摘要:

BackgroundOral lichen planus (OLP) is a chronic relapsing cell-mediated condition of unknown etiology. The purpose of this study was to ascertain if the human herpesviruses (HHVs) or human papillomaviruses (HPVs) act as possible factors or co-factors in the pathogenesis of OLP.MethodsThirty-eight histologically confirmed OLP and 20 normal control buccal mucosa tissue samples were analyzed. Polymerase chain reaction analysis was employed to detect members of the HHV and HPV families.ResultsThe Epstein-Barr virus and HHV-7 were detected in a small percentage of tissue samples. However, HPV-16 was detected in 26.3% of OLP samples and 0% of the normal control tissues. Theepidermodysplasia verruciformis-related HPV types were detected in 42% of OLP samples and 45% of normal control samples.ConclusionThe results of this study do not suggest a causative role for members of the HHV family in the pathology of OLP. However, a statistical association was found between HPV-16 presence and OLP.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundNumerous DNA-based tests are currently in use or under development for the detection of mutations associated with disease. Most of the current methods use PCR amplification technologies and detection after separation or chromatography of the products. We have developed a panel of standard reference materials consisting of 12 plasmid clones containing a 2.0kb region of theTP53gene, including exons 5–9. Eleven of these clones contain a single mutation within the mutational hot spots of theTP53gene, the twelfth is wild-type in this region of the gene. The mutations are amino acid (aa) 128: C to T; aa 175: G to A; aa 237: T to C; aa 245: G to A; aa 248: C to T; aa 248: G to A; aa 249: G to T; aa 273: C to T; aa 273: G to A; aa 282: C to T; and aa 328: T to C. These standard reference materials (SRMs), created by site-directed mutagenesis of wild-typeTP53from a human cell line, include the specific mutations most commonly found to be associated with cancer. Their use will improve disease detection by serving as validation materials to monitor errors in measurement methods, including PCR amplification, amplicon separation, and data analysis from different technology platforms.Methods and resultsThe single point mutations of the panel were validated by capillary electrophoresis single-strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, and denaturing high-performance liquid chromatography, as well as full sequence analysis of both DNA strands of the cloned material. For both heteroduplex analysis methods, the presence of the mutations was resolved for each SRM.ConclusionThe generation of a standardTP53reference panel and demonstration that the panel can successfully validate mutation detection across different mutation scanning technology platforms. Hence, this panel functions as an SRM to normalize results obtained from different laboratories using different techniques.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundA methylene tetrahydrofolate reductase (MTHFR) deficiency at siteC677Trenders the enzyme thermolabile and consequently represents a risk factor for vascular disease, neural tube defects, preeclampsia, and thrombosis. Highly specific identification techniques for genotyping are mandatory to give guidance for the diagnosis and monitoring of this deficiency.MethodsA new approach for performing genotyping has been introduced with the identification of single nucleotide polymorphisms of the human MTHFR. It is based on PCR followed by two-color cross-correlation fluorescence spectroscopy (FCS). Experiments were carried out with green- and red-tagged allele-specific primers, which were fully compatible with the two-color fluorescence cross-correlation setup at 488nm and 633nm excitation wavelengths.ResultsThe measured data of the amplification mixes (tubes) were normalized as the maximum correlation amplitude of each tube. Correlated and uncorrelated data were optically separated in the amplification mixes by their characteristic correlation times, which significantly differed from each other. The correlated data were generated in the presence of the proper mutated genotype template, whereas uncorrelated data were due to the absence of the proper genotype template. Furthermore, the specific association of the two-color fluorescence correlated signals with the target DNA was experimentally proven. Using this novel two-color cross-correlation approach, theMTHFRgenotypes, which were determined in 21 clinical samples, showed concordance with methods involving a PCR-based assay with hexachloro-6-carboxy-fluorescein (HEX)- and 6-carboxy-fluorescein (FAM)-tagged allele-specific primers and a subsequent separation step with capillary electrophoresis, yet are simpler to perform. There was no evidence of a central trend of false-positive or false-negative results. We demonstrated how the novel, ultrasensitive typing system could be applied to studies where researchers are trying to perfect their assays and are often working with the unknown, or application to problematic assays in a clinical environment for those involved in molecular diagnosis.ConclusionsWe present an alternative method to those commonly used in genotyping. Two-color cross-correlation FCS allows the detection of the fluorescence signals specifically associated with the heterozygous mutated, the homozygous mutated, and normal individuals, as exemplified in this study. The presence of nonspecific amplification products, which interfere with subsequent DNA analysis, could therefore highlight the need for two-color cross-correlation FCS as a means of discriminating between specific association of the fluorescence signals with the target DNA and DNA not related to the target.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundUniparental disomy (UPD) is defined by the inheritance of both homologous chromosomes from only one parent, resulting in an imbalance of the expression of imprinted genes. With the recent identification of several diseases associated with UPD, the diagnostic significance of this molecular finding is a focus of interest. Acrocentric chromosomes involved in Robertsonian translocations (RTs) are particularly prone to being affected by mis-segregation events, possibly resulting in UPD. While UPDs of chromosomes 13, 21, and 22 have no clinical consequences, and therefore have no diagnostic impact despite of homozygosity of recessive alleles, prenatal testing for UPDs 14 or 15 is becoming increasingly asked for.MethodsThirty-one fetuses with nonhomologous balanced RTs involving chromosome 14 were tested for UPD14 by microsatellite typing.ResultsNo cases of maternal UPD14 were detected among the 31 fetuses analyzed.ConclusionsBased on our own data from molecular testing in 31 prenatal RT cases and findings in the published literature, we delineated a risk of 0.3% for a UPD with clinical consequences for prenatally detected carriers of a nonhomologous RT. Prenatal UPD testing is not associated with any additional risk to the pregnancy once invasive prenatal testing has been carried out. However, the possibly conflicting consequences in the case of a prenatal UPD identification should be discussed in advance. Furthermore, risk figures in specific clinical cohorts, such as couples prior to intracytoplasmic sperm injection, as well as questions of prenatal diagnostic management, will be discussed.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

