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PubMed2005

The case of the unreliable SNP: recurrent back-mutation of Y-chromosomal marker P25 through gene conversion.

The Y-chromosomal binary marker P25 is a paralogous sequence variant, rather than a SNP: three copies of the P25 sequence lie within the giant palindromic repeats on Yq, and one copy has undergone a C to A transversion to define haplogroup R1b (designated C/C/A). Since gene conversion is known to be active in the palindromic repeats, we reasoned that P25 might be liable to back-mutation by gene conversion, yielding the ancestral state C/C/C. Through analysis of a set of binary markers in Y-chromosomes in two large samples from Great Britain and the Iberian Peninsula we show that such conversion events have occurred at least twice, and provide preliminary evidence that the reverse conversion event (yielding C/A/A) has also occurred. Because of its inherent instability, we suggest that P25 be used with caution in forensic studies, and perhaps replaced with the more reliable binary marker M269.

Chromosomes, Human, Y↗
PubMed2001

Molecular genetic analysis of appendiceal mucinous adenomas in identical twins, including one with pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is a clinical syndrome characterized by mucinous ascites and peritoneal lesions composed of histologically bland to low-grade adenomatous mucinous epithelium within pools of extracellular mucin, often with an associated mucinous adenoma of the appendix. There is evidence that the peritoneal lesions in PMP are clonally derived from the associated appendiceal adenoma. Little is known about the molecular genetic alterations or hereditary factors involved in the development of appendiceal mucinous tumors and PMP. We report the only known example of appendiceal mucinous adenomas in identical twin brothers, one of whom developed PMP. We analyzed the status of the K-RAS and APC genes in these tumors using digital polymerase chain reaction and digital single nucleotide polymorphism (SNP) assay. Identical K-RAS mutations were detected in the appendiceal adenoma and peritoneal tumor from the twin with PMP, whereas the adenoma from the other twin harbored a different mutation. Digital SNP analysis demonstrated loss of heterozygosity of APC only in the adenoma from the twin without PMP but not from the appendiceal or peritoneal tumors of the twin with PMP. The adjacent normal tissue in each case retained both APC alleles. The K-RAS mutational analysis supports the view that PMP is clonally derived from the associated appendiceal mucinous adenoma. The lack of loss of heterozygosity of APC in the adenoma and peritoneal tumor from the twin with PMP suggests that loss of heterozygosity of APC is not necessarily involved in the development of all appendiceal adenomas or PMP. The different types of mutations in K-RAS and the different allelic status of the APC locus in the tumors from both twins suggest that mutation in K-RAS and loss of heterozygosity of APC occurs somatically in adenomas and is independent of the identical genetic background of the twins.

Adult↗
PubMed2006

Truncated gamma-glutamyl carboxylase in rambouillet sheep.

A flock of Rambouillet sheep was examined because of increased lamb mortality due to ineffective hemostasis at parturition. Decreased activities of coagulation factors II, VII, IX, and X, and severely reduced hepatic gamma-glutamyl carboxylase activity with adequate vitamin K 2,3 epoxide reductase activity was determined.(1,)(21) Parenteral vitamin K(1) supplementation did not improve vitamin K-dependent coagulation factor activities in 3 affected lambs. Affected lamb gamma-glutamyl carboxylase deoxyribonucleic acid was sequenced, and 4 single nucleotide polymorphisms (SNPs 2-5) of the gamma-glutamyl carboxylase gene were identified. Single nucleotide polymorphism-4 results in an arginine to stop codon (UGA) substitution, which prematurely terminates the peptide at residue 686 (R686Stop). This genotype (GATT/GATT) has a strong association with the coagulopathy observed in clinically affected lambs, P < 0.001. The frequency of SNP-3 in exon 11 (R486H) within the MARC 1.1 database is high in the US sheep population overall. Gamma-glutamyl carboxylase activity in hepatic microsomes from a SNP-3 homozygous lamb lacking the SNP-4 mutation (GACC/GACC) was similar to control sheep homozygous for arginine at 486 and also lacking SNP-4 (TGCC/TGCC), indicating that the R486H does not measurably impact gamma-glutamyl carboxylase activity. The remaining two SNPs (2 and 5) are located within non-coding intron sequences. These 4 SNPs allowed for determining the genotype associated with the observed fatal coagulopathy. Screening for the premature truncation (SNP-4) based on the presence of a Bbv I restriction site in clinically normal lambs but not in the homozygous affected lambs allows for detection of the heterozygous state (GATT/GACC), because carrier animals are clinically normal.

