Discovery Of DNA Polymorphisms Research Articles

Discovery of DNA polymorphisms via genome-resequencing and development of molecular markers between two barley cultivars.

Genome resequencing uncovers genome-wide DNA polymorphisms that are useful for the development of high-density InDel markers between two barley cultivars. Discovering genomic variations and developing genetic markers are crucial for genetics studies and molecular breeding in cereal crops. Although InDels (insertions and deletions) have become popular because of their abundance and ease of detection, discovery of genome-wide DNA polymorphisms and development of InDel markers in barley have lagged behind other cereal crops such as rice, maize and wheat. In this study, we re-sequenced two barley cultivars, Golden Promise (GP, a classic British spring barley variety) and Hua30 (a Chinese spring barley variety), and mapped clean reads to the reference Morex genome, and identified in total 13,933,145 single nucleotide polymorphisms (SNPs) and 1,240,456 InDels for GP with Morex, 11,297,100 SNPs and 781,687 InDels for Hua30 with Morex, and 13,742,399 SNPs and 1,191,597 InDels for GP with Hua30. We further characterized distinct types, chromosomal distribution patterns, genome location, functional effect, and other features of these DNA polymorphisms. Additionally, we revealed the functional relevance of these identified SNPs/InDels regarding different flowering times between Hua30 and GP within 17 flowering time genes. Furthermore, we developed a series of InDel markers and validated them experimentally in 43 barley core accessions, respectively. Finally, we rebuilt population structure and phylogenetic tree of these 43 barley core accessions. Collectively, all of these genetic resources will facilitate not only the basic research but also applied research in barley.

Genome-wide discovery of DNA polymorphisms via resequencing of chickpea cultivars with contrasting response to drought stress.

Drought stress limits plant growth, resulting in a significant yield loss in chickpea. The diversification in genome sequence and selective sweep of allele(s) in different genotypes of a crop plant may play an important role in the determination of agronomic traits, including drought stress response. We investigated, via whole genome resequencing, the DNA polymorphisms between two sets of chickpea genotypes with contrasting drought stress responses (3 drought-sensitive vs. 6 drought-tolerant). In total, 36,406 single nucleotide polymorphisms (SNPs) and 3407 insertions or deletions (InDels) differentiating drought-sensitive and drought-tolerant chickpea genotypes were identified. Interestingly, most (91%) of these DNA polymorphisms were located in chromosomes 1 and 4. The genes harboring DNA polymorphisms in their promoter and/or coding regions and exhibiting differential expression under control and/or drought stress conditions between/within the drought-sensitive and tolerant genotypes were found implicated in the stress response. Furthermore, we identified DNA polymorphisms within the cis-regulatory motifs in the promoter region of abiotic stress-related and QTL-associated genes, which might contribute to the differential expression of the candidate drought-responsive genes. In addition, we revealed the effect of nonsynonymous SNPs on mutational sensitivity and stability of the encoded proteins. Taken together, we identified DNA polymorphisms having relevance in drought stress response and revealed candidate genes to engineer drought tolerance in chickpea.

Genome-wide discovery of DNA polymorphisms among chickpea cultivars with contrasting seed size/weight and their functional relevance

Seed size/weight is a major agronomic trait which determine crop productivity in legumes. To understand the genetic basis of seed size determination, we sought to identify DNA polymorphisms between two small (Himchana 1 and Pusa 362) and two large-seeded (JGK 3 and PG 0515) chickpea cultivars via whole genome resequencing. We identified a total of 75535 single nucleotide polymorphisms (SNPs), 6486 insertions and deletions (InDels), 1938 multi-nucleotide polymorphisms (MNPs) and 5025 complex variants between the two small and two large-seeded chickpea cultivars. Our analysis revealed 814, 244 and 72 seed-specific genes harboring DNA polymorphisms in promoter or non-synonymous and large-effect DNA polymorphisms, respectively. Gene ontology analysis revealed enrichment of cell growth and division related terms in these genes. Among them, at least 22 genes associated with quantitative trait loci, and those involved in cell growth and division and encoding transcription factors harbored promoter and/or large-effect/non-synonymous DNA polymorphisms. These also showed higher expression at late-embryogenesis and/or mid-maturation stages of seed development in the large-seeded cultivar, suggesting their role in seed size/weight determination in chickpea. Altogether, this study provided a valuable resource for large-scale genotyping applications and a few putative candidate genes that might play crucial role in governing seed size/weight in chickpea.

