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At least 181 records · Page 10Linked to original sources

Genetic heterogeneity of deafness phenotypes linked to DFNA4.

Mutations in the heavy chain of the class II nonmuscle myosin, MYH14, cause autosomal dominant hearing loss in families linked to the DFNA4 locus. Consistent with this discovery, we identified an S120L mutation in MYH14 in a large German family segregating deafness that links to DFNA4. However, complete screening of the American family that originally defined the DFNA4 locus revealed no mutations in this gene. Furthermore, haplotyping of a single nucleotide polymorphism (SNP) 5' to MYH14 excludes this gene from the critical region in this family. Our results imply that mutations in another gene result in deafness at the DFNA4 locus. The newly defined candidate region encompasses a region of approximately 19 cM. Several candidate genes have been screened for disease-causing mutations.

Amino Acid Sequence↗

Unravelling genetic data by arrayed primer extension.

We have developed a method for arrayed primer extension (APEX) on an oligonucleotide microchip together with the 4-color fluoresence imaging equipment and supporting software, that allows analysis of the DNA sequence and changes in it. Mutation analysis of BRCA1 gene and single nucleotide polymorphism (SNP) chip for genotyping were used as a model system. Chip surface chemistry, template preparation and APEX reaction conditions were optimised and the assay is ready to be implemented in variety of DNA analysis from SNP testing to DNA resequencing.

Alleles↗

A mutation-independent therapeutic strategem for osteogenesis imperfecta.

Given the genetically heterogeneous nature of many dominantly inherited disorders, it will be imperative to design mutation-independent therapeutic strategies to circumvent such heterogeneity. Intragenic polymorphism represents a genomic resource that may be harnessed in the development of allele-specific mutation-independent therapeutics. A hammerhead ribozyme, Rzpol1a1, selectively cleaves a common single-nucleotide polymorphism (SNP) of the human COL1A1 transcript (heterozygosity frequency of 2 pq = 0.4032, from Hardy-Weinberg equilibrium). One SNP variant contains a hammerhead ribozyme cleavage site, and the other does not. Kinetic evaluation shows Rzpol1a1 to be both specific and extremely efficient in vitro. Thus, a single efficient ribozyme has been characterized that should be valuable in the development of a gene therapy suitable for up to 1 in 5 dominant-negative osteogenesis imperfecta (OI) patients, where over 150 different mutations have been identified to date. Given the increasing characterization of intragenic SNP, it is predicted that such a mutation-independent strategy, based on selective silencing of mutant alleles at SNP, may become increasingly important in future genomics-driven drug development for many heterogeneous dominant disorders and complex traits.

Alleles↗

Analysis of the factor V Leiden mutation using the READIT Assay.

BACKGROUND: A variety of methods exist for the detection of single-nucleotide polymorphisms (SNPs) present in amplified segments of genomic DNA. We show the application of a novel SNP scoring tool for analysis of the factor V Leiden mutation. METHODS AND RESULTS: We have developed a novel method for analyzing SNPs. The luciferase-based technique, known as the READIT Technology (Promega Corp, Madison, WI), was used to analyze 510 residual human samples sent for factor V Leiden testing from three independent testing laboratories. A blinded retrospective analysis of the factor V Leiden mutation was used to determine the accuracy and throughput capabilities of the technology. One hundred percent concordance was observed between the READIT Assay and genotype assignments made in the testing laboratories. In addition, greater than 6 SDs of separation were observed between the means of wild-type and heterozygote sample populations. Repetitive sample measurements with representative wild-type, heterozygote, and mutant samples showed that greater than 9 SDs separated the means of heterozygote and homozygote sample populations. Confidence intervals based on the means of wild-type, heterozygote, and mutant sample populations were determined. CONCLUSION: Perfect concordance using the READIT Assay showed its effectiveness as a SNP scoring tool. The design of the factor V READIT Assay was straightforward, requiring the design of two unmodified oligonucleotides that differ at the 3' penultimate position to form perfect hybrids with the wild-type or Leiden form of the factor V sequence. The use of previously published amplification primers and conditions minimized the time needed to optimize and validate the assay. The READIT Calculator supplied with the assay allowed automated genotype assignments and statistical analysis from the READIT Assay data. Confidence-interval analysis validated the ability to distinguish between wild-type, heterozygote, and mutant samples using the READIT Assay.

