Search Results (226)

Background: Congenital diarrhea is persistent diarrhea that manifests during the neonatal period. Mutations in DGAT1, which is crucial for triglyceride synthesis and lipid absorption in the small intestine, are causal factors for congenital diarrhea. In this study, we aimed to determine the value of tissue RNA sequencing (RNA-seq) for assisting with the clinical diagnosis of some genetic variants of uncertain significance. Methods: We clinically evaluated a patient with watery diarrhea, vomiting, severe malnutrition, and total parenteral nutrition dependence. Possible pathogenic variants were detected using whole-exome sequencing (WES). RNA-seq was utilized to explore the transcriptional alterations in DGAT1 variants identified by WES with unknown clinical significance, according to the American College of Medical Genetics guidelines. Systemic examinations, including endoscopic and histopathological examinations of the intestinal mucosa, were conducted to rule out other potential diagnoses. Results: We successfully diagnosed a patient with congenital diarrhea and protein-losing enteropathy caused by a DGAT1 mutation and reviewed the literature of 19 cases of children with DGAT defects. The missense mutation c.620A>G, p.Lys207Arg located in exon 15, and the intronic mutation c.1249-6T>G in DGAT1 were identified by WES. RNA-seq revealed two aberrant splicing events in the DGAT1 gene of the patient’s small intestinal tissue. Both variants lead to loss-of-function consequences and are classified as pathogenic variants of congenital diarrhea. Conclusions: Rare DGAT1 variants were identified as pathogenic evidence of congenital diarrhea, and the detection of tissue-specific mRNA splicing and transcriptional effects can provide auxiliary evidence. Full article

Background: Osteodysplastic syndromes comprise a very diverse group of clinically and genetically heterogeneous disorders characterized by defects in bone and connective tissue development, as well as in bone density. Here, we report the case of a 48-year-old female with a complex medical history characterized by bone dysplasia, hyperostosis, and partial tooth agenesis. Methods: Genetic testing was performed using WES analysis and Sanger sequencing. Molecular modeling analysis and dynamics simulation explored the impact of detected pathogenic variants. Results: The genetic analysis detected multiple pathogenic variants in genes CREB3L1, SLCO2A1, SFRP4, LRP5, and LRP6, each of which has been associated with rare osteodysplastic syndromes. The patient was homozygous for the same rare alleles associated with three of the identified autosomal recessive disorders osteogenesis imperfecta type XVI, primary hypertrophic osteoarthropathy, and metaphyseal dysplasia Pyle type. She also had a variant linked to autosomal dominant endosteal hyperostosis and a variant previously associated with increased risk of osteoporosis and bone fractures. Two of the detected variants are predicted to cause abnormal splicing, while molecular modeling and dynamics simulations analysis suggest that the other three variants probably confer altered local secondary structure and flexibility that may have functionally devastating consequences. Conclusions: Our case highlights the rare coexistence of multiple osteodysplastic syndromes in a single patient that may complicate differential diagnosis. Furthermore, this case emphasizes the necessity for early genetic investigation of such complex cases with overlying phenotypic traits, followed by genetic counseling, facilitating orchestration of clinical interventions and allowing prevention and/or prompt management of manifestations. Full article

HIV-1 proviral landscapes were investigated using near-full-length HIV single-genome sequencing on blood samples from five children with vertically acquired infection and on ART for ~7–9 years. Proviral structures were compared to published datasets in children prior to ART, children on short-term ART, and adults on ART. We found a strong selection for large internal proviral deletions in children, especially deletions of the env gene. Only 2.5% of the proviruses were sequence-intact, lower than in the comparative datasets from adults. Of the proviruses that retained the env gene, >80% contained two or more defects, most commonly stop codons and/or gag start mutations. Significantly fewer defects in the major splice donor site (MSD) and packaging signal were found in the children on short or long-term ART compared to the adults, and tat was more frequently defective in children. These results suggest that different selection pressures may shape the proviral landscape in children compared to adults and reveal potentially different genetic regions to target for measuring the intact HIV reservoir and for achieving HIV remission in children. Full article

