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AIMS: Heterozygous mutations in the transcription factor Nkx2.5 indicate a genetic cause for congenital heart diseases (CHDs) in human beings. The present study aimed to assess the prevalence of NKX2.5 mutations in Italian patients with sporadic non-syndromic and syndromic CHD, as well as to appraise any genotype-phenotype correlations. METHODS: One hundred Italian patients affected with CHD (90 had sporadic non-syndromic CHD and 10 had syndromic CHD) were screened for NKX2.5 mutations. The coding region and flanking regions involved in gene splicing of the CSX/NKX2.5 gene were amplified from genomic DNA by PCR, and mutational analysis was performed using denaturing high performance liquid chromatography and DNA sequencing. RESULTS: One previously reported NKX2.5 mutation (c.73C>T, p.R25C) was identified in two of the 100 CHD patients (2%). We have detected the p.R25C alteration in a woman showing aneurysm of the membranous septum, aortic coarctation and bicuspid aortic valve, that was a different phenotype from those previously reported, and for the first time in a patient with syndromic CHD with Down's syndrome (posterior ventricular septal defect, atrial septal defect, left superior cava vein ' sinus, and patent ductus arteriosus). CONCLUSION: Our results confirm that NKX2.5 mutations are not a common cause of CHD; furthermore, the p.R25C variation may increase susceptibility to development of CHD in patients with and without chromosomal abnormalities.

IGFBP7 belongs to a family of insulin-like growth factor-1 regulatory binding proteins. IGFBP7 hypermethylation is associated with its down-regulation in various carcinomas. In prostate cancer IGFBP7 down-regulation has been widely reported but to our knowledge the mechanisms behind this event are unknown. We performed a denaturing high performance liquid chromatography screening and validation strategy to profile the methylation status of IGFBP7 in prostate cancer.We combined denaturing high performance liquid chromatography and bisulfite sequencing to examine IGFBP7 methylation in a panel of prostate cancer cell lines. Quantitative methylation specific polymerase chain reaction was used to determine methylation levels in prostate tissue specimens of primary prostate cancer, histologically benign prostate adjacent to tumor, high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. IGFBP7 gene expression was measured by quantitative methylation specific polymerase chain reaction in cell lines and tissue specimens.IGFBP7 was methylated in the 4 prostate cancer cell lines DU145, LNCaP, PC-3 and 22RV1. Quantitative methylation specific polymerase chain reaction analysis revealed that promoter methylation was associated with decreased IGFBP7 expression. Quantitative methylation specific polymerase chain reaction showed that IGFBP7 methylation was more frequently detected in prostate cancer (60% (31/52)) and high grade prostatic intraepithelial neoplasia (40% (6/15)) samples compared to histologically benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia (0%) samples.To our knowledge this is the first report of aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in prostate cancer cell lines. Results demonstrate that CpG methylation of IGFBP7 may represent a novel biomarker of prostate cancer and pre-invasive neoplasms. Thus, future examination of IGFBP7 methylation and expression in a larger patient cohort, including bodily fluids, is justified to further evaluate its role in a diagnostic and prognostic setting.

Microsatellite instability (MSI) is a characteristic molecular phenotype of tumors from the hereditary nonpolyposis colorectal cancer (Lynch) syndrome. Routine MSI screening of tumors in younger patients is an efficient prescreening tool for the population-based detection of Lynch syndrome in the absence of family cancer history. We describe here the optimization of a denaturing high performance liquid chromatography (DHPLC) assay for MSI analysis with the "Bethesda" panel of markers recommended by the National Cancer Institute and with a more recently proposed "pentaplex" panel of 5 mononucleotide repeat markers. By using various polymerase chain reaction primers and tumor DNA samples with known MSI status, each of the 3 standard DHPLC formats tested could correctly identify the MSI status without the "stutter peaks" inherent in the capillary electrophoresis (CE) methods that are currently in use. Dilution experiments showed that the detection limit for MSI using DHPLC was at least 1:100, thus avoiding the need for tumor enrichment by microdissection before analysis. Concordance between CE and DHPLC for the detection of instability in the Bethesda panel markers was 95%. Optimal DHPLC running conditions for the pentaplex mononucleotide panel are also described. In conclusion, DHPLC provides a sensitive and specific alternative for routine MSI analysis that is free of the stutter peaks observed with CE and which can be used with either the Bethesda or pentaplex mononucleotide marker panels.

