Evaluation of a PCR-RFLP-ITS2 assay for discrimination of Anopheles species in northern and western Colombia

Anopheles mosquitoes are routinely identified using morphological characters of the female that often lead to misidentification due to interspecies similarity and intraspecies variability. The aim of this work was to evaluate the applicability of a previously developed PCR–RFLP–ITS2 assay for accurate discrimination of anophelines in twelve localities spanning three Colombian malaria epidemiological regions: Atlantic Coast, Pacific Coast, and Uraba-Bajo Cauca-Alto Sinu region. The evaluation of the stability of the PCR–RFLP patterns is required since variability of the ITS2 has been documented and may produce discrepancies in the patterns previously reported. The assay was used to evaluate species assignation of 939 mosquitoes identified by morphology. Strong agreement between the morphological and molecular identification was found for species Anopheles albimanus, Anopheles aquasalis, Anopheles darlingi and Anopheles triannulatus s.l. (p ≥ 0.05, kappa = 1). However, disagreement was found for species Anopheles nuneztovari s.l., Anopheles neomaculipalpus, Anopheles apicimacula and Anopheles punctimacula (p ≤ 0.05; kappa ranging from 0.33 to 0.80). The ITS2–PCR–RFLP assay proved valuable for discriminating anopheline species of northern and western Colombia, especially those with overlapping morphology in the Oswaldoi Group.     

Restriction fragment length polymorphism (RFLP) of the human insulin receptor gene in Japanese: its possible usefulness as a genetic marker

Summary Restriction fragment length polymorphism of the human insulin receptor gene was analyzed with a 4.2 Kb cDNA probe in Japanese normal subjects and Type 2 (nonsulin-dependent) diabetic patients. Restriction endonuclease Rsa I digestion showed polymorphism of the human insulin receptor gene, with a band at 6.7 Kb, 6.2 Kb or 3.6 Kb. The frequency of the 6.7 Kb band was less than that in Caucasians. the Japanese subjects examined lacked a 3.6 Kb band, which is commonly found in Caucasians. We have also detected restriction fragment length polymorphism in the human insulin receptor gene by Pvu II or Stu I digestion. Although no significant association of restriction fragment length polymorphism with Type 2 diabetes was found in the present study, our results suggest that the restriction fragment length polymorphism in the human insulin receptor gene varies among ethnic groups, and that the restriction fragment length polymorphism linked to the human insulin receptor gene might be a useful marker for the linkage study of the genes located close to the human insulin receptor gene on chromosome 19.     

Association of IL-8 gene polymorphisms with non small cell lung cancer in Tunisia: A case control study

Interleukin 8 (IL-8), is a proinflammatory chemokine, has been reported to have angiogenic activity and to be responsible for tumor-associated angiogenesis in several cancers. In this study, we aimed to study the (IL-8) gene polymorphism in relation with risk development of non small cell lung cancer in Tunisian patient. Two single nucleotide polymorphisms (−251T/A [rs4073], +781C/T [rs2227306]) of the IL-8 gene were screened in 170 patients with NSCLC and 225 healthy controls by PCR–RFLP.     

Association of Interleukin-1β-31C/T, -511T/C and -3954C/T Single Nucleotide Polymorphism and Their Blood Plasma Level in Acquired Aplastic Anemia

Aplastic anemia (AA) is an immune-mediated disorder in which hematopoietic stem and progenitor cells are targeted by a number of cellular and molecular pathways. This case control study aims to investigate the association of interleukin-1beta (IL-1β) gene polymorphisms, (IL-1β-31, IL-1β-511 and IL-1β-3954) and their plasma levels with acquired AA. Genotyping was done by Restricted Fragment Length Polymorphism (PCR–RFLP) method and IL-1β plasma levels were evaluated in peripheral blood using ELISA. Increased level of IL-1β was reported to be significant in cases as compared to controls. The susceptibility of developing AA was higher in the cases for IL-1β-3954 genotype. IL-1β-511 genotype showed significant association with the severity groups of AA. No significant association was noticed in responder versus non-responder group. Plasma level of IL-1β gene was found to be significantly higher in severe and very-severe group of AA versus control group. Our findings suggest that IL-1β gene and its genotypes might be involved in the pathophysiology of AA and play a central role in the etiopathogenesis of AA.     

