Estudos moleculares de doenças genéticas humanas

Abstract

The molecular study of human genetic diseases has an important role in the detection, prevention, treatment and/or counseling genetic of diseases which are caused by chromosomal abnormalities and DNA sequence variants. The exponential increase of molecular genetic techniques has introduced new therapeutic and diagnostic opportunities to the medicine of the 21st century. In the present study, which consists of two independent parts, we used the technique of Real-Time quantitative PCR (qPCR) as primary methodology for the molecular study of four human genetic diseases. In the first part, we performed a case-control association study to identify single nucleotide polymorphisms (SNPs) associated with the risk of age-related macular degeneration (AMD) in Brazilian population. TaqMan® methodology was used to genotype eight SNPs located in the nuclear genome and RFLP technique was used to genotype one SNP located in the mitochondrial genome. Regression analyzes, which were performed with Golden Helix software, showed the significant association of four SNPs with AMD after adjusting to covariates: rs1061147, rs1329428, rs2230199 and rs3750847. Our results are consistent with the assertion that polymorphisms in CFH, C3 and ARMS2/LOC387715 loci are consistently associated with AMD, even in a population with extensive genetic admixture, such as Brazilian population. In the second part, we performed qPCR assays using SYBR® Green reagent for detection of microdeletions associated with velocardiofacial syndrome (VCFS), Williams-Beuren syndrome (WBS) and monosomy 1p36. We analyzed normal individuals and patients with clinical features suggestive of microdeletion syndromes which were chosen. ROC curve analyzes (Receiver Operating Characteristic curve), which were made using MedCalc software, showed 100% sensitivity and 100% specificity for the markers which were analyzed. The molecular screening of one patient using three markers (two target genes and one reference gene) would have a cost of approximately R$ 39.11 considering only the reagents involved in the process, from DNA extraction to qPCR. Our results show that the qPCR is a fast, sensitive, specific and cost-effective molecular screening technique, being an economically viable method to be implemented in public health services of developing countries for detection of chromosomal microdeletion syndromes.