Rapid molecular cytogenetic analysis of X-chromosomal microdeletions: Fluorescence in situ hybridization (FISH) for complex glycerol kinase deficiency

Diagnosis of X-chromosomal microdeletions has relied upon the traditional methods of Southern blotting and DNA amplification, with carrier identification requiring timeconsuming and unreliable dosage calculations. In this report, we describe rapid molecular cytogenetic identification of deleted DNA in affected males with the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency, CGKD) and female carriers for this disorder. CGKD deletions involve the genes for glycerol kinase, Duchenne muscular dystrophy, and/or adrenal hypoplasia congenita. We report an improved method for diagnosis of deletions in individuals with CGKD and for identification of female carriers within their families, using fluorescence in situ hybridization (FISH) with a cosmid marker (cosmid 35) within the glycerol kinase gene. When used in combination with an Xq control probe, affected males demonstrate a single signal from the control probe, while female carriers demonstrate a normal chromosome with two signals, as well as a deleted chromosome with a single signal from the control probe. FISH analysis for CGKD provides the advantages of speed and accuracy for evaluation of submicroscopic X-chromosomal deletions, particularly in identification of female carriers. In addition to improving carrier evaluation, FISH will make prenatal diagnosis of CGKD more readily available. © 1995 Wiley-Liss, Inc.     

Variable dystrophin expression in different muscles of a Duchenne muscular dystrophy carrier

The majority of Duchenne muscular dystrophy (DMD) female carriers show dystrophin immunostaining abnormalities, although a significant proportion of clinically non-manifesting carriers are normal following this analysis. We had the opportunity to study dystrophin immunostaining in two different muscles, the vastus lateralis and the rectus abdominis of a possible DMD carrier. While the vastus showed normal dystrophin immunostaining, pathological staining was detected in her rectus abdominis. These findings seem to indicate that dystrophin expression can vary in different muscle groups of a DMD carrier. The implications of these findings in DMD carrier detection and possible dystrophin function are discussed.     

Detection and analysis of gastrointestinal sounds in normal and small bowel obstructed rats

This study is aimed at detecting gastrointestinal sounds (GIS) and correlating their characteristics with gastrointestinal (GI) conditions. The central hypotheses are that GIS generation depends on the motility patterns and the mechanical properties of the gut, and that changes in those result in measurable differences in GIS. An animal model which included both healthy rats and those with small bowel obstruction (SBO) was developed. The acoustic bursts, of GIS were detected by amplitude thresholding the signal envelope. Three methods of envelope estimation were proposed and evaluated. Envelope estimation using a Hilbert transform was found to produce the best results in the current application. The duration and dominant frequency of each detected GIS event was estimated and clear differences between healthy and diseased rats were discovered. In the control state, GIS events were found to consistently be of relatively short duration (3–65ms). Although the majority of events in the SBO state had similar short duration, infrequent longer events were also detected and appeared to be pathognomonic. Long duration events (>100 ms) occurred in each of seven obstructed, but in none of 14 non-obstructed, cases (p<0.001). It is concluded that GIS analysis may prove useful in the non-invasive, rapid, and accurate diagnosis of SBO.     

The motivation of at-risk individuals and their partners in deciding for or against predictive testing for Huntington''s disease

Sixty-six percent of the at-risk persons and 74% of the partners in a large survey in Belgium have the intention of making use of predictive testing for Huntington's disease. One third of them, however, have expressed the intention of postponing the final decision for various reasons. The intention to be tested is not at all related to sociodemographic characteristics. A thorough exploration of the reasons for being in favour of or against taking the test reveals that the motivation inspiring this very personal decision is very complex. In the group of at-risk persons, less than half of the variation in the intention to be tested is explained by the role of a series of specific reasons as predictor variables in a regression analysis. The proportion of explained variation is slightly higher in the group of partners. 'To have certainty about my own future' and 'to make arrangements for the future' play a major part in the decision of the total group. 'Making decisions concerning children' and to a larger extent 'informing children about their risk status' are important factors in deciding in favour of the test.     

