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PubMed2006

A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay.

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are caused by a 1.5-Mb duplication and a deletion at chromosome 17p11.2-12 encompassing the peripheral myelin protein 22 gene (PMP22), respectively. We developed a rapid and reliable detection system for duplications/deletions of the PMP22 gene based on measurement of gene copy number. The method involves amplification of a test locus with unknown copy number and a reference locus of known copy number by multiplex PCR (MP), followed by denaturing high-performance liquid chromatography (DHPLC) or capillary electrophoresis detection to identify single copy changes. Thirty-two patients with CMT1A, 17 patients with HNPP, and 61 unaffected individuals were analyzed. Using the same competitive MP protocol, the measured PMP22 gene dosage revealed concordant results between DHPLC and capillary electrophoresis analysis. The results of the MP/DHPLC or the MP/capillary electrophoresis assay were all confirmed by PCR-restriction fragment length polymorphism analysis. We concluded that the MP/DHPLC assay is an efficient, accurate, and reliable technique for gene dosage determination of the PMP22 gene for CMT1A duplication and HNPP deletion. This technique further extends the application of DHPLC as an alternative method for the measurement of gene amplifications and heterozygous deletions in different genetic diseases.

Base Sequence↗

PubMed2006

Inflammation as a risk factor for myocardial infarction.

Myocardial infarction (MI) is one of the common diseases whose pathogenesis includes genetic factors. To reveal genetic backgrounds of this clinically heterogeneous disorder, we started our case-control association study by examining large-scale gene-based single nucleotide polymorphism (SNP) sets for approximately 1,000 patients and controls. As a core genotyping method, a combination of multiplex PCR and Invader method was used, and after genotyping approximately 65,000 SNPs, we found two SNPs located within lymphotoxin-alpha (LTA) gene showing significant association with MI. LTA is one of the cytokines produced in the early stages of vascular inflammatory processes. These SNPs seem to be involved in inflammation by both qualitatively and quantitatively modifying the function of LTA protein, thereby conferring a risk of MI. The genetic association was further confirmed by other researchers using white European trios. To further understand the roles of LTA protein in the pathogenesis of MI, we searched for proteins that interact directly with LTA protein and identified galectin-2 protein as a binding partner of LTA protein. It is a member of galactose-binding lectin family whose function has not been well characterized. Genetic association study again revealed that an SNP in LGALS2 encoding galectin-2 was also associated with susceptibility to MI. This genetic substitution seemed to affect the transcriptional level of galectin-2, which led to altered secretion of LTA, thereby affecting the degree of inflammation. Thus, our findings indicate the importance of inflammation, especially the LTA cascade, in the pathogenesis of MI. Also, combined strategy of genetic and molecular-cellular biological approaches may be useful for clarification of the pathogenesis of common diseases in general.

Case-Control Studies↗

PubMed2002

A rapid and easy method for multiple endocrine neoplasia type 1 mutation detection using conformation-sensitive gel electrophoresis.

Until now, the study of the multiple endocrine neoplasia type 1 (MEN1) gene in patients suspected of having the disease was expensive and laborious due to the large size of the gene. We have optimized the conformation-sensitive gel electrophoresis (CSGE) technique to analyze by four rather simple multiplex PCR reactions, and a single electrophoresis run, the entire coding region of the MEN1 gene, plus the exon-intron boundaries. This improvement of the CSGE technique was confirmed as an effective procedure for screening for the MEN1 gene by detecting ten previously known MEN1 gene mutations and four polymorphisms. The MEN1 gene of 12 patients with unknown mutations was then screened, and an abnormal CSGE profile was identified in 10/12 cases. Subsequent DNA sequencing demonstrated 3 of them to be novel mutations (E45K, 4479delACAG, 6073insC) and 7 to have been previously reported; in the remaining 2 patients, we confirmed the absence of any alteration of the coding sequence of MEN1. Mutation screening of the MEN1 gene using CSGE was demonstrated to be a fast, simple, and inexpensive method to study patients suspected of having MEN1 disease.

