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PubMed2001

Mitochondrial DNA mutations in black Americans with hypertension-associated end-stage renal disease.

For the majority of hypertensive cases, no gene or combination of genes and environmental factors clearly leading to hypertension has been identified. Studies to identify "hypertension" genes have focused on the assessment of markers and candidate genes in the nuclear genome. In this study, we have chosen to assess the mitochondrial genome as a site of mutations possibly contributing to susceptibility to hypertension in black Americans who have progressed to end-stage renal disease (H-ESRD). The mitochondrial genomes of 58 H-ESRD and 58 normotensive individuals were systematically analyzed by means of a high-resolution restriction analysis. After stratification by the presence or absence of an African continent-specific HpaI site gain at bp 3,592, differences in the frequencies of mitochondrial DNA (mtDNA) restriction variants in both groups were examined by chi-square analyses. A total of six variants was identified with significant differences in one or both cohorts. An A10398G DdeI mutation in the ND3 gene was significantly increased in the H-ESRD cohort (H-ESRD, P = 0.048; normotensives, P = 0.20), as was an HaeIII T6620C/G6260A double mutation in the CO1 gene (H-ESRD, P = 0.05; normotensives, P = 0.48). The remaining four variants were a G2758A mutation in the 16SrRNA gene (identified by RsaI), T10810C in the ND4 gene (identified by HinfI), a G7028A/T7055C double mutation in the CO1 gene (identified by AluI), and finally, a A10086G mutation in the ND3 gene (identified by TaqI; also causing an Asn-->Asp amino acid change). The RsaI and HinfI variants were in strong linkage disequilibrium with the HpaI site and not amenable to further analysis. After correction of all P values for multiple comparisons, the ND3 A10086G (Asn-->Asp) mutation shown by TaqI remained statistically significant (P = 0.0036) in the H-ESRD cohort, not in the normotensive cohort. To the best of our knowledge, this is the first report of an increased prevalence of mitochondrial gene variants in hypertensive individuals. In addition, we have identified single-nucleotide polymorphisms in flanking regions of these genes. Although replication and further assessment are necessary, the current results support our hypothesis that mtDNA may account for a portion of hypertensive cases in black Americans with ESRD.

Black or African American↗

PubMed2001

Three novel mutations of the RPGR gene exon ORF15 in three Japanese families with X-linked retinitis pigmentosa.

We describe three new mutations in a recently identified exon, ORF15, of the retinitis pigmentosa GTPase regulator gene (RPGR) in three unrelated Japanese families (Families 1-3) with X-linked retinitis pigmentosa (XLRP). The affected males had typical retinitis pigmentosa (RP), whereas the obligate carrier females showed a wide clinical spectrum, ranging from minor symptoms to severe visual disability. Some carrier females in Families 1 and 2 showed typical RP, most carriers manifested high myopia and astigmatism, and their corrected visual acuity was insufficient. They showed an impairment of cone function following the rod dysfunction and accompanied by refractive errors. Microsatellite analysis of Family 1 revealed that the RP in the family was linked to the RP3 locus. Although one patient in the family had no mutation in the previously published exons 1-19 including exon 15a, he had a single-nucleotide insertion in exon ORF15 (g.ORF15 + 753-754 insG). Likewise, patients in Families 2 and 3 had two-base insertion/deletion in the exon, i.e., g.ORF15 + 833-834delGG and g.ORF15 + 861-862insGG, respectively. These insertional/deletional mutations observed in the three families are all different and new, and are predicted to lead to a frameshift, resulting in a truncated protein. These findings may support the previous hypothesis that RPGR-ORF15 is a mutational hot spot.

Adult↗

PubMed2026

The causal relationship between multiple cardiovascular diseases and glioblastoma: A Mendelian randomization study.