DNA↗

[Screening of the delta-F508 mutation and analysis of two Single Nucleotide Polymorphism of the CFTR gene, in a sample of the general population of Valparaíso, Chile].

BACKGROUND: The Cystic Fibrosis (CF) carrier rate in Chile was estimated to be 1/40. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Delta F508 mutation is the most common in CF patients in Chile and worldwide. Delta F508 has linkage disequilibrium with two Single Nucleotide Polymorphisms (SNP), often used to define the haplotypic frameworks of CF mutations. AIM: To know the frequency of the delta F508 mutation and to establish the SNPs, M470V and T854T, haplotypic frequency, in a Valparaiso general population sample. SUBJECTS AND METHODS: Fifty subjects were studied. Genetic material was obtained from blood samples, amplified by PCR and analyzed by restriction fragment length polymorphism. RESULTS: Two of the 100 chromosomes analyzed, carried the delta F508 mutation. Therefore, the observed frequency carrier rate (0.02) was higher than the expected (0.01). Both carrier chromosomes had the same SNPs haplotypic framework (1-2). In normal chromosomes, the haplotype 2-1 was the most common. DISCUSSION: These results suggest that the chromosomes that bear delta F508 mutation have most likely a Mediterranean European origin, since this haplotypic framework has been reported in that region. We suggest that CF could be more common in Valparaiso than it was previously.

Base Sequence↗

[Study of molecular genetics of the size of reproductive organs and litter size in the sow].

The mutations of ESR, PRLR and FSHR genes were investigated by SNP; 103 F2 sows were slaughtered and their reproductive organs were determined; the litter size of sow was recorded. The linkage between mutations of genes and size of reproductive organ, litter size was analyzed. The results revealed: these sows that could provide more piglets with bigger reproductive organs usually carried genotype like as genotype BB on the sites of ESR, FSHRB and the genotype AA on the sites of ESRB and PRLR, that is, the genotype of BB on the sites of ESR or FSHRB in sow was not only helpful to promote growth of reproductive organs, but also benefit to improve litter size; The size of productive organs and litter size of the sow with genotype AA on the sites of ESRB or PRLR were significantly higher than those of sow with genotype AB or BB.

Animals↗

Single nucleotide polymorphisms and haplotypes of protein C and protein S genes in the Thai population.

Protein C (PC) and protein S (PS) play key roles in an anticoagulant pathway in order to control the haemostatic system. We identified single nucleotide polymorphisms (SNPs) and/or haplotypes in the promotor and exons of the whole PC and PS genes and in the 3'-untranslated region of the PS gene in 55 Thai individuals. The PC gene revealed 10 haplotypes. One synonymous SNP at 2196 was found in the normal Thai population with a minor allele frequency of 4.90%. One homozygous mutation in exon 7, R147W, co-segregated with the synonymous SNP 2196 (homozygote) of the PC gene, resulting in decreased PC activity and antigenic levels. The PS gene revealed three haplotypes with two frequent dimorphisms in exon 15 and the 3'-untranslated region. The most frequent haplotype in the PS gene was H3 (wild type). There was no correlation between the haplotypes of PC and PS genes with functional and antigenic levels of PC and PS.

Adult↗

Specific detection of bacillus anthracis using a TaqMan mismatch amplification mutation assay.

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation of Bacillus anthracis. The use of SNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene of B. anthracis. The assay permits specific, low-level detection (25 fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena of biodefense and microbial forensics.

Bacillus anthracis↗

Active microeletronic chip devices which utilize controlled electrophoretic fields for multiplex DNA hybridization and other genomic applications.

