Single Nucleotide Polymorphisms Polymorphisms Research Articles

SNP markers: Methods of analysis, ways of development, and comparison on an example of common wheat

SNPs (single nucleotide polymorphisms), which belong to the last-generation molecular markers, occur at high frequencies in both animal and plant genomes. The development of SNP markers allows to automatize and enhance tenfolds the effectiveness of genotype analysis. This review summarizes literature data on methods of SNP polymorphism analysis. Various methods of developing SNP markers are considered, taking common wheat Triticum aestivum L. as an example. These markers are compared to other DNA markers, in order to ensure adequate choice of marker type for solving various molecular genetic problems.

Molecular cloning and genetic mapping of perennial ryegrass casein protein kinase 2 α-subunit genes

The alpha-subunit of the casein protein kinase CK2 has been implicated in both light-regulated and circadian rhythm-controlled plant gene expression, including control of the flowering time. Two putative CK2alpha genes of perennial ryegrass (Lolium perenne L.) have been obtained from a cDNA library constructed with mRNA isolated from cold-acclimated crown tissue. The genomic organisation of the two genes was determined by Southern hybridisation analysis. Primer designs to the Lpck2a-1 and Lpck2a-2 cDNA sequences permitted the amplification of genomic products containing large intron sequences. Amplicon sequence analysis detected single nucleotide polymorphisms (SNPs) within the p150/112 reference mapping population. Validated SNPs, within diagnostic restriction enzyme sites, were used to design cleaved amplified polymorphic sequence (CAPS) assays. The Lpck2a-1 CAPS marker was assigned to perennial ryegrass linkage group (LG) 4 and the Lpck2a-2 CAPS marker was assigned to LG2. The location of the Lpck2a-1 gene locus supports the previous conclusion of conserved synteny between perennial ryegrass LG4, the Triticeae homoeologous group 5L chromosomes and the corresponding segment of rice chromosome 3. Allelic variation at the Lpck2a-1 and Lpck2a-2 gene loci was correlated with phenotypic variation for heading date and winter survival, respectively. SNP polymorphism may be used for the further study of the role of CK2alpha genes in the initiation of reproductive development and winter hardiness in grasses.

Efficient Genetic Mapping of Single Nucleotide Polymorphisms Based Upon DNA Mismatch Digestion

A single-strand specific (sss) nuclease, found in extracts of celery juice, can be used to digest heteroduplex DNA and hence identify heterozygous single nucleotide polymorphism (SNP) sites in PCR products. Here we show this method can be used to map specific genes with relative simplicity and low cost. A particular nucleotide substitution does not need to be identified, and in fact, a priori knowledge of the presence of a SNP is not required, as the entire length of the PCR product is interrogated for the presence of SNPs. This characteristic enables application of this technique to genomes that are not well characterized with regard to SNP polymorphism, and for rapidly placing particular genes onto linkage maps. While this technique is best suited for mapping markers in a backcross configuration, we show that in an F2 configuration, where alternative homozygotes cannot be discerned by this technique, data are still relatively informative about linkage.

Relationships between promoter polymorphisms in the thymidylate synthase gene and mRNA levels in colorectal cancers

Thymidylate synthase (TS) intratumoural expression may be a prognostic marker and predict outcome of 5-fluorouracil (5-FU)-based chemotherapy in colorectal cancer patients. The TS gene promoter enhancer region contains two different polymorphisms which can influence TS mRNA transcriptional and translational efficiency: a polymorphic tandem repeat sequence (2 or 3 repeats; 2R and 3R) and a single nucleotide polymorphism (SNP), G > C, within the second repeat of the 3R alleles. We studied the relationship between tumoural TS mRNA expression levels and TS gene polymorphisms in the colonic mucosa of 48 colorectal cancer patients. The 3R/3R genotype was characterised by higher TS mRNA levels in the tumour than the 2R/2R–2R/3R genotypes ( P = 0.071). Regarding the relationship with the SNP polymorphism, a statistically significant difference in TS gene expression between the 3RG/3RG genotype and 2R/2R–2R/3RC–2R/3RG genotype subset was observed ( P = 0.017). No statistically significant correlation was observed between experimental data and baseline clinical-pathological characteristics as well as clinical outcome in the relatively small patient series investigated. This is the first study reporting an association between the TS intra-repeat SNP and gene expression levels in colorectal cancer patients. These results suggest that in 3R/3R patients, the G > C polymorphism may be an important factor in determining TS mRNA expression levels, and warrant further investigation of the role of TS promoter polymorphisms as predictors of sensitivity to 5-FU-based chemotherapy in larger case series.

