Follicular fluid renin concentration and IVF outcome

Total renin protein concentration (TRC) was measured in stored follicular fluid (FF) samples from 42 women. Samples were selected according to their origin from follicles either without recovered ova ('empty', n = 38) or fertilized but with failed implantation ('failed', n = 36) or successful deliveries ('deliveries', n = 71). Ratios of number of embryos transferred to number of infants delivered were 2:1, 3:1 or 4:2 but 1:1 was not available. Non-parametric testing was applied to FF-TRC, volume and outcome. TRC was significantly higher in the delivery than the failed (P = 0.001) or empty (P = 0.002) categories. Assuming that the range of renin in failed follicles can identify the sub-population of unsuccessful follicles in the delivery category, then elevated FF-TRC was clearly associated with successful outcome. For individual women, the odds of infant delivery increased 17-fold as a function of average FF-TRC between 10,000 and 25,000 microIU/ml. For failed and delivery but not empty follicles, higher renin levels occurred in the smaller follicles, consistent with a burst of renin synthesis associated with the presence of an oocyte. The results suggest that FF-TRC relates to ovum viability with ovarian hyperstimulation and may have predictive use in IVF programmes.     

A PCR–restriction fragment length polymorphism assay to genotype human metapneumovirus

Human metapneumovirus (hMPV) genotypes A and B show epidemiological and probably clinical differences. This report describes a fast and simple PCR–restriction fragment length polymorphism (PCR-RFLP) assay, involving digestion of the fusion protein gene with Tsp 509I, that allows lineages A1, A2, B1 and B2 to be distinguished. The assay should help in elucidating the epidemiology of hMPV, and possibly in predicting the severity of clinical infection.     

Gene polymorphisms and serological markers of patients with active Crohn's disease in a clinical trial of antisense to ICAM-1

Serological profiles for anti-Saccharomyces cerevisiae antibodies (ASCA)/ perinuclear antineutrophil cytoplasmic antibodies (pANCA) and gene polymorphisms in tumour necrosis factor (TNF)-alpha and intercellular adhesion molecule-1 (ICAM-1) are associated with occurrence and/or outcome in Crohn's disease. The aim of the study was to characterize the ASCA/pANCA profile, soluble ICAM-1 expression and single nucleotide gene polymorphisms (SNPs) in TNF-alpha and ICAM-1 genes. Crohn's patients with moderate disease activity were enrolled in a clinical trial of Alicaforsen (ISIS 2302). Peripheral blood samples were collected prospectively for serum studies and for potential analysis of gene polymorphisms. A multivariate analysis was performed to compare treatment effect with the biomarkers studied. Serological testing for ASCA/pANCA was obtained for 257 patients at baseline: 37% were ASCA(+)/pANCA(-) (Crohn's pattern), 9% had both markers, 15% were ASCA(-)/pANCA(+) and 39% had neither marker. When the data were analysed by multiple regression analysis, a trend was found within the Alicaforsen-treated groups for greater rates of remission in the ASCA(+)/pANCA(-) subgroup versus all other serological profiles (25 versus 14%, P = 0.068), but not versus the placebo remission rate (18.8%). Gene polymorphisms were assessed in 64 patients, 21 from the placebo group. ICAM-1 assessment revealed no over-representation. However, three unique TNF-alpha SNPs were identified that correlated significantly with remission; sites 290 (P = 0.0253), -2735 (P = 0.0317) and -3090 (P = 0.0067). Although the overall clinical trial was negative, we have identified a trend towards clinical remission with Alicaforsen therapy in a subgroup of patients with Crohn's disease expressing ASCA(+)/pANCA(-). Furthermore, we have identified three TNF-alpha SNPs that may also predict a positive therapeutic outcome.     

Comparative study on deletions of the dystrophin gene in three Asian populations

The frequency and distribution of deletions of 19 deletion-prone exons clustered in two hot spots in the proximal and central regions of the dystrophin gene were compared in three populations from Singaporean, Japan, and Vietnam. DNA samples obtained from 105 Singaporean, 86 Japanese, and 34 Vietnamese Duchenne muscular dystrophy patients were examined by polymerase chain reaction amplification. Deletions of the examined exons were found in 51.2% of Japanese patients but in 40.0% or less of the Singaporeans and Vietnamese. About two thirds of the deletions were localized in the central region and the remaining deletions were clustered at the proximal region. The most commonly deleted exons at the central deletion hot spot were exon 50 in the Singaporean, exons 49 and 50 in the Japanese, and exon 51 in the Vietnamese population. At the proximal deletion hot spot, the most commonly deleted exons were exons 6 and 8 in the Singaporeans, exons 12 and 17 in the Japanese, and exons 8 and 12 in the Vietnamese. Two cases each from Singapore and Japan had large-scale gross mutations spanning both deletion hot spots. Our results suggest that, although the presence and frequency of the two deletion hot spots may be similar in the three Asian populations analyzed, the distribution and frequency of deletions among the different exons can vary as a result of population-specific intronic sequences that predispose individuals to preferential deletion breakpoints. Received: May 20, 2002 / Accepted: July 1, 2002     