Background At least five mismatch repair (MMR) genes, including hMSH2, hMLH1, hPMS, hPMS2, and hMSH6/GTBP, are associated with hereditary nonpolyposis colorectal cancer (HNPCC). More than 90% of families with HNPCC harbor the hMSH2 and hMLH1 gene mutations. We have analyzed the clinical features of HNPCC among Chinese patients and report the results of screening for mutations in the hMSH2 and hMLH1 genes. Methods The data concerning gender, site of colorectal cancer (CRC), age at diagnosis, history of synchronous and/or metachronous colorectal cancer, instance of extracolonic cancers, and histopathology of tumors for 126 patients from 28 independent families with HNPCC were collected. Fifteen of the families met the Amsterdam I criteria, and 13 met the Japanese clinical criteria for diagnosis. Genomic DNA was extracted from the peripheral lymphocytes. Polymerase chain reaction (PCR) and denaturing high-performance liquid chromatography (DHPLC) were used to screen the coding region of the hMSH2 and hMLH1 genes. Samples showing abnormal DHPLC profiles were sequenced. Results One hundred and seventy malignant neoplasms were found in the 126 patients, of whom 23 had multiple cancers. Ninety-eight of the patients (77.8%) had colorectal cancers, with an average age at onset of 45.9 years and a right-sided predominance. Eight hMSH2 or hMLH1 gene sequence variations were found in 12 families, and a germ-line G204X nonsense mutation in the third exon of hMSH2 was found, representing the first mutation in an MMR gene ever found in people of Chinese Mongolian ethnicity. Conclusions HNPCC is a typical autosomally dominant hereditary disease, characterized by early onset, a predominance of proximal colorectal cancer, and multiple synchronous and metachronous colorectal cancers. DHPLC is a powerful tool for detecting mutations in the hMSH2 and hMLH1 genes. Mutations in the first nine exons of the hMLH1 gene were more common in Chinese patients.

Objective Approximately 30–40% of grade III–IV toxicity to 5-FU has been associated with partial or profound deficiency in dihydropyrimidine dehydrogenase (DPD), the first of three enzymes in the catabolic pathway of fluoropyrimidines. There remains, however, a subset of patients presenting with 5-FU-associated toxicity despite normal DPD activity, suggesting possible deficiencies in enzymes downstream of DPD: dihydropyrimidinase (DHP), encoded by the DPYS gene, and/or β-ureidopropionase (BUP-1), encoded by the UPB1 gene. Previously, we reported the identification of inactivating mutations in the DPYS gene that could potentially alter the uracil catabolic pathway in healthy individuals with normal DPD enzyme activity. This study investigates the possible role of UPB1 genetic variations in the regulation of the uracil catabolic pathway in individuals presenting with a deficient uracil breath test (13C-UraBT) despite normal DPD enzyme activity. Methods This study included 219 healthy asymptomatic volunteers with known DPD enzyme activity and [2-13C]-uracil breath test (UraBT). All samples were genotyped for sequence variations in the UPB1 gene using denaturing high performance liquid chromatography (DHPLC) and Surveyor enzyme digestion with confirmation of detected sequence variants by direct sequencing. Results Seven novel and six previously reported sequence variations were identified, including one nonconservative mutation, which demonstrated 97.3% reduction in BUP-1 activity when expressed in the RKO cell line. Conclusion Data presented in this study demonstrate that alterations of uracil catabolism are not limited to DPD and/or DHP deficiency and that inactivating mutations in the UPB1 gene might impair uracil catabolism.

