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.

Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicals.

A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.

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.

Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells

Epithelial (E)-cadherin and its associated cytoplasmic proteins (α-, β-, and γ-catenins) are important mediators of epithelial cell–cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin–catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.

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 role of mismatch repair in the prevention of base pair mutations in Saccharomyces cerevisiae

In most organisms, the mismatch repair (MMR) system plays an important role in substantially lowering mutation rates and blocking recombination between nonidentical sequences. In Saccharomyces cerevisiae, the products of three genes homologous to Escherichia coli mutS—MSH2, MSH3, and MSH6—function in MMR by recognizing mispaired bases. To determine the effect of MMR on single-base pair mismatches, we have measured reversion rates of specific point mutations in the CYC1 gene in both wild-type and MMR-deficient strains. The reversion rates of all of the point mutations are similar in wild-type cells. However, we find that in the absence of MSH2 or MSH6, but not MSH3, reversion rates of some mutations are increased by up to 60,000-fold, whereas reversion rates of other mutations are essentially unchanged. When cells are grown anaerobically, the reversion rates in MMR-deficient strains are decreased by as much as a factor of 60. We suggest that the high reversion rates observed in these MMR-deficient strains are caused by misincorporations opposite oxidatively damaged bases and that MMR normally prevents these mutations. We further suggest that recognition of mispairs opposite damaged bases may be a more important role for MMR in yeast than correction of errors opposite normal bases.

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.

Molecular markers reveal that population structure of the human pathogen Candida albicans exhibits both clonality and recombination.

The life history of Candida albicans presents an enigma: this species is thought to be exclusively asexual, yet strains show extensive phenotypic variation. To address the population genetics of C. albicans, we developed a genetic typing method for codominant single-locus markers by screening randomly amplified DNA for single-strand conformation polymorphisms. DNA fragments amplified by arbitrary primers were initially screened for single-strand conformation polymorphisms and later sequenced using locus-specific primers. A total of 12 single base mutations and insertions were detected from six out of eight PCR fragments. Patterns of sequence-level polymorphism observed for individual strains detected considerable heterozygosity at the DNA sequence level, supporting the view that most C. albicans strains are diploid. Population genetic analyses of 52 natural isolates from Duke University Medical Center provide evidence for both clonality and recombination in C. albicans. Evidence for clonality is supported by the presence of several overrepresented genotypes, as well as by deviation of genotypic frequencies from random (Hardy-Weinberg) expectations. However, tests for nonrandom association of alleles across loci reveal less evidence for linkage disequilibrium than expected for strictly clonal populations. Although C. albicans populations are primarily clonal, evidence for recombination suggests that sexual reproduction or some other form of genetic exchange occurs in this species.

Multiplex genotype determination at a large number of gene loci.

To facilitate large-scale genotype analysis, an efficient PCR-based multiplex approach has been developed. For simultaneously amplifying the target sequences at a large number of genetic loci, locus-specific primers containing 5' universal tails are used. Attaching the universal tails to the target sequences in the initial PCR steps allows replacement of all specific primers with a pair of primers identical to the universal tails and converts the multiplex amplification into "uniplex." Simultaneous amplification of 26 genetic loci with this approach is described. The multiplex amplification can be coupled with genotype determination. By incorporating a single-base mismatch between a primer and the template into the target sequences, a polymorphic site can be converted into a desirable restriction fragment length polymorphism when it is necessary. In this way, the allelic PCR products for the polymorphic loci can be discriminated by gel electrophoresis after restriction enzyme digestion. In this study, 32 loci were typed in such a multiplex way.

