Zhangfa Song

Zhenyu Ju
email: zhenyuju@hotmail.com

K Lenhard

K-Lenhard Rudolph
email: lenhard.rudolph@uni-ulm.de

Cell intrinsic and extrinsic mechanisms of stem cell aging depend on telomere status

The function of adult stem cells declines during aging and chronic diseases. An understanding of the molecular mechanisms underlying these processes will help to identify targets for future therapies in order to improve regenerative reserve and organ maintenance. Telomere shortening represents a cell intrinsic mechanism inducing DNA damage in aging cells. Current studies in telomerase knockout mice have shown that telomere dysfunction induces cell intrinsic checkpoints and environmental alteration that limit stem cell function. While these phenotypes differ from wildtype mice with long telomere reserves, they appear to be relevant for human aging, which is associated with an accumulation of telomere dysfunction and DNA damage.

Introduction

There is growing evidence for a functional decline of adult stem cells during aging. In aging mammals, the functional reserve of adult stem cells decreases in the hematopoietic system [START_REF] Waterstrat | Mechanisms of Stem cell aging[END_REF], in pancreatic islets [START_REF] Krishnamurthy | p16INK4a induces an age-dependent decline in islet regenerative potential[END_REF], in areas of neurogenesis in the brain [START_REF] Molofsky | Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing[END_REF], in hair follicles [START_REF] Nishimura | Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche[END_REF], in intestinal crypts [START_REF] Martin | Altered stem cell regeneration in irradiated intestinal crypts of senescent mice[END_REF], and in muscle [START_REF] Brack | Intrinsic changes and extrinsic influences of myogenic stem cell function during aging[END_REF].

Several lines of evidence indicate that the decline in stem cell function during aging can involve both cell intrinsic mechanisms and environmental alterations [for review see: [START_REF] Ju | Telomere dysfunction and stem cell ageing[END_REF]]. In humans, it has been shown that both donor age [START_REF] Castro-Malaspina | Unrelated donor marrow transplantation for myelodysplastic syndromes: outcome analysis in 510 transplants facilitated by the National Marrow Donor Program[END_REF][START_REF] Kollman | Donor characteristics as risk factors in recipients after transplantation of bone marrow from unrelated donors: the effect of donor age[END_REF] and recipient age [START_REF] Kantarjian | Results of intensive chemotherapy in 998 patients age 65 years or older with acute myeloid leukemia or high-risk myelodysplastic syndrome: predictive prognostic models for outcome[END_REF][START_REF] Socie | Long-term survival and late deaths after allogeneic bone marrow transplantation. Late Effects Working Committee of the International Bone Marrow Transplant Registry[END_REF] influence patients' outcomes in response to hematopoietic stem cell (HSC) transplantation. These clinical data indicate that cell intrinsic and cell extrinsic (environmental) mechanisms could contribute to the functional decline of HSCs during human aging. The relative contribution of these mechanisms to the decline in function and maintenance of aging stem cells remains to be defined in different organ systems and genetic modifiers have yet to be identified. Overcoming these obstacles may provide a rational basis to begin therapeutic strategy development, which could improve regenerative reserve during aging. In this review, we focus on the influence of telomere dysfunction on cell intrinsic and cell extrinsic mechanisms limiting stem cell function during aging.

Telomere shortening and cellular aging

Telomeres consist of simple tandem DNA repeats and associated telomere binding proteins [START_REF] De Lange | Shelterin: the protein complex that shapes and safeguards human telomeres[END_REF]. The main function of telomeres is to cap chromosome ends thus preventing an activation of DNA damage responses and the evolution of chromosomal instability [START_REF] Blackburn | Switching and signaling at the telomere[END_REF]. Telomeres shorten with each round of cell division due to the 'end replication problem' of DNA polymerase and due to processing of telomeres during cell cycle [START_REF] Levy | Telomere end-replication problem and cell aging[END_REF][START_REF] Sfeir | Telomere-end processing the terminal nucleotides of human chromosomes[END_REF]. The enzyme telomerase is required to compensate for telomere shortening. Telomerase synthesizes telomeres de novo. In humans, telomerase is active during embryogenesis [START_REF] Wright | Telomerase activity in human germline and embryonic tissues and cells[END_REF], but it is suppressed in most somatic cells after birth. In adults, telomerase remains active in germ cells and certain stem cell compartments, like in intestinal stem cells and hematopoietic stem cells [START_REF] Broccoli | Telomerase activity in normal and malignant hematopoietic cells[END_REF] for review see: Zimmermann andMartens, 2005, Artandi, 2008) . In addition, telomerase is transiently up-regulated in stimulated lymphocytes [START_REF] Hiyama | Activation of telomerase in human lymphocytes and hematopoietic progenitor cells[END_REF]. Telomere shortening limits the lifespan of primary human cells to a finite number of cell divisions, which is 50-70 for human fibroblast. This limit is called after the discoverer -the 'Hayflick limit' [START_REF] Hayflick | The Limited in Vitro Lifetime of Human Diploid Cell Strains[END_REF]. Studies on over-expression of telomerase have proven that telomere shortening is the underlying mechanism limiting the lifespan of human cells [START_REF] Bodnar | Extension of life-span by introduction of telomerase into normal human cells[END_REF]. Since most primary human cells lack telomerase expression the lifespan of human cells is limited by telomere shortening. Studies on human fibroblasts have shown that telomere shortening induces two checkpoints. The first checkpoint is activated in response to low levels of telomere dysfunction [3-5 dysfunctional telomeres per cell, [START_REF] Zou | Does a sentinel or a subset of short telomeres determine replicative senescence?[END_REF]]. At this stage human fibroblasts enter a permanent cell cycle arrest, which is called replicative senescence [START_REF] Wright | The two-stage mechanism controlling cellular senescence and immortalization[END_REF]. Human fibroblasts can escape the senescence checkpoint when p53 and Rb checkpoints are abrogated [START_REF] Wright | The two-stage mechanism controlling cellular senescence and immortalization[END_REF]. However, during this extended lifespan period (ca. 10-20 cell divisions) telomeres continue to shorten and eventually reach a second checkpoint, which is called crisis [START_REF] Wright | The two-stage mechanism controlling cellular senescence and immortalization[END_REF]. Crisis is characterized by high levels of telomere dysfunction, massive chromosomal instability, and cell death.

