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Results for “Variant pathogenicity assessment”

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PubMed2026

Co-regulation of HIV control and cytomegalovirus pp65-specific IL-1β and TNF-α responses by genetic variants in the MHC region.

The spontaneous control of HIV infection in the absence of antiretroviral therapy, termed HIV control, is associated with genetic variation in the Major Histocompatibility Complex (MHC) locus. These variants are known to influence the immune response to HIV itself. However, people living with HIV are often co-infected with other pathogens that can also elicit immune responses, which might also be regulated by these variants. Here, we assessed whether genetic variants associated with HIV control influence cytokine responses to various co-pathogens. HIV-control-associated single nucleotide polymorphisms (SNPs) were enriched among variants regulating TNF-α and IL-1β production upon CMV pp65 peptide pool stimulation. The top enriched SNPs, rs1128175-A and rs2853971-A, were linked to lower odds of HIV control and increased cytokine responses to CMV. These SNPs were in linkage disequilibrium (LD) with classical HLA alleles HLA-B*07:02 and HLA-C*07:02. Intracellular cytokine staining showed CMV serostatus-dependent production of TNF-α by monocytes and CD8 T cells. The rs1128175-A/rs2853971-A/HLA-B*07:02/HLA-C*07:02 haplotype was associated with increased IFN-γ production by CD8 T cells upon CMV pp65 peptide pool stimulation, indicating an effect on memory responses. Quantitative trait locus (QTL) mapping showed that rs1128175 and rs2853971 influence HLA-B and HLA-C expression, DNA methylation levels and cell-type-specific cis-effects on chromatin accessibility, as well as CD8 T cell subset abundance. These QTL associations suggest that variants associated with poor HIV control are linked to heightened pro-inflammatory responses to CMV pp65 through effects on antigen presentation, epigenetic modifications, gene expression and immune cell repertoire, potentially negatively affecting HIV control status.

Humans↗

PubMed2006

Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing.

A substantial proportion of MLH1 and MSH2 gene mutations in hereditary nonpolyposis colon cancer syndrome (HNPCC) families are characterized by nucleotide substitutions, either within the coding sequence (missense or silent mutations) or in introns. The question of whether these mutations affect the normal function of encoding mismatch DNA repair proteins and thus lead to the predisposition to cancer is determinant in genetic testing. Recent studies have suggested that some nucleotide substitutions can induce aberrant splicing by disrupting cis-transcription elements such as exonic enhancers (ESEs). ESE disruption has been proposed to be the mechanism that underlies the presumed pathological missense mutations identified in HNPCC families. To investigate the prevalence of aberrant splicing resulting from nucleotide substitutions, and its relevance to predicted ESEs, we conducted a systematic RNA screening of a series of 60 patients who carried unrelated exonic or intronic mutations in MLH1 or MSH2 genes. Aberrant splicing was found in 15 cases, five of which were associated with exonic mutations. We evaluated the link between those splicing mutations and predicted putative ESEs by using the computational tools ESEfinder and RESCUE-ESE. Our study shows that the algorithm-based ESE prediction cannot be definitely correlated to experimental observations from RNA screening. By using minigene constructs and in vitro transcription assay, we demonstrated that nucleotide substitutions are the direct cause of the splicing defect. This is the first systematic screening for the effect of missense and silent mutations on splicing in HNPCC patients. The pathogenic splicing mutations identified in this study will contribute to the assessment of "unclassified variants" in genetic counseling. Our results also suggest that one must use caution when determining the pathogenic effect of a missense or silent mutation using ESE prediction algorithms. Analysis at the RNA level is therefore necessary.

Adaptor Proteins, Signal Transducing↗

PubMed2026

Challenges of Using Circulating Tumour DNA: Insights from Advanced Prostate Cancer.

Precision oncology relies on integrating tumour fraction, variant allele frequency, clonal haematopoiesis of indeterminate potential assessment, pathogenicity, and clinical context into next-generation sequencing interpretation, enabling biologically informed and clinically meaningful treatment decisions while reducing the risk of overinterpreting nontumour or nonactionable genomic alterations.

Editorial↗

PubMed2026

Visual Detection and Stratification of Pathogenic mtDNA SNV Heteroplasmy by Balancing FnCas12a Signal Output and Allelic Discrimination.