Comparison of SSCP and DHPLC for the detection of LDLR mutations in a New Zealand cohort

Familial hypercholesterolaemia (FH) is a common inherited disorder, associated with premature vascular disease. FH may be caused by many different mutations in the low density lipoprotein receptor (LDLR) gene, about 700 mutations have been described, most of which occur rarely and often only in single families. Although particular mutations are prevalent in certain ethnic groups, countries with heterogeneous population bases (such as NZ) may carry a wide variety of mutations; making a gene screening approach the appropriate first step for a mutation detection programme. We have compared SSCP with DHPLC to assess their effectiveness as methods for LDLR mutation detection. Although five novel LDLR mutations were detected by SSCP in patients with FH, DHPLC was more sensitive, with eight novel mutations detected. Six of these mutations (T392M, R419G, Y421N, 1206-1207delCT, 1872delC, and 1943delC) were clustered in exons 9 and 13 of the EGF precursor homology domain, one (679-680delAC) in the ligand binding domain (exon 4) and the eighth (P774H) in the membrane-spanning domain (exon 16). Twenty five mutations were identified in 35 patients in total. Of these, we were able to detect only 64% of mutations by SSCP even though all variants were detected by DHPLC. All patients are heterozygous for the mutations, which is consistent with the clinical phenotypes.     

An association of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene polymorphism and common carotid atherosclerosis

Plasma homocysteine (Hcy) concentration has been shown to be influenced by a mutation in the gene coding methylenetetrahydrofolate reductase (MTHFR). Although plasma Hcy is related to atherosclerotic disorders, conflicting results have been reported about the association between MTHFR gene polymorphism and sclerotic lesions of the common carotid arteries. The effect of age–gene interaction on carotid arterial remodeling was investigated in elderly subjects with several risk factors for atherosclerosis. We evaluated sclerotic lesions of the common carotid arteries by ultrasonography in 326 patients (mean age ± standard deviation, 73 ± 12 years) and studied relations among the known risk factors for atherosclerosis, including MTHFR gene polymorphism and its interactions with age and sex. Of the 326 subjects studied, 136 had MTHFR genotype CC, 136 genotype CT, and 54 genotype TT. The three groups did not differ with respect to background factors such as age, history of cigarette smoking, blood pressure, lipids or uric acid, or in the incidence of atherosclerotic diseases. Spearman's rank correlation revealed a significant relationship between gender, age, Brinkman index, systolic blood pressure, triglycerides, HDL-cholesterol (HDL-C), uric acid, and MTHFR gene polymorphism. Multiple regression analysis using intima-media complex thickness (IMT) as a criterion variable and risk factors, including MTHFR gene polymorphism as explanatory variables showed that MTHFR gene polymorphism (P = 0.039) was a significant independent explanatory variable for IMT, along with gender (male) (P < 0.001), age (P < 0.001), systolic blood pressure (SBP) (P = 0.047), total cholesterol (T-C) (P < 0.001), and HDL-C (P < 0.001). Furthermore, a general linear model analysis revealed that interaction between age and MTHFR gene polymorphism was significantly associated with IMT, independently of age, SBP, T-C, and HDL-C in male subjects. However, age–gene interaction was not observed in female subjects. The findings of the present study confirm an association between MTHFR gene polymorphism and common carotid atherosclerosis in the Japanese population and further support the role of risk factor–gene interaction in common carotid atherosclerosis. Received: May 14, 2001 / Accepted: June 8, 2001     

Physical separation of HLA-A alleles by denaturing high-performance liquid chromatography

Genomic typing of polymorphic loci may be hampered by ambiguous typing results. Moreover, robust methods for simultaneous sequencing of two alleles present in a given sample may be difficult to establish. We used denaturing high-performance liquid chromatography (DHPLC) for physical separation of HLA-A alleles before sequence-based genomic typing (SBT). Physical separation was achieved by resolution of heteroduplexes between the sample alleles and a modified reference probe by DHPLC followed by selective reamplification of the sample alleles present in heteroduplexes. Complementary strands of the reference probe and sample alleles for heteroduplex induction were obtained by lambda-exonuclease digestion. HLA-A genotyping of 101 individuals using DHPLC-SBT yielded better typing resolution compared with serological typing and genotyping by the sequence-specific primer-polymerase chain reaction (SSP-PCR) method. Physical separation of alleles using a modified reference probe allows for development of fully automated methods for genomic typing of highly polymorphic loci such as HLA.     