Microelectronic DNA chip devices that contain planar arrays of microelectrodes have been developed for multiplex DNA hybridization and a variety of genomic research and DNA diagnostic applications. These devices are able to produce almost any desired electric field configuration on their surface. This ability to produce well-defined electric fields allows charged molecules (DNA, RNA, proteins, enzymes, antibodies, nanobeads, and even micron scale semiconductor devices) to be electrophoretically transported to or from any microlocation on the planar surface of the device. Of key importance to the device function is the permeation layer which overcoats the microelectrodes. The permeation layer is generally a porous hydrogel material that allows water molecules and small ions (Na+, CI-, etc.) to freely contact the microelectrode surface, but impedes the transport of the larger analytes (oligonucleotides, DNA, RNA, proteins, etc.). The permeation layer prevents the destruction of DNA at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization reactions, and serves as a porous support structure for attaching DNA probes and other molecules to the array. In order to maintain rapid transport of DNA molecules, facilitate hybridization, and work within constrained current and voltage ranges, low conductance buffers and various electronic pulsing scenarios have also been developed. These active microelectronic array devices allow electrophoretic fields to be used to carry out accelerated DNA hybridization reactions and to improve selectivity for single nucleotide polymorphism (SNP), short tandem repeat (STR), and point mutation analysis.

Animals↗

Genome organization of the SARS-CoV.

Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves. Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions. The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs.

Amino Acid Substitution↗

Identification of a Haarlem genotype-specific single nucleotide polymorphism in the mgtC virulence gene of Mycobacterium tuberculosis.

MgtC is a virulence factor common to several intracellular pathogens, including Mycobacterium tuberculosis, that might have been acquired through horizontal gene transfer. In the present study, we investigated the polymorphism of mgtC in clinical isolates representative of the main epidemic groups of M. tuberculosis. MgtC appears to have a low polymorphism rate in M. tuberculosis that consists exclusively of nonsynonymous mutations. We identified a single nucleotide polymorphism (SNP) at mgtC codon 182 (mgtC182) specifically associated with the Haarlem genotype. A simple PCR assay, called the "on/off switch assay," using phosphorothioate-modified primers and Pfu polymerase allowed us to distinguish Haarlem from non-Haarlem strains based on the mgtC182 SNP. The amino acid change (H182R) associated with the mgtC182 SNP in Haarlem strains does not appear to procure a selective advantage. Our results offer a simple and rapid tool to distinguish between Haarlem and non-Haarlem strains. In addition, the on/off switch assay, which allows the detection of SNPs on chromosomal DNA and M. tuberculosis cultures, provides a novel approach for the screening of known SNPs in M. tuberculosis.

Bacterial Proteins↗

Whole-genome characterization of seven multidrug-resistant Neisseria gonorrhoeae isolates from a single tertiary center in Beijing.

BACKGROUND: To characterize the whole-genome features of Neisseria gonorrhoeae clinical isolates collected from a tertiary medical institution in Beijing, with a focus on the genomic basis of ceftriaxone non-susceptibility and multidrug resistance. METHODS: Clinical isolates were collected from April 2023 to November 2024. Of 14 collected isolates, seven were successfully subcultured after revival and included in subsequent analyses. Minimum inhibitory concentrations (MICs) were determined by the Etest method. Whole-genome data were obtained using a combination of second- and third-generation sequencing technologies. The isolates were combined with global and Chinese reference datasets to construct a core-genome single-nucleotide polymorphism (core-SNP) phylogenetic tree. Chromosomal resistance-associated mutations and plasmid characteristics were subsequently analyzed. RESULTS: The seven isolates displayed genomic diversity at the whole-genome level. Four isolates (8087, 8423, 8461, and 8801) carried penA 60.001 and belonged to distinct sequence types, including ST7365, ST8123, and ST7367. One additional isolate (8726) carried penA 273.001; both alleles encode PBP2 proteins sharing the core substitutions A311V, I312M, V316T, and T483S. All five isolates were non-susceptible to ceftriaxone (MIC 0.25-0.5 mg/L). Ceftriaxone non-susceptibility was associated with the co-occurrence of mutations at core penA positions and additional mutations in porB and ponA, with an mtrR mutation present in one isolate. Plasmid collinearity analysis revealed that several multidrug-resistant isolates simultaneously harbored an intact conjugative plasmid and an African-type resistance plasmid carrying bla TEM-1. CONCLUSION: The multidrug-resistant phenotype of Neisseria gonorrhoeae results from the co-existence of chromosomal multi-locus mutations and resistance plasmids. The penA 60.001 isolates in this study did not originate from a single source. This allele appeared in multiple local clonal lineages. This pattern is consistent with horizontal gene transfer of this resistance determinant into multiple endemic lineages.

