Human Mutation

IntroductionNeuromuscular disorders (NMDs) encompass a broad group of conditions that primarily affect the peripheral nervous system. They are often caused by genetic alterations that impair skeletal muscle function and result in debilitating symptoms. Obtaining an accurate molecular diagnosis remains a challenge, potentially because variants in genes that have yet to be identified as causal. We therefore used advanced computational methods to study the genetic architecture of NMDs and to identify key features that distinguish NMD genes from other genes in the broader genome. MethodsCurated genes implicated in NMDs (n = 639; GeneTable of NMDs) were obtained and merged with a comprehensive set of genomic features for human autosomal protein-coding genes. Machine-learning-based feature selection and ranking were performed using Boruta, along with complementary analytical approaches. These analyses were used to identify the most important genic features (n = 134, subcategories: gene complexity, genetic variation, expression patterns, and other general gene traits) for discriminating NMD genes from other genes in the genome ResultsNMD genes exhibit enriched expression in disease-relevant tissues, including skeletal muscle and heart. Additionally, compared with other protein-coding genes, these genes exhibit increased transcriptomic complexity (e.g., longer transcripts and more unique isoforms), contain more short tandem repeats, and show greater variation in conservation across model organisms. ConclusionsThis study identified several key genomic features that may distinguish NMD genes from the rest of the genome. This may enhance the identification of novel causal genes and could ultimately facilitate earlier diagnosis and medical management for affected individuals.

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.

Background: Short read sequencing technologies have dominated the field of human whole genome sequencing in the past years in terms of cost, throughput, and accuracy. However, thanks to recent technological evolution, long read approaches have become increasingly competitive and complementary to short reads. With the gap in the cost per genome closing slowly between both approaches, long reads might replace short read sequencing in future research and clinical applications. Still, comprehensive evaluation is necessary to conclude on the performance and general advantages of each technology. Results: In this study, we compared the latest chemistries of major suppliers of short and long read technologies: Illumina short reads, Illumina Complete Long Reads (ICLR), Pacific Biosciences HiFi reads (PacBio), and Oxford Nanopore Technologies long reads (ONT). Using the HG002 human reference sample and established bioinformatics guidelines, we assessed their variant calling performance against the latest available truth sets at different levels of coverage. For single nucleotide variant detection, all technologies were equivalent. Despite the latest improvements in chemistry, indel calling with ONT continues to lag in accuracy behind other technologies. In contrast, long reads delivered a clear advantage in structural variant detection, surpassing short reads in both accuracy and sensitivity. The hybrid ICLR approach achieved intermediate performance, narrowing the gap between short and long read sequencing. Furthermore, long reads enhanced haplotype-phasing resolution, enabling the phasing of over 80% of the genome. Conclusions: These findings highlight the specific strengths and limitations of recent sequencing technologies, aiding the decision-making in future research projects, technological platforms development, and clinical applications.

BackgroundInflammatory bowel disease (IBD) is associated with early structural changes in intestinal epithelial cells; however, the associated molecular alterations remain incompletely understood. The cytoskeletal protein keratin (K) 7 was recently found to be focally expressed in the colonic epithelium in IBD, while absent in the healthy colon. Here, we investigated the applicability of K7 as a noninvasive stool biomarker for pediatric IBD. MethodsIn this case-control study including adolescent patients with IBD (n=27) and healthy controls (n=15), stool lysates were analyzed by proteomics, immunoassay and qPCR to determine K7 protein and mRNA content, respectively. Additionally, stool mRNA levels of the simple epithelial keratins, K8, K18, K19 and K20, were measured. ResultsStool proteomic analysis identified focal K7 and K19 in IBD samples. Additionally, 23 differentially abundant proteins, of which 18 were higher in IBD, were identified and Gene Ontology enrichment analysis highlighted immune and inflammatory pathways. K7 specific immunoassay detected fecal K7 protein in all patients with active IBD, including both ulcerative colitis and Crohns disease, while K7 was near or below the detection limit in controls and IBD patients in remission (area under ROC curve=0.88, p<0.0001). While KRT7 mRNA levels were below the detection limit, KRT8 and KRT18 transcripts were elevated in IBD samples compared to controls (p<0.05). ConclusionsK7 protein is elevated in IBD patient stool, reflecting intestinal de novo expression and increased epithelial cell exfoliation. Fecal K7 may provide a novel, noninvasive marker for IBD diagnosis and monitoring.

