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PubMed2005

novoSNP, a novel computational tool for sequence variation discovery.

Technological improvements shifted sequencing from low-throughput, work-intensive, gel-based systems to high-throughput capillary systems. This resulted in a broad use of genomic resequencing to identify sequence variations in genes and regulatory, as well as extended genomic regions. We describe a software package, novoSNP, that conscientiously discovers single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (INDELs) in sequence trace files in a fast, reliable, and user-friendly way. We compared the performance of novoSNP with that of PolyPhred and PolyBayes on two data sets. The first data set comprised 1028 sequence trace files obtained from diagnostic mutation analyses of SCN1A (neuronal voltage-gated sodium channel alpha-subunit type I gene). The second data set comprised 9062 sequence trace files from a genomic resequencing project aiming at the construction of a high-density SNP map of MAPT (microtubule-associated protein tau gene). Visual inspection of these data sets had identified 38 sequence variations for SCN1A and 488 for MAPT. novoSNP automatically identified all 38 SCN1A variations including five INDELs, while for MAPT only 15 of the 488 variations were not correctly marked. PolyPhred detected far fewer SNPs as compared to novoSNP and missed nearly all INDELs. PolyBayes, designed for the sequence analysis of cloned templates, detected only a limited number of the variations present in the data set. Besides the significant improvement in the automated detection of sequence variations both in diagnostic mutation analyses and in SNP discovery projects, novoSNP also offers a user-friendly interface for inspecting possible genetic variations.

Animals↗
PubMed2006

Glucocerebrosidase mutations are not found in association with LRRK2 G2019S in subjects with parkinsonism.

Alteration G2019S in the leucine-rich repeat kinase 2 gene (LRRK2) has been identified in several populations of patients with parkinsonism, including Ashkenazi Jewish subjects with Parkinson disease. Mutations in glucocerebrosidase (GBA), the enzyme deficient in Gaucher disease, are also identified at an increased frequency among Parkinson probands, including those of Ashkenazi Jewish ancestry. A Taqman Assay-by-Design SNP genotyping strategy was utilized to establish whether G2019S was found in association with GBA mutations. Among 37 subjects with parkinsonism who were heterozygous for a GBA mutation, none carried G2019S. Furthermore, G2019S was not found in 18 patients with Gaucher disease who developed parkinsonian manifestations and 11 other Gaucher probands with parkinsonism in a first degree relative. Among 45 patients with Gaucher disease without a history of parkinsonism, one G2019S carrier was found. These findings suggest that GBA and LRRK2 mutations are discrete risk factors for parkinsonism in both Ashkenazi Jewish and non-Jewish subjects.

Amino Acid Substitution↗
PubMed2002

Translocation of telokin by cGMP signaling in smooth muscle cells.

Telokin is an acidic protein with a sequence identical to the COOH-terminal domain of myosin light chain kinase (MLCK) produced by an alternate promoter of the MLCK gene. Although it is abundantly expressed in smooth muscle, its physiological function is not understood. In the present study, we attempted to clarify the function of telokin by analyzing its spatial and temporal localization in living single smooth muscle cells. Primary cultured smooth muscle cells were transfected with green fluorescent protein (GFP)-tagged telokin. The telokin-GFP localized mostly diffusely in cytosol. Stimulation with both sodium nitroprusside (SNP) and 8-bromo-cyclic GMP induced translocation of GFP-tagged telokin to near plasma membrane in living single smooth muscle cells. The translocation was slow, and it took more than 10 min at room temperature. Mutation of the phosphorylation sites of telokin (S13A, S19A, and S13A/S19A) significantly attenuated SNP-induced translocation. Both KT-5823 (cGMP-dependent protein kinase inhibitor) and PD-98059 (mitogen-activated protein kinase inhibitor) diminished the telokin-GFP translocation. These results suggest that telokin changes its intracellular localization because of phosphorylation at Ser13 and/or Ser19 via the cGMP-signaling pathway.

