Journal of Gerontology: BIOLOGICAL SCIENCES 2004, Vol. 59A, No. 9, 886–889 Copyright 2004 by The Gerontological Society of America The Catalase -262C/T Promoter Polymorphism and Aging Phenotypes Lene Christiansen,1,2 Hans Christian Petersen,1 Lise Bathum,1,2 Henrik Frederiksen,1 Matt McGue,3 and Kaare Christensen1 Epidemiology, Institute of Public Health, University of Southern Denmark. Department of Clinical Biochemistry and Clinical Genetics, Odense University Hospital, Denmark. 3 Department of Psychology, University of Minnesota, Minneapolis. A low level of the central antioxidant enzyme catalase has been suggested to be a risk factor for diseases influenced by oxidative stress. In this study, we investigated the possible association of the catalase -262C/T polymorphism with survival, physical and cognitive functioning, and a number of oxidative stress-mediated disorders. The study population was 2223 Danish individuals, aged 45–93 years, drawn from three population-based surveys. The results suggest that the catalase -262C/T polymorphism is not associated with either survival, or the majority of the age-related phenotypes investigated. However, our data indicate a statistical significant association of TT homozygosity with improved physical functioning as well as a trend of the T allele conferring an improved general cognitive functioning, although these results did not remain significant after correcting for multiple testing. The results raise the hypothesis that the catalase -262T allele serves as protection against neurodegenerative and physical decline, although replication in other studies is warranted for confirmation of these findings. O XIDATIVE stress is suggested to play a major role in aging as well as in the pathogenesis of age-related diseases, thus emphasizing the potential importance of variations in antioxidant enzyme activity in such diseases (1,2). Catalase (CAT; MIM# 115500) is a central antioxidant enzyme constituting a primary defense against oxidative stress together with superoxide dismutases and glutathione peroxidases. Being widely expressed, CAT limits the toxic effect of H2O2 by catalyzing its decomposition into water and oxygen in all aerobic cells (3). Several rare CAT gene sequence variations have been identified in association with acatalasemia, which is characterized by severe catalase deficiency (4,5). Reports on the prevalence of possible oxidative stress-related diseases or other diseases in catalase-deficient individuals are scarce. However, one study has reported an increased frequency of diabetes in Hungarian patients with catalase deficiency compared with both healthy relatives and the background population (6). Previously, identification of a common polymorphism in the CAT promoter, -262C/T, which may be directly related to interindividual variations in the catalase level, has been reported (7). Expression studies using reporter gene assays have demonstrated that promoter constructs possessing a T in the polymorphic site had a significantly higher transcriptional activity than the -262C constructs. In the same study, it was furthermore found that the erythrocyte catalase level was significantly higher in CAT -262T allele carriers than in -262CC homozygotes (7). This may indicate that the -262T allele confers a protective effect against development of oxidative stress-related diseases. To our knowledge, only one previous study has explored the association of the CAT -262C/T variation with oxidative stress-related disease. In this study, no association was 886 found between the -262C/T polymorphism and Alzheimer’s disease, when comparing 137 patients with 130 controls, all of Caucasian origin (8). The present study was undertaken to investigate whether there is an association between the CAT -262C/T polymorphism and survival as well as susceptibility to several age-related pathological conditions. METHODS Participants The participants included in this study were drawn from three population-based nationwide surveys conducted at the University of Southern Denmark. From the Study of Middle-Aged Danish Twins (MADT) (9,10) we included 581 individuals aged 45–67 years; from the Longitudinal Study of Ageing Danish Twins (LSADT) (10,11), we included 472 individuals aged 70–90 years; and from the Danish 1905 cohort (12), we included 1170 individuals aged 93 years. All three surveys included extensive face-to-face interviews focusing on health and lifestyle issues, assessment of functional and cognitive abilities, and DNA sampling. All samples were randomly selected among participants with available DNA samples from each survey. For the twin studies (MADT and LSADT), only one twin from each pair was selected. We have previously shown that twins and singletons