Genome-wide discovery of DNA polymorphisms by whole genome sequencing differentiates weedy and cultivated rice

Analyzing the genome level DNA polymorphisms between weedy and cultivated rice is crucial to elucidate the molecular basis of weedy and agronomic traits, which in turn can enhance our ability to control weedy rice and its utilization for rice improvement. Here, we presented the genome-wide genetic variations between a weedy rice accession PSRR-1 and two cultivated rice accessions, Bengal and Nona Bokra, belonging to japonica and indica subspecies, respectively. The total number of SNPs and InDels in PSRR/Bengal was similar to that of Nona Bokra/Bengal, but was three times greater than that of PSRR/Nona Bokra. There were 11546 large-effect SNPs/InDels affecting 5673 genes, which most likely differentiated weedy rice from cultivated rice. These large effect DNA polymorphisms were mostly resulted in stop codon gain and least by start codon loss. Analysis of the molecular functions and biological processes of weedy rice specific SNPs/InDels indicated that most of these genes were involved in protein modification/phosphorylation, protein kinase activity, and protein/nucleotide binding. By integrating previous QTL mapping results with the DNA polymorphisms data, the candidate genes for seed dormancy and seed shattering were narrowed down. The genomic resource generated in this study will facilitate discovery of functional variants for weedy and agronomic traits.

Uncovering SNP and indel variations of tetraploid cottons by SLAF-seq

BackgroundCotton (Gossypium spp.), as the world’s most utilized textile fibre source, is an important, economically valuable crop worldwide. Understanding the genomic variation of tetraploid cotton species is important for exploitation of the excellent characteristics of wild cotton and for improving the diversity of cotton in breeding. However, the discovery of DNA polymorphisms in tetraploid cotton genomes has lagged behind other important crops.ResultsA total of 111,795,823 reads, 467,735 specific length amplified fragment (SLAF) tags and 139,176 high-quality DNA polymorphisms were identified using specific length amplified fragment sequencing (SLAF-seq), including 132,880 SNPs and 6,296 InDels between the reference genome (TM-1) and the five tetraploid cotton species. Intriguingly, gene ontology (GO) enrichment analysis revealed that a number of significant terms were related to reproduction in G. barbadense acc. 3–79. Based on the new data sets, we reconstructed phylogenetic trees that showed a high concordance to the phylogeny of diploid and polyploid cottons. A large amount of interspecific genetic variations were identified, and some of them were validated by the single-strand conformation polymorphism (SSCP) method, which will be applied in introgression genetics and breeding with G. hirsutum cv. Emian22 as the receptor and the other species as donors.ConclusionsUsing SLAF-seq, a large number of DNA polymorphisms were identified. The comprehensive analysis of DNA polymorphisms provided invaluable insights into the different tetraploid cotton species. More importantly, the identification of numerous interspecific genetic variations provides the basis and is very practical for future introgression breeding. The results presented herein provide a valuable genomic resource for new insights into the genetics and breeding of cotton.

Discovery of molecular markers for Fusarium wilt via transcriptome sequencing of chickpea cultivars

