Polymorphisms of Drug-Metabolizing Enzymes in Healthy Nonagenarians and Centenarians: Difference at GSTT1 Locus
ABSTRACT Drug metabolizing enzymes are involved in the detoxification of several drugs, environmental substances, and carcinogenic compounds, and their polymorphisms have been associated with risk for a variety of cancer. In this paper, we compared the frequency of polymorphisms in cytochrome P450-1A1 gene (CYP1A1), a phase 1 gene (oxidation, activation), and of two polymorphisms of glutathione S-transferase enzymes (GSTM1, GSTT1), two phase 2 genes (conjugation, detoxification). Two groups were studied and compared, i.e., 94 nonagenarians and centenarians and 418 control subjects of younger age. A significant difference in the proportion of nonagenarians and centenarians homozygotes for a GSTT1 deletion (28%) was observed in comparison to control subjects (19%, P = 0.03). The distribution of the other gene polymorphisms did not differ in the two groups. These findings on phase 2 drug-metabolizing enzyme gene polymorphisms may help in disentangling gene–environmental interactions which can have a role in successful aging and longevity, as well as in cancer incidence in the oldest old.
- SourceAvailable from: Kevin Morgan[Show abstract] [Hide abstract]
ABSTRACT: Genetics plays a crucial role in human aging with up to 30% of those living to the mid-80s being determined by genetic variation. Survival to older ages likely entails an even greater genetic contribution. There is increasing evidence that genes implicated in age-related diseases, such as cancer and neuronal disease, play a role in affecting human life span. We have selected the 10 most promising late-onset Alzheimer's disease (LOAD) susceptibility genes identified through several recent large genome-wide association studies (GWAS). These 10 LOAD genes (APOE, CLU, PICALM, CR1, BIN1, ABCA7, MS4A6A, CD33, CD2AP, and EPHA1) have been tested for association with human aging in our dataset (1385 samples with documented age at death [AAD], age range: 58-108 years; mean age at death: 80.2) using the most significant single nucleotide polymorphisms (SNPs) found in the previous studies. Apart from the APOE locus (rs2075650) which showed compelling evidence of association with risk on human life span (p = 5.27 × 10(-4)), none of the other LOAD gene loci demonstrated significant evidence of association. In addition to examining the known LOAD genes, we carried out analyses using age at death as a quantitative trait. No genome-wide significant SNPs were discovered. Increasing sample size and statistical power will be imperative to detect genuine aging-associated variants in the future. In this report, we also discuss issues relating to the analysis of genome-wide association studies data from different centers and the bioinformatic approach required to distinguish spurious genome-wide significant signals from real SNP associations.Neurobiology of aging 03/2012; 33(8):1849.e5-18. · 5.94 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Recently we have shown that the human life span is influenced jointly by many common single-nucleotide polymorphisms (SNPs), each with a small individual effect. Here we investigate further the polygenic influence on life span and discuss its possible biological mechanisms. First we identified six sets of prolongevity SNP alleles in the Framingham Heart Study 550K SNPs data, using six different statistical procedures (normal linear, Cox, and logistic regressions; generalized estimation equation; mixed model; gene frequency method). We then estimated joint effects of these SNPs on human survival. We found that alleles in each set show significant additive influence on life span. Twenty-seven SNPs comprised the overlapping set of SNPs that influenced life span, regardless of the statistical procedure. The majority of these SNPs (74%) were within genes, compared to 40% of SNPs in the original 550K set. We then performed a review of current literature on functions of genes closest to these 27 SNPs. The review showed that the respective genes are largely involved in aging, cancer, and brain disorders. We concluded that polygenic effects can explain a substantial portion of genetic influence on life span. Composition of the set of prolongevity alleles depends on the statistical procedure used for the allele selection. At the same