have similar mortality from age 6 and throughout life (13). Hence, the genetic study was population based with no exclusion criteria due to health status or residence. Furthermore, the minimal immigration into Denmark by the cohorts in these surveys and the high response rate in all Downloaded from https://academic.oup.com/biomedgerontology/article-abstract/59/9/B886/535287 by guest on 26 May 2020 1 2 CAT POLYMORPHISM AND AGING PHENOTYPES Table 1. Characteristics of the 2223 Included Participants From 3 Nationwide Surveys LSADT (N ¼ 472) 1905 (N ¼ 1170) 53.6 6 5.7 271/310 11 (2%) 16 (3%) 77.7 6 4.3 158/314 28 (6%) 57 (12%) 93 6 0 356/814 101 (9%) 200 (17%) 19 (3%) 82 (14%) 24 (4%) 52 (11%) 140 (30%) 29 (6%) 178 (15%) 237 (20%) 76 (7%) 29 (5%) 54 (11%) 134 (12%) 17 (3%) 46 (8%) 96 (17%) 13 (2%) 27 (5%) NA NA 60 (13%) 153 (32%) 39 (8%) 27 (6%) 25.9 6 3.8 89 (8%) 173 (15%) 384 (33%) 105 (9%) 101 (9%) 22.3 6 5.3 5.03 6 3.4 38.2 6 11.5 0.79 6 3.2 23.9 6 8.7
1.94 6 3.1 16.7 6 6.8 342/209/30 274/175/23 671/429/70 Notes: Data are presented as either mean 6 SD (standard deviation) or as the number of participants suffering from the mentioned disease. The diagnoses are self-reported physician-diagnosed diseases. *Angina and/or acute myocardial infarction; **asthma and/or chronic bronchitis; ***poor or very poor self-rated health as opposed to excellent, good, or fair self-rated health. NA ¼ not available; MADT ¼ Study of Middle-Aged Danish Twins; LSADT ¼ Longitudinal Study of Ageing Danish Twins; 1905 ¼ the Danish 1905 Cohort; MMSE ¼ Mini-Mental State Examination, only available for LSADT and the 1905 cohort. surveys minimized population stratification as well as selection bias within the population. Health Data Self-reported information regarding health issues was available for most participants from all surveys. As a part of the interview, the participants were asked whether they currently did or had previously suffered from various diseases. Only when the diagnosis had been made by a physician was a positive answer accepted as valid. Furthermore, the participants were asked to assess their overall health as excellent, good, fair, poor, or very poor. Cognitive functioning was assessed using the MiniMental State Examination (MMSE) (14) and a cognitive composite score that was generated by combining the scores of five cognitive measures: fluency (the number of animals the participant was able to name within 1 minute), forward and backward digit span, and a modified 12-word learning test (immediate and delayed recall). The cognitive composite score was computed by taking the sum of the standardized components (mean ¼ 0, standard deviation [SD] ¼ 1). Standardization was based on the means and SDs observed in the initial LSADT survey. Consequently, positive scores reflect performance that is better than the performance of the sample that completed the initial LSADT survey, while negative scores reflect poorer relative performance (15). The MMSE score was only available for LSADT and the 1905 cohort, since it has a ceiling effect among the middle-aged, i.e., nearly all middle-aged individuals have a maximum score. The quantification of hand-grip strength using a hand dynamometer was applied as a measure for physical functioning. Hand-grip strength was previously shown to be a good marker of total physical ability (16,17). The maximum grip strength value in kilograms of three measures with each hand in the MADT and LSADT studies and with the preferred hand in the 1905 cohort study was used in this study. Genotyping DNA was isolated from cheek swabs or blood spots using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA). The CAT -262C/T polymorphism was detected by a Taqmanbased allelic discrimination assay using the following probes: 59-FAM-ttcggctatcccgggcacc-39 and 59-TET-tcggctattccgggcaccc-39. The sense primer 59-agggcggcctgaaggat-39 and the antisense primer 59-agcaattggagagcctcgc-39 were used for DNA amplification. Primers and probes were designed using Primer Express software (Applied Biosystems, Foster City, CA). DNA was amplified in a total volume of 10 ll containing 5 ll Taqman Universal Master Mix (Applied Biosystems), 900 nM of each primer (DNA Technology, Aarhus, Denmark), 200 nM of each probe (MWG Biotech, Ebersberg, Germany), and ;10 ng template DNA. Polymerase chain reaction (PCR) was performed with the ABI Prism 7700 using the conditions recommended by the manufacturer (Applied Biosystems) and analyzed using the Sequence Detection System software. Statistics The Stata 8.2 statistical program (Stata Corp., College Station, TX) was used for statistical analyses. Deviations from Hardy-Weinberg equilibrium and comparison of CAT -262C/T genotype distribution in the three surveys were