Single-amino acid substitutions eliminate lysine inhibition of maize dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) catalyzes the first step in biosynthesis of lysine in plants and bacteria. DHPS in plants is highly sensitive to end-product inhibition by lysine and, therefore, has an important role in regulating metabolite flux into lysine. To better understand the feedback inhibition properties of the plant enzyme, we transformed a maize cDNA for lysine-sensitive DHPS into an Escherichia coli strain lacking DHPS activity. Cells were mutagenized with ethylmethanesulfonate, and potential DHPS mutants were selected by growth on minimal medium containing the inhibitory lysine analogue S-2-aminoethyl-L-cysteine. DHPS assays identified surviving colonies expressing lysine-insensitive DHPS activity. Ten single-base-pair mutations were identified in the maize DHPS cDNA sequence; these mutations were specific to one of three amino acid residues (amino acids 157, 162, and 166) localized within a short region of the polypeptide. No other mutations were present in the remaining DHPS cDNA sequence, indicating that altering only one of the three residues suffices to eliminate lysine inhibition of maize DHPS. Identification of these specific mutations that change the highly sensitive maize DHPS to a lysine-insensitive isoform will help resolve the lysine-binding mechanism and the resultant conformational changes involved in inhibition of DHPS activity. The plant-derived mutant DHPS genes may also be used to improve nutritional quality of maize or other cereal grains that have inadequate lysine content when fed to animals such as poultry, swine, or humans.

Nonsense mutation in the phosphofructokinase muscle subunit gene associated with retention of intron 10 in one of the isolated transcripts in Ashkenazi Jewish patients with Tarui disease.

Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family. Using a reverse transcription PCR assay, PFKM subunit transcripts differing by length were detected in skeletal muscle tissue of all three affected subjects. In the longer transcript, an insertion of 252 nucleotides totally homologous to the structure of the 10th intron of the PFKM gene was found separating exon 10 from exon 11. In addition, two single base transitions were identified by direct sequencing: [exon 6; codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; ACC (Thr) to ACT (Thr)] in either transcript. Single-stranded conformational polymorphism and restriction enzyme analyses confirmed the presence of these point substitutions in genomic DNA and strongly suggested homozygosity for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these patients, further expanding genetic heterogeneity of Tarui disease. Transcripts with and without intron 10 arising from identical mutant alleles probably resulted from differential pre-mRNA processing and may represent a novel message from the PFKM gene.

Mendel’s dwarfing gene: cDNAs from the Le alleles and function of the expressed proteins

The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA1, which is formed from GA20 by 3β-hydroxylation. This step, which limits GA1 biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3β-hydroxylase from lines of pea carrying the Le, le, le-3, and led alleles. The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le. The cDNA sequence from led, a mutant derived from an le line, contains both the le “mutation” and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in Escherichia coli. The expression product for the clone from Le converted GA9 to GA4, and GA20 to GA1, with Km values of 1.5 μM and 13 μM, respectively. The amino acid substitution in the clone from le increased Km for GA9 100-fold and reduced conversion of GA20 to almost nil. Expression products from le and le-3 possessed similar levels of 3β-hydroxylase activity, and the expression product from led was inactive. Our results suggest that the 3β-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.

β-Catenin mutations in cell lines established from human colorectal cancers

β-catenin has functions as both an adhesion and a signaling molecule. Disruption of these functions through mutations of the β-catenin gene (CTNNB1) may be important in the development of colorectal tumors. We examined the entire coding sequence of β-catenin by reverse transcriptase–PCR (RT-PCR) and direct sequencing of 23 human colorectal cancer cell lines from 21 patients. In two cell lines, there was apparent instability of the β-catenin mRNA. Five different mutations (26%) were found in the remaining 21cell lines (from 19 patients). A three-base deletion (codon 45) was identified in the cell line HCT 116, whereas cell lines SW 48, HCA 46, CACO 2, and Colo 201 each contained single-base missense mutations (codons 33, 183, 245, and 287, respectively). All 23 cell lines had full-length β-catenin protein that was detectable by Western blotting and that coprecipitated with E-cadherin. In three of the cell lines with CTNNB1 mutations, complexes of β-catenin with α-catenin and APC were detectable. In SW48 and HCA 46, however, we did not detect complexes of β-catenin protein with α-catenin and APC, respectively. These results show that selection of CTNNB1 mutations occurs in up to 26% of colorectal cancers from which cell lines are derived. In these cases, mutation selection is probably for altered β-catenin function, which may significantly alter intracellular signaling and intercellular adhesion and may serve as a complement to APC mutations in the early stages of tumorigenesis.