Animals↗
PubMed2002

Novel mutations and SNPs identified in CCR2 using a new comprehensive denaturing gradient gel electrophoresis assay.

A single nucleotide polymorphism (SNP) at codon 64 in the CC chemokine receptor 2 gene (CCR2 V64I) has been associated with a dominant effect of delaying disease progression from human immunodeficiency virus-1 (HIV-1) infection to acquired immunodeficiency syndrome (AIDS). The objective of our study was to design a comprehensive mutation detection assay for the entire coding region of the CCR2A and CCR2B gene transcripts, including all relevant splice site junctions and to identify novel mutations and SNPs within our predominantly African-based population, which could influence an individual's susceptibility to HIV-1 infection and/or progression to AIDS. The mutation detection assay, based on denaturing gradient gel electrophoresis (DGGE), allowed for the complete analysis of five individuals per denaturing gel. Our study cohort consisted of 102 HIV seropositive patients and 144 HIV seronegative controls from the diverse South African population. Application of the CCR2-DGGE assay resulted in the detection of two previously reported CCR2 polymorphisms, namely CCR2 V64I and CCR2 N260N, and 11 novel mutations, including seven SNPs occurring at high allelic frequencies within specific population groups of South Africa. The large number of novel mutations/SNPs identified, using the CCR2-DGGE assay, indicates the importance for comprehensive analysis of all candidate genes in host susceptibility to HIV-1 infection, specifically in the under-studied African-based populations.

Amino Acid Substitution↗
PubMed2003

Design of base-discriminating fluorescent nucleoside and its application to t/c SNP typing.

We report a novel method for base detection using a base-discriminating fluorescent (BDF) nucleoside. We developed BDF probes containing methoxybenzodeazaadenine MDA and methoxybenzodeazainosine MDI, which give strong fluorescence only when the base on the complementary strand is cytosine and thymine, respectively. Thus, the MDA- and MDI-containing ODNs can be used as a very effective BDF probe for the detection of single base alterations, such as SNPs and point mutations. The present method using BDF probes is a very powerful tool for SNP typing that does not require any enzymes and time-consuming steps, and can avoid hybridization errors. In addition, a combination of MDA- and MDI-containing BDF probes facilitates the T/C SNP typing of a heterozygous sample.

Adenine↗
PubMed2002

Novel mutations of the CYP2A6 gene in a Thai population with lowered capacity of coumarin 7-hydroxylation.

We explored genetic polymorphisms in a Thai population which exhibited a low capacity to metabolize coumarin. The following two silent single nucleotide polymorphisms (SNPs) were found: 1) SNP, 020228Kiyotani001; GENE NAME, CYP2A6; ACCESSION NUMBER, NT_011139; LENGTH, 25 base; 5'-AAACTACCTGCAG/TCTGAACACAGAG-3'. 2) SNP, 020228Kiyotani002; GENE NAME, CYP2A6; ACCESSION NUMBER, NT_011139; LENGTH, 25 base; 5'-AATCCCCAGCAC/TTTCCTGAATGAG-3'. These two mutations (G144A and C1245T), which were located in exon 1 and exon 8 of the CYP2A6 gene, were found in two subjects among nine poor metabolizers for coumarin.

Journal Article↗
PubMed2005

The use of real-time PCR methods in DNA sequence variation analysis.

BACKGROUND: Real-time (RT) PCR methods for discovering and genotyping single nucleotide polymorphisms (SNPs) are becoming increasingly important in various fields of biological sciences. SNP genotyping is widely used to perform genetic association studies aimed at characterising the genetic factors underlying inherited traits. The detection and quantification of somatic mutations is an important tool for investigating the genetic causes of tumorigenesis. In infectious disease diagnostics there is an increasing emphasis placed on genotyping variation within the genomes of pathogenic organisms in order to distinguish between strains. METHODS: There are several platforms and methods available to the researcher wishing to undertake SNP analysis using real-time PCR methods. These use fluorescent technologies for discriminating between the alternate alleles of a polymorphism. There are several real-time PCR platforms currently on the market. Two of the key technical challenges are allele discrimination and allele quantification. CONCLUSIONS: Applications of this technology include SNP genotyping, the sensitive detection of somatic mutations and infectious disease subtyping.

