2.3. Statistical analysis

Fisher’s exact probability tests were performed with Microstate software to assess the statistical significance of frequency differences observed between the aged and control groups. Where applicable, multiple comparisons were taken into consideration by applying Bonferroni’s correction, a¼ 1 2 0: 951=n (where n is equal to the number of comparisons).

Genomic DNA from all subjects was extracted from the buffy coats of peripheral blood by the salting out method (Miller et al., 1988).

2.2. KIR genotyping by PCR–SSOP

3. Results

PCR amplification was performed using the appropriate primer combinations for each target region (Table 1) in 100 ml reaction volumes containing 67 mM Tris–HCl pH 8.8; 16 mM (NH ) SO ; 2.0 mM MgCl ; 0.01%(w/v) 4 2 4 2 Tween; 200 mM of each dNTP; 0.2 mM of each primer; 3 units Taq polymerase (Bioline, London, UK); 0.1–0.5 mg DNA. After an initial denaturation step of 96 8C for 5 min, the cycling conditions specified in the legend of Table 1 were employed for each target region. After the final cycle, the samples were held at 72 8C for 5 min and then at 15 8C until removed from the PCR machine. Successful amplification

In the present study, diversity within KIR gene content was examined to determine its effect on ageing within the Irish population.

A high degree of variation was observed within the sample groups examined, with a total of 24 different genotypes being detected. The gene content of each of these genotypes and their frequency within each sample group analysed are displayed in Tables 3 and 4, respectively. Of the 24 genotypes identified during this study, 14 were

L.D. Maxwell et al. / Experimental Gerontology 39 (2004) 1223–1232

Table 2

Sequence specific oligonucleotide probes used for KIR gene assignment

Probe name (Specificity) 50 Sequence 30 Wash temp. (8C) Picomoles used Trans/cyt amplified domain NK1 (2DL1) TCTGCAGGAAACAGAAC 50 20 NK2DL1vR (2DL1*004) GGAGCACCAGCAATGAAGGAGA 64 100 NK2 (2DL2) TCGTGTACGCGGAACTT 54 20 NK3 (2DL3) CTGGTGCTGCAACAAA 48 20 NK2DL3/1350 CTGAGCCCTGATCCAAA 52 20 NK10 (2DL4) GCCTGCGGGACACAGAAC 60 20 NK2DL5 CATTGCTGCTGCTCCA 50 20 NK8 (3DS1) GGAACAGAAGTGAACAG 50 20 NK6 (3DL1) CAGAACAGCCAACAGCGA 56 20 NK7 (3DL2) GCCTGCGGGGGACAGAAC 62 20 D2 amplified domain Ig1 2DL2 CCATGAATGTAGGTTC 46 20 Ig1 2DL4 TTCGCTTACAGCCCGGC 56 40 Ig1 2DL5 TCGGCTCTCTCCATGAC 54 20 Ig1 2DS1aR (2DS1/2DL1*004) TTGACCTTGGTCCCTGCAGG 58 100 Ig1 2DS1bR (2DS1/2DL1*004) AGGTCCCTCCATGGGTGGCA 60 100 Ig1 2DS2 CCACGGTTTTGGCAGGA 56 40 Ig8 (2DS3) CCCAAGGAGGAACCTAC 54 40 Ig1 2DS3 ATCTATCCACGGAGGG 50 40 Ig1 2DS4 TCCGTGACGCTCCCTAC 56 40 Ig1 2DS4del (2DS4*003) CTGCAGCT–22bp–CCATCTAT 48 20 Ig1 2DS5n CCTCTGGACCCTGCC 48 40 D1 amplified domain Ig2 3DL2 TACAGATGTTATGGTTCT 48 20 Ig2 3DL3 CACGATGCGGGTTCCCAG 60 20 Ig2 3DS1 CACAAAGAGTGGATCTCT 52 30 Ig2 2DL1 CCTATCAGGTGTCAGCT 54 30 Ig2 2DL2 GTCTCCAAAGCCAACTT 50 30 Ig2 2DS1Agg (2DS1*001) CCATCAGTCGCATGAGGCAAG 66 100 Ig2 2DS1Aga (2DS1*003) CCATCAGTCGCATGAAGCAAG 64 50 Ig2 2DS2 GGGGAAGTATAAGGACA 48 30 Ig2 2DS3 CTATCAGTTTTCAGCTCC 52 20 Ig2 2DS4 TTCTCCATTGGTCCCAT 48 20 Ig2 2DS5a ACGTTTAACCACACTTTG 50 20 Ig2 X (3DP1) GAAGTTTAATGACACTTTG 50 20 Ig2 Z (2DP1) CCATGATGGAAGACCTG 52 30 D0 amplified domain p70-3 (3DL1*00101) GAATCCACATTCCCATC 50 20 p70-4 (3DL1*002) ATGGCAGATTATTCCAG 52 20 Ig3 3DL2 GACAGAAGCCACGTTC 50 20 Ig3 3DL3 CTGAAGGACAACATGTG 50 20 p70-1 (3DS1) TGCCTCGCGGAGGAC 52 20 Ig3 2DL4 CATCTTCACGCTGTAC 48 20 Ig3 2DL5 GGGTTTACCATCTTC 44 50 Ig3 X (3DP1) GAAACACCGTTTTCATAG 50 20 Ig3 Z (2DP1) CAGGGACGTACAGAT 44 20

KIR nomenclature is in accordance with that recently reported (Marsh et al., 2002). Probes were designed, where possible, to incorporate the polymorphic nucleotide(s) in a central position within the oligonucleotide. Underlined nucleotides represent the nucleotides conferring probe specificity.

encountered in both sample cohorts (1, 2, 3/9, 4, 5/10, 6, 7, 7a, 8, 12/17, 13, 15, 24, 27), five genotypes were observed to be unique to the controls (11, 19, 22, 23, 32), and five were detected only in the cohort of aged subjects (20, 28, 29, 30, 31). Within the 14 gene repertoires that were common to both the aged and control subsets, the same four KIR

genotypes were most frequent in both sample groups (1, 2, 3/9, 4). Although, these common KIR gene repertoires collectively account for the majority of genotypes encoun- tered in both the aged subjects and young controls (63 and 68%, respectively), rare KIR gene repertoires form a considerable proportion of the total genotype pool of both

L.D. Maxwell et al. / Experimental Gerontology 39 (2004) 1223–1232

Table 3

gene content of the genotypes detected within the aged and control subjects — et ol2DPI      

KIR gene content of the genotypes detected within the aged and control subjects

The genotype numbering system is in accordance with that previously reported (Middleton et al., 2002). Only a proportion of the KIR genotypes previously encountered within the Irish population (unpublished data) were detected during this study. Genotypes identified during this study are shown in italics. Solid boxes denote positivity for a KIR gene whilst white boxes denote their absence.

and accepted trends in the frequencies of certain KIR genes were also observed. In agreement with previous studies (Witt et al., 1999; Crum et al., 2000; Middleton et al., 2002), activating KIR genes were present at a much lower frequency within the sample groups investigated than genes encoding inhibitory receptors. In addition, the KIR2DL4, 3DL2, 3DL3 and 3DP1 genes were present in all individuals examined, whilst the frequencies of KIR2DL1 and KIR2DL2 were identical to KIR2DP1 and KIR2DS2, respectively.

the sample groups, with these repertoires displaying a marginally higher frequency in the aged subset relative to the controls (37 and 32%, respectively).