The wilt caused by Fusarium oxysporum f.sp. Ciceris is a major problem for chickpea. The large-scale discovery of DNA polymorphisms in different cultivars can provide insights into the genetic basis of phenotypic differences. In the present study, we sequenced the transcriptomes of four chickpea cultivars with contrasting response to Fusarium infection, including susceptible JG62 and resistant/tolerant ICCV2, K850 and WR315. Based on the analysis of their transcriptome sequences, we identified at least 303 polymorphic microsatellites, of which 64 % could be physically mapped on the chickpea genome. Further, we identified a total of 14,462 single nucleotide polymorphisms (SNPs) and 1864 insertions/deletions (InDels) among the chickpea cultivars analysed. More than 50 % of the SNPs and 19 % of the InDels were detected in the predicted coding regions, and about 46 % of these SNPs resulted in nonsynonymous changes. In addition, we identified at least 60 large-effect SNPs and 248 InDels that affected the integrity of the encoded protein. Several defence-related genes involved in different cellular processes were found to harbour nonsynonymous and/or large-effect SNPs and/or InDels. These data will provide functional markers and promising target genes for wilt resistance and present a valuable resource for molecular breeding for this important trait in chickpea.

Deep re-sequencing of a widely used maintainer line of hybrid rice for discovery of DNA polymorphisms and evaluation of genetic diversity

Genetic diversity within parental lines of hybrid rice is the foundation of heterosis utilization and yield improvement. Previous studies have suggested that genetic diversity was narrow in cytoplasmic male sterile (CMS/A line) and restorer lines (R line) for Three-line hybrid rice. However, the genetic diversity within maintainer lines (B line), especially at a genome-wide scale, remains largely unknown. In the present study, we performed deep re-sequencing of the elite maintainer line V20B (Oryza sativa L. ssp. indica). We then compared the V20B sequence with the 93-11 (Oryza sativa L. ssp. indica) genome sequence. 112.1 × 106 paired-end reads (PE reads) were generated with approximately 30-fold sequencing depth. The V20B PE reads uniquely covered 87.6 % of the 93-11 genome sequence. Overall, a total of 660,778 single-nucleotide polymorphism (SNPs) and 266,301 insertions and deletions (InDels) were identified, yielding an average of 2.1 SNPs/kb and 0.8 InDels/kb. Genome-wide distribution of the SNPs and InDels was non-random, and variation-rich and variation-poor regions were identified in all chromosomes. A total of 20,562 non-synonymous SNPs spanning 8,854 genes were annotated. Our results identified DNA polymorphisms at the genome-wide scale and uncovered the high level of genetic diversity between V20B and 93-11. Our results proved that next-generation sequencing technologies can be powerful tools to study genome-wide DNA polymorphisms, to query genetic diversity, and to enable molecular improvement efforts with Three-line hybrid rice. Further, our results also indicated that 93-11 could be used as core germplasm for the improvement of wild-abortive CMS lines and the maintainer lines.

Enrichment of genomic DNA for polymorphism detection in a non‐model highly polyploid crop plant

Large polyploid genomes of non-model species remain challenging targets for DNA polymorphism discovery despite the increasing throughput and continued reductions in cost of sequencing with new technologies. For these species especially, there remains a requirement to enrich genomic DNA to discover polymorphisms in regions of interest because of large genome size and to provide the sequence depth to enable estimation of copy number. Various methods of enriching DNA have been utilised, but some recent methods enable the efficient sampling of large regions (e.g. the exome). We have utilised one of these methods, solution-based hybridization (Agilent SureSelect), to capture regions of the genome of two sugarcane genotypes (one Saccharum officinarum and one Saccharum hybrid) based mainly on gene sequences from the close relative Sorghum bicolor. The capture probes span approximately 5.8 megabases (Mb). The enrichment over whole-genome shotgun sequencing was 10-11-fold for the two genotypes tested. This level of enrichment has important consequences for detecting single nucleotide polymorphisms (SNPs) from a single lane of Illumina (Genome Analyzer) sequence reads. The detection of polymorphisms was enabled by the depth of sequence at or near probe sites and enabled the detection of 270 000-280 000 SNPs within each genotype from a single lane of sequence using stringent detection parameters. The SNPs were present in 13 000-16 000 targeted genes, which would enable mapping of a large number of these chosen genes. SNP validation from 454 sequencing and between-genotype confirmations gave an 87%-91% validation rate.