Telomere shortening and human aging

There is ample evidence that telomeres shorten during human aging [START_REF] Jiang | Telomere shortening and ageing[END_REF]. This shortening also affects stem cell compartments, e.g., telomeres shorten in CD34+ hematopoietic cells during human aging [START_REF] Vaziri | Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age[END_REF], although human HSCs exhibit low level of telomerase activity. Together, it appears that telomerase expression contributes to the proliferative capacity of human HSCs but the level of telomerase expression is not sufficient to maintain stable telomeres into old age [START_REF] Zimmermann | Telomere dynamics in hematopoietic stem cells[END_REF].

In addition to telomere shortening during aging, various chronic diseases accelerate telomere shortening in humans [START_REF] Jiang | Telomere shortening and ageing[END_REF]. This has been reported for different forms of anemia, hypertension, coronary heart diseases, chronic HIV infection, ulcerative colitis, chronic liver disease, and others [START_REF] Wiemann | Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis[END_REF], Jiang et al. 2007). Moreover, telomere shortening during chronic diseases is often associated with disease progression [START_REF] Jiang | Telomere shortening and ageing[END_REF][START_REF] Wiemann | Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis[END_REF]. It has also been documented that telomere shortening correlates with age-related pathology and reduced human lifespan [START_REF] Cawthon | Association between telomere length in blood and mortality in people aged 60 years or older[END_REF][START_REF] Collerton | Telomere length is associated with left ventricular function in the oldest old: the Newcastle 85+ study[END_REF]. Lastly, mutations in human telomerase genes lead to telomere shortening, stem cell defects, impaired organ maintenance and a shortened lifespan [START_REF] Armanios | Telomerase mutations in families with idiopathic pulmonary fibrosis[END_REF][START_REF] Tsakiri | Adult-onset pulmonary fibrosis caused by mutations in telomerase[END_REF][START_REF] Vulliamy | Disease anticipation is associated with progressive telomere shortening in families with dyskeratosis congenita due to mutations in TERC[END_REF]. Together, these data strongly indicate that human telomere reserves are limited. New experimental data indicate that increasing levels of biomarkers of telomere dysfunction and DNA damage characterize human aging and aging-associated disease [START_REF] Jiang | Proteins induced by telomere dysfunction and DNA damage represent bio-markers of human aging and disease[END_REF]. Given the pivotal role of telomeres and adult stem cells for tissues maintenance and repair, it appears to be of utmost importance to understand the functional consequences of telomere dysfunction on stem cell levels. The characterization of stem cell aging in wild type mice cannot address this critical question since laboratory wild type mouse strains have very long telomeres compared to humans [START_REF] Prowse | Developmental and tissue-specific regulation of mouse telomerase and telomere length[END_REF] and inbred laboratory wildtype mice do not experience significant levels of telomere dysfunction during aging [START_REF] Jiang | Proteins induced by telomere dysfunction and DNA damage represent bio-markers of human aging and disease[END_REF]). In fact, there is experimental evidence that the decline in stem cell function in wild type laboratory mouse strains is mainly driven by telomere-independent mechanisms [START_REF] Allsopp | Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation[END_REF].

DNA damage signalling in response to telomere dysfunction

Critical telomere shortening leads to uncapping of chromosomal ends. These dysfunctional telomeric ends are recognized as DNA double strand breaks, that lead to activation of DNA-damage checkpoints (d' Adda di Fagagna et al., 2003;[START_REF] Takai | DNA damage foci at dysfunctional telomeres[END_REF]. DNA damage signals in response to telomere dysfunction are similar to those induced by γ-irradiation. The initial step of this DNA damage signalling is the formation of DNA damage foci at dysfunctional telomeres. These foci consist of proteins involved in DNA damage recognition and repair including γH2AX, 53bp1, NBS1, MDC1, among others (d' Adda di Fagagna et al., 2003;[START_REF] Satyanarayana | Mitogen stimulation cooperates with telomere shortening to activate DNA damage responses and senescence signaling[END_REF]. These DNA damage foci induce ATM/Chk2 and ATR/Chk1 kinase pathways (d' Adda di Fagagna et al., 2003) activating p53 [START_REF] Gire | DNA damage checkpoint kinase Chk2 triggers replicative senescence[END_REF][START_REF] Chin | p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis[END_REF] and its downstream target p21 [START_REF] Brown | Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts[END_REF]inducing cellular senescence [START_REF] Brown | Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts[END_REF]. In contrast to the senescence checkpoint, the crisis checkpoint is less well understood and may involve an activation of multiple p53-independent checkpoints inducing cell death.

There is a continuous need to investigate checkpoints in response to telomere dysfunction using in vivo models. It is conceivable that telomere dysfunction induces different checkpoint responses depending on the tissue type. In addition, checkpoint responses in stem cells may differ from somatic cells. In line with this assumption, it has been shown that embryonic stem cells lack efficient G1 cell cycle checkpoints in response to γ-irradiation [START_REF] Hong | Restoration of an absent G1 arrest and protection from apoptosis in embryonic stem cells after ionizing radiation[END_REF]. It is currently unknown whether similar differences are present in adult stem cells compared to somatic cells. Given the difficulties in culturing undifferentiated, adult stem cells, it is necessary to investigate consequences of telomere dysfunction using in vivo models.