APOA5 polymorphisms influence plasma triglycerides in young, healthy African Americans and whites of the CARDIA Study

Genetic variation in the apolipoprotein A-V gene (APOA5) has been associated with variation in plasma triglyceride (TG) levels in African American and white females and males older than 40 years and/or at increased risk of coronary artery disease. We have examined whether plasma TG levels are associated with 16 APOA5 polymorphisms in young (18-30 years) African American (1,075 females and 783 males) and white (1,041 females and 932 males) individuals of the Coronary Artery Risk Development in Young Adults (CARDIA) Study selected without regard to health. Plasma TG was significantly (P < 0.01) associated with markers 27376 and 28837 (-3A/G) in both white females and males, with 27709 (-1131T/C) and 29085 in white males, with 29009 (S19W) in African American females and white males, and with 30966 in African American females. No statistically significant associations were observed in African American males. These six single-nucleotide polymorphisms individually accounted for 0-0.78% of lnTG variation among white females, 0-2.46% among white males, and 0-0.69% among African American females. The results of our study suggest a small but replicable context-dependent influence of the APOA5 gene region on plasma TG levels in young, healthy individuals.

Acetohydroxyacid synthase mutations conferring resistance to imidazolinone or sulfonylurea herbicides in sunflower.

Wild biotypes of cultivated sunflower ( Helianthus annuus L.) are weeds in corn ( Zea mays L.), soybean ( Glycine max L.), and other crops in North America, and are commonly controlled by applying acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Biotypes resistant to two classes of AHAS-inhibiting herbicides-imidazolinones (IMIs) or sulfonylureas (SUs)-have been discovered in wild sunflower populations (ANN-PUR and ANN-KAN) treated with imazethapyr or chlorsulfuron, respectively. The goals of the present study were to isolate AHAS genes from sunflower, identify mutations in AHAS genes conferring herbicide resistance in ANN-PUR and ANN-KAN, and develop tools for marker-assisted selection (MAS) of herbicide resistance genes in sunflower. Three AHAS genes ( AHAS1, AHAS2, and AHAS3) were identified, cloned, and sequenced from herbicide-resistant (mutant) and -susceptible (wild type) genotypes. We identified 48 single-nucleotide polymorphisms (SNPs) in AHAS1, a single six-base pair insertion-deletion in AHAS2, and a single SNP in AHAS3. No DNA polymorphisms were found in AHAS2 among elite inbred lines. AHAS1 from imazethapyr-resistant inbreds harbored a C-to-T mutation in codon 205 ( Arabidopsis thaliana codon nomenclature), conferring resistance to IMI herbicides, whereas AHAS1 from chlorsulfuron-resistant inbreds harbored a C-to-T mutation in codon 197, conferring resistance to SU herbicides. SNP and single-strand conformational polymorphism markers for AHAS1, AHAS2, and AHAS3 were developed and genetically mapped. AHAS1, AHAS2, and AHAS3 mapped to linkage groups 2 ( AHAS3), 6 ( AHAS2), and 9 ( AHAS1). The C/T SNP in codon 205 of AHAS1 cosegregated with a partially dominant gene for resistance to IMI herbicides in two mutant x wild-type populations. The molecular breeding tools described herein create the basis for rapidly identifying new mutations in AHAS and performing MAS for herbicide resistance genes in sunflower.

Comparative radiation hybrid map of canine chromosome 1 incorporating SNP and indel polymorphisms

We report a comparative map of canine chromosome 1 (CFA1) incorporating single nucleotide polymorphisms (SNPs) and insertion/deletion (indel) polymorphisms, developed by using cross-species primers, radiation hybrid analysis, and pool-and-sequence identification of genetic variations. Fifty-five genes were chosen with relatively even spacing (approximately 3 Mb between the human homologues) and were mapped to CFA1, with 49 of these being new assignments. Evolutionary chromosomal breakpoints between CFA1 and the corresponding human chromosomes (HSA6, HSA9, HSA18, and HSA19) were located within 1 to 5 Mb based upon the human genome sequence. The process of identifying the evolutionary chromosomal breakpoints between CFA1 and the relevant human chromosomes led to an improvement in the comparative maps of CFA7, CFA12, and CFA29 through the mapping of 21 additional genes. A manual pool-and-sequence method was used to identify 79 SNPs, 9 small indels, 7 simple tandem repeats, and 2 polymorphic SINE insertions within the genes mapped. The cross-species primers can also be used in the manner described here to improve the comparative maps for other mammalian species.