Assessment of pathogenic mitochondrial DNA (mtDNA) single-nucleotide variant (SNV) heteroplasmy is important for molecular diagnostics, yet rapid visual profiling remains analytically challenging because an assay must combine single-nucleotide allelic discrimination, mutant-fraction-associated readout, and suitable target access. Herein, we report VISTA (visual identification and stratification of targeted mtDNA alleles), a broad-PAM FnCas12a assay that rebalances trans-cleavage signal output and mutant-wild-type discrimination for visual mtDNA SNV heteroplasmy analysis. VISTA uses unmodified FnCas12a with relaxed TTN PAM recognition and integrates crRNA spacer-length engineering with PEG8000/acBSA reaction tuning to improve the practical signal-discrimination balance without nuclease engineering. At the m.3243A>G model locus, spacer truncation enhanced mutant-wild-type discrimination, while molecular-dynamics simulations identified spacer-dependent differences between matched and mismatched complexes at the crRNA-DNA interface. The optimized assay resolved defined synthetic m.3243A>G heteroplasmy gradients by fluorescence imaging and was further adapted to lateral-flow detection. In locus-specific analyses of a deidentified collection of 74 peripheral-blood samples, fluorescence and lateral-flow readouts achieved ROC AUC values above 0.9 for mutant-allele classification after target-region amplification. Fluorescence supported heteroplasmy-associated profiling, whereas lateral flow provided a visual, semiquantitative readout for relative ranking based on the T/C ratio rather than absolute heteroplasmy measurement. VISTA therefore provides an accessible dual-readout analytical strategy for visual detection and heteroplasmy-associated profiling by tuning the FnCas12a signal output and allelic discrimination.

DNA, Mitochondrial↗

PubMed2026

A recurrent CCDC82 frameshift variant associated with syndromic neurodevelopmental disorder in a consanguineous Pakistani family.

BACKGROUND: Intellectual disabilities (IDs) are part of neurodevelopmental disorders (NDDs) and are genetically heterogeneous conditions characterized by impairments in cognition, learning, and adaptive functioning. Despite advances in gene discovery, many individuals, particularly those from understudied populations, remain without a molecular diagnosis. Recent reports implicate CCDC82 (HGNC: 26282) as an autosomal recessive ID gene, although the phenotypic spectrum and biological context remain incompletely defined. METHODS: Exome sequencing (ES) was performed in a consanguineous Pakistani family (PKMR06A) with four affected individuals presenting with moderate to severe ID. Variant segregation was confirmed by Sanger sequencing. In silico analyses, including pathogenicity prediction, protein structural modeling, and domain intolerance assessment, were used to evaluate the functional consequences of the identified variant. Spatiotemporal gene expression patterns were examined using bulk and single-cell human brain transcriptomic datasets. RESULTS: Clinically, affected individuals of family PKMR06A presented with early childhood global developmental delay, speech delay, hypotonia, gait abnormalities, spasticity, and mild facial dysmorphism. Genetic screening revealed a recurrent rare homozygous frameshift variant in CCDC82 (NM_024725.4): c.373del; p.(Asp125Ilefs*6), segregating with disease in all available affected individuals of the family. The identified c.373del variant was absent from the gnomAD database and was classified as pathogenic (PVS1, PM2, and PP1) based on ACMG/AMP criteria. The c.373del variant is predicted to introduce a premature termination codon, p.(Asp125Ilefs*6), leading to deletion of essential coiled-coil domains from the encoded protein, supporting a loss-of-function mechanism. In silico, transcriptomic analyses demonstrated preferential CCDC82 expression during prenatal human brain development, providing developmental context for the neurodevelopmental phenotype associated with the identified truncating variant. CONCLUSIONS: This study expands the mutational landscape of CCDC82 and provides additional clinical and molecular evidence supporting its role in autosomal recessive NDD. The findings reinforce the importance of CCDC82 in human neurodevelopment and highlight the value of genomic investigation in underrepresented populations.