Telomere shortening and stem cell aging

Inbred laboratory mice have long telomeres (25 -60 kb compared to 10-12 kb in humans) and a short lifespan (24-30 months) compared to 75-80 years in humans [START_REF] Cawthon | Association between telomere length in blood and mortality in people aged 60 years or older[END_REF][START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF][START_REF] Prowse | Developmental and tissue-specific regulation of mouse telomerase and telomere length[END_REF]. Using an unbiased proteomic approach we have recently identified biomarkers of telomere dysfunction. These markers show an increased expression in various tissues and blood plasma of aging telomere dysfunctional mice but not in wildtype controls. The same biomarkers significantly increase during human aging and aging-associated disease [START_REF] Jiang | Proteins induced by telomere dysfunction and DNA damage represent bio-markers of human aging and disease[END_REF]. Together these results support the hypothesis that accumulation of telomere dysfunction and DNA damage contribute to human aging, but have little impact on aging of wild type mice with long telomere reserves. Thus, telomerase knockout mice provide a unique experimental model to study the in vivo consequences of this important aspect of human aging.

Current experimental evidence indicates that telomere shortening influences stem cell aging. In some wild type mouse strains (e.g., C57Bl/6J) the number of HSCs increases during aging, whereas other mouse strains, such as DAB2 and BalbC, show a decrease in HSC number during aging [START_REF] Morrison | The aging of hematopoietic stem cells[END_REF][START_REF] Waterstrat | Mechanisms of Stem cell aging[END_REF]. Interestingly, the number of HSCs has been associated with murine lifespan [START_REF] Henckaerts | Quantitative genetic variation in the hematopoietic stem cell and progenitor cell compartment and in lifespan are closely linked at multiple loci in BXD recombinant inbred mice[END_REF][START_REF] Van Zant | Genotype-restricted growth and aging patterns in hematopoietic stem cell populations of allophenic mice[END_REF]. Although HSCs in wildtype C57Bl/6J mice acquire defects in long-term repopulating potential during aging, the total stem cell activity remains fairly constant due to the increase in stem cell number in this strain [START_REF] Chambers | Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation[END_REF]. Moreover, C57BL/6J mice rarely develop hematopoietic failure during aging. This scenario appears to be very different from human aging, which is characterized by a decline in HSC number [START_REF] Waterstrat | Mechanisms of Stem cell aging[END_REF], and frequent occurrence of bone marrow failure and anemia [START_REF] Beghe | Prevalence and outcomes of anemia in geriatrics: a systematic review of the literature[END_REF]). It appears likely that shorter telomeres and longer lifespan in humans represent fundamental differences affecting stem cell aging in humans but not in C57BL/6J mice. In agreement with this assumption, heterozygous mutations in telomerase gene impair telomerase activity by haplo-insufficiency and represent risk factors for bone marrow failure in human aplastic anemia and dyskeratosis congenita (DC) (Vulliyami et al., 2004). In contrast, heterozygous mutations of telomerase in C57BL/6J mice with long telomeres have little effect on stem cell function during aging [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF]. However, the heterozygous deletion of telomerase in a mouse strain with short telomeres (CAST/EjI) induced stem cell defects and impairment of organ maintenance similar to the human situation [START_REF] Hao | Short telomeres, even in the presence of telomerase, limit tissue renewal capacity[END_REF]. Together, these results indicate that laboratory mouse strains with long telomeres lack telomere shortening, which represent one important aspect of human aging.

Studies on telomerase knockout mice (Terc-/-) have provided first experimental evidence that telomere shorting has a causal role in limiting organ maintenance and lifespan [START_REF] Rudolph | Longevity, stress response, and cancer in aging telomerase-deficient mice[END_REF]. Terc-/-mice with short telomeres showed premature aging phenotypes, specifically affecting organ systems with high rate of cell turnover [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF]Ju et al., 2007;[START_REF] Lee | Essential role of mouse telomerase in highly proliferative organs[END_REF]. Aging phenotypes of hematopoietic stem cells in Terc-/-mice are different from HSC aging in wild type mice (Table 1). Given the prevalence of telomere shortening in human aging (see above) studies on Terc-/-mice may allow a better understanding of telomere dependent mechanisms of stem cell aging in humans.

In contrast to wild type C57BL/6J mice (see above), the number of long-term HSCs does not significantly expand in aging Terc-/-C57BL/6J mice with dysfunctional telomeres, but rather shows a decrease compared to age-matched wildtype controls [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF]. Moreover, Terc-/-mice more frequently develop hematopoietic failure, which also occurs during human aging, namely anemia and a reduction in lymphocytes and total white blood counts [START_REF] Rudolph | Longevity, stress response, and cancer in aging telomerase-deficient mice[END_REF]. In addition, telomere dysfunction impaired the function of HSCs in transplantation experiments (see below). In addition to the defects in the hematopoietic system, telomere dysfunctional mice exhibited a stem cell depletion phenotype affecting germ cells, intestinal stem cells, and neuronal stem cells [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF]Ju et al., 2007;[START_REF] Wong | Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing[END_REF]. Together, these findings suggest that adult stem cells are the most sensitive cell compartment affected by telomere dysfunction.

In human, two important pathophysiological features of the aged hematopoietic system are a decrease in lymphopoiesis and immune function and an increase in myeloproliferative disease. Evidence from humans HSC transplantation (see above) and mice experiments (see below) indicate that stem cell intrinsic and extrinsic mechanisms contribute to the age-dependent decline in stem cell function. In wild type mice, a similar skewing of hematopoiesis (decreased lymphoiesis, increased myelopoiesis) occurs during aging. This phenotype in wild type mice is transplantable and is characterised by cell intrinsic changes in gene expression suggesting that this phenotype can be induced by cell intrinsic mechanisms [START_REF] Chambers | Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation[END_REF][START_REF] Rossi | Cell intrinsic alterations underlie hematopoietic stem cell aging[END_REF]. However, there is also evidence that cell extrinsic alteration may contribute to impaired b-lymphopoiesis in aging wild type mice [START_REF] Labrie | Bone marrow microenvironmental changes underlie reduced RAG-mediated recombination and B cell generation in aged mice[END_REF][START_REF] Stephan | Impaired ability of bone marrow stromal cells to support B-lymphopoiesis with age[END_REF]. Moreover, it was shown that the aging environment impairs the homing of transplanted HSCs [START_REF] Liang | Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells[END_REF]. In aging telomere dysfunctional Terc-/-C57BL/6J mice, the skewing of hematopoiesis is accelerated compared to wild type mice (Ju et al., 2007). Interestingly, the skewing of hemato-lymphopoiesis in Terc-/-mice is mainly induced by cell extrinsic alterations, whereas stem cell intrinsic checkpoints affect self-renewal and repopulation capacity of telomere dysfunctional HSCs (Ju et al., 2007, Choudhury et al., 2007).

