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PubMed2001

Carbamoyl phosphate synthetase I deficiency: molecular genetic findings and prenatal diagnosis.

We report a Japanese boy who died at Day 28 of life because of severe carbamoyl phosphate synthetase I (CPS1) deficiency that was proven by enzyme assay. By analysis of cDNA and genomic DNA, he was shown to be a compound heterozygote with two point mutations of the CPS1 gene, 840G>C leading to an aberrant splicing and 1123C>T (predicting Q375X). The 840G>C was a mutation described in another Japanese family. Since his parents carried each mutation heterozygously, we performed prenatal diagnosis at 16 weeks of his mother's next gestation by multiplex PCR and melting curve analysis in a single capillary containing two-color fluorescent (LC-Red 640 and LC-Red 705) probes on LightCycler. We analyzed genomic DNA extracted from amniotic cells and found that the fetus was homozygous for the wild-type alleles. At term a healthy girl was born without hyperammonemia.

Carbamoyl-Phosphate Synthase I Deficiency Disease↗

PubMed2006

First-trimester prenatal diagnosis in haemophilia A and B families--10 years experience from a centre in India.

During the last 10 years (1995-2005) we have offered diagnosis in 438 families out of 502 families referred, by chorionic villus sampling procedure between 10 and 12 weeks of pregnancy in severe haemophilia A and B families. Sixty four families could not be offered a diagnosis in the first trimester either due to non-informativeness with the available techniques or due to the absence of affected members in the family and were subsequently offered diagnosis in the second trimester by direct analysis of factor VIII/IX clotting activity and antigen in the fetal blood samples. For first-trimester diagnosis in the chorionic villus samples, we have used both direct and indirect methods, that is, intron 22 and 1 inversions in the factor VIII gene, a multiplex PCR for the detection of gross deletions in the factor IX gene and RFLP analysis using a battery of markers within and outside the factor VIII/IX gene. The informativeness of all these techniques was found to be 92.21% in case of haemophilia A and 83.91% in case of haemophilia B. We followed up 122 children out of 326 (diagnosed unaffected prenatally) after birth and the diagnosis was adequately reconfirmed by both factor assays and by DNA analysis. Only one case of misdiagnosis was detected so far (0.22%), where the diagnosis was based only on the extragenic marker of the factor VIII gene.

Chorionic Villi Sampling↗

PubMed2006

Experience in preimplantation genetic diagnosis for exclusion of homozygous alpha degrees thalassemia.

OBJECTIVE: To report our experience in preimplantation genetic diagnosis (PGD) for the exclusion of homozygous alpha degrees thalassemia. PATIENTS AND METHODS: PGD was performed on nine couples with alpha degrees thalassemia genotype undergoing assisted reproduction. Oocytes were aspirated after ovarian stimulation and fertilized by intracytoplasmic sperm injection. One or two blastomeres were biopsied from the six- to eight-cell embryo. Single cell multiplex PCR of the normal and alpha degrees thalassemia alleles was performed for first round, followed by semi-nested PCR of the respective alleles using 5'-end labelled fluorescent primers. Only those embryos with a blastomere diagnosed as having at least one normal allele were selected for transfer. RESULTS: One hundred and twenty-six blastomeres from 82 embryos were analyzed. The rates of allele dropout was 10.2% and PCR failure 12.7%. Fifty-eight embryos (70.7%) had at least one normal allele, of which 31 were transferred to 13 prepared cycles and one triplet pregnancy achieved. The triplets showed no ultrasound features of homozygous alpha degrees thalassemia at 18 weeks and were delivered in healthy condition by caesarean section at 34 weeks. Their genotypes were confirmed by cord blood analysis. CONCLUSIONS: PGD for alpha degrees thalassemia is possible by single cell PCR. The transfer and successful implantation of unaffected embryos ensure birth of disease-free babies.

Alleles↗

PubMed1995

Homozygous deletions of the MTS1 gene are rare in non-astrocytic brain tumors.

Several studies support a role for the multiple tumor suppressor gene (MTS1) in the malignant progression of different tumor types. In this study we have examined the status of the MTS1 gene in a variety of non-astrocytic tumors of the central nervous system. It was not possible, using multiplex PCR with primers for MTS1 and D9S196, a chromosome 9q marker, to demonstrate deletions of MTS1 in 59 primary non-astrocytic tumors. Two out of 5 (40%) secondary tumors showed evidence of homozygous deletion of MTS1. The results obtained here for primary non-astrocytic tumors contrast with those previously described for astrocytic tumors where a high frequency of deletions of MTS1 was associated with tumor progression.

