Mutational analysis of the human dihydropyrimidine dehydrogenase gene by denaturing high-performance liquid chromatography

Mutations in the DPYD gene, which encodes dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in the catabolism of pyrimidines, are responsible for an inborn error of metabolism associated with thymine-uraciluria and neurological symptoms. Because the antimetabolite 5-fluorouracil (5-FU) is metabolized by the same enzyme, deficient DPYD alleles may also constitute a risk factor for severe toxicity following treatment with this anticancer drug. The aim of this study was to develop a comprehensive and rapid method to detect sequence variations within the DPYD gene. Using polymerase chain reaction (PCR) amplification and denaturing high-performance liquid chromatography (DHPLC), we established a protocol that makes it possible to screen all 23 exons of the DPYD gene and their exon-intron boundaries for both known and unknown mutations under identical conditions. A novel one-step PCR mutagenesis procedure was developed to generate heterozygous mutant amplicons as positive controls to optimize DHPLC detection of any sequence variation. DHPLC analysis was shown to result in mutation-specific elution profiles and to be able to distinguish different base changes within the same exon or different heterozygous combinations of mutations within the same exon. By analyzing the DPYD gene in 16 affected individuals, a total of 47 base changes were detected, representing eight known mutations and three novel intronic base changes. Sequence analysis confirmed all base changes detected. This method will be useful in identifying patients at risk for toxicity prior to 5-FU treatment, as well as in the analysis of individual patients with thymine-uraciluria.     

Mutation detection in the human HSP7OB' gene by denaturing high-performance liquid chromatography

Variances, particularly single nucleotide polymorphisms (SNP), in the genomic sequence of individuals are the primary key to understanding gene function as it relates to differences in the susceptibility to disease, environmental influences, and therapy. In this report, the HSP70B' gene is the target sequence for mutation detection in biopsy samples from human prostate cancer patients undergoing combined hyperthermia and radiation therapy at the Dana-Farber Cancer Institute, using temperature-modulated heteroduplex analysis (TMHA). The underlying principles of TMHA for mutation detection using DHPLC technology are discussed. The procedures involved in amplicon design for mutation analysis by DHPLC are detailed. The melting behavior of the complete coding sequence of the target gene is characterized using WAVEMAKER software. Four overlapping amplicons, which span the complete coding region of the HSP70B' gene, amenable to mutation detection by DHPLC were identified based on the software-predicted melting profile of the target sequence. TMHA was performed on PCR products of individual amplicons of the HSP70B' gene on the WAVE Nucleic Acid Fragment Analysis System. The criteria for mutation calling by comparing wild-type and mutant chromatographic patterns are discussed.     

Detection of novel ALAD gene polymorphisms using denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DH-PLC), which is based on the separation of mismatched DNA heteroduplexes, is one of the most promising techniques for detecting nucleotide polymorphisms. Lead is an important environmental toxicant that can impair the cardiovascular, central nervous, renal, reproductive, and hematologic systems. Here we compare the sensitivity and efficiency of DNA polymorphism detection in the delta-aminolevulinate dehydratase (ALAD) gene encoding the principal lead-binding protein in humans by means of DHPLC and direct DNA sequencing of polymerase chain reaction amplicons. In a sample of 48 unrelated Chinese women, five novel mutations were discovered in intron 6 (G13298C). exon 7 (C13348T), intron 8 (C13847T), intron 12 (C15096T), and the 3' untranslated region of exon 13 (A15762C). The allele frequencies of C13298, T13348, T13847, T15096, and C15762 alleles were 21.3%, 2.3%. 82.1%, 62.5%, and 1.1%, respectively. All five mutations were detected by both DHPLC and direct DNA sequencing. No previously reported missense ALAD mutations were found in this Chinese population. Our study confirms that DHPLC provides an accurate method for the rapid identification of single nucleotide polymorphisms.     

Genomic organization and polymorphisms detected by denaturing high-performance liquid chromatography of porcine SLC11A1 gene.