Given the evidence for a functional role of telomere dysfunction during human aging (see above) the following section will provide a summary of cell intrinsic and extrinsic alterations that limit stem cell function in response to telomere dysfunction.

Cell intrinsic checkpoints limiting stem cell function in response to telomere dysfunction

Experiments in late generation mTerc-/-mice revealed first experimental evidence that cell intrinsic checkpoints contribute to the decline in stem cell function in response to telomere dysfunction and aging. Comparing studies on wild type and telomerase knockout mice it becomes clear that DNA damage checkpoint genes can have protective or detrimental effects on stem cell function depending on telomere status (Table 2). (i) Exonuclease-1 (Exo1): Exo1 is a 5-3 prime exonuclease involved in the DNA mismatch repair pathway [START_REF] Amin | exo1-Dependent mutator mutations: model system for studying functional interactions in mismatch repair[END_REF]. Exo1 deletion rescued the induction of senescence in response to telomere dysfunction in yeast cells [START_REF] Bertuch | EXO1 contributes to telomere maintenance in both telomerase-proficient and telomerase-deficient Saccharomyces cerevisiae[END_REF][START_REF] Hackett | End resection initiates genomic instability in the absence of telomerase[END_REF][START_REF] Maringele | Telomerase-and recombination-independent immortalization of budding yeast[END_REF] . In Terc-/-mice, Exo1 deletion improved the maintenance of intestinal stem cells and rescued the skewing of hemato-/lymphopoiesis in aging telomere dysfunctional mice. The study revealed that Exo1 deletion prevented the formation of single stranded DNA and the activation of ATR-p53-p21 signaling pathway [START_REF] Schaetzlein | Exonuclease-1 deletion impairs DNA damage signaling and prolongs lifespan of telomere-dysfunctional mice[END_REF]. This rescue in DNA damage signaling was associated with improved cell cycle activity and reduced induction of apoptosis in intestinal stem and progenitor cells in response to telomere dysfunction. Studies on PMS2 have also shown a role of mismatch repair in DNA damage signaling in response to telomere dysfunction [START_REF] Siegl-Cachedenier | Deficient mismatch repair improves organismal fitness and survival of mice with dysfunctional telomeres[END_REF]. In contrast to the rescue of intestinal stem cells in response to deletion of mismatch repair genes in telomere dysfunctional mice, the deletion of the mismatch repair gene MSH2 diminished the function of HSCs in wild tpe mice [START_REF] Reese | Repopulating defect of mismatch repair-deficient hematopoietic stem cells[END_REF]. (ii) Ataxia Telangiectasia Mutated (ATM): Deletion of ATM did not rescue the activation of p53-signalling in telomere dysfunctional mice indicating that ATM-independent pathways can activate p53 in response to telomere dysfunction [START_REF] Wong | Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing[END_REF]. Paradoxically the deletion of ATM enhanced aging phenotypes in mTERC -/-mice [START_REF] Wong | Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing[END_REF]. A potential explanation is that ATM itself has a role in telomere capping and ATM deletion may increase telomere dysfunction [START_REF] Bi | Telomere protection without a telomerase; the role of ATM and Mre11 in Drosophila telomere maintenance[END_REF]. In addition, ATM plays a crucial role in reactive oxygen species (ROS) metabolism and the increase in ROS level in ATM-/-cells may cooperate with telomere dysfunction to aggravate aging phenotypes. Indeed, ATM deficiency affected the function of HSCs in wild type mice with long telomeres [START_REF] Ito | Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells[END_REF]. However, these defects only occurred in response to stress and not under homeostatic conditions in aging mice. Moreover, the effects of ATM mutations in humans (e.g. impaired organ homeostasis including neurodegeneration, immunodeficiency, and predisposition to malignancy) were not fully reproduced in mTerc +/+ mice with long telomere but to a greater extent in telomere dysfunctional mice [START_REF] Wong | Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing[END_REF]. These data suggest that telomere shortening can cooperate with other genetic lesions to induce aging related pathology. In line with this assumption, Wrn knockout mice with long telomere reserves did not display typical cellular or clinical phenotypes of human Werner syndrome [START_REF] Lombard | Mutations in the WRN gene in mice accelerate mortality in a p53-null background[END_REF]. However, in late generation Terc -/-mice, deletion of Wrn accelerated the overall attrition of telomere length, resulting in phenotypes resembling human Werner syndrome, including early onset of age-related disorders, increased incidence of mesenchymal cancers, and premature death [START_REF] Chang | Essential role of limiting telomeres in the pathogenesis of Werner syndrome[END_REF][START_REF] Du | Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes[END_REF]. (iii) p53: p53 appears to have a more complicated role in stem cell aging. First, in Terc +/+ mice, p53 seems to have a dose effect on organismal aging and stem cell function. Mice with a hypermorphic form of p53 showed reduced longevity, osteoporosis, generalized organ atrophy, and impaired stress tolerance [START_REF] Tyner | p53 mutant mice that display early ageing-associated phenotypes[END_REF]. Moreover, overactive p53 decreased the number and function of HSCs, whereas heterozygous deletion of p53 increased proliferation of HSCs during mouse aging [START_REF] Dumble | The impact of altered p53 dosage on hematopoietic stem cell dynamics during aging[END_REF]. In contrast, mice containing an extra copy of naturally regulated p53 and p19ARF(under control of the natural promoters of these genes) did not show increased tissue atrophy but an elongated lifespan [START_REF] Matheu | Delayed ageing through damage protection by the Arf/p53 pathway[END_REF]. The authors proposed that naturally regulated p53 is preferentially activated in damaged cells thus protecting the organisms from an accumulation of damaged cells. However, introducing an extra copy of p53 did not improve the lifespan of Terc-/-mice, although the load of damaged cells is higher in the context of telomere dysfunction in Terc-/-mice compared to wild type mice [START_REF] Garcia-Cao | Increased p53 activity does not accelerate telomere-driven ageing[END_REF]. A possible explanation for this difference could be that an extra copy of p53 can only delay aging in a context of relatively mild accumulation of DNA and telomeric damage. In contrast, in the context of high levels of DNA or telomeric damage beneficial effects of higher checkpoint control may be counteracted by the fact that the number of surviving cells falls under a critical threshold. The consequences of increased activity of naturally regulated p53 on human aging remain speculative. The above discussed similarities between human aging and aging of telomere dysfunctional mice suggest that increased activity of p53 may not improve organismal fitness. (iv) p21: Deletion of p21 improved organ homeostasis and elongated lifespan of telomere dysfunctional mice without accelerating cancer formation [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF]. A possible explanation is that apoptosis checkpoints remained intact preventing an increase in chromosomal instability and cancer formation. Interestingly, deletion of p21 improved self renewal and repopulation capacity of HSCs in aging telomere dysfunctional mice [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF], but impaired stem cell maintenance and repopulation capacity of HSCs in wildtype mice [START_REF] Cheng | Hematopoietic stem cell quiescence maintained by p21cip1/waf1[END_REF]. However, the latter effect appears to show mouse strain specificity and may only be relevant in the context of replication stress [START_REF] Van Os | A Limited role for p21Cip1/Waf1 in maintaining normal hematopoietic stem cell functioning[END_REF]. Together, p21 could have a dual role in aging stem cells (Ju et al., 2007). In non-damaged stem cells of wildtype mice, p21 contributes to the maintenance of stem cell quiescence thus preventing abnormal cycling and stem cell depletion. In contrast, p21 impairs self renewal and function of stem cells accumulating telomere dysfunction. (v) p16/p19: Experiments in wild type mice showed that p16-deletion improved regenerative capacity in different stem cell compartments including HSCs, neuronal stem cells, and pancreatic islets. These data indicate that p16 might be one of the causal factors limiting stem cell function in aging wild type mice with long telomere reserves [START_REF] Janzen | Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a[END_REF][START_REF] Krishnamurthy | p16INK4a induces an age-dependent decline in islet regenerative potential[END_REF][START_REF] Molofsky | Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing[END_REF]. In line with this hypothesis, expression of Bmi-1 (a repressor of p16) promotes stem cell self-renewal in wildtype mice [START_REF] Hosen | Bmi-1-green fluorescent protein-knock-in mice reveal the dynamic regulation of bmi-1 expression in normal and leukemic hematopoietic cells[END_REF]. In contrast to aging of wild type mice, p16 and p19ARF deletion did not improve organ maintenance and lifespan of telomere dysfunctional mice indicating that this pathway is not a major component limiting stem cell function in response to telomere dysfunction [START_REF] Khoo | Ink4a/Arf tumor suppressor does not modulate the degenerative conditions or tumor spectrum of the telomerase-deficient mouse[END_REF] .