Observational studies suggest an association between glioblastoma (GBM) and cardiovascular diseases (CVDs), but a causal relationship remains unestablished. This study aimed to investigate the causal link between multiple CVDs and GBM risk. The inverse variance weighted method indicated that all 18 CVDs had significant causal associations with GBM (P < .05). Genetically predicted CVDs were uniformly associated with a lower risk of GBM (odds ratio < 1), identifying them as potential protective factors. Sensitivity analyses confirmed the absence of significant heterogeneity or horizontal pleiotropy, and the MR-Steiger test validated the correct causal direction. This Mendelian randomization (MR) study provides evidence that a range of CVDs are causally associated with a decreased risk of developing GBM. These findings suggest shared biological pathways and offer new insights for understanding GBM etiology. We conducted a 2-sample MR analysis using publicly available genome-wide association study data. GBM was the outcome, and 18 cardiovascular-related traits (including coronary artery disease, myocardial infarction, and venous thromboembolism) were exposures. Instrumental variables were single-nucleotide polymorphisms significantly associated with exposures (P < 5 × 10-8). The primary analysis used the inverse variance weighted method, supplemented with MR-Egger, weighted median, and weighted mode methods. Sensitivity analyses, including Cochran Q test, MR-Egger intercept test, leave-one-out analysis, and MR-Steiger directionality test, were performed to ensure robustness.

Causality↗

PubMed1998

Mapping of complex traits by single-nucleotide polymorphisms.

Molecular geneticists are developing the third-generation human genome map with single-nucleotide polymorphisms (SNPs), which can be assayed via chip-based microarrays. One use of these SNP markers is the ability to locate loci that may be responsible for complex traits, via linkage/linkage-disequilibrium analysis. In this communication, we describe a semiparametric method for combined linkage/linkage-disequilibrium analysis using SNP markers. Asymptotic results are obtained for the estimated parameters, and the finite-sample properties are evaluated via a simulation study. We also applied this technique to a simulated genome-scan experiment for mapping a complex trait with two major genes. This experiment shows that separate linkage and linkage-disequilibrium analyses correctly detected the signals of both major genes; but the rates of false-positive signals seem high. When linkage and linkage-disequilibrium signals were combined, the analysis yielded much stronger and clearer signals for the presence of two major genes than did two separate analyses.

Biosensing Techniques↗

PubMed2000

DNA polymerase beta: contributions of template-positioning and dNTP triphosphate-binding residues to catalysis and fidelity.

The specific catalytic roles of two groups of DNA polymerase beta active site residues identified from crystal structures were investigated: residues possibly involved in DNA template positioning (Lys280, Asn294, and Glu295) and residues possibly involved in binding the triphosphate moiety of the incoming dNTP (Arg149, Ser180, Arg183, and Ser188). Eight site-specific mutants were constructed: K280A, N294A, N294Q, E295A, R149A, S180A, R183A, and S188A. Two-dimensional NMR analysis was employed to show that the global conformation of the mutants has not been perturbed significantly. Pre-steady-state kinetic analyses with single-nucleotide gapped DNA substrates were then performed to obtain the rate of catalysis at saturating dNTP (k(pol)), the apparent dissociation constant for dNTP (K(d)), catalytic efficiency k(pol)/K(d), and fidelity. Of the three template-positioning residues, Asn294 and Glu295 (but not Lys280) contribute significantly to k(pol). Taken together with other data, the results suggest that these two residues help to stabilize the transition state during catalysis even though they interact with the DNA template backbone rather than directly with the incoming dNTP or the opposite base on the template. Furthermore, the fidelity increases by up to 19-fold for N294Q due to differential k(pol) effects between correct and incorrect nucleotides. Of the four potential triphosphate-binding residues, Ser180 and Arg183 contribute significantly to k(pol) while the effects of R149A are relatively small and are primarily on K(d), and Ser188 appears to play a minimal role in the catalysis by Pol beta. These results identify several residues important for catalysis and quantitate the contributions of each of those residues. The functional data are discussed in relation to the prediction on the basis of available crystal structures.

Animals↗

PubMed2024

Association of different milk fat content with coronary artery disease and myocardial infarction risk: A Mendelian randomization study.