Astrocytoma↗

PubMed2000

PPAR-gamma is selectively upregulated in Caco-2 cells by butyrate.

The peroxisome proliferator-activated receptor (PPAR)-gamma is a nuclear lipid-activable receptor controlling the expression of genes involved in lipid metabolism and adipocyte differentiation. In order to investigate the possible role of PPAR-gamma in the differentiation of intestinal epithelial cells, we examined its expression in the human colon carcinoma cell line Caco-2, which undergoes rapid cell differentiation in the presence of butyrate. PPARs were quantified on mRNA level by RT competitive multiplex PCR, the corresponding proteins were determined by Western blot. In contrast to PPAR-alpha and PPAR-beta, PPAR-gamma mRNA and protein increased significantly in butyrate-treated Caco-2 cells in a dose- and time-dependent manner. This effect was butyrate-specific, since no change in PPAR-gamma expression could be observed after incubation with propionate or valerate. Activation of PPAR-gamma by ciglitazone further increased butyrate-induced cell differentiation dose-dependently. These data demonstrate a role for PPAR-gamma in the regulation of cell differentiation in Caco-2 cells.

Alkaline Phosphatase↗

PubMed1999

Trypanosoma rangeli: discrimination from Trypanosoma cruzi based on a variable domain from the large subunit ribosomal RNA gene.

306-314. Three synthetic oligonucleotides corresponding to sequences within the D7a divergent domain of the large subunit ribosomal RNA gene have been used to amplify the total DNA of Trypanosoma rangeli and Trypanosoma cruzi, two morphologically similar protozoa with overlapping geographical distribution and hosts. The two organisms may be distinguished by the electrophoretic mobilities of their respective amplification products. For T. rangeli a 210-bp product was obtained. The presence of this fragment was confirmed in 14 T. rangeli strains. For T. cruzi two possible amplification products were originated: a 265-bp DNA fragment for strains typed as lineage 1 and a 250-bp fragment for lineage 2 strains. Eleven unidentified trypanosome stocks, recently isolated from Amazonian vectors, could be discriminated using the proposed assay. The potential field application of multiplex PCR was further demonstrated by identification of the two parasite species in samples containing intestinal tract and feces of triatomines. In the present study we have also amplified the D7a domain of several trypanosomatids employing primers complementary to the conserved flanking regions. Size and sequence polymorphisms were observed, indicating that this region could also be explored as a target for specific detection of other members of the Trypanosomatidae family.

Algorithms↗

PubMed2001

An 800-kb region of deletion at 13q14 in human prostate and other carcinomas.

Deletions of regions at 13q14 have been detected by various genetic approaches in human cancers including prostate cancer. Several studies have defined one region of loss of heterozygosity (LOH) at 13q14 that seems to reside in a DNA segment of 7.1 cM between genetic markers D13S263 and D13S153. To define the smallest region of overlap (SRO) for deletion at 13q14, we first applied tissue microdissection and multiplex PCR to detect homozygous deletion and/or hemizygous deletion at 13q14 in 134 prostate cancer specimens from 114 patients. We detected deletions at markers D13S1227, D13S1272, and A005O48 in 13 (10%) of these tumor specimens. Of the 13 tumors with deletions, 12 were either poorly differentiated primary tumors or metastases of prostate cancer. To fine-map the deletion region, we then constructed a high-resolution YAC/BAC/STS/EST physical map based on experimental and database analyses. Several markers encompassing the deletion region were analyzed for homozygous deletion and/or hemizygous deletion in 61 cell lines/xenografts derived from human cancers of the prostate, breast, ovary, endometrium, cervix, and bladder, and a region of deletion was defined by duplex PCR assay between markers A005X38 and WI-7773. We also analyzed LOH at 13q14 in the 61 cell lines/xenografts using the homozygosity mapping of deletion approach and 26 microsatellite markers. We found 24 (39%) of the cell lines/xenografts to show LOH at 13q14 and defined a region of LOH by markers M1 and M5. Combination of homozygous or hemizygous deletion and LOH results defined the SRO for deletion to be an 800-kb DNA interval between A005X38 and M5. There are six known genes located in or close to the SRO for deletion. This region of deletion is at least 2 Mb centromeric to the RB1 tumor-suppressor gene and the leukemia-associated genes 1 and 2, each of which is located at 13q14. These data suggest that the 800-kb DNA segment with deletion contains a gene whose deletion may be important for the development of prostate and other cancers. This study also provides a framework for the fine-mapping, cloning, and identification of a novel tumor-suppressor gene at 13q14.

