Polymerase Chain Reaction-single Strand Conformation Polymorphism Method Research Articles

Frequent Novel Variations Within MSH2 and MLH1 Genes in a Subset of Iranian Families With Hereditary Non-Polyposis Colorectal Cancer Shadi

Abstract- Hereditary non-polyposis colorectal cancer (HNPCC) is the most frequent autosomal dominant predisposition for development of colorectal cancer (CRC) caused by germline defects in mismatch repair (MMR) genes. Current study was aimed to find genetic variations in MSH2 and MLH1 genes and their correlation with the serum levels of Carcinoembryonic Antigen (CEA) in seven Iranian HNPCC families. Seven unrelated Iranian families including 11 HNPCC patients and 7 affected family members were selected. They were initially screened for mutations in exons 7 of MSH2 and exon 15 of MLH1 gene through polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Positive PCR results were further analyzed through exon sequencing. Serum CEA level was determined using the ELISA test. PCRSSCP was positive in 8 out of 18 patients (44%) for exons 7 of MSH2 gene, whereas two samples (11%) demonstrated to bear a mutation in exon 15 of the MLH1 gene. Sequencing analysis of both amplified exons in positive and negative samples have confirmed no mutation in negative samples while revealed 5 and 7 novel mutations in exons 7 and 15, respectively. The mean serum concentration of CEA had a significant difference between HNPCC patients and their healthy family members. Our results demonstrated that the PCR-SSCP method has high specificity and sensitivity in the first step of mutation screening of HNPCC families. High frequency of novel alterations found in the current assay may revise the mutation screening of MSH2 and MLH1 genes and abet further assessment of their frequency among individual HNPCC patients.

Heterozygous p.I171V mutation of the NBN gene as a risk factor for lung cancer development.

The NBN gene, also known as NBS1, is located on the chromosome band 8q21.3, and encodes a 754-amino acid-long protein named nibrin. This protein is a member of the MRE1-RAD50-NBN nuclear complex, and is involved in numerous cell processes essential for maintaining genomic stability. Heterozygous variants in the NBN gene, including p.I171V, c.657del5 and p.R215W, have been described as risk factors for the development of several malignancies. However, there is no report regarding the association of these mutations with lung cancer thus far. Therefore, the present study aimed to evaluate whether there is an association between the heterozygous p.I171V, c.657del5 and p.R215W variants of the NBN gene and the risk of developing lung cancer. The frequency of these variants was estimated in a group of 453 adults diagnosed with non-small cell lung cancer (NSCLC) and in healthy controls (2,400 for p.I171V, 2,090 for c.657del5 and 498 for p.R215W). The p.I171V variant was assessed by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR) products, using MunI (MfeI) restriction enzyme, whereas the c.657del5 and p.R215W variants were assessed by the PCR single-strand conformation polymorphism method. A significantly increased risk of developing lung cancer was observed for the p.I171V variant, which was present in 17 (3.75%) of the 453 cases of lung cancer and in 12 (0.5%) of the 2,400 healthy individuals (odds ratio, 7.759; P<0.0001). The results obtained indicated an association between the p.I171V mutation and the development of lung cancer. Therefore, this variant may be considered a risk factor for NSCLC. Prospective studies with larger groups of patients may reveal the potential impact of the p.I171V variant in the occurrence of lung cancer.

Analysis of methanogens in the bovine rumen by polymerase chain reaction single-strand conformation polymorphism

The polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method reported by Schwieger and Tebbe (1998) was used to analyze the diversity of methanogens inhabiting the rumen. Partial 16S rRNA gene fragments were amplified from DNA extracted from rumen contents by PCR with archaea-specific primers, Ar1000F and Ar1500R, or methanogen-specific primers, M301F and M915R, with one primer phosphorylated at the 5′ end. The amplified DNA fragments were analyzed by SSCP gel electrophoresis after the phosphorylated strands of the PCR products were digested with λ exonuclease. When we analyzed samples collected from the six Holstein cows used in a previous study, in which cows were given feed with or without α-cyclodextrin-horseradish oil complex (CD-HR), nine and six bands were identified in the profiles generated by PCR products amplified with archaea-specific and methanogen-specific primers, respectively. While dendrogram analysis based on SSCP gel profiles found that the methanogens from each rumen showed a particular composition of methanogens, the profiles of the methanogens isolated from two of three cows fed with CD-HR fell into the same branch in the dendrogram constructed from the profiles. Therefore, this study demonstrates the potential of the PCR-SSCP method in the methanogenic community analysis of the rumen and in investigating changes in the methanogenic community due to the addition of CD-HR to the rumen.