Spondylocostal dysostosis (SCDO) is a group of rare genetic disorders characterized by segmental vertebral defects and rib deformities due to congenital misalignment, fusion, or reduction in the number of ribs. The causes of the disease have been found in seven genes, including DLL3 (SCDO1, OMIM 602768), MESP2 (SCDO2, OMIM 608681), LFNG (SCDO3, OMIM 609813), HES7 (SCDO4, OMIM 608059), TBX6 (SCDO5, OMIM 602427), RIPPLY2 (SCDO6, OMIM 616566), and DLL1 (SCDO7). Among these, SCDO4, characterized by a short trunk, short neck, and mild nonprogressive scoliosis, is a rare form of reported cases. SCDO4 is identified as caused by homozygous or compound heterozygous variants in the HES7 gene (NM_001165967.2; NP_001159439.1). This study reports a novel homozygous HES7 splice variant (c.43-9T>A) detected in an SCDO4 patient by whole-exome sequencing and confirmed by Sanger sequencing. This variant was evaluated as an acceptor loss variant in intron 1 in the HES7 transcript by in silico analysis and was inherited from the patient’s parent. This study also reviews previous reports to provide a comprehensive overview of SCDO and help us to understand the pathogenesis to develop future treatment strategies. Full article

Reinforced concrete columns constructed prior to the 1970s often exhibit deficient lap splices at the base, characterized by insufficient splice lengths. In response to the urgent need for an efficient seismic assessment of these vulnerable structural elements, this study proposed a modelling method for lap-spliced columns. Typically, numerical simulations of columns with lap splices require the cross-sections of the lap-spliced and non-lap-spliced zones to be established, a process that is complex and time-consuming. This paper proposes an equivalent distribution of curvature along the height of the column to represent the effect of lap splice defects on the mechanical behavior of columns, thereby reducing the modelling complexity of such components. Four large-scale column specimens with varying lap splice lengths were subjected to monotonic pushover loading to investigate the effect of splice length on failure modes, strain distribution, and displacement ductility. An active strengthening method was employed to improve the performance of columns with deficient lap splices. Applying lateral prestress to the strengthening devices improves the mechanical behavior of columns. The experimental results revealed that insufficient splice lengths lead to reduced ductility and stress-transfer capacity. The strengthened specimen demonstrated significantly improved ductility and enhanced stress-transfer efficiency, indicating a marked improvement in mechanical performance. The proposed equivalent plastic hinge model was established in OpenSees. A database was created to verify the accuracy of the model. The results showed the modelling method to be accurate. Full article

The tumor suppressor protein p53 prevents the malignant transformation of cells by responding to DNA damage, oncogene activation, and abnormal growth signals including ribosome assembly defects. Under normal conditions, p53 activity is controlled by the regulatory proteins MDM2 and MDM4, which suppress its function through ubiquitin-mediated degradation and transcriptional inhibition. A subset of ribosomal proteins initiates the p53 response to impaired ribosome biogenesis. The ability of some ribosomal proteins to control MDM2 and MDM4 activities, and thereby p53, underscores an intriguing aspect of cell biology: proteins primarily known for their roles in ribosome function can exert extra-ribosomal functions. One notable example is the cellular RNA-protein complex involving RPL5, RPL11, and 5S rRNA (5S RNP) which inhibits MDM2 and stabilizes p53. Another RP, RPL22, is frequently mutated in cancers with microsatellite instability and its paralog RPL22L1 is often amplified. Recent studies have revealed that RPL22 directly modulates the alternative splicing of MDM4 to promote p53 activation, suggesting that the ribosomal protein-p53 relationship is more complex than previously thought. Cellular responses to ribosome biogenesis inhibition extend beyond general alterations in transcription and translation to actively determine cancer cell fate by selectively engaging tumor-suppressor pathways. RPL22’s effect on MDM4 and other mRNA splicing events is a striking example. A better understanding of the mechanisms involved could guide the development of improved cancer treatments. Full article

This study presents the results of an investigation into the potential use of the DiagBelt+ magnetic diagnostic system for assessing the quality of conveyor belt splices. Splices in conveyor belts are susceptible to damage and irregularities resulting from assembly errors, improper vulcanization parameters, or unfavorable operational conditions. Detecting geometric deviations from the reference standard after splice fabrication can serve as a component of QA/QC systems. Later deviations may indicate material or fabrication defects. To date, applications of the DiagBelt+ system have been limited to locating damage within the belt and its splices. Recently, efforts have been made to extend the system’s functionality to include splice diagnostics. This study was conducted under laboratory conditions on an ST2500 belt featuring five splices (three bias and two straight splices). Data acquisition was performed under various configurations of measurement parameters, including sensor-to-belt distance, belt travel speed, and system sensitivity threshold. For each splice, the signal width was measured and analyzed as a potential indicator of splice geometry and quality. The results indicate that the DiagBelt+ system can be effectively used for splice diagnostics. Work has commenced on automating the splice quality assessment process. Full article