Together the above studies show that cell intrinsic DNA damage checkpoints have very different effects on stem cell aging depending on the telomere status.

Cell extrinsic checkpoints limiting stem cell function in response to telomere dysfunction

Cell extrinsic mechanisms contribute to the decline in stem cell function in aging mice [START_REF] Liang | Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells[END_REF][START_REF] Stephan | Impaired ability of bone marrow stromal cells to support B-lymphopoiesis with age[END_REF][START_REF] Labrie | Bone marrow microenvironmental changes underlie reduced RAG-mediated recombination and B cell generation in aged mice[END_REF]. Telomere dysfunction increased the influence of environmental alterations limiting the function of HSCs in aging mice. Experimental evidence for an accumulation of telomere dysfunction in aging humans [START_REF] Jiang | Proteins induced by telomere dysfunction and DNA damage represent bio-markers of human aging and disease[END_REF] and clinical data (Table 1) support the hypothesis that environmental alterations induced by telomere dysfunction could contribute to the decline in stem cell function in human aging.

One hallmarks of HSC aging is the skewing of hemato-/lymphopoiesis (decreased lymphopoiesis and increased myelopoiesis) occurring during human and mouse aging [START_REF] Kim | Major age-related changes of mouse hematopoietic stem/progenitor cells[END_REF][START_REF] Morrison | The aging of hematopoietic stem cells[END_REF][START_REF] Rossi | Cell intrinsic alterations underlie hematopoietic stem cell aging[END_REF][START_REF] Sudo | Age-associated characteristics of murine hematopoietic stem cells[END_REF]. The skewing of hemato-/lymphopoiesis occurs in aging wild type mice and in Terc-/-mice with dysfunctional telomeres [START_REF] Rossi | Cell intrinsic alterations underlie hematopoietic stem cell aging[END_REF][START_REF] Rudolph | Longevity, stress response, and cancer in aging telomerase-deficient mice[END_REF]Ju et al., 2007). However, in Terc-/-mice, the phenotype is accelerated and appears earlier in life compared to wild type mice (Ju et al. 2007).