time, there is a core set of longevity alleles that are selected with all statistical procedures. Functional relevance of respective genes to aging and major diseases supports causal relationships between the identified SNPs and life span. The fact that genes found in our and other genetic association studies of aging/longevity have similar functions indicates high chances of true positive associations for corresponding genetic variants.Rejuvenation Research 04/2012; 15(4):381-94. · 2.92 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Successful aging (SA) is a multidimensional phenotype involving living to older age with high physical function, preserved cognition, and continued social engagement. Several domains underlying SA are heritable, and identifying health-promoting polymorphisms and their interactions with the environment could provide important information regarding the health of older adults. In the present study, we examined 263 cognitively intact Amish individuals age 80 and older (74 SA and 189 "normally aged") all of whom are part of a single 13-generation pedigree. A genome-wide association study of 630,309 autosomal single nucleotide polymorphisms (SNPs) was performed and analyzed for linkage using multipoint analyses and for association using the modified quasi-likelihood score test. There was evidence for linkage on 6q25-27 near the fragile site FRA6E region with a dominant model maximum multipoint heterogeneity LOD score = 3.2. The 1-LOD-down support interval for this linkage contained one SNP for which there was regionally significant evidence of association (rs205990, p = 2.36 × 10(-5)). This marker survived interval-wide Bonferroni correction for multiple testing and was located between the genes QKI and PDE10A. Other areas of chromosome 6q25-q27 (including the FRA6E region) contained several SNPs associated with SA (minimum p = 2.89 × 10(-6)). These findings suggest potentially novel genes in the 6q25-q27 region linked and associated with SA in the Amish; however, these findings should be verified in an independent replication cohort.Age 07/2012; · 6.28 Impact Factor
Polymorphisms of Drug-Metabolizing Enzymes
in Healthy Nonagenarians and Centenarians:
Difference at GSTT1 Locus
Emanuela Taioli,*,1Daniela Mari,† Claudio Franceschi,‡ Massimiliano Bonafe `,§ Daniela Monti,¶
Stefano Bertolini,? Daniela Marinelli,** and Seymour Garte**
*Laboratorio Epidemiologico, Ospedale Policlinico IRCCS, Milan, Italy; †Institute of Internal Medicine, University of
Milan, Milan, Italy; ‡Italian National Research Centers on Aging, INRCA, Ancona, Italy; §Department of Experimental
Pathology, University of Bologna, Bologna, Italy;
Florence, Florence, Italy; ?Atherosclerosis Prevention Centre, Department of Internal Medicine, University of Genoa,
Genoa, Italy; and **Genetics Research Insitute, Strada della Carita ` 10, 20135 Milan, Italy
¶Department of Experimental Pathology and Oncology, University of
Received J anuary 5, 2001
Drug metabolizing enzymes are involved in the detox-
ification of several drugs, environmental substances,
and carcinogenic compounds, and their polymorphisms
have been associated with risk for a variety of cancer. In
this paper, we compared the frequency of polymor-
phisms in cytochrome P450-1A1 gene (CYP1A1), a phase
1 gene (oxidation, activation), and of two polymorphisms
of glutathione S-transferase enzymes (GSTM1, GSTT1),
two phase 2 genes (conjugation, detoxification). Two
groups were studied and compared, i.e., 94 nonagenari-
ans and centenarians and 418 control subjects of
younger age. A significant difference in the proportion
of nonagenarians and centenarians homozygotes for a
GSTT1 deletion (28%) was observed in comparison to
control subjects (19%, P ? 0.03). The distribution of the
other gene polymorphisms did not differ in the two
groups. These findings on phase 2 drug-metabolizing en-
zyme gene polymorphisms may help in disentangling
gene–environmental interactions which can have a role
in successful aging and longevity, as well as in cancer
incidence in the oldest old.
Key Words: ageing; longevity; centenarians; genetics
of ageing; genetics of longevity; cancer; cancer and
ageing; environmental carcinogens.
© 2001 Academic Press
Human longevity can be regarded as a multifactorial
trait, highly dependent on the interaction between ge-
netic and environmental factors.
