Consistence of miniexon polymerase chain reaction-restriction fragment length polymorphism and single-copy gene sequence analyses in discriminating Leishmania genotypes

Recently, a new polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP)-based assay had been developed using the miniexon sequences for genotyping Leishmania isolates. We had used this method for rapid diagnosis and genotyping of visceral and cutaneous leishmaniasis with the combination of microcapillary cultivation. In this study, we have evaluated this approach by examining genomic DNAs from 47 independent isolates, which were grouped into 19 genotypes of Leishmania subgenus complexes by sequence polymorphism of single-copy genes. Results obtained provide miniexon RFLP configurations specific to Leishmania enriettii, Leishmania tarentolae, and Leishmania gerbilli for the first time. Altogether, 92% of the results from miniexon PCR-RFLP are in agreement with those based on the sequence database of single-copy genes from the same isolates. The miniexon PCR-RFLP method is simple, sensitive, and specific method useful for routine diagnosis of different Leishmania.     

A PCR-RFLP assay for identification of Anisakis simplex from different geographical regions

Anisakis simplex, a nematode from the family Anisakidae, is a parasite of fish and mammals. It is a casual agent of a human disease called anisakiosis. We found that the assay based on PCR amplification of the ITS-1–5·8 S–ITS-2 fragment of rDNA and subsequent restriction fragment length polymorphism, previously described on the basis of A. simplex isolated solely from one geographical region, can be used as a general test for identification of this worm species. The restriction patterns analysed for four restriction enzymes were found to be identical in the case of allA. simplex individuals isolated from as different geographical regions as Baltic Sea, Norwegian Sea, Bering Sea and Sea of Okchotsk. Moreover, our results support the previously proposed hypothesis, based on the studies of isoenzymes, that there is a remarkable genetic homogeneity within A. simplex from different geographical regions.     

DNA analysis in a paternity case involving a triploid fetus

BACKGROUND: A parentage testing laboratory was asked to perform testing in a case of sexual assault that resulted in the conception of a child. Samples submitted to the laboratory included blood from the mother, the alleged father, and the fetus. CASE REPORT: DNA typing was used to determine if the suspect in this sexual assault was the father of the expected child. DNA extracted from these samples was subjected to both restriction fragment length polymorphism and polymerase chain reaction/short-tandem repeat analysis at a total of 13 genetic loci. Examination of DNA profiles for selected markers suggested that the fetus was triploid. Triploidy was confirmed through the use of fluorescent in situ hybridization of chromosomes, employing three chromosome-specific alpha satellite probes and fetal trophoblast nuclei. Statistical interpretation of the test results required identifying a method for calculation that would consider two transmitted paternal genes. Attempts to modify the standard method of calculating a paternity index were unsuccessful, because it was not possible to distinguish between dispermy and diandry as the mechanism of conception. Therefore, the likelihood ratio was calculated as the reciprocal of the random men not excluded value or the proportion of the population that possesses all of the paternal markers observed in the triploid fetus. CONCLUSION: Calculation of a likelihood ratio employing the exclusionary power of a collection of DNA markers appears to be the only method suitable for assigning weight to the significance of DNA matches between an alleged father and a child who is triploid.     

Novel polymerase chain reaction method for detecting cutaneous human papillomavirus DNA

There is no simple test to identify the human papillomavirus (HPV) genotypes that cause cutaneous warts. A new polymerase chain reaction (PCR) method, called SK-PCR, was developed for this purpose. This PCR amplifies 210-238 base pairs of L1 DNA of 17 HPV types (HPV-1a, -2a, -3, -4, -7, -10, -27, -28, -29, -40, -57, -60, -63, -65, -77, -91, and -94), which are thought to cause various cutaneous warts, including common, flat, butcher's, punctate, and pigmented warts. The method is novel because the location of these primers is completely different from that of any previous PCR method for HPV. The target sequences are specific to alpha-, gamma-, and mu-papillomaviruses (PVs), but not to beta-PVs. Furthermore, direct sequencing and restriction fragment length polymorphism (RFLP) were used to determine the HPV genotypes. Fifty of samples of plantar warts were examined, and HPV-27 was identified in 22 warts, HPV-57 in 15 warts, and HPV-2a in 9 warts. These PVs, which are alpha species 4, were the most common. HPV-4 and -65 (gamma-PVs) and HPV-1a and -63 (mu-PVs) were detected in one case each. A single HPV type was identified in all of these warts. This method appears to be useful for genotyping the HPVs causing skin warts, and for distinguishing between HPV-induced warts and warty lesions unrelated to HPV infection.     

Polymorphisms of genes related to endothelial cells are associated with primary biliary cirrhosis patients of Cretan origin

Background

Primary biliary cirrhosis (PBC) is an organ specific autoimmune disease of still unidentified genetic etiology. We have shown that endothelins (ETs), produced by the liver endothelial cells are increased in PBC and may play a major pathogenetic role.

Aims

To study gene polymorphisms related to the endothelial cells (eNOS, EDN-1 genes) and, to investigate whether the previously reported association of CTLA4 gene polymorphisms is replicated in a genetically homogeneous Greek population.