Experimental data suggest that skewing of hemato-lymphopoiesis in aging wild type mice is induced by both cell-intrinsic and cell extrinsic mechanisms (see above and [START_REF] Labrie | Bone marrow microenvironmental changes underlie reduced RAG-mediated recombination and B cell generation in aged mice[END_REF][START_REF] Rossi | Cell intrinsic alterations underlie hematopoietic stem cell aging[END_REF][START_REF] Stephan | Impaired ability of bone marrow stromal cells to support B-lymphopoiesis with age[END_REF]. In contrast, the skewing of hemato-/lymphopoiesis in aging mTerc-/-mice with dysfunctional telomeres was mainly due to cell extrinsic mechanisms. Skewing of hemato-lymphopoiesis was rescued when Terc-/-HSCs were transplanted into a wildtype environment. Moreover, young wildtype HSC showed skewing when transplanted into telomere dysfunctional recipients (Ju et al., 2007). Together, these results indicate that telomere dysfunction increases the influence of cell extrinsic factors on stem cell aging (Table 1).

The defects of the stem cell environment in telomere dysfunctional mice were age-dependent and correlated with progressive telomere shortening in bone marrow stromal cells (Ju et al. 2007).

Telomere dysfunctional mice showed an impaired capacity of bone marrow stromal cells to maintain functional HSCs, and an increased expression of various cytokines, including granulocyte colony-stimulating factor (G-CSF) in blood serum and in supernatant of primary bone marrow stromal cells. Administration of G-CSF to wild-type mice mimicked some of the defects seen in aging telomere dysfunction mice, including impairment of b-lymphopoiesis and HSC engraftment. Conversely, inhibition of G-CSF improved HSC engraftment in aged telomere dysfunction mice.

Proteomic analysis on cell extrinsic proteins in culture supernatant of bone marrow from telomere dysfunctional mice has identified a new set of biomarker showing increased expression in response to telomere dysfunction in aging mTerc-/-mice [START_REF] Jiang | Proteins induced by telomere dysfunction and DNA damage represent bio-markers of human aging and disease[END_REF]. Interestingly, these biomarkers showed increased expression in human cells in response to telomere dysfunction and irradiation induced DAN damage. Moreover, the expression levels of these proteins in blood serum were robust indicators for human aging and age related diseases indicating that similar cell extrinsic alterations occur in aging humans and telomere dysfunctional mice.

There is accumulating evidence that cell extrinsic alterations contribute to the decline in stem cell function in various organ systems during aging [START_REF] Conboy | Rejuvenation of aged progenitor cells by exposure to a young systemic environment[END_REF]Ryu et al., 2006, Mackall and[START_REF] Mackall | Thymic aging and T-cell regeneration[END_REF]Kempermann et al., 2002[START_REF] Collins | A population of myogenic stem cells that survives skeletal muscle aging[END_REF]. Emerging data suggest that systemically acting factors and alterations in cell-to-cell contact in stem niches impinge on stem cell aging [START_REF] Boyle | Decline in self-renewal factors contributes to aging of the stem cell niche in the Drosophila testis[END_REF][START_REF] Pan | Stem cell aging is controlled both intrinsically and extrinsically in the Drosophila ovary[END_REF][START_REF] Zhao | Age-related changes of germline stem cell activity, niche signaling activity and egg production in Drosophila[END_REF] . The relative contribution of telomere dysfunction to these mechanisms of stem cell aging remains yet to be defined across different organ systems.

Outlook

In summary, current data suggest that self-renewing stem cells age because of cell intrinsic events and environmental alterations. Several lines of evidence suggest that the relative contributions of these routes of stem cell aging are highly context dependent. In mice, there are fundamental differences in aging wild type strains compared to mutant strains modeling the human situation of an age dependent accumulation of telomere dysfunction. Future studies focusing on model organisms and human aging should attempt to identify molecular mechanisms of stem cell aging relevant to human aging and disease. Such studies should ultimately lead to the identification of possible targets for future therapies aiming to improve regenerative reserve and organ maintenance in response to human aging and disease.

Table 1

Aging of hematopoietic stem cell is influenced by cell intrinsic and cell extrinsic mechanisms. In mice, telomere dysfunction changes the relative contribution of cell extrinsic and intrinsic mechanisms to HSC aging (Table 1).

Stem cell aging Humans

Wildtype mice Telomere dysfunctional mice

HSC self renewal

Repopulation capacity

Skewing of hematopoiesis

Decreased CD34+ HSCs with human aging (Waterstrat, et.al., 2008.). The contribution of cell intrinsic or extrinsic mechanisms is unknown.

Cell intrinsic: Telomere shortening and declined repopulating capacity with increasing donor age [START_REF] Widmann | Sustained telomere erosion due to increased stem cell turnover during triple autologous hematopoietic stem cell transplantation[END_REF][START_REF] Castro-Malaspina | Unrelated donor marrow transplantation for myelodysplastic syndromes: outcome analysis in 510 transplants facilitated by the National Marrow Donor Program[END_REF][START_REF] Kollman | Donor characteristics as risk factors in recipients after transplantation of bone marrow from unrelated donors: the effect of donor age[END_REF][START_REF] Yakoub-Agha | Allogeneic marrow stem-cell transplantation from human leukocyte antigen-identical siblings versus human leukocyte antigen-allelic-matched unrelated donors (10/10) in patients with standard-risk hematologic malignancy: a prospective study from the French Society of Bone Marrow Transplantation and Cell Therapy[END_REF].

Cell extrinsic: Long-term survival of bone marrow transpanted patients is significantly reduced in older recipients. [START_REF] Socie | Long-term survival and late deaths after allogeneic bone marrow transplantation. Late Effects Working Committee of the International Bone Marrow Transplant Registry[END_REF]) [START_REF] Kantarjian | Results of intensive chemotherapy in 998 patients age 65 years or older with acute myeloid leukemia or high-risk myelodysplastic syndrome: predictive prognostic models for outcome[END_REF] Loss of immune function and increased incidence of myeloid proliferative disorders including acute myeloid leukemia, indicating a skewed hematopoiesis with age. The contribution of cell intrinsic or extrinsic mechanisms are unknown.

An increase in absolute number of HSC in the C57Bl/6J strain but a decrease in the DBA and Balb/C strain. [START_REF] Morrison | The aging of hematopoietic stem cells[END_REF][START_REF] Sudo | Age-associated characteristics of murine hematopoietic stem cells[END_REF][START_REF] Rossi | Cell intrinsic alterations underlie hematopoietic stem cell aging[END_REF][START_REF] Waterstrat | Mechanisms of Stem cell aging[END_REF].