A significant proportion of centenarians are in good
health (category A and B, as defined by Franceschi et
al., 2000) either from a physical or a cognitive point of
view (1). They are the best example of successful aging,
and represent a valuable model for studying the ge-
netic factors impinging on survival (risk factors for
cancer and other major age-related diseases) (2). In
fact, some risk factors for age-related diseases are hy-
porepresented in centenarians (3), whilst others are
unexpectedly present in healthy centenarians at equal
or even increased frequency, in comparison to controls
(4–7). Nevertheless, a number of studies revealed that
in centenarians important changes occur in genetic
variability of loci which are not risk factors for specific
diseases, but are rather responsible for modulating the
individual capacity to cope with stress, such as Ty-
rosine Hydroxilase and mitochondrial DNA (8, 9). In
this scenario, enzymes which are involved in the me-
tabolism of potentially dangerous endogenous and ex-
ogenous compounds, are likely to play a major role in
human longevity. A variety of polymorphisms have
been reported in these genes, and have been associated
with susceptibility tocancer (10) and other age-related
diseases such as Parkinson disease (11). Among these
candidate genes, there are Phase 1 (oxidation, activa-
tion) and Phase 2 (conjugation, detoxification) drug
metabolizing genes, such as cytochrome P450 genes
and glutathione S-transferase enzymes (GST). Cyto-
chrome P450 genes are involved in the first step of the
metabolism of polycyclic aromatic hydrocarbons con-
tained in tobaccosmoke, whilst GST genes areinvolved
in the detoxification of several drugs, environmental
substances, and carcinogenic compounds. In particu-
lar, common polymorphisms leading to complete dele-
tion of the gene cause a loss of GST functional activity.
Individuals with such gene deletions exhibit decreased
conjugation activity, and are consequently exposed to
higher internal doses of potentially toxic intermediary
1To whom correspondence should be addressed at Laboratorio
Epidemiologico, Ospedale PoliclinicoIRCCS, Via F. Sforza 28, 20122
Milano, Italy. Fax: ?39 02 55038207. E-mail: firstname.lastname@example.org.
Biochemical and Biophysical Research Communications 280, 1389–1392 (2001)
doi:10.1006/bbrc.2001.4280, available online at http://www.idealibrary.com on
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
metabolites from xenobiotic agents. Data on the fre-
quency of polymorphisms in some phase 1 genes
(CYP2E1 (12), CYP2C19 (14), CYP2D6 (13, 14)) and
phase 2 genes (NAT2 (13, 14), GSTM1 (13)), as well as
other polymorphisms associated with cancer suscepti-
bility, i.e., p53 (15, 16), did not show any difference
between centenarians and the general population. In
this study, wefocussed our attention on other members
of phase 1 gene family (CYP1A1), and phase 2 genes
(GSTT1 and GSTP1) never studied before in centenar-
ians, as well as on a gene which has been studied in
French centenarians with negative results (GSTM1)
(13). Thehypothesis was that thefrequency of theleast
favorable polymorphisms should be lower in centenar-
ians than that observed in the general population.
MATERIALS AND METHODS
Nonagenarians and centenarians in good health, i.e., belonging to
category A and B according toFranceschi et al. (1) were identified in
Northern Italy (Milan, Modena, Parma, Genoa), as part of an ongo-
ing multicentric study in Italy with the aim of studying the mecha-
nisms responsible for successful aging (1). A total of 94 subjects were
included in the present study. The mean age of the group was
100.2 ? 2.1 years (range 95–105 years; median 100 years), 22% of
whom were males and 88% females. The male tofemale ratiowas in
agreement with the recent data on the nation-wide study on Italian
centenarians (1). None was a current smoker. In the rest of the
present paper, for simplicity, these very old people are collectively
indicated as “centenarians”.
