United States, but they have limited representation in genetic studies of pancreatic ductal adenocarcinoma (PDAC). To address this gap, we performed admixture mapping and genome-wide association analysis (GWAS) in genetically inferred African ancestry individuals (1,030 cases and 889 controls). Admixture mapping identified three regions with a significantly higher proportion of African ancestry in cases compared to controls (5q33.3, 10p1, 22q12.3). GWAS identified a genome-wide significant association at 5p15.33 (CLPTM1L, rs383009:T>C, T Allele Frequency=0.51, OR:1.45, P value=1.24x10-8), a locus previously associated with PDAC. Known loci at 5p15.33, 7q32.3, 8q24.21 and 7q25.1 also replicated (P value <0.01). Multi-ancestral fine-mapping identified two potential causal SNPs (rs3830069 and rs2735940) at 5p15.33. Collectively these findings identified novel PDAC risk loci and expanded our understanding of this deadly cancer in underrepresented populations, emphasizing the multifactorial nature of PDAC risk including inherited genetic and non-genetic factors. Statement of SignificanceTo understand how genetic variation contributes to PDAC risk in Black people in North American, we studied individuals of genetically-inferred African ancestry. We identified novel risk loci and differences in the contribution of known loci. This demonstrates that ancestry-informed genetic analyses improve our understanding of PDAC risk and enhances discovery.

The spinocerebellar ataxias (SCAs) are a clinically heterogenous group of neurodegenerative disorders that affect movement, vision, speech and balance. Here, we reassign the linkage of SCA30 to 14q32.13 based on a cumulative LOD score >12. Within this interval we identified a 331 kb duplication, absent in population controls and not observed in >800 unrelated individuals with genetically unresolved cerebellar ataxia. RNASeq analysis of patient-derived lymphoblastoid cell lines revealed a splice-mediated chimeric transcript resulting from the duplication event. This transcript joined exon 1 of CLMN to exon 2 of SYNE3. In silico translation predicted that this chimeric transcript would produce a short N-terminal peptide corresponding to exon 1 of CLMN and the usually untranslated region of exon 2 of SYNE3 fused to the complete and in-frame SYNE3 protein. Transient overexpression of SYNE3 or the CLMN::SYNE3 fusion protein, in both HeLa cells and mouse primary cortical neurons, resulted in equivalent cellular outcomes including altered nuclear morphology and chromosomal DNA fragmentation. SYNE3 forms part of the linker of nucleoskeleton and cytoskeleton complex and is not usually expressed in cerebellar Purkyn[e] neurons while, CLMN has a Purkyn[e] specific expression pattern within the brain. Our data suggests that ectopic expression of SYNE3 in cerebellar Purkyn[e] neurons, mediated by the CLMN promoter, leads to cerebellar atrophy and causes spinocerebellar ataxia in the SCA30 family. This is an example of Mendelian disease arising from a novel, chimeric transcript with a likely dominant negative effect. Chimeric transcripts are commonly associated with cancers, but they are not often associated with monogenic disorders. Detection of chimeric transcripts as part of structural variant analysis could increase the genetic diagnostic yield of Mendelian disorders.

Eukaryotes typically maintain telomere length within a defined range. While short telomeres are known to activate DNA damage responses and limit cell proliferation, long telomeres are associated with extended proliferative capacity. The broader cellular consequences of long telomeres are comparatively less well understood. In budding yeast Saccharomyces cerevisiae, long telomeres have been shown to influence gene expression at specific loci, but whether long telomeres affect transcription genome-wide has not been reported. Here, we analysed transcriptomes in a lineage that inherited long telomeres (originally due to a rif2{Delta} mutation). Transcriptomes were assessed over two rounds of mitosis and meiosis in the absence of the rif2{Delta} mutation. We show that strains with long telomeres exhibit a distinct gene expression profile, including upregulation of membrane transporters and downregulation of a smaller subset of genes. Both up- and down-regulated genes were distributed across the genome, arguing against a purely telomere-proximal effect on gene expression. Affected genes were enriched for Rap1 binding sites, consistent with a model in which long telomeres sequester telomere-associated transcriptional regulators, such as Rap1, and thereby affect gene expression at non-telomeric binding sites for these regulators. Accordingly, the magnitude of transcriptional changes was greatest in strains with the longest telomeres. Together, our findings demonstrate that long telomeres induce a genome-wide transcriptional response that can accompany inherited long telomeres across generations. Similar effects of long telomeres are likely to occur in other eukaryotes, including humans, where long telomeres are associated with disease. Article summaryTelomeres protect chromosome ends, and their length is tightly regulated. While short telomeres are known to be harmful, the effects of long telomeres are less well understood. Using budding yeast, we show that inherited long telomeres alter the expression of dozens of genes across the genome, particularly membrane transporters. These changes are consistent with a model in which long telomeres sequester regulatory proteins away from other loci. Our findings may have broader implications in more complex organisms, including humans.