Variations in the butyrylcholinesterase (BCHE) gene can prolong neuromuscular block after the administration of the neuromuscular blocking drugs succinylcholine and mivacurium. The most frequent variations in the BCHE gene are the atypical (A) and Kalow (K) variants. We, therefore, developed a detection approach for these most common variants based on the method of denaturing high-performance liquid chromatography (dHPLC).Forty-six subjects were included. Sixteen were known to carry either the normal sequence or different combinations of A- and K-variants and 30 test subjects were randomly selected from the general population. Three samples of the 16 were used to establish the dHPLC protocols. Results from dHPLC were blindly compared with the automated sequencing data.All A- and K-variants in their heterozygous and homozygous forms were unequivocally identified by dHPLC. For all 86 tested alleles, dHPLC results were 100% concordant with DNA sequencing results. Additional genetic variations were also detected, which included G344A encoding for the silent variant (S) and a known polymorphism, A1914G.The described dHPLC protocols offer a rapid and accurate screening method for the most common variants of BCHE. This screening tool might be useful in pharmacokinetic studies investigating drugs metabolized by BCHE, such as mivacurium and succinylcholine.

BACKGROUND Checkpoint kinase 2 (CHK2) is a DNA damage-activated protein kinase which is involved in cell cycle checkpoint control. CHK2 gene could be a candidate gene for colorectal cancer susceptibility. But there are few systematic reports on mutation of CHK2 in colorectal cancer. METHODS The mutations of all 14 exons of CHK2 in 56 colorectal cancer cell lines were screened systematically, using denaturing high-performance liquid chromatography (DHPLC) to screen the mismatches of the CHK2 exons amplified products, and then the suspected mutant cell lines were scanned by nucleotide sequence analysis. RESULTS VACO400 in CHK2 exon 1a was suspected to have mutation by DHPLC and confirmed by sequence, but this was nonsense mutation. C106, CX-1, HT-29, SK01, SW480, SW620 and VACO400 in CHK2 exon 1b were confirmed to have the same nonsense mutation in 11609 A > G. DLD-1 and HCT-15 in CHK2 exon 2 were confirmed to have missense mutation R145W, which was heterozygous C > T missense mutation at nucleotide 433, leading to an Arg > Trp substitution within the FHA domain. CONCLUSIONS The CHK2 mutation in colorectal cancer is a low frequency event. There are just 10 cell lines to have sequence variations in all the 14 exons in 56 colorectal cancer cell lines and only DLD-1/HCT-15 had heterozygous missense mutation. These findings may give useful information of susceptibility of colorectal cancer as single nucleotide polymorphysim.

Dihydropyrimidine dehydrogenase (DPD) deficiency accounts for approximately 43% of grade 3-4 toxicity to 5-fluorouracil. There, however, remain a number of patients presenting with 5-fluorouracil-associated toxicity despite normal DPD enzyme activity, suggesting possible deficiencies in dihydropyrimidinase (DHP), encoded by the DPYS gene, and/or beta-ureidopropionase (BUP-1), encoded by the UPB1 gene. This study investigates the role of DPYS sequence variations in individuals with unexplained molecular basis of altered uracil catabolism.This study included 219 asymptomatic healthy volunteers with known DPD enzyme activity and [2-13C]-uracil breath test (UraBT) profiles. All samples were genotyped for sequence variations in the DPYS gene using denaturing high-performance liquid chromatography (DHPLC) and Surveyor enzyme digestion with confirmation by direct sequencing. Site-directed mutagenesis and expression analysis were performed to determine the effect of the identified nonconservative mutations on DHP enzyme activity.Seven previously reported and 11 novel sequence variations were identified, including three nonconservative mutations; two of which (L7V and 1635delC) demonstrated decreased DHP activity when expressed in the RKO cell line (P=0.25). The P values were not significant due to the small sample size (n=3); however, a modified [2-13C]-uracil breath test, the 13C-dihydrouracil breath test, was administered to four volunteers to confirm that the 1635delC mutation does in fact reduce in-vivo DHP activity.Data presented in this study demonstrate that alterations of uracil catabolism are not limited to DPD deficiency, and that inactivating mutations in DHP might impair uracil catabolism in cases of normal DPD activity.

