| Background: Angiotensin I -converting enzyme, encoded by ACE, also known as DCP1 (MIM 106180), which controls fluid-electrolyte balance and blood pressure by catalyzing the angiotensin I to the physiologically active peptide IgA nephropathy angiotensin II, plays a key role in the rennin-angiotensin- system (RAS). Many associated studies have been carried with ACE, almost all studies have related with the presence (insertion, I) or absence (deletion, D) of a 287bp Alu repeat element within intron 16, the I/D polymorphism which had been shown to be associated with the incidence and progression of IgA nephropathy and the predictor of IgA nephropathy to the therapy with ACE inhibitor, but the finding had been conflicting.IgA nephropathy is the most common glomerulonephritis in the world among patients undergoing renal biopsy, longitudinal follow-up studies have revealed that 9% to 50% patients with IgAN progress to the end stage renal disease within 20 years of disease onset. Both environmental and genetic factors likely contribute to the development and progression of IgA nephropathy. Our experimental strategy was to first identify the complete genomic sequence of ACE which about 24kb in length, consists of 26 extrons, 16% of whole gene is coding region from 12 Chinese individuals (8 patients with IgA nephropathy and 4 controls) for sequence variation in human ACE and then to utilize this information to select a subset of polymorphisms for testing by TaqMan PCR assay in a larger cohort of patients and controls to identifyhow much nucleotide diversity exists and whether or not the pattern of variation is involved in the predisposition of IgA nephropathy in Chinese.Methods: 2ml EDTA blood was taken from 8 Chinese patients who have biopsy-proven primary IgA nephropathy and who were first seen at renal medicine department of Singapore General Hospital between the year of 1978 and 2000 (criteria for renal biopsy were abnormal serum creatinine, hypertension, total urinary protein >lg/day and nephritic syndrome). 4 unrelated healthy Chinese individuals were also enlisted. Genomic DNA was extracted from 0.2ml EDTA blood by using the QIAamp DNA blood extraction kit (Qiagen, Germany). ACE complete sequences were obtained from GeneBank (accession number AF118569). overlapping primers sets spanning the whole genomic sequence of ACE were chosen on the basis of size and overlap of PCR amplicons (average size,840± 180bp, average overlap, 260±57bp ). All samples were amplified from genomic DNA (10-100ng) in reactions (25-50ul) containing 10 × PCR Buffer, 5.0ul; 2 mM dNTP mix, 0.2mM of each; Primer F (Forward), 0.4uM; Primer R (Reward), 0.4uM; Taq DNA polymerase, 1 unit/50ul; 25 mM MgC12, 1.5mM. The amplification program consisted of an initial denaturation at 94 ℃ for 3 minutes, followed by 35 cycles at 95 ℃ for 45seconds, 53 ℃ to 68 ℃ for 45seconds and 72 ℃ for 2minutes, with a final extension at 72 ℃ for 10 minutes before holding at 4 ℃. Identity of PCR products: Products were separated in 2% agarose gel and visualized by ethidium bromide (0.5ug/ml) staining and photographed under UV Tran illumination. Purification of PCR products: PCR products were purified with the use of QIAquick PCR purification kit (Qiagen, Germany) and QIAquick gel Extraction kit (Qiagen, Germany). DNA fragments (the above PCR products) were sequenced with the use of BigDye Terminator v3.1 Cycle Sequencing Kit. The 10ul reaction mixtures contain: Terminator Ready Reaction Mix, 4.0ul; primer, 3.2pmol; DNA, 5-20ng. Cycle Sequencing carried .on the PCR System 9700. The amplification program consisted of an initial denaturation at 96 ℃ for 1 minutes, followed by 25 cycles at 96 ℃ for 10 seconds, 50 ℃ for 5seconds and 60 ℃ for 4 minutes, hold at 4 ℃ until ready to purify. Automated capillary gel electrophoresis were carried on the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, USA). Polymorphisms were identified bythe comparison of sequences from 24 chromosomes using DNA Star sequence analysis software. SNPs found on one chromosome (a sing...
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