Common sex chromosome aneuploidies (SCAs) often present with cognitive and cardiovascular dysfunction in humans. To address SCA effects on gene expression and DNA methylation in relevant cell types, we differentiated neural precursor cells (NPCs) and cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) with different numbers of sex chromosomes, including isogenic and independent lines. As expected, the expression of genes that escape X inactivation (escapees) mostly increases with the number of inactive X chromosomes (Xi). However, allelic analysis shows dampening of escapees specifically on the Xi in XXY compared to XX in both NPCs and CMs, revealing a novel type of dosage compensation in SCA. In contrast, Y-linked gene expression is higher in XXY versus XY NPCs, but the opposite is observed in CMs. This may explain the greater number of differentially expressed autosomal genes in NPCs versus CMs with an added Y chromosome, while effects of added X chromosomes are similar between cell types. Concordantly, changes in autosomal DNA methylation are mainly driven by the presence of a Y chromosome. These findings highlight the cell-type specificity of sex-linked and autosomal gene regulation in SCA conditions. HighlightsO_LISex chromosome aneuploidy induces cell-type specific changes in gene expression C_LIO_LIDampening of the inactive X chromosome in XXY alleviate X overexpression C_LIO_LIHigh Y-linked gene expression in XXY neuronal precursor cells but not cardiomyocytes C_LIO_LISex chromosome aneuploidy disrupts sex biases in autosomal gene expression C_LI

EHMT1 and EHMT2 genes encode human euchromatin histone lysine methyltransferase 1 and 2 (EHMT1 alias GLP; EHMT2 alias G9a) that form heteromeric GLP/G9a complexes with essential roles in epigenetic regulation of gene expression. While EHMT1 haploinsufficiency has been established as the cause of Kleefstra syndrome 1, the pathogenesis of G9a dysfunction in human disease remains largely unknown. We identified seven de novo EHMT2 variants in patients with clinical presentation, episignatures, histone modifications and transcriptomic profiles similar to those of Kleefstra syndrome 1. In vitro studies revealed that these variants encode for structurally stable G9a proteins that are catalytically incompetent due to aberrant interactions either with histone H3 tail or with S-adenosylmethionine. Heterozygous mice carrying a patient-derived variant exhibited growth retardation, facial/skull dysmorphia and aberrant behavior. Here we report pathogenic EHMT2 variants that likely exert dominant-negative effect on GLP/G9a complexes and thus genocopy the EHMT1 haploinsufficiency via a distinct molecular mechanism, defining an autosomal dominant EHMT2-related Kleefstra syndrome.

Scleraxis (Scx), a basic helix-loop-helix (bHLH) transcription factor, is a primary marker for tendon and ligament lineages. Consequently, mouse models utilizing Cre recombinase under the control of the Scx locus represents a powerful tool for control of gene expression in tendon. The constitutive ScxCre mouse line is widely used for tendon-specific genetic manipulation. In this study, we demonstrate that ScxCre exhibits undesired significant off-target activity in the male germline, leading to ubiquitous recombination of floxed alleles in all tissues of the resulting offspring. This inheritance of recombined LoxP alleles occurs independently of Cre inheritance, indicating that ScxCre-induces recombination occurs prior to meiosis in diploid germ cells. This off-target activity is not observed in female germline. These findings highlight a critical need for stringent parental sex selection when using ScxCre lines to ensure tissue-specific targeting and avoid unintentional global gene deletion or transgene activation.

