Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology最新文献

Introduction: The kallikrein-kinin system plays an important role in blood pressure homeostasis and renal sodium regulation, and some studies have reported that the kinins have a protective effect against hypertension and the development of renal disease. The B2-bradykinin receptor (B2R) mediates the majority of physiological actions of bradykinin. We investigated the effect of the C181-->T polymorphism in exon 2 of the B2R gene in patients with end-stage renal disease (ESRD).

Methods: This study involved 790 patients with ESRD and 510 healthy controls. All participants were genotyped for the B2R C181-->T polymorphism by PCR followed by digestion of a PCR product with TaqI restriction endonuclease. DNA fragments were separated by agarose gel electrophoresis. Genotype and allele frequencies were compared between the groups. All calculations were performed using SPSS 5.0 for Windows.

Results: B2R genotype distribution in patients and controls was in accordance with Hardy-Weinberg equilibrium. The frequency of the T allele was higher in ESRD patients than in controls. The significant difference was observed in the age at onset of renal disease; for patients with the T allele the mean age at onset was 36.8 years, compared with 52.4 years for those carrying only the C allele (p<0.001). The frequencies of the T allele and carrier genotypes were not associated with gender, presence of hypertension, or underlying kidney disease.

Conclusion: Our results suggest that the B2R polymorphism has a potential role in the earlier development of chronic renal failure in susceptible individuals. We did not confirm the previously published reports that the B2R gene polymorphism has a protective role in the development of ESRD.

Introduction: Genetic factors in the immune system are widely suspected to have a role in the etiology of glaucoma. In this study, we evaluated the association between primary open-angle glaucoma (POAG) and the transporter associated with antigen processing (TAP) gene polymorphisms. The TAP gene polymorphisms we evaluated were TAP1-1 codon 333 A/G (Ile-Val), TAP1-2 codon 637 (Asp-Gly), TAP2-1 codon 379 (Val-Ile), TAP2-2 codon 665 (Thr-Ala) and codon 687 (Stop-Gln), and TAP2-3 codon 565 (Ala-Tht). Due to the lack of predictive markers for glaucoma, many glaucoma patients are only diagnosed when their visual acuity and visual field has irreversibly deteriorated. Our aim was to confirm whether or not the TAP1 and TAP2 genes can be used to identify suspectability to glaucoma.

Methods: Sixty-six patients with POAG and 105 healthy volunteers were enrolled in this case-control study. We resolved the TAP1 and TAP2 gene polymorphisms by PCR-based analysis.

Results: There was a significant difference in the distribution of TAP1-1 codon 333 and TAP1-2 codon 637 gene polymorphisms (p = 0.0375 and 0.01, respectively) between POAG patients and healthy controls. However, there was no significant difference between the two groups in TAP2-1 codon 370, TAP2-2 codon 665, and TAP2-3 codon 565 (p = 0.273, 0.19, and 0.131, respectively). In TAP1-1 codon 333, there was a significant difference between the "GG" homozygote and "GA" heterozygote (OR 4.32; 95% CI 1.336, 13.969). In TAP1-2 codon 637, there was a significant difference between the "GG" homozygote and "GA" heterozygote (OR 15; 95% CI 1.733, 129.860). There was also a significant difference between "GG" homozygote and "AA" homozygote (OR 10.8; 95% CI 1.286, 91.880). Therefore, TAP1-1 codon 333 and TAP1-2 codon 637 gene polymorphisms were useful genetic markers of POAG. The prevalence of the TAP1-2 "GG" homozygote differs significantly between POAG patients and healthy controls, although in the alleles of the both TAP genes, there were no significant differences between the two groups.

Conclusion: The immune system acts as an arbiter to help determine whether under stress a neuronal cell will survive or sacrifice itself to injuries. TAP1-1 and TAP1-2 play an important role in the immune system. TAP1-1 and TAP1-2 gene polymorphisms may, by way of post-transcriptional changes and altered regulation of gene expression, be involve the immune system in the development of POAG.

Background: Approximately one-third of new cases of Duchenne muscular dystrophy (DMD) can be attributed to sporadically arising new mutations, however in the majority of cases the DMD mutation has been inherited from the mother. These female carriers can have either a constitutive or mosaic mutation.

Aim: The aim of this study was to determine the segregation of the at-risk haplotype and to find a deletion in the dystrophin gene of patients.