Comparative radiation hybrid map of canine chromosome 1 incorporating SNP and indel polymorphisms*1

We report a comparative map of canine chromosome 1 (CFA1) incorporating single nucleotide polymorphisms (SNPs) and insertion/deletion (indel) polymorphisms, developed by using cross-species primers, radiation hybrid analysis, and pool-and-sequence identification of genetic variations. Fifty-five genes were chosen with relatively even spacing (approximately 3 Mb between the human homologues) and were mapped to CFA1, with 49 of these being new assignments. Evolutionary chromosomal breakpoints between CFA1 and the corresponding human chromosomes (HSA6, HSA9, HSA18, and HSA19) were located within 1 to 5 Mb based upon the human genome sequence. The process of identifying the evolutionary chromosomal breakpoints between CFA1 and the relevant human chromosomes led to an improvement in the comparative maps of CFA7, CFA12, and CFA29 through the mapping of 21 additional genes. A manual pool-and-sequence method was used to identify 79 SNPs, 9 small indels, 7 simple tandem repeats, and 2 polymorphic SINE insertions within the genes mapped. The cross-species primers can also be used in the manner described here to improve the comparative maps for other mammalian species.

Myeloperoxidase

Myeloperoxidase (MPO) is an haemoprotein that metabolizes tobacco smoke procarcinogens such as benzo(a)pyrene and aromatic amines into highly reactive intermediates. A polymorphism in the MPO promoter (−463G>A), resulting in lower expression, was previously associated with a decrease in lung cancer risk. In this study, the MPO gene was analysed for new polymorphisms by denaturing high-performance liquid chromatography and sequencing on a panel of 48 subjects. Subsequently, the association between newly described polymorphisms, the −463G>A single nucleotide polymorphism (SNP) and lung cancer risk was investigated in a case–control study comprising 245 Caucasian cases and 249 Caucasian controls. Genetic polymorphisms were found in exons 2 (V53F), 6 (M251T), 7 (A332V), 11 (I642L) and 12 (I717V), with allelic frequencies of 6.0%, 1.8%, 1.8%, 0.2% and 1.8%, respectively. A new polymorphism located in the intron 11 3′ splice site was found (0.4%). The known −463G>A polymorphism (24.0%) and four new polymorphisms in promoter region were also detected. No correlation between these new polymorphisms and lung cancer risk was found. For the −463G>A polymorphism, no modification of lung cancer risk was observed for either the G/A genotype [odds ratio (OR) 1.19; 95% confidence interval (CI) 0.8–1.8] or the A/A genotype (OR 0.97; 95% CI 0.4–2.6). Linkage analysis showed a strong disequilibrium between −463G>A polymorphism and exonic SNPs. However, the distribution of reconstructed haplotypes did not differ significantly between cases and controls. Further studies need to be performed to investigate the role of these polymorphisms in others diseases.

A single nucleotide polymorphism (SNP) marker linked to the fragrance gene in rice ( Oryza sativa L.)

The whole rice genome sequence was used to assist in the identification of a single nucleotide polymorphism (SNP) marker linked to the fragrance gene ( fgr) in rice. Genes flanked by restriction fragment length polymorphism and microsatellite markers known to be linked to the fragrance gene were identified by DNA sequence alignment of EST sequences against BAC clones covering this region of chromosome eight. Re-sequencing and comparison of parts of these genes derived from a fragrant and a non-fragrant cultivar revealed only one SNP (a C/T transition) in more than 6 kbp of sequence from 14 genes. Ten of eleven fragrant genotypes and six of 14 non-fragrant genotypes tested carried the C allele. This approach indicated a generally low level of SNP polymorphism in cultivated rice suggesting that association of SNP with phenotypes should be an efficient path to gene discovery in cultivated rice.