Animals↗
PubMed2002

SNP genotyping with fluorescence polarization detection.

When a fluorescent molecule is excited by plane polarized light, the fluorescence emitted is also polarized. The degree of fluorescence polarization (FP) detected, under constant temperature and solvent viscosity, is proportional to the molecular weight of the dye molecule. By monitoring the FP of a fluorescent dye, one can detect significant changes in the molecular weight of the molecule without separation or purification. Because the size of the probe is altered in the course of a number of single nucleotide polymorphism (SNP) genotyping reactions, FP is therefore an excellent detection mechanism for these assays. Indeed, FP detection can be used in SNP genotyping with the primer extension TaqMan((R)) and Invader((R)) assays. Use of FP detection makes it possible to reduce the cost of TaqMan((R)) and Invader((R)) probes by abrogating the need for a fluorescence quencher. Moreover, inexpensive, unpurified, and unlabeled probes are used in the primer extension reaction with FP detection. As an end-point detection mechanism, FP detection is suitable for high-throughput SNP genotyping.

DNA Mutational Analysis↗
PubMed2003

On the power to detect SNP/phenotype association in candidate quantitative trait loci genomic regions: a simulation study.

We use coalescent methods to investigate the ability of linked neutral "markers" to reveal in simulated population samples the presence of one or more single nucleotide polymorphisms that is contributing to a trait having a complex genetic basis (QTN: quantitative trait nucleotide). Realistic mutation and recombination rates in our simulations allow us to generate SNP data appropriate for analyzing human variation across short chromosomal intervals corresponding to approximately 100 kilobases. We investigate the performance of both single marker and multiple-marker (haplotype) data for several ad hoc procedures. Our results with single SNP markers indicate that (1) the density of SNP markers need not be much higher than 10% in order to achieve near-maximal detection of a QTN; (2) a higher density of markers does not improve much on the ability to localize a QTN within an interval unless the recombination rate is high. Haplotype-based tests were investigated for the case in which more than one QTN is present in the studied interval. Larger sample sizes improve both the probability of detecting the haplotype with the largest number of QTNs, as well as the ability to infer correct haplotypes from genotypic data. Testing a series of short haplotypes across a longer interval can also be beneficial. The rate of false positives (i.e., when the most significant haplotype does not contain the greatest number of QTNs in the sample) can be very high when the contribution of individual QTNs to a trait is small. The elimination of low-frequency haplotypes does not substantially reduce the probability of detecting the haplotype with the largest number of QTNs but it can reduce the rate of false positives.

Computational Biology↗
PubMed2005

Gene SNPs and mutations in clinical genetic testing: haplotype-based testing and analysis.

Haplotype-based analysis using high-density single nucleotide polymorphism (SNP) markers have gained increasing attention in evaluating candidate genes in various clinical situations. For example, haplotype information is useful for predicting the severity and prognosis of certain genetic disorders. The intragenic cis-interactions between the common polymorphisms and the pathogenic mutations of prion protein (PRNP) and cystic fibrosis transmembrane conductance regulator (CFTR) genes greatly influence the phenotypes and the disease penetrance of hereditary Creutzfeldt-Jakob disease and cystic fibrosis. Merits of haplotype study are more evident in the fine mapping of complex diseases and in identifying genetic variations that influence individual's response to drugs. Knowledge-based approaches and/or linkage analyses using SNP tagged haplotypes are effective tools in detecting genetic associations. For example, haplotype studies in the inflammatory bowel disease susceptibility loci revealed diverse cis and trans gene-gene interactions, which can affect the clinical outcomes. Although currently, we have very limited knowledge on haplotype-phenotypic characterizations of most genes, these examples demonstrate that increased understanding of the clinically relevant haplotypes will provide better results in the diagnosis and possibly in the treatment of both monogenic and polygenic diseases.