Method: We analyzed individuals from two families with a history of DMD in order to predict the carrier status of related females. In one of these cases the mother had two affected sons, while in the other one son and two grandchildren were affected; therefore we predict that the mother would be an obligatory carrier.

Results: Haplotype analysis of the DMD loci revealed that in the two families both the healthy and affected brothers had inherited the same X maternal chromosome. However, the affected brother carried a deletion, which was absent in the unaffected sibling.

Conclusion: These findings suggested that the mothers in the two families were germline mosaics for the DMD gene. The results of this study demonstrate the usefulness of the methodology that combine the haplotype analysis with the identification of the mutation in order to detect hidden germline mosaicisms and, thus, improve genetic counseling.

The pace of development in novel technologies that promise improvements in the early diagnosis of disease is truly impressive. One such technology at the forefront of this revolution is mass spectrometry. New capabilities in mass spectrometry have provided the means for the development of proteomics, and the race is on to find innovative ways to apply this powerful technology to solving the problems faced in clinical medicine. One area that has garnered much attention over the past few years is the use of mass spectral patterns for cancer diagnostics. The use of these so-called 'proteomic patterns' for disease diagnosis relies fundamentally on the pattern of signals observed within a mass spectrum rather than the more conventional identification and quantitation of a biomarker such as in the case of cancer antigen-125- or prostate-specific antigen. The inherent throughput of proteomic pattern technology enables the analysis of hundreds of clinical samples per day. Currently, there are two primary means by which proteomic patterns can be acquired, surface-enhanced laser desorption/ionization (SELDI) and an electrospray ionization (ESI) method that has been popularized under the name, OvaCheck. In this review, an historical perspective on the development of proteomic patterns for the diagnosis of early-stage cancers is described. In addition, a critical assessment of the overall technology is presented with an emphasis on the steps required to enable proteomic pattern analysis to become a viable clinical tool for diagnosing early-stage cancers.

Background: Differences in gene expression are frequently encountered in malignant tissues, and have been intensively studied as they can reflect different experimental or clinical conditions. Quantification of the often subtle changes in messenger RNA content is performed through comparison with the expression of endogenous controls. The appropriate choice of these endogenous controls (e.g. housekeeping genes) is critical for meaningful quantitative RNA analysis. The most important characteristics of housekeeping genes are that they are present in all cells and that their expression levels remain relatively constant in different experimental conditions. However, no single housekeeping gene always manifests stable expression levels under all experimental conditions. Therefore, it is necessary to characterize the suitability of various housekeeping genes to serve as internal RNA controls under particular experimental conditions where transcription effects are being tested.

Aim: It was the aim of this study to determine the validity of a number of housekeeping genes for their use as internal standards in cancer research.

Methods: The expression of the housekeeping genes porphobilinogen deaminase (PBGD) and mitochondrial ATP synthase 6 (mATPsy6), were compared with the expression of the more commonly used glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We examined a number of cell lines and tumor versus matched normal tissue samples using real-time quantitative (RTq)-PCR.

Results: Our findings suggest that in cell lines, all three of the studied housekeeping genes can be used as an internal control. When comparing tumor tissue samples with matched normal tissue samples, we validated mitochondrial ATPsy6 (mATPsy6) as the best choice for a housekeeping gene.

Conclusion: Since gene expression studies are becoming increasingly important in the clinical environment, especially in cancer diagnosis and treatment, the use of an reliable housekeeping gene in these studies to normalize gene expression is essential. We conclude that a bad choice of housekeeping gene may lead to errors when interpreting experiments involving quantitation of gene expression. Our study demonstrated the usefulness of mATPsy6 as an endogenous control when comparing tumor tissue samples with normal tissue samples.

Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumor. It is usually asymptomatic in the early stages and tends to be intravascularly and intrabiliary invasive. Therefore, most patients present with incurable disease at the time of detection and early diagnosis of HCC is critical for a good prognosis. The imaging-based diagnosis of small tumors is relatively inaccurate, as cirrhotic and dysplastic nodules mimic HCC radiologically. The availability of a suitable serological marker to distinguish between HCC and benign liver lesions would, therefore, be very useful for early diagnosis. The only serological marker currently widely used for the diagnosis of HCC is alphafetoprotein (AFP). However, the sensitivity of this marker is limited (41-65%). Given the high heterogeneity of HCC, it is currently thought that an optimal serological test for HCC will be based on the simultaneous measurement of two or three highly specific serological markers.Several laboratories have recently reported that glypican-3 (GPC3), a membrane-bound proteoglycan, is expressed by a large proportion of HCCs, but is undetectable in normal hepatocytes and non-malignant liver disease. Furthermore, various studies demonstrated that GPC3 could be used as a serological test for the diagnosis of patients with HCC. Although the specificity of the test was very high in the context of a population with chronic liver disease, the sensitivity was limited (within the same range as AFP). Interestingly, in most cases, elevated GPC3 values did not correlate with elevated AFP values. As a consequence, the serological level of at least one of the two markers was elevated in a large majority of HCC patients. These results suggest that the sensitivity of the diagnostic test can be significantly improved without compromising specificity with the simultaneous measurement of both GPC3 and AFP.

