In a previous work, we described the establishment of a multiplex realtime PCR method enabling quantification of gene copy number, which easily discriminate carriers of one or two GSTM1 and GSTT1 alleles (Brasch-Andersen et al., 2004). In the present study, we have used this method to investigate the influence of the GSTT1 and GSTM1 gene deletion polymorph- isms on mortality in a longitudinal study of 681 elderly Danish twins (234 men and 447 women), thereby circumventing the potential problems of the cross sectional approach. The included subjects were drawn from the 1997 wave of the population-based survey ‘‘the Longitudinal Study of Ageing Danish Twins’’ comprising all Danish twins age 70 years and older (Skytthe et al., 2002). Information on survival status was retrieved from the Danish Central Population Register, which is continuously updated. The mean follow-up time for survivors was 7.6 years (range 7.1–8.2 years). Only all-cause mortality was recorded as information on cause-specific mortality was unavailable for the main part of the follow-up time included. Genotyping was performed as described (Brasch-Andersen et al., 2004) and all samples were done in triplicates. Mortality risk was estimated using the Cox proportional hazard model, adjusting for age. To account for the non-independence of twins, twin pairs where both members participated were analyzed in clusters of two, using the cluster option of the statistical software Stata 8.0.

functional gene is somewhat paradoxical, given the generally accepted assumption that the null genotype is a disease risk factor. However, our data are in accordance with the findings of Taioli et al. (2001) and with a recent study by Geisler et al. (2005) who found that the presence of the GSTT1 gene was a predictor of both overall mortality and disease-specific mortality for patients diagnosed with carcinomas of the head and neck. Furthermore, reports on the influence of GSTT1 null genotypes on cancer susceptibility have not been as unambig- uous as expected, and the presented results may thus reflect a dual role of GSTT1 in detoxification of some hazardous compounds and activation of other toxic substrates occurring with different preference at various stages of life. It is well known that GSTT1 participates in bioactivation of certain compounds, like e.g. dihalogenated alkanes (Sherratt et al., 1997; Shokeer et al., 2005; van Bladeren, 2000), thus suggesting that deletion of the GSTT1 gene may be beneficial under some circumstances.

The basic characteristics shown in Table 1 demonstrate an increased mortality in female carriers of both one and two copies of the GSTT1 gene compared to individuals with the null genotype and this result was confirmed in the Cox regression model provided in Table 2. Furthermore, we found a tendency towards a decreased mortality restricted to individuals carrying two copies of the GSTM1 gene. The latter association, however, did not reach statistical significance, probably due to the rather low number of individuals with two GSTM1 gene copies.

One possible concern is that the GSTT1 genotype distribu- tion in the elderly men deviates from Hardy-Weinberg equilibrium. The observed GSTT1 null allele frequency is somewhat lower than the frequency reported for most younger populations of European origin (19–21% in e.g. Gaspari et al., 2003; Pesch et al., 2004; Taioli et al., 2001), although comparable with the frequency reported in the most similar age range (14% in Pesch et al., 2004). Since the null genotype is

Table 1

Basic characteristics of the study population

Men (N = 234) Women (N = 447) Genotype Age at intake Deaths during Genotype Age at intake Deaths during distribution (%) (years   S.D.) follow-up (%) distribution (%) (years  S.D.) follow-up (%) GSTM1a   137 (58.5) 76.5   3.1 68 (50) 228 (51.0) 77.1   3.9 87 (38) (cid:2)/+ 84 (35.9) 77.4   3.9 49 (58) 185 (41.4) 77.5   3.9 77 (42) +/+ 13 (5.6) 76.0 + | 1.5 3 (23) 34 (7.6) 77.9 ) +: 3.9 10 (29) GSTT1a   21 (9.0) 77.3   3.3 12 (57) 60 (13.4) 77.4 $+ ¢ 4.1 13 (22) (cid:2)/+ 129 (55.1) 76.6   3.1 65 (50) 228 (51.0) 77.4 $+ ¢ 4.1 98 (43) +/+ 84 (35.9) 76.9   3.7 43 (51) 159 (35.6) 77.3   3.7 63 (40)

a ((cid:2)/(cid:2)) Double deletion (null genotype), ((cid:2)/+) heterozygous for deletion, (+/+) two functional

L. Christiansen et al./Mechanisms of Ageing and Development 127 (2006) 597–599

dose on lung cancer risk in a Swedish population. Cancer Epidemiol. Biomarkers Prevent.

,

–

Ates, N.A., Tamer, L., Ates, C., Ercan, B., Elipek, T., Ocal, K., Camdeviren H.,

Glutathione S-transferase M

, T

, P

genotypes and risk for devel- opment of colorectal cancer. Biochem. Genet.

,

–

Bellizzi, D., Rose, G., Cavalcante, P., Covello, G., Dato, S., De Rango, F., Greco, V., Maggiolini, M., Feraco, E., Mari, V., Franceschi, C., Passarino, G., De Benedictis, G.,

A novel VNTR enhancer within the SIRT

gene, a human homologue of SIR

, is associated with survival at oldest ages. Genomics

,

–

Brasch-Andersen, C., Christiansen, L., Tan, Q., Haagerup, A., Vestbo, J., Kruse, T.A.,

Possible gene dosage effect of glutathione-S-transferases on atopic asthma: using real-time PCR for quantification of GSTM

and GSTT

gene copy numbers. Hum. Mutat.

,

–

Christiansen, L., Bathum, L., Frederiksen, H., Christensen K.,

Paraox- onase

polymorphisms and survival. Eur. J. Hum. Genet.

,

–

Gaspari, L., Pedotti, P., Bonafe, M., Franceschi, C., Marinelli, D., Mari, D., Garte, S., Taioli, E.,

Metabolic gene polymorphisms and p

muta- tions in healthy centenarians and younger controls. Biomarkers

,

–

Geisler, S.A., Olshan, A.F., Cai, J., Weissler, M., Smith, J., Bell, D.,

Glutathione S-transferase polymorphisms and survival from head and neck cancer. Head Neck

,

–

Hayes, J.D., Flanagan, J.U., Jowsey, I.R.,

Glutathione transferases. Annu. Rev. Pharmacol. Toxicol.

,

–