Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic

Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high‐risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non‐radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high‐risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761‐3762delGA and 2616‐2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073‐5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA‐binding protein. © 2000 Wiley‐Liss, Inc.     

Accumulation of somatic hypermutation and antigen-driven selection in rapidly cycling surface Ig+ germinal center (GC) B cells which occupy GC at a high frequency during the primary antihapten response in mice

Well-developed germinal centers (GC) contain rapidly dividing surface immunoglobulin-negative (sIg^-) B cells (centroblasts), and most of their progeny are sIg⁺ B cells (centrocytes) in a resting state. It has been predicted that somatic hypermutation occurs in centroblasts, whereas antigen-driven selection takes place in centrocytes. The present analysis indicates that murine GC B cells bearing sIg with specificity for an immunizing antigen are in a rapidly cycling state and increase exponentially in number to occupy spleen GC at high frequency during the 1st week after primary immunization; however, the number of these cells is significantly reduced in the 2nd week of immunization. During that period, these proliferating sIg⁺ GC B cells accumulate somatic hypermutations with nucleotide exchanges indicative of affinity maturation. These sIg⁺ GC B cells co-express B7-2, ICAM-1, and LFA-1, and have potent antigen-presenting activity which results in T cell activation in vitro. These observations indicate that the sIg⁺ GC B cells accumulate somatic hypermutations and undergo antigen-driven selection through proliferation, probably upon activation by T cells. This sIg⁺ GC B cell population may represent cell cycling centrocytes; however, the possibility that these may represent centroblasts undergoing re-expression of sIg could not be excluded.     

Retrospective analysis of antiretroviral HIV treatment success based on medical history or guided by the reverse hybridisation LiPA HIV genotyping system

The changes in viral load and CD4(+) count at 3 and 6 months in a group of 166 HIV-infected patients was evaluated. The new therapy was chosen based on the medical history procedures for 70 patients, and in 96 patients it was guided by the partial or complete result of the line probe assay (LiPA) HIV RT and Protease resistance tests. The absolute difference from the baseline of the log viral load at 3 and 6 months was significantly different between the two groups when adjusted for baseline viral load (P < 0.0001) and stayed significant when intention-to-treat analysis was carried out (P < 0.001). The absolute difference of the CD4(+) count was not significantly different when adjusted for baseline CD4(+) (P = 0.854, 3 months; P = 0.06, 6 months). The proportion of patients with a viral load 相似文献   

Subtelomeric deletions detected in patients with idiopathic mental retardation using multiplex ligation-dependent probe amplification (MLPA)

Subtelomeric rearrangements are responsible for 5% to 10% of cases of unexplained mental retardation. Despite their clinical relevance, methods to screen for these cytogenetically invisible abnormalities on a routine base are scarce. We screened patients with idiopathic mental retardation for subtelomeric aberrations using multiplex ligation-dependent probe amplification (MLPA). This recently developed technique is based on PCR amplification of ligated probes hybridized to chromosome ends. Currently, 41 telomeres can be screened in just two multiplex reactions. Four subtelomeric rearrangements (5.3%) were detected in a group of 75 patients with mild to severe mental retardation in combination with dysmorphic features and/or a familial history of mental retardation: two terminal 1p deletions, a terminal 1q deletion, and a terminal 3p deletion. Deletions could be verified by FISH and marker analysis. In one case the MLPA indicated a terminal 21q deletion due to a 3-bp deletion at the site of the probe, giving a false-positive rate of 1.3%. This study demonstrates that MLPA is a fast and reliable screening method, potentially suitable for use in routine diagnostics.     

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.     

Clinical,immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: an Italian multicenter study

A questionnaire-based retrospective clinical and immunological survey was conducted in 73 males with a definite diagnosis of X-linked agammaglobulinemia based on BTK sequence analysis. Forty-four were sporadic and 29 familial cases. At December 2000, the patients' ages ranged from 2 to 33 years; mean age at diagnosis and mean duration of follow-up were 3.5 and 10 years respectively. After the mid-1980s all but 2 were on intravenous immunoglobulin (IVIG) substitution therapy, with residual IgG >500 mg/dl in 94% of the patients at the time of enrollment. Respiratory infections were the most frequent manifestation both prior to diagnosis and over follow-up. Chronic lung disease (CLD) was present in 24 patients, in 15 already at diagnosis and in 9 more by 2000. The cumulative risk to present at diagnosis with CLD increased from 0.17 to 0.40 and 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years respectively. For the 9 patients who developed CLD during follow-up, the duration of follow-up, rather than age at diagnosis; previous administration of intramuscular immunoglobulin; and residual IgG levels had a significant effect on the development of CLD. Chronic sinusitis was present in 35 patients (48%), in 15 already at diagnosis and in 20 by 2000. Sistemic infections such as sepsis and meningitis/meningoencephalitis decreased over follow-up, probably due to optimal protection provided by high circulating IgG levels reached with IVIG.     

