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PubMedDate not supplied

Generation, characterization, and molecular cloning of the Noerg-1 mutation of rhodopsin in the mouse.

We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12-14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A non-recombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.

Amino Acid Substitution↗
PubMed2003

Sodium channels SCN1A, SCN2A and SCN3A in familial autism.

Autism is a psychiatric disorder with estimated heritability of 90%. One-third of autistic individuals experience seizures. A susceptibility locus for autism was mapped near a cluster of voltage-gated sodium channel genes on chromosome 2. Mutations in two of these genes, SCN1A and SCN2A, result in the seizure disorder GEFS+. To evaluate these sodium channel genes as candidates for the autism susceptibility locus, we screened for variation in coding exons and splice sites in 117 multiplex autism families. A total of 27 kb of coding sequence and 3 kb of intron sequence were screened. Only six families carried variants with potential effects on sodium channel function. Five coding variants and one lariat branchpoint mutation were each observed in a single family, but were not present in controls. The variant R1902C in SCN2A is located in the calmodulin binding site and was found to reduce binding affinity for calcium-bound calmodulin. R542Q in SCN1A was observed in one autism family and had previously been identified in a patient with juvenile myoclonic epilepsy. The effect of the lariat branchpoint mutation was tested in cultured lymphoblasts. Additional population studies and functional tests will be required to evaluate pathogenicity of the coding and lariat site variants. SNP density was 1/kb in the genomic sequence screened. We report 38 sodium channel SNPs that will be useful in future association and linkage studies.

Autistic Disorder↗
PubMed2005

Evaluation of the paraoxonases as candidate genes for stroke: Gln192Arg polymorphism in the paraoxonase 1 gene is associated with increased risk of stroke.

BACKGROUND AND PURPOSE: The paraoxonases are involved in protecting low-density lipoprotein (LDL) from lipid oxidation. Paraoxonase 1 (PON1) was implicated in susceptibility to coronary artery disease and stroke in previous studies. We evaluated, in a comprehensive way, all 3 paraoxonase genes for association with stroke observed in the Cholesterol and Recurrent Events (CARE) trial. METHODS: Over 2500 subjects enrolled in the CARE trial were genotyped for 14 single nucleotide polymorphisms, including 7 newly identified in this study, in the 3 paraoxonase genes. RESULTS: A glutamine (Gln)/arginine (Arg) polymorphism at amino acid residue 192 in PON1 was significantly associated with stroke (P=0.003 in multivariate analysis, including age, sex, LDL, hypertension, diabetes, smoking, and pravastatin treatment as covariates). The odds ratios were 2.28 (95% CI, 1.38 to 3.79) for Gln/Arg heterozygotes and 2.47 (95% CI, 1.18 to 5.19) for Arg/Arg homozygotes compared with Gln/Gln homozygotes. These results are consistent with 2 of 3 other published studies. In combined analysis of all 4 studies, the association between Gln192Arg SNP and stroke was highly significant (chi2(8df)=45.58, P<0.000001). Sequence analysis of the PON1 gene from seventy stroke cases revealed a novel nonsense mutation at codon 32 in one stroke case, which was not detected in over 2500 unaffected individuals. Polymorphisms in the PON2 and PON3 genes were not associated with stroke. CONCLUSIONS: These results suggest that Gln192Arg genotype is an important risk factor for stroke.

Arginine↗
PubMed2005

Rapid single nucleotide polymorphism mapping in C. elegans.

BACKGROUND: In C. elegans, single nucleotide polymorphisms (SNPs) can function as silent genetic markers, with applications ranging from classical two- and three-factor mapping to measuring recombination across whole chromosomes. RESULTS: Here, we describe a set of 48 primer pairs that flank SNPs evenly spaced across the C. elegans genome and that work under identical PCR conditions. Each SNP in this set alters a DraI site, enabling rapid and parallel scoring. We describe a procedure using these reagents to quickly and reliably map mutations. We show that these techniques correctly map a known gene, dpy-5. We then use these techniques to map mutations in an uncharacterized strain, and show that its behavioral phenotype can be simultaneously mapped to three loci. CONCLUSION: Together, the reagents and methods described represent a significant advance in the accurate, rapid and inexpensive mapping of genes in C. elegans.

Animals↗
PubMed2002

[Single nucleotide polymorphism analysis in chicken leptin receptor exon 9].