Amino Acid Substitution↗

PubMed2007

Emergence of rare sapovirus genotype among infants and children with acute gastroenteritis in Japan.

A total of 1,154 fecal specimens from infants and children with acute gastroenteritis in five cities in Japan (Maizuru, Tokyo, Sapporo, Saga, and Osaka), collected from July 2003 to June 2005, were tested for the presence of diarrheal viruses by reverse transcriptase multiplex PCR. Overall, 469 of 1,154 (40.6%) were positive for diarrheal viruses, of which 49 (10.4%) were positive for sapovirus. The peak of sapovirus infection shifted from April-June in 2003-2004 to October-December in 2004-2005. The observations show that maximum sapovirus prevalence can occur during warmer seasons. Sapovirus was subjected to molecular genetic analysis by sequencing. The results indicated that sapovirus genogroup I was a dominant group (100%). Sapovirus strains detected in this study were further classified into four genotypes (GI/1, GI/4, GI/6, and GI/8). Of these, sapovirus GI/1 was the most predominant, followed by sapovirus GI/6; these accounted for 93% (13 of 14) and 7% (1 of 14), respectively, in 2003-2004. However, it was noteworthy that sapovirus GI/6 suddenly emerged to become the leading genotype, accounting for 77% (27 of 35) of isolates in 2004-2005. This is believed to be the first report of the changing distribution of sapovirus genotypes and of the emergence of the rare sapovirus GI/6.

Adolescent↗

PubMed2004

Determination of genetic status in a closed colony of rhesus monkeys (Macaca mulatta).

The long-term management of breeding colonies requires some measure of genetic diversity in the animal population. For the maintenance of breeding colonies of monkeys used for biomedical research, known pedigrees supply precise data to determine the genetic status of colonies. We present data of genetic analyses in an old closed colony of rhesus macaques (Macaca mulatta) that was established in 1932 with 100 animals. For more than 40 years, the animals were kept on an isolated island and, in 1980, single-male breeding groups were established. A total of 333 DNA samples of these animals were typed to 20 microsatellite markers using multiplex PCR in order to verify inbreeding coefficient (alpha) and level of heterozygosity. We found an average heterozygosity of 64% and obtained alpha=-0.03293 (+/-0.00573). Our results indicate that the reproductive strategy used was effective because consanguineous breeding was avoided. A continuous genetic program must be carried out in order to obtain better quality primates for biomedical research.

Animals↗

PubMed2006

Germline mutations of the hMLH1 and hMSH2 mismatch repair genes in Belgian hereditary nonpolyposis colon cancer (HNPCC) patients.

BACKGROUND: Hereditary nonpolyposis colon cancer (HNPCC-Lynch syndrome) is caused by mutations in genes involved in DNA mismatch repair (MMR), mostly in the hMLH1 and hMSH2 genes. The mutation spectrum in the Belgian population is still poorly documented. AIM: To report our experience on the mutation screening in Belgian familial colorectal cancer (CRC) patients, including the investigation of the pathogenicity of the missense and splice mutations. To increase the mutation detection rate by selecting the target population. METHODS: Two hundred and twenty five Belgian patients with familial clustering of CRC were genetically tested. Point mutations in the hMLH1 and hMSH2 genes were screened by denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing. Genomic deletions and duplications were assessed by multiplex ligase dependent probe amplification (MLPA) and multiplex PCR. Missense mutations were examined for pathogenicity by means of cosegregation of the mutation with the disease, microsatellite instability (MSI) in tumors, immunohistochemical staining of tumors and determination of the population frequency of the particular mutation. RESULTS: Twenty five pathogenic mutations were identified from which 16 were novel: 7 frameshifts, one in frame deletion, 5 genomic deletions, 5 splice defects, 4 nonsense (stop) mutations and 3 missense mutations which were classified as pathogenic (out of 10 missense mutations). In retrospect, a mutation detection rate of 71% was obtained if MSI was used as a supplementary selection criterion in addition to familial clustering. CONCLUSION: Different types of pathogenic mutations in the hMLH1 and hMSH2 genes were identified in a Belgian CRC group with familial clustering. The mutation detection yield drastically increased by preliminar selection of those familial CRC patients with a microsatellite instable tumor. Considerable attention went to the assessment of the pathogenicity of the missense mutations. In practice, the cosegregation with the disease was the most relevant criterion.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Accuracy of BRCA1 and BRCA2 founder mutation analysis in formalin-fixed and paraffin-embedded (FFPE) tissue.