Mutations in the spliceosomal gene PRPF8 are associated with a range of human diseases. Studies in mouse and zebrafish suggest that Prpf8 also has a developmental function. Here, using a Prpf8 mutant mouse line isolated from a chemical induced mutagenesis screen, we uncover a previously unrecognised and essential role for Prpf8 in heart development, consistent with the embryonic lethality observed in Prpf8N1531S homozygous mutants. Prpf8N1531S mutant embryos display severe defects in ventricular trabeculation and compact zone formation, accompanied by increased cardiomyocyte proliferation specifically in the compact zone. Mutant embryonic hearts also exhibit disrupted cellular organisation, altered cytoskeletal architecture and changes in extracellular matrix protein expression. Notably, these cardiac abnormalities were exacerbated in embryos exhibiting cardiac looping defects. Transcriptomic analysis identified multiple aberrantly spliced transcripts in Prpf8N1531S mutant embryos, among which the cardiac transcription factor Tead1 was selected as a key functional candidate due to it known role in cardiac ventricle wall developemnt. Tead1 mis-splicing generated an in-frame, lower molecular weight protein isoform, associated with reduced overall TEAD1 expression. The Tead1 mis-spliced isoform showed altered nuclear localisation and dysregulation of TEAD1-dependent gene network important for heart development, including known cardiac sarcomeric genes. In addition, we observed reduced levels of the intracellular domain of the NOTCH1 receptor (NICD1), indicating impaired Notch signalling.. These findings suggest that impaired TEAD1-dependent transcription and Notch signalling contribute to abnormal cardiac trabeculation and compact zone development, highlighting a critical role for Prpf8 in maintaining proper heart development through the regulation of cardiac transcription factor expression and associated signalling networks. This study offers new mechanistic insights into congenital heart diseases linked to spliceosomal gene mutations.

BackgroundEpidemiological studies show an inverse association between cigarette smoking and Parkinsons disease (PD), suggesting a potential protective effect of smoking on PD incidence, despite the well-established and overwhelming harms of smoking to human health. We integrated genomic and proteomic approaches to investigate the causality and molecular basis of this potential relationship. MethodsWe analyzed summary statistics from genome-wide association studies (GWAS) of smoking initiation (SmkInit), smoking intensity, and PD. Two-sample Mendelian randomization (MR) tested whether genetic liability to smoking behaviors causally influences PD risk. Shared genomic architecture was quantified using MiXeR, and conjunctional false discovery rate (conjFDR) analysis identified loci jointly associated with smoking and PD, which were then mapped to genes and tested for tissue enrichment. To identify mediating proteins, we integrated dorsolateral prefrontal cortex proteomic data with GWAS using proteome-wide association studies (PWAS), summary-based MR, heterogeneity in dependent instruments testing, and colocalization. Finally, the druggability of convergent genes was evaluated. ResultsMR analyses indicated a protective effect of genetic liability to SmkInit on PD risk (OR = 0.78, 95% CI: 0.67-0.91, P = 1.5 x 10-3), which was consistent across sensitivity analyses and not suggestive of directional pleiotropy. However, no significant effect of genetic liability to cigarettes per day (CigDay) on PD risk was found. MiXeR revealed modest polygenic overlap between SmkInit and PD (13.9%; genetic correlation rg = -0.16) and between CigDay and PD (22.9%; rg = -0.09). ConjFDR identified 95 shared loci for SmkInit-PD and 26 for CigDay-PD. SmkInit-PD loci mapped to genes involved in neurotrophic signaling, synaptic organization, microglial modulation, and mitochondrial stress responses, with expression enriched in substantia nigra, basal ganglia, and interconnected cortical regions. PWAS identified 11 proteins shared by PD and SmkInit and 5 shared with CigDay, several of which (AKT3, MAPT, RIT2, EXD2, and PPP3CC) were supported by both genomic and proteomic analyses. Druggability assessment highlighted six proteins with existing pharmacologic modulation potential, spanning neurotrophic, microglial, proteostatic, and ion-channel pathways. ConclusionsGenetic liability to smoking initiation appears to confer modest protection against PD. Integrative genomic and proteomic evidence converges on neurotrophic, synaptic, microglial, and mitochondrial pathways as shared mechanisms, identifying biologically coherent potential therapeutic targets for advancing smoke-free neuroprotective strategies in PD.