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.

PurposeDiagnostic yield from exome and genome sequencing varies widely across studies. It remains unclear how much of this variation reflects patient-level factors (e.g., sex, clinical features, race/ethnicity, genetic ancestry) versus site-level practices such as sequencing modality or variant interpretation workflows. We aimed to quantify the contributions of these factors to diagnostic outcomes across five U.S. clinical sequencing sites. MethodsWe performed a cross-sectional analysis of 3,008 prenatal, neonatal, and pediatric cases from the NHGRI Clinical Sequencing Evidence-Generating Research (CSER) consortium (2017-2023). Clinical indications spanned neurodevelopmental, neurological, immunological, metabolic, craniofacial, skeletal, cardiac, prenatal, and oncologic presentations. Genetic ancestry was inferred from sequencing data, and variants were interpreted using ACMG/AMP guidelines to classify DNA-based diagnoses. Generalized linear mixed models were used to estimate associations between diagnostic yield and fixed effects (sex, prenatal status, isolated cancer, number of clinical indications, sequencing modality, race/ethnicity, and genetic ancestry), while modeling study site as a random effect to quantify between-site variation. ResultsThe overall diagnostic yield was 19.0%. Multiple clinical indications (OR=1.47, 95% CI 1.20-1.80, p<0.001) were associated with higher diagnostic yield, and male sex (OR=0.80, 95% CI 0.66-0.96, p=0.017) and prenatal status (OR=0.63, 95% CI 0.44-0.90, p=0.012) were associated with lower yield. Sequencing modality, race/ethnicity, genetic ancestry, and isolated cancer were not statistically significantly associated with diagnostic outcomes.. A model without fixed effects attributed [~]10% of variance in diagnostic yield to between-site differences. After adjusting for covariates, site-level variance decreased to 5.7%, indicating consistent variation across sites not explained by measured patient factors. ConclusionAcross five sites, patient-level clinical features influenced diagnostic yield, but substantial site-level variation remained even after adjustment. Differences in variant interpretation, or case-classification practices may contribute to this residual variability. Further efforts to increase consistency in exome- and genome-sequencing diagnostic workflows may help reduce inter-site differences.

Huntingtons disease is caused by expansion of a CAG repeat in the human HTT gene, producing a mutant huntingtin protein that misfolds and forms intracellular aggregates. Although Huntingtons disease is primarily characterized as a neurodegenerative disorder, mutant huntingtin is ubiquitously expressed, and peripheral tissues such as skeletal muscle exhibit pathological abnormalities. To define the muscle-intrinsic consequences of pathogenic huntingtin expression, we expressed caspase-6 truncated pathogenic human huntingtin in body wall muscle of Drosophila melanogaster larvae and performed quantitative structural and functional analyses. Aggregate analysis revealed that fluorescence intensity increased with aggregate size while aggregate morphology became more irregular. Delaying transgene expression until later stages of larval development dramatically reduced aggregate number, demonstrating a strong temporal dependence of aggregate formation. Myonuclei were enlarged, misshapen, and exhibited significantly reduced fluorescence intensity, consistent with altered chromatin organization. Notably, huntingtin aggregates were observed within the nucleus, indicating that nuclear proteostasis is directly perturbed by pathogenic huntingtin in muscle cells. Despite these intracellular defects, muscle fiber shape and sarcomere organization were preserved, suggesting that contractile apparatus assembly is not overtly disrupted. In contrast, mitochondrial organization was severely affected, with extensive mitochondrial aggregation throughout muscle fibers, consistent with altered organelle homeostasis. Functional analyses demonstrated that pathogenic huntingtin expression significantly impaired neuromuscular performance. Larvae exhibited reduced excitatory junctional potentials and diminished muscle contractile force, indicating compromised synaptic transmission and muscle function. Together, these findings demonstrate that pathogenic human huntingtin expression in skeletal muscle is sufficient to drive widespread protein aggregation, nuclear and mitochondrial abnormalities, and functional deficits despite the absence of overt structural changes. Our results highlight the importance of muscle-intrinsic pathogenic mechanisms and provide a quantitative framework for understanding how mutant huntingtin disrupts cellular organization and physiology outside the nervous system.