Neisseria gonorrhoeae↗

Parallel optimization and genotyping of multiple single-nucleotide polymorphism markers by sample pooling approach using cycling-gradient CE with multiple injections.

Increasing importance of single-nucleotide polymorphisms (SNPs) in determination of disease susceptibility or in prediction of therapy response brings attention of many molecular diagnostic laboratories to simple and low-cost SNP genotyping methodologies. We have recently introduced a mutation detection technique based on analysis of homo- and heteroduplex PCR fragments resolved in cycling temperature gradient conditions on a conventional multicapillary-array DNA sequencer. The main advantage of this technique is in its simplicity with no requirement for sample cleanup prior to the analysis. In this report we present a practical application of the technology for genotyping of SNP markers in two separate clinical projects resulting in a combined set of 44 markers screened in over 500 patients. Initially, a design of PCR primers and conditions was performed for each SNP marker. Then, optimization of CE running conditions (limited just to the proper selection of temperature cycling) was performed on pools of 20 DNA samples to increase the probability of having each of the two allele types represented in the sample. After selecting the optimum conditions, screening of markers in patients was performed using a multiple-injection approach for further acceleration of the sample throughput. The rate of successful optimization of experimental conditions without any pre-selection based on the SNP sequence or melting characteristics was 80% from the initial SNP marker candidates. By studying the failed markers, we attempt to identify critical factors enabling successful typing. The presented technique is very useful for low to medium sized SNP genotyping projects mostly applied in pharmacogenomic research as well as in clinical diagnostics. The main advantages include low cost, simple setup and validation of SNP markers.

Electrophoresis, Capillary↗

Identification of mutations from phenotype-driven ENU mutagenesis in mouse chromosome 7.

We have used the new high-throughput mutation-scanning technique temperature-gradient capillary electrophoresis (TGCE) for the identification of point mutations induced by N-ethyl-N-nitrosourea (ENU) in the mouse genome. TGCE detects the presence of heteroduplex molecules formed between a wild-type gene segment and the corresponding homologous segment containing an induced mutation or a naturally occurring single nucleotide polymorphism (SNP). Partially denatured heteroduplex molecules are resolved from homoduplexes by virtue of their differential mobilities during capillary electrophoresis conducted in a finely controlled temperature gradient. Simultaneous heteroduplex analysis of 96 amplicons ranging from 150 to 600 bp in size is achieved in approximately 45 min without the need for predetermining the melting profile of each fragment. Initially, we exploited known mouse mutations to develop TGCE protocols for analyzing unpurified PCR samples amplified from crude tail-DNA preparations. TGCE was then applied to the rapid identification of three new ENU-induced mutations recovered from regional mutagenesis screens of a segment of mouse Chromosome 7. Enzyme assays and quantitative reverse transcription-PCR (qRT-PCR) methods validated these new mutations. Our data demonstrate that rapid mutation scanning with TGCE, followed by sequence verification only of detected positives, is an efficient approach to the identification of point mutations in the mouse genome.

Animals↗

A novel mechanism for upregulation of the Escherichia coli K-12 hmp (flavohaemoglobin) gene by the 'NO releaser', S-nitrosoglutathione: nitrosation of homocysteine and modulation of MetR binding to the glyA-hmp intergenic region.