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

Co-mutation of p53,K-rasgenes and accumulation of p53 protein and its correlation to clinicopathological features in rectal cancer

To determine the accuracy of p53 gene mutations predicted by overexpression of p53 protein immunohistochemically, and to investigate the co-mutation of p53 and K-ras genes in rectal cancer and its effect on promoting malignant biologic behaviors of tumors. Ninety-seven specimens of rectal cancer were surgically resected in our hospital from August 1996 to October 1997. The hot mutation areas of p53 gene (in exons 5-8) and K-ras gene (in codon 5/12 and 13) were detected with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and overexpression of p53 protein was detected with immunohistochemistry (IHC) in the 97 specimens of rectal cancer. Correlation between gene mutations and tumor clinicopathologic factors was studied, and survival analysis was penfomed as well. There were 36 cases of p53 gene mutations in 61 p53 protein positive cases, and 21 cases of p53 gene non-mutation in 36 p53 protein negative cases respectively. The coincidence rate of p53 gene mutation by IHC method with PCR-SSCP method was 58.8% (57/97). The mutation rate of p53 gene was 52.6% (51/97), while K-ras gene mutation was observed in codons 12 and 13 in 61 cases with a mutation rate of 62.9% (61/97). Single gene mutation of p53 or K-ras was found in 32 cases. Both p53 and K-ras gene mutation were found in 48 cases. Statistical analysis showed that p53 and K-ras gene mutations were not related to the clinicopathologic factors, including tumor size, gross tumor type, histological classification, differentiation, invasion to intestinal veins, lymphatics and nerves, invasive depth to wall, lymph node metastasis, and Dukes' stages (P>0.05). The survival in patients with no gene mutation, single gene mutation and both gene mutations were similar (P>0.05). IHC has a certain false positive and false negative rate in detecting p53 gene mutations. Malignant biological behaviours of rectal cancer are not enhanced by p53 and K-ras gene mutations. Co-mutation of p53 and K-ras gene has neither synergic carcinogenesis-promoting effect, nor prognostic effect on rectal cancer.

Assessment of the Mutations of p53 Suppressor Gene and Ha- and Ki-ras Oncogenes in Malignant Mesothelioma in Relation to Asbestos Exposure: A Study of 12 American Patients.

In our previous study, we found no genetic alteration in exons 1 and 2 of Ha- and Ki-ras oncogenes nor in exons 5 to 9 of the p53 suppressor gene in seven Japanese malignant mesothelioma patients exposed to asbestos. To examine further whether malignant mesothelioma due to asbestos has genetic alterations in the p53 suppressor gene and in Ha- and Ki-ras oncogenes, we analyzed point mutations of these genes in paraffin embedded operative open biopsied samples of the primary tumor of malignant mesothelioma in twelve American patients. The genetic analysis was conducted by the PCR-SSCP (polymerase chain reaction single-strand conformation polymorphism) method in all patients and by sequencing analysis of DNA bases in the two patients with suspected gene mutation. The analysis of the p53 suppressor gene showed an amino acid converting mutation of exon 7 in one patient and a polymorphism of exon 6 in another patient; the former patient was a heavy smoker with a biphasic cell type. No genetic alteration was found in exons 1 and 2 of Ha- and Ki-ras oncogenes in any of the patients. The results suggest that the effects of asbestos on the p53 suppressor gene and Ha- and Ki-ras oncogenes in malignant mesothelioma are negligible. Further studies are needed to examine whether the observed mutation of the p53 suppressor gene is due to the combined effects of asbestos and smoking or to other unknown factors.

MHC (major histocompatibility complex)-DRB genes and polymorphisms in common marmoset.