Amyloid↗
PubMed2003

Mutation detection in rice waxy mutants by PCR-RF-SSCP.

PCR-RF-SSCP (PRS), which combines cleaved amplified polymorphic sequence (CAPS) and single-strand conformation polymorphism (SSCP), is expected to be a useful technique for DNA polymorphism analysis. We evaluated the ability of PRS to detect single nucleotide polymorphism (SNP) using the Waxy gene, Wx, of rice, and subsequently were able to identify point mutations in wx mutant lines. The approximately 6-kb Wx gene was divided into five regions for PCR amplification. Two regions, in which most of the point mutations of the wx mutants have been identified, were amplified by PCR and cloned into a vector, and those clones containing SNPs produced as a result of the inherent inaccuracy of PCR were used for the evaluation of PRS. The efficiency of PRS in the detection of SNPs of these clones was over 70%. PRS analysis of the wx genes in 18 waxy mutants was carried out in the five regions using two different restriction endonucleases and two gel conditions, with and without glycerol. Of the 18 lines tested, 17 showed band patterns different from that of the wild type. Most of the mutations identified in this study were nucleotide changes in exons, which result in amino acid changes. One mutation generated an in-frame stop codon, and another was a frame shift mutation by one-base deletion. Two mutations found at a splice site were considered to inhibit normal splicing of mRNA. These results show that PRS is a useful technique for detecting point mutations in large plant genes.

Chromosome Mapping↗
PubMed2005

Parallel minisequencing followed by multiplex matrix-assisted laser desorption/ionization mass spectrometry assay for beta-thalassemia mutations.

Beta-thalassemia is a common monogenic disease caused by mutations in the human beta-globin gene (HBB), many of which are differentially represented in human subpopulations stratified by ethnicity. This study describes an efficient and highly accurate method to screen for the eight most-common disease-causing mutations, covering more than 98% of HBB alleles in the Taiwanese population, using parallel minisequencing and multiplex assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS was optimized for sensitivity and resolution by "mass tuning" the PinPoint assay for eight HBB SNPs. Because of the close proximity and clustering of mutations in HBB, primer extension reactions were conducted in parallel. Efficient sequential desalting using POROS and cationic exchange chromatography allowed for an unambiguous multiplex genotyping by MALDI-TOF MS. The embellishing SNP assay allowed for highly accurate identification of the eight most-common beta-thalassemia mutations in homozygous normal control, carrier, and eight heterozygous carrier mixtures, as well as the diagnosis of a high-risk family. The results demonstrated a flexible strategy for rapid identification of clustering SNPs in HBB with a high degree of accuracy and specificity. It can be adapted easily for high-throughput diagnosis of various hereditary diseases or to establish family heritage databases for clinical applications.

Base Sequence↗
PubMed2004

RNAi of COL1A1 in mesenchymal progenitor cells.

Given that mutant COL1A1 is known to cause Osteogenesis Imperfecta (OI), tools to modulate COL1A1 expression are likely to be of significant therapeutic value. In this context, we have evaluated RNA interference (RNAi) as a means to downregulate COL1A1 expression in Cos-7 cells and in human mesenchymal progenitor stem cells (MPCs), the latter cells giving rise to bone and therefore representing a target cell type for collagen-related disorders. In addition, allele-specificity, a key factor to the success of RNAi-based suppression, was explored with a view to developing a mutation-independent RNAi-based therapeutic for OI by targeting an intragenic SNP within transcripts derived from the COL1A1 gene. Preferential suppression of individual polymorphic alleles that differed by a single nucleotide was observed.

Alleles↗
PubMed2003

Phenotype- and gene-driven approaches to discovering the functions of mammalian genes.