BACKGROUND: Numerous observational studies have investigated on the correlation of whole, semi-skimmed, and skimmed milk with coronary artery disease (CAD) and myocardial infarction (MI) risk; However, no consensus has been reached and evidence on any causal links between these exposures and outcomes remains unclear. This study aimed to conduct univariate and multivariate Mendelian randomization (MR) analyses, using publicly released genome-wide association study summary statistics (GWAS) from the IEU GWAS database, to ascertain the causal association of milk with various fat content with CAD and MI risk. METHODS: For the exposure data, 29, 15, and 30 single-nucleotide polymorphisms for whole milk, semi-skimmed milk, and skimmed milk, respectively, obtained from 360,806 Europeans, were used as instrumental variables. CAD and MI comprised 141,217 and 395,795 samples, respectively. We used inverse variance weighted (IVW), weighted median, MR-Egger regression, and MR Pleiotropy Residual Sum and Outlier analyses to determine whether pleiotropy and heterogeneity could skew the MR results. Sensitivity tests were conducted to verify the robustness of the results. RESULTS: After adjusting for false discovery rates (FDR), we discovered proof that skimmed milk intake is a genetically predicted risk factor for CAD (odds ratio [OR] = 5.302; 95% confidence interval [CI] 2.261-12.432; P < 0.001; FDR-corrected P < 0.001) and MI (OR = 2.287; 95% CI 1.218-4.300; P = 0.010; FDR-corrected P = 0.009). Most sensitivity assessments yielded valid results. Multivariable MR for CAD and MI produced results consistent with those obtained using the IVW method. There was no causal relationship between whole or semi-skimmed milk, and CAD or MI. CONCLUSION: Our findings indicate that the consumption of skimmed milk may increase the risk of CAD and MI. This evidence may help inform dietary recommendations for preventing cardiovascular disease. Further studies are required to elucidate the underlying mechanisms.

Humans↗

PubMed2002

Genotyping Cryptosporidium parvum with an hsp70 single-nucleotide polymorphism microarray.

We investigated the application of an oligonucleotide microarray to (i) specifically detect Cryptosporidium spp., (ii) differentiate between closely related C. parvum isolates and Cryptosporidium species, and (iii) differentiate between principle genotypes known to infect humans. A microarray of 68 capture probes targeting seven single-nucleotide polymorphisms (SNPs) within a 190-bp region of the hsp70 gene of Cryptosporidium parvum was constructed. Labeled hsp70 targets were generated by PCR with biotin- or Cy3-labeled primers. Hybridization conditions were optimized for hybridization time, temperature, and salt concentration. Two genotype I C. parvum isolates (TU502 and UG502), two C. parvum genotype II isolates (Iowa and GCH1), and DNAs from 22 non-Cryptosporidium sp. organisms were used to test method specificity. Only DNAs from C. parvum isolates produced labeled amplicons that could be hybridized to and detected on the array. Hybridization patterns between genotypes were visually distinct, but identification of SNPs required statistical analysis of the signal intensity data. The results indicated that correct mismatch discrimination could be achieved for all seven SNPs for the UG502 isolate, five of seven SNPs for the TU502 isolate, and six of seven SNPs for both the Iowa and GCH1 isolates. Even without perfect mismatch discrimination, the microarray method unambiguously distinguished between genotype I and genotype II isolates and demonstrated the potential to differentiate between other isolates and species on a single microarray. This method may provide a powerful new tool for water utilities and public health officials for assessing point and nonpoint source contamination of water supplies.

Animals↗

PubMed2001

A transmission/disequilibrium test that allows for genotyping errors in the analysis of single-nucleotide polymorphism data.

