中国东北地区未经抗病毒治疗的HIV/AIDS患者HIV毒株的耐药基因变异研究

目的　研究我国东北地区未接受抗逆转录病毒治疗的HIV AIDS患者HIV毒株的逆转录酶和蛋白酶耐药变异情况 ,为开展大规模临床抗病毒药物治疗提供本底数据。方法　RT PCR和套式PCR扩增HIVpol区基因 ,双脱氧法测定逆转录酶和蛋白酶基因序列 ,与国际耐药数据库比对辨别耐药变异。结果　 (1) 5 3例患者毒株亚型分析结果 :B′亚型 4 7例 ,B′ C亚型 4例 ,A、B亚型各 1例 ;(2 )未发现逆转录酶和蛋白酶原发耐药变异存在 ,但发现存在逆转录酶抑制剂继发变异 :M4 1L(1.9% )、I6 3M (1.9% )、L74I (1.9% )、S6 8G (1.9% )、V75L (3.8% )、V10 6I (1.9% )、I135L T (5 .7% )、V179D (7.5 % )和V189I (1.9% ) ,无症状感染者RT继发耐药变异出现率为 11.8% ,而艾滋病患者为5 2 .6 % (P <0 .0 1)。存在大量蛋白酶耐药继发变异V77I (88.7% )、L6 3P (86 .8% )、E35D (81% )、A71V(2 4 .5 % )、R4 1K (15 .1% )、L10I (9.4 % )、R5 7K (9.4 % )、D6 0E (9.4 % )、N37D (5 .7% )、G16E (3.8% )、I15V (1.9% )、M36I (1.9% )、K5 5R (1.9% )和L89M (1.9% )。未发现明显的亚型特异性耐药变异。结论　在中国东北地区未接受抗逆转录病毒治疗的HIV AIDS患者中未发现毒株逆转录酶和蛋白酶耐药原发变异 ,但大量继发耐药变异的存在提     

Novel mutations in the EXT1 gene in two consanguineous families affected with multiple hereditary exostoses (familial osteochondromatosis)

Multiple hereditary exostoses (HME) is an autosomal dominant developmental disorder exhibiting multiple osteocartilaginous bone tumors that generally arise near the ends of growing long bones. Here, we report two large consanguineous families from Pakistan, who display the typical features of HME. Affected individuals also show a previously unreported feature--bilateral overriding of single toes. Analysis using microsatellite markers for each of the known EXT loci, EXT1, EXT2, and EXT3 showed linkage to EXT1. In the first family, mutation analysis of the EXT1 gene revealed that affected individuals were heterozygous for an in-frame G-to-C transversion at the conserved splice donor site in intron 1. This mutation is predicted to disrupt splicing of the first intron and produce a frameshift that leads to a premature termination codon. In the second family, an insertion of an A in exon 8 is predicted to produce a frameshift at codon 555 followed by a premature termination, a further 10 codons downstream. In both families, an increased number of affected male subjects were observed. In affected females in family 2, phenotypic variability and incomplete penetrance were noted.     

Comprehensive molecular screening of the FBN1 gene favors locus homogeneity of classical Marfan syndrome

In order to estimate the contribution of mutations at the fibrillin-1 locus (FBN1) to classical Marfan syndrome (MFS) and to study possible phenotypic differences between patients with an FBN1 mutation vs. without, a comprehensive molecular study of the FBN1 gene in a cohort of 93 MFS patients fulfilling the clinical diagnosis of MFS according to the Ghent nosology was performed. The initial mutation screening by CSGE/SSCP allowed identification of an FBN1-mutation in 73 patients. Next, sequencing of all FBN1-exons was performed in 11 mutation-negative patients, while in nine others, DHPLC was used. This allowed identification of seven and five additional mutations, respectively. Southern blot analysis revealed an abnormal hybridization pattern in one more patient. A total of 23 out of the 85 mutations identified here are reported for the first time. Phenotypic comparison of MFS patients with cysteine-involving mutations vs. premature termination mutations revealed significant differences in ocular and skeletal involvement. The phenotype of the eight patients without proven FBN1 mutation did not differ from the others with respect to the presence of major cardiac, ocular, and skeletal manifestations or positive familial history. Most likely, a portion of FBN1-mutations remains undetected because of technical limitations. In conclusion, the involvement of the FBN1-gene could be demonstrated in at least 91% of all MFS patients (85/93), which strongly suggests that this gene is the predominant, if not the sole, locus for MFS.     

A mutation analysis of the<Emphasis Type="Italic"> BRCA1</Emphasis> gene in 140 families from southeast France with a history of breast and/or ovarian cancer

A mutation analysis of the BRCA1 gene in 140 French families with a history of breast cancer or breast-ovarian cancer revealed several deleterious germline mutations, as well as rare sequence variants. The 19 genetics variants were of 15 different types, two of which had not been reported in the Breast cancer Information Core (BIC) database. Five distinct truncating mutations, leading to putative nonfunctional proteins, were identified out of 140 index cases (3.5%). One novel nonsense mutation, C4491T, was reported, whereas the four other BRCA1 deleterious mutations identified consisted of frequent frameshifts in the nucleotide sequence. One splice variant (331+3A>G) and thirteen missense variations leading to amino acid substitutions of unknown structural and functional importance were identified. Among these, two BRCA1 missense mutations, A120G and T243C could be considered as suspected deleterious. The first missense mutation modified the initiation codon (M1V) and the second (C39R) may have consequences on the structure and functioning of the BRCA1 protein by modifying cysteine ligands from the RING finger domain. As expected BRCA1 gene alteration, including missense mutations of unknown biological significance, were more frequent in families with a history of breast-ovarian-cancer (32%) than in breast-cancer-only families (12%).     

Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations

Although various genetic factors have been implicated in human male infertility, the causative genes for the different types of idiopathic male infertility have not been elucidated. Protamines, which are the major DNA-binding proteins in the sperm nucleus, package the DNA into the sperm head. Analysis of the human protamine-1 (PRM1) and -2 (PRM2) gene sequences in 226 sterile male patients and in 270 proven-fertile male volunteers revealed four single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and one SNP in the PRM2 gene, which produced translation termination. We also observed one SNP in the 3' non-coding region of the PRM1 gene, and two SNPs within the intron of the PRM2 gene. The prevalence of these SNPs was similar in both infertile patients and in proven-fertile volunteers, except that the c248t alteration in the PRM2 gene induced a nonsense codon under conditions of heterozygosity in one infertile patient. Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene that induces translation termination may result in male infertility due to haploinsufficiency of PRM2.     

A novel STK11 germline mutation in two siblings with Peutz-Jeghers syndrome complicated by primary gastric cancer

Patients with Peutz-Jeghers syndrome (PJS) are known to be at risk of gastric cancer (GC), and the STK11 gene is a susceptibility gene for PJS. However, as no cases of PJS with GC in which a STK11 germline mutation has been identified have ever been reported and other susceptibility genes have also been suggested to be involved in PJS, the relation between STK11 germline mutations and GC in PJS is still unknown. In this study, we used sequencing analysis to investigate the STK11, CDH1, and TP53 loci for a germline mutation in two siblings with PJS with primary GC. A novel type of the STK11 germline mutation, c.890delG, encoding a truncated protein (p.Arg297fsX38) was identified, but no germline mutations of the CDH1 and TP53 genes were detected. No inactivation of the wild-type allele by somatic mutation or chromosomal deletion or hypermethylation at the 5'-CpG site of STK11 was detected in the GC. This is the first report of a STK11 germline mutation in a PJS patient with GC and should contribute to establishing correlations between the STK11 germline mutations and GC in PJS patients.     

DHPLC screening of cystic fibrosis gene mutations

Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromatography (IPRPC) columns is a technique for the screening of gene mutations. In order to evaluate the potential utility of this assay method in a clinical laboratory setting, we subjected the PCR products of 73 CF patients known to bear CFTR mutations to this analytic technique. We used thermal denaturation profile parameters specified by the MELT program tool, made available by Stanford University. Using this strategy, we determined an initial analytic sensitivity of 90.4% for any of 73 known CFTR mutations. Most of the mutations not detected by DHPLC under these conditions are alpha-substitutions. This information may eventually help to improve the MELT algorithm. Increasing column denaturation temperatures for one or two degrees above those recommended by the MELT program allowed 100% detection of CFTR mutations tested. By comparing DHPLC methodology used in this study with the recently reported study based on Wavemaker 3.4.4 software (Transgenomic, Omaha, NE) [Le Marechal et al., 2001) and with previous SSCP analysis of CFTR mutations [Ravnik-Glavac et al., 1994] we emphasized differences and similarities in order to refine the DHPLC system and discuss the relationship to the alternative approaches. We conclude that the DHPLC method, under optimized conditions, is highly accurate, rapid, and efficient in detecting mutations in the CFTR gene and may find high utility in screening individuals for CFTR mutations. Hum Mutat 19:374-383, 2002. Published 2002 Wiley-Liss, Inc.     

Increased stem cell somatic mutation in the non-neoplastic colorectal mucosa of patients with familial adenomatous polyposis

Colorectal tumorigenesis in familial adenomatous polyposis (FAP) results from somatic mutation of either the normal APC allele or another growth control gene in epithelial cells bearing a germline APC defect. The rate at which tumors develop is therefore dependent on the somatic mutation frequency; it is not known whether this is normal or elevated in FAP. We aimed to quantify stem cell somatic mutation in FAP, comparing it with hereditary nonpolyposis colorectal cancer (HNPCC) and Crohn's disease (CD). Stem cell somatic mutation frequency was studied in 47 FAP patients, 5 HNPCC patients, and 13 CD patients, all younger than 49 years, by quantifying crypt-restricted loss of O-acetyltransferase activity in sections of morphologically normal colonic mucosa from individuals heterozygous for this monogenically inherited polymorphism. Median stem cell somatic mutation frequency was significantly higher in FAP than HNPCC (4.2 × 10⁻⁴v 1.4 × 10⁻⁴, Mann-Whitney U, P < .02). The level in CD (4.0 × 10⁻⁴) was similar to FAR Mutated crypts occurred in groups more frequently in FAP (22%) than HNPCC (12%) or CD (10%), suggesting an increase in stem cell division associated with crypt fission in FAP. We conclude that stem cell somatic mutation frequency is raised in non-neoplastic colorectal mucosa in FAR This is probably related to increased stem cell proliferation and contributes to the high rate of tumor formation in this condition.     

Ehlers-Danlos syndrome type IV caused by Gly400Glu, Gly595Cys and Glyl003Asp substitutions in collagen III: clinical features, biochemical screening, and molecular confirmation

Three patients with Ehlers-Danlos syndrome type IV (EDS IV) and biochemical evidence of structural defects in collagen III were investigated for mutations within the collagen III gene ( COL3A1 ). Single strand conformation polymorphism analysis of α1(III) cDNA indicated the presence of different heterozygous sequence changes in each of the patients. Nucleotide sequencing revealed mutations leading to the substitution of glycine 400 with glutamic acid, glycine 595 with cysteine, and glycine 1003 with aspartic acid. EDS IV is a life-threatening disorder which, as the clinical histories of our patients and their families show, still often escapes diagnosis. Biochemical and molecular studies can clarify the diagnosis and help provide appropriate management and counselling.     

中国汉族雄激素过多症女性21-羟化酶缺陷症携带者基因检测

目的　初步了解中国汉族女性雄激素过多症患者中2 1-羟化酶缺陷症(2 1- hydroxylasedeficiency,2 1- OHD)携带者发生率,探讨促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)兴奋试验结果与基因突变检测结果的相关性。方法　82例汉族女性雄激素过多症患者及14名健康女性进行ACTH兴奋试验,并应用PCR扩增产生限制性酶切位点方法检测已知的9个2 1- OHD常见突变位点。结果　雄激素过多组(n=82 ) F0 显著高于正常对照组(P<0 .0 1) ;17- OHP0 及17- OHP6 0 也显著高于对照组(P<0 .0 1) ,而F6 0 差异没有统计学意义(P>0 .0 5 )。比较17- OHP净增值及17- OHP净增值/ F净增值,雄激素过多组也均显著高于正常对照组(P<0 .0 1)。正常对照组未检测出细胞色素P4 5 0 (cytochrome P4 5 02 1,CYP2 1)基因突变。发现雄激素过多组4例CYP2 1基因突变携带者(4/ 82 ,4 .9% ) ,分别携带V2 81L(2例) ,i2 g及Q318X(各1例) ,携带者的ACTH兴奋试验结果与正常对照及未检出突变的雄激素过多症患者的结果存在一定的交叉。结论　82例汉族雄激素过多症女性中2 1- OHD携带者为4例,占4 .9%。ACTH兴奋试验不能用以发现携带者,应进行基因检测确定。