Genome-wide association studies of idiopathic pulmonary fibrosis (IPF) have identified 35 common genetic risk loci associated with IPF susceptibility. In this study, we evaluated the effects of the reported variants in clinically curated non-European individuals. Despite limited sample sizes, we observed partial replication, limited transferability of some variants and evidence of ancestry-specific effects. The MUC5B promoter variant rs35705950 emerged as the dominant and most consistent signal across ancestries. Our findings highlight the need for larger, well-characterised studies in understudied populations to support robust discovery and translation.

BackgroundAgeing is a sex-specific process characterised by a progressive decline in physiological integrity. DNA methylation represents a primary epigenetic hallmark of ageing, yet sex-specific patterns of epigenetic ageing within and across tissues remain poorly understood. This study aims to address these gaps through an integrated analysis of sex-moderated epigenetic ageing across eight human tissues. MethodsA total of 137 DNA methylation datasets comprising over 36,000 individuals aged 10-114 years were analysed using a meta-analytic workflow to identify age-associated differentially methylated positions (aDMPs) and regions (aDMRs), meta-regression to assess sex moderation, and pathway enrichment analyses to interpret functional relevance. FindingsIndividual tissues displayed distinct age-related methylation trajectories, but some DMP sites showed consistent hyper- or hypomethylation across tissues. Across tissues, we identified 68,630 aDMPs (10%) robustly associated with ageing. Age-associated changes at the regional level were less common, with only 80 robust age-associated aDMRs detected across tissues, representing 0.09% of analysed regions. Sex moderation was observed for only 16 aDMPs (0.002%), indicating that sex effects on age-associated DNA methylation are largely tissue-specific rather than shared across tissues. InterpretationOur findings indicate that age-associated DNA methylation changes predominantly occur at isolated CpG sites rather than extended genomic regions and are strongly dependent on tissue and genomic context. The minimal overlap of sex-moderated methylation signals across tissues suggests that age-related sex differences at the epigenetic level are more likely attributable to tissue- and cell-type-specific variation rather than to broadly conserved epigenetic mechanisms shared across tissues. FundingThis study was funded by an Australian Research Council (ARC) Discovery project (DP200101830). Severine Lamon was funded by an ARC Future Fellowship (FT210100278). Nir Eynon was funded by NHMRC Investigator Grant (APP1194159), and a Hevolution/AFAR New Investigator Award in Aging Biology and Geroscience Research. Mandhri D. Abeysooryia was supported by an Australian Government Research Training Program (RTP) Scholarship. Research in context Evidence before this studyDNA methylation is widely recognised as a central epigenetic hallmark of ageing. Previous research has demonstrated that some age-related methylation changes are conserved across tissues, forming the basis of pan-tissue epigenetic clocks. Most studies to date have primarily examined age effects in isolation. Although biological sex influences ageing trajectories and susceptibility to nearly all age-related diseases, sex-moderated epigenetic ageing has received limited investigation. Specifically, pan-tissue clocks, including GrimAge and PhenoAge, are "sex-aware" but were trained and validated in mixed-sex cohorts, limiting their capacity to disentangle tissue-specific sex effects. Consequently, it remains unclear whether sex-moderated epigenetic ageing signals are shared across tissues or are tissue-specific. Added value of this studyThis study provides a large-scale, comprehensive multi-tissue analysis of sex-moderated epigenetic ageing, integrating 137 DNA methylation datasets across eight human tissues and more than 36,000 male and female individuals spanning the lifespan. Our findings show that while age-associated methylation changes are widespread at the CpG level, sex-moderated effects are rare and largely tissue-specific, with minimal overlap across tissues. Implications of all the available evidenceTogether, the available evidence indicates that epigenetic ageing is predominantly driven by shared, conserved age-related methylation changes, whereas sex differences in epigenetic ageing are modest and context dependent. These sex-related effects are more likely to reflect tissue- and cell-type-specific variation rather than widespread, shared mechanisms. This underscores the need to develop sex-specific epigenetic clocks and to conduct longitudinal cohort and intervention studies to more precisely characterise sex-specific dynamics of epigenetic ageing across tissues.