SLC11A1 (also known as Natural Resistance Associated Macrophage Protein1, NRAMP1) plays a crucial role in resistance of inbred mice to infection with several intracellular pathogens such as Mycobacterium, Leishmania and Salmonella. In this study, PCR amplification and sequencing were performed to obtain the genomic organization and sequence of porcine SLC11A1 gene by comparative genomic analysis. Results showed that porcine SLC11A1 gene consists of 15 exons and 14 introns, which is consistent with that of mice and human. All introns were sequenced and their nucleotide sequences were submitted to GenBank. The exon/intron boundaries were determined by comparing cDNA sequence with amplified genomic DNA sequences. Mutational analysis was performed on exonic and neighboring intronic region by denaturing high-performance liquid chromatography (DHPLC) and sequencing confirmation. Forty polymorphisms were identified; six are located in exons and thirty-four in introns. Two exonic polymorphisms are nonsynonymous changes (D6H and V175I), three are synonymous changes (S23, G33 and I155), and one is in 3' UTR. The availability of the fine genomic organization and identification of the polymorphisms will facilitate the evaluation of porcine SLC11A1 functional role in diseases resistance or susceptibility.     

Comprehensive screening of the thiopurine methyltransferase polymorphisms by denaturing high-performance liquid chromatography

The drug-metabolizing enzyme thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathiopurine, which are used as immunosuppressants and in the treatment of acute lymphoblastic leukemia and rheumatoid arthritis. TPMT enzymatic activity is a polymorphic trait, and poor metabolizers may develop life-threatening bone marrow failure. To avoid such adverse effects, the TPMT enzymatic activity in patients' red blood cells (RBCs) is routinely measured prior to thiopurine administration in a limited number of oncology clinics. In the present study, we took advantage of a highly sensitive and specific automated denaturing high-performance liquid chromatography (dHPLC) technique that not only detects known polymorphic alleles, but also identifies previously uncharacterized sequence variants. We developed a dHPLC-based protocol to analyze the entire coding region and validated the protocol to detect all 16 previously described variant alleles. We further analyzed the entire coding region of the TPMT gene in 288 control samples collected worldwide and identified two novel amino acid substitutions Arg163Cys (487C>T) and Arg226Gln (677G>A) within exons 7 and 10, respectively. The clinical application of this comprehensive screening system for examining the entire TPMT gene would help to identify patients at risk for bone marrow failure prior to 6-mercaptopurine therapy.     

Profiling and monitoring of microbial populations by denaturing high-performance liquid chromatography

We describe a new molecular technique for the analysis of microbial species and complex microbial populations based on the separation of PCR-amplified 16S rDNA fragments by denaturing high-performance liquid chromatography (DHPLC). Using marine bacterial samples, we determined the optimum conditions for the analysis of bacterial species and the examination of complex bacterial assemblages obtained from different environments. The incorporation of a 40-bp GC clamp into the amplification primer was essential to effectively discriminate genetic differences in DHPLC-primers with a 20-, 10-, or 0-bp GC clamp length were less efficient. A 64.5 degrees C column temperature in DHPLC allowed optimal separation of species in a complex bacterial population. PCR-DHPLC analysis of bacterial assemblages demonstrated profiles with distinguishable peaks, which constituted the different populations and their degree of abundance. Fraction collection and DNA sequencing from profile peaks enabled bacterial identification. PCR-DHPLC analysis can also provide opportunities for describing bacterial communities, cloning bacteria, and monitoring bacterial populations in environments of interest.     