Natural resistance-associated macrophage protein 1 gene polymorphisms and response to vaccine against or challenge with Salmonella enteritidis in young chicks

Salmonella enteritidis (SE) contamination of poultry products is of global food-safety concern. The natural resistance-associated macrophage protein 1 (NRAMP1) affects host innate immunity to intracellular bacteria because of its ability to transport divalent cations in late endosome/lysosomes. Studying the association of the NRAMP1 gene and chicken innate immune response to SE can, therefore, aid understanding and enhancement of chicken genetic resistance to SE. The chicken NRAMP1 gene was investigated as a candidate gene for SE response in a unique resource population. Outbred broiler sires and three diverse, highly inbred dam lines (two major histocompatibility complex-congenic Leghorn and one Fayoumi line) produced F1 progeny that were evaluated as young chicks for either bacterial load in spleen and cecum after pathogenic SE inoculation or antibody level after SE vaccination. Thirty-seven single nucleotide polymorphisms (SNP) were identified in 3.1 kb of genomic DNA of the NRAMP1 gene. A PCR-RFLP assay was developed to identify a SNP in a conserved transport motif. The sire NRAMP1 gene SNP was associated (P < 0.02) with antibody level to SE vaccine for Sire 8170 offspring in the two Leghorn crosses. In Sire 8296 offspring, NRAMP1 was associated (P < 0.02) with spleen bacterial load in the combined dam-line crosses. This study demonstrated the association of a SNP polymorphism in a highly conserved region of NRAMP1 with SE vaccine and pathogen challenge response in young chicks, indicating that either NRAMP1 or a linked gene controls these SE-response traits.

Polymorphism of CCR5 affecting HIV disease progression in the Japanese population.

Among several factors associated with HIV-1 disease progression, genetic polymorphism of CCR2, CCR5, and CXCR4 in HIV-1 infection has been found. Single-nucleotide polymorphisms (SNPs) in the CCR2, CCR5, and CXCR4 genes as well as a 32-base pair deletion in the open reading frame of the CCR5 gene are associated with HIV disease progression among Caucasians and African-Americans in North America and Europe. However, in populations other than Caucasians and African-Americans, SNPs have not been fully examined. In our study SNPs in CCR2 coding and CCR5 regulatory regions have been examined in 98 Japanese HIV-positive individuals. The alleles of CCR5 regulatory regions at -2135T and -2086G are associated with late onset of AIDS (p < 0.05; odds ratio for the early onset of AIDS, 0.502 and 0.404, respectively). In contrast to this, the allele of CCR5 at -2086A is associated with the early onset of AIDS (p < 0.05; odds ratio for the early onset of AIDS, 2.133). A haplotype including two alleles at -2135G and -2086G is associated with the late onset of AIDS (p < 0.05; odds ratio for the early onset of AIDS, 0.372). Thus we found that a CCR5 SNP and haplotype polymorphism affect HIV disease progression even in the Japanese population. This indicates that the CCR5 genetic polymorphism affecting disease progression should be studied in a wider range of population.

SSCP analysis of cDNA markers provides a dense linkage map of the Aedes aegypti genome.

An intensive linkage map of the yellow fever mosquito, Aedes aegypti, was constructed using single-strand conformation polymorphism (SSCP) analysis of cDNA markers to identify single nucleotide polymorphisms (SNPs). A total of 94 A. aegypti cDNAs were downloaded from GenBank and primers were designed to amplify fragments <500 bp in size. These primer pairs amplified 94 loci, 57 (61%) of which segregated in a single F(1) intercross family among 83 F(2) progeny. This allowed us to produce a dense linkage map of one marker every 2 cM distributed over a total length of 134 cM. Many A. aegypti cDNAs were highly similar to genes in the Drosophila melanogaster genome project. Comparative linkage analysis revealed areas of synteny between the two species. SNP polymorphisms are abundant in A. aegypti genes and should prove useful in both population genetics and mapping studies.

