Single Strand Conformation Polymorphism (SSCP) as a quick and reliable method to genotype M235T polymorphism of angiotensinogen gene.
OBJECTIVE
Conflicting results on the relationship between M235T polymorphism of angiotensinogen (AGT) gene and diabetic nephropathy are reported in the literature, probably due to the small number of subjects, to different inclusion criteria and the different genotype analysis methods used. The aim of the present study was to set up a fast, cheap and reliable method to allow the genotyping of M235T polymorphism in a large number of subjects.


METHODS
We developed in our laboratory a new specifically designed PCR-SSCP method for M235T genotyping whose specificity was compared with that of Allele Specific PCR (ASPCR) and Mutagenically Separated PCR (MS-PCR). The exact M235T genotype was estabilished by direct sequencing. The new PCR-SSCP method was then used to genotype a population of 1171 hypertensive, normoalbuminuric type II diabetes mellitus patients. The patients were also genotyped for ACE I/D polymorphism. For comparison a group of hypertensive non diabetic patients (n = 88) were also screened.


RESULTS
The PCR-SSCP method identified the M235T polymorphism with no misinterpretation at variance with ASPCR and MS-PCR methods that showed a preferential amplification of the T allele. The rare Y248C polymorphism of the AGT gene was also detected by PCR-SSCP. In diabetic hypertensive patients the prevalence of TT genotype was higher than in normotensive healthy controls and equivalent to that found in hypertensive non diabetic patients.


CONCLUSIONS
The PCR-SSCP method for detection of M235T polymorphism is a powerful and sensitive tool for rapid, cheap and efficient screening of a large number of samples. The results obtained with this method demonstrate an association of the TT genotype of AGT gene with hypertension, both in diabetic and non diabetic patients.
Bettinaglio
Paola
P
Mario Negri Institute for Pharmacological Research, Clinical Research Center for Rare Diseases Aldo e Cele Daccò, Villa Camozzi-Ranica, Italy.
Galbusera
Andrea
A
Caprioli
Jessica
J
Orisio
Silvia
S
Perna
Annalisa
A
Arnoldi
Federica
F
Bucchioni
Sara
S
Noris
Marina
M
D000328:Adult; D000368:Aged; D000483:Alleles; D000808:Angiotensinogen / Q000235:genetics*; D001483:Base Sequence; D003924:Diabetes Mellitus, Type 2 / Q000150:complications / Q000235:genetics; D003928:Diabetic Nephropathies / Q000235:genetics; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D006801:Humans; D006973:Hypertension / Q000150:complications / Q000235:genetics; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D018401:Sample Size; D012680:Sensitivity and Specificity; D013997:Time Factors
D000808:Angiotensinogen