Search Results (110)

Peripheral artery disease (PAD) is a global health burden affecting over 200 million individuals and is frequently complicated by limb-threatening ischemia, leading to major amputations. Despite known clinical risk factors, the genetic basis underlying amputation risk in PAD remains poorly defined. In this study, we performed a multi-pronged genome-wide association study (GWAS) to identify genetic variants associated with lower extremity amputation in patients with PAD, using data from the All of Us Research Program. Two analytical strategies were employed: a targeted GWAS using ClinVar variants on the full cohort and a comprehensive genome-wide association study using Allele Count/Allele Frequency (ACAF) data on a balanced subset of the cohort. The ClinVar analysis of 118,871 variants in 7558 PAD patients (405 with amputation, 7153 without) identified 3 suggestive associations with a genomic inflation factor of 1.046. The ACAF analysis of 7,784,837 quality-controlled variants in 804 balanced samples (399 cases, 405 controls) yielded 35 suggestive associations (p < 1 × 10⁻⁵) with a genomic inflation factor of 1.017. No variants achieved suggestive significance in both analyses. These findings highlight candidate loci for further validation and may inform future development of risk prediction tools and targeted interventions to reduce limb loss in PAD. All associations are exploratory and require independent replication. Full article

Missense single nucleotide variants (SNVs) represent one of the most common forms of genetic variation and account for a substantial proportion of variants of uncertain significance in clinical databases. Accurate computational classification of these variants remains an important challenge in precision medicine and genomic research. In this study, we present PathoPredictor, an interpretable machine-learning framework designed to distinguish pathogenic from benign missense variants using curated clinical variant data and functional annotations. High-confidence variants were obtained from the November 2023 ClinVar release and annotated using dbNSFP v5.1 (GRCh37). After data filtering, imputation, and normalization, 59,302 expert-reviewed missense variants were retained for model development. Six machine-learning algorithms were evaluated under identical cross-validation conditions applied to the training set. Among the evaluated models, LightGBM demonstrated the strongest overall performance and was selected as the final PathoPredictor classifier, achieving a mean ROC–AUC of 0.93 ± 0.004, accuracy of 0.90 ± 0.006, and Matthew’s correlation coefficient of 0.80 ± 0.008 across five cross-validation folds. Model interpretability was examined using SHAP (SHapley Additive exPlanations), enabling both global feature ranking and variant-level explanation of predictions. Temporal validation using ClinVar variants submitted after November 2023 showed consistent predictive performance on previously unseen submissions within the same database ecosystem (ROC–AUC = 0.91). While the framework demonstrates strong discrimination and structured interpretability, potential limitations include training data bias and partial circularity associated with the inclusion of existing meta-predictors. Overall, PathoPredictor provides a reproducible and interpretable computational framework for integrating functional annotations in missense variant prioritization, supporting research and genomic analysis workflows. Full article

Background: X-linked immunodeficiency with magnesium defect, Epstein–Barr virus (EBV) infection, and neoplasia (XMEN) disease is a rare inborn error of immunity caused by loss-of-function mutations in MAGT1, leading to impaired N-linked glycosylation. Although chronic EBV viremia is a hallmark of XMEN disease, the mechanisms underlying its marked clinical heterogeneity remain poorly understood. Methods: We performed an in-depth clinical, immunological, and genetic characterization of two siblings carrying a pathogenic MAGT1 variant (c.369_370insCC; p.Gly124fs), validated and deposited in ClinVar (SCV007293792). Assessments included whole-exome sequencing, multiparametric flow cytometry focusing on NKG2D expression, and longitudinal clinical follow-up. Results: Despite shared absence of NKG2D expression, the siblings exhibited strikingly divergent phenotypes. One sibling developed progressive neurodegeneration with central nervous system atrophy. The other presented with a complex immuno-hematologic phenotype, including EBV-positive Hodgkin lymphoma, recurrent autoimmune cytopenias, and lymphoma-associated thrombotic microangiopathy, representing a novel clinical association in XMEN disease. Comparative immunophenotyping revealed shared defects in B-cell maturation but distinct T-cell differentiation patterns. To contextualize neurological variability, we propose a descriptive, hypothesis-generating three-category conceptual classification comprising early-onset neurodevelopmental forms, adult-onset neurodegenerative manifestations, and secondary immune-mediated or vascular involvement of the nervous system. Conclusions: These findings demonstrate profound intrafamilial heterogeneity in XMEN disease and suggest a model in which modifier-sensitive factors influence organ-specific disease expression. The observation of lymphoma-associated thrombotic microangiopathy and the proposed descriptive neurological classification provide a conceptual framework that may help guide tailored, multidisciplinary surveillance beyond the primary genetic defect. Full article