Background: Abnormal sodium-dependent hexose reabsorption in the proximal tubule, accompanied by a functional decrease in sodium and water reabsorption under conditions of increased volemia, may be attributed to a dysfunction of primary transporters related to a genetic defect in the Na,K-ATPase gamma subunit. Methods: We examined two sisters, aged 6 and 8 years, who presented with hypercalciuria, glucosuria, fructosuria, galactosuria, and atypical proteinuria. Primary diabetes, galactosemia, and fructosemia were excluded, suggesting a defect in cellular hexose transport in the proximal tubule. We conducted tests on the family members to assess the impact of gradually increasing volemia, using a water-loading test, on tubular H⁺ transport and urinary excretion of calcium, citrate, endothelin-1 (ET-1), and atypical proteins. Whole-exome sequencing was performed in the affected patients to identify the genetic basis of this phenotype. Results: Extended investigations revealed a complex defect in tubular H⁺ transport, calcium and citrate handling, and atypical proteinuria, resulting from water load-driven overproduction of endothelin-1 (ET-1). Genetic analysis identified a heterozygous pathogenic variant, c.80G>A, p.(Arg27His), in the FXYD2 gene, which encodes the gamma subunit of sodium/potassium-transporting ATPase. Conclusions: Our findings provide evidence that a defect in FXYD2 (splice form a) leads to functional impairment of proximal tubular hexose reabsorption. This is the first report on the metabolic consequences of a pathogenic FXYD2 variant affecting the gamma subunit of sodium/potassium-transporting ATPase in humans. The genotype–phenotype correlation in two siblings with a sodium-dependent defect in fructose, galactose, and glucose renal reabsorption allowed us to characterize a disease with a distinct clinical course and biochemical profile, not previously reported in the medical literature or genetic databases. Analysis of this condition was crucial for the early introduction of reno-protective treatment aimed at slowing the progression of nephropathy and for risk assessment in family members, which was essential for genetic counseling. Full article

Metastatic prostate cancer (mPCa) remains a significant cause of cancer-related mortality in men. Advances in molecular profiling have demonstrated that the androgen receptor (AR) axis, DNA damage repair pathways, and the PI3K/AKT/mTOR pathway are critical drivers of disease progression and therapeutic resistance. Despite the established benefits of hormone therapy, chemotherapy, and bone-targeting agents, mPCa commonly becomes treatment-resistant. Recent breakthroughs have highlighted the importance of identifying actionable genetic alterations, such as BRCA2 or ATM defects, that render tumors sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Parallel efforts have refined imaging—particularly prostate-specific membrane antigen (PSMA) positron emission tomography-computed tomography—to detect and localize metastatic lesions with high sensitivity, thereby guiding patient selection for PSMA-targeted radioligand therapies. Multi-omics innovations, including liquid biopsy technologies, enable the real-time tracking of emergent AR splice variants or reversion mutations, supporting adaptive therapy paradigms. Nonetheless, the complexity of mPCa necessitates combination strategies, such as pairing AR inhibition with PI3K/AKT blockade or PARP inhibitors, to inhibit tumor plasticity. Immuno-oncological approaches remain challenging for unselected patients; however, subsets with mismatch repair deficiency or neuroendocrine phenotypes may benefit from immune checkpoint blockade or targeted epigenetic interventions. We present these pivotal advances, and discuss how biomarker-guided integrative treatments can improve mPCa management. Full article

