Background:Accurate clinical variant interpretation depends on the transcript used for annotation and consequence assessment. Transcript-aware reasoning is also incorporated into existing ClinGen guidance for loss-of-function, splicing, functional and computational evidence and into gene- and disease-specific specification. However, limited guidance exists when unresolved transcript context warrants escalation beyond routine annotation, how heterogeneous transcript-relevance evidence should be integrated or how the resulting determination should be documented. Methods:We propose TRACE (Transcript Relevance Assessment for Clinical Evaluation), a four-tiered gated framework. Applicable gene- or disease-specific Variant Curation Expert Panel (VCEP) specifications take precedence where they resolve the transcript question. Otherwise, TRACE begins with standard transcript annotation, escalates to focused transcript-relevance assessment only when transcript context could materially alter molecular consequence or criterion applicability and reserves targeted developmental, RNA, protein or functional evidence for unresolved cases. Results:TRACE separates transcript-context assessment from criterion-specific evidence assignment. Transcript context may establish whether the biological prerequisite for PVS1, PM1, PM4, PS3/BS3 or PP3/BP4 assessment is satisfied; established ClinGen SVI or gene-specific guidance then determines whether the criterion is applied and at what strength. Representative mechanisms include exon utilisation in TTN, poison-exon regulation in SCN1A, promoter-specific isoforms in DMD and regulatory transcript architecture in FKRP. A YAP1 example demonstrates withholding PVS1 when an alternative transcript preserves a downstream product, whereas CDKL5 illustrates a historical diagnosis missed through transcript selection and now governed by gene-specific expert curation. Conclusions:TRACE is an escalation and documentation framework, not a parallel evidence-weighting system. Its primary aim is to make clinically material transcript determinations explicit, reproducible and auditable while preserving established SVI/VCEP rules for evidence application and strength. Whether TRACE improves classification accuracy, interlaboratory concordance or diagnostic yield requires empirical validation.
Background:Loeys-Dietz syndrome (LDS) is a heritable connective tissue disorder caused by pathogenic variants in genes of the transforming growth factor-β (TGF-β) signaling pathway. Although genotype-phenotype correlations have been suggested, comprehensive comparative data across LDS subtypes remain limited. Improved understanding of these correlations is essential to guide individualized surveillance and management strategies. Methods:We conducted a retrospective cohort study of adults with genetically confirmed LDS evaluated between 2018 and 2024 across Mayo Clinic sites. Patients with pathogenic, likely pathogenic, or suspicious variants in TGFBR1, TGFBR2, or SMAD3 were included. Clinical characteristics, physical examination findings, cardiovascular and noncardiovascular manifestations, surgical interventions, and mortality were compared across genotypes. Categorical variables were analyzed using chi-square tests and continuous variables using parametric or nonparametric methods as appropriate. Results:A total of 93 patients were included (29 TGFBR1, 33 TGFBR2, 31 SMAD3). Demographics and mortality did not differ significantly between groups. Patients with TGFBR1 and TGFBR2 demonstrated trends toward higher rates of ascending aortic aneurysm and aortic dissection compared with SMAD3 patients, though these differences were not statistically significant. Renal artery aneurysms were significantly more common in TGFBR1 patients (13.8%, p = 0.010). SMAD3 patients had significantly higher rates of mitral regurgitation (54.8%, p = 0.04), peripheral neuropathy (29.0%, p = 0.018), and trends toward increased atrial fibrillation and osteoarthritis. A novel association was identified between TGFBR1 variants and migraine, which was significantly more prevalent in this group (62.1%, p = 0.017). The rates of major cardiovascular surgical interventions were high and comparable across all genotypes. Conclusion:Distinct genotype-phenotype associations exist among LDS subtypes. TGFBR1 and TGFBR2 variants are associated with a greater burden of aggressive vascular disease, whereas SMAD3 variants are linked to mitral valve disease, peripheral neuropathy, and osteoarthritis. We also identified a novel association between TGFBR1 genotype and migraine. These findings reinforce the importance of comprehensive genetic testing to inform personalized surveillance and management strategies in LDS.