BACKGROUND: A major limitation in counseling unaffected women from families with inherited breast and ovarian cancer is that a "true-negative" interpretation of wild type BRCA analysis of the proband cannot be inferred in the absence of demonstration of a BRCA mutation segregating in the kindred. Documentation of familial BRCA mutations from paraffin-derived DNA of deceased patients has been limited due to reports of technical complications leading to lack of reproducibility of BRCA testing of archival material. METHODS: DNA was extracted from formalin-fixed paraffin-embedded (FFPE) morphologically normal tissue of 161 blinded, coded samples from women previously genotyped for the three Ashkenazi Jewish BRCA founder mutations from lymphocyte-derived DNA. Multiplex PCR followed by denaturing polyacrylamide gel electrophoresis was performed for the three founder mutations to determine if analysis on FFPE tissue could produce results concordant with those of the lymphocyte-derived DNA. RESULTS: After disclosure of the sample codes, the results were compared with the original lymphocyte-derived DNA genotypes. Excluding one sample unevaluable due to PCR failure, there was 100% concordance of 160 genotypes (120 mutation samples) derived from DNA from archival FFPE tissue compared to peripheral lymphocytes. CONCLUSIONS: The method described reliably detected BRCA founder mutations in archival DNA derived from FFPE tissue. These results suggests that this technique may be useful in clinical settings to inform wild type BRCA results of unaffected probands, leading to avoidance of unnecessary intensified surveillance or risk-reducing surgery. With further validation this approach can also be applied to other populations where founder mutations are observed.

DNA↗

PubMed2006

Screening for Y microdeletions in men with testicular cancer and undescended testis.

PURPOSE: To investigate a possible association between testicular cancer or undescended testis and Y microdeletions. METHODS: It was designed as a retrospective clinical study. A total of 225 men with testicular cancer or undescended testis were included to study. Fertile men (n = 200) were investigated as a control. Genomic DNA, which was extracted from blood samples were investigated with a fluorescent multiplex PCR protocol for screening for Y microdeletions. RESULTS: A single STS missing was found in eight men; one from the control group (sY153), seven from the patients group. The positive cases showed a single STS missing of marker sY153 and sY139 in testicular cancer (6/185) and undescended testis (1/40) patients, respectively. CONCLUSIONS: Since no contiguous, real Y microdeletions were found in the study population, it seems that Y microdeletions are not a likely common etiological cause of poor spermatogenesis in testicular cancer and undescended testis. However, it remains to be determined whether men having a single STS missing have a risk of developing testis cancer or having undescended testis.

Adult↗

PubMed2005

Atypical molecular background of glioblastoma and meningioma developed in a patient with Li-Fraumeni syndrome.

We observed three neoplasms with completely different histologies: malignant fibrous histiocytoma (MFH), atypical meningioma (AM), and glioblastoma (GB), developing in a patient with Li-Fraumeni syndrome. By using a combined molecular approach we performed molecular characterization of all three tumours. Data obtained showed an interesting molecular background of the AM and GB. AM showed TP53mutations and a 22q loss of heterozygosity (LOH). GB showed epidermal growth factor receptor (EGFR) amplification and TP53 mutations, whereas P16, PTEN, Rbwere intact in terms of LOH and/or multiplex PCR (polymerase chain reaction) analysis. Additionally, GB has a 1q LOH, which is an extremely rare alteration in glioblastomas. Identical 1q LOH was also observed in MFH.