The present study assesses the effects of genotyping errors on the type I error rate of a particular transmission/disequilibrium test (TDT(std)), which assumes that data are errorless, and introduces a new transmission/disequilibrium test (TDT(ae)) that allows for random genotyping errors. We evaluate the type I error rate and power of the TDT(ae) under a variety of simulations and perform a power comparison between the TDT(std) and the TDT(ae), for errorless data. Both the TDT(std) and the TDT(ae) statistics are computed as two times a log-likelihood difference, and both are asymptotically distributed as chi(2) with 1 df. Genotype data for trios are simulated under a null hypothesis and under an alternative (power) hypothesis. For each simulation, errors are introduced randomly via a computer algorithm with different probabilities (called "allelic error rates"). The TDT(std) statistic is computed on all trios that show Mendelian consistency, whereas the TDT(ae) statistic is computed on all trios. The results indicate that TDT(std) shows a significant increase in type I error when applied to data in which inconsistent trios are removed. This type I error increases both with an increase in sample size and with an increase in the allelic error rates. TDT(ae) always maintains correct type I error rates for the simulations considered. Factors affecting the power of the TDT(ae) are discussed. Finally, the power of TDT(std) is at least that of TDT(ae) for simulations with errorless data. Because data are rarely error free, we recommend that researchers use methods, such as the TDT(ae), that allow for errors in genotype data.

Alleles↗

PubMed2001

IL10.G microsatellites mark promoter haplotypes associated with protection against the development of reactive arthritis in Finnish patients.

OBJECTIVE: To investigate the association of microsatellites and single-nucleotide promoter polymorphisms (SNPs) in the gene for the cytokine interleukin-10 (IL-10) with susceptibility to and outcome of reactive arthritis (ReA). METHODS: From genomic DNA, IL-10 microsatellites G and R and IL-10 promoter polymorphisms at positions -1087 and -524 were typed by polymerase chain reaction, automated fragment length analysis, and restriction fragment digestion in 85 Finnish patients with ReA and 62 HLA-B27-positive Finnish controls. ReA patients had been followed up for 20 years. Genotypes and haplotypes of IL-10 were correlated with distinct features of the disease course, such as triggering agent, chronic arthritis, development of ankylosing spondylitis, and other chronic features. RESULTS: There was a significant decrease in the promoter alleles G12 (allele frequency 0.206 versus 0.033; corrected P < 0.001, odds ratio 0.14) and G10 (0.183 versus 0.092; P < 0.05, odds ratio 0.44) in the ReA group compared with the HLA-B27-positive controls. Chronic arthritis developed significantly more frequently in the B27-positive subjects than in the B27-negative subjects (P < 0.05) as well as in patients with [corrected] the IL10.G8 allele. No associations were observed for either SNP or for the IL10.R microsatellite polymorphism. CONCLUSION: IL10.G12 and G10 microsatellite alleles show a strong protective effect against the development of ReA in Finnish subjects. Since these polymorphic markers themselves do not have direct functional implications, they most likely mark promoter haplotypes with distinct functional properties, suggesting that differential production of IL-10 is an important susceptibility factor for the development of ReA.

Adult↗

PubMed2026

Robust error-minimization in the genetic code across physicochemical metrics and variant codes: A graph-theoretic analysis in GF(2)6.

The standard genetic code reduces the impact of point mutations, but the robustness of this property across physicochemical metrics, naturally occurring variant codes, and codon-reassignment mechanisms remains incompletely quantified. Embedding the 64 codons in GF(2)6 represents the hypercube Q6 as a coordinate-dependent subgraph of the encoding-independent single-nucleotide mutation graph H(3,4), and enables continuous ρ-interpolation between the two. Under a quartet-pattern shuffle null (n=10,000), the standard code is significantly low-cost across four established, code-independent physicochemical distance metrics with partially overlapping content (Grant ham p=0.0062; Miyata p<0.001; Woese polar requirement p=0.003; Kyte-Doolittle hydropathy p=0.001), and the signal strengthens monotonically as ρ moves Q6→H(3,4). A structure-aware sensitivity analysis under the alignment-derived ProtSub matrix (Jia & Jernigan 2021) yields the most extreme percentile of any measure tested (p=0.0004; all five p-values pass Bonferroni at α=0.05). Across the 27 NCBI translation tables, near-optimality is preserved: 11 of 12 informative-distance variants retain top-5% placement after BH-FDR correction. Natural codon reassignments avoid disrupting codon-family connectivity: under the encoding-independent H(3,4) adjacency, observed events are topology-breaking at relative risk 0.32 versus the candidate landscape (permutation p≤10-4). The H(3,4) result is stable by construction; the Q6 decomposition is representation-specific and fails to show depletion under 8 of 24 base-to-bit encodings, so we report H(3,4) as the primary test and Q6 as a sensitivity. Event-level conditional-logit modelling shows that topology avoidance and local physicochemical cost provide complementary, only weakly correlated signal (rs=0.15), and that topology adds explanatory value beyond physicochemistry under both Q6 and encoding-independent H(3,4) adjacency. Retrospective reanalysis of nine genome-recoding datasets is consistent with codon-family topology operating as an evolutionary-trajectory constraint distinct from acute engineering fitness. The contribution is the second axis: code evolution is jointly constrained by physicochemical smoothness and codon-family topological integrity, and these two constraints are partly independent.