Widespread genomic sequencing efforts have characterized the molecular foundations of the different cancers. By combining these genomic data in a manner proportional to the population-level abundances of these different cancers, we estimate the overall abundances of each observed missense and nonsense mutation within the U.S. cancer patient population. We find BRAF V600E (5.2%) is the most common mutation in the cancer patient population, TP53 R175H (1.5%) is the most common tumor suppressor mutation, and APC R876X (0.4%) is the most common nonsense mutation. These values differ largely and significantly from what would be found in a typical pan-cancer analysis, where different cancer types are included out of proportion to population level incidence. We present the full ordered lists of population-level abundances for specific missense and nonsense mutations, and we demonstrate the value of these data by further analyzing high priority genes (e.g., TP53, KRAS, BRAF) and pathways (e.g., RTK/RAS, PI3K, and WNT/{beta}-catenin). Overall, this information is a resource that should benefit the basic science, translational, and clinical cancer research communities.

Abstract Background Spontaneous coronary artery dissection (SCAD) is a well-recognised cause of acute coronary syndrome particularly among women without conventional cardiovascular risk factors. Increasing evidence indicates a genetic contribution; however, the underlying genetic architecture of SCAD remains insufficiently understood. Objective The aim of this study was to assess the prevalence of rare variants in previously reported SCAD associated genes and to explore the potential presence of novel genetic alterations in well-characterised Swedish patients with SCAD. Methods The study comprised 201 patients enrolled in SweSCAD, a national project examining the clinical characteristics, aetiology, and outcomes of SCAD. All individuals had a confirmed diagnosis based on invasive coronary angiography. Comprehensive exome sequencing was performed to identify rare variants contributing to disease susceptibility. Results Genetic variants that have been associated with SCAD according to current clinical genetics practice for variant reporting were identified in approximately 4 % of patients. In addition, rare potentially relevant variants were detected in almost 60 % of patients in genes associated with vascular integrity and vascular remodelling. Conclusion This study supports SCAD as a genetically complex arteriopathy, driven by rare high?impact variants together with broader polygenic susceptibility. Variants in collagen, vascular extracellular matrix, and oestrogen?responsive pathways provide biologically plausible links to female?predominant disease. Although the diagnostic yield of clearly actionable variants is modest, these findings support broader genomic evaluation beyond overt syndromic presentations and highlight the need for larger integrative genomic and functional studies to refine risk stratification and management.

S-acylation is the addition of fatty acids to cysteine residues to regulate protein function and localization. S-acylation is catalyzed by the ZDHHC (Asp-His-His-Cys) family of protein S-acyltransferases (PATs), which S-acylate protein substrates by first auto-S-acylating the catalytic cysteine of the DHHC active site followed by transfer to the substrate. ZDHHC13 and ZDHHC17 are related ankyrin repeat domain (ANK) PATs that S-acylate multiple neuronal proteins, including huntingtin (HTT), the protein mutated in Huntington disease. However, unlike ZDHHC17 and other human PATs, ZDHHC13 possesses a non-canonical DQHC active site. As the first histidine is essential for auto-S-acylation, it is unclear if ZDHHC13 is catalytically active. Our phylogenetic analysis of eukaryotic ANK-containing PATs shows that ZDHHC13 orthologues are more divergent compared to ZDHHC17. While the ZDHHC17 DHHC is highly conserved, the motif varies among ZDHHC13 orthologues, with some vertebrate lineages containing a serine in place of the catalytic cysteine. Interestingly, we found that the ZDHHC13 S-acylation is lower than that of ZDHHC17, but the ZDHHC13 catalytic cysteine is indeed S-acylated. While expression of wild type (WT) ZDHHC13 in ZDHHC13 deficient HEK293T cells increased S-acylation of a HTT1-588 fragment, surprisingly, expression of catalytically dead DQHS ZDHHC13 was still able to facilitate HTT1-588 S-acylation equally. This suggests the ZDHHC13 catalytic cysteine is not required for S-acylation of target proteins, suggesting ZDHHC13 may coordinate another PAT. Indeed, we identified ZDHHC13 in high-molecular weight complexes. Our results indicate that ZDHHC13 is a likely pseudoenzyme that may function via a non-conventional mechanism reliant on other PATs. This work broadens our understanding of the function of this non-canonical PAT.