Adult↗

PubMed2006

Prevalence, molecular characterization and antimicrobial resistance of thermophilic campylobacter isolates from cattle, hens, broilers and broiler meat in south-eastern Italy.

Eleven cattle farms, 8 layer farms, 7 broiler farms and 30 broiler meat samples were investigated in south-eastern Italy throughout 2003 to evaluate the prevalence, the molecular type and antimicrobial resistance of thermophilic Campylobacters. A total of 398 samples were analysed. One Campylobacter isolate for each positive faecal swab and three isolates per positive broiler meat sample were selected for further analysis. Multiplex PCR was performed for species-level identification and PCR-RFLP of the flagellin A gene for genotyping. Resistance to 14 antimicrobials was studied in 188 Campylobacter isolates. Prevalence of campylobacters was high both on farms (100%) and in food samples (73%). On 4/11 cattle farms and on 10/15 poultry farms more than one species was isolated. The presence of more than one genotype was found on 8/11 cattle farms, on 10/15 poultry farms and in 8/22 Campylobacter-positive food samples. High rates of resistance to quinolone were observed: 9/31 (29%) C. jejuni bovine isolates, 4/22 (18%) C. jejuni poultry isolates, and 14/26 (54%) C. coli poultry isolates. Resistance to sulphamethoxazole-trimethoprim was also observed frequently: 18/26 (69%) of the avian C. coli strains, 25/31 (80%) of the C. jejuni strains isolated from poultry and 15/22 (68%) of those isolated from cattle were resistant. There was a significant difference between the rate of resistance to macrolides of C. coli and C. jejuni isolated in poultry, which amounted to 23% and 3%, respectively. This study provided data on the prevalence and antimicrobial resistance of thermophilic campylobacters in south-eastern Italy and confirmed that flaA-typing is an efficient tool to study the epidemiology of Campylobacter strains in short-term investigations.

Animals↗

PubMed1996

Mutation induction in gamma-irradiated primary human bronchial epithelial cells and molecular analysis of the HPRT- mutants.

We have examined various radiobiological parameters using commercially-available primary normal human bronchial epithelial (NHBE) cells, which can be subcultured more than 20 population doublings, and have established the mutation system in order to characterize the molecular changes in gamma-irradiated primary cells. The survival curve, obtained after irradiation of cells with 137Cs gamma-rays, indicates that the D0, Dq, and n values are 1.34 Gy, 1.12 Gy, and 2.3, respectively. The induction of HPRT- mutation was dose-dependent and the mutant fraction increased in a non-linear fashion. Since the doubling number of NHBE cells is limited, DNA was extracted directly from the single mutant colonies and alteration in the HPRT gene locus was analyzed using multiplex PCR technique. Among spontaneous mutants, the proportion with total and partial deletions of the gene was 10.0% (2/20) and 60.0% (12/20), respectively, while 30.0% (6/20) did not have any detectable changes in the nine exons examined. On the other hand, the fraction of total deletion increased by more than 2-fold among mutants induced by gamma-rays in that 26.3% (10/38) of them showed the total gene deletions. Twenty-five out of 38 gamma-induced mutants (65.8%) had partial deletions and 3 mutants (7.9%) had no detectable alteration. The present results showed that gamma-irradiation efficiently induced HPRT gene mutation in primary human epithelial cells and that most of the induced mutants suffered larger deletions compared to that observed in spontaneous mutants. This system provides an useful tool for determination of mutagenicity and understanding the molecular mechanisms of environmental carcinogens in primary human bronchial cells.

Bronchi↗

PubMed1996

The molecular nature of spontaneous mutations at the hprt locus in the radiosensitive CHO mutant xrs-5.

