Restriction Enzyme-based Method for Transforming Growth Factor-β1 Genotyping: Nonisotopic Detection of Polymorphisms in Codons 10 and 25 and the 5′-Flanking Region

Abstract

Transforming growth factor-β1 (TGF-β1) modulates a wide spectrum of biological activities, including cell proliferation, differentiation, and adhesion as well as extracellular matrix formation. The involvement of TGF-β1 in inflammation and tissue repair has been established in the past few years, suggesting a crucial role for this cytokine in pathologies of the cardiovascular system, kidney (chronic allograft rejection in kidney transplantation, glomerulonephritis, and diabetic nephropathy), bones (osteoporosis), skin (systemic sclerosis), lung (idiopathic lung fibrosis and asthma), and liver (cirrhosis). The human TGF-β1 gene (TGFB1) , located on chromosome 19q13, contains seven exons that give rise to a precursor protein of 390 amino acids (1), which is proteolytically processed to generate the mature protein of 112 amino acids. The promoter for TGF-β1 contains two major sites for the initiation of transcription and multiple regulatory motifs, but it lacks the typical TATA or CAAT boxes (2). Seven TGFB1 polymorphisms recently have been reported as single-stranded conformational polymorphisms and applied to a multicenter study population, the Etude Cas-Temoin de L’Infarctus du Myocarde (ECTIM) study (3). Three of the seven allelic variations were localized in the 5′-flanking region of the TGFB1 gene (at positions −988, −800, and −509), three were in the coding region (codons 10, 25, and 263), and an insertion was also described in the 5′-untranslated region at position +72. The analysis of candidate genes and/or polymorphisms has been widely applied in clinical genetics to study human diseases. A rapid and easy method for genotyping is essential in such studies, in which large numbers of cases and controls are needed. The ECTIM study used allele-specific oligonucleotides (ASOs). After amplification by PCR, products were blotted into a nylon membrane and hybridized with an ASO probe. The major disadvantage of this method is that it its time-consuming and requires …