Coexpression of Gene Polymorphisms Involved in Bilirubin Production and Metabolism

The potential for genetically determined conditions to modulate the risk for developing neonatal hyperbilirubinemia is increasingly being recognized. The aims of this investigation were to (1) develop genotyping assays for an expanded panel of mutations and polymorphisms across 3 genes that are involved in bilirubin production and metabolism (glucose-6-phosphate dehydrogenase [G6PD], uridine diphosphate glucuronosyl transferase 1A1 [UGT1A1], and organic anion transporter polypeptide 1B1 [OATP1B1]) and (2) determine their allele frequencies in 450 anonymous DNA samples of US residents with ancestry from all of the major regions of the world. Genotyping assays were developed on the basis of allele-specific hybridization and melting peak analysis of the probe set and the match or mismatch template. Allele frequencies and the complexity of coinheritance of multiple genetic variants across G6PD, UGT1A1, and OATP1B1 genes in DNA samples from the DNA Polymorphism Discovery Resource of the National Human Genome Research Institute were determined by using this expanded panel. Genetic polymorphisms of the UGT1A1 promoter, specifically the T-3279G phenobarbital responsive enhancer module and the (thymidine-adenine)(7) dinucleotide repeat TATAA box variants, were common. Similarly, OATP1B1 coding sequence gene variants were frequently observed. Moreover, in more than three quarters of the samples, >or=2 variants were detected, reflecting a high degree of polymorphism coexpression across these genes, including those who carried the African A(-) G6PD mutation. We conclude that this expanded panel of mutations and polymorphisms can serve as an effective instrument to study the genetic architecture of hyperbilirubinemia and speculate an important role for genetic polymorphism coinheritance in determining hyperbilirubinemia risk in newborns.

Utilizing tiling microarrays for whole‐genome analysis in plants

The recent explosion in available genome sequence data has ushered in an era in which analysis of a whole genome can be performed in a single experiment. While DNA microarrays have long been the established technology for measuring gene expression levels, standard expression arrays use relatively few probes for each gene and are typically biased toward known and predicted gene structures. Recently, with the availability of complete genome sequences for many organisms, very-high-density oligonucleotide-based microarrays that span the entire genome have emerged as the preferred platform for genomic analysis. Whole-genome tiling microarrays can be employed for a myriad of purposes, including empirical annotation of the transcriptome, chromatin immunoprecipitation-chip studies, analysis of alternative RNA splicing, characterization of the methylation state of cytosine bases throughout a genome (methylome), and DNA polymorphism discovery. Here, we review several applications of whole-genome technology to obtain a variety of genomic-scale information in plants.

The impact of sequencing the bovine genome

Sequencing the bovine genome is the culmination of more than a decade of international collaboration to bring together resources to chart the genome of an economically important and biologically interesting species. Although considerable sequence is available at the publication of these proceedings, much work remains in annotation of the genome and the discovery of DNA polymorphisms within and between breeds. Nonetheless, the public availability of this sequence has already enhanced our ability to identify genes underlying phenotypes and to understand evolutionary relationships with other mammalian species. The accelerated rate of gene discovery in humans and laboratory animals following the sequencing of their genomes promises an exciting post-sequencing era for bovine genomics.

Genetic Association, Post-translational Modification, and Protein-Protein Interactions in Type 2 Diabetes Mellitus

Type 2 diabetes mellitus is a complex disorder with a strong genetic component. Inherited complex disease susceptibility in humans is most commonly associated with single nucleotide polymorphisms. The mechanisms by which this occurs are still poorly understood. Here we focus on analyzing the effect of a set of disease-causing missense variations of the monogenetic form of Type 2 diabetes mellitus and a set of disease-associated nonsynonymous variations in comparison with that of nonsynonymous variations without any experimental evidence for association with any disease. Analysis of different properties such as evolutionary conservation status, solvent accessibility, secondary structure, etc. suggests that disease-causing variations are associated with extreme changes in the value of the parameters relating to evolutionary conservation and/or protein stability. Disease-associated variations are rather moderately conserved and have a milder effect on protein function and stability. The majority of the genes harboring these variations are clustered in or near the insulin signaling network. Most of these variations are identified as potential sites for post-translational modifications; certain predictions have already reported experimental evidence. Overall our results indicate that Type 2 diabetes mellitus may result from a large number of single nucleotide polymorphisms that impair modular domain function and post-translational modifications involved in signaling. Our emphasis is more on conserved corresponding residues than the variation alone. We believe that the approach of considering a stretch of peptide sequence involving a polymorphism would be a better method of defining the role of the polymorphism in the manifestation of this disease. Because most of the variations associated with the disease are rare, we hypothesize that this disease is a "mosaic model" of interaction between a large number of rare alleles and a small number of common alleles along with the environment, which is little contrary to the existing common disease common variant model.