All of us are involved in discovery science as we pursue the genes, networks, cellular processes and biophysical principles that govern our chosen biological question. For those of us who choose to proceed using plant or animal models to dissect the elements of our favorite biological system, there are many classical and newer approaches available for our use, including two complementary strategies by which the discovery process is proceeding at the Oak Ridge National Laboratory (ORNL). The ORNL has been known for six decades for its investigations of the effects of radiation and chemicals in inducing heritable mutations in mouse germ cells, and for using mouse mutations as tools for the cloning and characterization of mammalian genes. Our history and experience in making mouse models are being applied via these two complementary strategies: 1), a phenotype-driven approach, in which mice carrying random chemically-induced mutations are screened for abnormal phenotypes; and 2) a gene-driven approach in which heritable single nucleotide polymorphisms (SNP) in preselected genes already thought likely to influence a biological system of choice can be recovered in live mice. The SNP-carrying mice can then be phenotyped for alterations in one's target biology. Both approaches have value and are necessary; while we can use mutations in genes that we already know to be of interest in our favorite biology to discover gene function, we also know that biology is full of surprise genes whose effects on our favorite biology would not be predicted and which will be identified only through phenotype screening.

Animals↗
PubMed2004

Bovine mu-calpain (CAPN1) gene: new SNP within intron 14.

The calpain system originally comprised molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. This proteolytic system plays a key role in the tenderisation process that occurs during post-mortem storage of meat under refrigerated conditioning. Their polymorphism is examined from the point of view of their effect on corresponding production traits. The calpain genes are investigated as potential candidate genes for a quantitative trait locus (QTL) affecting meat tenderness. In this study a new single nucleotide polymorphism (SNP) was found within intron 14 of the bovine CAPN1 gene, being transition C --> T at position 4685 nt (consensus sequence - GenBank No. AF 248054), as this mutation creates a new FokI restriction site detected with PCR-RFLP analysis. This sequence fragment of the SNP position has already been deposited in the GenBank database under accession No. AY639597. The RFLP-FokI polymorphism was studied in 141 bulls of seven breeds, including the native Polish Red (PR, preserved), and Polish Black-and White (BW) breed. The frequency of alleles T and C varied between the breeds considered, the mean reaching 0.38 and 0.62, respectively. Associations between CAPN1/FokI gene polymorphism and meat production traits were studied in BW (n = 84) young bulls. In the animals of the TT genotype the lean share in valuable cuts (%) was found more favourable than in CC animals.

Animals↗
PubMed2005

[Relationship of the HSG gene single nucleotide polymorphism and hypertension].

OBJECTIVE: To investigate the single nucleotide polymorphism (SNP) of the novel hyperplasia suppressor gene (HSG) to uncover the relationship between HSG SNP and hypertension. METHODS: Totally, 74 normotensive people (38 men and 36 women), 51 patients with essential hypertension (27 men and 24 women) and 20 hypertensive patients with family history of essential hypertension (9 men and 11 women) were chosen, with mean ages of (54 +/- 8) years, (57 +/- 8) years and (38 +/- 22) years, respectively. Peripheral venous blood specimen was collected from each of them and then DNA was extracted. The right primers were designed for DNA amplification with PCR. Each of the PCR-products from different groups was sequenced by ABI PRISM 377-DNA sequencer and their base components and characteristics of the same fragment were compared each other. RESULTS: Blood levels of creatinine (CRE) and urea nitrogen (BUN) were significantly higher in the hypertensives than in the normotensives (P < 0.01). Systolic and diastolic blood pressures were significantly higher in the hypertensives and the hypertensives with family history of essential hypertension than in the normotensives (P < 0.01 and P < 0.05). There existed three kinds of SNP in the HSG 12th intron (1q82139 G/A, 82153C/G and 82273G/-), and there was significant difference in 1q82153C/G and 82273G/- SNP between the hypertensives and normotensives (P < 0.05 and P < 0.01) and between the hypertensives with family history and the normotensives (P < 0.01 and P < 0.01). And, the similar difference in C/G allele and G deletion could be found in different populations. Moreover, the CC genetype of 1q82153 was common in the population (P < 0.01) and G deletion was more common in Chinese Han people with family history of essential hypertension. There was no significant difference existed in 1q82139 G/A mutation among the three groups. CONCLUSIONS: Measurements of renal function indicators (CRE and BUN) could probably reflect earlier advance of hypertension and damage to target organs. There existed three kinds of mutation in the 12th intron of the HSG 1q82139 G/A, 82153 C/G and 82273 G/-. The 1q82139 G/A could be a nonsense mutation and there was significant difference in the 1q82153 C/G and 1q82273 G/- SNP and gene frequencies between different Chinese Han populations, which could be independent risk factors for essential hypertension.

