A common variant of the p16INK4a genetic region is associated with physical function in older people

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Abstract

p16INK4a is active in cell senescence, ageing and tumor suppression. Deletion of the small p16INK4a/ARF/p15INK4b region occurs in many cancers. We screened 25 common polymorphisms across the region and three related genes for associations with physical functioning in older people.

In an initial sample of 938 (aged 65–80 years) from the EPIC study (Norfolk, UK), the rs2811712 SNP minor allele (located between the shared p16INK4a/ARF locus and p15INK4b) was associated with reduced physical impairment. This association remained after testing an additional 1319 EPIC-Norfolk samples (p-value = 0.013, total n = 2257), and on independent replication in the InCHIANTI study (n = 709, p = 0.015), and at one-sided significance in Iowa-EPESE (n = 419, p = 0.079). Overall (n = 3372), the prevalence of severely limited physical function was 15.0% in common homozygotes and 7.0% in rare homozygotes (per minor allele odds ratio = 1.48, 95% CI: 1.17–1.88, p = 0.001, adjusted for age, sex and study). This estimate was similar excluding screening set 1 (OR = 1.45, 95% CI: 1.09–1.92, p = 0.010, n = 2434).

These findings require further replication, but provide the first direct evidence that the p16INK4a/ARF/p15INK4b genetic region and the senescence machinery are active in physical ageing in heterogeneous human populations. The mechanism involved may be via greater cellular restorative activity and reduced stem cell senescence.

Introduction

Several recent findings suggest that cancer protection pathways leading to cell cycle arrest and cell senescence are important in ageing (Campisi, 2005, Beausejour and Campisi, 2006). A key protein in triggering cell senescence is p16INK4a, which regulates the Rb cancer suppressor pathway (Sharpless, 2004, Herbig et al., 2006). p16INK4a is coded for by the CDKN2a locus, situated on chromosome 9p21 in humans (Kim and Sharpless, 2006), but unusually this locus also codes for a second product, ARF (called p19ARF in mice), which regulated the p53 cancer suppressor (Quelle et al., 1995).

The small region (<42 kb in HapMap II) including p16INK4a/ARF and p15INK4b is amongst the most frequent lost in human cancer (Kim and Sharpless, 2006) and germline mutations of p16INK4a/ARF are linked to melanoma and other malignancies (Murphy et al., 2004, Lang et al., 2005).

p16INK4a expression and cell senescence can be triggered by various major biological stresses including DNA damage and oxidative stress (Ben-Porath and Weinberg, 2005, Massague, 2004). Expression of p16INK4a increases markedly with age (Nielsen et al., 1999, Zindy et al., 1997), including many tissues in rodents (Krishnamurthy et al., 2004), in the skin of aged baboons (Herbig et al., 2006) and in human kidney tissue (Melk et al., 2004). In addition, transgenic models have recently shown that increased expression of p16INK4a causes some aspects of ageing in several stem cell and self-renewing compartments, including neural stem cells (Molofsky et al., 2006), hematopoietic stem cells (Janzen et al., 2006) and pancreatic beta-cells (Krishnamurthy et al., 2006).

In addition to p16INK4a/ARF (CDKN2a) and p15INK4b (CDKN2b) there are related cyclin dependent kinase inhibitors involved at various points linked to the p53 or Rb pathways (Satyanarayana and Rudolph, 2004).

While the most popular approach to identifying ‘ageing genes’ in humans involves comparisons of long lived with younger groups (Melzer et al., 2007), there is increasing interest in markers of age-related functioning (Barzilai and Shuldiner, 2001, Karasik et al., 2005, Martin, 2005), from early old age onward. Declines in functional abilities (e.g. slow walking speed, impaired ability to rise from the sitting position, etc.) offer good summary measures of health in older people, and are predictive of disability progression and mortality (Guralnik et al., 1989, Guralnik et al., 1995, Guralnik and Ferrucci, 2003). Many aspects of physical functioning in older people show substantial heritability (Leinonen et al., 2005), including muscle strength (Reed et al., 1991, Frederiksen et al., 2002, Tiainen et al., 2004), and gait speed (Carmelli et al., 2000). Even the self reported SF36 health questionnaire physical functioning sub-score (based on limitations in walking, climbing stairs and certain activities of daily living) had an additive genetic component of 30% (95% CI: 27–45%) in middle-aged male twins in the USA (Romeis et al., 2005). Measured functional impairments have been shown to be a sensitive phenotype in older people for the ApoE e4 polymorphism (Melzer et al., 2005) and for a polymorphism in the inflammatory marker interleukin-18 (Frayling et al., 2007).

In this study, we tested the hypothesis that common genetic variants (minor allele frequency (MAF) >0.05) in the p16INK4a/ARF/p15INK4b region and three related cyclin dependent kinase inhibitor genes involved in triggering cell senescence are associated with differences in physical functioning in older people.

Section snippets

Materials and methods

Three separate studies of white Europeans were used. An initial sample of 938 participants aged 65–80 years from the Norfolk site of the EPIC study (Day et al., 1999) was genotyped for the 25 single nucleotide polymorphisms (SNPs) chosen as tagging the known common variants (Table 1) in the genetic regions covering CDKN1a, CDKN1b, CDKN2a, CDKN2b and CDKN2c.

This initial screening set formed the older part of the control group for a breast cancer gene study (Anglian Breast Cancer Study Group, 2000

Results

We examined associations between 25 selected SNPs tagging variation in five CDK inhibitors and the SF36 physical performance subscale (SF36-PF), in an initial set of 938 EPIC-Norfolk participants. Genotyping frequencies did not deviate significantly from Hardy–Weinberg equilibrium. Of the SNPs tested, two (rs2811712 and rs3218005) were associated with the continuous SF36-PF score. The rare alleles of each SNP (G allele in both cases) were associated with higher SF36 PF scores, reflecting better

Discussion

In a survey of 25 SNPs tagging variation in five cyclin dependent kinase inhibitors, we identified the rs2811712 SNP (which lies 3.5 kb proximal to the p16/ARF locus) as significantly related to physical function in a heterogeneous older human population. Our screening findings were then supported by further samples from the EPIC-Norfolk study, and from the independent InCHIANTI and Iowa-EPESE population studies. Using either the replication samples alone (p = 0.010 excluding the multiply tested

Conclusions

Increased p16INK4a activity plays a pivotal role in triggering cell senescence and protection from malignancy, but such protection is also associated with acceleration of some aspects of ageing in laboratory models. We have shown that a common inherited (germline) variant of the p16INK4a/ARF/p15INK4b genetic region is strongly associated with reduced limitation in physical function in people aged 65–80 years, although further replication is needed in independent populations. These findings

Conflicts of interest

None.

Acknowledgements

This work is supported in part by NIH/NIA grant R01 AG24233-01, and by the Intramural Epidemiology, Demography and Biometry Laboratory, National Institute on Aging. DM is supported by a NHS Executive National Public Health Career Scientist Awards, Ref: PHCSA/00/002.

EPIC-Norfolk is supported by programme grants from the Medical Research Council and Cancer Research UK, with additional support from the Stroke Association, Research Into Ageing and British Heart Foundation.

The InCHIANTI study was

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