O0071 GENETIC VARIABILITY OF MULTI-DRUG RESISTANCE PROTEIN 3: POSSIBLE IMPACT ON FUNCTION

Introduction: Multi-drug Resistance Protein 3 (MRP3) is important in the intestinal absorption of many agents including anti-neoplastic, anti-biotic, anti-retroviral, and cardiac drugs. It also is an important mediator of the enterohepatic circulation of bile salts. Inter-individual variability in these processes is suggestive of genetic differences, but such variability is not well understood. Methods: Single Nucleotide Polymorphism (SNP) analysis of MRP3 is being performed using 24 samples from the Coriell DNA Polymorphism Discovery Resource(1), which was established to discover common variants in human DNA. Targeted genomic regions are being amplified by the polymerase chain reaction followed by high throughput sequencing using Sanger methodology. Validation of identified SNP’s is being performed on 600 DNA samples representing three distinct ethnic groups. Functional impact on absorption will be studied using MDCKII cell stable transfectants of genetic variants and measuring methotrexate transport. This study has been deemed exempt from review by the Institutional Review Board. Results: At the time of submission, 2 SNP’s have been identified. A g.+41428AT SNP was identified in 3/48 alleles and introduces a premature stop codon resulting in a truncated protein that is missing 476 amino acids (AA) relative to the wild type (1527 AA). A second SNP, g.+41112TA, is a missense variant, resulting in a S to T substitution at position 1009. The g.+41428AT variant will likely have a significant impact on transporter activity as it appears to be relatively common and results in the loss of two transmembrane (TM)- spanning helices and a nucleotide binding fold. The significance of the g.+41112TA variant is unclear at present. Although it is located in a TM, it has minimal impact on predicted protein structure or hydrophilicity. Discovery of additional variants is expected with further analysis which will be followed by an assessment of their prevalence and functional impact on MTX transport. Conclusion: Two common MRP3 polymorphisms have been discovered. Although one of the variants is predicted to have a significant impact on function, additional study is needed to define the relevance of both variants on drug absorption and enterohepatic circulation of bile salts.

Efficient discovery of DNA polymorphisms in natural populations by Ecotilling.

We have adapted the mutation detection technology used in Targeting Induced Local Lesions in Genomes (TILLING) to the discovery of polymorphisms in natural populations. The genomic DNA of a queried individual is mixed with a reference DNA and used to amplify a target 1-kbp region of DNA with asymmetrically labeled fluorescent primers. After heating and annealing, heteroduplexes are nicked at mismatched sites by the endonuclease CEL I and cut strands are visualized using Li-cor gel analyzers. Putative polymorphisms detected in one fluorescence channel can be verified by appearance of the opposite cut strand in the other channel. We demonstrated the efficiency of this technology, called Ecotilling, by the discovery in 150+ individuals of 55 haplotypes in five genes, ranging from sequences differing by a single nucleotide polymorphism to those representing complex haplotypes. The discovered polymorphisms were confirmed by sequencing and included base-pair changes, small insertions and deletions, and variation in microsatellite repeat number. Ecotilling allows the rapid detection of variation in many individuals and is cost effective because only one individual for each haplotype needs to be sequenced. The technology is applicable to any organism including those that are heterozygous and polyploid.

Genetic variability at the human FMO1 locus: significance of a basal promoter yin yang 1 element polymorphism (FMO1*6).