CD38 and Interleukin 6 Gene Polymorphism in Egyptians with Diffuse Large B-Cell Lymphoma (DLBCL)

Given the importance of understanding the genetic variations involved in the pathogenesis of non-Hodgkin’s lymphoma (NHL), this pilot study was designed to investigate the impact of CD38 (184C/G; rs6449182) and IL-6 (?174 G/C; rs1800795) gene polymorphism on susceptibility of Egyptians to diffuse large B cell lymphoma (DLBCL); major types of NHL. To the best of our knowledge, this study is the first one that examines CD38 polymorphism in the NHL. Genotyping polymorphism is performed using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) for CD38 and Mutagenically separated PCR (MS-PCR) for IL-6 in 100 Egyptian NHL patients with DLBCL subtype and 119 normal controls. The serum level of IL-6 was measured using Enzyme-linked immunosorbent assay (ELISA). CD38 (184C/G) genotype is significantly increased in NHL patients (p?<?0.01), while the GG genotype is significantly increased in controls (p?<?0.05). Only two genotypes were found (GG and GC) in IL-6 (?174), no CC in our NHL patients and only one case in the controls. Insignificant change in IL-6 (?174 G/C) genotypes was recorded. Significantly increased serum IL-6 (p?<?0.05) was positively correlated (r?=?0.17; p?<?0.05) with the disease. Taken together, our data stressed the importance of CD38 gene polymorphism in developing DLBCL. Our pilot study indicates that CD38 (184) CG genotype might play a role in DLBCL susceptibility in Egyptians. Additional prospective studies on larger population are needed to confirm our findings.     

上海地区汉族健康人群PAF乙酰水解酶基因4号外显子275位点G/A单核苷酸多态性

目的了解血小板活化因子乙酰水解酶(PAF-AH)基因4号外显子275位点G/A位点单核苷酸多态性(SNP)在中国上海地区汉族健康人群中的分布及与其他种族比较的特点。方法采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法,对100名上海地区汉族健康者PAF-AH4号外显子275位点G/A位点的SNP进行了检测,计算其基因型和等位基因频率,并结合文献进行了不同种族间的分析比较。结果上海地区汉族健康人群GG基因型频率最高(88%),GA基因型次之(12%),未发现AA基因型。与英国人相比,上海地区汉族人群G等位基因频率较高。结论上海地区汉族健康人群G→A位点碱基突变率较低,其SNP与英国人相比分布存在明显差异。     

Polymorphisms in <Emphasis Type="Italic">methionine synthase</Emphasis> (A2756G) and <Emphasis Type="Italic">cystathionine β-synthase</Emphasis> (844ins68) and susceptibility to carcinomas of the upper gastrointestinal tract

Purpose

Folate deficiency is considered to increase the risk for the development of malignant tumors such as prostate and colorectal cancer. Methionine synthase (MTR) and cystathionine ß-synthase (CBS) are enzymes that play a central role in folate metabolism, thereby affecting DNA methylation and synthesis. A single A→G substitution at nucleotide 2756 of the MTR and a 68 bp CBS insertion polymorphism in exon 8 have been associated with decreased enzyme activity. The purpose of this study is to compare the association of the MTR A2756G polymorphism and CBS insertion polymorphism with susceptibility to carcinomas of the upper gastrointestinal tract.

Methods

Using the restriction fragment length polymorphism (RFLP)-PCR, the prevalence of MTR A2756G and CBS insertion polymorphism was determined in healthy controls (n = 257) and in patients with esophageal squamous cell carcinoma (ESCC) (n = 263), Barrett’s esophagus-associated esophageal adenocarcinoma (BC) (n = 89), cardiac carcinoma (CC) (n = 144), or gastric carcinoma (GC) (n = 221) from German Caucasian subjects.

Results

No significant difference in MTR A2756G genotype distribution was observed between controls (A/A 66.9%, A/G 29.8%, G/G 3.3%) and patients with ESCC (A/A 61.7%, A/G 36.3%, G/G 2.1%), BC (A/A 69.2%, A/G 26.9%, G/G 3.9%), CC (A/A 51.8%, A/G 44.6%, G/G 3.6%), or GC (A/A 73.4%, A/G 20.9%, G/G 5.7%). Similarly, the CBS genotype (I: allele with 68 bp insertion; N: allele without insertion) distribution among German patients with ESCC (N/N 86.8%, I/N 13.2%), BC (N/N 90.2%, I/N 9.8%), CC (N/N 90.1%, I/N 9.9%) or GC (N/N 91.3%, I/N 8.7%) was not different from healthy controls (N/N 90.4%, I/N 9.6%). The gene allele constellation I/I was not present.

Conclusions

The current study suggests that there is no association between MTR A2756G polymorphism and the CBS (844ins68) insertion polymorphism and cancer of the upper gastrointestinal tract.

     

Detection of c-Ki- ras point mutation from pancreatic juice

Summary Cytological diagnosis of pancreatic carcinoma sometimes poses difficulties in distinguishing malignant from benign cells. Recent molecular study of pancreatic carcinoma has revealed a very high incidence of a point mutation of the c-Ki-ras oncogene at codon 12 in this neoplasm. To take advantage of this technique for the diagnosis of pancreatic carcinoma, we attempted to amplify the c-Ki-ras gene from endoscopically obtained pancreatic juice by isolation of DNA and polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP). PCR was possible in approx 70% of the cases. A point mutation was nonradioisotopically detected in 4 of 6 pancreatic carcinomas and in one intraductal papillary neoplasm, whereas no mutation was detected in other cases. Thus, this method was thought to be useful for the diagnosis of pancreatic carcinoma.