Control subjects were a sample of 418 healthy volunteers of the
same ethnic group as the nonagenarians and centenarians, recruited
in Northern Italy to study the geographic distribution of drug-
metabolizing enzymes polymorphisms. They were recruited among
blood donors (n ? 98), or health screening programs (n ? 320). All
the subjects gave informed consent to participate in the study. The
mean age of the control subjects was 46.0 ? 11.3 years (range 4–82
years; median 47 years), 46% males and 54% females. As by study
design, sex ratio was significantly different in the two groups.
Genomic DNA was extracted from peripheral blood lymphocytes,
and polymorphisms were assessed by PCR method, as previously
described (17, 18). A multiplex PCR method was used to detect the
presence or absence of the GSTM1 and GSTT1 genes in genomic
DNA samples. This method uses both GST primer sets (GSTM1, 5?
GAACTCCCTGAAAAGCTAAAGC 5? GTTGGGCTCAAATATACG-
GTGG; and GSTT1, 5? TCCTTACTG GTCCTCACATCTC, 5? TCAC-
CGGATCATGGCCAGCA) in the same PCR and includes a third
primer set for albumin (5? GCCCTCTGCTAACAAGTCCTAC, 5? GC-
CCTAAAAAGAAAATCGCCAATC), and uses 30 cycles with denatur-
ing at 94°C for 1 min, annealing at 64°C for 1 min, and extension at
72°C for 1 min. For analysis of CYP1A1 the following primers were
used: 5? TTAGGAGTCTTGTCTCATGCCT and 3? CAGTGAAGAG-
GTGTAGCCGCT for analysis of the Msp1 RFLP using PCR condi-
tions as previously described (17).
Nomenclature used: for CYP1A1, the term “wild type” refers tothe
homozygotes for the common allele (absence of the restriction site),
“heterozygous” refers to the presence of the polymorphism on one
allele, “homozygous” refers to the presence of the restriction site on
both alleles. For GSTM1 and GSTT1, “null” refers tohomozygotes for
deletion allele, while “present” refers to both homozygotes and het-
erozygotes (which cannot be distinguished by the experimental
Chi-square was used to compare the frequency of each polymor-
phism and of the combined genotype, in centenarians and in control
subjects. Monte Carlosimulation was used (19), totake intoaccount
for cells with small numbers. Data were adjusted for sex, in order to
control for a possible confounding factor, by multiple logistic regres-
sion, using the SAS package version 6.12. Linear regression analysis
was used to test the association between age and each genotype.
The frequency of the polymorphisms analyzed in this
study is reported in Table 1.
A significant differencein theproportion of centenar-
ians homozygotes for GSTT1 deletion was observed, in
comparison to younger subjects (28% vs 19%: ?2?
4.18; P ? 0.03). Linear regression analysis confirmed
the independent association between age and GSTT1
deletion (F ? 4.61, P ? 0.032). When the analysis
was restricted to control subjects below the age of 60
years (n ? 386), the results did not change (data not
shown). The distribution of the other gene polymor-
phisms did not differ in the two groups (CYP1A1*2A:
?2? 0.94, P ? 0.68; GSTM1: ?2? 0.10, P ? 0.74).
When the multiloci genotypes for CYP1A1, GSTM1,
and GSTT1 were examined (Table 2), a statistical bor-
derline difference was observed between centenarians
and control subjects (?2? 15.67; P ? 0.079). The
difference was mainly due to the CYP1A1-GSTM1-
GSTT1 combinations 1-0-2 and 1-2-2 (Table 2), both
containing the combination of CYP1A1 heterozygotes
and GSTT1 deletion genotypes (8% in centenarians vs
Genotype Frequency of Drug-Metabolizing Gene
Polymorphisms in Centenarians and Younger Subjects
(n ? 94)
(n ? 418)
n (%)n (%)
Note. Statistical analysis: CYP1A1, ?2? 0.94, P ? 0.68; GSTM1,
?2? 0.10, P ? 0.74; GSTT1, ?2? 4.18, P ? 0.03.