Adult↗

PubMed2001

High-speed, multicolor fluorescent two-dimensional gene scanning.

Two-dimensional gene scanning (TDGS) is a method for analyzing multiple DNA fragments in parallel for all possible sequence variations, using extensive multiplex PCR and two-dimensional electrophoretic separation on the basis of size and melting temperature. High throughput application of TDGS is limited by the prolonged time periods necessary to complete the second-dimension electrophoretic separation step--denaturing gradient gel electrophoresis--and the current need for gel staining. To address these problems, we constructed a high-voltage, automatic, two-dimensional electrophoresis system and used this in combination with thinner gels to reduce two-dimensional electrophoresis time about 80%. Instead of gel staining, we used three different fluorophores to simultaneously analyze three samples in the same gel. These improvements greatly increase TDGS speed and throughput and make the method highly suitable for large-scale single-nucleotide polymorphism discovery and genetic testing.

Adaptor Proteins, Signal Transducing↗

PubMed1998

No evidence exists for methylation inactivation of the p16 tumor suppressor gene in ovarian carcinogenesis.

The p16ink4/CDKN2/MTS1 tumor suppressor gene encodes a cyclin-dependent kinase inhibitor which plays an important role in regulation of the G1/S phase cell cycle checkpoint. Loss of heterozygosity (LOH) at the p16 locus, 9p21, has been documented in a wide variety of tumors including ovarian carcinoma. However, inactivating mutations of the remaining allele and homozygous deletions are relatively infrequent events in primary tumors, even in cases where expression of p16 at the mRNA and protein level is clearly absent. These findings initially cast doubt on the role of p16 as a tumor suppressor gene in vivo. Recently, an alternative mechanism of p16 inactivation involving methylation of the CpG island in the 5' region of the gene has been demonstrated in a number of malignancies and cell lines. In this study we have analyzed the methylation status of four CpG dinucleotides in a panel of 23 ovarian tumors using a multiplex PCR approach to correlate our findings with the LOH data in this region. Using the microsatellite markers D9S171 and D9S1679 LOH was demonstrated in 4/22 (18%) informative cases. All 23 tumors showed no evidence of methylation at the p16 locus including the 4 tumors demonstrating LOH at 9p21. These results suggest that methylation inactivation of the p16 gene does not play an important role in ovarian carcinogenesis.

Alleles↗

PubMed1997

Differentiation of Campylobacter jejuni and Campylobacter coli by polymerase chain reaction.

A multiplex PCR assay was developed using two primer sets for the identification and differentiation of Campylobacter coli and Campylobacter jejuni. Primer Set I amplifies a 460-bp fragment present in C. coli and C. jejuni. Set II amplifies a 160-bp target unique to C. jejuni. When the assay was performed on reference strains, amplification of C. coli yielded only the 460-bp fragment. Amplification of C. jejuni generated both the 160- and 460-bp fragments. Campylobacter field strains (n = 85) isolated from raw poultry were identified by PCR and by conventional biochemical methods. Species determination by the two methods agreed for 83 of the 85 isolates examined. By PCR, 23 were identified as C. coli and 62 as C. jejuni. One isolate was unidentifiable by biochemical testing. The PCR assay identified this isolated as C. coli. In addition, one strain which was identified as C. coli by biochemical testing was determined to be C. jejuni by PCR. The PCR assay offers an alternative to traditional biochemical typing methods for the identification and differentiation of C. coli and C. jejuni isolated from poultry. It is accurate, simple to perform, and can be completed within 8 h.

Animals↗

PubMed1997

Rapid, sensitive, microbial detection by gene amplification using restriction endonuclease target sequences.

The use of primers synthesized to eight class II restriction endonuclease target sequences, from Haemophilus parainfluenzae, Escherichia coli, Staphylococcus aureus, Salmonella infantis, Rhodobacter sphaeroides, Klebsiella pneumoniae, Bacillus amyloliquefaciens and Proteus vulgaris for single and multiplex PCR identification of the organisms is discussed. Results indicate that the method is sensitive and specific enough to detect single cells and attogram amounts of target DNA. It has also been demonstrated that the primers can be used in whole cell PCR for identification and whole cell PCR product recovery could be enhanced by the addition of gelatin or DMSO to PCR reaction mixtures. Other results have indicated that the method can be used for the definite identification of specific individuals present in mixed cultures or suspensions of organisms. The applicability of the method for detection of a specific strain within a group of closely related organisms has also been investigated and for that sequence/organism the results suggest that the proposed method is indeed very specific and discriminative. It is suggested that as more information becomes available regarding such sequences and their distribution, this approach could form the basis of a widescale, rapid, simple and cheap identification and/or typing system for bacteria.