A New World monkey, the common marmoset (Callithrix jacchus), will be used as a preclinical animal model to study the feasibility of cell and gene therapy targeting immunological and hematological disorders. For elucidating the immunogenetic background of common marmoset to further studies, in the present study, polymorphisms of MHC-DRB genes in this species were examined. Twenty-one Caja-DRB exon 2 alleles, including seven new ones, were detected by means of subcloning and the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) methods followed by nucleotide sequencing. Based on the alignment of these allele sequences, we designed two pairs of specific primers and established a PCR-SSCP method for DNA-based histocompatibility typing of the common marmoset. According to the family segregation data and phylogenetic analyses, we presumed that Caja-DRB alleles could be classified into five different loci. Southern blotting analysis also supported the existence of multiple DRB loci. The patterns of nucleotide substitutions suggests that positive selection operates in the antigen-recognition sites of Caja-DRB genes.

The cardiac beta-myosin heavy chain gene is not the predominant gene for hypertrophic cardiomyopathy in the Finnish population.

The aim of the study was to screen 36 unrelated patients with hypertrophic cardiomyopathy (HCM; 16 familial and 20 sporadic cases) from a genetically homogeneous area in eastern Finland for variants in the cardiac beta-myosin heavy chain (beta-MHC) and alpha-tropomyosin (alpha-TM) genes. Mutations in the beta-MHC and alpha-TM genes have been reported to be responsible for 30% to 40% and less than 5% of familial HCM cases, respectively. However, most genetic studies have included patients from tertiary care centers and are subject to referral bias. Exons 3-26 and 40 of the beta-MHC gene and the nine exons of the alpha-TM gene were screened with the PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) method. Linkage analyses between familial HCM locus and two intragenic polymorphic markers (MYO I and MYO II) of the beta-MHC gene were performed in 16 familial HCM kindreds. A previously reported Arg719Trp (arginine converted to tryptophan in codon 719) mutation of the beta-MHC gene was found in one proband and two relatives. In addition, a novel Asn696Ser (asparagine converted to serine in codon 696) substitution was found in one HCM patient. No linkage between familial HCM and the beta-MHC gene was observed in 16 familial kindreds. A previously reported Aspl75Asn (aspartic acid converted to asparagine in codon 175) mutation of the alpha-TM gene was found in four probands and 16 relatives. Mutations in the beta-MHC and alpha-TM genes accounted for 6% and 25% familial HCM cases and 3% and 11% of all cases, respectively. Our results indicate that the beta-MHC gene is not the predominant gene for HCM in the Finnish population, whereas HCM caused by the Aspl75Asn mutation of the a-TM gene is more common than previously reported.

Nine novel germline mutations of STK11 in ten families with Peutz-Jeghers syndrome.

Peutz-Jeghers Syndrome (PJS) is an autosomal dominant hereditary disease characterized by hamartomatous polyposis involving the entire bowel. Recently STK11, a gene bearing a mutation responsible for PJS, was isolated. We investigated the entire coding region of STK11 in 15 unrelated PJS families by the PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) method and PCR-direct sequence analysis, and found nine different, novel mutations among ten of those families. One nonsense mutation and five different frameshift mutations (two families carried the same mutation), all of which would cause truncation of the gene product, were found in seven families; mutations found in five families were clustered within exon 6. Among these five mutations, three occurred at the mononucleotide-repeat region (CCCCCC) of codons 279-281, suggesting that this region is likely to be a mutational hotspot of this gene. One of the remaining three families carried a 3-bp in-frame deletion that would eliminate an asparagine residue within a kinase domain of the product; the other two carried intronic mutations at or adjacent to the consensus dinucleotide sequences of splice-acceptor or -donor sites, which were likely to lead to aberrant splicing.