Intestinal epithelial cells are central players in mucosal barrier integrity and host-microbe interactions. Genetic studies have revealed that epithelial dysfunction is a key contributor to the pathogenesis of inflammatory bowel disease. Non-SMC condensin II complex subunit D3 (NCAPD3) is essential for chromatin organization and stability. NCAPD3 also promotes antimicrobial defense and autophagy responses in vitro. NCAPD3 expression is decreased in intestinal epithelial cells from patients with ulcerative colitis; however, it is not known whether loss of NCAPD3 expression drives intestinal barrier dysfunction or is a result of disease-associated inflammation. To investigate this relationship in vivo, a tissue-specific approach was required, as global constitutive knockout of NCAPD3 is embryonic lethal. Therefore, a transgenic mouse line with doxycycline-inducible expression of a short hairpin RNA targeting NCAPD3 restricted to villin-expressing cells was generated (NCAPD3KD mice) to enable the study of NCAPD3 function in the intestinal epithelium. Treatment of NCAPD3KD mice with 9-tert-butyl doxycycline resulted in [~]75% reduction of NCAPD3 protein in EpCAM intestinal cells. Short-term epithelial NCAPD3 knockdown did not induce spontaneous colitis but was associated with increased serum amyloid A and a trend towards increased intestinal permeability. Upon dextran sodium sulfate or Salmonella enterica serovar Typhimurium {Delta}AroA challenge, NCAPD3KD mice exhibited exacerbated weight loss, higher disease activity, increased histopathological damage, abnormal colonic cytokines and chemokines, and significantly increased intestinal permeability. These results indicate that NCAPD3 expression in the intestinal epithelium is required for optimal barrier maintenance and antimicrobial defense under chemical or microbial stress. These findings support prior in vitro observations and solidify NCAPD3 as a regulator of intestinal epithelial barrier function and mucosal host defense. Author SummaryNCAPD3 is a multifunctional protein with established roles in chromatin organization, genome stability, mitochondrial function, and antimicrobial defense. Dysregulated NCAPD3 is implicated in human diseases, such as inflammatory bowel disease (IBD) and microcephaly; however, due to its essential role in cellular division, determination of whether NCAPD3 loss drives these pathologies in vivo has been lacking. Using a new transgenic mouse model that selectively reduces NCAPD3 expression in intestinal epithelial cells, our study establishes NCAPD3 as an epithelial regulator of the mammalian intestine that enhances epithelial barrier resilience and antimicrobial defense during stress. Although dispensable for short-term basal homeostasis, NCAPD3 function becomes critical during epithelial injury and enteric infection. Reduced NCAPD3 expression may therefore lower the threshold for inflammatory disease by weakening barrier integrity, amplifying inflammatory cascades, and impairing antimicrobial defenses. These findings position NCAPD3 as a potential modulator of IBD susceptibility and highlight chromatin organization as an important, previously underappreciated layer of intestinal epithelial regulation.

Objective The decline of the perinatal demise rate is slowing and demises are often unexplained. Significant research has been done regarding diagnostic yield and genetic causes of demise, but little is known about how Geneticist involvement impacts outcomes. The goal of the study was to evaluate post-mortem genetic testing practices and effects of the geneticists involvement. Methods Retrospective data from 111 perinatal demise cases was examined, including rates of prenatal genetic counseling, post-delivery genetics consult, genetic testing, and autopsy investigation. Results In this cohort 54% received genetic testing and 25% received a genetics consult. When compared to those without, cases with genetic specialist involvement were associated with significant increases in testing uptake (p=0.007), diagnostic yield (p<0.001), and patient education (p<0.001). Second trimester stillbirths and those with fewer ultrasound (US) abnormalities were less likely to receive genetic testing (both p values <0.001) and consults (p<0.001, p=0.020). Conclusion Though it was not possible to avoid ascertainment bias, this data demonstrates that geneticist involvement correlates with a higher rate of testing, greater diagnostic yield, and more thorough counseling. These findings underscore the importance of integrating genetics providers into perinatal postmortem healthcare teams.