Bacteria↗

PubMed2002

SNP genotyping using a simple and rapid single-tube modification of ARMS illustrated by analysis of 6 SNPs in a population of males with FRAXA repeat expansions.

Microsatellites have been used extensively in gene mapping, linkage and association studies but with the near completion of the human genome project (HGP) single nucleotide polymorphisms (SNP) have become the marker of choice. However, for association studies to be useful large numbers of SNPs must be analysed. To make these studies cost effective a simple and non-labour intensive method for SNP genotyping is essential. This work describes a single-tube modification of the amplification refractory mutation system (Biallelic-ARMS). Control amplimers flanking the SNP were amplified in a single-tube multiplex PCR with two SNP specific primers that prime in opposite directions. The SNP allele was identified on the basis of PCR product size after gel electrophoresis. Biallelic-ARMS was used to analyse six SNPs within 300 kb of the FRAXA repeat, two from the HGP SNP Database (ATL1 and FMRb) and four novel SNPs (WEX1, WEX10, WEX17 and WEX28). The study population consisted of 649 males with a range of FRAXA (10 to >200) repeat sizes. Each SNP correlated with distinct haplogroups, as identified by DXS548, FRAXAC1 and FRAXAC2 flanking microsatellite repeat patterns and confirmed the initial choice of haplogroups for FRAXA repeat stability defined by Enniset al.

Alleles↗

PubMed1997

A search for cholera toxin (CT), toxin coregulated pilus (TCP), the regulatory element ToxR and other virulence factors in non-01/non-0139 Vibrio cholerae.

Twenty-four selected non-O1/non-O139 Vibrio cholerae strains were examined for the presence of virulence associated genes like ctxA, tcpA, toxR and the repetitive sequence (RS element). Seventeen of these were isolated from diarrhoeal stool samples while the remaining seven were of local environmental origin. Nine and four respectively of these strains were positive for ctxA and tcpA by Multiplex PCR analysis. The majority (16 out of 18 tested) of the strains (including the four tcpA + strains) contained toxR sequences as determined by another PCR assay. The presence of RS element was demonstrable in ctxA+ strains only. Interestingly, three of these non-O1/non-O139 strains were shown to contain all the three virulence associated genes (ctxA, tcpA and toxR) as well as the RS element. Two of these belonged to serogroups 037 (V2) and 064 (CG15) while the third one (V315-1) was untypable. These three strains also produced cholera toxin, expressed toxin coregulated pilus (TCP) and/or TcpA related antigens when grown under appropriate culture conditions. Southern hybridization analysis of their chromosomal DNA fragments using DNA probes representing ctxA, zot, ace and RS element revealed that the strains V2 and CG15 contained, at least, two complete copies of the CTX genetic element, while the strain V315-1 had three or more copies of the same. Presence of the RS element in these strains led to tandem duplication of the CTX genetic element in the chromosome of V2 and V315-1, but not in CG15 where the copies were likely to be present at different loci. These results also indicate the presence of additional copies of incomplete "core region' with zot and ace genes, but not ctxA, in strains V2 and CG15. The significance of these results in terms of the pathogenic and epidemic potential of V. cholerae strains is discussed.

Animals↗

PubMed2002

Increased frequency of intestinal Escherichia coli carrying genes for S fimbriae and haemolysin in IgA-deficient individuals.

Persons with selective IgA deficiency carry an increased risk of coeliac disease, inflammatory bowel disease and perhaps also gastrointestinal malignancies. Inflammatory bowel disease is associated with an increased carriage of adherent and haemolytic Escherichia coli in the intestinal microflora. This study was designed to investigate whether IgA-deficient individuals carry E. coli with virulence-associated properties in their gut flora. The last free-lying colony of E. coli isolates obtained from rectal flora of 25 IgA-deficient and 20 age-matched control individuals was assayed by multiplex PCR for genes for the following adhesins or virulence determinants: P, type 1 and S fimbriae, Dr haemagglutinin, haemolysin, aerobactin and the capsular types K1 and K5. E. coli strains from the intestinal microflora of IgA-deficient individuals more often had the gene for S fimbriae (36% of the strains compared with 0% in control subjects, P=0.003) as well as for haemolysin (40 vs 10% of the strains, P=0.040). IgA-deficient individuals had instead lower frequencies of E. coli carrying genes for type 1 fimbriae in their microflora (68 vs 90%, P=0.14). The results suggest that IgA-deficient individuals carry an increased frequency of E. coli with potentially inflammatogenic properties in their microflora, which may contribute to the development of gastrointestinal disorders such as inflammatory bowel diseases.

Adolescent↗

PubMed1992

Non-radioactive detection of the most common mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex allele-specific polymerase chain reaction.

A rapid, simple, nonradioactive method for detection of four common mutations causing cystic fibrosis (CF) has been developed combining multiplexing with allele-specific polymerase chain reaction amplification. This approach (MASPCR) provides an easy assay for direct genotyping of normal and mutant CF alleles in homozygotes and heterozygotes. The strategy involves multiplex PCR of exons 10, 11, and 21 within the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a single reaction containing three common oligoprimers and either the four normal or four mutant oligos corresponding to the delta F508, G551D, G542X, and N1303K mutations. Primers are chosen so that the size of the four PCR products differ, thereby facilitating detection on agarose gels following amplification in the same reaction. Patient samples are primed with either four normal or four mutant oligo mixtures, and PCR products run in parallel on gels to detect band presence or absence. This approach provides a simple and potentially automated method for cost-effective population screening.

Alleles↗

PubMed1996

Sequence analysis and allelic designation of the two short tandem repeat loci D18S51 and D8S1179.

This paper reports the sequences of eleven D8S1179 and twenty one D18S51 alleles. The D8S1179 alleles ranged in size from 162 bp to 202 bp and increased in size by regular 4 bp increments. They were shown to possess a compound repeat region composed of the tetranucleotides TCTA and TCTG. Alleles at the D18S51 locus ranged in size from 271 bp to 343 bp and possessed a simple repeat region composed of the tetranucleotide AGAA. The majority of alleles increased in size by 4 bp increments corresponding to the addition of one tetranucleotide repeat unit. However, three alleles differed in size by 2 bp from the 4 bp increment as a result of a dinucleotide insertion within the 3' flanking region. These alleles also exhibited an altered 3' flanking sequence in the first four nucleotides following the repeat region. The allelic designations proposed for these loci on the basis of this sequence data are currently being used in a multiplex PCR profiling system employed in a National DNA database in the United Kingdom.

Alleles↗

PubMed1994

Characterization of spontaneous and induced mutations in SV40-transformed normal and ataxia telangiectasia cell lines.

Spontaneous and induced mutations at the HPRT locus were analyzed in one normal (MRC5CV1) and one ataxia telangiectasia (AT5BIVA) SV40-transformed cell line derived from male donors. Multiplex PCR and Southern analyses revealed a high frequency of spontaneous deletion mutations that may be a consequence of the SV40 transformation. Four mutagens (ethyl methanesulfonate, bromodeoxyuridine, bleomycin, adriamycin), which differ in their types of primary DNA lesions, caused specific patterns of mutations. By using fluorescence in situ hybridization (FISH) techniques, we were able to show that more than 90% of the AT5BIVA cells contained two X chromosomes with HPRT alleles, while in more than 90% of the MRC5CV1 cells genomic hemizygosity for the HPRT gene was found. Taking into account these findings we found that the AT5BIVA cell line possesses spontaneous hypermutability as well as hypersensitivity and hypermutability to bleomycin (BLM) and adriamycin (AM). Both mutagens induced deletion mutations in both cell lines, but more complex mutations and larger deletions were found in AT5BIVA cells.

Ataxia Telangiectasia↗

PubMed2002

Detection of Shiga toxins, intimin and enterohemolysin in Escherichia coli strains isolated from children in eastern Slovakia.

Fifty Escherichia coli strains isolated from stool samples of 51 healthy children, 143 strains isolated from stool samples of 327 children with diarrhea and 24 strains isolated from stool samples of 21 children with suspected hemolytic uremic syndrome were examined for the presence of Shiga toxin-producing E. coli virulence factors (shiga toxin 1 and 2, intimin and enterohemolysin) and their genes. Vero-cell assay and latex agglutination were used for detection of Shiga toxin 1 and 2, TSB agar with washed erythrocytes was used for detection of enterohemolysin; genes encoding shiga toxin 1 and 2, intimin and enterohemolysin were detected using multiplex PCR. The presence of E. coli strains harboring genes encoding shiga toxin 1 and 2 (12 strains), intimin (34 strains) and enterohemolysin (12 strains) was demonstrated.

Adhesins, Bacterial↗