Animals↗
PubMed2004

High sensitive approach for point mutation detection based on electrochemiluminescence.

An electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for point mutation detection has been developed. The target is amplified using a tris (bipyridine) ruthenium (TBR)-labeled forward and a biotinylated reverse primer. The amplification products are digested with specific restriction enzyme, then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. The established technique was further applied to detect a specific point mutation in H-ras oncogene in T24 cell line. The results show that the system has a low detection limit of 100 fmol and a linear range of more than 3 orders of magnitude for H-ras amplicon; the two genotypes can be reliably discriminated. In summary, the mutant specific ECL-PCR method can be used to detect a point mutation that creates or destroys a restriction site in any gene. It is useful in single nucleotide polymorphism (SNP) and mutation detection due to its safety, high sensitivity and simplicity.

Biomarkers, Tumor↗
PubMed2004

Polymorphism in wild-type p53 modulates response to chemotherapy in vitro and in vivo.

A single-nucleotide polymorphism (SNP) in exon 4 results in expression of either arginine (72R) or proline (72P) at codon 72 of p53. We demonstrate that the in vitro response of cells exposed to anticancer agents is strongly influenced by this SNP in wild-type p53. In inducible systems and in cells expressing the endogenous protein, expression of 72P wild-type p53 results in a predominant G1 arrest, with only a minor apoptosis, at drug concentrations causing extensive apoptosis in cells expressing the 72R wild-type variant. The superior apoptosis-inducing activity of the 72R form correlates with more efficient induction of specific apoptosis-associated genes, and is maximal in the presence of serine 46 (S46). In vivo, the outcome of chemo-radiotherapy of squamous carcinomas is more favourable in cancers retaining a wild-type 72R allele, such cases having higher response rates and longer survival than those with wild-type 72P. Together, these results reveal that this SNP is an important determinant of response to anticancer agents in cells expressing wild-type p53. Analysis of complete p53 genotype (mutation and SNP) merits detailed investigation as a simple means for prediction of treatment response and survival in clinical oncology.

Antineoplastic Agents↗
PubMed2005

InSNP: a tool for automated detection and visualization of SNPs and InDels.

Availability of high quality SNP data is a rate-limiting factor in understanding the impact of genetic variability on gene function and phenotype. Although global projects like HAPMAP generate large numbers of SNPs in an even spacing throughout the human genome, many variation studies have a more focused approach: in the follow-up of positional association findings, candidate gene studies, and functional genomics experiments, knowledge of all variations in a limited amount of sequence (e.g., a gene) is needed. This leads to a large number of resequencing experiments, for which there is a surprising lack of analysis software. We have thus developed specialized software (InSNP) for targeted mutation detection and compared its performance to Polyphred and Mutation Surveyor using 28 amplicons. Out of a total of 579 (InSNP), 644 (Polyphred), and 526 (Mutation Surveyor) SNP predictions, 39 SNPs were confirmed by human expert inspection, with five SNPs missed by Polyphred and one missed by InSNP using the default settings. For InDel detection, out of 70 (InSNP), 28 (Polyphred), and 693 (Mutation Surveyor) InDel predictions, two InDels were confirmed by human expert inspection, with one InDel missed by Polyphred. InSNP provides a user-friendly interface with better functionality for mutation detection than general-purpose sequence handling software. It provides similar SNP detection sensitivity and specificity as the public domain and commercial alternatives in the investigated dataset. We hope that InSNP lowers the barriers to the use of automated mutation detection software and aids in the improvement of the efficiency of such experiments. The Windows installer (setup) program and sample datasets are available at www.mucosa.de/insnp/.