Codon reassignment↗

PubMed2026

Advances in CRISPR Base Editing: From Molecular Evolution to Therapeutic Applications in Genomic Medicine.

CRISPR-Cas9 systems revolutionized gene editing, but inherent drawbacks, namely DNA double-strand breaks (DSBs) and the difficulty of achieving precise repairs (due to low HDR efficiency), led researchers to invent new, more accurate gene editing tools. Base editing represents a significant leap forward, enabling targeted single-nucleotide conversions directly on the DNA without DSBs or donor templates. The core technology involves fusing catalytically dead or nickase Cas proteins to DNA deaminase enzymes. Cytosine base editors (CBEs) convert C•G to T•A pairs, while adenine base editors (ABEs) change A•T to G•C. These editors exploit the deaminase function within the R-loop structure formed by Cas binding and co-opt endogenous DNA repair mechanisms for precision. While offering improved efficiency and editing precision, base editing faces persistent challenges, such as off-target effects, bystander edits, delivery and ethical concerns. Continuous engineering efforts have refined these tools, enhancing accuracy, expanding targetability and reducing unwanted edits. The base editing arsenal has also broadened to include C-to-G base editors (CGBEs), dual A&C editors and versions targeting organelles. Successful preclinical studies demonstrating the correction of mutations responsible for the disease have paved the way for clinical trials, which are now testing therapies for conditions like sickle cell disease, β-thalassaemia and hypercholesterolemia using various delivery systems. This review explores CRISPR base editing's origins, mechanisms of action, potential therapies and current restrictions, pointing to its broadening impact on medical genetics.

Humans↗

PubMed2002

Variation in the calpain-10 gene affects blood glucose levels in the British population.

Variation in the calpain-10 gene (CAPN10) has been shown to be associated with type 2 diabetes in Mexican-Americans and in at least three Northern European populations. Studies in nondiabetic Pima Indians showed that one of the at-risk DNA polymorphisms, single-nucleotide polymorphism (SNP)-43, in CAPN10 was associated with insulin resistance, and individuals with the G/G-genotype had significantly higher fasting plasma glucose and 2-h insulin concentrations after a 75-g oral glucose tolerance test (OGTT). We have examined the effect of variation in CAPN10 on plasma glucose and insulin levels in a group of 285 nondiabetic British subjects after a 75-g OGTT. The results showed that subjects with G/G genotype at SNP-43 had higher 2-h plasma glucose levels than the combined G/A + A/A group (P = 0.05). We also examined the SNP-43, -19, and -63 haplotype combination 112/121, which is associated with an approximately threefold increased risk of diabetes. Subjects with the 112/121 haplotype combination (n = 29) had increased fasting (P = 0.004) and 2-h plasma glucose levels (P = 0.003) compared with the rest of the study population after correction for age, sex, and BMI. The 112/121 haplotype combination was also associated with a marked decrease in the insulin secretory response, adjusted for the level of insulin resistance (P = 0.002). We conclude that genetic variation in the CAPN10 gene influences blood glucose levels in nondiabetic British subjects and that this is due, at least in part, to the effects of calpain-10 on the early insulin secretory response.

