Mutations in HLA DRB1*0101 Exon 2 in a Sub-population of Latvia.

Aims: To find out whether ongoing missense mutations in the exon 2 of DRB1*01:01 affect the operation of this protective allele in HIV patients. Place and Duration of Study: the Clinical Immunology and Immunogenetics Laboratory of Riga Stradiņš University, Riga Eastern Clinical University Hospital, “Infectology Center of Latvia”, between May 2006 and December 2011. Methodology: The study includes 200 HIV-infected patients. DNA was isolated from venous blood samples using the Qiagen QIAamp DNA kit reagents and the exon 2 nucleotide sequence of HLA was determined by the automatic sequencing – “Big Dye Terminator mix” (Applied Biosystems, USA). Statistical analysis was performed using Microsoft Excel, DOS StatCalc programs. The significance of the differences in indicators was evaluated according to reliability p£0.05. The odds ratio was calculated according to Wolf’s method. Results: We found missense: at codon 47– in 80% of cases; at codon 67– in 20% of cases; at codon 75 – in 11% of cases; at codon 82– in 10% of cases; at codon 86– in 10% of cases (p<0.05) (See Table 3). One of the HIV patients had a STOP-codon (codon 13). Besides, a balance between nucleotide transversion and transition has been observed, suggesting mutations in the exon 2 (transversion in a human genome is rare) (OR 0.05, 95% CI 0.00-0.053). Conclusion: The results of the study are not complete in order to be able to say conclusively that the existing mutations in the exon 2 of HLA-DRB1 *01:01 gene cause wrong immune response, thus the protective functions of this allele are not fulfilled. For a fuller understanding of the importance of ongoing mutations in the exon 2 in the development of HIV/AIDS, it is necessary to increase the study group.

Gene Polymorphism of Adiponectin in Han Nationality with Type 2 Diabetes Mellitus from Guangxi / 中国药师

Objective:To evaluate the relationship between single nucleotide polymorphism (SNP) +45T→G in adiponectin gene exon 2 and type 2 diabetes mellitus (T2DM), obesity and insulin resistance (IR) in Han population in Guangxi. Methods:Polymer-ase chain reaction-restriction fragment length polymorphism ( PCR-RFLP) was performed to identify the T-G polymorphism of adiponec-tin (exon 2) in 313 unrelated Han people from Guangxi including 95 normal glucose tolerance,103 impaired glucose tolerance and 115 type 2 diabetes mellitus. Results:In Han people of Guangxi, the SNP +45 GG genotype in adiponectin gene had significantly lower HDL-C levels than TT and TG genotypes in Han people(P<0. 05). The subjects with the GG genotype at position 45 had a signifi-cantly increased risk of IR (odds ratio of 2. 876[95%CI:1. 141-7. 248]) compared with those with the TT genotype at position 45. Conclusion:In Han people of Guangxi, the SNP +45 TT genotype in adiponectin gene has significantly higher HDL-C levels than GG genotype. The subjects with the GG genotype at position 45 have a significantly increased risk of IR compared with those with the TT genotype, while obesity and T2DM are independent on SNP+45 of adiponectin gene.

Mutational analyses of K-ras exon 2 in Thailand colorectal cancer tissue samples.

Objective: The main purpose of this study was to elucidate the genotype of K-ras gene in Thailand colorectal cancer tissue samples, especially in exon 2, which has never before been reported. Methods: 106 patients’ samples in formalin fixed paraffin embedded tissue blocks were investigated in this study. DNA was extracted and PCR was performed by using primers specific for the K-ras gene at exon 2. Direct sequencing was performed in a Genetic Analyser ABI3130 with specific software. Results: The mutation of K-ras exon 2 gene in Thailand colorectal cancer samples accounted for 37.7% of the total. The most common mutation found in this series was the G"A transition which accounted for 70%. Conclusion: The incidence of K-ras exon 2 mutation in Thailand colorectal cancer samples was remarkedly similar to previous reports.

Analysis of the association between lactotransferrin (LTF) gene polymorphism and dental caries

OBJECTIVE: The present study evaluated the association between lactotransferrin (LTF) gene polymorphism (exon 2, A/G, Lys/Arg) and dental caries. MATERIAL AND METHODS: A convenience sample of 110 individuals, 12 years old, was divided into: group 1, 48 individuals without caries experience (DMFT=0), and group 2, 62 subjects with caries experience (DMFT>1). DNA was obtained from a mouthwash with 3 percent glucose solution, followed by a scrapping of the oral mucosa. After DNA purification, polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) was performed to access the study polymorphism. The LTF A/G (Lys/Arg) polymorphism had been previously reported as located in exon 1. RESULTS: Allele 1 of the study polymorphism was associated with low DMFT index and showed a protective effect against caries experience (OR=0.16, IC=0.03-0.76, p=0.01). CONCLUSIONS: Lactotransferrin A/G (exon 2, Lys/Arg) polymorphism was associated with susceptibility to dental caries in 12-year-old students.

Repeating exon 2 mutation caused by trans-splicing dystrophin gene in Duchenne muscular dystrophin (DMD) patient

The dystrophin gene is the largest human gene. Mutations in this gene cause Duchenne muscular dystrophin (DMD) disease. This is complex genomic unit exhibiting many errors splicing during mRNA process. More than 10 alternative splicing products have been identified in the 5' region of the dystrophin gene. In this study, two dystrophin transcripts including one containing exon 2 and exon X duplications, other one containing single exon 2 duplication were identified in peripheral blood lymphocytes of DMD case. Interestingly, genomic Southern blot analysis ruled out the hypothesis of duplication of dystrophin at exon 2. Therefore, these data suggested that exon 2 duplication transcripts were likely generated by trans-splicing event that occurring during the mARN maturation in which RNA segments of two independent transcripts are spliced together to generate a new mRNA species. However, the mechanisms modulating the trans-splicing activity of the dystrophin exon 2 remain to be clarified.

Expression of Exon 2 of Type II Procollagen mRNA in Primary Culture of Rat Chondrocyte / 대한정형외과연구학회지

Type II procollagen mRNA exists in two forms, type II A and II B, by alternative splicing of exon 2. Type II A procollagen mRNA has been known to be expressed in chondroprogenitor, periosteal and perichondrial cells, whereas type II B procollagen mRNA in chondrocytes. Previous reports indicate that both types of procollagen mRNA exist in primary culture of rat chondrocyte. In this study, the time-dependent change in the type II A and II B mRNA expression in the primarily cultured rat chondrocytes was analyzed using reverse transcription polymerase chain reaction (RT-PCR) and Northern hybridization. Most chondrocytes in the early stage of primary cell culture were oval or polygonal in their shape. However, since 2 to 3 weeks of primary cell culture, most cells changed their shapes into fibroblast-like or ameboid cells. RT-PCR revealed that until 1 week of culture, the expression of the type II B mRNA was greater than that of the type II A. After 2 weeks of culture, the expression of the type II A mRNA became greater. Northern blot analysis showed a gradual decrease in the expression of the total type II procollagen mRNA with time of culture. On the other hand, the expression of the type II A procollagen mRNA increased significantly at 2 weeks and the increased level was maintained thereafter. This study confirmed that in primary culture of chondrocyte, the cells changed their morphological and molecular characteristics at the relatively early stage. This study implicates that in order to delineate the changes of phenotypes of chondrocytes, quantitative analysis of both type II A and II B procollagen mRNA is necessary instead of performing analysis of total type II procollagen mRNA.