Nonsmall cell lung cancer (NSCLC) is characterized by substantial heterogeneity in driver mutation backgrounds and tumor microenvironment (TME) phenotypes, yet the transcriptional regulatory states linking genotype context to immune-stromal variation remain incompletely defined. In this study, we integrated mutation, transcriptomic, and clinical data from TCGA-LUAD/LUSC to construct a mutation-expression-matched NSCLC cohort and evaluated RUNX1 as a mutation context-associated transcriptional/regulatory state rather than as a recurrently mutated driver. Driver contexts were assessed using a prespecified six-gene panel comprising EGFR, KRAS, TP53, STK11, KEAP1, and BRAF in the overall NSCLC cohort, the primary LUAD analysis, and a descriptive LUSC sensitivity analysis. The prespecified driver-context-negative operational group was defined as tumors without mutations in any of these six genes. RUNX1 mutation status was reported separately and did not contribute to this definition. Analyses included RUNX1 distributions, six-gene overlap, the RUNX1 cutoff, comutation burden, model sample-size audits, and adjusted context models. RUNX1 expression and inferred RUNX1 target-gene-set activity were evaluated across mutation contexts, whereas TME features were characterized using MCP-counter, xCell, ssGSEA, and ESTIMATE. Single-cell RNA sequencing and Visium spatial transcriptomic datasets were further used to localize RUNX1 expression, inferred target projections, and candidate TME-supporting genes. RUNX1 was prioritized within a prespecified candidate-screening framework and was significantly associated with the inferred RUNX1 target-gene-set readout. RUNX1 expression differences required interpretation in relation to histology and comutation structure. In the primary LUAD analysis, the KRAS-mutant context was associated with higher RUNX1 expression, whereas the KEAP1-mutant context was associated with lower expression. LUSC findings were retained as a descriptive sensitivity analysis and were not given the same inferential weight. Six-gene overlap, comutation burden, the RUNX1 cutoff, and model sample-size audits further showed that driver labels were nonmutually exclusive and that the prespecified driver-context-negative group was an operational reference rather than a biologically homogeneous subtype. RUNX1-high and RUNX1-low tumors displayed mutation context-dependent immune and stromal differences, involving stromal/cancer-associated fibroblast, myeloid/dendritic cell, endothelial, immune-score, and tumor-purity axes. Single-cell analysis localized RUNX1-associated signals and target-gene projections across epithelial-like and myeloid compartments, and spatial transcriptomics supported concordance between RUNX1 and selected TME-supporting genes in NSCLC tissue sections. Public immune checkpoint inhibitor cohorts were limited by small sample size and incomplete annotations and did not support RUNX1 as a stable immunotherapy-response predictor. Overall, these findings propose a mutation-context-aware RUNX1-TME stratification framework and generate testable hypotheses for mechanistic validation and mutation-annotated immunotherapy studies.
Purpose:Prenatal phenotypic manifestations of genetic disorders associated with NSD2 variants remain poorly characterized. This study presents our institutional experience with the prenatal diagnosis of NSD2-associated genetic disorders, specifically Rauch-Steindl syndrome (RAUST), aiming to improve understanding of both the molecular and clinical features of RAUST. Methods:We performed a retrospective analysis of six fetuses and one adult diagnosed with RAUST at our institution and thoroughly reviewed the prenatal ultrasound reports of six fetuses. Prenatal and postnatal phenotypes of RAUST cases were summarized alongside findings from previously published literature. Correlations between NSD2 variant locations, variant types, and phenotypes were analyzed. Additionally, protein modeling was used to visualize structural changes in NSD2 protein before and after C-terminal variants. We integrated single-cell transcriptomic and gene expression data from multiple public databases to investigate spatiotemporal expression patterns of NSD2 during human fetal development. Results:Fetal growth restriction (FGR) was the most prevalent prenatal manifestation in RAUST fetuses, followed by microcephaly. Bilateral renal hypoplasia emerged as a novel prenatal ultrasonographic feature. Postnatally, speech and motor developmental delays were the most commonly reported phenotypes, followed by physical developmental delays and intellectual disability. Genotype-phenotype correlation analysis revealed an association between N-terminal truncating variants in NSD2 and impaired fetal growth parameters. Notably, C-terminal truncating variants-predicted not to directly impact NSD2 functional domains-also exerted disease-causing effects. Conclusion:This study provides a comprehensive analysis of prenatal phenotypes in RAUST cases, enriching the prenatal phenotypic spectrum of the disease and facilitating early diagnosis and clinical management of RAUST. Furthermore, our genotype-phenotype correlation findings lay a foundational basis for future research into the complex molecular mechanisms underlying NSD2-associated genetic disorders.