The flavohaemoglobin gene, hmp, of Escherichia coli is upregulated by nitric oxide (NO) in a SoxRS-independent manner. We now show that hmp expression is also upregulated by S-nitrosoglutathione (GSNO, widely used as an NO releaser) and sodium nitroprusside (SNP, which is a NO+ donor). Elevated homocysteine (Hcy) levels, achieved either by adding Hcy extracellularly or using metE mutants, decreased hmp expression. Conversely, metC mutants (defective in Hcy synthesis) had higher levels of hmp expression. Mutations in metR abolished hmp induction by GSNO and SNP, and hmp expression became insensitive to Hcy. We propose that the previously documented modulation by Hcy of MetR binding to the glyA-hmp intergenic regulatory region regulates hmp transcription. Although two MetR binding sites are present in this region, only the higher affinity site proximal to hmp is required for hmp induction by GSNO and SNP. GSNO and SNP react with Hcy in vitro under physiologically relevant conditions of pH and temperature generating S-nitrosohomocysteine, although in the latter case this would be co-ordinated to the Fe in SNP as a stable species. The free S-nitrosocysteine generated in the reaction with GSNO breaks down to release NO more readily than via homolysis of GSNO. As GSNO and SNP upregulate hmp similarly, the NO released in the former case on reaction with homocysteine cannot be involved in hmp regulation.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

Independent mutational events are rare in the ATM gene: haplotype prescreening enhances mutation detection rate.

Mutations in the ATM gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). Many different mutations have been identified using various techniques, with detection efficiencies ranging from 57 to 85%. In this study, we employed short tandem repeat (STR) haplotypes to enhance mutation identification in 55 unrelated A-T families of Iberian origin (20 Spanish, 17 Brazilian, and 18 Hispanic-American); we were able to identify 95% of the expected mutations. Allelic sizes were standardized based on a reference sample (CEPH 1347-2). Subsequent mutation screening was performed by PTT, SSCP, and DHPLC, and abnormal regions were sequenced. Many STR haplotypes were found within each population and six haplotypes were observed across several of these populations. Single nucleotide polymorphism (SNP) haplotypes further suggested that most of these common mutations are ancestrally related, and not hot spots. However, two mutations (8977C>T and 8264_8268delATAAG) may indeed be recurring mutational events. Common haplotypes were present in 13 of 20 Spanish A-T families (65%), in 11 of 17 Brazilian A-T families (65%), and, in contrast, in only eight of 18 Hispanic-American families (44%). Three mutations were identified that would be missed by conventional screening strategies. In all, 62 different mutations (28 not previously reported) were identified and their associated haplotypes defined, thereby establishing a new database for Iberian A-T families, and extending the spectrum of worldwide ATM mutations.

Ataxia Telangiectasia↗

Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression.

Dysregulation of central serotonin neurotransmission has been widely suspected as an important contributor to major depression. Here, we identify a (G1463A) single nucleotide polymorphism (SNP) in the rate-limiting enzyme of neuronal serotonin synthesis, human tryptophan hydroxylase-2 (hTPH2). The functional SNP in hTPH2 replaces the highly conserved Arg441 with His, which results in approximately 80% loss of function in serotonin production when hTPH2 is expressed in PC12 cells. Strikingly, SNP analysis in a cohort of 87 patients with unipolar major depression revealed that nine patients carried the mutant (1463A) allele, while among 219 controls, three subjects carried this mutation. In addition, this functional SNP was not found in a cohort of 60 bipolar disorder patients. Identification of a loss-of-function mutation in hTPH2 suggests that defect in brain serotonin synthesis may represent an important risk factor for unipolar major depression.

Adult↗

Intraspecific nucleotide variation in Anopheles gambiae: new insights into the biology of malaria vectors.

The Anopheles gambiae genome sequence, together with the recent development of molecular tools for genome-wide analysis, promises new insights into the biology of the malaria vector. These insights should help define the best possible breakdown point for interrupting transmission in the mosquito vector. A survey of the intraspecific nucleotide diversity in coding regions of three different mosquito strains showed an average of one single nucleotide polymorphism (SNP) every 125 coding base pairs. High levels of nucleotide polymorphism were observed in mosquito immune-related genes and pathogen recognition receptors harbored higher replacement substitutions. Genotyping at SNP loci in natural populations of An. gambiae from three malaria foci showed contrasting patterns. The distribution of mutation Y443H in the thioester-containing protein 3 (TEP3) gene suggested this mutational event has occurred under selective constraints. Our results show that SNP-based studies will be valuable in identifying the sequence variation associated with phenotypic traits shaping vector competence.

Animals↗