GSTT1, GSTP1 and XPC genes are associated with longevity in an Italian cohort

Abstract Longevity is a complex process controlled by environmental and genetic factors. We evaluated the association of seven drug metabolising and DNA-repair gene polymorphisms with longevity in an Italian cohort. A sample of 756 subjects aged 18-98 was genotyped for CYP1A1 (rs1048943, A>G), GSTM1 (rs 1183423000, presence/absence), GSTT1 (rs1601993659, presence/absence), GSTP1 (rs1695, A>G), XRCC1 (rs1799782, C>T), XRCC1 (rs25489, A>G) and XPC (rs2228001, A>C) gene polymorphisms. The association between the studied gene polymorphisms and longevity was evaluated by dividing the sample into three age groups: 18–50, 51–85, and 86–98. We observed a significant decrease in the frequency of the GSTT1 null, GSTP1 G and XPC C alleles in the oldest group with respect to the youngest one. We also obtained the same results when dividing the sample into 18–85 and 86–98 age groups. The general linear model analyses confirmed a significant decreasing trend with age of the above mentioned alleles. We hypothesised that these minor alleles, being important in the sensitivity against the development of different types of cancer, may reflect a reduced life-expectancy in carrier subjects and may explain their significantly lower frequency observed among subjects belonging to the oldest age group.


Introduction
Longevity is a complex trait controlled by both environmental and genetic factors. Most of the published papers about the genetic basis of longevity are principally focussed on those gene polymorphisms related to susceptibility to cardiovascular diseases, such as FTO, ACE and ApoE (Santovito et al. 2019), while the relationship between drug metabolising and DNA-repair gene polymorphisms and longevity has been poorly studied (Debrabant et al. 2014;Dato et al. 2018;Kim et al. 2018).
Drug metabolising enzymes (DMEs) are involved in the metabolism and elimination of xenobiotic compounds to which humans are exposed, and include phase I and phase II metabolising enzymes and phase III transporter enzymes (Xu and Wei 2015). The first two classes of enzymes allow the biotransformation of lipid-soluble xenobiotic compounds into a more hydrophilic metabolite through oxidation reactions (phase I cytochrome P450 (CYP) superfamily enzymes) and conjugation reactions (phase II glutathione S-transferases (GST) enzymes) (Wauthier et al. 2007;Xu and Wei 2015). At the end of the process, phase III transporters help in transferring the more hydrophilic products outside the cells (Almazroo et al. 2017).
In order to prevent the deleterious consequences of mutagenic processes induced by environmental xenobiotics, cells evolved different mechanisms of DNA repair, depending on the specific type of DNA damage. DNA-repair genes, being responsible for maintaining the integrity of the genome, may be considered as longevity-associated genes and the fact that a reduced repair capacity has been reported to be associated with cancer development should also be considered (Langie et al. 2015).
Given these premises, in the present study we decided to evaluate both the frequencies of seven polymorphic drug metabolising and DNA-repair genes and the variation of these frequencies in different age groups in a sample of a Northern Italian population. We considered the following most studied cancer-associated gene polymorphisms: CYP1A1 (rs1048943), GSTM1 (rs1183423000), GSTT1 (rs1601993659), GSTP1 (rs1695), X-ray repair cross-complementing group 1 (XRCC1) (rs1799782 and rs25489) and Xeroderma pigmentosum complementation group C (XPC) (rs2228001).
The CYP1A1 gene is highly polymorphic and plays a central role in the metabolism of polycyclic aromatic hydrocarbons (Androutsopoulos et al. 2009). In this study we considered the CYP1A1 rs1048943 single nucleotide polymorphism (SNP) in which adenine is replaced by guanine, resulting in the amino acid substitution Ile(462)Val (Wei and Hu 2015).
GST genes codify for one of the major groups of phase II detoxifying enzymes, evolved to protect organisms against toxic substances (Nebert and Vasiliou 2004). The polymorphisms of GSTM1 and GSTT1 genes consist of a deletion of a segment of DNA, with the subsequent lack of protein synthesis in homozygous individuals (Nebert and Vasiliou 2004). GSTP1 is the major isoenzyme expressed in human lung tissue. The SNP changes an adenine with a guanine in codon 105, resulting in the replacement of an isoleucine with a valine in the enzyme (L opez-Cima et al. 2012).
The XRCC1 gene, involved in the Base Excision Repair (BER) pathway, encodes enzymes having an important role in the repair of single-strand DNA breaks associated with exposure to free oxygen radicals, radiation, ultraviolet, and alkylating agents (Caldecott 2019). In the present study, we evaluated the frequencies of the two most common SNPs, rs1799782 and rs25489.
Finally, an important key gene belonging to the Nucleotide Excision Repair (NER) pathway is XPC encoding a 940 amino-acid protein involved in DNA damage recognition and apoptosis (Fontana et al. 2008). We analysed one of the most common polymorphisms of this gene, represented by A>C transition in exon 15 (rs2228001), resulting in a lysineto-glutamine substitution at position 939 (Fontana et al. 2008).
Given the above-mentioned role of metabolic and DNArepair gene polymorphisms in the modulation of genomic damage and in the maintenance of DNA integrity, the aim of this work was to evaluate the possible role of these gene polymorphisms in longevity. The hypothesis was that some variants of these genes, being associated with a different efficiency in both metabolising potentially harmful environmental xenobiotics and in repairing genomic damage, may be differentially represented in elderly individuals, giving them an advantage or a disadvantage in terms of longevity.