Background/Objectives: FAM111A is a trypsin-like serine protease that has emerged as a regulator of DNA replication, and is directly related to genome stability, protein homeostasis, antiviral defense and cancer progression. Pathogenic variants in FAM111A are correlated with genetic syndromes such as Kenny–Caffey syndrome type 2 (KCS2) and gracile bone dysplasia/osteocraniostenosis (GCLEB/OCS). This study focuses on the evolutionary, genetic, and structural analysis of FAM111A, in order to identify key regions and candidate pharmacological targets that are related to this enzyme’s function. Methods: The methodology of this in silico study includes separate analyses at the sequence, structural and functional levels. Initially, data mining was carried out using NCBI/Protein (2025), and then data filtering was performed in order to identify representative FAM111A sequences for several species. Sequence analysis was then executed through multiple alignments and phylogenetic analyses. Through this, conserved domains and motifs were identified. For structural analysis, human pathogenic mutations and protein structures were identified through searches in biological databases including PDB and ClinVar, and then all data were analyzed in order to identify candidate pharmacological targets related to FAM111A function. Results: Approximately 1850 FAM111A protein sequences were retrieved for several species, and after filtering processes a dataset of 85 representative sequences was generated. Evolutionary analysis indicates that FAM111A originated in early metazoans, with progressive domain specialization leading to mammal-restricted acquisition of regulatory elements, including the PIP-box PCNA (proliferating cell nuclear antigen) interacting peptide and UBL (ubiquitin-like) domains. The ubiquitin-like/DNA binding domain and catalytic serine protease domain (SPD) are the most conserved, containing seven highly conserved motifs. The structural analysis was based on two protein structures and 34 critical mutations that accumulate in two distinct regions. Finally, by combining the results, six pharmacological targets and 100 inhibitors are proposed. Conclusions: Advancing the structural and function characterization of FAM111A, coupled with pharmacological target identification and evolutionary insights, will be critical to validate this underexplored protease as a therapeutic genetic target in genetic disorders, cancer, and antiviral responses. Full article

Background: Exome (ES) and genome sequencing (GS) are powerful tools for diagnosing neurodevelopmental disorders (NDDs), yet sequencing coverage failures can leave clinically relevant variants undetected. Analyzing the distribution of coverage gaps across sequencing approaches and batches is therefore informative for diagnostic accuracy. Methods: We analyzed sequencing data from 43 NDD patients across four ES runs, including 14 individuals sequenced by both ES (Twist Human-Core-Exome-v1.3) and GS. Low-coverage regions (LCRs) were defined as target intervals with mean depth <20 x, and z-scores < −1.96 were used to identify batch-specific systematic LCRs. LCRs were clinically annotated using OMIM and SysNDD databases. Results: LCR patterns were highly consistent within each ES batch but were characterized by extreme variability between batches. Higher global mean coverage increased intra-batch consistency, but batches sequenced at a commonly accepted yield in clinical sequencing (>100 x mean coverage) showed thousands of batch-specific LCRs. LCR patterns substantially diverged between ES and GS, displaying preferential impact on different genes. Although a restricted group of genes accumulates LCRs disproportionately, most LCRs are broadly dispersed throughout the genome. LCRs were not systematically associated with features such as GC content and genomic location (e.g., exon 1). Interestingly, LCRs affected OMIM/SysNDD genes and occasionally overlapped ClinVar pathogenic variants, indicating potential impact on diagnostic sensitivity. Conclusion: The global distribution of coverage gaps appears strongly influenced by batch-specific effects, making the occurrence of LCRs partly unpredictable even within clinically relevant gene sets. These findings support systematic assessment of LCRs as a component of quality evaluation in diagnostic sequencing workflows. Full article