Background:The microcephalin 1/BRITI1 (MCPH1) gene has been implicated in microcephaly (MCPH), primary ciliary dyskinesia (PCD) syndrome, enlarged cranial volume, and pancreatic and breast cancers. It functions as a tumor suppressor and shows reduced expression in various cancer types. Therefore, the identification of MCPH1 polymorphisms is of paramount importance. Aims and Objectives:Considering the multifaceted role of MCPH1, this study is aimed at examining the structural and functional impact of nonsynonymous single nucleotide polymorphisms (nsSNPs) on MCPH1 using in silico tools to understand its potential role in pathogenesis. Methodology:In the present investigation, a wide array of in silico tools was deployed to explicate putative pathogenic missense/nsSNPs that might impact the structural integrity and molecular functions of MCPH1. The mining of damaging nsSNPs was carried out based on the evolutionary conservation of sequence information, prediction of protein stability, interaction analysis, and their potential impact was assessed using structure prediction and 3D modeling, and interaction with other genes/pathways. Results:In the initial screening, SIFT, PolyPhen-2, SNAP2, Align GVGD, PhD-SNP, PANTHER, and SNPs&GO revealed 23 potential deleterious SNPs in MCPH1. Population frequency analysis using gnomAD revealed that the prioritized variants were extremely rare in the global population. I-mutant and ConSurf further revealed that 16 nsSNPs exhibited a decrease in protein stability due to changes in size and charge. I-TASSER was used to predict wild-type and mutant protein 3D structures. The potential effect of variation on 3D protein structure was assessed using the Chimera tool, which presented the probable structural loss in eight variants, including p.A11D, p.H49D, p.T59I, p.W60S, p.W60C, p.L74P, p.C79G, and p.L781P. Most of these potential variants resided in the N-terminal BRCT domain, which is implicated in neurodevelopment, and thus may lead to the pathogenesis of primary microcephaly. Furthermore, single-cell transcriptomic mapping of MCPH1 against dataset of 33,206 cells from malformations of cortical development detected MCPH1 expression across multiple cellular populations. This broad distribution suggested potential MCPH1 function across several neural and glial cell populations. Conclusion:The potentially deleterious nsSNPs predicted in this study can be of great significance in understanding MCPH1-related diseases and may serve as potential targets for future functional validation and therapeutic interventions.
The TTN gene encodes a crucial structural protein within cardiac sarcomeres, and its variants may contribute to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy; however, phenotype and genotype are different. Whole-exome sequencing (WES) was conducted on a Chinese proband diagnosed with HCM. In silico splicing prediction tools and minigene assays were employed to investigate the impact of the identified variant on mRNA splicing. A literature review was performed to retrieve and analyze previously reported splicing variants in the TTN gene associated with HCM. A 42-year-old male proband presented with nonobstructive HCM and paroxysmal atrial arrhythmias. A novel homozygous TTN variant was found, predicted to cause a 14-base pair deletion at a splice acceptor site. Two asymptomatic offspring were found to carry the heterozygous variant. Based on variant interpretation guidelines, the variant met the PM2_supporting criterion and was classified as a variant of uncertain significance (VUS). The predicted aberrant splicing effect was subsequently confirmed by the minigene splicing assay, demonstrating altered pre-mRNA splicing leading to an in-frame insertion/deletion (p.Arg32498_Glu32504delinsGln). Functional verification confirmed that this mutation conforms to the PM4 criterion, suggesting that it can be reclassified as a tepid VUS (scoring 3 points). The functional result might provide pathogenic evidence. We also reviewed 28 previously reported splicing variants in TTN associated with HCM. Of these, 57.1% (16/28) localized to the I-band region, whereas 21.4% (6/28) were situated in the A-band domain of titin. Notably, 21.4% (6/28) co-occurred with pathogenic variants in other sarcomeric genes (MYH7 or MYBPC3), correlating with more severe clinical phenotypes. Reclassification and reinterpretation of the variants revealed that none met the level of likely pathogenic or higher. The present case contributes a homozygous splice-site variant with experimentally confirmed aberrant splicing and an in-frame protein alteration in titin. The focus of this report is the Mendelian genetic basis of the proband's cardiomyopathy phenotype, and our data provide additional case-level and functional evidence for a possible role of specific TTN splicing defects in HCM.
Background MYC is a central oncogenic driver in lung adenocarcinoma (LUAD), coordinating proliferation, metabolism, and immune evasion. However, LUAD lacks a MYC‐anchored prognostic framework that jointly relates survival risk to pathway activity, immune contexture, and potential therapeutic response. This study is aimed at developing and validating such multidimensional model and determining whether it captures clinically and biologically relevant heterogeneity. Methods We first obtained LUAD transcriptome and clinical data from public databases, along with the HALLMARK_MYC_TARGETS_V1 and HALLMARK_MYC_TARGETS_V2 gene sets from the Molecular Signatures Database (MSigDB). Subsequently, enrichment analysis was performed using gene set enrichment analysis (GSEA), and differentially expressed genes (DEGs) were screened using the DESeq2 package. A MYC‐related prognostic model was constructed using univariate Cox analysis and LASSO, followed by multivariate Cox regression. Immune infiltration, pathway enrichment, TIDE‐based immunotherapy prediction, and GDSC2‐based drug sensitivity were compared between high‐ and low‐risk groups. Seurat was used to process single‐cell RNA sequencing (scRNA‐seq) data and map cell type‐specific expression of key genes. Finally, we validated the expression and potential functions of the screened key genes using in vitro LUAD cells lines. Results We identified 41 genes that are differentially expressed and associated with MYC in LUAD, and from these, we constructed a three‐gene prognostic model driven by MYC, comprising lactate dehydrogenase A ( LDHA ), poly(A)‐binding protein cytoplasmic 1 ( PABPC1 ), and polo‐like kinase 1 ( PLK1 ). High‐risk tumors were enriched for MYC targets, E2F targets, G2/M checkpoint, DNA replication, and chromosome‐segregation programs. They also showed distinct immune infiltration patterns, higher TIDE scores, and differential predicted sensitivity to multiple anticancer agents. Single‐cell analysis revealed heterogeneous expression of LDHA and PABPC1 across tumor and stromal compartments, whereas PLK1 was not detectably expressed. In vitro, LDHA , PABPC1 , and PLK1 were upregulated in A549 cells compared with BEAS‐2B cells, and LDHA knockdown significantly impaired A549 cell invasion and migration. Conclusion The MYC‐related three‐gene model provides a discovery‐stage framework for linking prognosis with pathway activity, immune features, and predicted therapeutic response in LUAD. Prospective cohorts and clinical‐grade analytical validation are required before the model can be considered for clinical decision‐making.