Subjects
We recruited healthy subjects (persons not hospitalised on the date of the sampling and without familiar history of genetic pathologies), who were natives of Piedmont localities for at least two generations, and voluntarily joined the project. In order to avoid selection bias, subjects were sampled without a priori exclusions, for example in terms of age and sex. The nutritional status was evaluated by the body mass index (BMI). Since obesity is considered as a pathological status (Lu et al. 2018), we excluded obese subjects from the sampling (subjects with BMI ! 30 Kg/m 2 ).
All data from each participant, including ancestry, were collected during an interview in an open-ended manner. We excluded the subjects who claimed to be natives of other Italian regions or who belonged to religious ethnic groups. All subjects received detailed information about the study and gave their written informed consent. Personal data were collected in an anonymous manner and participants were informed about their right to privacy and about the management procedures of their personal data.
In order to evaluate the possible association of some alleles with longevity, we decided to divide our sample into three age groups: 18-50, 51-85, and 86-98, representing individuals in reproductive, post-reproductive and long-lived phases, respectively (Santovito et al. 2019). The study was conducted with the formal approval of the Bioethical Committee of the University of Turin ethical committee and was performed in accordance with the ethical standards laid down in the 2013 Declaration of Helsinki.

Statistical analysis
All statistical analyses were performed using the SPSS software statistical program (version 25.0, SPSS Inc., Chicago, USA). Pearson's v2 test contingency table was used to evaluate the Hardy-Weinberg equilibrium (HWE), whereas comparison of genotype frequencies between age groups was carried out using the Fisher's exact test. The correlation between age and a specific genotype was analysed using a multivariate general linear model with Bonferroni's correction. All p values were two-tailed, and the level of statistical significance was set at p < 0.05 for all tests.
The sample was also subdivided into two age groups, 18-85 and 86-98, and we obtained the same results, with the oldest age group showing a significant decrease of the GSTT1 null (p ¼ 1.05 Â 10 À2 ), GSTP1 G (p ¼ 1 Â 10 À4 ) and XPC C (p ¼ 3.4 Â 10 À3 ) allele frequencies with respect to the 18-85 age group.