The ability of enzymes to recognize and process structurally diverse substrates is fundamental to metabolic flexibility and biological regulation. In melanin biosynthesis, human tyrosinase (Tyr) catalyzes the oxidation of several chemically distinct intermediates, including L-tyrosine, L-DOPA, DHICA, and DHI. Although its catalytic chemistry is well established, the structural basis of substrate selectivity and how it is altered by disease-associated mutations remains unclear. Using molecular docking and molecular dynamics simulations, we mapped the Tyr active site and identified 23 evolutionarily conserved residues that mediate multi-substrate recognition and binding. Across all substrates, binding induces coordinated conformational responses, particularly within an anchoring region (334–347) that provides electrostatic and hydrophobic steering, and a flexible gating loop (374–386) that modulates access and stabilizes bound intermediates. The OCA1B-associated P406L mutation, although distant from the catalytic core, disrupts long-range dynamic coupling and impairs loop flexibility, while 25 ClinVar-listed genetic variants at substrate-interacting residues weaken active-site organization, underscoring the sensitivity of Tyr’s dynamic network to perturbation. Integrating these findings, we propose an ordered multi-substrate binding mechanism in which substrates are first guided by the anchoring region, then aligned by the universal triad, and finally refined through loop-mediated, substrate-specific contacts. Our work suggests a dynamic framework that could be useful for understanding human tyrosinase catalysis, genetic mutation impact, and future engineering strategies. Full article

The accumulation of somatic mutations contributes to clonal evolution and biological properties of cancers. Acquired mutations in mitochondrial (mt)DNA have been studied, but with the exception of those in isocitrate dehydrogenase genes, no comprehensive assessment of mutations in nuclear mitochondrial genes has been reported in sequential glioblastoma (GBM). We obtained ten pairs of GBM samples at diagnosis (GBM-P) and at recurrence (GBM-R). Extracted DNA was subjected to whole exome and mtDNA sequencing. After filtering out germline variants, bioinformatics analysis was performed using a mitochondrial gene panel of 483 nuclear-encoded, and 37 mtDNA-encoded genes. Variant classification was performed using established clinical- and molecular criteria, integrating population-frequency data, bioinformatic predictions, functional evidence, segregation information, and curated entries from the Mitomap and ClinVar databases. Benign single nucleotide variants in mtDNA-encoded genes of RNR1, RNR2, ATP6, CYB, CO2, TV, ATP8, and ND2 were detected, which changed little over time. However, three variants in TI, ND5 and ND1 with possible or likely pathogenic significance were found in the GBM-R samples. In contrast, pathogenic or likely pathogenic variants in 29 nuclear genes were found in GBM-P and GBM-R samples. Not only the overall number, but also the number of protein-truncating variants in nuclear genes increased over time. Conclusions: This study sheds light on the accumulation of mutations in nuclear genes of mitochondrial proteins in sequential GBM samples. As such variants may influence metabolic, proliferative and invasive properties as well as the necrotic propensity of the tumor, a comprehensive analysis of these genes merits further studies. Full article