Autosomal recessive↗

PubMed2026

SOD1 Variants in Patients With Amyotrophic Lateral Sclerosis in Central Eastern Europe: From Genetic Testing to SOD1 Targeted Therapy.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is one of the most devastating fatal motor neuron diseases, characterized by progressive degeneration of motor neurons in the brain and spinal cord. A significant advance in ALS therapy was achieved with the recent European Medicines Agency approval of Tofersen, the first antisense oligonucleotide (ASO) specifically targeting SOD1 mRNA, a key genetic determinant of the disease. Yet, despite its clinical relevance, data on SOD1-ALS in Central Eastern Europe remain scarce. METHODS: Here, we present a multicentric study across six countries-Austria, Czechia, Poland, Hungary, Slovakia, and Slovenia-representing approximately 16% of the European Union's population. We report all pathogenic, likely pathogenic, and uncertain SOD1 variants, along with the phenotypic features, including heritability, age, site of onset, and survival. We also assessed the availability of genetic testing, counseling, and access to Tofersen therapy across the region. RESULTS: Out of 1200 patients with confirmed ALS, we identified 24 distinct pathogenic SOD1 variants in a total of 67 patients (median age at onset 47 [40-55] years), of whom 65.7% had familial ALS (fALS) and 34.3% had sporadic ALS (sALS). We characterized the associated phenotypes and reported that 42 patients are currently receiving Tofersen therapy. CONCLUSION: This study provides the first comprehensive overview of SOD1-ALS in Central Eastern Europe. Our findings underscore the importance of genetic testing and counseling, as well as equitable access to targeted therapies such as Tofersen to advance patient-specific care in this region.

Humans↗

PubMed2006

Evidence for adaptive selection acting on the tRNA and rRNA genes of human mitochondrial DNA.

In order to identify putative adaptive human mitochondrial DNA (mtDNA), transfer RNA (tRNA), and ribosomal RNA (rRNA) variants, we assembled a sequential mutational tree from 2,460 human mtDNA coding sequences, thus providing the relative age of all mtDNA sequence variants. Deleterious mutations affect evolutionarily conserved nucleotides and have been eliminated from the older internal branches of the tree by purifying selection, while beneficial mutations also alter conserved nucleotides but have been enriched in the internal branches of the tree by adaptive selection. Neutral polymorphisms alter poorly conserved nucleotides and are distributed throughout the tree. Stem nucleotides are more constrained than loop nucleotides. The functional importance of both types of nucleotide variants was assessed by comparison to the average evolutionary conservation index (CI) of all known pathogenic tRNA mutations, thus permitting discrimination between internal branch neutral and adaptive tRNA variants. This revealed that 19% of the stem and 13% of the loop internal branch tRNA variants were potentially adaptive. Since few pathogenic rRNA mutations are known, evidence for adaptive rRNA variation was revealed by higher stem to loop variant ratios and elevated CIs in the internal branches vs. external branches. Moreover, variants among stem noncanonical apposition bases predominantly created new Watson-Crick (WC) base pairs, thus also suggesting adaptive selection. Among the putative adaptive tRNA and rRNA polymorphisms, a number were found to occur at the base of the branches of the tree, to have recurred multiple times, and to be associated with altered human phenotypes. Therefore, a significant portion of ancient tRNA and rRNA polymorphisms appear to have been adaptive, and these are affecting human health today.

Animals↗

PubMed1998

Identification of missense and truncating mutations in the BRCA1 gene in sporadic and familial breast and ovarian cancer.

The cloning of the breast and ovarian cancer susceptibility gene, BRCA1, allows direct estimation of the proportion of these cancers in the general population which can be attributed to germline mutations in this gene. We have used a combination of SSCP, heteroduplex analysis, and chemical cleavage of mismatch to screen the BRCA1 gene for mutations in the germline of 42 patients with breast or ovarian cancer who either have a moderate family history of these cancers, or have no family history of malignancy but a very early onset of the disease. A total of 30 sequence variants were observed, eight of which have not been described previously. Three sequence changes detected by chemical cleavage or heteroduplex analysis were missed by SSCP. The variants included 13 missense mutations, which were assessed for their pathogenic implications. Two of these (M18T and A1708E) are nonconservative substitutions which are located in evolutionarily conserved regions of the gene: M18T lies just upstream of the RING finger motif, and A1708E abolishes the transcriptional transactivation activity of the carboxy-terminal region of BRCA1. Mutations were observed in eight patients overall (19.0%), and protein-truncating mutations occurred in five of 27 (18.5%) families with 1-3 cases of breast or ovarian cancer. The data suggest that a significant proportion of patients with a modest or no family history of these cancers may carry germline mutations in BRCA1.

Adult↗

PubMed1986

Susceptibility to other antiherpes drugs of pathogenic variants of herpes simplex virus selected for resistance to acyclovir.