Fibroblast growth factor receptor 2 (FGFR2): genomic sequence and variations

Fibroblast growth factor receptors (FGFRs) play an important role in development and tumorigenesis. Mutations in FGFR2 cause more than five craniosynostosis syndromes. The FGFR2 genomic structure is the largest of the FGFR family. We have refined and extended the genomic organization of the FGFR2 gene by sequencing more than 119 kb of PACs, cosmids, and PCR products and assembling a region of approximately 175 kb. Although the gene structure has been reported to include only 20 exons, we have verified the presence of at least 22 exons, some of which are alternatively spliced. The sizes of six exons differed from those reported previously. Comparison of our sequence and those in the NCBI database detected more than 300 potential single nucleotide polymorphisms (SNPs). However, sequencing regions containing 52 of these potential SNPs verified only 14 in PCR products generated from 16 CEPH alleles. In contrast, direct sequencing of the CEPH DNAs revealed 21 other polymorphisms. Only one SNP was found in the 2,926 bp of coding sequence. Twenty-seven SNPs, two insertion polymorphisms and five microsatellite polymorphisms are contained in approximately 16.6 kb of non-coding sequence. These data yield an average of one polymorphism for approximately 488 bp of non-coding sequence examined. This collection of SNP, insertion, and repeat polymorphisms will aid future association studies between the FGFR2 gene and human disease and will enhance mutation detection.

Association between Novelty Seeking and the -521 C/T polymorphism in the promoter region of the DRD4 gene.

Association between the human personality trait 'Novelty Seeking' and the polymorphism of the DRD4 gene was first reported by Ebstein and Benjamin in 1996. This was soon followed by replication studies in various ethnic groups and by studying the role of other neurotransmitter receptor and transporter genes in the genetic determination of human temperament. More recently, several polymorphic sites of the upstream regulatory region of the DRD4 gene have been described. Among these the -521 C/T single nucleotide polymorphism (SNP) was shown to be associated with the Novelty Seeking (NS) scores of the Temperament and Character Inventory (TCI) in a Japanese male population. We have investigated the -521 C/T SNP polymorphism in a Caucasian (Hungarian) population, and here we report a replication of the Japanese findings, in an association study involving 109 healthy Hungarian volunteers. We found a weak association between NS and CC vs CT or TT genotypes (P < 0.06). Examination of this relation in male and female sex groups, however, strengthened the association for females (P < 0.01), but showed no genotypic effect for males.

DbSNP—Database for Single Nucleotide Polymorphisms and Other Classes of Minor Genetic Variation

A key aspect of research in genetics is associating sequence variations with heritable phenotypes. The most common variations are single nucleotide polymorphisms (SNPs), which occur approximately once every 500–1000 bases in a large sample of aligned human sequence. Because SNPs are expected to facilitate large-scale association genetics studies, there has recently been great interest in SNP discovery and detection. In collaboration with the National Human Genome Research Institute (NHGRI), the National Center for Biotechnology Information (NCBI) has established the dbSNP database (http://www.ncbi.nlm. nih.gov/SNP) to serve as a central repository for molecular variation. Designed to serve as a general catalog of molecular variation to supplement GenBank (Benson et al. 1999) database submissions can include a broad range of molecular polymorphisms: single base nucleotide substitutions, short deletion and insertion polymorphisms, microsatellite markers, and polymorphic insertion elements such as retrotransposons. Although the name dbSNP is a slight misnomer given the variations represented, SNP polymorphisms are the largest class of variation in the database, and the name dbSNP, selected at the request of NHGRI, reflects this fact. For the sake of brevity, we elected to use the term SNP as a shorthand for “variation” in the database notation and documentation (http://www.ncbi.nlm.nih.gov/ SNP/get_html.cgi?whichHtml=how_to_ submit). Thus terms used in the documentation like “submitted SNP” or “reference SNP” refer to all classes of variation in the database and should be regarded as meaning “a submitted report of variation” and “a reference report of variation.” Furthermore, it should be noted that in serving its role as the variation complement to GenBank, dbSNP does not restrict submissions to only neutral polymorphisms. Submissions are welcome on all classes of simple molecular variation, including those that cause rare clinical phenotypes. Submissions to dbSNP come from a variety of sources including individual laboratories, collaborative polymorphism discovery efforts, large-scale genome sequencing centers, and private industry. The data collected range from the tightly focused characterization of particular genes to broadly sampled levels of variation from random genomic sequence. The distribution of reported marker density across the genome is thus expected to be mixed, with an expected minimum density of 1/3000 bases in regions of random genomic sequence, and local regions of higher density around well-characterized genes. Each variation submitted to dbSNP must have an identifier provided by the submitter (called a “local” identifier by dbSNP), and each is issued a unique identifier, formatted as an integer prefixed with ss (for submitted SNP), for example, ss334. An ss number is thus permanently associated with the submitter’s identifier, and it can be treated as a formal accession number by the scientific publishing community.