Algorithms↗
PubMed2000

Signature-based analysis of MET proto-oncogene mutations using DHPLC.

Research tools which improve mutation detection, SNP discovery, and allele characterization will facilitate studies of cancer, inherited disease, and genomic evolution. Denaturing High-Performance Liquid Chromatography (DHPLC) is a recently developed methodology for detection of heteroduplexes formed in DNA samples containing mismatches between wild type and mutant strands. In an effort to develop a rapid, sensitive mutation detection method for studies of families with inherited kidney cancer, we evaluated DHPLC for detection and analysis of MET proto-oncogene mutations in papillary renal carcinomas (PRC). We found DHPLC to be 100% accurate in detecting 15 known disease-associated MET mutations. Significantly, each MET mutation and two novel SNPs generated a characteristic chromatographic profile or signature with reproducible distinguishing features. Standardization of DHPLC reagents and improved methods design were critical to the reliability and accuracy of mutation prediction. Improvements included addition of a 75% acetonitrile wash followed by a rejuvenating gradient, and detailed analysis of signature shape, retention time (RT), RT differences (DeltaRT), and temperature-dependent (melt) profiling. We used signatures to predict mutations in new PRC samples, mutation carriers in asymptomatic hereditary PRC family members, and in a blind study of previously characterized DNAs. Application to SNP discovery is discussed. Wiley-Liss, Inc.

Carcinoma, Renal Cell↗
PubMed2006

RAI1 point mutations, CAG repeat variation, and SNP analysis in non-deletion Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation disorder characterized by distinct craniofacial features and neurobehavioral abnormalities usually associated with an interstitial deletion in 17p11.2. Heterozygous point mutations in the retinoic acid induced 1 gene (RAI1) have been reported in nine SMS patients without a deletion detectable by fluorescent in situ hybridization (FISH), implicating RAI1 haploinsufficiency as the cause of the major clinical features in SMS. All of the reported point mutations are unique and de novo. RAI1 contains a polymorphic CAG repeat and encodes a plant homeo domain (PHD) zinc finger-containing transcriptional regulator. We report a novel RAI1 frameshift mutation, c.3103delC, in a non-deletion patient with many SMS features. The deletion of a single cytosine occurs in a heptameric C-tract (CCCCCCC), the longest mononucleotide repeat in the RAI1 coding region. Interestingly, we had previously reported a frameshift mutation, c.3103insC, in the same mononucleotide repeat. Furthermore, all five single base frameshift mutations preferentially occurred in polyC but not polyG tracts. We also investigated the distribution of the polymorphic CAG repeats in both the normal population and the SMS patients as one potential molecular mechanism for variability of clinical expression. In this limited data set, there was no significant association between the length of CAG repeats and the SMS phenotype. However, we identified a 5-year-old girl with an apparent SMS phenotype who was a compound heterozygote for an RAI1 missense mutation inherited from her father and a polyglutamine repeat of 18 copies, representing the largest known CAG repeat in this gene, inherited from her mother.

Abnormalities, Multiple↗
PubMed2005

Prevalent SLC26A4 mutations in patients with enlarged vestibular aqueduct and/or Mondini dysplasia: a unique spectrum of mutations in Taiwan, including a frequent founder mutation.