Salla disease is caused by a genetic mutation in sialin, a lysosomal membrane transporter, which exports sialic acid from lysosomes. Substrate translocation occurs via a rocker-switch mechanism that alternately exposes the substrate-binding site to the lysosomal lumen and the cytosol. The pathogenic mutation R39C found in most Salla disease patients decreases the lysosomal localisation and the transport activity. In this study, we used computational and mutagenesis approaches to elucidate the molecular effects of the R39C mutation. Using three-dimensional models of human sialin in the lumen-open (LO) and cytosol-open (CO) states combined with the mutagenesis of selected residues, we identify a critical "triplet" motif comprising R39, E194, and E262, which is associated with an ionic lock formed between K197 and D350 in the LO conformation. Molecular dynamics simulations suggest that the electrostatic triplet negatively modulates the ionic lock, and are consistent with a strengthened ionic lock in R39C sialin, potentially favouring the LO state. To assess the global effects of the R39C mutation, we computed dynamic cross-correlation matrices and identified correlation patterns consistent with an allosteric coupling between the ionic lock K197/D350 and the region surrounding the sialic acid binding site in wild-type sialin, whereas in the LO state of R39C sialin, this communication preferentially bypasses this region. Therefore, the R39C mutation may impede the LO to CO conformational transition required for sialic acid transport, providing a plausible mechanistic framework for the decreased transport activity, and possibly the decreased lysosomal localisation, observed in Salla disease. HighlightsO_LIThe R39 residue participates in an interaction triplet, which negatively regulates an ionic lock stabilising the lumen-open conformation C_LIO_LIThe R39C mutation is associated with a stronger ionic lock in the simulations, and may favour the lumen-open state C_LIO_LICorrelation network analysis suggests an allosteric coupling between the ionic lock and the region surrounding the sialic acid binding site C_LIO_LIThe R39C mutation alters the inferred allosteric coupling between the ionic lock and the region surrounding the sialic acid binding site C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/719580v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1ed0f72org.highwire.dtl.DTLVardef@913798org.highwire.dtl.DTLVardef@1d8e5adorg.highwire.dtl.DTLVardef@cf0060_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundTau aggregation is the defining feature of tauopathies, however, the mechanisms by which distinct tau strains drive disease-specific responses remain unclear. Existing models largely rely on recombinant tau seeding or tau overexpression, which fail to capture the biochemical diversity of pathological tau. The aim of this study was to develop a robust and reproducible human cell-based model of disease-specific tau pathology and to use this model to determine how tau from unique diseases impact tau accumulation and lysosomal dysfunction. MethodsPatient-derived tau aggregates were enriched from post-mortem brain tissue obtained from sporadic Alzheimers disease (AD), Picks disease (PiD), progressive supranuclear palsy (PSP), and control cases using phosphotungstic acid precipitation. Patient-derived tau preparations were biochemically characterised by immunoblotting and mass spectrometry and normalised for tau content prior to seeding. Patient-derived tau aggregates were seeded into multiple human immortalised cell lines (SH-SY5Y, M03.13, U-87 MG, and U-118 MG cells) and iPSC-derived astrocytes. Tau seeding efficiency, aggregate morphology, and integrity of the autophagy-lysosomal pathway was assessed using quantitative imaging approaches. ResultsPatient-derived tau seeds retained disease-specific phosphorylation patterns and isoform composition and led to reproducible, dose-dependent insoluble tau accumulation in all cell lines tested. Despite equivalent tau input and similar background protein composition, PiD-derived tau had the most aggressive pathological signature, showing the highest number of tau aggregates per cell and inducing system wide disruptions in the autophagy lysosomal system including increased SQSTM1 puncta and lysosomal damage markers. Seeding with AD-derived tau led to a high number of tau aggregates per cell and more specifically depleted the lysosomal protease CTSD and uniquely co-seeded A{beta} pathology. Seeding with PSP-derived tau resulted in only a moderate number of tau aggregates per cell and uniquely caused increased lysosomal biogenesis. ConclusionsTogether, these results demonstrate that intrinsic properties of human tau strains drive disease-specific cellular responses and establish a scalable, physiologically relevant platform for dissecting tau-cell interactions and screening therapeutics across tauopathies.

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.

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.

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.