Adult↗
PubMed2005

Comparative genomics and gene expression analysis identifies BBS9, a new Bardet-Biedl syndrome gene.

Bardet-Biedl syndrome (BBS) is an autosomal recessive, genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS genes representing all known mapped loci have been identified. Mutation analysis of the known BBS genes in BBS patients indicate that additional BBS genes exist and/or that unidentified mutations exist in the known genes. To identify new BBS genes, we performed homozygosity mapping of small, consanguineous BBS pedigrees, using moderately dense SNP arrays. A bioinformatics approach combining comparative genomic analysis and gene expression studies of a BBS-knockout mouse model was used to prioritize BBS candidate genes within the newly identified loci for mutation screening. By use of this strategy, parathyroid hormone-responsive gene B1 (B1) was found to be a novel BBS gene (BBS9), supported by the identification of homozygous mutations in BBS patients. The identification of BBS9 illustrates the power of using a combination of comparative genomic analysis, gene expression studies, and homozygosity mapping with SNP arrays in small, consanguineous families for the identification of rare autosomal recessive disorders. We also demonstrate that small, consanguineous families are useful in identifying intragenic deletions. This type of mutation is likely to be underreported because of the difficulty of deletion detection in the heterozygous state by the mutation screening methods that are used in many studies.

Alleles↗
PubMed2005

Polymorphisms in exon 13 and intron 14 of the RET protooncogene: genetic modifiers of medullary thyroid carcinoma?

CONTEXT: Single-nucleotide polymorphisms (SNPs) of the RET protooncogene (RET) could modify disease susceptibility and clinical phenotype in patients with sporadic or familial medullary thyroid carcinoma (FMTC). OBJECTIVE/DESIGN OF THE STUDY: Because frequencies of RET SNPs have not yet been evaluated in patients with elevated serum concentrations of calcitonin (hCt), a biochemical marker for medullary thyroid carcinoma (MTC), we studied RET SNPs in patients with FMTC (n = 22), patients with sporadic MTC (n = 45), and 71 subjects presenting with moderately elevated hCt concentrations (basal, >10 pg/ml; pentagastrin stimulated, > 50 < 100 pg/ml) in comparison with an age- and gender-matched control group (n = 79) with basal hCt concentrations in the normal range (<5 pg/ml). METHODS: After DNA extraction from citrated whole blood, RET exons 10, 11, 13, 14, 15, and 16 and exon/intron boundaries were analyzed by PCR-based cycle sequencing for RET germ line mutations, exonic (G691S, L769L, S836S, S904S) and intronic (IVS13+158; NCBI rs2472737 = IVS14-24) SNPs. RESULTS: In FMTC patients, the F791Y mutation was found to be associated (P = 0.001) with the L769L SNP. The exonic SNPs (G691S, L769L, S836S, and S904S) were not different among the four groups. The intron 14 SNP (IVS14-24), however, was more frequent in individuals with elevated hCt serum concentrations (P = 0.016) and patients with sporadic MTC (P < 0.001) when compared with the control group. CONCLUSIONS: These data suggest that the exon 13 (L769L) and the intron 14 (IVS14-24) SNPs could act as genetic modifiers in the development of some forms of hereditary and sporadic MTC, respectively.

Adult↗