Haplotype analysis of the BRCA2 9254delATCAT recurrent mutation in breast/ovarian cancer families from Spain

A frame-shift 9254del5 mutation was independently identified in 12 families, eleven of them with Spanish ancestors, in a BRCA2 screening performed in 841 breast and/or ovarian cancer families and in 339 women with breast cancer diagnosed before the age of 40 at different centers in France and Spain. We sought to analyze in detail the haplotype and founder effects of the 9254del5 and to estimate the time of origin of the mutation. Eight polymorphic microsatellite markers and two BRCA2 polymorphisms were used for the haplotype analyses. The markers were located flanking the BRCA2 gene spanning a region of 6.1 cM. Our results suggest that these families shared a common ancestry with BRCA2 9254del5, which is a founder mutation originating in the Northeast Spanish, with an estimated age of 92 (95% CI 56-141) generations.     

母系遗传耳聋大家系的线粒体基因组全序列分析

目的 在江苏淮阴一母系遗传非综合征型耳聋大家系中，寻找线粒体基因组上可能影响1555（A→G）突变表型的其他位点突变。方法 采用聚合酶链反应-限制性片段长度多态性分析（PCR-restriction fragment length polymorphism，PCR-RFLP）和测序技术。检测了核心分支家系中27名母系成员的线粒体DNA上1555位点和7445位点的碱基变化，进而对该家系2名母系成员的线粒体全基因组和其他25名母系成员线粒体12S rRNA基因MTRNR1和tRNA-Ser^（UCN）基因MTTS1进行了全长测序。结果 再次证明了1555（A→G）突变是该家系成员致聋的分子生物学基础之一；并发现该家系27名母系成员的线粒体基因组中除1555（A→G）突变外，还同时存在有955-960（insC）同质型突变，两突变共分离。另外，新发现一个线粒体DNA突变——7449（insG），但该突变仅在2名母系成员中存在。结论 推测955-960（insC）突变可能通过改变12S rRNA基因的高级结构，并与1555（A→G）突变协同作用，提高了突变携带者对氨基糖甙类药物的敏感性；同时该突变可能也会导致线粒体蛋白质的合成缺陷。从而提高1555（A→G）突变致聋的外显率。     

Novel Brugada syndrome‐causing mutation in ion‐conducting pore of cardiac Na+ channel does not affect ion selectivity properties

Aim: Brugada syndrome is an inherited cardiac disease with an increased risk of sudden cardiac death. Thus far Brugada syndrome has been linked only to mutations in SCN5A, the gene encoding the α‐subunit of cardiac Na⁺ channel. In this study, a novel SCN5A gene mutation (D1714G) is reported, which has been found in a 57‐year‐old male patient. Since the mutation is located in a segment of the ion‐conducting pore of the cardiac Na⁺ channel, which putatively determines ion selectivity, it may affect ion selectivity properties. Methods: HEK‐293 cells were transfected with wild‐type (WT) or D1714G α‐subunit and β‐subunit cDNA. Whole‐cell configuration of the patch‐clamp technique was used to study biophysical properties at room temperature (21 °C) and physiological temperature (36 °C). This study represents the first measurements of human Na⁺ channel kinetics at 36 °C. Ion selectivity, current density, and gating properties of WT and D1714G channel were studied. Results: D1714G channel yielded nearly 80% reduction of Na⁺ current density at 21 and 36 °C. At both temperatures, no significant changes were observed in V_1/2 values and slope factors for voltage‐dependent activation and inactivation. At 36 °C, but not at 21 °C, D1714G channel exhibited more slow inactivation compared with WT channel. Ion selectivity properties were not affected by the mutation at both temperatures, as assessed by either current or permeability ratio. Conclusion: This study shows no changes in ion selectivity properties of D1714G channel. However, the profoundly decreased current density associated with the D1714G mutation may explain the Brugada syndrome phenotype in our patient.     

马凡综合征微纤维蛋白1基因突变检测及单倍型连锁分析

目的：检测中国人马凡综合征（Marfan syndrome,MFS)患者微纤维蛋白1（fibillin-1,FBN1)基因的突变及对马凡综合征患者的家系成员进行症状前诊断。方法：应用聚合酶链反应－单链构象多态性技术和测序方法，对汉族9个家系中共17个MFS患者进行基因突变检测；运用FBN1基因内4个内含子中的可变串联重复序列构建染色体单倍型，进行家系单倍型连锁分析和基因诊断。结果：发现MFS（A）家系Ⅱ1患者有单链构象改变，测序证实为位于FBN1基因第25号外显子3243－3256核苷酸之间有I个13bp的小片段缺失，为新位点基因移码突变，其序列为gcctctgcaccca;单倍型连锁分析发现MFS（B）家系Ⅲ1是1个无症状期患者。结论：中国人FBN1基因突变可以引起马凡综合征，应用突变检测与单倍型连锁分析方法能为马凡综合征基因诊断提供依据。     

Alterations of plasma antioxidants and mitochondrial DNA mutation in hair follicles of smokers

The effects of long-term smoking on mitochondrial DNA (mtDNA) deletions in hair follicles were investigated in subjects with different antioxidant capacity. Twenty-two male smokers with a smoking index of greater than 5 pack-years and without any known systemic diseases were recruited for this study. Forty healthy nonsmoking males were included as controls. We found that the concentrations of ascorbate and alpha-tocopherol and the activities of glutathione S-transferase (GST) and glutathione peroxidase in blood plasma were significantly decreased in smokers. The levels of glutathione and protein thiols in whole blood and the incidence of a 4,977 bp deletion of mtDNA (dmtDNA) in hair follicles were significantly increased in smokers. A significantly higher incidence of the 4,977 bp dmtDNA was found in smokers with plasma GST activity less than 5.66 U/l (OR = 7.2, P = 0.020). Using multiple covariate ANOVA and logistic regression, we found that age and low plasma GST activity were the only two risk factors for the 4,977 bp dmtDNA. These results suggest that smoking depletes antioxidants and causes mtDNA deletions and that plasma GST may play an important role in the preservation of the mitochondrial genome in tissue cells of smokers.     

Significance of beta-catenin and pRB pathway components in malignant ovarian germ cell tumours: INK4A promoter CpG island methylation is associated with cell proliferation

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.     

A novel C202F mutation in the connexin26 gene (GJB2) associated with autosomal dominant isolated hearing loss

Mutations in the GJB2 gene encoding connexin26 (CX26) account for up to 50% of cases of autosomal recessive hearing loss. In contrast, only one GJB2 mutation has been reported to date in an autosomal dominant form of isolated prelingual hearing loss. We report here a novel heterozygous 605G→T mutation in GJB2 in all affected members of a large family with late childhood onset of autosomal dominant isolated hearing loss. The resulting C202F substitution, which lies in the fourth (M4) transmembrane domain of CX26, may impair connexin oligomerisation. Finally, our study suggests that GJB2 should be screened for heterozygous mutations in patients with autosomal dominant isolated hearing impairment, whatever the severity of the disease.


Keywords: C202F mutation; connexin26 gene (GJB2); autosomal dominant hearing loss     

Alteration of the p53 gene of lung carcinomas with sarcomatous transformation (spindle cell carcinoma): analysis of four cases

Lung carcinoma with sarcomatous transformation (LCST) is highly aggressive and characterized by local invasion and/or distant metastasis, which leads to a shorter survival than ordinary lung carcinomas. Therefore, to elucidate whether the malignant potential of the spindle cell element in LCST is associated with the alteration of the p53 gene, four cases were examined by analyses of overexpression of the p53 oncoprotein, mutation of the p53 gene and loss-of-heterozygosity (LOH) at chromosome 17p. In two cases overexpression of the p53 oncoprotein of the spindle cell component showed a higher degree of staining than that of the carcinoma component; LOH was identified in both carcinoma and sarcomatous components in one case, while in contrast, another case showed LOH in the sarcomatous component only. Mutations were clearly detected in two cases; one showed a CTT to CGT transversion in codon 194 of exon 6 in both components, whereas the other showed a CTG to CAG transversion in codon 265 of exon 8 in the sarcomatous component only. On the basis of these observations, it suggested that the sarcomatous component shows a higher frequency of p53 gene abnormalities in comparison to the carcinoma component. These results also suggested that the acquisition of malignant potential in the sarcomatous component, or the morphological alteration of carcinoma cells, is correlated with abnormalities associated with the p53 gene.     

Prenatal exclusion of choroideremia

We performed prenatal testing to predict the inheritance of choroideremia (CHM) using a linked polymorphic DNA marker, DXS95. DNA analysis of chorionic villi at the 12th week of pregnancy indicated that the allele at risk had not been passed from the heterozygous mother to the fetus. This prenatal exclusion of choroideremia was confirmed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. © 1992 Wiley-Liss, Inc.     

Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype

PEX7 encodes the cytosolic receptor for the set of peroxisomal matrix enzymes targeted to the organelle by the peroxisome targeting signal 2 (PTS2). Mutations in PEX7 cause rhizomelic chondrodysplasia punctata (RCDP), a distinct peroxisome biogenesis disorder. In previous work we described three novel PEX7 mutant alleles, including one, L292X, with a high frequency due to a founder effect. We have now extended our analysis to 60 RCDP probands and identified a total of 24 PEX7 alleles, accounting for 95% of the mutant PEX7 genes in our sample. Of these, 50% are L292X, 13% are IVS9+1G>C, and the remainder are mostly private. IVS9+1G>C occurs on at least three different haplotypes and thus appears to result from recurrent mutation. The phenotypic spectrum of RCDP is broader than commonly recognized and includes minimally affected individuals at the mild end of the spectrum. To relate PEX7 genotype and phenotype, we evaluated the consequence of the disease mutation on PEX7 RNA by Northern analysis and RT/PCR. We evaluated the function of the encoded Pex7 protein (Pex7p) by expressing selected alleles in fibroblasts from RCDP patients and assaying their ability to restore import of a PTS2 marker protein. We find that residual activity of mutant Pex7p and reduced amounts of normal Pex7p are associated with milder and variant phenotypes.