(1) Aims and hypothesisLoss-of-function mutations in SLC30A8, encoding the zinc ion (Zn2+) transporter ZnT8 in pancreatic beta cells, lower type 2 diabetes risk dose-dependently, but the underlying mechanisms remain unclear. Here, we combine proteomic, transcriptomic and functional approaches in human stem cell-derived islet-like clusters bearing common alleles or the inactivating variant R138X. We hypothesized that this variant protects against the deleterious effect of Zn2+ depletion on cell survival and function. (2) MethodsHuman embryonic stem cells INS(GFP/w) (MEL1), and CRISPR/Cas9-derived heterozygous or homozygous R138X lines were differentiated into stem cell-derived islet-like clusters. Intracellular Zn2+ levels were reduced using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-1,2-ethanediamine (TPEN). Apoptosis was assessed by TUNEL staining and protein expression by immunofluorescence. Glucose-stimulated calcium (Ca2+) dynamics were measured using the intracellular probe (Cal590) and insulin secretion by homogenous time-resolved fluorescence. Transcriptomic profiling was performed by bulk mRNA sequencing and proteomics by liquid chromatography-tandem mass spectrometry. (3) ResultsIntracellular Zn2+ depletion increased apoptosis in wild-type islet-like clusters, whereas R138X clusters were protected. R138X heterozygous clusters showed a mild increase in GCG+ cells and R138X homozygous clusters exhibited increased NKX6.1+ cells, without affecting polyhormonal populations. These changes were reversed under Zn2+ depletion. Transcriptomic and proteomic analyses, assessing genotype effects while accounting for Zn2+ depletion, showed that R138X clusters (versus wild-type) exhibited upregulation of genes and proteins involved in vesicle trafficking, secretion, Ca{superscript 2} signaling and mitochondrial metabolism, consistent with enhanced glucose-stimulated insulin secretion in homozygous clusters. Conversely, genes and proteins associated with extracellular matrix remodeling, metal-ion handling, apoptosis and cellular stress were downregulated. R138X clusters displayed altered Ca2+ signaling, with decreased area under the curve and oscillation amplitude, but increased frequency. These differences were reversed by TPEN, while Zn2+ depletion impaired Ca2+ response in wild-type clusters. Despite lowered overall activity, R138X homozygous clusters showed enhanced overall cell-cell connectivity, reversed by TPEN treatment. The opposite effects were observed in R138X heterozygous clusters, showing improved connectivity and activity under Zn2+ depletion. (4) Conclusion and interpretationIntracellular Zn2+ depletion compromises islet-like cluster identity and function, while the R138X variant confers protection against these effects. Under Zn2+-depleted conditions, ZnT8 deficiency promotes a more mature and metabolically active state of the R138X clusters, with enhanced Ca2+ signaling and insulin secretion, supported by a structural remodeling and the downregulation of apoptosis and cellular stress. These findings highlight the therapeutic potential of targeting ZnT8 in type 2 diabetes and support its relevance for further improving cell-based therapies. Research in ContextO_ST_ABSWhat is already know about this subject?C_ST_ABSO_LIRare inactivating mutations in the insulin granule-associated zinc transporter gene, SLC30A8/ZnT8, drive lowered type 2 diabetes risk. C_LIO_LIPrevious studies have indicated that apoptosis is lowered, and glucose-stimulated insulin secretion enhanced, after ZnT8 inactivation. C_LIO_LIThe molecular mechanisms underlying these changes are unclear. C_LI What is the key question?O_LIHow do inactivating mutations in SL30A8/ZnT8 lead to lowered apoptosis and enhanced insulin secretion from stem cell-derived islet-like clusters, and is altered susceptibility to intracellular zinc depletion involved? C_LI What are the new findings?O_LIThe rare inactivating R138X mutation in SLC30A8 leads to gene dose-dependent changes in the transcriptome and proteome of islet-like clusters. C_LIO_LIChanges include upregulation of maturity and downregulation of immaturity genes. C_LIO_LIDepletion of intracellular Zn2+ exaggerates the protective effects of the inactivating mutation on apoptosis and insulin secretion C_LI How might this impact on clinical practice in the foreseeable future?O_LIOur findings suggest that careful monitoring of both dietary zinc intake and of circulating levels of zinc ions, whose effects are mitigated in SLC30A8 mutation carriers, may be helpful in some populations to lower diabetes risk. C_LI