Cross-resistance data for a group of nine acyclovir-resistant variants of herpes simplex virus type 1 are reported. These mutants, which express either altered thymidine kinase (TK) or DNA polymerase, were all derived from the same wild-type (wt) strain after exposure to acyclovir in tissue culture. Furthermore, all variants have pathogenic properties similar to the wt parental strain as assessed using mouse model systems (G. Darby, H.J. Field, and S.A. Salisbury, Nature (London) 289:81-83, 1981; B.A. Larder and G. Darby, Virology 146:262-271, 1985). Two groups of antiherpes compounds were used: those requiring activation by TK and those whose action is independent of that enzyme. The TK substrate-specificity mutants were generally resistant to the TK-activated drugs but showed wt susceptibility to phosphonoacetic acid, 9-beta-D-arabinofuranosyladenine, and aphidicolin. The DNA polymerase mutants were relatively susceptible to most TK-activated drugs, although two were resistant to 5-(trifluoromethyl)-2'-deoxyuridine. The polymerase mutants showed a more complex pattern of susceptibility, however, to those compounds whose mode of action is independent of TK. In general, these variants showed similar responses to phosphonoacetic acid, phosphonoformate, and 9-beta-D-arabinofuranosyladenine, a particular variant being either resistant, susceptible, or hypertensive to all three. The response of each variant to aphidicolin, however, appeared to be the inverse of its response to the other three drugs. The cross-resistance patterns are discussed, and their implications for combined or successive therapies are considered.

Acyclovir↗

PubMed2026

Early-Onset Retinopathy in Patients With Variants in SLC6A6 Leading to Impaired Taurine Transport.

IMPORTANCE: Inherited retinal dystrophies are a group of disorders that may lead to progressive vision loss. Improved knowledge of their molecular genetics is important for accurate diagnosis or development of targeted therapies. OBJECTIVE: To identify pathogenic variants in the SLC6A6 gene (encoding TauT, the main transporter for taurine) and assess their role in the molecular pathogenesis of hereditary early-onset retinal dystrophy (EORD) in affected individuals from diverse ethnic backgrounds. DESIGN, SETTING, AND PARTICIPANTS: This was a retrospective, multicenter observational study conducted between June 2019 and March 2025, involving 7 affected and 10 unaffected individuals from 4 unrelated families recruited in Pakistan, Italy, the US, and France. EXPOSURE: Pathogenic variants in SLC6A6 in individuals with EORD. MAIN OUTCOMES AND MEASURES: Genetic, clinical, and functional outcomes of pathogenic variants in SLC6A6 in individuals with Leber congenital amaurosis (LCA) and EORD. All patients underwent standard clinical examinations, including visual acuity, full-field electroretinography, and multimodal retinal imaging, followed by measurement of fasting plasma taurine levels. In vitro and ex vivo taurine transport and membrane trafficking assays in human embryonic kidney (HEK)-293 cells, as well as patient-derived fibroblasts, were also performed. RESULTS: All 7 affected individuals exhibited LCA/EORD, with extraocular findings in some. Genetic analysis identified homozygous pathogenic SLC6A6 variants in all affected individuals, while unaffected relatives were heterozygous carriers. Families 1 and 2 carried missense variants p.(Thr249Ile) and p.(Ala294Thr), while families 3 and 4 carried truncating variants-a deletion of exon 11 and p.(Thr113Ter), respectively. Functional studies demonstrated that both missense variants are associated with complete loss of taurine transport in HEK-293 cells and patient-derived fibroblasts. Additionally, irrespective of the variants considered, plasma taurine levels in affected individuals were reduced compared with heterozygous carriers (difference between means, -31.7 µmol/L; 95% CI, -42.7 to -20.8; P < .001) and healthy control individuals (difference between means, -37.7 µmol/L; 95% CI -41.6 to -33.8; P < .001). CONCLUSIONS AND RELEVANCE: These findings confirm and expand the role of biallelic variants in SLC6A6 in association with LCA/EORD due to impaired taurine transport. These findings suggest that patients with a diagnosis of SLC6A6-related LCA/EORD may be candidates for investigational oral taurine supplementation.

Humans↗

PubMed2025

Genotype-first assessment of presentation and penetrance of neurofibromatosis type 1, autosomal dominant polycystic kidney disease, and Marfan syndrome within the All of Us research program cohort.