The flavin-containing monooxygenases (FMOs) are important for the disposition of a variety of toxicants, therapeutics, and dietary components. Although FMO1 is the dominant isoform in fetal liver and adult kidney and intestine and despite up to a 10-fold intersubject variation in expression, a paucity of information is available on FMO1 genetic variability. To address this issue, 24 samples from the Coriell DNA Polymorphism Discovery Resource Panel were sequenced revealing 10 common single nucleotide polymorphisms (SNPs): four located upstream of the structural gene; three within exonic sequences; one within the intron 1 splice donor site; and two with the 3'-untranslated region. Six of these variants are novel. Compared with other FMO loci within the chromosome 1q23-25 cluster, FMO1 seems more highly conserved. Of the identified FMO1 SNPs, only a C>A transversion 9536 base pairs upstream of the exon 2 ATG start codon (g.-9536C>A) would likely affect function, because it lies within the conserved core binding sequence for the yin yang 1 (YY1) transcription factor. Electrophoretic mobility shift assays demonstrated that the g.-9536C>A transversion eliminated YY1 binding. Furthermore, data from transient expression assays in HepG2 cells suggested this SNP could account for a 2- to 3-fold loss of FMO1 promoter activity. Genotype analysis revealed a g.-9,536A allele (FMO1*6) frequency of 13 and 11% in African- and northern European-Americans, respectively, but a significantly higher frequency of 30% in Hispanic-Americans. Thus, the FMO1*6 variant may account for some of the observed interindividual variation in FMO1 expression.

Development of a quantitative competitive reverse transcriptase polymerase chain reaction for the quantification of growth hormone gene expression in pigs

After the advent of the genome projects, followed by the discovery of DNA polymorphisms, basic understanding of gene expression is the next focus to explain the association between polymorphisms and the level of gene expression, as well as to demonstrate the interaction among genes. Among the various techniques for the investigation of transcriptional profiling involving patterns of gene expression, quantitative PCR is the simplest analytical laboratory technique. The objective of this work was to analyze two strategies of a competitive PCR technique for the quantification of the pig growth hormone (GH) gene expression. A pair of primers was designed targeting exons 3 and 5, and two competitive PCR strategies were performed, one utilizing a specific amplicon as a competitor, and the other utilizing a low-stringency PCR amplicon as a competitor. The latter strategy proved to be easier and more efficient, offering an accessible tool that can be used in any kind of competitive reaction, facilitating the study of gene expression patterns for both genetics and diagnostics of infectious diseases.

DNA Sequence Variation in a 3.7-kb Noncoding Sequence 5′ of the CYP1A2 Gene: Implications for Human Population History and Natural Selection

CYP1A2 is a cytochrome P450 gene that is involved in human physiological responses to a variety of drugs and toxins. To investigate the role of population history and natural selection in shaping genetic diversity in CYP1A2, we sequenced a 3.7-kb region 5' from CYP1A2 in a diverse collection of 113 individuals from three major continental regions of the Old World (Africa, Asia, and Europe). We also examined sequences in the 90-member National Institutes of Health DNA Polymorphism Discovery Resource (PDR). Eighteen single-nucleotide polymorphisms (SNPs) were found. Most of the high-frequency SNPs found in the Old World sample were also found in the PDR sample. However, six SNPs were detected in the Old World sample but not in the PDR sample, and two SNPs found in the PDR sample were not found in the Old World sample. Most pairs of SNPs were in complete linkage disequilibrium with one another, and there was no indication of a decline of disequilibrium with physical distance in this region. The average +/- SD nucleotide diversity in the Old World sample was 0.00043+/-0.00026. The African population had the highest level of nucleotide diversity and the lowest level of linkage disequilibrium. Two distinct haplotype clusters with broadly overlapping geographical distributions were present. Of the 17 haplotypes found in the Old World sample, 12 were found in the African sample, 8 were found in Indians, 5 were found in non-Indian Asians, and 5 were found in Europeans. Haplotypes found outside Africa were mostly a subset of those found within Africa. These patterns are all consistent with an African origin of modern humans. Seven SNPs were singletons, and the site-frequency spectrum showed a significant departure from neutral expectations, suggesting population expansion and/or natural selection. Comparison with outgroup species showed that four derived SNPs have achieved high (>0.90) frequencies in human populations, a trend consistent with the action of positive natural selection. These patterns have a number of implications for disease-association studies in CYP1A2 and other genes.