华南、华北人群HLA-DQB1等位基因多态性

目的从基因水平调查了中国华南、华北地区人群HLA-DQB1等位基因频率，并研究比较两地区人群HLA-DQB1多态性分布。方法采用深圳益生堂生物企业有限公司研制开发的“HLA-DQB1低分辨率分型基因芯片检测试剂盒”，应用聚合酶链反应．序列特异性引物＋序列特异性寡核苷酸探针芯片检测技术，对700名南方地区的中国人和320名北方地区的中国人进行基因分型。结果鉴定了10个HLA-DQB1等位基因，获得了一组准确、科学的统计数据。结论得到了中国华南、华北地区人群HLA-DQB1等位基因频率差异的数据，证明中国人群HLA-DQB1＊02，05，0601，0602，0603的分布南北差异有统计学意义（P〈0．05），为疾病相关性研究、人文科学研究提供了可靠的遗传学数据。     

Real-time PCR method for the quantitative analysis of human T-cell receptor gamma and beta gene rearrangements

Analyzing the status of T-cell receptor (TCR) gene rearrangements has been an essential part of deciphering the stages of thymocyte development, understanding the β vs. γδ lineage decision, and characterizing T-cell leukemias. Methods such as PCR and quantitative Southern blotting provide useful information, but also have significant shortcomings such as lack of quantitation in the case of PCR and technical challenges in the case of Southern blotting. Here we describe a real-time PCR method that overcomes many of these shortcomings. This new method shows comparable results for the fraction of unrearranged TCRγ and TCRβ genes in human thymocytes and peripheral blood T cells as Southern blotting, and has the advantages of being simple to perform, highly quantitative, and requiring nanogram quantities of DNA. We also describe a real-time PCR method to quantitate T-cell receptor excision circles formed during TCRβ rearrangements.     

Characterization of a galactose-1-phosphate uridylyltransferase gene from the marine red alga Gracilaria gracilis

The metabolism of D-galactose is a major feature of red-algal physiology. We have cloned and sequenced a gene from the red alga Gracilaria gracilis that encodes a key enzyme of D-galactose metabolism, galactose-1-phosphate uridylyltransferase (GALT). This gene, designated GgGALT1, is apparently devoid of introns. A potential TATA box, four potential CAAT boxes, and a repeated sequence occur in the 5′-flanking region. The predicted 369-aa peptide shares significant sequence similarity with GALTs from other organisms (human, 47%; Saccharomyces cerevisiae, 49%; Solanum tuberosum, 49%). Southern-hybridization analysis reveals two related, but apparently not identical, GALT genes in the nuclear genome of G. gracilis. Sequence analysis indicates that the GgGALT1 enzyme lacks a rubredoxin “knuckle” motif, which in bacterial and fungal GALTs is involved in binding zinc. An open reading frame encoding a potential peptidyl tRNA hydrolase occurs 179 bp downstream from the GgGALT1 gene. Received: 6 April / 2 June 1998     

Gene-deletion and carrier detections,and prenatal diagnosis of Duchenne muscular dystrophy by analysis of the dystrophin gene amplified by polymerase chain reaction

Polymerase chain reaction (PCR)-based diagnosis was carried out in 62 patients (57 probands) with Duchenne or Becker muscular dystrophy (DMD or BMD) and 226 members in 57 families. The PCR studies were also performed for carrier detection in 57 mothers and 58 sisters, and prenatal diagnosis of 4 fetuses at risk of DMD. The PCR with 7 sets of primers, which amplify 7 different exon-sequences of the dystrophin gene, detected gene deletion of at least one exon in 49% of the probands. The PCR with the other 4 primer sets, which amplify 3 intragenic loci, and subsequent endonuclease digestion detected in 84% of the mothers a heterozygous pattern in at least one such locus/segment. Using the same primer sets, carrier detection was successful in 5 sisters of familial DMD cases, while recombination between the ERT87 and the 3 end intragenic loci was observed in 11% of family members studied. Prenatal diagnosis was made in all the 4 fetuses; two males were affected, one male fetus non-affected, and the remaining one female fetus a carrier. Thus, the PCR study and the primers used in the present study are useful and convincing for rapid diagnosis of DMD and/or BMD.     

Cloning of a human immunoglobulin gene fragment containing both VH-D and D-JH rearrangements: Implication for VH-D as an intermediate to VH-D-JH formation

In an Epstein-Barr virus-transformed human B cell line we found an unusual immunoglobulin heavy chain gene rearrangement. Restriction mapping and sequencing analysis led us to conclude that V_H-D and D-J_H recombination took place in a single allele. Both V_H-D and D-J_H complexes still had their recombination signal sequences adjacent and the DNA sandwiched by these two complexes retained a germ line configuration, suggesting the potential for a secondary rearrangement resulting in a V_H-D(-D)-J_H formation. With this finding, we propose a novel pathway, in which the V_H-D complex is an intermediate in the formation of a functional V_H exon.