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, affecting approximately 1 in 1000 individuals. This genetically heterogeneous condition is primarily caused by monoallelic pathogenic or likely pathogenic variants in the PKD1 and PKD2 genes, accounting for 78% and 15% of typical cases, respectively. Recently, the application of NGS methods has led to the identification of additional genes associated with ADPKD, which have been incorporated into routine diagnostic testing for detecting phenocopies of the disease. Methods: In this study, targeted NGS (tNGS) analysis of the main cystogenes associated with classic and atypical ADPKD was performed in a cohort of 218 patients clinically diagnosed with cystic nephropathies. Results: Genetic testing identified variants in 175 out of 218 cases (80.3%). Among these, 133 probands (76%) harbored likely pathogenic or pathogenic variants in one or more genes of the panel, while 42 individuals (24%) had a variant of unknown significance (VUS). Specifically, one or more class 4/5 variants in PKD1, PKD2, or both were identified in 111 (83.5%) probands. Remarkably, a pathogenic variant in the IFT140 gene was identified in 14 index cases (8% of positive individuals, 6.4% of the global cohort): 10 distinct loss-of-function (LoF) variants were identified (including four frameshift variants, four nonsense variants, and two splice site defects); one individual carried a second IFT140 missense variant classified as VUS. Furthermore, five affected family members were found to carry a P/LP LoF variant in IFT140. Conclusions: Our data support that IFT140 heterozygous IFT140 LoF variants result in an atypical, mild form of ADPKD, consisting of bilateral kidney cysts and renal functional decline at older ages. Furthermore, we describe the second pediatric patient with a mild form of ADPKD due to an IFT140 variant and discuss hyperuricemia as a previously unappreciated feature of this condition. Full article

Alzheimer’s disease (AD) is the most common form of dementia, characterized by β-amyloid (Aβ) plaques and neurofibrillary tangles, leading to neuronal loss and cognitive impairments. Recent studies have reported the dysregulation of RNA splicing in AD pathogenesis. Our previous transcriptomic study demonstrated the neuroprotective effect of the phytocannabinoid cannabinerol (CBNR) against the cell viability loss induced by Aβ in differentiated SH-SY5Y cells. This study also highlighted the deregulation of genes involved in mRNA splicing after Aβ exposure or CBNR pre-treatment. Here, we investigated whether CBNR could restore the splicing defects induced by Aβ in an AD in vitro model. Using the rMATS computational tool for detecting differential alternative splicing events (DASEs) from RNA-Seq data, we obtained 96 DASEs regulated in both conditions and, remarkably, they were all restored by CBNR pre-treatment. The pathway analysis indicated an over-representation of the “Alzheimer’s disease–amyloid secretase pathway”. Additionally, we observed that Aβ exposure increased the frequency of retained introns (RIs) among the shared DASEs, and that this frequency returned to normality by CBNR pre-treatment. Interestingly, most of these RIs contain a premature in-frame stop codon within the RNA sequence. Finally, analyzing the DASE regions for miRNA hybridization, we found 33 potential DASE/miRNA interactions that were relevant in AD pathogenesis. These findings revealed a novel trans-gene regulation by CBNR, potentially explaining part of its neuroprotective role. This is the first study demonstrating the involvement of a cannabinoid in the regulation of mRNA splicing in an AD model. Full article

The premature aging disease Hutchinson–Gilford Syndrome (HGPS) is caused by defined mutations in the LMNA gene, resulting in the activation of a cryptic splice donor site, which leads to a defective truncated prelamin A protein called progerin. Notably, progerin expression has also been detected in non-mutated healthy individuals, and therefore, its involvement in the physiological aging process has been widely discussed. Since diabetes mellitus is associated with premature aging and increased cardiovascular mortality, we aimed to investigate the role of progerin expression in patients with diabetic retinopathy (DR). mRNA expression of progerin was analyzed in blood samples from 140 patients with DR who received anti-vascular endothelial growth factor (VEGF) therapy. Progerin mRNA levels were significantly lower in female compared to male patients (n = 42 vs. n = 98; 0.67 ± 0.19 vs. 0.89 ± 0.51, p = 0.006) and higher in patients with non-proliferative (NP)DR (n = 87 vs. n = 53; 0.9 ± 0.51 vs. 0.71 ± 0.29, p = 0.013) compared to those with proliferative (P)DR. Additionally, a positive correlation was found between progerin mRNA expression and the number of intravitreal anti-VEGF applications (n = 139, r = 0.21, p = 0.015), central macula thickness (CMT), (n = 137, r = 0.18, p = 0.036) and nicotine consumption (n = 105, r = 0.235, p = 0.002). The nuclear localization and significant upregulation of progerin mRNA and protein levels in dermal fibroblasts from HGPS donors emphasize its role in cellular aging mechanisms. Progerin mRNA levels were higher in patients with NPDR. CMT, number of intravitreal anti-VEGF therapy treatments, and cigarette consumption were positively related to progerin mRNA, suggesting an association with disease progression and premature aging. Full article