Moving from an association signal to a clinically credible biomarker requires several links that are often conflated: verified variant identity, aligned allelic effects, reproducible gene-level association, relevant cellular expression, and a plausible functional consequence. We developed a source-aware multiomics framework to assess MPHOSPH6 in lung adenocarcinoma (LUAD) while keeping those evidence classes separate. Six prespecified rsIDs were recovered from the harmonized TRICL LUAD dataset, of which five reached p < 5 × 10 - 8. Only rs112333466 and rs76474922 were available with alignable alleles in FinnGen R10, and both showed concordant directions. Fixed-effect estimates were OR = 1.592 for rs112333466-T (95% CI, 1.401-1.809; p = 9.91 × 10 - 13) and OR = 0.819 for rs76474922-C (95% CI, 0.773-0.867; p = 1.03 × 10 - 11). In a prespecified two-variant GTEx v8 lung model, genetically predicted MPHOSPH6 expression was positively associated with LUAD in TRICL (Z = 3.341, p = 8.35 × 10 - 4) and FinnGen (Z = 2.697, p = 0.0070). This gene-level result did not establish colocalization or connect MPHOSPH6 to the six susceptibility rsIDs. Patient-level analysis of 89,241 immune cells from six paired tumor and normal-adjacent lung samples found no significant difference in MPHOSPH6 pseudobulk abundance (exact paired Wilcoxon p = 0.3125). None of 688 lung-lineage pharmacogenomic tests remained significant after false-discovery-rate correction. Ten recorded MPHOSPH6 missense alleles, including five ClinVar variants of uncertain significance, were curated; structural analysis identified I58 at an experimental RNA-exosome interface and defined a focused perturbation series. MPHOSPH6 is therefore supported as a human-variation-informed candidate for functional evaluation, not as a validated LUAD biomarker, pathogenic gene, drug-response predictor, or therapeutic target.
Glioblastoma (GBM) remains one of the most aggressive brain tumors, with limited treatment options and poor prognosis. Advances in computational biology have enabled the identification of novel biomarkers for improved diagnosis and therapy. In this study, we investigate the role of antigen presentation gene HLA‐DMB in GBM using a multiomics approach integrating single‐cell RNA sequencing, bulk transcriptomics, survival analysis, and machine learning. Differential expression analysis identified HLA‐DMB as a key gene associated with GBM. Machine learning algorithms, including LASSO regression and random forest, consistently ranked HLA‐DMB as a top feature. Survival analysis revealed that high HLA‐DMB expression is independently associated with poor progression‐free interval (PFI), whereas associations with overall survival and disease‐specific survival did not reach statistical significance. Functional enrichment analysis linked HLA‐DMB to apoptosis, cell cycle regulation, and immune‐related pathways. Single‐cell analysis demonstrated that HLA‐DMB is primarily expressed in M1 macrophages, suggesting a role in tumor immunity. Immune infiltration analysis further confirmed its association with tumor‐associated macrophages, fibroblasts, and T‐cell activation. Drug sensitivity analysis using Connectivity Map (CMAP) identified potential compounds targeting HLA‐DMB. Overall, our findings identify HLA‐DMB as a candidate biomarker for GBM, providing insights that may inform future prognostic and therapeutic investigations.
