Restriction endonuclease fingerprinting enhanced conformation sensitive gel electrophoresis (REF-CSGE) in the analysis of BRCA1 exon 11 mutations in a high-risk breast cancer cohort

Efficient genetic analysis of large exonic regions containing heterozygous mutations and common polymorphisms can be difficult. We have analyzed 30 patients for inherited susceptibility mutations (ISM) within exon 11 of the BRCA1 gene as part of an ongoing genetic epidemiological study of high-risk breast cancer (HRBC). A novel combination of restriction endonuclease fingerprinting (REF) and conformation sensitive gel electrophoresis (CSGE) was developed for rapid and efficient screening of mutations. This method (REF-CSGE) was compared side-by-side with standard CSGE and evaluated for both efficiency and sensitivity of detection. REF-CSGE detected 100% of the alterations found by CSGE. However, one variant was only detectable by REF-CSGE. All samples with variant bands were sequenced to confirm the nature of the alteration. In total, two small deletions (frameshifts) and 62 point mutations (60 known polymorphisms and two variants of unknown significance) were found in our cohort. The majority of the exon 11 polymorphisms detected are inherited as a linked haplotype. Point mutations that comprise these haplotypes could be simultaneously detected on a single gel by REF-CSGE, thereby decreasing the number of sequencing reactions necessary to elucidate heteroduplex patterns seen on CSGE gels. An analysis of the overall efficiency of both techniques revealed that REF-CSGE required 67% fewer confirmatory sequencing reactions, resulting in savings in both reagents and technician time.     

Mutations in activation-induced cytidine deaminase in patients with hyper IgM syndrome

Recent studies have shown that mutations in a newly described RNA editing enzyme, activation-induced cytidine deaminase (AID), can cause an autosomal recessive form of hyper IgM syndrome. To determine the relative frequency of mutations in AID, we evaluated a group of 27 patients with hyper IgM syndrome who did not have defects in CD40 ligand and 23 patients with common variable immunodeficiency. Three different mutations in AID were identified in 18 patients with hyper IgM syndrome, including 14 French Canadians, 2 Lumbee Indians, and a brother and sister from Okinawa. No mutations were found in the remaining 32 patients. In the group of patients with hyper IgM syndrome, the patients with mutations in AID were older at the age of diagnosis, were more likely to have positive isohemagglutinins, and were less likely to have anemia, neutropenia, or thrombocytopenia. Lymphoid hyperplasia was seen in patients with hyper IgM syndrome and normal AID as well as the patients with hyper IgM syndrome and defects in AID.     

Prevalence, geographical distribution and genealogical investigations of mutation 188 of lipoprotein lipase gene in the French Canadian population of Québec

Familial lipoprotein lipase deficiency (FLD) is of particular interest to the French Canadian population of Québec since the largest concentration of homozygotes and carriers of this genetic disease in the world resides in this area. We have previously described a missense mutation (M-188) in the lipoprotein lipase (LPL) gene which was present in FLD patients belonging to different ancestries, including a number of French Canadians (Monsalve MV et al. J Clin Invest 1990: 86: 728-734). In the present report, we show that this mutation, although found in largest absolute numbers among French Canadians as compared to other groups in the world, accounts for only a small proportion (24%) of all the LPL mutant alleles in this population. The M-188 occurs either in the homozygote state or as a compound heterozygote with another LPL mutation. Analysis of geographic distribution indicates that the M-188 is more prevalent in western Québec, with the highest carrier rate in the Mauricie region. Genealogical reconstruction leads to the recognition of four founders for M-188, all emigrants from France to Québec in the 17th century.     

线粒体DNA继发突变对Leber遗传性视神经病变的影响

Leber遗传性视神经病变是一种多发于青壮年的母系遗传性疾病,可导致双眼严重的急性无痛性、且一般是永久性的双侧中心视野缺失.3个线粒体DNA(mitochondrial DNA,mtDNA)原发突变ND4 G11778A、ND1 G3460A和N06 T14484C是其致病的主要因素,但家族问及家族内不同成员之间的表型差异表明存在其他的修饰因子,包括核及线粒体遗传修饰因子、环境因素.本综述主要阐述mtDNA继发突变对LHON表型表达的影响.     