Background/Objectives: The nuclear factor (NF)-kB essential modulator (NEMO) has a crucial role in the NFκB pathway. Hypomorphic IKBKG pathogenic variants cause ectodermal dysplasia with immunodeficiency (EDA-ID) in affected males. However, heterozygous amorphic IKBKG variants could be responsible for Incontinentia Pigmenti (IP) in female carriers. Typically, IP patients do not exhibit immunodeficiency, although hypomorphic variants might lead to immunodeficiency in female IP patients. Here, we report the case of an IKBKG female carrier, with no IP but an unexpected picture of immunodeficiency. She had a positive family history for the same genetic condition. Methods: We performed immunological, molecular, and functional analysis to evaluate NEMO contribution. Results: The patient was healthy until the age of 25 when severe asthma and Hashimoto thyroiditis occurred. She had HLAB27-positive ankylosing spondylitis, non-tubercular mycobacteriosis, and pulmonary aspergillosis infections. We found CD19+ B cell lymphopenia and T cell subset alterations. Sanger sequencing revealed a heterozygous IKBKG variant at position +1 of the 5′ UTR of the gene which disrupted the normal pre-mRNA splicing. We observed a decreased NEMO protein expression, a reduced level of mRNA, and a defective NF-κB pathway. Conclusions: These findings suggest a possible correlation between the partial loss of NEMO function and the immunodeficiency observed in this patient. This case could expand our understanding of NEMO deficiency in female carriers. Full article

Background/Objectives: Metabolic diseases in humans, such as obesity or type 2 diabetes, arise from defects in the body’s ability to take in and store nutrients such as carbohydrates and triglycerides. Previous studies in the fruit fly, Drosophila melanogaster, have identified SR proteins, mRNA splicing factors that regulate splice-site selection, as regulating lipid storage in the fly fat body. However, whether SR proteins function in other tissues to regulate nutrient metabolism is not known. Methods: We focused on studying the role of SR proteins in intestines by decreasing their levels in the fly gut and measuring the concentrations of lipids and glycogen. Results: We further characterized the intestinal functions of 9G8, an SR protein, which displayed an increase in organismal lipid levels when knocked down in the intestine but had less triglyceride storage in isolated intestines. Interestingly, decreasing 9G8 in the intestine resulted in increased intestinal expression of five fatty acid synthesis/elongation enzyme genes, as well as four triglyceride lipase genes, which may contribute to the triglyceride phenotypes we observed in 9G8-RNAi flies. Conclusions: These data suggest that 9G8 regulates whole body and intestinal lipid homeostasis by altering the expression of lipid metabolic enzyme genes in the fly intestine. Full article

Background: An estimated 10–15% of all genetic diseases are attributable to variants in noncanonical splice sites, auxiliary splice sites and deep-intronic variants. Most of these unstudied variants are classified as variants of uncertain significance (VUS), which are not clinically actionable. This study investigated two novel splice-altering variants, CHM NM_000390.4:c.941-11T>G and CACNA1F NM_005183.4:c.2576+4_2576+5del implicated in choroideremia and cone dystrophy (COD), respectively, resulting in significant visual loss. Methods: Next-generation sequencing was employed to identify the candidate variants in CHM and CACNA1F, which were confirmed using Sanger sequencing. Cascade analysis was undertaken when additional family members were available. Functional analysis was conducted by cloning genomic regions of interest into gateway expression vectors, creating variant and wildtype midigenes, which were subsequently transfected into HEK293 cells. RNA was harvested and amplified by RT-PCR to investigate the splicing profile for each variant compared to the wildtype. Novel variants were reclassified according to ACMG/AMP and ClinGen SVI guidelines. Results: Midigene functional analysis confirmed that both variants disrupted splicing. The CHM NM_000390.4:c.941-11T>G variant caused exon 8 skipping, leading to a frameshift and the CACNA1F NM_005183.4:c.2576+4_2576+5del variant caused a multimodal splice defect leading to an in-frame insertion of seven amino acids and a frameshift. With this evidence, the former was upgraded to likely pathogenic and the latter to a hot VUS. Conclusions: This study adds to the mutational spectrum of splicing defects implicated in retinal degenerations by identifying and characterising two novel variants in CHM and CACNA1F. Our results highlight the importance of conducting functional analysis to investigate the consequences of intronic splice-altering variants and the significance of reclassifying VUS to confirm a genetic diagnosis. Full article