Background:Nonobstructive azoospermia (NOA), the most severe form of male infertility, has approximately 30% of its cases attributed to genetic factors. However, current genetic research only explains a small fraction of NOA cases. Methods:Here, 156 infertile Chinese patients diagnosed with idiopathic male infertility were recruited. Whole exome sequencing and subsequent bioinformatic analysis were performed to find candidate pathogenic variants. Sanger sequencing was used to validate the candidate variants. Quantitative reverse-transcript PCR (RT-qPCR) was performed to detect the mRNA expression of TDRD12 and related piRNA genes. Hematoxylin and eosin staining and immunofluorescence staining were performed on testicular sections obtained from patients' biopsies. Intracytoplasmic sperm injection (ICSI) was applied for assisted fertilization. Additionally, small RNA sequencing was performed to identify the abnormalities of piRNA biogenesis. Results:We identified eight variants in PET (PIWI-EXD1-TDRD12) complex encoding genes TDRD12 and EXD1 from three NOA patients and one oligoasthenozoospermia patient. These variants, which were rare or absent in public human databases, were predicted to be deleterious. Notably, TDRD12 mRNA was absent in three NOA patients. Testicular histopathological analyses revealed that spermatogenesis was arrested at zygotene stage in patients carrying biallelic TDRD12 variants. Further immunofluorescence staining of L1ORF1p and small RNA sequencing revealed disruptions in piRNA biogenesis and subsequent derepression of retrotransposons. Further assisted reproduction outcomes showed that one of the individuals conceived successfully after ICSI. Conclusion:Overall, this study significantly expands the mutational phenotype of PET complex-encoding genes and significantly advances our understanding of spermatogenic failure and male infertility. Our findings provide direct clinical and functional evidence that the function of the PET complex and piRNA biogenesis is highly conserved between humans and mice.
Cisplatin response in gastric cancer is shaped by malignant cell programs, stromal states, and mutation-immune crosstalk, but the CAF subtypes linked to platinum tolerance and their epithelial effectors remain incompletely defined. We integrated single-cell RNA sequencing, epithelial CNV-like inference, fibroblast reclustering, CellChat analysis, signature scoring, hdWGCNA, CAF-epithelial coupling, exploratory GSE14209 evaluation, TCGA-STAD mutation/copy-number contextualization, and DepMap CRISPR virtual knockout analysis, followed by experimental validation in AGS and HGC-27 cells. IGF1+ CXCL12+ CAFs showed resistance-supportive stromal features, and CAF-epithelial coupling prioritized ARPC1A as a tumor epithelial candidate associated with this stromal program. In TCGA-STAD, ARPC1A mutations were rare and consisted of three missense and two frameshift variants without a recurrent hotspot; the mutant tumors were confined to the MSI subtype and showed an immune-activated, high TMB/MSI context, indicating mutation-immune crosstalk rather than a clear mutation-specific CAF, EMT, drug resistance, or cisplatin response phenotype. By contrast, ARPC1A copy-number gain/amplification was more frequent and tracked with ARPC1A expression, CIN enrichment, aneuploidy, and fraction of genome altered. DepMap CRISPR data indicated mostly weak-to-mild baseline ARPC1A dependency in upper GI and gastric/GEJ models. Experimentally, ARPC1A knockdown increased cisplatin sensitivity, promoted cisplatin-induced apoptosis, and suppressed migration and invasion. These findings identify a CAF-associated ARPC1A epithelial program linked to cisplatin response and support a mutation-immune/copy-number framework in which ARPC1A is interpreted mainly through immune-contextual mutation patterns and expression dosage rather than recurrent gain-of-function mutation.
Introduction:Genome-wide association studies (GWAS) have identified over 200 germline risk loci for colorectal cancer (CRC), yet the causal variants and genes behind most GWAS signals remain unknown and the link between inherited risk and tumor outcome is largely unexplored. Connecting germline single nucleotide polymorphisms (SNPs) to gene expression through expression quantitative trait loci (eQTL) and to clinical outcome is needed to interpret this inherited risk. Methods:We combined CRC GWAS summary data (73,149 cases and 112,467 controls of European ancestry) with GTEx v8 eQTL from colon sigmoid, colon transverse, small intestine terminal ileum, and whole blood, integrating causal transcriptome-wide association study (cTWAS) with SuSiE fine-mapping, Bayesian colocalization, MAGMA SNP-to-gene analysis, and AlphaGenome variant-effect prediction. Prioritized proteins were assessed by western blot. Four experimentally selected regulatory variants were genotyped in HCT116, SW480, RKO, and NCM460 cells; genotype-protein associations were tested across cell-line means, and cis-regulatory effects were assessed by allele-specific expression (ASE) and reference-versus-alternate dual-luciferase assays. Prognostic relevance was evaluated in TCGA colorectal tumors. Results:cTWAS identified seven genes with posterior inclusion probability (PIP) > 0.50, and colocalization across 78 gene-tissue pairs revealed 20 associations with PP.H4 > 0.80. Four genes showed convergent evidence: SMAD9 (PIP = 0.916, PP.H4 = 0.981), MAP3K2 (PIP = 0.762, PP.H4 = 0.827), FADS1 (PIP = 0.632, PP.H4 = 0.942), and ACTR1B (PIP = 0.566, PP.H4 = 0.994). All four proteins were reduced in CRC cells. Alt-allele dosage was inversely associated with SMAD9 (Pearson r = -0.985, BH-adjusted p = 0.029) and FADS1 protein abundance (r = -0.995, BH-adjusted p = 0.020), but not with MAP3K2 or ACTR1B. Reporter and ASE assays detected the clearest allele-specific effects at SMAD9 and FADS1, whereas MAP3K2 and ACTR1B were null in the tested systems. Lower SMAD9 expression was nominally associated with poorer survival (log - rank p = 0.022 to 0.050), but these associations did not survive Benjamini-Hochberg correction across 12 tests. Conclusions:Integrating statistical genetics, regulatory prediction, and locus-directed experiments prioritized SMAD9, MAP3K2, FADS1, and ACTR1B as CRC susceptibility genes. Functional evidence was strongest and most directionally coherent for SMAD9, demonstrated allele-specific but context-dependent regulation at FADS1, and placed experimental bounds on the proposed MAP3K2 and ACTR1B mechanisms.