CACNA1H基因G773D突变对钙通道功能的影响

目的研究儿童失神癫痫（childhood absence epilepsy，CAE）患儿CACNA1H基因G773D突变对钙通道功能的影响。方法用定点突变重叠延伸聚合酶链反应（polymerase chain reaction，PCR）方法构建G773D突变体，脂质体法将突变体和野生型人Cav3．2a cDNA分别转染HEK-293细胞，获得稳定表达细胞株，全细胞膜片钳法研究其电生理变化。结果突变体和野生型细胞钙通道激活和失活动力学差异无统计学意义，但突变体G773D钙电流密度明显高于野生型。结论CACNA1H基因G773D突变可使其编码通道电流增加，并可能引起神经元兴奋性增加。     

Public health impact of isoniazid-resistant Mycobacterium tuberculosis strains with a mutation at amino-acid position 315 of katG: a decade of experience in The Netherlands

A previous limited study demonstrated that Mycobacterium tuberculosis isolates with a mutation at amino-acid position 315 of katG (Delta315) exhibited high-level resistance to isoniazid and were more frequently resistant to streptomycin. In the present study, isoniazid-resistant M. tuberculosis isolates from 8,332 patients in The Netherlands (1993-2002) were screened for the Delta315 mutation. Isoniazid resistance was found in 592 (7%) isolates, of which 323 (55%) carried Delta315. IS6110 restriction fragment length polymorphism analysis showed that Delta315 isolates occurred in clusters, suggesting recent transmission, at the same frequency as isoniazid-susceptible isolates. In contrast, other isoniazid-resistant isolates clustered significantly less frequently. Delta315 isolates were high-level isoniazid-resistant, streptomycin-resistant and multidrug-resistant significantly more often, and may have a greater impact on public health, than other isoniazid-resistant isolates.     

Triple A syndrome: genotype-phenotype assessment

The triple A or Allgrove syndrome is an autosomal-recessive disease (MIM*231550) characterized by the triad of achalasia, alacrima and adrenocorticotropic hormone (ACTH)-resistant adrenal insufficiency. Associated features of the syndrome are neurological and dermatological abnormalities. Until the discovery of the AAAS gene as the responsible gene in triple A syndrome, the diagnosis was based on characteristic clinical features. Here we present the clinical and molecular genetic data which demonstrated the marked phenotypic variability in three unrelated patients with triple A syndrome. The final diagnosis of triple A syndrome was confirmed by molecular analysis. In one patient with isolated achalasia, the diagnosis of triple A syndrome could only be made on the basis of the molecular genetic analysis of the AAAS gene. We therefore suggest that the diagnosis of triple A syndrome should be considered in patients who exhibit only one or two of the main symptoms (i.e. alacrima, achalasia or adrenal insufficiency). These patients require careful neurological investigation, and mutation analysis of the AAAS gene should be performed.     

Identification of a common variant in the lipoprotein lipase gene in a large Utah kindred ascertained for coronary heart disease: the -93G/D9N variant predisposes to low HDL-C/high triglycerides

Defects in the lipoprotein lipase (LPL) gene are associated with dyslipidemia in the general population. Several rare mutations in the gene, as well as two common coding region polymorphisms, D9N and N291S, exhibit deleterious effects on circulating lipid levels. Using a linkage-based approach, we have identified a large Utah kindred segregating the D9N variant in the LPL gene. The kindred was ascertained for premature coronary heart disease and was expanded based on familial dyslipidemia. A genomic scan identified a region of linkage including LPL, and mutation screening identified the segregating variant. In the kindred, the variant shows high penetrance for a hypoalphalipoproteinemia phenotype, but is also associated with hypertriglyceridemia and elevated insulin levels. The strength of linkage was dependent on the combination of phenotype definition and model parameters, favoring the use of a MOD score approach. Most other studies of LPL have proceeded by mutation screening of randomly chosen individuals or selected affected probands; this is the first example identifying a segregating LPL mutation using direct linkage.     

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys⁺ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37°C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys ^- Lys⁺ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23°C or 37°C. Therefore, when the proof-reading activity of DNA polymerase is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced.