ScienceDirect: Mechanisms of Ageing and Development 127 (2006) 817-820 www.elsevier.com/locate/mechagedev Variation in the CNDP1 gene and its lack of association with longevity and coronary heart disease Johannes Zschocke, Almut Nebel, Kate Wicks, Verena Peters, Nour Eddine El Mokhtari, Michael Krawczak, Fokko van der Woude, Bart Janssen, Stefan Schreiber of Human Genetics, Ruprecht-Karls-University, Heidelberg, Germany Department of Pediatrics, Ruprecht-Karls-University, Heidelberg, Germany Received 24 May 2006; received in revised form 25 July 2006; accepted 1 August 2006 Available online 11 September 2006 Abstract Carnosine, a cytoprotective dipeptide found at very high concentrations in skeletal muscle, heart and brain, is cleaved in blood by serum carnosinase which is encoded by the CNDP1 gene. We recently found that homozygosity of a 5-leucine variant in the leader peptide of this enzyme protects diabetes mellitus patients against nephropathy. Hypothesizing that the same allele could also be associated with longevity or a reduced incidence of cardiovascular problems, we examined the frequency of CNDP1 alleles in German centenarians, patients with premature coronary heart disease, and matched controls. A total of 1382 individuals were investigated. The 5-leucine allele was the most common allele in all groups investigated. There was no difference in allele or genotype frequency between centenarians and their control group or between cardiovascular patients and their control group. The recently identified functional carnosinase variant therefore does not contribute to longevity nor protect against coronary heart disease in our probands. In addition to the known trinucleotide repeat alleles in the CNDP1 gene, we detected a rare 8-leucine allele, a rare duplication, p.L13_V15dup, and a more common frameshift deletion, L17fsX20. Homozygosity for L17fsX20, estimated to have a prevalence of approximately 1:20,000, would be expected to cause carnosinaemia, an autosomal recessive trait with uncertain clinical relevance. Keywords: Carnosinase; CNDP1; Mutation; Ageing; Coronary heart disease 1. Introduction Carnosine (beta-alanyl-histidine), a dipeptide found at very high concentrations in skeletal muscle, heart and brain, has been shown to have a range of antioxidant or cytoprotective properties including scavenging of free radicals, formation of complexes with transition metals and anti-glycating effects (Boldyrev, 2000). The protein has been reported to extend the life span of cultured cells (McFarland and Holliday, 1994), to increase the survival of rodents (Boldyrev and Severin, 1990) and to raise the lifespan of male Drosophila flies to that of females (Yuneva et al., 2002). Hence, carnosine has been widely suggested as a naturally occurring anti-aging substance in men. In addition, a range of beneficial effects on the cardiovascular system, including a protective role in ischemic heart disease, has been reported (Roberts and Zaloga, 2000). Carnosine is frequently offered as a nutritional supplement. Two enzymes in humans are able to cleave carnosine: a secreted (serum) carnosinase and a non-specific dipeptidase (previously denoted tissue carnosinase). Serum carnosinase is encoded by the CNDP1 gene that acts on imidazole dipeptides including carnosine and homocarnosine. In human and higher primates serum carnosinase is found at high concentrations in blood. Other species do not have a secreted CNDP1 gene product. The non-specific dipeptidase encoded by the CNDP2 gene is less active on carnosine and is found in the cytosol of many organs (Teufel et al., 2003). In blood, carnosine is rapidly metabolized and present at very low concentrations, while a high intracellular concentration in some organs is maintained by the absence of carnosinase activity. Recently we found a functionally relevant trinucleotide repeat polymorphism in exon 2 of the CNDP1 gene (D18S880) that codes for a variable number of leucine residues in the leader peptide. Presence of five (as opposed to six or seven) leucines is associated with reduced serum carnosinase activity and confers a lower risk of diabetic nephropathy (Janssenidentified in the present study. The 5-leucine allele codes for reduced activity of carnosinase and is thought to act as a recessive allele, with a strong functional impact only in the homozygous state. Previous studies by us (Janssen et al., 2005), independently confirmed by others (Barry Freedman, personal communication), showed that homozygosity for the 5-leucine allele protects against the development of nephropathy in diabetes mellitus. Considering the well-established cytoprotective effects of carnosine, we hypothesized that a similar beneficial effect would be likely regarding longevity or the risk of developing cardiovascular disease. However, there was no significant difference between centenarians or CHD patients and their respective controls in terms of the frequency of neither the 5-leucine allele itself nor of homozygotes for it. Fisher's exact test showed a difference in allele frequency spectrum between the longevity target and control groups which reached statistical significance (P = 0.021), owing to a higher frequency of the 7-leucine allele among centenarians. There is no conceivable biological explanation for a specific impact of this allele on the likelihood of reaching older age, and sequence analysis of the whole CNDP1 coding region did not reveal a genetic variant in linkage disequilibrium with the 7-leucine allele that could be assumed to influence carnosinase activity. The association may therefore be spurious.