Background: Heart failure (HF) is a major and growing public health problem, and prior studies support a meaningful genetic contribution to HF susceptibility. Clinically, HF is commonly categorized into the major clinical sub-types of HF with reduced ejection fraction (HFrEF) and HF with preserved ejection fraction (HFpEF), which differ in pathophysiology and clinical profiles. However, previous genome-wide association studies have focused on autosomal variation and have routinely excluded the X chromosome, leaving X-linked genetic contributions to HF and its subtypes under-characterized. Methods: We performed X-chromosome wide association study (XWAS) utilizing directly genotyped data from 590,568 Million Veteran Program participants, including 90,694 HF cases across European, African, Hispanic, and Asian Americans. Sex- and ancestry-stratified logistic regression was used with XWAS quality control measures, adjusting for age and population structure, followed by fixed-effects multi-ancestry meta-analysis. Functional annotation, gene-based testing, fine-mapping, and colocalization were performed. We replicated genetic associations with all-cause HF in the UK Biobank. Results: In the multi-ancestry meta-analysis, we identified five X-chromosome-wide significant loci for all-cause HF, five for HFrEF, and one locus for HFpEF in males. No loci reached significance in female-specific analyses. In sex-combined analyses, we identified six loci for all-cause HF and four for HFrEF. The strongest and most emphasized signals mapped to genes were BRWD3, FHL1, and CHRDL1. Ancestry-specific analyses revealed additional loci, including NDP and WDR44 in African ancestry and PHF8 in Hispanic ancestry. One locus, BRWD3, was replicated in UK Biobank HF cohort. Integrated post-GWAS analyses (fine-mapping, colocalization and pleiotropy trait association studies) reinforced the biological plausibility of the X-linked signals. Conclusions: This multi-ancestry, sex-stratified XWAS identifies X-linked genetic contributions to HF and its subtypes and highlights the role of X-chromosome in heart failure pathogenesis.

Gene-environment interactions (GEI) contribute to circulating polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid (MUFA) profiles. GEI may partly explain differences in trait variance across genotype groups. To identify GEI for circulating unsaturated fatty acids, we adopted a two-stage strategy. First, we detected quantitative trait loci associated with trait variance (vQTLs). Second, we tested these vQTLs for interaction with fish oil supplements (FOS). We performed genome-wide vQTL screens for 14 plasma PUFA and MUFA phenotypes in a UK Biobank subset of 200,478 participants. At the genome-wide significance threshold (p < 5.0 x 10-8), we identified 172 vQTL-trait pairs across all 14 traits, and 16 of these vQTLs had no marginal genetic effect on the corresponding trait. We found 46 non-overlapping loci across all phenotypes, with an average of 12 vQTLs per trait. Omega-6% and PUFA% had the most independent vQTLs (N = 24) while DHA% and Omega-3% had the least (N = 1 and 2, respectively). For each of the 172 vQTL-trait pairs, we tested the interaction effect of the vQTL with FOS on the corresponding trait. We found six significant interaction signals in DHA, DHA%, Omega-3, Omega-3%, LA, and Omega-6/Omega-3 ratio around the FADS1/2, ZPR1, and SUGP1/TM6SF2 genes. Our results provide a comprehensive resource of vQTLs and gene-FOS interactions shaping the circulating levels of unsaturated fatty acids.

