2.3. Genotyping and statistical analysis

The leaderpeptidetrinucleotiderepeat polymorphismD18S880inexon2 of CNDP1 gene was genotyped on a fluorescent DNA analysis system (ABI 3100, Applied Biosystems, Darmstadt, Germany) as previously described (Janssen et al., 2005). In selected samples, the exact sequence of exon 2 was determined by sequence analysis using the forward primer gtgtttggggaaggacgtag, the reverse primer tctcacccaaataccaaagg and the same equipment.

Hardy–Weinberg equilibrium at the CNDP1 gene leucine repeat, using a Monte-Carlo Markov-Chain (MCMC) exact test (Guo and Thompson, 1992). Sincenoneofthetestsyieldedasignificantresult,theleucinerepeatwastestedfor a possible association with either CHD or longevity by comparing the respective allele frequencies in the target and control group. The observed differences were assessedforstatisticalsignificancebymeansofanin-houseMCMCimplementa- tion of Fisher’s exact test. Both MCMC-based procedures comprised a burn-in phase of 20,000 iterations and an evaluation phase of 500,000 iterations. Con- sidering that alleles coding for higher activity of an enzyme (such as CNDP1 alleles with 6 or more leucines) dominate over alleles coding for reduced activity (such as the CNDP1 5-leucine allele), and that the protective effect in diabetic nephropathy was mediated by the 5-leucine allele (Janssen et al., 2005), we also chose to compare the respective proportion of individuals homozygous for the 5- leucine allele between target and control groups.

2. Methods

A sample of 330 unrelated German centenarians was studied. All indivi- duals were between 100 and 110 years of age at the time of recruitment (mean age: 101.4 years). The gender ratio in this sample was 80.6% females to 19.4% males. The 368 German control subjects were between 60 and 75 years of age (mean age: 67.2 years) and matched the long-lived individuals by ancestry (all Germans), geographical origin within the country, and gender. A detailed description of the samples and the recruitment procedure has been previously published (Nebel et al., 2005).

2.2. Coronary heart disease patients and control subjects

3. Results

The coronary heart disease patients and the corresponding control subjects are part of the PopGen biobank (Krawczak et al., 2006). The patient sample comprised 350 unrelated individuals with premature coronary heart disease (CHD). Cases were required to have coronary catheterization demonstrating

DNA samples from a total of 1382 individuals were examined in this study. These included 330 centenarians as well

Table 1

Frequency of CNDP1 alleles and genotypes in centenarians (330 individuals) and individuals with coronary heart disease (CHD, 350 individuals), together with the respective control groups (368 for the centenarians, 334 individuals for CHD)

Centenarians Controls CHD patients Controls Total n % n % n % n % n % Alleles Five leucines 408 61.8 455 61.8 418 59.7 387 57.9 1668 60.35 Six leucines 214 32.4 256 34.8 244 34.9 252 37.7 966 34.94 Seven leucines 34 5.2 17 2.3 35 5.0 23 3.5 109 3.9 Eight leucines 1 0.1 0 0 0 0 0 0 1 0.04 L13_V15dup 0 0 1 0.1 0 0 0 0 1 0.04 Null mutation 3 0.5 7 1.0 3 0.4 6 0.9 19 0.69 Total 660 736 700 668 2764 Genotypes Homozygous 5 leucines 121 36.7 142 38.6 120 34.3 118 35.3 501 36.3 Others 209 63.3 226 61.4 230 65.7 216 64.7 881 63.7 Total 330 368 350 334 1382

n: number of chromosomes or probands.

J. Zschocke et al./Mechanisms of Ageing and Development 127 (2006) 817–820

Fig.1. Schematicrepresentationofthedifferentallelesobserved intheleucine-repeat regionoftheserum-carnosinasegene.Shownarethenucleotideandaminoacid sequences. The 5-leucine allele is regarded as thewild type, with the first leucine in the repeat representing amino acid residue p.L16 encoded by nucleotides c.46–48.

as 350 individuals with premature coronary heart disease (CHD) and corresponding sex- and age-matched control groups. The results of the analyses in the four groups are shown in Table 1. The 5-leucine allele was the most common allele in all groups investigated. In addition to the previously reported 5-, 6- and 7-leucine alleles, we found a repeat of eight leucines on one chromosome. Furthermore, we observed an allele with a fragment length similar to the 8-leucine allele which however carried a novel duplication of three amino acids (LAV) preceding a 5-leucine repeat (p.L13_V15dup). There were 19 samples in which a shift in the peak position corresponding to the 7-leucine allele became apparent. Sequence analysis revealed that these samples contained a novel frameshift mutation L17fsX20 (c.48_49insTGCTG) caused by an insertion of five bases after the third base of the trinucleotide repeat in the reference sequence (Fig. 1). This mutation is predicted to change the amino acid sequence and to introduce a stop codon at position 36 of the amino acid chain. It is therefore expected to represent a null-allele that completely removes serum-carnosinase function. The null allele was infrequent in both the proband and the control groups, and no homozygote for this allele was observed.