Alkaline-mediated differential interaction (AMDI): A simple automatable single-nucleotide polymorphism assay

The key requirements for high-throughput single-nucleotide polymorphism (SNP) typing of DNA samples in large-scale disease case-control studies are automatability, simplicity, and robustness, coupled with minimal cost. In this paper we describe a fluorescence technique for the detection of SNPs that have been amplified by using the amplification refractory mutation system (ARMS)-PCR procedure. Its performance was evaluated using 32 sequence-specific primer mixes to assign the HLA-DRB alleles to 80 lymphoblastoid cell line DNAs chosen from our database for their diversity. All had been typed previously by alternative methods, either direct sequencing or gel electrophoresis. We believe the detection system that we call AMDI (alkaline-mediated differential interaction) satisfies the above criteria and is suitable for general high-throughput SNP typing.

A single nucleotide polymorphism in the RAD51 gene modifies cancer risk in BRCA2 but not BRCA1 carriers

BRCA1 and BRCA2 carriers are at increased risk for both breast and ovarian cancer, but estimates of lifetime risk vary widely, suggesting their penetrance is modified by other genetic and/or environmental factors. The BRCA1 and BRCA2 proteins function in DNA repair in conjunction with RAD51. A preliminary report suggested that a single nucleotide polymorphism in the 5′ untranslated region of RAD51 (135C/G) increases breast cancer risk in BRCA1 and BRCA2 carriers. To investigate this effect we studied 257 female Ashkenazi Jewish carriers of one of the common BRCA1 (185delAG, 5382insC) or BRCA2 (6174delT) mutations. Of this group, 164 were affected with breast and/or ovarian cancer and 93 were unaffected. RAD51 genotyping was performed on all subjects. Among BRCA1 carriers, RAD51-135C frequency was similar in healthy and affected women [6.1% (3 of 49) and 9.9% (12 of 121), respectively], and RAD-135C did not influence age of cancer diagnosis [Hazard ratio (HR) = 1.18 for disease in RAD51-135C heterozygotes, not significant]. However, in BRCA2 carriers, RAD51-135C heterozygote frequency in affected women was 17.4% (8 of 46) compared with 4.9% (2 of 41) in unaffected women (P = 0.07). Survival analysis in BRCA2 carriers showed RAD51-135C increased risk of breast and/or ovarian cancer with an HR of 4.0 [95% confidence interval 1.6–9.8, P = 0.003]. This effect was largely due to increased breast cancer risk with an HR of 3.46 (95% confidence interval 1.3–9.2, P = 0.01) for breast cancer in BRCA2 carriers who were RAD51-135C heterozygotes. RAD51 status did not affect ovarian cancer risk. These results show RAD51-135C is a clinically significant modifier of BRCA2 penetrance, specifically in raising breast cancer risk at younger ages.

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205–2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

A common polymorphism associated with antibiotic-induced cardiac arrhythmia

Drug-induced long QT syndrome (LQTS) is a prevalent disorder of uncertain etiology that predisposes to sudden death. KCNE2 encodes MinK-related peptide 1 (MiRP1), a subunit of the cardiac potassium channel IKr that has been associated previously with inherited LQTS. Here, we examine KCNE2 in 98 patients with drug-induced LQTS, identifying three individuals with sporadic mutations and a patient with sulfamethoxazole-associated LQTS who carried a single-nucleotide polymorphism (SNP) found in ≈1.6% of the general population. While mutant channels showed diminished potassium flux at baseline and wild-type drug sensitivity, channels with the SNP were normal at baseline but inhibited by sulfamethoxazole at therapeutic levels that did not affect wild-type channels. We conclude that allelic variants of MiRP1 contribute to a significant fraction of cases of drug-induced LQTS through multiple mechanisms and that common sequence variations that increase the risk of life-threatening drug reactions can be clinically silent before drug exposure.

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP–Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.

Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination

One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of ≈6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.

Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of the linear chromosomal DNA of Streptomyces ambofaciens

The chromosomal DNA of the bacteria Streptomyces ambofaciens DSM40697 is an 8-Mb linear molecule that ends in terminal inverted repeats (TIRs) of 210 kb. The sequences of the TIRs are highly variable between the different linear replicons of Streptomyces (plasmids or chromosomes). Two spontaneous mutant strains harboring TIRs of 480 and 850 kb were isolated. The TIR polymorphism seen is a result of the deletion of one chromosomal end and its replacement by 480 or 850 kb of sequence identical to the end of the undeleted chromosomal arm. Analysis of the wild-type sequences involved in these rearrangements revealed that a recombination event took place between the two copies of a duplicated DNA sequence. Each copy was mapped to one chromosomal arm, outside of the TIR, and encoded a putative alternative sigma factor. The two ORFs, designated hasR and hasL, were found to be 99% similar at the nucleotide level. The sequence of the chimeric regions generated by the recombination showed that the chromosomal structure of the mutant strains resulted from homologous recombination events between the two copies. We suggest that this mechanism of chromosomal arm replacement contributes to the rapid evolutionary diversification of the sequences of the TIR in Streptomyces.

The nucleotide changes governing cuticular hydrocarbon variation and their evolution in Drosophila melanogaster

The cuticular hydrocarbon (CH) pheromones in Drosophila melanogaster exhibit strong geographic variation. African and Caribbean populations have a high ratio of 5,9 heptacosadiene/7,11 heptacosadiene (the “High” CH type), whereas populations from all other areas have a low ratio (“Low” CH type). Based on previous genetic mapping, DNA markers were developed that localized the genetic basis of this CH polymorphism to within a 13-kb region. We then carried out a hierarchical search for diagnostic nucleotide sites starting with four lines, and increasing to 24 and 43 lines from a worldwide collection. Within the 13-kb region, only one variable site shows a complete concordance with the CH phenotype. This is a 16-bp deletion in the 5′ region of a desaturase gene (desat2) that was recently suggested to be responsible for the CH polymorphism on the basis of its expression [Dallerac, R., Labeur, C., Jallon, J.-M., Knipple, D. C., Roelofs, W. L. & Wicker-Thomas, C. (2000) Proc. Natl. Acad. Sci. 97, 9449–9454]. The cosmopolitan Low type is derived from the ancestral High type, and DNA sequence variations suggest that the former spread worldwide with the aid of positive selection. Whether this CH variation could be a component of the sexual isolation between Zimbabwe and other cosmopolitan populations remains an interesting and unresolved question.

Codon repeats in genes associated with human diseases: fewer repeats in the genes of nonhuman primates and nucleotide substitutions concentrated at the sites of reiteration.

Five human diseases are due to an excessive number of CAG repeats in the coding regions of five different genes. We have analyzed the repeat regions in four of these genes from nonhuman primates, which are not known to suffer from the diseases. These primates have CAG repeats at the same sites as in human alleles, and there is similar polymorphism of repeat number, but this number is smaller than in the human genes. In some of the genes, the segment of poly(CAG) has expanded in nonhuman primates, but the process has advanced further in the human lineage than in other primate lineages, thereby predisposing to diseases of CAG reiteration. Adjacent to stretches of homogeneous present-day codon repeats, previously existing codons of the same kind have undergone nucleotide substitutions with high frequency. Where these lead to amino acid substitutions, the effect will be to reduce the length of the original homopolymeric stretch in the protein.