Patients and methods

Genomic DNA was extracted from 100 PBC patients (83 females, 93% AMA+, 74/100 Ludwig stage I–II) and 158 healthy controls. eNOS, CTLA4 and ET1 polymorphisms were determined by PCR–RFLPs analysis.

Results

Both eNOS intron4 VNTR and eNOS exon7 G894T SNP were significantly associated with increased risk in PBC. EDN-11 rs2071942 “A” and rs5370 “T” alleles appeared a tendency for association with disease progression. No association was found between PBC and the CTLA4 SNPs analyzed.

Conclusions

We demonstrated that eNOS, a gene related to the liver endothelium function is associated with PBC. Contrarily, the important in adaptive immunity gene CTLA4 was not associated with the disease in the homogeneous population analyzed. These results are compatible partially with our previous hypothesis that defects of the liver endothelial system, leading to endothelin overproduction, may be a fundamental early pathogenetic mechanism in PBC.     

A novel ENG mutation causing impaired co-translational processing of endoglin associated with hereditary hemorrhagic telangiectasia

Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal dominant vascular dysplasia caused by mutations in mainly the endoglin gene (ENG) or activin-like kinase receptor 1 (ALK1) gene (ACVRL1). We investigated the molecular basis of HHT in a Japanese patient, and identified a novel missense mutation in ENG (c.38 T > A, p.Leu13Gln) located in the signal peptide's hydrophobic core, but not in ACVRL1. In experiments in COS-1 cells, the Leu13Gln (L13Q) mutant endoglin appeared to be expressed as a precursor form, probably due to impaired protein processing. Flow cytometry analyses of the COS-1 cells transiently expressing recombinant endoglins revealed that the wild-type endoglin was detected on the cell surface, but the L13Q mutant was not. We also analyzed expression patterns of the recombinant endoglins by immunofluorescent staining, and found that the wild-type co-localized with the endoplasmic reticulum (ER), but the L13Q mutant did not. These results implied that the L13Q mutant endoglin fails to insert into the ER, probably due to destruction of the hydrophobic core structure in the signal peptide to be recognized by signal recognition particles. Thus, the Leu13 in the signal peptide of endoglin might be essential for correct protein processing through the ER and cell-surface expression. Taken together, the novel c.38 T > A mutation in ENG would impair co-translational processing of the endoglin, and could be responsible for HHT in this patient.     

沙眼衣原体垂直传播基因水平的快速检测

应用细胞培养法对８６名孕妇及其新生儿进行沙眼衣原体（ＣＴ）的分离，然后采用聚合酶链反应（ＰＣＲ）结合限制性片段长度多态性（ＲＦＬＰ）分析法，对ＣＴ阳性的母婴标本进行基因鉴定。结果显示，８６例孕妇ＣＴ阳性２０例，阳性孕妇的亲手儿有８例感染，垂直传播率４０．０％。８对母婴相对标本鉴定后证实每对母婴感染的ＣＴ基因型一致。提示ＣＴ有很高的垂直传播率，应早期进行孕期筛查，降低新生儿ＣＴ感染率。     

幽门螺杆菌的分子分型及其分子流行病学研究

目的：在基因分型基础上探讨不同基因型别的幽门螺杆菌（Hp）与疾病之间的关系。方法：采用随机扩增的多态性DNA(RAPD)和PCR-RFLP法对50株来自不同病人的Hp进行分析，在此基础上运用聚类分析进行基因分型，并用病例对照研究探讨不同基因型别的Hp与疾病之间的关系。结果：RAPD结果显示，不同基因型别的Hp与不同的疾病结局有关。PCR-RFLP结果显示，单个基因的分析结果均未能说明不同基因型别的Hp与不同的疾病结局有关，而多个基因的综合分析结果则说明不同基因型别的Hp确实与不同的疾病结局有关。将RAPD和PCR-RFLP的分析结果进行综合分析，也从多方法水平说明不同基因型别的Hp确实与不同的疾病结局有关。结论：对于Hp来说，多基因性状数值分类法的分型效率高于单基因数值分类法，这为建立Hp的多基因、多方法的数值分类系统打下了基础。     

Carrier detection for hemophilia B: evaluation of multiple polymorphic sites

DNA analysis was performed in families with hemophilia B. Restriction fragment length polymorphisms (RFLPs) produced by endonucleases Taql, Xmnl, and Ddel were studied by two factor IX genomic probes, F9(VIII) and F9(XIII). Fifty-seven subjects from ten families were investigated; of them, 31 were carriers (11 obligate and 20 potential). Of the potential carriers, ten displayed laboratory features allowing for a phenotypic diagnosis of heterozygosity. Segregation analysis of the markers was informative in 19/20 potential carriers, which belong to nine of the ten studied families. Among the potential carriers, Taql allowed the carriership assessment in 15 (78.9%), Xmnl in 15 (94.7%), and Ddel in two (10.4%). Diagnosis was not possible in one family since a homozygosity in the key individuals with all the employed enzymes (Taql, Xmnl, Ddel, + BamHI) was found. Hemophilia B syndrome in two families likely results from a new mutation. In one family, a first-trimester prenatal diagnosis was performed. The use of RFLP analysis allowed us to improve genetic counseling as compared with the phenotypic evaluation by clotting factor assays. Indeed, evaluation of RFLP increased by 26% the carriership assessment of the potential carriers of the hemophilia B trait.