tested using the chi-square test. The disease variables were all treated as dichotomous dependent variables. The same applies to self-evaluated heath, with poor or very poor in one group and fair, good, or excellent in the other. These variables were analyzed by logistic regression in order to obtain odds ratios. The cognitive variables, MMSE, and cognitive composite scores, as well as grip strength, were treated as continuous dependent variables. These were analyzed by multiple regression in order to obtain regression coefficients. In all regression analyses, sex and age (in three categories) and genotype (three categories, with CC as reference) were included, and all possible two-way interactions were tested. The ‘‘desmat’’ and ‘‘destest’’ program for Stata (18) were used for these regression analyses. RESULTS The characteristic features of the included participants from the three surveys are summarized in Table 1. The CAT -262C/T genotype distribution did not differ between the Downloaded from https://academic.oup.com/biomedgerontology/article-abstract/59/9/B886/535287 by guest on 26 May 2020 Age Male/female Stroke Heart failure Ischemic heart disease* Hypertension Diabetes Cancer including skin Cancer excluding skin Pulmonary disease** Osteoarthritis Osteoporosis Poor health*** MMSE score Cognitive composite score Grip strength (kg) CAT -262 genotype CC/CT/TT MADT (N ¼ 581) 887 CHRISTIANSEN ET AL. 888 Table 2. Odds Ratios for Self-Reported Physician-Diagnosed Disease Variable 95% CI p Value 0.67 0.99 0.28–1.57 0.56–1.76 .35 .97 .63 .93 .78 0.57 0.93 0.52 0.28–1.15 0.58–1.48 0.19–1.45 .12 .75 .21 0.68–1.24 .59 1.24 0.70–2.20 .46 0.43–1.05 0.79–1.34 0.87–1.28 0.76–1.52 0.60–1.19 .08 .83 .60 .68 .33 0.98 0.96 0.75 1.0 0.65 0.44–2.21 0.55–1.70 0.48–1.16 0.49–2.08 0.34–1.23 .96 .90 .20 .99 .19 95% CI 0.78 1.09 0.54–1.13 0.83–1.43 .19 .53 0.93 1.01 0.95 0.71–1.24 0.81–1.26 0.65–1.34 0.92 0.67 1.03 1.05 1.08 0.84 Notes: Age and sex adjusted odds ratio (OR) and 95% confidence intervals (CI). The CAT -262CC genotype is the reference group. *Angina and/or acute myocardial infarction; **asthma and/or chronic bronchitis; ***poor or very poor self-rated health as opposed to excellent, good, or fair self-rated health. three surveys, indicating that this polymorphism is not related to survival in the Danish population. The genotype distribution in all three populations was in accordance with the predicted values assuming Hardy-Weinberg equilibrium. Table 2 provides the age and sex-adjusted odds ratios (ORs) for having a number of age-associated pathological conditions. We found a trend toward a modest protective effect of the T allele for some of the conditions considered, but none of these reached statistical significance. Results of regression analysis, exploring the association between the CAT -262C/T polymorphism and cognitive as well as physical functioning, are shown in Table 3. The data shows that -262TT homozygosity was significantly associated with slightly better physical functioning. This result, however, did not remain significant after applying Bonferroni correction for multiple testing. Furthermore, a small positive association of the TT homozygosity with cognition assessed with both MMSE and the cognitive composite score was indicated. This was, however, not statistically significant. DISCUSSION Catalase has been suggested to play a central role in the protection against severe oxidative stress, and a low level of catalase activity may thus be considered a risk factor for diseases influenced by reactive oxygen species (ROS). This study aimed to examine the possible association of the catalase level-related CAT -262C/T polymorphism with survival, physical and cognitive functioning, as well as several oxidative stress-mediated disorders in a large sample of Danish individuals. Our data do not provide evidence for an association between the CAT -262C/T polymorphism and either survival or the majority of the diseases investigated, although a nonsignificant trend toward a protective effect of the T allele is seen in several of the investigated conditions. The presented data, however, indicate a modest association between CAT -262TT homozygosity and improved Variable MMSE score* Cognitive composite score** Grip strength (kg) p -262TT Value b 95% CI p Value 0.51
0.54–1.57 .341 .726 0.44
0.15–1.03 .146 .880 1.31 0.15–2.47 .027 -262CT b 95% CI 0.06
0.44–0.57 .803 0.05
0.23–0.33 0.04