Hereditary spherocytosis (HS) is a common inherited hemolytic anemia characterized by spherical erythrocytes, splenomegaly, and increased osmotic fragility, with ANK1 mutations being the most frequent cause. We identified a novel ANK1 mutation (c.5096G>A, p.R1699K) in a patient with classic HS phenotypes and a family history of hemolytic anemia. To explore its pathogenicity, we performed a series of hematological and morphological analyses. Compared with wild-type mice, the Ank1 c.5198G>A knock-in mice displayed typical HS features. Hematologically, they showed reduced MCV, MCH, and MCHC. The EMA fluorescence intensity was significantly decreased, and RBCs exhibited increased osmotic fragility. Morphologically, peripheral smears revealed abundant spherocytes, and electron microscopy showed a transformation from normal biconcave discs to spherical erythrocytes. Additionally, the Ank1 c.5198G>A knock-in mice presented with significant splenomegaly. Mechanistically, coimmunoprecipitation analysis indicated a significantly weakened association between ankyrin-1 and β-spectrin in the Ank1 c.5198G>A knock-in mice. This study provides in vivo evidence that the Ank1 R1733K mutation impairs erythrocyte membrane stability and cytoskeletal integrity, recapitulating the HS phenotype in mice. Our findings support the pathogenicity of the ANK1 c.5096G>A mutation and offer a useful model for further investigating the molecular mechanisms of HS.
Background:Osteogenesis imperfecta (OI) is a clinical and genetic disorder characterised by bone fragility, growth deficiency and skeletal deformity. Ninety per cent of OI cases are attributable to autosomal dominant variants in the COL1A1 and COL1A2 genes. Methods:Candidate variants were identified and verified through trio whole-exome sequencing (trio-WES), copy number variation sequencing (CNV-seq) and Sanger sequencing. Minigene splicing assays were performed in HeLa and HEK293T cells with pcDNA3.1 and pcMINI-C vectors to investigate the function of the candidate variants. A systematic review of COL1A1 splicing variants and the corresponding genotype-phenotype spectrum was performed. Results:Trio-WES revealed a novel heterozygous variant in the C-terminal region of the COL1A1 gene: NM_000088.4:c.3423+5G>A. Sanger sequencing confirmed the variant in both the proband (II-2) and her foetus (III-1) who were clinically suspected of having OI. The c.3423+5G>A variant causes complete skipping of Exon 46, as demonstrated by a minigene splicing assay. We retrieved 419 COL1A1 splicing variants from PubMed, excluded 15 without phenotypic data and 2 linked to Ehlers-Danlos syndrome and stratified the remaining 402 variants into three types on the basis of splice site location: (1) Variants at canonical splicing sites (77.8%, 313/402) mostly cause mild phenotypes, whereas a minority may be severe. (2) Intron variants in other locations, such as splice region variants (17.9%, 72/402), usually cause mild clinical phenotypes, and deep intronic splice variants (0.4%, 2/402) that may result in severe phenotypes. (3) Other variants (3.7%, 15/402), such as exon variants or fragment loss, are extremely rare. We also preliminarily discuss the mechanisms underlying phenotypic variability and the characteristics of C-terminal variants. Conclusions:This intron variant in COL1A1 was classified as likely pathogenic and was confirmed to disrupt COL1A1 expression. The summary analysis results also revealed a correlation among splicing variants, C-terminal region variants and disease, suggesting that variant location provides a useful framework for prognosis prediction.