Mutation analysis of MLH1 and MSH2 genes performed by denaturing high-performance liquid chromatography

Mutation analysis of large genes, such as MSH2 and MLH1, is time-consuming and expensive. We investigated the sensitivity and specificity of DHPLC analysis for the detection of mutations within both MSH2 and MLH1. Studies included a series of 46 patients affected by colorectal cancer from HNPCC families. We confirmed 19 changes previously identified by DNA sequencing and, in a blind study, an additional 16 rare alterations including four mutations not previously described. Generally, false negative results were not observed. Elution profiles were highly characteristic for a given change and in 98.5% cases allowed the distinction between novel alterations and previously identified mutations and polymorphisms. For the detection of changes in almost all amplicons, it was sufficient to use just one denaturing temperature. DHPLC was confirmed to be highly sensitive, specific and a cost-effective technique with particularly high potential for the detection of MSH2 and MLH1 gene mutations in the diagnostic setting.     

Identification by denaturing high-performance liquid chromatography of numerous polymorphisms in a candidate region for multiple sclerosis susceptibility

Genetic association analysis of candidate regions where evidence of linkage has accumulated is becoming a key issue in the study of complex diseases. A high density of markers, at least one per centimorgan, is required to improve the chances of observing linkage disequilibrium with disease alleles. A recently available single nucleotide polymorphism (SNP) map designed to cover the whole genome provides an average density of one marker per 2 cM. In the present study we show that the number of markers can be approximately doubled in a selected region, thus reaching a density suitable for association studies, by applying a completely automated technique for polymorphism detection, denaturing high-performance liquid chromatography (DHPLC). A systematic search for SNPs was performed in the region 5ptel-q13, where weak but convergent evidence for linkage with multiple sclerosis has accumulated. Screening for polymorphisms was performed on 124 sequence tagged sites (STSs) in the 3'UTR ends of expressed sequence tags totaling about 30,000 bp. Thirty SNPs in 28 STSs were found with less than 10% overlap with the markers already detected in the same region. The data confirm the validity of the approach using DHPLC on expressed gene sequences tagged by a set of standard commercially available primers.     

DNA methylation profiling to assess pathogenicity of BRCA1 unclassified variants in breast cancer

Germline pathogenic mutations in BRCA1 increase risk of developing breast cancer. Screening for mutations in BRCA1 frequently identifies sequence variants of unknown pathogenicity and recent work has aimed to develop methods for determining pathogenicity. We previously observed that tumor DNA methylation can differentiate BRCA1-mutated from BRCA1-wild type tumors. We hypothesized that we could predict pathogenicity of variants based on DNA methylation profiles of tumors that had arisen in carriers of unclassified variants. We selected 150 FFPE breast tumor DNA samples [47 BRCA1 pathogenic mutation carriers, 65 BRCAx (BRCA1-wild type), 38 BRCA1 test variants] and analyzed a subset (n=54) using the Illumina 450K methylation platform, using the remaining samples for bisulphite pyrosequencing validation. Three validated markers (BACH2, C8orf31, and LOC654342) were combined with sequence bioinformatics in a model to predict pathogenicity of 27 variants (independent test set).  Predictions were compared with standard multifactorial likelihood analysis. Prediction was consistent for c.5194-12G>A (IVS 19-12 G>A) (P>0.99); 13 variants were considered not pathogenic or likely not pathogenic using both approaches. We conclude that tumor DNA methylation data alone has potential to be used in prediction of BRCA1 variant pathogenicity but is not independent of estrogen receptor status and grade, which are used in current multifactorial models to predict pathogenicity.     

Analysis of genetic variations of lamin A/C gene (LMNA) by denaturing high-performance liquid chromatography

The human LMNA gene, when mutated, has been shown to cause at least 7 human diseases: dilated cardiomyopathy, Emery Dreifuss muscular dystrophy, limb girdle muscular dystrophy, familial partial lipodystrophy, Charcot Marie tooth disease type II, mandibuloacral dysplasia, and Hutchinson-Gilford Progeria (OMIM #176670). This article describes a high-throughput method for screening the human lamin A/C (LMNA) gene for genetic mutations and sequence variation using denaturing high-performance liquid chromatography (DHPLC). In the present study, 76 patients with dilated cardiomyopathy were screened for mutations using DHPLC and sequence analysis. Abnormal elution profiles were identified and sequenced on an ABI 377 automatic sequencer. Heterozygous LMNA mutations were detected in 8% of the affected patients. In addition, a number of intronic and exonic single nucleotide polymorphisms were identified. LMNA mutations are clinically relevant in at least 6 human diseases. This study provides a protocol for high-throughput LMNA analysis applicable both in the research and in the clinical diagnostic setting.     