Cystic fibrosis mutations and immotile cilia syndrome

The immotile cilia syndrome (ICS) presents with autosomal recessive inheritance and is a chronic respiratory disease supposed to be caused by different genetic determinants. The hypothesis that cystic fibrosis (CF) heterozygotes may have a predisposition to develop bronchial or respiratory diseases other than CF prompted us to look for CF mutations in patients with ICS. Five patients, as well as the parents and two healthy brothers of one patient were tested for 12 CF mutations, for the polymorphic GATT repeat in intron 6a and for the CF gene flanking markers XV-2c, KM19, MP6d-9, J3.11. None of the 12 mutations at the CF locus have been detected in the ICS patients and no linkage was found between ICS and the polymorphic markers. Thus, based on our data, ICS and CF seem to be two different clinical entities.     

p53 protein overexpression and K-rascodon 12 mutation in pancreatic ductal carcinoma: Correlation with histologic factors

The correlation of p53 protein overexpression and the K-ras codon 12 mutatlon wlth histologlc type, grade of cytologic atypla, depth of lnvasion and other histologlc prognostic factors was studied In paraffin sectlons from 43 ductectatic-and 70 solid-type pancreatic ductal carcinomas. Overexpression of p53 was found in 23.3% (10143) of ductectatic carcinomas (17.2% of intraductal and 35.7% of lnvaslve carclnomas) and in 61.4% (43/70) of solid carcinomas. In ductectatic cancers, p53 overexpression was detected In 14.8% (4/27) of carcinomas wlth lowgrade atypla (CAL), 50.0% (5110) of carcinomas wlth high-grade atypla (CAH) and in 16.7% (In) of mixed low- and hlgh-grade cancers. In the last group, expression was restricted to an area of CAH. In solld cancers, p53 overexpression did not dlffer by histologic type or grade. Overexpresslon of p53 and K-ras mutatlons did not correlate with histologlc prognostic factors (lymphatic, venous and perineural Invasion, and lymph node metastasls) in ductectatlc and solld cancers or depth of invasion of solld carclnomas. Our data suggest that p53 alteratlon occurs at an early intraductal stage of solld carcinoma, irrespectlve of cellular atypla, but Is low in ductectatic CAL and becomes hlgher In ductectatlc CAH. K-ras mutatlon, present In a high percentage of tumors of all groups and not correlating with the factors above, showed no changes In frequency with tumor progression.     

Generation and expression of a Hoxa11eGFP targeted allele in mice

Hox genes are crucial for body axis specification during embryonic development. Hoxa11 plays a role in anteroposterior patterning of the axial skeleton, development of the urogenital tract of both sexes, and proximodistal patterning of the limbs. Hoxa11 expression is also observed in the neural tube. Herein, we report the generation of a Hoxa11eGFP targeted knock‐in allele in mice in which eGFP replaces the first coding exon of Hoxa11 as an in‐frame fusion. This allele closely recapitulates the reported mRNA expression patterns for Hoxa11. Hoxa11eGFP can be visualized in the tail, neural tube, limbs, kidneys, and reproductive tract of both sexes. Additionally, homozygous mutants recapitulate reported phenotypes for Hoxa11 loss of function mice, exhibiting loss of fertility in both males and females. This targeted mouse line will prove useful as a vital marker for Hoxa11 protein localization during control (heterozygous) or mutant organogenesis. Developmental Dynamics 237:3410–3416, 2008. © 2008 Wiley‐Liss, Inc.     

精神分裂症与KCNN3基因1137～1140缺失移码突变传递不平衡研究

目的 探讨KCNN3基因1137～1140的4个碱基缺失所致移码突变与精神分裂症的关系。方法 95个核心家系共289名家庭成员，包含107例精神分裂症患者纳入本研究。精神分裂症采用CCMD-Ⅱ-R诊断标准。KCNN3基因1137～1140的4个碱基缺失基因型检测使用PCR技术和限制性内切酶DdeI消化方法。精神分裂症与KCNN3基因1137～1140缺失4个碱基的关联分析采用基于单倍型的单倍型相对风险率和传递／不平衡检验。结果 患者组与正常父母组比较，KCNN3基因1137～1140的4个碱基缺失的基因型频数和等位基因频率分布差异无统计学意义（χ^2=0．253，P〉0．05和χ^2＝0．010，P〉0．05）。基于单倍型的单倍型相对风险分析发现父母传递与不传递给患者的KCNN3基因等位基因之间差异无统计学意义（χ^2=0．042，P〉0．05）。传递不平衡检验结果发现KCNN3基因等位基因传递不存在连锁不平衡（χ^2=3．000，P＝0．0833）。结论 本组研究对象中，KCNN3基因第1外显子1137～1140的4个碱基缺失的发生率少；分析结果提示KCNN3基因第1外显子1137～1140的4个碱基缺失的等位基因与精神分裂症无关联。