Background/Objectives: ASXL1 is a chromatin-associated gene implicated in both hematologic malignancies and neurodevelopmental disorders, including Bohring–Opitz syndrome (BOS). Although many ASXL1 variants are well classified, a substantial proportion remain variants of uncertain significance (VUS), complicating molecular diagnosis and genetic counseling. The objective of this study was to evaluate whether structural context can inform the interpretation of selected ASXL1 missense variants in a clinical setting. Methods: We describe a 17-year-old female with clinical features consistent with BOS carrying the heterozygous ASXL1 variant p.Q1448R, currently classified as benign under ACMG/AMP guidelines. Three-dimensional in silico structural modeling was performed using AlphaFold3 and available crystallographic data. Three additional ASXL1 missense variants classified as VUS in ClinVar (p.R265H, p.T297M, and p.Y358C) were also analyzed. Evolutionary conservation, domain localization, and residue-level interactions were assessed. Results: Structural modeling indicated that the p.Q1448R substitution alters polar interactions and introduces a steric constraint near a conserved PHD-type zinc finger domain. Variants p.R265H and p.T297M affected stabilizing interactions within the DEUBAD, which is involved in BAP1 activation, while p.Y358C altered a polar microenvironment adjacent to a chromatin-interacting region. All analyzed variants, except p.T297M, localized to evolutionarily conserved regions. Conclusions: This study demonstrates that in silico structural analysis can provide complementary, domain-level insights for the interpretation of ASXL1 missense variants that remain classified as benign, likely benign or VUS under current frameworks. Such approaches may assist in prioritizing variants for further functional evaluation and refining molecular interpretation when experimental data are limited. Full article

Background: Ankylosis of the temporomandibular joint (TMJ) is a rare developmental disorder that involves fibrous or bony fusion within the joint. It is a severe structural and functional disorder. Typically, the phenotype manifests as joint immobilization and results in facial deformity and trismus. To date, ankylosis is rarely diagnosed as congenital and its occurrence mechanism has not been thoroughly understood. We observed a female patient who as a newborn showed slight facial asymmetry and impaired mandibular retraction. In addition, non-uniform occlusal fissures were noted; the lower part of the left earlobe was slightly smaller than the right earlobe. The aim of the work was the identification of pathogenic variants in the genome related to ankylosis. Ankylosis has no known causative gene yet; thus, Whole Exome Sequencing (WES) was performed. Materials and Methods: We observed a female patient with facial asymmetry and impaired mandibular retraction from birth. No phenotypic abnormalities were noted on the head or elsewhere on the body. A diagnostic computed tomography (CT) scan of the head performed at five months of age led to the diagnosis of congenital zygomatic-coronoid ankylosis. Genomic DNA samples were subjected to WES. Library preparation was carried out using the Twist Library Preparation EF Kit 2.0, followed by target enrichment with the Twist Exome 2.0 Plus Comprehensive Exome. Sequencing reads were aligned to the human reference genome (GRCh38), and variant calling was performed using standard bioinformatics workflows. Variants were subsequently filtered, annotated, and interpreted using VariantStudio. Assessment of variant pathogenicity was primarily based on comparisons with public databases, including ClinVar and VarSome, and was supported by in silico prediction tools such as SIFT and PolyPhen-2. Results: In genes responsible for disorders of the I and II pharyngeal arches, three pathogenic variants were identified: in the genes TCOF1 and POLR1B, responsible for the development of Treacher Collins syndrome (TCS), and one in the DHODH gene, responsible for Miller syndrome. Additionally, in genes that have not been linked so far with rare facial disorders, 42 variants were identified, of which 8 are listed as pathogenic. We present the first described patient with congenital ankylosis, who, although showing no phenotypic features of these syndromes, has identified pathogenic variants in genes responsible for craniofacial dysostosis. Conclusions: Variants in TCOF1, POLR1B and DHODH may represent candidate genetic factors associated with susceptibility to ankylosis. WES analysis is an appropriate method in the case of patients with congenital diseases with unknown genetic origin. In this study we provide a comprehensive list of all identified pathogenic variants. This might be useful for scientists searching for the genetic background of skeletal system issues, one of which could be bone and fibrous tissue remodeling. Full article