Adult↗

PubMed2001

READIT: a novel technology used in the interrogation of nucleic acid sequences for single-nucleotide polymorphisms.

The ability to perform analyses for single-nucleotide polymorphisms (SNPs) has become routine in many molecular diagnostic laboratories. While various procedures and technologies are available to do so, we evaluated a novel technology for SNP analysis using the Factor V Leiden polymorphism as an example. The Factor V Leiden polymorphism G1691A is the most common genetic abnormality associated with hereditary thrombophilia. Current testing methods remain highly accurate yet their main disadvantage is gel-based detection. The READIT System (Promega Corp., Madison, WI) is a novel approach to SNP analysis that utilizes DNA polymerase-mediated pyrophosphorolysis to release dNTPs, which are converted to ATP and used in a luciferase detection reaction. We screened 280 DNA specimens, previously analyzed using the PCR/MnlI restriction digest assay, to evaluate READIT System capabilities along with the KingFisher robotic magnetic particle processor (ThermoLabsystems). Concordant results were obtained for 278/280 (99%) specimens. One discordant result was due to an equivocal relative light unit while the other was indeterminate. Both specimens gave correct results upon repeat analysis. The READIT System offers several advantages including: (1) rapid SNP analysis, (2) accuracy and precision, (3) cost effectiveness, (4) decreased turn-around times, (5) high throughput, and (6) excellent analysis software.

DNA↗

PubMed2026

No receptor-binding domain adaptation detected in within-host H5N1 surveillance of 4,559 US dairy outbreak sequences.

BACKGROUND: The 2024-2026 US H5N1 clade 2.3.4.4b dairy cattle outbreak has been characterised primarily through consensus-level phylogenetics. Whether mammalian-adaptation variants are emerging at sub-consensus frequencies within infected hosts, particularly at the haemagglutinin receptor-binding domain (RBD), remains unknown because no systematic within-host variant analysis of the public sequencing corpus has been performed. METHODS: We conducted a pre-registered, corpus-wide intrahost single-nucleotide variant (iSNV) analysis of all publicly available H5N1 cattle, feline-spillover, and retail-milk sequences on the NCBI Sequence Read Archive (4559 samples across 7 BioProjects). A dual-caller concordance pipeline (iVar + LoFreq) with empirically determined allele frequency (AF) threshold (3%, set via four-criterion validation including synthetic spike-in controls) was applied to an 11-site Tier 1 mammalian-adaptation panel spanning the polymerase complex, haemagglutinin RBD, and accessory proteins. Within-host nucleotide diversity was compared across host categories. RESULTS: The HA RBD sites Q226L and G228S (H3 numbering) showed zero detections across >4300 adequately sequenced samples at all AF thresholds tested (1-5%), despite the pipeline detecting other non-synonymous variants at these exact codon positions (upper 95% CI for prevalence: 0.08%). Seven of eleven adaptation sites carried statistically significant iSNV signals after Bonferroni correction (corrected α = 0.00417), though all at low prevalence (≤2.95%). Genotype stratification showed that most polymerase-site detections reflected genotype structure rather than within-host emergence: the apparent PB2 631 L→M "reversion" was largely the ancestral avian state of the D1.1 genotype (20 of 23 detections), which never acquired the 631L mammalian adaptation, with only two genuine sub-consensus events in the B3.13 background, while consensus-level PB2 701N was a fixed feature of the D1.1 genotype (10 of 14 detections) rather than independent sub-consensus emergence. Cattle exhibited significantly higher within-host nucleotide diversity than feline-spillover samples (π = 1.59 × 10-4 vs 6.11 × 10-5; Kruskal-Wallis p = 6.6 × 10-15), a finding that persisted after depth-matching (p = 4.6 × 10-5); this may reflect prolonged mammary-gland infection, though sampling differences and host biology cannot be excluded. CONCLUSIONS: We did not detect HA receptor-switching adaptation (the acquisition of human-type α2,6 receptor binding via Q226L/G228S) at any tested allele frequency in the US dairy H5N1 outbreak. Sub-consensus mammalian-adaptation signals exist at polymerase-complex sites but at low prevalence, are genotype-structured rather than independently recurrent, and require functional characterisation before informing risk assessment.