PURPOSE: Phenotype-based ascertainment of probands in studies of Mendelian disorders may exclude individuals with mild phenotypes or that lack health care access. We explore this premise in All of Us Research Program participants with pathogenic variation causal for 3 Mendelian conditions: autosomal dominant polycystic kidney disease (ADPKD), Marfan syndrome, and neurofibromatosis type 1 (NF1). METHODS: We identified All of Us Research Program participants with putatively pathogenic variation in NF1, FBN1, PKD1, and PKD2. Concept terms were extracted from electronic health records to assess participant diagnosis and phenotype. Variant annotation and participant surveys were evaluated to identify biological and social factors differentiating diagnosed and undiagnosed individuals. RESULTS: Large proportions of individuals with pathogenic variation in NF1, FBN1, or PKD1/PKD2 lack the associated diagnosis of NF1 (47%), Marfan syndrome (58%), or ADPKD (52%), respectively. Pathogenic variants in diagnosed individuals have greater inferred deleteriousness for NF1 and ADPKD, and undiagnosed individuals had less severe phenotypes compared with diagnosed individuals for all 3 conditions. CONCLUSION: A genotype-first ascertainment of individuals in genomic research allows for a more comprehensive assessment of Mendelian disease and removes biases that confound our understanding of the penetrance and presentation of these conditions.

Humans↗

PubMed2003

Pathogenic potential of environmental Vibrio cholerae strains carrying genetic variants of the toxin-coregulated pilus pathogenicity island.

The major virulence factors of toxigenic Vibrio cholerae are cholera toxin (CT), which is encoded by a lysogenic bacteriophage (CTXPhi), and toxin-coregulated pilus (TCP), an essential colonization factor which is also the receptor for CTXPhi. The genes for the biosynthesis of TCP are part of a larger genetic element known as the TCP pathogenicity island. To assess their pathogenic potential, we analyzed environmental strains of V. cholerae carrying genetic variants of the TCP pathogenicity island for colonization of infant mice, susceptibility to CTXPhi, and diarrheagenicity in adult rabbits. Analysis of 14 environmental strains, including 3 strains carrying a new allele of the tcpA gene, 9 strains carrying a new allele of the toxT gene, and 2 strains carrying conventional tcpA and toxT genes, showed that all strains colonized infant mice with various efficiencies in competition with a control El Tor biotype strain of V. cholerae O1. Five of the 14 strains were susceptible to CTXPhi, and these transductants produced CT and caused diarrhea in adult rabbits. These results suggested that the new alleles of the tcpA and toxT genes found in environmental strains of V. cholerae encode biologically active gene products. Detection of functional homologs of the TCP island genes in environmental strains may have implications for understanding the origin and evolution of virulence genes of V. cholerae.

Animals↗

PubMed2026

Utility of High-Throughput Genomic Analysis for Genetic Counseling in Large Family with Wilson Disease Carrying a Novel 28-bp ATP7B Splice-Junction Deletion.

Background/Objectives: Wilson disease (WD) is an autosomal recessive disorder of copper metabolism caused by pathogenic variants in the ATP7B gene. Early diagnosis and appropriate treatment are essential for preventing irreversible complications. This study demonstrated the clinical utility of integrated high-throughput genomic analysis for molecular diagnosis and genetic counseling in a large Thai family affected by WD. Methods: A 32-year-old woman with clinical features suggestive of WD underwent clinical, biochemical, and molecular genetic evaluations, including sequencing of the entire ATP7B gene and SNP microarray. Fluorescent PCR followed by capillary electrophoresis was used for segregation analysis in available family members. SNP microarray analysis and whole-exome sequencing were performed on the proband's husband to identify pathogenic variants in the ATP7B gene and other disease-associated genes for reproductive risk assessment. Results: The proband presented with hepatic dysfunction, Kayser-Fleischer rings, low serum ceruloplasmin, and a family history of fatal liver disease. She also developed progressive weakness, with nerve conduction findings consistent with axonal sensorimotor polyneuropathy predominantly affecting the lower limbs. Sequencing identified a novel homozygous 28-bp splice-junction deletion, c.4022-24_4025del, which disrupted the canonical splice acceptor site at the intron 19/exon 20 boundary and was classified as pathogenic variant. Segregation analysis confirmed carrier status in the proband's father and identified heterozygous carrier or homozygous wild-type status among her living siblings. SNP microarray analysis revealed a 46.7 Mb copy-neutral long contiguous stretch of homozygosity (CN-LCSH) encompassing ATP7B, with CN-LCSH regions accounting for 2.046% of the total autosomal genome. These findings potentially reflected segmental uniparental isodisomy or identity by descent, while the overall homozygosity pattern did not support recent consanguinity. Combined genomic analyses of the proband's husband revealed no pathogenic or likely pathogenic ATP7B variants. Based on the available testing, all offspring are expected to be heterozygous carriers, and the risk of an affected child is considered very low. Conclusions: This study highlights the value of integrated genomic analysis for molecular diagnosis, cascade testing, and reproductive risk counseling. Further functional studies should be conducted to validate their pathogenicity.