Peripherin (PRPH) is a class III intermediate filament protein expressed in peripheral nerves and upregulated during axon outgrowth and regeneration. In this study, we developed a transgenic Xenopus laevis line for long-term in vivo visualization of the peripheral nervous system. Deletion and motif analyses identified cis-regulatory regions within the promoter and intron 1 that are important for neuronal expression of the X. laevis prph gene. Stable lines exhibited robust EGFP reporter activity in developing neural primordia in embryos and in the peripheral nerves of tadpoles. Transgenic tadpoles enabled in vivo imaging of peripheral nerves throughout limb development. During larval limb regeneration, we observed modest early nerve entry into the blastema, recapitulating that seen in early limb development. In contrast, post-metamorphic limb blastemas displayed extensive innervation from the early phase of regeneration. Moreover, increased reporter activity in the nerves of the regenerating adult forelimb suggests regeneration-associated regulation of peripheral innervation and its potential role in blastema formation. This transgenic line will serve as a versatile tool for analyzing such large-scale neural remodeling across development, metamorphosis, and regeneration.

Phenome-wide association studies (PheWAS) enable systematic exploration of relationships between genetic variants and clinical phenotypes derived from electronic health records (EHRs). Conventional regression-based PheWAS treats phenotypes separately and relies on binary phenotype representations, which limits statistical power for rare variants and rare phenotypes and reduces the ability to detect associations with phenotypes that are distributed across clinical codes. To address this limitation, we first developed EmbedPheScan, a phenotype embedding-based prioritization framework that summarizes the phenotypic profiles of rare loss-of-function variant carriers in a continuous embedding space. We then proposed EA-PheWAS by combining these embedding-derived signals with conventional regression-based PheWAS results using the aggregated Cauchy association test. Using the UK Biobank whole-exome sequencing and EHR data, we show that the proposed methods maintain appropriate false-positive control. We then performed genome-wide phenome scans across all genes and across biologically defined gene classes to evaluate EA-PheWAS relative to conventional PheWAS and EmbedPheScan, consistently finding that EA-PheWAS outperformed the other two methods. We illustrate the utility of EA-PheWAS focusing on four genes representing distinct scenarios, including strong-effect disease genes (PKD1, PKD2), genes with large numbers of rare LoF carriers (NF1), and genes with extremely sparse carrier counts (FBN1).

Background: Diabetic retinopathy (DR) is the leading cause of preventable blindness in working-age adults. In Chile, despite GES coverage since 2006, screening reaches only ~21% of the diabetic population under control. Chilean evidence shows that autonomous AI screening platforms have produced heterogeneous field results (sensitivity 40.8-100%, specificity 55.4%), while Ophthalmic Medical Technologists (TMOs) consistently achieve >97% sensitivity, suggesting AI is most effective as structured support for trained professionals rather than as an autonomous filter. Objective: We present DIRD+ (Diabetic Integrated Retinal Diagnosis), an open-source clinical platform that performs complete DR clinical workflows - patient management, AI-assisted lesion detection, clinical classification, annotation, and report generation - entirely within the web browser using WebAssembly-based inference, without transmitting patient data to any server. This work describes the system architecture and a preliminary technical validation. Methods: DIRD+ implements a six-stage inference pipeline using ONNX Runtime Web (v1.23) with SIMD and multi-thread optimizations, a pluggable clinical guideline engine (ICDR 2024, MINSAL Chile 2017), and a human-in-the-loop annotation workflow. A YOLOv26n detection model was trained on 500 pseudo-labeled APTOS 2019 images using the Annotix framework [11] and evaluated on the IDRiD test set (n=81 images). Results: Optic disc detection - the spatial calibration landmark - achieved AP=1.000 on IDRiD (IoU=0.1). Soft exudate detection achieved AP=0.243 (F1=0.364). Internal validation mAP50=0.578. Browser-based inference averaged 0.297 s/image (3.4 images/second) on CPU without GPU. Lesion detection performance reflects a first-generation model trained on 500 images; progressive improvement through collaborative annotation is ongoing. Conclusions: DIRD+ demonstrates that a complete offline-first DR clinical workflow can be deployed at zero cost within a standard web browser without server infrastructure or GPU. The pluggable guideline engine and human-in-the-loop architecture make DIRD+ a viable tool for TMO-assisted screening in connectivity-limited primary care settings.