Cell intrinsic: genetic factors modify HSC self renewal [START_REF] Yang | Rho GTPase Cdc42 coordinates hematopoietic stem cell quiescence and niche interaction in the bone marrow[END_REF]).

The contribution of Cell extrinsic mechanisms is unknown Cell intrinsic: Decline in repopulating capacity on a per cell basis during aging [START_REF] Chambers | Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation[END_REF].

Cell extrinsic: Diminished homing and engraftement of transplanted cells depending on recipient age (Laird et al., 2008;[START_REF] Liang | Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells[END_REF] Impaired Lymphopoiesis and increase myelopoiesis in aging mice.

Cell intrinsic: Gene expression changes in aging

HSCs favor myelopoiesis and impair lymphopoiesis [START_REF] Chambers | Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation[END_REF][START_REF] Rossi | Cell intrinsic alterations underlie hematopoietic stem cell aging[END_REF] Reduced number in aged telomere dysfunction mice compared to age-matched wildtype control mice. [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF], Ju et al., 2007) Cell intrinsic: Reduced self renewal of transplanted HSCs with dysdfucntional telomeres (Ju et al., 2007, Rossi et al., 2007) Cell extrinsic: Telomere dysfunction impairs the capacity of stromal cells to maintain functional HSCs (Ju et al., 2007) Cell intrinsic: DNA damage checkpoints induced by telomere dysfunction diminished the number and self-renewal capacity of LT-HSC [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF][START_REF] Allsopp | Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation[END_REF] Cell extrinsic: Telomere shortening of the donor impairs engraftment of transplanted wildtype HSCs (Ju et al., 2007) Telomere dysfunction accelerates skewing of hematopoiesis in Terc -/- mice compared to wildtype mice Cell intrinsic: no evidence for contribution of cell intrinsic mechanisms. Skewing is completely measured after transplantation of Terc -/-HSCs into wildtype recipient mice. (Ju et al.,2007)

Atm

Ataxia telangiectasia Shortened lifespan [START_REF] Shiloh | ATM and related protein kinases: safeguarding genome integrity[END_REF] Neurodegeneration, ataxia. (Shiloh,2003) Chromosomal instability. [START_REF] Shiloh | ATM and related protein kinases: safeguarding genome integrity[END_REF] Radiosensitivity. (Shiloh,2003) Immunodeficiency. [START_REF] Shiloh | ATM and related protein kinases: safeguarding genome integrity[END_REF] Shortened lifespan due to cancer formation [START_REF] Elson | Pleiotropic defects in ataxia-telangiectasia protein-deficient mice[END_REF][START_REF] Barlow | Atm-deficient mice: a paradigm of ataxia telangiectasia[END_REF] Impaired repopulation capacity of HSCs due to increased reactive oxygen species. [START_REF] Ito | Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells[END_REF] Modest neurodegeneration, no functional defect [START_REF] Eilam | Late degeneration of nigro-striatal neurons in ATM-/-mice[END_REF].

Low level of chromosomal instability [START_REF] Callen | ATM prevents the persistence and propagation of chromosome breaks in lymphocytes[END_REF].

Radiosensitivity. [START_REF] Barlow | Atm-deficient mice: a paradigm of ataxia telangiectasia[END_REF]Ch'ang et al., 2005;[START_REF] Peter | Elevated Cu/Zn-SOD exacerbates radiation sensitivity and hematopoietic abnormalities of Atm-deficient mice[END_REF] Immunodeficieny. [START_REF] Peter | Elevated Cu/Zn-SOD exacerbates radiation sensitivity and hematopoietic abnormalities of Atm-deficient mice[END_REF][START_REF] Xu | Targeted disruption of ATM leads to growth retardation, chromosomal fragmentation during meiosis, immune defects, and thymic lymphoma[END_REF][START_REF] Bagley | Bone marrow transplantation restores immune system function and prevents lymphoma in Atm-deficient mice[END_REF] Osteoporosis. [START_REF] Rasheed | Atm-deficient mice: an osteoporosis model with defective osteoblast differentiation and increased osteoclastogenesis[END_REF][START_REF] Hishiya | Ataxia telangiectasia mutated (Atm) knockout mice as a model of osteopenia due to impaired bone formation[END_REF] Shortened lifespan due to premature aging. (Wong et al., 2003) Impaired colony formation of hematopoietic cells, [START_REF] Wong | Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing[END_REF] impaired proliferation of neuronal stem cells. [START_REF] Wong | Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing[END_REF] Increased neurodegeneration, hypokinesia.

High level of chromosomal instability. [START_REF] Wong | Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing[END_REF] Not investigated.

Not investigated.

Not investigated.

p53

Shortened lifespan due to cancer formation (Li Fraumeni Syndrome) [START_REF] Soussi | Shaping genetic alterations in human cancer: the p53 mutation paradigm[END_REF] Radioresistence [START_REF] Sproston | Fibroblasts from Li-Fraumeni patients are resistant to low dose-rate irradiation[END_REF] Chromosomal instability.

Shortened lifespan due to cancer formation [START_REF] Donehower | Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours[END_REF] Increased self renewal, increased repopulation capacity, but impaired long term function of HSCs [START_REF] Strasser | DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2[END_REF][START_REF] Dumble | The impact of altered p53 dosage on hematopoietic stem cell dynamics during aging[END_REF][START_REF] Tekippe | Expansion of hematopoietic stem cell phenotype and activity in Trp53-null mice[END_REF] Radioresistence. [START_REF] Merritt | The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice[END_REF] Chromosomal instability. [START_REF] Donehower | Deficiency of p53 accelerates mammary tumorigenesis in Wnt-1 transgenic mice and promotes chromosomal instability[END_REF] Shortened lifespan due to cancer formation. [START_REF] Artandi | Telomere dysfunction promotes non-reciprocal translocations and epithelial cancers in mice[END_REF] Rescue of apoptosis in germ cells and intestinal stem-/progentior cells, HSC function not analyzed. [START_REF] Chin | p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis[END_REF] Not investigated.