Dairy cattle↗

PubMed2001

A high-throughput SNP typing system for genome-wide association studies.

One of the most difficult issues to be solved in genome-wide association studies is to reduce the amount of genomic DNA required for genotyping. Currently available technologies require too large a quantity of genomic DNA to genotype with hundreds or thousands of single-nucleotide polymorphisms (SNPs). To overcome this problem, we combined the Invader assay with multiplex polymerase chain reaction (PCR), carried out in the presence of antibody to Taq polymerase, as well as using a novel 384-well card system that can reduce the required reaction volume. We amplified 100 genomic DNA fragments, each containing one SNP, in a single tube, and analyzed each SNP with the Invader assay. This procedure correctly genotyped 98 of the 100 SNP loci examined in PCR-amplified samples from ten individuals: the genotypes were confirmed by direct sequencing. The reproducibility and universality of the method were confirmed with two additional sets of 100 SNPs. Because we used 40 ng of genomic DNA as a template for multiplex PCR, the amount needed to assay one SNP was only 0.4 ng; therefore, theoretically, more than 200,000 SNPs could be genotyped at once when 100 microg of genomic DNA is available. Our results indicate the feasibility of undertaking genome-wide association studies using blood samples of only 5-10 ml.

Alleles↗

PubMed2026

SeqQC-former: A sequence-quality fusion framework for QC-aware review prioritization of candidate somatic SNVs in cancer genomics.

The accurate prioritization of candidate somatic single-nucleotide variants (SNVs) remains a challenge due to the substantial variability in sequencing quality across genomic loci. SeqQC-Former is a sequence-quality fusion framework that integrates the local nucleotide context with read-level quality-control (QC) covariates derived from matched tumor-normal sequencing data. This integration generates QC-aware prioritization scores for the downstream review of candidate variants. Unlike conventional variant callers, SeqQC-Former is designed not to infer biological truth but to support post-calling review and prioritization under heterogeneous sequencing conditions. The framework was trained and evaluated on a SEQC2-derived dataset comprising 89,447 candidate loci, including 1378 positive and 88,069 negative loci. In chromosome-held-out validation, which aims to reduce potential genomic-position leakage, SeqQC-Former demonstrated strong discrimination (AUROC = 0.9479; AUPRC = 0.9448), indicating good generalization to previously unseen chromosomes. Given that the SEQC2-derived labels contain QC-associated information; these results should be interpreted as an evaluation of QC-aware prioritization capability rather than an independent validation of biological variant correctness. Ablation analyses revealed that structured QC covariates provided the dominant predictive signal under the current SEQC2-derived labeling regime. SeqQC-Former achieved a significantly higher AUROC than classical machine-learning baselines, as determined by DeLong's test (p < 0.01). Application to 53,164 glioblastoma variants demonstrated that external predictions were sensitive to QC scaling and threshold selection, underscoring that model outputs should be interpreted as QC-dependent prioritization scores rather than calibrated probabilities or definitive biological classifications. Overall, SeqQC-Former offers a reproducible post-calling QC-aware prioritization framework for large-scale somatic SNV review and underscores the importance of explicitly modeling sequencing-quality information when interpreting structured cancer genomics datasets.

Humans↗

PubMed2000

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry.

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.

Amino Acid Sequence↗

PubMed2001

Loss of DNA polymerase beta stacking interactions with templating purines, but not pyrimidines, alters catalytic efficiency and fidelity.

Structures of DNA polymerases bound with DNA reveal that the 5'-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, alpha-helix N of DNA polymerase beta is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions.

Arginine↗