Background/Objectives: Interpreting missense variants in intrinsically disordered proteins (IDPs) remains a major challenge, as these proteins lack stable structure and are under-represented in experimental and clinical annotations. Variants occurring in IDPs are disproportionately classified as variants of uncertain significance (VUS), reflecting the absence of appropriate predictive tools rather than true biological neutrality. Here, we address this challenge using a curated dataset of neurodevelopmental disorder (NDD)-associated proteins. Methods: We integrated curated and predicted disorder annotations from DisProt and MobiDB to characterize the structural landscape of 339 NDD-associated proteins. To quantify a regional genetic constraint, we recalculated the Missense Tolerance Ratio (MTR) using a published framework adapted to the recent gnomAD release (v4.1.0). Integration with 33,124 ClinVar-reported missense variants revealed that, while mean constraint levels differ only modestly across structural states, ordered and structural transition regions show the strongest depletion of missense variation. Results: MTR identifies localized low-tolerance subregions within IDRs, indicating that these regions are not uniformly permissive and can harbor functionally essential elements. Conclusions: Overall, our results demonstrate that missense constraint in NDD proteins is highly localized and context-dependent, and that integrating high-quality disorder annotations with updated MTR profiles can improve the prioritization and interpretation of missense variants in IDRs and IDPs. Full article

Pathogenic variants in the thyroid-stimulating hormone receptor gene (TSHR) contribute to a wide spectrum of thyroid dysfunctions, ranging from congenital hypothyroidism to thyrotropin resistance. With the advancement of bioinformatics algorithms for variant effect prediction, assessing the pathogenic potential of variants has become increasingly important. This study aimed to investigate the pathogenic effects of TSHR variants classified as variants of uncertain significance (VUSs) in the gnomAD v4.1.0 database. TSHR variants listed in gnomAD v4.1.0 were retrieved and filtered to select missense VUSs based on ClinVar classifications. Multiple bioinformatics tools were used to assess the secondary and three-dimensional structures of the TSHR, as well as protein stability, evolutionary conservation, and molecular dynamics simulations. A total of 2760 TSHR variants were found in gnomAD v4.1.0, including 75 frameshifts, 80 splice-sites, 265 in the 3′ and 5′ untranslated regions, 422 synonymous, 892 others, and 1026 missense variants. Among these, 68 missense VUSs were identified and selected for bioinformatics analysis. Three variants (p.Cys29Trp, p.Leu57Pro, and p.Phe97Ser) were consistently predicted to be pathogenic by all the bioinformatics tools used. All three variants were located within the leucin-rich repeat domain extracellular region of the TSHR and within a highly conserved region across species. Molecular dynamics simulations for mutant proteins (p.Cys29Trp, p.Leu57Pro, and p.Phe97Ser) reveal structural instability in comparison to the wild protein. Comprehensive bioinformatics analysis revealed that three TSHR missense VUSs exhibited pathogenic potential. These variants may contribute to thyroid dysfunction by affecting the receptor’s structural and signalling integrity. Full article

In genetic disease assessment centers, DNA sequencing can produce results irrelevant to the genetic examination’s purpose. The American College of Medical Genetics and Genomics (ACMG) recommends evaluating and reporting 81 genes discovered using clinical genomic sequencing. While population studies on large cohorts can provide statistics on the prevalence of secondary findings (SFs), no studies have been published yet on large cohorts in Turkiye. We investigated ACMG SF by evaluating clinical exome sequencing data in 1600 individuals from different regions in Turkiye. We detected SF variants reported in ClinVar in 86 individuals (5.375%). Of the SFs, 30% were cardiovascular, 26% were cancer, 16% were neonatal metabolic disorders, and 28% were variants associated with various genetic diseases. In addition, we identified 212 different variants in 226 individuals and 45 different genes, which were not reported in ClinVar. When our results are compared with the Turkish National Genome and Bioinformatics Project database and studies in the literature, the studies vary in terms of participant characteristics, sequencing techniques, and versions of the ACMG SF list. Our findings highlight the importance of expanding and tailoring SF reporting guidelines in populations with high consanguinity and limited cohort-based data. Full article