The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell, is defective in DNA double-strand break rejoining ability and in V(D)J recombination. The radiosensitivity and defective repair phenotype are complemented by the 80-kDa subunit of the Ku protein. We determined the nature of the mutations that develop spontaneously at the hprt locus in this cell line using both multiplex PCR deletion screening and DNA sequencing. Ninety-two independent spontaneous mutants were analyzed and the results were compared to the mutation spectrum of 64 previously analyzed hprt spontaneous mutants isolated from the parental CHO-K1 cell line. More than 50% of the spontaneous xrs-5 mutants had lost one or more exons while less than 25% of spontaneous CHO-K1 mutants had lost one or more exons. Most of the deletions in xrs-5 cells involved the loss of multiple exons while single exon deletions predominated in CHO-K1. There was also a nonrandom distribution of breakpoints in both CHO-K1 and xrs-5. Most of the deletion breakpoints were 3' to exon 9, around exons 4-6, or near exon 1. Although the frequency of base substitutions was lower in xrs-5, the spectrum of base substitutions was qualitatively similar to that of CHO-K1. There was no significant difference in the spontaneous mutant frequency in xrs-5 and CHO-K1. The results suggest that in certain regions of the hprt gene, base alterations can be converted to large deletions, and that alterations in the Ku protein complex can influence this process.

Animals↗

PubMed1994

Adriamycin induces large deletions as a major type of mutation in CHO cells.

Adriamycin (ADR), a commonly used cancer chemotherapy antibiotic, exhibits a variety of genotoxicities. In this study, we have examined the mutagenicity of ADR at the hypoxanthine-guanine phosphoribosyltransferase gene (hprt) in Chinese hamster ovary (CHO) cells and the xanthine-guanine phosphoribosyltransferase locus (gpt) in a pSV2gpt-transformed CHO cell line, AS52. Although ADR induced a dose-dependent increase of mutant frequency at both loci, it was more mutagenic to the gpt gene than to the hprt locus. Multiplex PCR analysis revealed that 35% of the 103 independent ADR-induced HPRT-deficient mutants carried large deletions. Among these deletion mutants, 33% were total gene deletions, 22% affected multiple exons, and 42% involved a single exon, of which most (9/15) were exon 1. The majority (63%) of ADR-induced AS52 mutants had a total deletion of the gpt gene. These observations indicate that ADR induces large deletions as a major type of gene mutation in mammalian cells, suggesting the involvement of reactive oxygen species as one mutagenic pathway in the mutagenesis of ADR.

Animals↗

PubMed1996

Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs.

Adenoviruses and herpes simplex virus (HSV) can cause clinically indistinguishable episodes of acute eye disease. Adenovirus infection is associated with nosocomial outbreaks and HSV may result in episodes of recurrent ocular inflammation. In a comparison of multiplex PCR for the two viral DNAs and virus isolation in cell culture, identical results were obtained for 18 of 20 specimens (positive for adenovirus in 5, HSV in 5, and negative in 8). One specimen was falsely negative for each viral DNA. Inclusion of human beta-globin primers in the adenovirus-HSV reaction was precluded by a consequential 10--100-fold reduction in sensitivity for the two viral targets and by the failure of beta-globin DNA amplification at the annealing temperature (45 degrees C) required to ensure detection of adenoviruses of serotypes 7 and 11 with the selected adenovirus primers. A single-target beta-globin PCR gave positive results with 19 of the 20 specimens prepared by treatment with proteinase K lysis buffer, indicating the effectiveness of this simple DNA extraction procedure. Nonetheless, the availability of effective antiviral therapy for HSV made monitoring for extraction failure using human primers crucial to avoid false-negative results for HSV DNA. Adenovirus-HSV PCR has considerable potential for the rapid diagnosis of viral eye disease particularly if beta-globin primers can be included in the reaction.

Adenovirus Infections, Human↗

PubMed1995

Analysis of pfmdr1 and drug susceptibility in fresh isolates of Plasmodium falciparum from subsaharan Africa.