ATP7B↗

PubMed2026

Optimization of a Semi-nested PCR Protocol for Amplifying the Entire Spike Protein Region to Identify SARS-CoV-2 Variants in Wastewater.

Wastewater-based monitoring of SARS-CoV-2 and other pathogens is a widely adopted tool for assessing epidemic dynamics. While quantitative assays are commonly used to estimate infection levels in catchment populations, phylogenetic information-such as identifying circulating variants-is also crucial for public health. However, applying the widely used ARTIC protocol for full-genome sequencing to wastewater samples has proven challenging, likely due to the limited specificity and sensitivity of multiplex RT-PCR in such complex matrices. In this study, we developed and optimized a semi-nested RT-PCR assay targeting the full S-protein coding region (~4000 bases) for phylogenetic characterization of SARS-CoV-2 in wastewater. By reducing multiplexing and using single-plex reactions for both RT and PCR steps, we successfully amplified ~2000 bp fragments. Amplicons were sequenced using the Flongle Flow Cell platform. The optimized method-consisting of reverse transcription with specific primers followed by three parallel single-plex semi-nested PCRs-yielded over 1,000 SARS-CoV-2-like reads per primer set in 30 out of 39 wastewater samples in treatment plants in Japan, including those with <10 copies per analyte. Variant proportions were estimated using a newly developed approach based on single-nucleotide variant pattern matrix, revealing the presence of multiple co-circulating variants, including XBB lineages, JN.1, and notably BA.2.75, which was undetected in domestic clinical surveillance. These results highlight the effectiveness of our approach for detecting temporal shifts in SARS-CoV-2 variants, even at low RNA concentrations.

Polymerase Chain Reaction↗

PubMed2001

Generation of cellular immunity to lymphocytic choriomeningitis virus is independent of CD1d1 expression.

CD1 molecules are cell surface glycoproteins, structurally similar to major histocompatibility complex (MHC) class I molecules. The murine CD1d1 molecule has been shown to be essential for the positive selection of a unique subpopulation of T cells [the natural killer (NK) T cells], as CD1d1-deficient mice lack NK T cells. These cells have recently been suggested to play an important role in the induction of innate immunity (i.e. NK cells) and the regulation of immune homeostasis. As such, it was asked whether NK T cells were necessary for the generation of cellular immunity to an acute virus infection. In these studies, the Armstrong strain of lymphocytic choriomeningitis virus (LCMV), a classic inducer of NK cells, and its pathogenic variant clone 13 were used. When NK-cell activity was assessed on day 3 post-LCMV infection, surprisingly, it was found that CD1d1-deficient mice could generate NK-cell activity at wild-type levels. Likewise, LCMV-specific cytotoxic T-lymphocyte (CTL) activity in CD1d1-deficient mice was indistinguishable from that generated in wild-type mice. Additionally, viral titres in the spleen (LCMV Armstrong) and blood (LCMV clone 13) of infected CD1d1-deficient mice were at comparable levels to those found in wild-type mice, as were virus infection-induced increases in cell surface H-2Kb in the spleen. Therefore, these results suggest that the LCMV-induced generation of NK-cell and virus-specific CTL activity, as well as viral clearance, are independent of CD1d1 expression.

Animals↗

PubMed1999

The acidic region and conserved putative protein kinase C phosphorylation site in Nef are important for SIV replication in rhesus macaques.