Leptin receptor is a type I cytokine super family member and plays an important role in leptin functioning signal transduction. This study was designed to investigate the single nucleotide polymorphism (SNP) of OBR gene in various breeds, including Fatness Line (FL), Leanness Line (LL), Beijing Youji, Baierji, Shiqiza, Dwarf Yellow Chickens, Mini Yellow Chickens, Huiyang Huxuji, Recessive White Chickens and Hyline Layer. The primers for exon 9 in OBR gene were designed from the database of chicken genomic sequence and the SNPs were detected by PCR-SSCP method. One SNP (C/A at 1167 in cds) was found among individuals within all breeds. However, the amino acid was not changed because it was a silence mutation. The result of population genetics analyses showed that the frequency of AA genotype in Beijing Youji was significantly higher than that in other lines. Also, the frequency of A allele in FL was significantly higher than that in LL.

English Abstract↗
PubMed2005

In B-CLL, the codon 72 polymorphic variants of p53 are not related to drug resistance and disease prognosis.

BACKGROUND: A common sequence polymorphism at codon 72 of the p53 gene encoding either arginine or proline was recently shown to be functionally relevant for apoptosis induction in vitro. In B-type chronic lymphocytic leukemia (B-CLL), p53 gene mutations occur in a subset of patients and are associated with impaired survival and drug resistance. Here, we address the functional relevance of the codon 72 single nucleotide (SNP) polymorphism for cell death sensitivity following exposure to clinically employed cytotoxic drugs and gamma-irradiation. METHODS: 138 B-CLL samples were analysed by SSCP-PCR and sequencing for single nucleotide polymorphism at codon 72 of the p53 gene. The in vitro cytotoxicity assay (DiSC-assay) was performed with 7 drugs (chlorambucil, mafosfamide, fludarabine phosphate, methylprednisolone, doxorubicin, vincristine) or gamma-irradiation. RESULTS: Of the 138 B-CLL samples, 9 samples were homozygous for proline (Pro/Pro), 78 samples homozygous for arginine (Arg/Arg), and 49 samples heterozygous (Arg/Pro). No differences were found for patient survival and cell death triggered by 7 cytotoxic drugs or gamma-irradiation. CONCLUSION: These data indicate that polymorphic variants of p53 codon 72 are not clinically relevant for apoptosis induction or patient survival in B-CLL.

Aged↗
PubMed2005

Mutation screening of a haplotype block around the insulin degrading enzyme gene and association with Alzheimer's disease.

Genetic and biological studies point to a role for insulin-degrading enzyme (IDE) in Alzheimer's disease (AD). Two SNP-based studies recently reported evidence for association with AD using markers in a approximately 270 kb haplotype block on chromosome 10q. This haplotype block region harbors three known genes; insulin-degrading enzyme (IDE), kinesin family member 11 (KIF11), and hematopoietically expressed homeobox (HHEX). In an attempt to search for susceptibility variants we have sequenced all coding exons, 2 kb of 5' and 3'-flanking sequence, and all regions showing a high degree of human-mouse conservation in these three genes in 30 individuals. We found a total of 40 single nucleotide polymorphisms and 8 insertion/deletion polymorphisms. No coding variants were identified in any of the three genes. Nine polymorphisms in IDE and four polymorphisms in KIF11 situated in conserved regions or near coding exons were subsequently genotyped in a set of AD cases and controls. Two markers in KIF11 yielded borderline significant results in the ApoE4 non-carrier subgroup, but the results were otherwise not significant in this small set of samples. This study of multiple new markers in the region will facilitate further association studies in this important AD region.

Alzheimer Disease↗
PubMed2006

Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension-application to the variants of mannose-binding lectin gene.

Mannose-binding lectin (MBL) is a key component of the innate immune system, and its deficiency is associated with increased susceptibility to various infections and autoimmune disorders. Since several nucleotide variations in the mannose-binding lectin 2 gene (MBL2) have been associated with the functional deficiency of MBL, there is a growing need to screen its allelic variants and develop genotyping methods for MBL2. In this context we propose a rapid, robust, cost-efficient, and automatable method for detecting all known allelic variants of MBL2. This report introduces for the first time the photoprotein aequorin as a reporter in genotyping by primer extension (PEXT) reactions. The method involves a single PCR amplification of a genomic region that spans all six variant nucleotide sites, i.e., three structural mutations in exon 1 (c.154C>T, pArg52Cys; c.161A>G, p.Gly54Asp; and c.170A>G, p.Gly57Glu), two single nucleotide polymorphisms (SNPs) at positions c.-619G>C and c.-290G>C (promoter region), and one SNP at position c.-66C>T of the 5' untranslated region. PCR is followed by PEXT reactions for each site. Biotin-dUTP is incorporated in the extended primer. The genotyping primers contain a poly(dA) segment at their 5' end. The products are captured by hybridization on the surface of microtiter wells that are coated with a poly(dT)-albumin. The extended primers only are detected by reaction with a streptavidin-aequorin conjugate. The bound photoprotein aequorin is measured within 3 sec by simply adding Ca2+. We carried out extensive optimization studies of the PEXT reaction and genotyped the six nucleotide variant sites using blood specimens from 27 normal DNA samples. The results of the proposed method agreed entirely with the sequencing data.