Resistance of Plasmodium falciparum to many therapeutic agents is an increasing problem in most endemic areas. The role of the mdr-like gene products of P. falciparum in resistance to quinoline-containing compounds is not clear. The purpose of this study was to further examine the role of pfmdr1 in drug resistance in fresh clinical isolates originating from Africa. Drug susceptibility testing (chloroquine, mefloquine, halofantrine and quinine) and a molecular analysis of pfmdr1 was completed for 51 fresh clinical isolates. A statistical association between the chloroquine sensitivity phenotype and an intragenic allele of pfmdr1 was noted at a position, amino acid 86, which was previously associated with chloroquine resistance. There was little variation in the other intragenic alleles previously associated with chloroquine resistance. No correlation between pfmdr1 intragenic allelic variation and susceptibility to mefloquine, halofantrine or quinine was found. There was no association between gene copy number of pfmdr1 and any drug resistant phenotype in an analysis of selected isolates. This, along with other data, suggests that mefloquine resistance may have arisen by two different mechanisms in African and Southeast Asian isolates. Much more variability in the polyasparaginated region of the pfmdr1 gene was noted in this study than previously reported. In addition, fingerprint analysis using multiplex PCR revealed considerable genetic variability among these isolates.

Africa South of the Sahara↗

PubMed1991

Nearly 80% of cystic fibrosis heterozygotes and 64% of couples at risk may be detected through a unique screening of four mutations by ASO reverse dot blot.

A technique allowing the simultaneous screening of the four main CF mutations in the French population (delta F508, delta I507, G542X, S549N) has been developed by means of allele sequence-specific oligonucleotide (ASO) reverse dot blot. Using a strategy proposed by R. K. Saïki et al. (1989, Proc. Natl. Acad. Sci. USA 86: 6230-6234) for HLA-DQA, the seven ASOs for normal and mutant CF alleles were given a homopolymer T tail with terminal deoxyribonucleotidyltransferase and then immobilized on a nylon membrane. T-tail homopolymers were preferentially bound to the nylon, leaving the specific ASO sequences free to hybridize with amplified and radiolabeled exons 10 and 11 of a patient. These exons were simultaneously coamplified by a multiplex PCR and radiolabeled by random priming. ASO reverse dot blot currently appears to be the most efficient, rapid, and economic means of screening the population for CF mutations. This screening can detect nearly 80% of carriers and 64% of couples at risk and could prevent the birth of CF-affected infants in these families.

Alleles↗

PubMed1995

Kainate receptor subunits expressed in single cultured hippocampal neurons: molecular and functional variants by RNA editing.

To determine the kainate receptor subunits that are found in native kainate receptors, we have applied a multiplex PCR of cDNAs reverse transcribed from mRNA harvested from single cultured hippocampal neurons after electrophysiological recording. We found that all the cells showing rapidly desensitizing currents in response to kainate express the GluR6 subunit mRNA, and that some of them also express the GluR5 subunit mRNA. No GluR7, KA-1, or KA-2 subunit mRNAs were detected. Analysis of the editing sites of the GluR6 mRNA demonstrated that the three editing sites present in these subunits are edited to a different extent. Predominant expression of the unedited variant (Q) was observed, but edited and unedited variants may coexist in the same cell. In addition, we show that the Q/R site from the GluR6 subunit controls functional properties of native kainate receptors.

Animals↗

PubMed1993

Very small dystrophin molecule in a family with a mild form of Becker dystrophy.

The genetic defect in a family with a mild form of Becker dystrophy was characterized by immunocytochemical, immunoblot and genomic DNA analysis in two patients and a carrier. Immunocytochemical localization on muscle preparations with a series of antibodies against different regions of the dystrophin molecule showed normal dystrophin expression with all the antibodies except anti-30 kDa antiserum. In the carrier's muscle, mosaicism was observed only with the anti-30 kDa. Immunoblot analysis revealed a band of about 250 kDa in the patients' muscles and a double band of normal and of reduced weight protein in carrier muscle. In the patients Multiplex-PCR (M-PCR) and Southern blot revealed deletions from exon 13 to exon 41. The study confirms that very mild Becker muscular dystrophy can be associated with a large intragenic deletion from the dystrophin gene.

Adult↗