WAC is an autism-associated gene involved in neurodevelopment. However, the effects of reduced WAC function on behavior and synaptic regulation in vivo remain unclear. Taking cues from the previous studies on the wac gene and the C. elegans model of ASD, we elucidated the effects of wac gene deletion on food-leaving behavior, a known parameter linked to ASD associated genes along with the cholinergic pathway. wac-deficient worms exhibited curtailed food-leaving behavior. Notably, observed phenotype was similar to that exhibited by nematodes with mutation in ASD related gene, neuroligin. In addition, wac-deficient worms showed impaired growth, reduced pharyngeal pumping, and lifespan. To examine potential synaptic mechanisms, we analyzed expression of genes related to cholinergic signaling across all developmental stages (L1-L4) through young adult (YA). Stage-specific transcriptional changes were observed, with increased expression of ace-1 and acr-3 at L1, acr-3 at L3, and acr-3, cha-1, lev-1, and lev-10 at L4. The transcriptomic alteration was most prominent at YA stage, exhibiting upregulation of ace-1, cha-1, cho-1, lev-1, lev-10, unc-17, unc-29, unc-38, and unc-50. To identify specific suppressor of upmodulated Ach signaling, RNAi of the upregulated genes was performed. cho-1 was identified as a specific suppressor of elevated Ach signaling. cho-1 encodes a high-affinity choline transporter responsible for choline uptake in the pre-synapse. These studies identify the molecular mechanisms pertaining to up-modulation of cholinergic signaling in wac mutant worms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/719318v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1bdf8a9org.highwire.dtl.DTLVardef@1104825org.highwire.dtl.DTLVardef@1f09682org.highwire.dtl.DTLVardef@293b08_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hereditary hearing loss is highly genetically heterogeneous, with emerging overlap between genes implicated in early-onset and age-related hearing loss. We report a consanguineous family with autosomal recessive, non-syndromic hearing loss in which the proband harbors a homozygous splice-site variant in PALM3 (NM_001145028.2:c.314+1G>A) and a homozygous missense variant in OTOA. A minigene assay for the PALM3 variant demonstrated aberrant splicing with exon skipping, resulting in a frameshift and a large inframe deletion, both consistent with loss of function and impacting all known transcripts. While the organ of Corti from 12-month-old heterozygous Palm3 mice showed preserved overall architecture, published Palm3 knockout mice exhibit auditory dysfunction, supporting an auditory phenotype with loss of function. Although a dual molecular diagnosis cannot be excluded, the combined genetic, functional, and comparative data support PALM3 as a strong candidate gene for autosomal recessive hearing loss.

Background: Type 2 diabetes mellitus (T2DM) has long been considered a risk factor for cerebral small vessel disease (cSVD), yet the exact relationship between glycemic markers and cSVD remains unclear. This study explores the genetic overlap and causal associations between T2DM, glycemic indices, and cSVD phenotypes using genome-wide association studies (GWAS). Methods: Using large consortium-based GWAS data, we examined relationships between T2DM, glycemic indicators (glycated hemoglobin, fasting glucose, 2-hour glucose after oral challenge, and fasting insulin), and cSVD phenotypes (white matter hyperintensity volume, lacunar stroke, cerebral microbleeds, and enlarged perivascular spaces). Our multi-level genomic strategy included: 1) identifying pleiotropic single nucleotide polymorphisms (SNPs) through PLEIO and eQTL analysis, 2) assessing genome-wide genetic correlations using LDSC and GNOVA, and 3) determining causal relationships with two-sample and multivariable Mendelian randomization analyses. Results: We identified 14 pleiotropic SNPs with significant shared associations among T2DM, glycemic indicators, and cSVD phenotypes. Notably, MICB gene expression was elevated in brain, vascular, and pancreatic tissues, while three HLA genes (HLA-DQA1, HLA-DRB1 and HLA-DRB5) showed reduced expression. Genetic correlation analysis revealed positive correlations between T2DM, fasting glucose, and postprandial glucose with multiple cSVD phenotypes including WMH, lacunar stroke, and perivascular spaces. Mendelian randomization demonstrated that T2DM, 2-hour glucose, and HbA1c level causally increased lacunar stroke risk (OR 1.16 [1.09-1.23], OR 1.46 [1.20-1.77], OR 1.52 [1.04-2.23], respectively). Multivariable Mendelian randomization analysis confirmed that T2DM and postprandial glucose maintained a robust direct effect on lacunar stroke independent of other cSVD phenotypes, while HbA1c did not retain significance after conditioning on cSVD imaging markers. Conclusions: Our multi-level genomic analysis reveals links between T2DM, glycemic traits, and cSVD through specific genetic variants, genome-wide correlations, and causal relationships. The involvement of immune-related genes suggests potential biological mechanisms. The causal effect of postprandial glucose on lacunar stroke suggests that impaired glucose tolerance may be a relevant therapeutic target for lacunar stroke prevention.