5' Untranslated Regions↗
PubMed2004

Single-molecule analysis for molecular haplotyping.

In the genome era, there is great hope that genetic approaches such as linkage equilibrium mapping can be used to study common human disorders using a case-control population association study design. Ideally, the parental chromosomes are marked so that chromosomal regions in the form of haplotypes are compared in these studies to increase the power of association. Determining the haplotypes in a diploid individual is a major technical challenge in genetic studies of complex traits. A molecular approach to haplotyping is therefore highly desirable. Recent advances in DNA preparation, separation, labeling, and image analysis provide hope that a strategy of using a three-dye system coupled with DNA distance measurements between alleles will yield haplotype information of sufficiently high quality for genetic studies. In this work, we present the outline of the major challenges one must meet in developing a robust strategy for SNP detection and molecular haplotyping using single molecule analysis.

Alleles↗
PubMed2005

Single-nucleotide polymorphism genotyping in DNA pools.

To undertake partial, or complete, genome screens by association-based methodology for quantitative trait loci, multiple individuals have to be screened for large numbers of genetic markers. Consequently, much recent interest has focused on methods enabling accurate allele quantification in pooled deoxyribonucleic acid (DNA) samples. Microsatellites were the favored markers in initial studies, but the extraordinary wealth of data concerning single-nucleotide polymorphisms (SNPs) has turned attention to the quantification of SNP alleles in pools. All such approaches require accurate estimation of DNA concentrations, followed by the preparation of replicate pools, their validation, and application of procedures for determining allele frequencies. This chapter describes the important steps in preparing pools and surveys a variety of techniques that have been proposed for SNP detection. Finally, we describe the application of a generic approach using pooled DNA for detection of allele frequency differences between case and control populations based on primer extension protocols and outline a strategy for estimating SNP allele frequencies employing microarrays.

DNA↗
PubMed2005

High-density single nucleotide polymorphism array defines novel stage and location-dependent allelic imbalances in human bladder tumors.

Bladder cancer is a common disease characterized by multiple recurrences and an invasive disease course in more than 10% of patients. It is of monoclonal or oligoclonal origin and genomic instability has been shown at certain loci. We used a 10,000 single nucleotide polymorphism (SNP) array with an average of 2,700 heterozygous SNPs to detect allelic imbalances (AI) in 37 microdissected bladder tumors from 17 patients. Eight tumors represented upstaging from Ta to T1, eight from T1 to T2+, and one from Ta to T2+. The AI was strongly stage-dependent as four chromosomal arms showed AI in > 50% of Ta samples, eight in T1, and twenty-two in T2+ samples. The tumors showed stage-dependent clonality as 61.3% of AIs were reconfirmed in later T1 tumors and 84.4% in muscle-invasive tumors. Novel unstable chromosomal areas were identified at chromosomes 6q, 10p, 16q, 20p, 20q, and 22q. The tumors separated into two distinct groups, highly stable tumors (all Ta tumors) and unstable tumors (2/3 muscle-invasive). All 11 unstable tumors had lost chromosome 17p areas and 90% chromosome 8 areas affecting Netrin-1/UNC5D/MAP2K4 genes as well as others. AI was present at the TP53 locus in 10 out of 11 unstable tumors, whereas 6 had homozygous TP53 mutations. Tumor distribution pattern reflected AI as seven out of eight patients with additional upper urinary tract tumors had genomic stable bladder tumors (P < 0.05). These data show the power of high-resolution SNP arrays for defining clinically relevant AIs.

Allelic Imbalance↗
PubMed2006

[The relationship between resting heart rate and single nucleotide polymorphism at 3 sites of nitric oxide synthase gene in the Chinese Han nationality population].

