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Mechanisms of Ageing and Development 127 (2006) 817–820

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variation in the CNDP1 gene and its lack of association with longevity and coronary heart disease

Johannes Zschocke , Almut Nebel , Kate Wicks , Verena Peters , d e f Nour Eddine El Mokhtari , Michael Krawczak , Fokko van der Woude , a c Bart Janssen , Stefan Schreiber

of Human Genetics, Ruprecht-Karls-University, Heidelberg, Germany bDepartment of Paediatrics, 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. Hypothesising 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 was 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 neither 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

life span of cultured cells (McFarland and Holliday, 1994), to increase the survival of rodents (Boldyrev and Severin, 1990) and to raise the life-span 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.

Carnosine (b-alanyl- -histidine), a dipeptide found at very L 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

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

* Corresponding author at: Institute of Human Genetics, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany. Tel.: +49 6221 56 33762; fax: +49 6221 56 33762.

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significant CHD (at least a 70% stenosis in one major epicardial coronary vessel) before the age of 55 years. The average age of disease onset was 48.2 years. The majority of subjects (90.3%) had undergone a coronary revascular- ization procedure (percutaneous coronary intervention or coronary artery bypass grafting) and a subset of 243 individuals (66%) had suffered myocardial infarction. The patients were between 35 and 63 years of age at the time of recruitment (mean age: 54.8 years). The gender ratio in the entire sample was 63.3% males to 36.7% females. A total of 334 unrelated control individuals were included in the study who matched the CHD patients by ancestry (all Germans) and gender. The controls were drawn from the same age range as the CHD patients, with an average age of 50.0 years.

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 metabolised and present at very low concentrations, whilst 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 (Janssen et al., 2005). Considering the known cytoprotective and anti-aging effects of carnosine, we hypothesised that the same allele might also be associated with longevity and a reduced incidence of cardiovas- cular problems. We therefore assessed the frequency of CNDP1 alleles in large samples of German centenarians, coronary heart disease (CHD) patients, and appropriate control individuals.

There was no overlap between the control groups in the two cohorts. All subjects gave informed, written consent prior to participation. The study was approved by the Ethics Committee of the University Hospital Schleswig- Holstein in Kiel.

2.3. Genotyping and statistical analysis