Common allelic variants of cytochrome P4503A4 and their prevalence in different populations.

Marked interindividual variability in expression of CYP3A4 influences the disposition of many endo- and xenobiotics, including the metabolism of steroids, environmental toxins and therapeutically useful drugs. The present study was designed to determine the genetic basis of CYP3A4 variability. We analysed DNA from 82 individuals with known CYP3A4 phenotype including 53 Caucasians and 21 African-American liver donors, seven individuals who were outliers in CYP3A4 metabolism and five individuals in a family of a poor nifedipine metabolizer. In addition, we analysed DNA from the eight person DNA Polymorphism Discovery Resource subset (Coriell Institute) and 89 individuals representing nine ethnic groups. Five non-synonymous mutations in the coding region of CYP3A4 were observed. CYP3A4*14 (T44C) in exon 1 resulted in an L15P change; CYP3A4*15 (G14387A) in exon 6 resulted in a R162Q substitution; CYP3A4*10 (G14422C) in exon 6 resulted in a D174H substitution; CYP3A4*16 (C15721G) in exon 7 resulted in a T185S amino acid substitution; and CYP3A4*12 (C22002T) in exon 11 resulted in a L373F change in the CYP3A4 protein. An additional six single nucleotide polymorphisms (SNPs) in the 5'-UTR, 13 SNPs in the introns and three SNPs in the 3'-UTR were observed. Extensive population differences were observed in the frequencies of various CYP3A4 alleles. None of the 28 CYP3A4 SNPs identified in CYP3A4 phenotyped persons (most individuals being heterozygous for any CYP3A4 variant) was associated with low hepatic CYP3A4 protein expression or low CYP3A4 activity in vivo.

A Polymorphism in the BCL-6 Gene is Associated with Follicle Center Lymphoma

Follicle center lymphoma (FCL) accounts for approximately 40% of all non-Hodgkin's lymphomas (NHL). The genetic-environmental interactions involved in the etiology and pathogenesis of this disease are unknown. In our previous study a single nucleotide polymorphism (SNP) (397C) in the regulatory untranslated first intron region of the BCL-6 gene was found in four of the eight FCL patients but in none of the 10 healthy controls. To further evaluate the potential association between the 397C allele of the BCL-6 gene and FCL, we performed a case-control study. Genomic DNA was isolated from 85 FCL patients, from 98 control cases without a previous history of malignancy, treated at Stanford University Medical Center for non-malignant disorders and from 90 samples from the DNA Polymorphism Discovery Resource. The 397G and the 397C polymorphic alleles were identified by a PCR-RFLP method. To evaluate the possible effect of this polymorphism on gene expression, BCL-6 mRNA levels in nine FCL tumors with the 397G-G genotype and in nine FCL tumors with the 397G-C genotype were measured by quantitative real-time RT-PCR.The 397C polymorphic allele was found in 32 FCL cases (37.6%), in 20 controls (20.4%) and in 17 (18.9%) samples from the DNA Polymorphism Discovery Resource. The prevalence of the 397G-C and 397C-C genotypes was significantly higher in FCL cases than in control group (p = 0.01). No difference in BCL-6 gene expression was observed between FCL cases with 397G-G and 397G-C genotypes. The present study demonstrates a possible association between the 397C allele of the BCL-6 proto-oncogene and FCL. The similar levels of BCL-6 mRNA expression in 397G-G and in 397G-C FCL cases suggests that any possible oncogenic effect of the polymorphic allele would not simply be related to a direct effect on BCL-6 gene expression and suggests the existence of other FCL susceptibility genes that are in linkage disequilibrium with the 397C allele of the BCL-6 gene.

Ethical-legal problems of DNA databases in criminal investigation

Advances in DNA technology and the discovery of DNA polymorphisms have permitted the creation of DNA databases of individuals for the purpose of criminal investigation. Many ethical and legal problems...