OBJECTIVE: To study the relationship between resting heart rate and single nucleotide polymorphisms (SNP) at 3 sites of nitric oxide synthase (NOS) gene including NOS3 -922A/G, NOS3 894G/T and NOS2 -1173C/T SNPs. METHODS: Genomic DNA was gained from 211 Chinese Han nationality population. The SNPs of NOS3 -922A/G, NOS3 894G/T and NOS2 -1173C/T were genotyped by allele-specific primer-polymerase chain reaction (ASP-PCR) technique. RESULTS: The distribution frequencies of GG, GT and TT genotypes of NOS3 894G/T and AA, AG and GG genotypes of NOS3 -922A/G and CC, CT and TT genotypes of NOS2 -1173C/T were consistent with Hardy-Weinberg equilibrium (P> 0.05). The resting heart rate of Chinese Han nationality population with AA genotypes was higher than that with GG genotype of NOS3 -922A/G (P< 0.01). The resting heart rate of the sub-population with GG genotype was higher than that with TT genotype of NOS3 894G/T (P< 0.05). There were no difference among the resting heart rates of the sub-populations with the allele genotypes of NOS2 -1173C/T. CONCLUSION: The resting heart rate of Chinese Han nationality population with mutated genotype GG of NOS3 -922A/G and with mutated genotype TT of NOS3 894G/T were lower than those with wild genotype of NOS3 -922A/G and NOS3 894G/T. The finding suggests that resting heart rate is associated with SNP of NOS3 -922A/G and NOS3 894G/T.

Adult↗
PubMed2006

Mitochondrial DNA mutation detection by electrospray mass spectrometry.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations cause a large spectrum of clinically important neurodegenerative, neuromuscular, cardiovascular, and endocrine disorders. We describe the novel application of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) to the rapid and accurate identification of pathogenic mtDNA variants. METHODS: In a blinded study, we used ESI-FTICR MS to analyze 24 unrelated samples of total cellular DNA containing 12 mtDNA variants and compared the results with those obtained by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and gel electrophoresis. RESULTS: From the 24-sample blinded panel, we correctly identified 12 of the samples as bearing an mtDNA variant and found the remaining 12 samples to have no pathogenic variants. The correlation coefficient between the 2 methods for mtDNA variant detection was 1.0; there were no false positives or false negatives in this sample set. In addition, the ESI-FTICR method identified 4 single-nucleotide polymorphisms (SNP) that had previously been missed by standard PCR-RFLP analysis. CONCLUSIONS: ESI-FTICR MS is a rapid, sensitive, and accurate method for the identification and quantification of mtDNA mutations and SNPs.

DNA, Mitochondrial↗
PubMed2006

Family-based case-control study of MAOA and MAOB polymorphisms in Parkinson disease.

Monoamine oxidase (MAO) is an enzyme regulating metabolism of neurotransmitters such as dopamine. Two distinct forms of enzyme, encoded by genes MAOA and MAOB located on the X chromosome, have been considered as possible factors in the pathogenesis of Parkinson disease (PD). Previous association studies of PD and MAO genes reported inconsistent results. In this study, we used a large family-based data set to test associations between MAO genes and a risk of PD. The data set includes 298 female discordant sibpairs and 348 male discordant sibpairs. For this study, all subjects analyzed were white and families with known parkin mutations were removed. We analyzed 15 single nucleotide polymorphisms (SNPs) and a dinucleotide repeat marker in the MAO genes. Association was found with the intron 13 SNP of MAOB in the female subset (P = 0.02). No significant association was found in the male subset. Our results add to the evidence of involvement of MAOB in PD and suggest that the effect may be stronger in women.

Adult↗
PubMed2005

The adiponectin gene SNP+276G>T associates with early-onset coronary artery disease and with lower levels of adiponectin in younger coronary artery disease patients (age <or=50 years).

Adiponectin, an adipocyte-derived protein, is an essential modulator of insulin sensitivity and several studies suggest an important role of adiponectin in the processes leading to atherosclerosis, thus indicating the adiponectin gene as a potential candidate for coronary artery disease (CAD). In the present study we have studied the association between two single nucleotide polymorphisms (SNPs) (+45T>G and +276 G>T) of the adiponectin gene and CAD, looking also into the possible influence of these SNPs on adiponectin plasma levels. The SNPs were analysed in a first cohort of 595 subjects, 325 with CAD and 270 matched controls. We observed a significant association (p<0.001) between the SNP +276G>T in the adiponectin gene and CAD. In multivariate analysis, carriers of the +276G>T SNP had an odds ratio (OR) for CAD of 4.99 (p<0.0007). A strong interaction between the +276G>T SNP and age was also present (OR, 1.03; p<0.0001). The increase in CAD risk was most evident among individuals with early-onset CAD (age T SNP was analysed in a second cohort of CAD and controls. The difference between CAD and controls in the +276G>T SNP frequencies showed a similar trend as before, although not significant. The combination of the two cohorts (1,046 subjects: 580 CAD and 466 controls) showed a statistically significant association, particularly in CAD subjects with early-onset of disease. In addition, we confirmed that in younger CAD subjects the SNP was a significant determinant of lower levels of adiponectin. In view of these results, it could be speculated that the adiponectin gene variant, or a mutation in linkage with it, determines lower adiponectin gene expression, causing in turn an increased risk to develop insulin resistance, atherosclerosis and cardiovascular disease. The significant association of the adiponectin gene in subjects with early-onset CAD also suggests that that genetic factors for late-onset diseases may exert a greater influence in younger persons, when other risk factors are not as prevalent as in older age groups.