In 1992, a family with hereditary macrothrombocytopenia and progressive sensorineural hearing impairment without renal dysfunction was described. Recently, mutations in MYH9, a nonmuscle myosin heavy chain, have been found in several forms of hereditary macrothrombocytopenia.The hereditary macrothrombocytopenia and hearing loss in the previously reported family is due to a mutation in MYH9 gene.Genomic DNA was extracted from the affected proband. Mutation screening of all MYH9 coding exons was carried out using denaturing high-performance liquid chromatography. Abnormal results were followed by direct sequencing of the exon and comparison of the sequence with the normal MYH9 sequence.The results of denaturing high-performance liquid chromatography suggested a potential sequence alteration in exon 30 of MYH9. Direct sequence analysis of this exon in the affected individual identified a G to A single base pair transition at nucleotide 4270 altering codon 1424. This mutations leads to an amino acid change from aspartate (D) to asparagine (N) in the highly conserved coiled-coil domain.A single base pair transition in MYH9, resulting in an amino acid substitution D1424N, is responsible for macrothrombocytopenia and hearing loss in the kindred under study. The presence of hearing impairment and the absence of renal symptoms, as reported in other families with the same mutation MYH9, further highlights the role of genetic background in expression and modification of the affected phenotype.

PURPOSE Investigators have noted that primary open-angle glaucoma (POAG) in West Africa has an earlier age of onset and appears to be more clinically severe than in the United States and Europe. Primary open-angle glaucoma patients with mutations in myocilin have a similar phenotype. Therefore, we investigated the role of mutations in myocilin in patients with POAG in a West African population. MATERIALS AND METHODS Patients seen at the Emmanuel Eye Clinic in Accra, Ghana, were recruited for this study. Informed consent was obtained from all study patients. Glaucoma specialists from the sponsoring institution (PC, LWH, or RRA) ascertained all POAG and control patients. Age-matched unaffected controls were obtained in patients with an IOP < 22 mm Hg and normal-appearing optic nerves. PCR amplification of each of the three myocilin exons was performed. Denaturing high-performance liquid chromatography (Transgenomics Corp.) was used to detect allelic differences and samples demonstrating a mobility shift were sequenced in both directions. RESULTS Ninety unrelated affecteds with POAG and 76 control patients were recruited. Four individuals with severe POAG were found to have novel missense mutations in exon 3. Two exhibit an Asp380Asn mutation and two an Arg342Lys mutation. These changes were not detected in 152 ethnically matched control chromosomes. Fourteen affected individuals and eight controls exhibit a translationally silent polymorphism in codon 325 (Thr325Thr). CONCLUSIONS A total of 4.4% of patients with POAG have novel disease-associated mutations in myocilin. Mutations in myocilin appear to play a limited role in the pathogenesis of POAG in this region of West Africa.

Sulfotransferase 4A1 (SULT4A1) is a novel sulfotransferase expressed almost exclusively in the brain. The gene is located on chromosome 22q13.3, a region implicated in predisposition to schizophrenia. Recently, a variable microsatellite region located upstream of SULT4A1 was found to be associated with an increase in schizophrenia risk. We hypothesised that if functional dysregulation of SULT4A1 was involved in the aetiology of schizophrenia, then genetic variants in the coding sequence of SULT4A1 might be identified in cases compared with controls. To test this, we carried out a mutation analysis of the coding region (exons 2-7) in 71 Australian schizophrenia cases and 69 controls. We found no mutations, either synonymous or nonsynonymous, in either cohort. However, intronic variants (IVS5+12 C>T and IVS5+28 G>C) were identified, the frequency of which was not statistically different between cases and controls. The lack of polymorphisms in the coding region of the SULT4A1 gene is highly unusual and, along with its high conservation between species, suggests that SULT4A1 may have an important function in vivo. However, our findings do not support the hypothesis that germline mutations in the coding region of SULT4A1 contribute to susceptibility to schizophrenia.

Hereditary spastic paraplegias (HSPs) are characterized by progressive lower extremity spasticity due to an axonal degeneration of motor and sensory neurons. We report a four-generation pedigree segregating an autosomal dominant phenotype for HSP and showing a linkage to the SPG10 locus, coding for Kinesin family member 5A. Subsequent to a denaturing high performance liquid chromatography (dHPLC) mutation screening we found a new missense mutation 838C>T (R280C) at an invariant arginine residue in a region involved in the microtubule binding activity.