Use of polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis to detect a point mutation in the catalase-peroxidase gene (katG) ofMycobacterium tuberculosis

We have previously reported that a significant percentage (44%) of isoniazid-resistantMycobacterium tuberculosisstrains carry an arginine to leucine mutation in codon 463 (R463L) in the catalase-peroxidase gene (katG). For the current study, we compared the utility of one mutation screening method, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, with a reference method, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), to detect this mutation. The PCR-SSCP method detects mutations by electrophoretic mobility shifts of single-stranded DNA in nondenaturing polyacrylamide gels. The RFLP method detects a loss in anMspI restriction site which occurs when the R463L is present. Eighty oneM. tuberculosisstrains, including the wild type strain H37Rv, with isoniazid susceptibility in the range <0·12 to >32 μg ml−1were evaluated. The results for the PCR-SSCP method were in complete agreement with the PCR-MspI RFLP reference method. Of 81M. tuberculosisstrains analysed, 13 showed mobility shifts by the PCR-SSCP method and all of those strains carried the R463L as detected by the PCR-MspI RFLP method. All of the remaining 54 strains had PCR-SSCP and PCR-MspI RFLP results identical to the wild type (R463)M. tuberculosisstrain, H37Rv. It is concluded that the described PCR-SSCP is a reliable method for screeningM. tuberculosisstrains for thekatGR463L mutation.

Quantitation of heteroplasmy of mitochondrial tRNA(Leu(UUR)) gene using PCR-SSCP.

We have devised a novel method for quantitative analysis of the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) tRNA(Leu(UUR)) mutation of mitochondrial DNA using a PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) method, and compared the results obtained using the PCR-SSCP method with those obtained using other methods including Southern blotting, last one cycle hot PCR, and conventional PCR-RFLP (restriction fragment length polymorphism). The standard curve obtained using the PCR-SSCP method is linear, with a correlation coefficient of 0.999; it was determined that this method is more accurate than other methods for quantitative analysis. The PCR-SSCP method does not require restriction digestions, thereby avoiding potential problems of partial digestions or heteroduplex formation during PCR. The method is quite simple and should have a broad range of application for quantitation of mutant mtDNAs in various mitochondrial encephalomyopathies. We applied the method for quantitation of mutant mitochondrial DNA carrying a single base substitution in the tRNA(Leu(UUR)) gene in two autopsied cases of MELAS. In both cases, the mutant mtDNA is abundantly present (82-95%) withd little variation among tissues.

DNA-based typing of human platelet antigen systems by polymerase chain reaction-single-strand conformation polymorphism method.

Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method has been established to discriminate genotypes for the human platelet antigen (HPA) systems HPA-1, HPA-2, HPA-3, HPA-4, and HPA-5. Gene fragments which contain polymorphic sequences corresponding to the HPA-1, HPA-2, HPA-3, HPA-4, and HPA-5 systems were PCR-amplified with specific primers. The amplified DNA was denatured and subjected to non-denaturing polyacrylamide gel electrophoresis followed by silver staining. The results obtained by the PCR-SSCP method were in good agreement with those of the allotypes determined by serological typing. Furthermore, the results agreed with those obtained by other DNA-based typing methods such as PCR-allele-specific restriction enzyme analysis and PCR-sequence-specific primer. These results indicate that PCR-SSCP is a simple and sensitive method for determining HPA genotypes and identifying unknown polymorphisms.

Simultaneous Use of the PCR-SSCP Method and Immunohistochemistry for Increasing the Detection Efficacy of p53 Abnormalities in Human Lung Cancer

Malignant neoplasms possess multiple genetic abnormalities. Among those, p53 gene abnormalities are the most frequent in human neoplasms. To screen for p53 abnormalities, both immunohistochemistry (IHC) and the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) methods are commonly used, but neither can detect specimens simultaneously by the two methods, 12 abnormal cases (34%) were detected by IHC and 9 abnormal cases (26%) were detected by PCR-SSCP. Six abnormal (17%) and 20 normal (57%) cases showed concordant results between the methods, although 9 cases (26%) showed discordance, including 6 IHC-positive cases (17%) and 3 SSCP-positive cases (9%). Because it is known that some p53 abnormalities are detected only by IHC or only by PCR-SSCP, discordant cases should be assessed as possessing abnormalities. Fifteen cases (43%) were finally assessed as abnormal. These results suggest that the two methods together can increase the sensitivity of screening for p53 abnormalities.