Variants of the pathogenic SIVmac239 clone with changes in Nef were analyzed to assess the functional relevance of two highly conserved regions in Nef in vitro and in vivo. Changes in a region with an acidic charge (Aci-Nef), or a potential protein kinase C phosphorylation site (PKC-Nef), impaired the ability of Nef to down-regulate CD4 and MHC class I surface expression and to alter CD3-initiated signal transduction in Jurkat T cells. The Aci-Nef, but not the PKC-Nef, associated with the previously described p65 phosphoprotein. SIV containing Aci-Nef, but not SIV containing PKC-Nef, showed reduced infectivity and replication in cell culture systems. One of two rhesus macaques infected with the PKC-Nef mutant virus showed rapid reversion and progressed to disease. In the second animal no reversions and nonprogressive infection was observed. In one of two macaques infected with the Aci-Nef variant, the mutations were stable during the first 40 weeks after infection. Thereafter, variants evolved in which up to six of the eight mutated positions in Nef were reverted and functional activity in vitro was partially restored. These changes occurred concomitantly with increasing viral load and disease progression. The second animal infected with the Aci-Nef variant showed no reversions and remained asymptomatic. Our study suggests that the acidic region and conserved PKC phosphorylation site in Nef are important for SIV replication in rhesus macaques and for several in vitro Nef functions. An almost wild-type activity in in vitro infectivity and replication assays seems insufficient to confer a full nef-positive phenotype in vivo.

Amino Acid Sequence↗

PubMed2007

The additive effect of p53 Arg72Pro and RNASEL Arg462Gln genotypes on age of disease onset in Lynch syndrome patients with pathogenic germline mutations in MSH2 or MLH1.

p53 and the prostate-cancer-susceptibility gene RNASEL are tumour suppressor genes involved in apoptosis. We have previously reported that the common, functionally different variants Arg72Pro in p53 and Arg462Gln in RNASEL are associated with the age of disease onset of colorectal cancer in Lynch syndrome patients. To assess the combined effect of both variants, we screened 246 unrelated Lynch syndrome patients with a pathogenic germline mutation either in MSH2 (n=138) or in MLH1 (n=108) and colorectal cancer as first tumour, and 245 healthy controls. The global log rank test revealed significant differences in the age of disease onset for the genotypes of each variant (p=0.0176 for p53 and p=0.0358 for RNASEL) and for the combined genotypes of both variants (p=0.0174). The highest difference in median age of disease onset was seen between homozygotes for the wild-types in both genes (42years [range 22-75]) and homozygotes for the variant alleles in both genes (30years [range 26-47]). A multivariate Cox regression model indicated that only the p53 and RNASEL genotypes had a significant influence on age of disease onset (p=0.016 for p53 and p=0.014 for RNASEL) in an additive mode of inheritance, and that the effects of both variants are purely additive, which supports the notion that the p53 and RNaseL pathways do not interact. These findings may be relevant for preventive strategies in Lynch syndrome.

Adaptor Proteins, Signal Transducing↗

PubMed2025

Deep learning-based assessment of missense variants in the COG4 gene presented with bilateral congenital cataract.

OBJECTIVE: We compared the protein structure and pathogenicity of clinically relevant variants of the COG4 gene with AlphaFold2 (AF2), Alpha Missense (AM), and ThermoMPNN for the first time. METHODS AND ANALYSIS: The sequences of clinically relevant Cog4 missense variants (one novel identified p.Y714F and three pre-existing p.G512R, p.R729W and p.L769R from Uniprot Q9H9E3) were imported into AF2 for protein structural prediction, and the pathogenicity was estimated using AM and ThermoMPNN. Different pathogenicity metrics were aggregated with principal component analysis (PCA) and further analysed at three levels (amino acid position, substitution and post-translation) based on all possible Cog4 missense variants (n=14 915). RESULTS: Localised protein structural impact including change of conformation and amino acid polarity, breakage of hydrogen bond and salt-bridge, and formation of alpha-helix were identified among clinically relevant Cog4 variants. The global structural comparison with multidimensional scaling demonstrated variants with similar protein structures (AF2) tended to exhibit similar clinical and biological phenotypes. The Cog4 p.Y714F variant exhibited greater protein structural similarity to mutated Cog4 found in Saul‒Wilson syndrome (p.G512R) and shared similar clinical phenotype (congenital cataract and psychomotor retardation). PCA of included pathogenic metrics demonstrated p.Y714F occurred at a critical position in Cog4 amino acid sequence with disrupted post-translational phosphorylation. CONCLUSION: Deep learning algorithms, including AF2, AM and ThermoMPNN, can be useful for evaluating variant of uncertain significance (VUS) by structural and pathogenicity prediction. Despite classified as VUS (American College of Medical Genetics and Genomics criteria: PM1, PP4), the pathogenicity in this Cog4 variant cannot be ruled out and warrants further investigation.

Mutation, Missense↗