The placenta is often described as the “window to the brain” due to its crucial role in fetal neurological development. In this study, we investigated a family where the older male offspring exhibited severe neurodevelopmental and mild motor coordination disorders. His brother displayed emotional and behavioral dysregulation along with mild motor coordination disorders. The father was asymptomatic, while the mother and daughter showed mild learning disabilities. Whole exome sequencing (WES) identified a disruptive X-linked pathogenic variant, c.1088_1089del p.Asp363GlyfsTer2, within the calpain-6 (CAPN6) gene. We have submitted this variant to the ClinVar database (RCV005234146.2). The variant was found in hemizygous condition in the affected male offspring and in heterozygous condition in both the mother and daughter. As predicted, the variant undergoes nonsense-mediated mRNA decay (NMD), preventing the translation of the CAPN6 gene into a functional protein. CAPN6 is a critical gene predominantly expressed in placental and trophoblast tissues. Although its function is not well characterized, CAPN6 is also expressed in several regions of the developing brain. Recent studies have shown that genetic variants in CAPN6 significantly influence vascular endothelial growth factor (VEGF) activity, thereby affecting angiogenesis and the blood supply essential for fetal growth and development. Although CAPN6 lacks an MIM phenotype code, we hypothesize that it might be enumerated as a novel candidate gene contributing to neurodevelopmental disorders. Functional studies are imperative to elucidate the role of CAPN6 in placental function and its potential implications for neurodevelopmental processes. This work aims to inspire further research into the role of CAPN6 in placental biology and its relevance to neurodevelopmental disorders. Full article

Based on PCR with complementary primer pairs and Pfu DNA polymerase, QuickChange site-directed mutagenesis has been widely employed, but its efficiency varies from mutation to mutation. An alternative strategy relies on partially overlapping primer pairs with 3′-overhangs, and this strategy has led to the recent development of P3a and P3b site-directed mutagenesis, in which the use of SuperFi II and Q5 polymerases raises the mutagenesis efficiency to ~100%. It is unclear whether these two DNA polymerases also improve the QuickChange method. Herein, we have evaluated this possibility by engineering 46 mutations on seven expression plasmids, two of which possess extremely GC-rich sequences. As Pfu DNA polymerase is a slow enzyme, its replacement with SuperFi II and Q5 polymerases reduced PCR length. Moreover, the average efficiency for each of the seven plasmids ranged from 48% to 69%, thereby outperforming the original QuickChange method. However, this efficiency is still lower than that from the P3a and P3b methods, supporting the superiority of primer pairs with 3′-overhangs. Analysis of the incorrect plasmids from the improved QuickChange method revealed frequent insertions at primer sites. The insertions were derived from primers and varied from mutation to mutation, with certain sites much more prone to such insertions. In comparison, these insertions occurred at a much lower frequency with the P3a and P3b methods, suggesting that primer pairs with 3′-overhangs enhance mutagenesis efficiency by reducing the likelihood to introduce insertions at primer sites. Thus, this study improves the QuickChange mutagenesis method, supports the superiority of the P3a and P3b methods, and uncovers a novel molecular mechanism by which the efficiency of PCR-based mutagenesis with completely overlapping primer pairs is negatively affected. Full article

Autoinflammatory diseases involve recurrent systemic inflammation caused by dysregulated innate immunity, arising from genetic or multifactorial mechanisms, as seen in periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome. About 10% of PFAPA patients show autosomal dominant inheritance. We describe a three-generation family with a PFAPA-like recurrent fever syndrome displaying clear autosomal dominant transmission. All affected individuals tested negative on a diagnostic panel of 13 known autoinflammatory genes. Whole-exome sequencing was performed in two distantly related affected members, followed by variant filtering, segregation analysis, and phenotype-based prioritization. A single heterozygous missense variant in RXFP1, c.154G>A p.(Asp52Asn), co-segregated with disease in all affected relatives. This variant is extremely rare in population databases, absent from ClinVar, present in COSMIC, and predicted as damaging by REVEL and CADD. RXFP1, not previously implicated in autoinflammatory or innate immune disorders, encodes the relaxin family peptide receptor 1, a G protein–coupled receptor involved in extracellular matrix regulation, anti-fibrotic pathways, and modulation of inflammatory cytokine production. Protein network analysis showed interactions with RLXN1-3, inflammatory mediators, PTGDR, ADORA2B, and C1QTNF8, supporting an immunomodulatory function. This is the first report linking RXFP1 variation to a hereditary recurrent fever syndrome, identifying relaxin signalling as a potential immune regulatory pathway. Full article