Discussion
Although longevity is due to a complex interaction of genetic and environmental factors, a strong genetic component appears to have an important role in survival to extreme ages (Franceschi et al., 2020;Brooks-Wilson, 2013;Gems and Partridge 2013).
In the present paper we found a negative association of GSTT1 rs1601993659 null, GSTP1 rs1695 G and XPC rs2228001 C alleles with human longevity in an Italian sample. A possible explanation for our findings is that polymorphisms of the metabolic and DNA-repair genes could play an important role in determining the incidence of diseases associated with genomic damage and, consequently, in modulating the life span. For example, many studies have shown that variants of GSTs genes are significantly associated with disease risk, including cancer, cardiovascular and allergic diseases (Di Pietro et al. 2010;Esalmi and Sahebrak 2014;Dar et al. 2017). The lowest frequency of GSTT1 null genotypes found in subjects aged between 86 and 98 years seems to suggest a selective disadvantage of the possible detrimental genotypes in this age group.
Vice versa, this association with longevity was not observed for GSTM1 polymorphism. This result is not surprising and agrees with what other authors have observed. For example, a lack of association between human longevity and GSTM1 gene polymorphism was already observed by Muiras et al. (1998). Similarly, Ruiz et al. (2012), in a Spanish sample, observed a significantly higher frequency for the GSTT1-positive allele in a sample of centenarians (age range 100-108) than in a control group (age range 19-43), while the frequency of the GSTM1-positive allele was similar in both groups.
Differently to GSTT1 and GSTM1 gene polymorphisms, no data are present in the literature about the relationships between GSTP1 rs1695 (A>G) gene polymorphism and longevity. However, like GSTT1, GSTP1 also has a corresponding rationale for an association with age. In subjects carrying the variant G-allele, substrate-specific catalytic activity becomes less effective, resulting in increased risk for the development of chronic and malignant diseases, such as cardiovascular diseases and neoplasms (Khabaz 2014;Chielle et al. 2016) , and, consequently, in a reduced longevity.
In the literature a relationship between CYP1A1 variants and cancer was observed (Liu et al. 2016), however we found no differences between age groups in the frequency of CYP1A1 rs1048943 genotypes. These data seem to be concordant with other studies, such as the one conducted by Taioli et al. (2001), that did not find any difference in the frequency of CYP1A1 alleles in centenarians, as compared to younger control subjects. However, published data evaluating the possible association between phase I enzyme polymorphisms and ageing reported conflicting results (Wauthier et al. 2007;Seripa et al. 2010).
Several investigations have demonstrated that subjects showing compromised repair capacity have increased mutation rates, an increased risk of cancer and a reduced lifeexpectancy (Tian et al. 2017;Kim et al. 2018). We hypothesised that polymorphisms in the XPC gene, being important in the sensitivity against the development of different types of cancer (Bahceci et al. 2015), may reflect a reduced lifeexpectancy in carrier subjects and may explain the significant lower frequency of XPC exon 15 C-allele we observed among subjects belonging to oldest age group.
Conversely, there is no consensus regarding the possible association between XRCC1 rs1799782 and rs2228001 polymorphisms with different types of cancer (Moghaddam et al. 2016;Dylawerska et al. 2017), data that may explain the lack of association of these gene polymorphisms with longevity, as observed in our study.
In conclusion, with this study we highlighted a role for GSTT1, GSTP1 and XPC gene polymorphisms in longevity. We hypothesised that the minor alleles of these metabolic genes, playing an important role in the susceptibility to different types of cancer, may reflect a reduced life-expectancy in carriers, explaining their significantly lower frequency observed among subjects belonging to the oldest age group. Conversely, an optimum activity of repair processes may favour longevity, justifying the significant reduced frequency of XPC C allele in elderly subjects.
Finally, we would like to emphasise that, considering that the number of centenarians in the world will drastically increase in the next few decades (Nijiati et al. 2013), studies on longevity will play an important role, and will also allow us to analyse any pleiotropic effects of analysed genes.

Informed consent
Written informed consent was obtained from all subjects involved in the study. Participants were also informed about their right to privacy and about the management procedures of their personal data, as required by the Italian law (art. 13 of Legislative Decree n. 196/2003).

Author contributions
Manuel Scarf o, Chiara Sciandra and Stefano Ruberto extracted DNA, performed genotyping and drafted manuscript. Alfredo Santovito designed the project, collected the samples, performed genotyping, drafted and edited the manuscript.

Disclosure statement
The Authors declare that they have no competing financial interests in relation to the present work.

Funding
This study was supported by University of Turin with local 2015-2018 grants.

Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.