ELISPOT,MHC肽五聚体和ICCD三种检测特异性细胞免疫应答方法的比较研究

目的 比较三种特异性细胞免疫应答的检测方法，寻求一种简便、重复性好、易于质控的评价疫苗细胞免疫的检测方法。方法采用HIV DNA疫苗肌内注射BALB／c小鼠，第6周用艾滋病重组MVA痘苗病毒腹腔注射加强免疫，于第10天制备脾脏细胞悬液，并用酶联免疫斑点法（Enzymeunked Immune Spot，EusPOT）、MHC肽五聚体法和胞内细胞因子染色分析法（Intra—cellular cytokinede tection，ICCD）分别检测细胞免疫应答。结果 ELISPOT、MHC肽五聚体染色以及胞内细胞因子染色分析法检测疫苗组细胞免疫应答的阳性率分别为5／5、4／5和3／5；而且ELISPOT和MHC肽五聚体染色法之间有一定相关性（r=0、647），而ELISPOT和胞内因子染色法之间以及MHC肽五聚体染色和胞内因子染色之间无相关性。结论ELISPOT试验操作相对简单，灵敏度高、重复性好，是评价疫苗细胞免疫的有效方法。     

High-throughput single strand conformation polymorphism mutation detection by automated capillary array electrophoresis: validation of the method

Capillary array electrophoresis (CAE) is a novel technique, which allows for high throughput analysis of DNA fragments. When screening for mutations in whole populations or large patient groups it is necessary to have robust and well-characterized setups for high throughput analysis. For large-scale mutation screening, we have developed procedures for single strand conformation polymorphism (SSCP) assays using CAE (CAE-SSCP) whereby we may increase both the sensitivity and the throughput compared to conventional SSCP analysis. In this study we have validated CAE-SSCP by 1) comparing detection by slab-gel based SSCP with CAE-SSCP of mutations in the MYH7, MYL2, and MYL3 genes encoding sarcomere proteins from patients suffering from hypertrophic cardiomyopathy; and 2) by constructing a series of 185 mutants having substitution mutations, as well as insertion/deletion mutations, or some combinations of these, in different sequence contexts in four exons and different positions relative to the end of the amplicon (three from the KCNQ1 gene, encoding a cardiac potassium channel, and one from the TNNI3 gene encoding cardiac troponin I). The method identified 181 out of 185 mutations (98%), and the data suggest that the position of mutation in the fragment had no effect on the sensitivity. Analysis of the specificity of the method showed that only very few mutants could not be distinguished from each other and there were no false positives.     

人颈动脉粥样硬化斑块环氧化物酶-2及Ⅰ型前列腺素合成酶的表达

目的探讨环氧化物酶-2(cyclooxygenase type 2,COX-2)及Ⅰ型前列腺素合成酶(membrane associated prostaglandin E-1,mPGES-1)在人颈动脉粥样硬化斑块中的表达变化及作用机制。方法收集24例人颈动脉粥样硬化斑块标本和10例肠系膜动脉标本做对照组,应用免疫组织化学及逆转录PCR方法测定COX-2及mPGES-1mRNA表达水平,Western印记方法检测COX-2及mPGES-1的蛋白表达水平。比较不同程度动脉粥样硬化组织间COX-2、mPGES-1 mRNA表达水平及蛋白表达水平。结果颈动脉粥样硬化斑块组的免疫组织化学染色检测COX-2和mPGES-1呈阳性表达,斑块组COX-2 mRNA和mPGES-1 mRNA表达与对照组相比上调,差异有统计学意义(P<0.05);COX-2及mPGES-1 mRNA上调水平相关(P<0.05);颈动脉粥样硬化斑块的COX-2蛋白表达上调水平与对照组相比差异有统计学意义(P<0.05);颈动脉粥样硬化斑块COX-2、mPGES-1 mRNA及蛋白表达水平与病理损害程度有关,差异有统计学意义(P<0.05)。结论COX-2及mPGES-1基因表达水平上调可能是进展性动脉粥样硬化损害的关键因素。