IntroductionEndothelial nitric oxide synthase (ecNOS) is a key regulator of vascular nitric oxide production. Polymorphism in intron 4 of theecNOSgene is implicated in cardiovascular and renal diseases. We investigated a potential involvement of this polymorphism in the development of type 2 diabetes mellitus and its renal complications.MethodsThis preliminary study involved 410 individuals with type 2 diabetes and 330 healthy control subjects. From the diabetes group 178 patients had diabetic nephropathy. All subjects were genotyped for theecNOS4polymorphism by the polymerase chain reaction (PCR) followed by gel electrophoresis. Genotype and allele frequencies were compared between diabetes patients with and without nephropathy and the control group. All calculations were performed using the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL, USA) for Windows 5.0. The chi-square test and Fisher’s exact test were used for case-control comparisons. The Kruskal-Wallis test was used for the comparison of subgroups of patients with diabetes.ResultsThe analysis revealed that patients with diabetes, regardless of their nephropathy status, were significantly different in genotype distribution and4aallele frequencies compared with controls (p < 0.05). The frequency ofaagenotype was 8.2% in diabetic patients without nephropathy, 8.4% with those with nephropathy and 1.2% in controls. The4aallele showed a significant effect on diabetic nephropathy, with odds ratio of 2.24 (95% confidence interval 1.12–3.40). There were no significant differences in the4aallele frequency between the normotensive and hypertensive patients with diabetes.ConclusionOur results suggest that theecNOSgene polymorphism can serve as a useful genetic marker of increased susceptibility to type 2 diabetes and its renal complications.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundWe report on a novel mutation (S270T) in the M2 domain of theGLRA1(α subunit of the glycine receptor) gene causing autosomal dominant hyperekplexia in a neonate, the mother and maternal uncle. All affected members showed the typical clinical features of the disorder. This novelS270T(T1188A) mutation is located in the boundary of the transmembrane M2 domain of theGLRA1protein, close to other previously reported mutations. Mutations in this ‘hot spot’ domain ofGLRA1are frequent in autosomal dominant hyperekplexia but are not usually seen in the autosomal recessive form of the disease in which both the M1 and the carboxy terminal domains have been implicated.MethodsGenomic DNA was extracted by standard procedures from peripheral blood leukocytes and exon 6 of theGLRA1gene was amplified using primers and PCR conditions. A complete sequence analysis of the fragment was performed. DNA sequences were analyzed both by direct observation of the electropherogram and by comparison with the published sequence.ResultsThe proband had metabolic acidosis, which was probably related to continuous contractions of somatic muscles and intractable hypertonia. Data seem to show a direct relationship between the mechanism of inheritance of the disorder and the location of the molecular defect. The patients showed almost all the clinical signs of hyperekplexia: exaggerated startle response, muscle hypertonia in response to unexpected tactile and/or auditory stimuli, hyperexcitability, and sudden falls.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

Maturity onset diabetes of the young (MODY) is caused by mutations in at least six different genes, including the glucokinase gene (MODY 2) and genes encoding the tissue-specific transcription factors (MODY 1 and MODY 3–6). To determine the presence of mutations in MODY 2 in four members of a family who have the clinical characteristics of MODY, we performed polymerase chain reaction and single strand conformation polymorphism screening, followed by DNA sequencing. We found a novel mutation which consisted of the deletion of a cytosine in the position 2 of the exon 5 codon 168. This mutation produced a frame shift which determines a stop codon at position 203 in exon 6. The identification of a mutation in glucokinase gene and transcription factor genes in patients with early-onset diabetes confirms the diagnosis of MODY and has important implications for clinical management.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS