Search for genetic variants in the p66Shc longevity gene by PCR-single strand conformational polymorphism in patients with early-onset cardiovascular disease

Abstract

Background: Among the possible candidate genes for atherosclerosis experimental data point towards the longevity gene p66Shc. The p66Shc gene determines an increase of intracellular reactive oxygen species (ROS), affecting the rate of oxidative damage to nucleic acids. Knock-out p66Shc-/- mice show reduction of systemic oxidative stress, as well as of plasma LDL oxidation, and reduced atherogenic lesions. Thus, p66Shc may play a pivotal role in controlling oxidative stress and vascular dysfunction in vivo.

Methods: We searched for sequence variations in the p66Shc specific region of the Shc gene and its upstream promoter by PCR-SSCP in a selected group of early onset coronary artery disease (CAD) subjects (n. 78, mean age 48.5 +/- 6 years) and in 93 long-living control subjects (mean age 89 +/- 6 years).

Results: The analysis revealed two variant bands. Sequencing of these variants showed two SNPs: -354T>C in the regulatory region of p66Shc locus and 92C>T in the p66 specific region (CH2). Both these variants have never been described before. The first substitution partially modifies the binding consensus sequence of the Sp1 transcription factor, and was detected only in two heterozygous carriers (1 CAD subjects and 1 control subject). The 92C>T substitution in the CH2 region consists in an amino acid substitution at codon 31 (proline to leucine, P31L), and was detected in heterozygous status only in one CAD subject. No subjects homozygous for the two newly described SNPs were found.

Conclusion: Only two sequence variations in the p66Shc gene were observed in a total of 171 subjects, and only in heterozygotes. Our observations, in accordance to other studies, suggest that important variations in the p66Shc gene may be extremely rare and probably this gene is not involved in the genetic susceptibility to CAD.

Figure 1

Mutation detection with SSCP and sequence electropherograms of p66^Shc. PCR-SSCP and sequence analysis of the regulatory region of p66^Shc locus (A) and of the p66^Shc specific region CH2 (B). In the sequence electropherograms, arrows indicate the nucleotide substitution in heterozygous subjects as shown by the presence of the 2 superimposed peaks representing the normal and abnormal base. A) SSCP analysis of the 5' regulatory region of p66 locus (T/T subjects with wild-type sequence, T/C subjects heterozygous for the variant). B) SSCP analysis of the CH2 region: the 92C>T substitution consisted in an amino acid substitution P31L (C/C subjects with wild-type sequence, C/T subject heterozygous for the variant).