Detection of sequence variations in the adenomatous polyposis coli (APC) gene using denaturing high-performance liquid chromatography

We have evaluated the usefulness of denaturing high performance liquid chromatography (dHPLC) for scanning the adenomatous polyposis coli (APC) gene for point mutations, small deletions, and insertions. Our assay consists of 28 sets of primers to amplify the 15 exons of the APC gene. All PCR reactions were amplified simultaneously using the same reaction conditions in a 96-well format and then analyzed by dHPLC, using empirically determined optimum temperatures for partial fragment denaturation. Previously studied DNA specimens from 47 familial adenomatous polyposis (FAP) patients were analyzed by dHPLC and all mutations were correctly identified and confirmed by sequence analysis. This approach identified a single-base substitution in exon 6 and a 2-bp insertion in exon 15 that initially had not been detected by single-strand conformational polymorphism (SSCP) analysis. A novel mutation in exon 15 of the APC gene, 2065delG (codon 689) that had previously been undetected by the protein truncation test (PTT) was also identified by dHPLC. We present our validation studies of dHPLC technology for APC gene analysis in terms of sensitivity and specificity and compare it to current standard scanning technologies including PTT, SSCP, and conformational sensitive gel electrophoresis (CSGE).     

Genetic variability of immunomodulatory genes in ectromelia virus isolates detected by denaturing high-performance liquid chromatography

The genetic variability of nine genes in 12 isolates and strains of ectromelia virus, which causes a smallpox-like disease (mousepox) in mice, was determined and allows for classification of ectromelia viruses. The low genetic variability suggests that evolutionary pressure maintains the activity of immunomodulatory genes in natural poxvirus infections.     

Application of denaturing high-performance liquid chromatography for rice variety identification and seed purity assessment

Recent DNA sequencing projects and the establishment of high-throughput assays have provided an abundance of sequence information and data on nucleotide polymorphisms in rice. Based on previously identified single-nucleotide polymorphisms (SNPs) and insertions/deletions, we employed denaturing high-performance liquid chromatography (DHPLC) to genotype rice varieties using a chromatographic pattern-based strategy. In this study, 12 amplicons harboring multiple and informative SNPs were screened. PCR products of the 12 amplicons from 47 rice varieties were analyzed by DHPLC and DNA sequencing. Each homozygous sample with a single peak pattern in the initial DHPLC analysis was mixed with zhenshan97 for a second DHPLC analysis. The 12 amplicons were found to be polymorphic across the hybrids, and mixed homozygous samples with 43 distinct DHPLC elution profiles detected. Sequence analysis confirmed that the distinct DHPLC patterns corresponded to different DNA sequences. A set of distinct characteristic profiles in six amplicons differentiated between all of the hybrids, inbred lines, and restorer lines and produced unique a fingerprint for these lines. In addition, we found that the DHPLC pattern of the hybrid was in accordance with the results obtained by DHPLC analysis of a mixed sample of the two parents. These results demonstrate that DHPLC can be efficiently applied for the rapid and automated identification of diverse rice varieties and could possibly be utilized for seed genetic purity testing on a high-throughput scale.     