Familial adenomatous polyposis (FAP) is an autosomal dominant colorectal cancer predisposition syndrome most commonly caused by pathogenic variants in the APC gene. Although targeted gene panel sequencing is routinely used, some FAP cases remain genetically unresolved due to limitations in detecting structural and deep intronic variants. We investigated two unrelated kindreds with clinically evident but genetically unexplained FAP using targeted panel sequencing and whole exome sequencing. Pedigree-based whole genome sequencing (WGS) was performed to detect cryptic variants, with in silico splice prediction and transcript-level validation using RT-PCR, Sanger sequencing, and RT-qPCR of blood-derived RNA. WGS uncovered two novel pathogenic APC variants missed by prior testing. In Kindred 1, a heterozygous large deletion encompassing exon 10 was detected and confirmed to cause exon skipping, resulting in a frameshift and premature termination codon. In Kindred 2, a deep intronic complex deletion-insertion variant was identified between exons 11 and 12, predicted to create novel splice sites. Transcript analysis demonstrated aberrant pseudoexon activation with a 2-base-pair deletion, leading to a frameshift and premature termination. Both variants segregated with disease within the respective families and were classified as pathogenic according to ACMG guidelines based on loss-of-function effects and functional RNA evidence. Our findings demonstrate that cryptic pathogenic APC variants beyond the detection limits of routine genetic testing can be resolved through WGS combined with transcript-level validation. This study highlights the importance of transcript-aware variant interpretation and supports the integration of genome-wide sequencing and RNA-based analyses into the diagnostic evaluation of genetically unexplained FAP.
Congenital nemaline myopathies are rare genetic disorders that typically manifest at birth or in childhood, with muscle weakness and respiratory distress. They are characterized by the presence of rod-like structures on the muscle biopsy, or a mix of rods with cores, focal areas with disorganization of oxidative activity. Pathogenic variants in the NEB gene represent a main cause of these conditions. However, significant phenotypic variability exists, hampering disease prognosis, healthcare, and genetic counseling. This study is aimed at further characterizing the clinical and genetic features of nebulin-related congenital myopathies and exploring genotype-phenotype correlations in a novel large cohort of patients. In a total of 49 patients from 48 families with nebulin-related myopathies, a substantial proportion (23/49) exhibited a typical form of the disease, with childhood onset and preserved ambulation beyond 11 years of age. Respiratory insufficiency was observed in 33/49 patients, with a median age of 16 years for the initiation of ventilatory support. Facial muscle weakness was common (32/49), whereas severe bulbar symptoms were less frequent, with only 14/49 requiring nutritional support, all in cases where respiratory assistance was necessary. Patients with rods (n = 28) or core-rods (n = 11) exhibited no significant clinical differences. Genetic analysis identified 76 NEB variants, including 7 recurrent, with a mutational hotspot detected between Exons 169 and 175 in 25/49 patients. Furthermore, our analyses suggest an association between the type of variant and five groups of patients with different levels of clinical severity. Additionally, our data raise the possibility that ophthalmoplegia may be preferentially observed in patients harboring variants located in Exon 143. Overall, these findings will contribute to facilitating and expediting genetic diagnosis in future cases with suspected NEB involvement, ultimately improving patient care and management.
Cleidocranial dysplasia (CCD) is a genetic disorder characterized by delayed cranial suture closure, hypoplastic clavicles, and dental anomalies, with varying severity. Most cases are linked to RUNX2 variants; however, rare CCD-like phenotypes can arise from other genetic alterations, including variants in MSX2, a critical skeletal development gene. Here, we describe two patients with persistent anterior fontanelles and microduplications upstream of MSX2, a region previously associated with CCD-like phenotypes. Microarray analysis revealed narrower duplication ranges in these patients than in two earlier reported cases. To elucidate the underlying mechanisms, we performed in silico analyses using publicly available datasets. Epigenetic characterization of the minimal overlapping duplicated region (chr5:173848716-173888281) identified two transcriptionally active subregions showing high chromatin accessibility in osteoblasts. These findings suggest a regulatory role in osteogenic differentiation. Differential chromatin accessibility analysis using ATAC-seq data demonstrated preferential accessibility in osteoblasts over chondrocytes in both human and mouse models, further implicating this region in bone development. The duplicated region was fully contained within a single topologically associating domain (TAD), suggesting that intra-TAD duplications may disrupt spatial and temporal MSX2 regulation, contributing to the CCD-like phenotype. Phenotypic variations, such as synpolydactyly in a previously reported patient, likely arise from additional regulatory elements outside the minimal overlapping region. Our findings establish a causal link between MSX2 upstream duplications and CCD-like phenotypes, emphasizing the role of epigenetic mechanisms and TAD structures in regulating skeletal development. Further studies, including long-read sequencing and ChIP-seq, are needed to clarify the precise regulatory pathways involved.