Background: Hyperekplexia, also known as startle disease or stiff-person syndrome, is a neurological condition characterized by neonatal hypertonia and a highly exaggerated startle reflex. Genetic studies have linked mutations in the gene encoding glycine receptor alpha1 (GLRA1) with hereditary hyperekplexia.

Methods: We analyzed four Turkish families with a history of hyperekplexia. Genomic DNA was obtained from members of these families, and the entire coding sequence of GLRA1 was amplified by PCR followed by the sequencing of PCR products. DNA sequences were analyzed by direct observation using an electropherogram and compared with a published reference sequence.

Results: We identified three novel mutations in GLRA1. These included a large deletion removing the first 7 of 9 exons, a single-base deletion in exon 8 that results in protein truncation immediately after the deletion, and a missense mutation in exon 7 causing a tryptophan-to-cysteine change in the first transmembrane domain (M1). These mutant alleles have some distinct features as compared to previously identified GLRA1 mutations. Our data provides further evidence for mutational heterogeneity in GLRA1. The new mutant alleles reported here should advance our understanding of the etiology of hyperekplexia.

Background: The routine prenatal determination of fetal RhD blood group would be very useful in the management of pregnancies in RhD-negative women, as up to 40% of these pregnancies bear a RhD-negative fetus. The fetal DNA present in maternal plasma offers an opportunity for risk-free prenatal diagnosis.

Aim: This study focused on the feasibility and accuracy of large-scale RhD fetal diagnosis in non-immunized and anti-D immunized RhD-negative women.

Methods: Plasma DNA was extracted from 893 RhD-negative pregnant women and amplified in exons 7 and 10 of the RHD gene using conventional and real-time PCR. The results were then compared with the RHD fetal genotype determined on amniotic cells and/or the RhD phenotype of the red blood cells of the infants at birth.

Results: After exclusion of 42 samples from women exhibiting a nonfunctional or rearranged RHD gene, fetal RhD status was predicted with a 99.5% accuracy. A strategy is also proposed to avoid the small number of false-positive and -negative results.

Conclusion: Fetal RHD genotyping from maternal plasma DNA in different clinical situations may be used with confidence.

Background: In the last decade, microarray technology has been extensively used to evaluate gene expression profiles and genome imbalances. We have developed a microarray-based comparative genomic hybridization (CGH) approach to identify MYCN gene amplification and 1p36 chromosome loss, two markers of tumor aggressiveness in neuroblastoma.

Aim: The aim was to use microarray CGH technology to detect the two major prognostic markers for neuroblastoma, MYCN amplification and 1p36 chromosome deletion, in neuroblastoma patients and, therefore, confirm the usefulness of this approach in this cancer.

Methods: DNA was purified from 16 tumors containing at least 90% malignant neuroblasts and collected at the onset of disease. Pooled fluorescent-labeled reference and neuroblastoma tumor genomic DNA was hybridized to epoxide-coated glass slides on laboratory-made complementary DNA microarray. The microarray contained cDNA mapped at the 1p36.33-36.1 chromosomal region and MYCN gene. cDNA from the 2q33-q34 and 12p13 chromosomes was used as a control and Arabidopsis thaliana DNA was spotted to control unspecific hybridization. Fluorescence in situ hybridization analysis was also performed to validate results from the microarray CGH.

Results: Both MYCN amplification and 1p36 chromosome deletion were detected by microarray CGH. The sensitivity and specificity for 1p36 loss detection were 66.7% and 90.0%, respectively. The method had a sensitivity of 66.7% and specificity of 90.9% to detect MYCN amplification.

Discussion: Our results demonstrated that the microarray CGH can be efficiently applied to study DNA gain and loss of specific chromosome regions.