OBJECTIVES/HYPOTHESIS: The purpose of the study is to elucidate the mutation spectrum of SLC26A4 among patients with enlarged vestibular aqueduct and/or Mondini dysplasia in Taiwan and to explore the origin of the most common mutation, IVS7-2A>G. The correlation between the genotypes and the phenotypes is also investigated, with special emphasis placed on comparison between the genotypes and hearing levels. STUDY DESIGN: A 3-year prospective clinical genetic study at a tertiary care university hospital. METHOD: Mutations on SLC26A4 were screened in 38 families that fulfilled the criteria of enrollment, and single nucleotide polymorphisms (SNPs) in the vicinity of IVS7-2A>G were typed. The presence of goiter, radiologic findings, and audiologic results of the probands were then compared according to the genotypes. RESULTS: A total of eight mutations were detected in 33 families, and IVS7-2A>G accounted for 84% (48/57) of the mutated alleles. SNP analysis confirmed the founder effect of IVS7-2A>G. Meanwhile, no obvious correlation was observed between SLC26A4 genotypes and phenotypes. CONCLUSION: The present study disclosed the unique SLC26A4 mutation spectrum in Taiwan, confirmed that IVS7-2A>G arose from a common ancestor, and demonstrated the lack of correlation between genotypes and phenotypes. High prevalence of certain SLC26A4 mutations in East Asians, as revealed here and previously, might largely facilitate mutation screening and genetic counseling in these areas.

Cochlea↗
PubMed2005

Influence of endothelial nitric oxide synthase T-786C single nucleotide polymorphism on aneurysm size.

OBJECT: Among patients with aneurysms, those with heterozygous (T/C) endothelial nitric oxide synthase (eNOS) T-786C single nucleotide polymorphism (SNP), a mutation reducing endothelial nitric oxide synthesis, are reported to have larger ruptured intracranial aneurysms (IAs) than those with homozygous (C/C or T/T) genotype. The authors tested patients harboring aneurysms for eNOS T-786C SNP in two populations--Japanese and Korean. METHODS: The eNOS T-786C SNP was genotyped through direct sequencing in genomic DNA obtained from 336 Japanese and 191 Korean patients with lAs and 214 Japanese and 191 Korean control volunteers. Differences in genotype frequencies among the various aneurysm sizes were evaluated using the Fisher exact test. There was no significant difference in heterozygous (T/C) eNOS T-786C SNP between aneurysms 5 mm or smaller and those from 6 to 9 mm, and between lesions 5 mm or smaller and those 10 mm or larger in 336 Japanese patients harboring aneurysms--220 with ruptured and 116 with unruptured lesions--and in 191 Korean patients with ruptured aneurysms. CONCLUSION: The eNOS T-786C SNP genotype does not influence the size of aneurysms.

Aged↗
PubMed2000

Depletion of intracellular glutathione reduces mutations by nitric oxide-donating drugs.

The Mutatect system is a mouse tumor line in which mutations at the hypoxanthine phosphoribosyltransferase (Hprt) locus can be readily detected both in vitro and in vivo. We have previously shown that the nitric oxide-generating drugs, glyceryl trinitrate (GTN) and sodium nitroprusside (SNP), can induce mutations that are readily detected in these cells. In the present report, we have tested the effect of glutathione depletion by buthionine sulfoximine (BSO) on cytotoxicity and mutagenicity by these two drugs. Exposure for 24 h to either drug (123 microM GTN; 500 microM SNP) induced mutations with relatively little cytotoxicity. Pretreatment with 50 microM BSO for 24 h, and then removal at the time of GTN or SNP addition, enhanced cytotoxicity to a modest extent. However, mutagenicity induced by both GTN and SNP was largely abolished. BSO did not affect nitrite accumulation in the medium over a 24-h period, indicating no inhibition of bioactivation of GTN or SNP. Maintaining BSO in the medium for 24 h prior and throughout the period of exposure to GTN or SNP produced a similar effect on mutations. N-Acetylcysteine and oxothiazolidine-4-carboxylate, drugs that are used to increase intracellular glutathione, also blocked mutations. We postulate that a product of the reaction between nitric oxide and intracellular glutathione, such as GSNO or some species derived from it, is promutagenic.

Acetylcysteine↗
PubMed2025

Attomolar Detection of HIV-1 with Label-Free RCA-rCRISPR on Smartphone.

Human Immunodeficiency Virus-1 (HIV) remains a major global public health challenge, having led to over 42.3 million deaths since its discovery in the early 1980s. Despite progress in prevention and treatment, around 60% of people with HIV (PWH) remain undiagnosed in resource-limited regions, disproportionately affecting vulnerable populations and underserved communities across the world. This illustrates the critical need for accessible, accurate, and equipment-free diagnostic tools to enhance detection and thus provide opportunities to curb its spread. Here, we developed a low-cost, robust, and label-free rolling circle amplification (RCA)-rCRISPR diagnostic platform for detecting HIV viral load with minimal instrumentation. Our strategy, combining the integration of RNA-detecting RCA reaction with plasmid reporter-based ratiometric CRISPR (rCRISPR), enables sensitive detection of unprocessed RNA targets without the need for intensive sample pre-treatment. This label-free RCA-rCRISPR diagnostic platform detected HIV RNA down to single-digit aM sensitivity (~3000 copies/mL) from PWH-derived HIV samples ex vivo. Unlike typical RCA, which requires sample fragmentations to break long RNA target sequences, our design harnesses the triple functions of the phi29 DNA polymerase (namely exonuclease activity, polymerization, and strand displacement), enabling the detection of the entire HIV genome without pre-fragmentation. For point-of-care (POC) applications, we constructed an all-in-one smartphone-based minigel electrophoresis device to facilitate equipment-free HIV viral load testing, making it accessible to resource-limited communities. Additionally, the assay has demonstrated the ability for point mutation detection (BRAF mutation in canine urothelial carcinoma), showcasing the robustness of our strategy for broad disease diagnostic applications.

HIV↗
PubMed2004

Genetic study of the CD36 gene in a French diabetic population.

OBJECTIVES: CD36 is a multifunctional membrane receptor widely expressed in different tissues which binds and internalizes oxidized low-density lipoprotein. In rodents, CD36 gene variations modulate glucose homeostasis and contribute to metabolic syndrome associated with type 2 diabetes but the effects in human are unknown. METHODS: We screened the entire coding sequence of the CD36 gene in 272 individuals and we genotyped both rare and frequent variants in 454 T2D subjects and 221 controls. RESULTS: We detected five mutations, P191P and N247S were only found each in one family and did not segregate with diabetes, the three others (A/C-178 in the promoter, A/G-10 in intron 3 and (GGGTTGAGA) insertion in intron 13) being equally frequent in diabetic subjects and in controls. However, adiponectin levels, a marker for insulin sensitivity, were significantly associated with the -178 A/C promoter variant allele (p=0.003, p corrected for multiple testing=0.036), possibly reflecting association with insulin-resistance in the French population. CONCLUSION: Thus, the -178 A/C SNP promoter mutation in the CD36 gene represents a putative genetic marker for insulin-resistance in the French population, although it does not appear to contribute to the genetic risk for T2D.

Adiponectin↗
PubMed2004

Optimum sample medium for single-nucleotide polymorphism and mutation detection by capillary electrophoresis.

Capillary electrophoresis (CE) is a versatile analytical platform widely used for nucleic acids analysis. Its applications in research and diagnostics include scanning and screening for mutations and polymorphisms by such reliable methods as single-strand conformation polymorphism (SSCP), heteroduplex analysis (HA), and combined SSCP/HA. This study, aimed at the further development of these methods, is focused on detailed sample-media characteristics. Factors affecting single-strand conformer stability and DNA intake efficiency were analyzed. The sample media optimal for efficient mutation or SNP detection were determined, and complex SSCP-CE patterns arising from unpurified PCR products were explained. It turns out, that the nondenaturing aqueous media assure both efficient DNA intake, and single-strand conformers stability required for the SSCP and combined SSCP/HA. The results of this study are applicable to all these areas of biomedical research, in which capillary electrophoresis is used for the characterization of nucleic acids.

DNA↗
PubMed2002

GENOTRACE: cDNA-based local GENOme assembly from TRACE archives.

UNLABELLED: GENOTRACE identifies the genomic organization for a cDNA using raw data from genome sequencing projects in progress (trace archives). Local genomic contigs are generated, allowing for example the design of PCR primers in intronic sequences to amplify coding regions of a gene, needed for example for mutation or SNP detection. AVAILABILITY: The package and examples of output files can be downloaded from http://rat.niob.knaw.nl/GENOTRACE

Archives↗
PubMed2007

HMDB: the Human Metabolome Database.

The Human Metabolome Database (HMDB) is currently the most complete and comprehensive curated collection of human metabolite and human metabolism data in the world. It contains records for more than 2180 endogenous metabolites with information gathered from thousands of books, journal articles and electronic databases. In addition to its comprehensive literature-derived data, the HMDB also contains an extensive collection of experimental metabolite concentration data compiled from hundreds of mass spectra (MS) and Nuclear Magnetic resonance (NMR) metabolomic analyses performed on urine, blood and cerebrospinal fluid samples. This is further supplemented with thousands of NMR and MS spectra collected on purified, reference metabolites. Each metabolite entry in the HMDB contains an average of 90 separate data fields including a comprehensive compound description, names and synonyms, structural information, physico-chemical data, reference NMR and MS spectra, biofluid concentrations, disease associations, pathway information, enzyme data, gene sequence data, SNP and mutation data as well as extensive links to images, references and other public databases. Extensive searching, relational querying and data browsing tools are also provided. The HMDB is designed to address the broad needs of biochemists, clinical chemists, physicians, medical geneticists, nutritionists and members of the metabolomics community. The HMDB is available at: www.hmdb.ca.

Databases, Factual↗
PubMed2004

Hairpin primers for simplified single-nucleotide polymorphism analysis of Mycobacterium tuberculosis and other organisms.

We describe a novel, simple, rapid, and highly sensitive method to detect single-nucleotide polymorphisms (SNPs) in Mycobacterium tuberculosis and other organisms. Amplification refractory mutation (ARMS) SNP assays were modified by converting the SNP-detecting linear primers in the ARMS assay to hairpin-shaped primers (HPs) through the addition of a 5' tail complementary to the 3' end of the linear primer. The improved ability of these primers to detect SNPs in M. tuberculosis was compared in a real-time PCR with SYBR-I green dye. Linear primers resulted in incorrect or indeterminate allele designation for 6 of the 13 SNP alleles tested in seven different SNP assays, while HPs determined the correct SNP in all cases. We compared the cycle threshold differences (DeltaC(t)) between the reactions containing primer-template matches and the reactions containing primer-template mismatches (where a larger DeltaC(t) indicates a more robust assay). The use of HPs dramatically improved the mean DeltaC(t) values for the SNP assays (7.6 for linear primers and 11.2 for HPs). We designed 98 different HP assays for SNPs previously associated with resistance to the antibiotic isoniazid to test the large-scale utility of the HP approach. Assay design was successful in 72.4%, 83.7%, 88.8%, and 92.9% of the assays after one to four rounds of assay design, respectively. HP SNP assays are simple, sensitive, robust, and inexpensive. These advantages favor the application of this technique for SNP assays of M. tuberculosis and other organisms.

Base Sequence↗
PubMed2006

Two-temperature LATE-PCR endpoint genotyping.

BACKGROUND: In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. RESULTS: Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the same alleles. CONCLUSION: SNP genotyping via Two-Temperature LATE-PCR takes place in a homogeneous closed-tube format and uses a single hybridization probe per SNP site. These assays are convenient, rely on endpoint analysis, improve the options for construction of multiplex assays, and are suitable for SNP genotyping, mutation scanning, and detection of DNA duplication or deletions.

Alleles↗