One hundred and eighty control samples (68% female, 32% male with an age-range of 19–45 years; mean age 29 years) were chosen at random from an anonymous DNA bank of normal healthy Caucasian individuals from the Northern Ireland population. The study involved 180 aged samples (71% female, 29% male with an age-range of 80–97 years; mean age 89 years) from the Belfast Elderly Longitudinal Free-living Ageing Study (Rea et al., 2000). All samples were from healthy Caucasian individuals from the Northern Ireland population displaying no signs of age- related disorders, still living independently and approximate SENIEUR status (Ligthart et al., 1984, 1990). Ethical approval was obtained from the Queen’s University of Belfast ethics committee.

In the present study, the frequency of the deleted from the KIR2DS4 gene was examined in a cohort of healthy aged Irish subjects in comparison to a younger control group

2DS4*001022D84*0032D84*001022D84*0032DS4*001022D84*0032D84*001022D84*0032D84*001022D84*003FRAMESHIFT2D84*001022D84*0032D84*001022D84*0032D84*001022D84*0032D84*001022D84*0032D84*001022D84*0032D84*001022D84*003+1newa EXON4 (D1)CATGGCGTGTGTTGGGTTCTTCTTGCTGCAGGGGGCCTGGCCACAGGAGGGAGTCCACAGAAAACCTTCCTTCCTGGCCCTCCCAGGTCACCTGGTGAAA 220TCAGAAGAGACAGTCATCCTGCAATGTTGGTCGGATGTCATGTTTGAGCACTTCCTTCTGCACAGAGAGGGGAAGTTTAACAACACTTTGCACCTCATTG 320GAGAGCACCATGATGGGGTTTCCAAGGCCAACTTCTCCATTGGTCCCATGATGCCTGTCCTTGCAGGAACCTACAGATGCTACGGTTCTGTTCCTCACTC EXONS (D2) 420CCCCTATCAGTTGTCAGCTCCCAGTGACCCTCTGGACATGGTGATCATAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTCAG 520GCAGGAGAGAATGTGACCTTGTCCTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAGGGGGAGGCCCATGAACGTAGGCTCCCTGCAG 620 c AAS TEHS R PTFLWALPPTEGP TDA SAL SV TL PEXON6 EXON7CGAGTGGTCAAACTCGAGTGATCCACTGCTTGTTTCCGTCACAGGAAACCCTTCAAATAGTTGGCCTTCACCCACTGAACCAAGCTCCAAAACCGGTAACTSGQTRVIHCLUFPSQETLQIVGELHPLNQAPKPV TE 820  1020TCACTGTGTTTTCACACAGAGAAAAATCACTCGCCCTTCTGAGAGGCCCAAGACACCCCCAACAGATACCAGCATGTACATAGAACTTCCAAATGCTGAG 1110CCCAGATCCAAAGTTGTCTTCTGTCCACGAGCACCACAGTCAGGCCTTGAGGGGATCTTCTAGGGAGACAACAGCCCTGTCTCAAAACCG<<

Fig. 1. Representative alignment of KIR2DS4 deletion variant sequence (KIR2DS4*003) with undeleted KIR2DS4*00102 sequence. Numbering initiates on the first nucleotide of the ATG initiation codon of the 2DS4 coding sequence—only the cDNA sequence from position 21–1110 of the coding sequence was targeted in our PCR design. Dashes indicate complete identity while an * indicates a nucleotide deletion. Arrows highlight the location and direction of the two PCR primers (2DS4f and 3UTNKex) used to amplify the KIR cDNA sequence, and spaces are left under the PCR primer sequences as these sequences were not determined. The amino acid sequence of the new open reading frame accessed by 2DS4del due to the 22 bp deletion is depicted under its nucleotide sequence and terminates at the first in frame stop codon (V). The exon start positions are also highlighted. The only other nucleotide difference found in this 2DS4 deletion variant sequence is the presence of the G-A (nt489) change just downstream of the 22 bp deletion site, which in this particular instance would no longer be silent due to the resulting frameshift at the deletion site and concomitant R-K change in the predicted protein coding sequence representing the carboxyl end of the 2DS4 deletion variant.

Ross et al. / Experimental Gerontology 39 (2004) 801–805

(FMC BioProducts, Maine, USA) for greater resolution, stained with ethidium bromide (0.5 mg/ml), at 200 v for 45 min. A representative gel image is shown in Fig. 2. Samples which failed to amplify were confirmed as 2DS4 negative (2ve) samples by employing a sequence specific oligonucleotide probe KIR gene typing system (Crum et al., 2000; Middleton et al., 2002).

deletion product(199 base pairs)full length product(221 base pairs) 

3. Results and discussion

Fig. 2. Representative gel image for genotyping of the 2DS4 polymorph- ism. A representative Metaphor agarose gel image for the genotyping of the KIR2DS4 amplicons. Lane M contains a DNA size marker, f £ 174 RF Hae III digest; Lanes 1 and 5 display the results obtained for a heterozygous individual; Lanes 2 and 6, show the presence of the homozygous deleted 2DS4 form only; Lanes 3, 4 and 7, displays the results for homozygous individuals displaying the full length 2DS4 form only.

The present study investigated the frequency of a common 22 bp deletion in the killer immunoglobulin-like receptor gene 2DS4 that predicts a soluble KIR molecule. A simple PCR methodology was employed to genotype 180 aged and 180 young control subjects from the Irish population. The observed frequencies of the deleted and full-length forms of the 2DS4 gene are displayed in Table 1.

2.2. PCR amplification

The 2DS4-specific oligonucleotide primers employed for the amplification of the 221/199 bp region of the 2DS4 gene were:

No significant associations were observed for the 2DS4 deletion variant with either age group or with respect to gender. The 2DS4 gene was present in the vast majority of samples examined, with only approximately 5% of each sample group appearing negative for the gene. The deleted form of the 2DS4 gene was identified at a high frequency (70–80%) within the Irish population as previously reported (Maxwell et al., 2002), and this raises the question concerning the possible function of this KIR variant. We previously reported the presence of mRNA for this gene (Maxwell et al., 2002) and studies are presently underway in an attempt to identify the hypothetical protein product in blood plasma. If the presence of this soluble KIR can be confirmed, it will be important to elucidate its role in the immune response to infection/disease and autoimmunity, and its potential effects on ageing.