Neural circuit assembly relies on different neuronal subtypes coming together to form a functional circuit. The question of how the appropriate number of each subtype is integrated into an emerging circuit remains relatively unknown. To answer this question, we used the mouse retina to uncover the molecular mechanisms responsible for neuron subtype integration in a developing circuit. In the mammalian retina, bipolar neurons are a class of interneurons that relay visual information from photoreceptors to ganglion cells. Extensive studies have shown there are 15 distinct bipolar subtypes: 6 types of OFF cone bipolars, 8 types of ON cone bipolars, and 1 type of rod bipolar. During retinal development, bipolar neurons are born in excess and through programmed cell death, a precise number of each subtype remains to give rise to the retinal circuit. Although this process has been well-described, little is known about the key molecules responsible for bipolar subtype integration in the developing retina. Our work uncovered a new role for the autism-associated risk gene, Protocadherin 9 (Pcdh9) in bipolar subtype integration. Deletion of Pcdh9 using a floxed allele leads to loss of OFF and ON cone bipolars; however, disruption in the extracellular binding of Pcdh9 leads to selective loss of ON cone bipolars but not rod bipolars. Moreover, we found this later function of Pcdh9 is mediated by homophilic interactions between ON cone bipolars and their known synaptic partners. Taken together, our work revealed a new role for Pcdh9 in bipolar subtype integration during retinal development. SUMMARY STATEMENTNeural circuits are comprised of multiple neuronal subtypes where a specific number need to come together to give rise to a functional circuit. Although this is a critical process during neurodevelopment, little is known about the molecular mechanisms that determines the precise number of each subtype during circuit development. In the present study, we identified the autism risk gene, Protocadherin 9 as a critical molecule in subtype integration of bipolar neurons within the developing mouse retina. Using newly generated mouse lines, we found distinct requirements of Pcdh9 to promote survival in different bipolar subtypes during retinal circuit assembly. The significance of this work is that it shed lights into how different neuronal subtypes are integrated in nascent neural circuits.

Background: Elevated lipoprotein(a) [Lp(a)] is a known risk factor for several cardiovascular-related diseases established from multiple genetic and observational studies. However, the underlying mechanisms mediating the effects of Lp(a) levels on cardiovascular disease risk and major adverse cardiovascular events (MACE) are unclear. The aim of this study was to identify proteins downstream of Lp(a) using mendelian randomization (MR) - a genetic causal inference approach. Methods: A two-sample MR was performed by initially identifying Lp(a) genetic instruments based on data from genome wide association studies (GWAS) of Lp(a) blood concentrations. These instruments were then tested for association with proteins from proteomic pQTL data (Olink from UK Biobank, 2940 proteins and SomaScan from deCODE, 4907 proteins). Results: A total of 521 proteins associated with Lp(a) were identified. Using pathway enrichment analysis, the following MACE-relevant pathways were identified comprising a total of 91 Lp(a) downstream proteins: oxidized phospholipid-related, chemotaxis of immune cells and endothelial cell activation, pro-inflammatory monocyte activation, neutrophil activity, coagulation, and lipid metabolism. Conclusion: The results suggest that the influence of Lp(a) treatments is primarily through modifying inflammation rather than lipid-lowering, thus providing insight into the mechanistic framework which mediates the effects of elevated Lp(a) on atherosclerotic cardiovascular disease.

The identification of genetic factors influencing cardiac senescence in natural populations is central to our understanding of cardiac aging and to identify the etiology of associated cardiac disorders in human populations. However, the genetic underpinning of complex traits in human is almost impossible, due to the infeasibility to control genetic background and gene-environment interactions. Drosophila has striking similarities in cardiac aging with humans, highlighting the conserved nature of cardiac aging for organisms with a heart. Leveraging on a large collection of inbred lines from the Drosophila Genetic Reference Panel (DGRP), we provide an accurate analysis of cardiac senescence in a natural population of flies. This permitted the discovery of an unprecedented number of variants and associated genes significantly associated to the natural variation of cardiac aging. We focused on the function of the PAR-domain bZIP transcription factor Pdp1 for which several variants were found associated with natural variation of the aging of multiple cardiac functional traits. We demonstrated that Pdp1 cell autonomously plays a central role in cardiac senescence and might do so by regulating mitochondria homeostasis. Overall, our work provides a unique resource regarding the genetics of cardiac aging in a natural population.