Faculty Bibliography

BACKGROUND: Single nucleotide polymorphism association studies among cases and controls have been widely used for genetic analysis. The pyrosequencing method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. It has the advantages of accuracy, flexibility, automatization and speed when compared with PCR-RFLP method. AIM: To develop a protocol for allele frequency determination using pyrosequencing technology in the detection of the polymorphism at position -31 of the interleukin-1beta (IL-1beta) gene. METHODS: 162 patients (F/M = 0.93) who were enrolled at the Hospital Universitario Dr 'Jose Eleuterio Gonzalez' were studied. 123 patients had non-ulcer dyspepsia and 39 had histologically confirmed gastric cancer (GC). The polymorphism of IL-1beta -31 was determined by both RFLP and pyrosequencing methods. PCR-RFLP method used Alul restriction endonuclease. The same specific primers for PCR-RFLP and pyrosequencing were used for initial amplification and an additional biotinylated specific primer was designed for sequencing. RESULTS: 157 (96.9%) samples were clearly typed by the pyrosequencing method and the results were in accordance with the results of the PCR-RFLP method. The results of 5 samples (3.1%) were not in accordance between both methods. Two of them were T/T and 2 were C/T by sequencing method and all four were C/C by RFLP. Another sample was C/ C by sequencing and T/T by RFLP. CONCLUSION: The pyrosequencing method is not only suitable for the IL-1beta -31 genotyping but is a fast and unexpensive way of genotyping since requires smaller amounts of DNA, and required significantly less time in the generation of results than the RFLP technique. The protocol developed is useful for the typing of the IL-1beta -31 polymorphism

Helicobacter pylori colonizes the stomachs of half of the world's population and usually persists in the gastric mucosa of human hosts for decades or life. Although most H. pylori-positive people are asymptomatic, the presence of H. pylori is associated with increased risk for the development of peptic ulcer disease, gastric adenocarcinoma and gastric lymphoma. The development of a sustained gastric inflammatory and immune response to infection appears to be pivotal for the development of disease. During its long co-existence with humans, H. pylori has evolved complex strategies to maintain a mild inflammation of the gastric epithelium while limiting the extent of immune effector activity. In this review, the nature of the host immune response to H. pylori infection and the mechanism employed by the bacterium to evade them is considered. Understanding the mechanisms of colonization, persistence and virulence factors of the bacterium as well as the innate and adaptive immune responses of the host are critically important for the development of new strategies to prevent the development of H. pylori-induced gastroduodenal disease

We review the current information concerning the role of cytokine polymorphisms and the risk of develop distal gastric cancer in different populations. We have included populations colonized with Helicobacter pylori as well as populations without colonization. We found that the study of polymorphisms alone seems insufficient to assess gastric cancer risk and it is necessary to examine environmental factors in different ethnic groups and geographic areas along with the study of H. pylori strains to define better the risk factors associated with distal gastric cancer

BACKGROUND: This study was undertaken in order to analyze the genetic incidence of human lymphocyte antigen diabetic retinopathy (HLA-DR) and its influence in proliferative diabetic retinopathy (PDR). METHODS: We designed a case-control study in which 127 mestizo Mexican patients with DM II and diabetic retinopathy were studied. DNA was extracted and HLA-DR regions were amplified using PCR. Alleles were determined by DNA hybridization. Diagnosis was assessed clinically and by fluorescein angiography. Incidence of HLA-DR alleles in patients was compared with an ethnically matched control group of healthy subjects (n = 98). Statistical significance was established with non-parametric tests. RESULTS: Patients with diabetic retinopathy showed less frequency of HLA-D11 compared with the control group (p = 0.043). NPDR patients with 10 or more years of DM II showed an increase of HLA-DR7 (p = 0.01). CONCLUSIONS: Our results suggest that the presence of HLA-DR7 protects against the development of proliferative disease in the diabetic Mexican population.

The major histocompatibility (MHC) genes including TNF-alpha, HSP70 and HLA genes have been associated with systemic lupus erythematosus (SLE) in several populations. In this study we analyze the polymorphism of TNF-alpha promoter in 51 Mexican Mestizo SLE patients and 55 ethnically-matched healthy controls by polymerase chain reaction methods. No statistically significant differences were observed in the TNF -308 allele and genotype distribution between patients and healthy controls. However, we found a significant increase in the TNF G/A -238 genotype and in the TNFA -238 allele frequencies in the SLE group when compared with healthy controls (Pc = 0.03, OR = 4.77 and Pc = 0.02, OR = 3.62, respectively). DRB1 analysis showed a similar distribution in patients and controls. Linkage disequilibrium was observed for five haplotypes: DRB1*1401-TNFA-238 (D = 0.84; D' = 1.0; P = 0.015); DRB1*0301-TNFA-238 (D = 1.38; D' = 0.41; P = 0.042); DRB1*1106-TNF2-308 (D = 0.9; D' = 1.0; P = 0.0006); DRB1*1104-TNF2-308 (D = 0.83; D' = 0.45; P = 0.02) and DRB1*1406-TNF2-308 (D = 0.83; D' = 0.45; P = 0.02). Our data suggest that the association between the TNF-alpha -238 polymorphism and SLE could play a major role in disease susceptibility.

Takayasu arteritis (TA) is characterized by a 'pulseless' condition and occurs frequently in young females from Asian and South American countries. It has been associated with Mayor Histocompatibility Complex (MHC) genes in different populations. Recent data indicate direct participation of HLA-B alleles in the susceptibility to the disease. This fact was explored in an associative study with TA to establish if some region in the exon 2, intron 2 or in the exon 3 of HLA-B alleles is common in the alleles associated with TA and at the same time to know if a specific sequence or an epitope, more than an allele, would be responsible for the susceptibility to this vasculitis. We studied HLA-B alleles of 12 Mexican patients with TA using PCR-SSP and sequencing. The analysis by PCR-SSP in 12 patients showed that five of them showed the B*15 allele, three the B*40 allele and two the B*39 allele, the remaining two presented the B*44 allele. Sequence analysis enabled us to define that the B*39 subtypes are B*3908; B*15 subtypes are B*1510, B*1515, B*1522 and B*1531; and the B*40 subtypes are B*4005 and B*4008. An individual with B*51 (B*5107) and another with B*52 (B*5201) alleles were also identified. The sequences of the intron 2 seem be heterogeneous. Analysis at the 63 and 67 positions of HLA-B alleles showed that 9 of them have similarity in some of these positions with the residues detected in the B*5201 and B*3902 alleles associated with TA in Asian populations. The results indicate that there is heterogeneity in the alleles associated with TA in Mexicans but, in spite of that heterogeneity, the alleles associates can be separated into three groups: B*39, B*15 and B*40, whose subtypes are rare and apparently of recent generation in Mexico, probably by recombination events at intron 2 level. The sequences analysis also shows that most of the alleles detected in the Mexican patients share two epitopes described in the susceptibility alleles in Asian populations, suggesting that these epitopes could be responsible for the susceptibility to develop the disease in spite of the allele in which are found.