a“Wild type” refers to the homozygotes for the common allele
(absenceof therestriction sitepolymorphism), heterozygous refers to
thepresenceof thepolymorphism on oneallele, homozygous refers to
the presence of the polymorphism on both alleles.
bTotals may vary due to missing values.
c“Null” refers to homozygotes for deletion allele, while “Present”
refers to both homozygotes and heterozygotes which cannot be ex-
Vol. 280, No. 5, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.5% in control subjects). According to the strong in-
teraction between CYP1A1 and GSTT1, a significant
differencein GSTT1 distribution between centenarians
and control subjects occurred in CYP1A1 heterozygotes
(?2? 11.278; P ? 0.003), but not in CYP1A1 wild
type genotypes (?2? 1.249; P ? 0.26).
The frequency of the GSTP1 polymorphism did not
show any difference between centenarians and control
subjects (data not shown).
In this study we confirm the lack of association be-
tween GSTM1 and longevity, as previously described
in a French population of centenarians (3), and we
report new data for twopolymorphisms in drug metab-
olizing enzymes which have never been tested in cen-
tenarians, namely CYP1A1 and GSTT1. We did not
find any difference in the frequency of CYP1A1 in
centenarians, as compared to control subjects of
younger age. A significant increase in the GSTT1 ho-
mozygous deletion in centenarians was observed, and
this is the first report of a significant difference regard-
ing drug metabolizing enzyme gene polymorphisms in
centenarians. This finding is rather unexpected, as the
deletion of the GST genes is considered a risk factor for
cancer, causing the lack of conjugation of toxic com-
pounds of environmental origin. Accordingly, a de-
crease in frequency of the GSTT1 deletion in centenar-
ians would be expected. However, this finding is not
totally surprising and it is not the first description of
an increased risk factor in centenarians. Indeed, an
increased frequency of alleles and genotypes conferring
susceptibility to cardiovascular diseases and thrombo-
sis, such as PAI-1 and ACE, have been reported in
centenarians (4, 7, 20). In order to explain this para-
dox, which emerged from theresearch on centenarians,
we have proposed a mathematical model suggesting
that theover-crossing of themortality curves at certain
ages can explain these phenomena (21, 22). This model
assumes that a genetic risk factor can confer an in-
creased risk to die until a certain age, above which its
impact is nullified or reversed. This phenomenon may
occur because those people who survived at very ad-
vanced ages have been selected by mortality forces,
and thus could benefit from those genetic variants
which are detrimental for younger people. For exam-
ple, a profile predisposing to hypercoagulability could
be beneficial at very advanced ages, but could predis-
pose to cardiovascular diseases in the middle aged
people (4, 20). Accordingly, the lack of GSTT1 (or other
phase 2 genes) could be related todeleterious effects in
young age (high risk of cancer) but it could exert long
term beneficial effects on survival, by avoiding the
catabolism of important substances, which have che-
mopreventive effect on cancer and other diseases. In-
deed, a recent study, shows that among subjects con-
suminga diet rich in
carcinogen inhibitor), those with deletion of GSTM1
and/or GSTT1 have higher levels of this compound in
the urine than subjects with the wild type genotype
(23). Another possibility is that the association be-
tween GSTT1 locus and longevity regards indeed a
proximate locus of unknown nature whose variants are
in linkage disequilibrium with the one we studied.
Further studies are needed to clarify this point.
In conclusion, GSTT1 can be added to the list of
candidate longevity genes which have been identified
so far, and it can open a new perspective on a possible
“pharmacogenetics of longevity”.
This work was supported by grants from AIRC “Healthy centenar-
ians as a model to study genetic and cellular factors involved in
M.U.R.S.T. Projects “Genetic determinants of human longevity” and
“Genetic factors involved in human aging and longevity”, Ministry of
Health Project “Marcatori genetici e biologici di invecchiamentonor-
male e patologico” to C.F., and M.U.R.S.T. Project “Immunogenetics
of Longevity” to D. Monti.
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