Adiponectin↗
PubMed2005

Analysis of the genetic diversity of the Plasmodium falciparum multidrug resistance gene 5' upstream region.

Recent findings indicating a low level of polymorphism in the Plasmodium falciparum genome have led to the hypothesis that existent polymorphisms are likely to have functional significance. We tested this hypothesis by developing a map of the polymorphism in the P. falciparum multidrug resistance 1 (pfmdr1) gene 5' upstream region and assaying its correlation with drug resistance in a sample of field isolates from Dakar, Senegal. A comparison of six geographically diverse laboratory strains showed that the 1.94-kb 5'-untranslated region is highly monomorphic, with a total of four unique single nucleotide polymorphisms (SNPs) being identified. All of the mutations were localized to a 462-basepair region proximal to the transcription start point. Analysis of this region in field isolates shows the prevalence of one SNP throughout the entire population of parasites, irrespective of drug resistance status. The SNP frequency of the pfmdr1 upstream region is lower than that found in the noncoding region of other genes.

ATP-Binding Cassette Transporters↗
PubMed2003

DHPLC scoring of a SNP between promoter sequences of HMW glutenin x-type alleles at the Glu-D1 locus in wheat.

The promoter regions of HMW glutenin x-type genes at the Glu-D1 locus were surveyed for SNPs within a subpopulation of German bread wheat cultivars. On the basis of the promoter sequences of HMW glutenin subunit genes Glu-A1-x1, Glu-A1-x2, Glu-B1-x1, Glu-B1-x7, Glu-D1-x2, and Glu-D1-x5, an amplification refractory mutation system assay was designed to selectively amplify Dx-specific PCR fragments. Comparative sequence analysis among seven Glu-D1-x2 and seven Glu-D1-x5 wheat cultivars only confirmed a G-A transition in the promoter sequence to be a true polymorphism. SNP scoring by DHPLC of 95 German bread wheat cultivars, with the exception of cv. Anemos, showed that the transition completely agreed with the presence of HMW glutenin subunits 1Dx5 + 1Dy10 in SDS-PAGE. Therefore, the developed DHPLC assay is suitable for high-throughput genotyping to assist the selection of HMW glutenin genes in wheat quality breeding programs.

Alleles↗
PubMed2000

A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays.

This study describes a practical system that allows high-throughput genotyping of single nucleotide polymorphisms (SNPs) and detection of mutations by allele-specific extension on primer arrays. The method relies on the sequence-specific extension of two immobilized allele-specific primers that differ at their 3'-nucleotide defining the alleles, by a reverse transcriptase (RT) enzyme at optimized reaction conditions. We show the potential of this simple one-step procedure performed on spotted primer arrays of low redundancy by generating over 8000 genotypes for 40 mutations or SNPs. The genotypes formed three easily identifiable clusters and all known genotypes were assigned correctly. Higher degrees of multiplexing will be possible with this system as the power of discrimination between genotypes remained unaltered in the presence of over 100 amplicons in a single reaction. The enzyme-assisted reaction provides highly specific allele distinction, evidenced by its ability to detect minority sequence variants present in 5% of a sample at multiple sites. The assay format based on miniaturized reaction chambers at standard 384-well spacing on microscope slides carrying arrays with two primers per SNP for 80 samples results in low consumption of reagents and makes parallel analysis of a large number of samples convenient. In the assay one or two fluorescent nucleotide analogs are used as labels, and thus the genotyping results can be interpreted with presently available array scanners and software. The general accessibility, simple set-up, and the robust procedure of the array-based genotyping system described here will offer an easy way to increase the throughput of SNP typing in any molecular biology laboratory.

Alleles↗