0.50–0.58 Notes: Age and sex adjusted regression coefficients (b) and 95% confidence intervals (CI). The CAT -262CC genotype is the reference group. *MMSE ¼ Mini-Mental State Examination, only available for LSADT and the 1905 cohort; **comprising a test of fluency, forward and backward digit span, and a modified 12-word learning test. physical performance as assessed by grip strength. It has previously been suggested that oxidative stress is associated with age-related physical decline. Recently, Cesari and colleagues (19) showed a positive association between physical performance and plasma antioxidant concentration as well as dietary antioxidant intake in a study of elderly Italian individuals. It can thus be hypothesized that an increased level of an antioxidant enzyme such as catalase, perhaps in part caused by -262TT homozygosity, has a beneficial effect on physical performance. The results furthermore suggest a minor, but nonsignificant, positive association between the general cognitive status and CAT -262TT homozygosity. It is generally recognized that oxidative stress is implicated in the development of neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and stroke. The physiological catalase activity in the brain seem to be relatively low, and, although not consistently, previous studies have indicated that the catalase level is further decreased in the brain tissues of patients with such diseases (20,21). Furthermore, synthetic H2O2 scavengers exhibiting catalase activity have been demonstrated to have a neuroprotective effect in, for example, animal stroke models (22,23). In a previous study, Forsberg and colleagues (7) showed that carriers of the CAT -262T allele displayed a significantly higher erythrocyte catalase level compared to CC homozygotes, which is in accordance with a protective effect of the T allele, although it is not yet known whether this promoter-directed variation in erythrocyte catalase level is reflected in the brain. There are some limitations to this study that need to be considered. First, the participants included in this work were drawn randomly among participants from nationwide surveys. Consequently, the number of individuals affected by the various diseases investigated is relatively low compared with a classical case-control study. This is especially true for MADT, where the prevalence of agerelated disease by nature is minimal. Second, the fact that parts of our results rely on self-reported disease status necessitates cautious interpretation, as the diagnosis of some of these diseases may be somewhat uncertain. However, the introduced misclassification is likely to be nondifferential, which, in general, would lead to a bias toward the null hypothesis. Therefore, we cannot rule out that we underestimate the association between the CAT genotype and Downloaded from https://academic.oup.com/biomedgerontology/article-abstract/59/9/B886/535287 by guest on 26 May 2020 Stroke Heart failure Ischemic heart disease* Hypertension Diabetes Cancer including skin Cancer excluding skin Pulmonary disease** Osteoarthritis Osteoporosis General health*** p -262TT Value OR -262CT OR Table 3. Regression Analysis of Cognitive and Physical Functioning CAT POLYMORPHISM AND AGING PHENOTYPES Conclusion We found no major effects of the CAT -262C/T polymorphism with regard to a large number of phenotypes important for successful aging. The suggested tendency toward an association between the CAT -262C/T polymorphism and physical and cognitive performance should be confirmed by replication in other studies. 8. 9. 10. 11. 12. 13. 14. 15. ACKNOWLEDGMENTS We thank Shuxia Li for technical assistance. This work was supported by research grants from the National Institute on Aging (NIA-PO1-AG08761), the Danish Medical Research Council, and the GENOMEUTWIN Project (European Union Contract No. QLG2-CT-2002-01254). Address correspondence to Lene Christiansen, Department of Clinical Biochemistry and Clinical Genetics, Odense University Hospital, Sdr. Boulevard 29, 5000 Odense C, Denmark. E-mail: lchristiansen@health.sdu.dk 16. 17. 18. 19. 20. REFERENCES 1. Mates JM, Perez-Gomez C, Nunez DCI. Antioxidant enzymes and human diseases. Clin Biochem. 1999;32:595–603. 2. Stadtman ER. Protein oxidation in aging and age-related diseases. Ann N Y Acad Sci. 2001;928:22–38. 3. Mates M. 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Smith, PhD Downloaded from https://academic.oup.com/biomedgerontology/article-abstract/59/9/B886/535287 by guest on 26 May 2020 most diseases. Finally, we cannot rule out that the shown tendency toward a protective effect of the -262C/T polymorphism is a consequence of multiple testing, and applying Bonferroni correction for multiple testing accordingly left none of the results as being significant. However, the fact that this trend is seen for several of the conditions usually considered to be influenced by oxidative stress indicates a possible, although modest, relevance of this polymorphism in protection against oxidative stress-mediated physical and cognitive decline. 889