Rapid detection of gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae by denaturing high-performance liquid chromatography

The detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that is used to detect mutations in human DNA. This is the first report that this technique is used as a tool to detect mutations in genes encoding fluoroquinolone resistance in Neisseria gonorrhoeae. Eighty-one strains of N. gonorrhoeae were used in this study. Genomic DNA from each strain was subjected to PCR amplification of 225 bp in gyrA and 166 bp in parC spanning the fluoroquinolone-resistance determining regions (QRDRs). After we performed DNA sequencing of these amplicons and identification of mutations in the QRDRs, DHPLC was undertaken to investigate whether its results correlate the distinctive chromatogram with their DNA mutations pattern. The profilings detected by DHPLC completely corresponded to the results of the DNA sequencing in mutation patters in gyrA and parC genes. They resulted in the following amino acid substitutions: Ser-91Phe, Asp-95Gly, and Asp-95Asn in gyrA; and Gly-85Asp, Asp-86Asn, Ser-87Arg, and Ser-88Pro in parC, respectively. These mutations existed alone or as combinations, and we identified five mutations patterns in gyrA and six in parC including wild-type. These mutations and their patterns could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This novel detection system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run), and reliable technique for characterizing fluoroquinolone resistance in N. gonorrhoeae.     

应用变性高效液相色谱技术建立胸苷酸合成酶基因多态检测平台

TS基因5′非翻译区(5′ untranslation region, 5′UTR)增强子区域(TS enhancer region, TSER)存在28 bp的2次(2R)、3次(3R)的串联重复多态, 在3R等位基因第二次重复中还存在一个G→C的单核苷酸多态性(single nucleotide polymorphisms, SNPs), 同时在3′非翻译区(3′ untranslation region, 3′ UTR)存在6个碱基片段缺失/插入多态。这些多态形式的存在影响了TS基因mRNA的稳定和翻译效率, 并可导致不同TS基因型肿瘤患者对以5-fuorouracil (5-FU)为基础的化疗疗效产生差异。为提高TS基因型临床检测的效率和准确性, 方便、快捷、准确和自动化区分各种纯合及杂合基因型, 设计多重PCR反应, 同时扩增TS基因5′ 和3′ 非翻译区多态所处片段。利用DHPLC技术建立TS基因多态性检测平台, 在非变性条件下, 通过优化DHPLC 洗脱梯度, 同时检测5′ TSER区的串联重复多态和3′ UTR片段长度多态; 在变性条件下, 检测5′ TSER区单核苷酸多态。同时采用PCR-RFLP和DNA 测序方法, 验证DHPLC分析结果。     

Analysis of single nucleotide polymorphisms in the promoter region of interleukin-10 by denaturing high-performance liquid chromatography.

Interleukin-10 (IL10), an anti-inflammatory cytokine, has been implicated in a variety of immune- and inflammatory-related diseases. We investigated the following SNPs: -1082, -819, -592 in the promoter region of IL10 in a normal (control) population and selected diseases: breast cancer (BrCa), systemic lupus erythematosus (SLE), and B-cell chronic lymphocytic leukemia (B-CLL) by denaturing high-performance liquid chromatography (DHPLC) and found distinct genotype and haplotype patterns. DHPLC was performed using the Transgenomic WAVE instrument, a mutational discovery tool that allows for high throughout analysis of SNPs. The principle of DHPLC is based on separation of homo- and heteroduplex formation of individual polymerase chain reaction products at specific melting temperatures and set gradients. The melting temperature selected for each SNP was based on size and sequence of the polymerase chain reaction product (for -1082, 57 degrees C; for -819, 58 degrees C; and for -592, 59.2 degrees C). Before fragment mutational analysis, all samples were denatured at 95 degrees C and slowly reannealed to allow for reassociation of different strands. Heteroduplex samples were easily distinguished from homoduplex samples. In order to identify wild type from homozygous mutant, two homoduplex polymerase chain reaction samples had to be mixed together, denatured at 95 degrees C and reannealed. The homozygous mutant, when combined with wild type, displayed a double peak on chromatogram. Once distinct chromatograms were established for each of the SNPs and the nucleotide changes confirmed by sequencing, genotype and haplotype frequencies were tabulated for the groups studied.