Patients with suspected monogenic disorders often remain undiagnosed after exome sequencing. We report a family with two sisters affected by a complex spastic paraplegia. Initial exome sequencing had identified monoallelic pathogenic nonsense variants in AP4S1 and AP4B1, subunits of the adaptor protein complex 4 (AP-4), suggesting digenic inheritance. As digenic inheritance has not been established for AP-4-associated disorders, we applied a multiomics approach including genome sequencing, RNA sequencing and proteomics to clarify the genetic cause. By RNA sequencing a predicted synonymous variant (NM_006594.5:c.969G > A), compound heterozygous to the nonsense variant in AP4B1 and previously considered as benign, was re-prioritized as aberrant splicing was demonstrated. Proteomics showed reduced abundance of AP-4 components AP4B1 and AP4M1 and an upregulation of the cargo protein ATG9A, confirming AP-4 deficiency. Although the AP4S1 variant resulted in nonsense-mediated decay, the identification of biallelic causative variants in AP4B1 established the diagnosis of monogenic "Spastic paraplegia 47, autosomal recessive" while the initial hypothesis of digenic inheritance was refuted. This study illustrates the value of multiomics approaches in the diagnostic workflow of rare diseases and the potential for pathogenicity of synonymous variants.
Objective:FBXO43 is a known inhibitor of the anaphase-promoting complex/cyclosome (APC/C), a key E3 ubiquitin ligase that controls meiotic cell cycle progression. However, how different FBXO43 mutations affect APC/C inhibition and lead to divergent clinical phenotypes remains unclear. This study is aimed at clarifying how different FBXO43 mutations produce divergent clinical phenotypes in male infertility and to investigate their preliminary molecular mechanisms, thereby providing evidence-based guidance for precision-assisted reproduction in affected individuals. Methods:Two infertile patients carrying distinct compound heterozygous FBXO43 variants-missense mutations in a macrozoospermia case and truncating mutations in a nonobstructive azoospermia (NOA) case-were identified through whole-exome sequencing. Sperm morphology, chromatin status, and aneuploidy were assessed, and a testicular biopsy was performed for the NOA case. Functional consequences of each mutation were evaluated using in vitro HEK 293T cell models, including protein stability, ubiquitination, and APC/C subunit interactions. Clinical outcomes of assisted reproductive technology (ART) were also reviewed. Results:Missense variants (NM_001029860, p.Pro641Leu/p.Arg660Gln) in the macrozoospermia patient allowed completion of meiosis but led to severe sperm head enlargement, chromatin condensation defects, and markedly elevated aneuploidy, resulting in repeated ICSI failure. In contrast, the truncating variants (p.Trp532 ∗/p.Ser577Leufs ∗11) in the NOA patient abolished the C-terminal functional domain and caused meiotic arrest with complete absence of mature sperm. Mechanistically, all mutations reduced FBXO43 protein stability and disrupted its specific binding to the APC/C substrate recognition subunit APC3, whereas interactions with APC2/6/8 remained intact. Loss of APC3 binding likely impairs APC/C inhibition, disturbing meiotic chromosome segregation in a mutation-severity-dependent manner. Conclusions:Different types of FBXO43 mutations generate distinct infertility phenotypes through a dose-dependent mechanism: Truncating mutations cause profound meiotic arrest and NOA, whereas hypomorphic missense mutations permit meiotic completion but result in severe teratozoospermia with high aneuploidy. These findings link FBXO43 to a continuous phenotypic spectrum and highlight its relevance for precision reproductive counseling. Based on the identified mutation-specific risks, preimplantation genetic testing for aneuploidy (PGT-A) is recommended for affected couples to improve embryo selection and optimize ART outcomes.
Mutations in mitochondrial tRNA (mt-tRNA) are found to be associated with hypertrophic cardiomyopathy (HCM), but their molecular mechanisms remain largely undetermined. In this study, we investigated the contribution of a novel HCM-related mt-tRNASer(AGY) 12234A > G mutation to the phenotypic expression of the mt-tRNAIle 4263A > G mutation in two genetically unrelated Han Chinese pedigrees. Strikingly, the penetrance and expressivity of one pedigree (HCM2) with both m.4263A > G and m.12234A > G mutations are much higher than another pedigree (HCM1) with only m.4263A > G mutation. By molecular level, the homoplasmic m.4263A > G mutation is located at the processing site for the tRNAIle 5 '-end precursor, disrupting a conserved Watson-Crick base pairing (1A-69T) which is believed to cause mitochondrial dysfunction. Moreover, the heteroplasmic m.12234A > G mutation occurs at an extremely conserved nucleotide in the anticodon stem of tRNASer(AGY), a position which is critical for tRNA structure and function. Using trans-mitochondrial cell models, we demonstrated that cybrids with both mt-tRNA mutations exhibited more severe mitochondrial dysfunctions than cybrids with only the m.4263A > G mutation. Furthermore, a marked decrease in mt-RNA transcripts was observed in cells harboring both m.4263A > G and m.12234A > G mutations. Taken together, our study indicated that the m.12234A > G mutation acted in synergy with the m.4263A > G mutation, triggering mitochondrial dysfunctions and contributing to a high penetrance of HCM in a pedigree harboring both mtDNA mutations.