Increased chromosomal instability (Artandi et al

Table 2 Consequences of DNA damage checkpoint mutations in humans, wildtype mice, and telomere dysfunctional mice Gene Deletion/Mutation in Human Diseases/Cancer Deletion in Wildtype mice Deletion in telomere dysfunctional mice

 2

Thymus involution

Reduced thymus output and altered structure of the aging thymus (for review see Sutherland et al.) The contribution of cell intrinsic mechanisms is unknown. . Cell extrinsic: Rescue of thymus involution by androgen depletion [START_REF] Sutherland | Activation of thymic regeneration in mice and humans following androgen blockade[END_REF] Cell extrinsic: Impaired lymphopoiesis depending on recipient age [START_REF] Labrie | Bone marrow microenvironmental changes underlie reduced RAG-mediated recombination and B cell generation in aged mice[END_REF][START_REF] Stephan | Impaired ability of bone marrow stromal cells to support B-lymphopoiesis with age[END_REF] Reduced thymocyte output and altered structure of the aging thymus [START_REF] Aw | Architectural changes in the thymus of aging mice[END_REF][START_REF] Gray | Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells[END_REF] [START_REF] Sutherland | Activation of thymic regeneration in mice and humans following androgen blockade[END_REF] Cell extrinsic: Appears to be dominant mechanisms, HSCs of young wildtype show skewing after transplantation into old Terc -/-mice with dysfunctional telomere, but not after transplantation in age matched wildtype mice. (Ju et al.,2007) Premature involution of the thymus The contribution of cell intrinsic or extrinsic mechanisms are largely unknown.

Extra copy of naturally regulated p53: delayed aging [START_REF] Matheu | Delayed ageing through damage protection by the Arf/p53 pathway[END_REF] Overactive mutant p53: premature aging, impaired self renewal and function of HSCs [START_REF] Tyner | p53 mutant mice that display early ageing-associated phenotypes[END_REF][START_REF] Donehower | Does p53 affect organismal aging?[END_REF]

2000)

Extra copy of naturally regulated p53: no rescue of premature aging [START_REF] Garcia-Cao | Increased p53 activity does not accelerate telomere-driven ageing[END_REF] p21

Mutations are not associated with human diseases or cancer Increased cycling of HSCs (under debate), Exhaustion of HSCs when exposed to chemotherapy and replicative stress. [START_REF] Cheng | Hematopoietic stem cell quiescence maintained by p21cip1/waf1[END_REF] but under steady state the role of p21 n HSCsfuncton epends on strain background. [START_REF] Van Os | A Limited role for p21Cip1/Waf1 in maintaining normal hematopoietic stem cell functioning[END_REF] Increased neurogenesis and neuronal regeneration, exhaustion of neuronal stem cells during aging. [START_REF] Pechnick | p21Cip1 restricts neuronal proliferation in the subgranular zone of the dentate gyrus of the hippocampus[END_REF][START_REF] Kippin | p21 loss compromises the relative quiescence of forebrain stem cell proliferation leading to exhaustion of their proliferation capacity[END_REF][START_REF] Qiu | Regenerative response in ischemic brain restricted by p21cip1/waf1[END_REF] Elongated lifespan. [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF] Improved maintenance and function of HSCs. [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF] Improved maintenance of intestinal stem and progenitor cells. [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF] No rescue of apoptosis in intestinal progenitor cells and germ cells. [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF]

p16/p19

Polymorphisms are associated with aging [START_REF] Saxena | Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels[END_REF][START_REF] Scott | A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants[END_REF][START_REF] Zeggini | Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes[END_REF][START_REF] Melzer | A common variant of the p16(INK4a) genetic region is associated with physical function in older people[END_REF]) P16-deletion: improved regenerative capacity in different stem cell compartments: HSCs, neuronal stem cells, pancreatic islets. [START_REF] Molofsky | Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing[END_REF][START_REF] Krishnamurthy | p16INK4a induces an age-dependent decline in islet regenerative potential[END_REF][START_REF] Janzen | Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a[END_REF][START_REF] Stepanova | A limited role for p16Ink4a and p19Arf in the loss of hematopoietic stem cells during proliferative stress[END_REF] No improvement in organ maintenance and lifespan in p16Ink4A, P19ARF compound mutant mice [START_REF] Khoo | Ink4a/Arf tumor suppressor does not modulate the degenerative conditions or tumor spectrum of the telomerase-deficient mouse[END_REF] Shortened lifespan due to lymphoma formation in mixed genetic background [START_REF] Wei | Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility[END_REF] Shortened lifespan due to lymphoma at early age. (De wind et al.,1995) Reduced repopulation capacity of HSCs. [START_REF] Reese | Repopulating defect of mismatch repair-deficient hematopoietic stem cells[END_REF] Increased lifespan [START_REF] Schaetzlein | Exonuclease-1 deletion impairs DNA damage signaling and prolongs lifespan of telomere-dysfunctional mice[END_REF]. Improved maintenance of intestinal stem cells [START_REF] Schaetzlein | Exonuclease-1 deletion impairs DNA damage signaling and prolongs lifespan of telomere-dysfunctional mice[END_REF] Rescue in skewing of hemato-lymphopiesis Impaired DNA damage signaling. (Schaetzlein et al., 2007)


Arno Lingenhel

Barbara Kollerits

Johannes P Schwaiger

Steven C Hunt

Richard Gress

Paul N Hopkins

Veit Schoenborn

Iris M Heid

MD Florian Kronenberg
email: florian.kronenberg@i-med.ac.at

Serum bilirubin levels, UGT1A1 polymorphisms and risk for coronary artery disease

Keywords: Bilirubin, UGT1A1 bilirubin, coronary artery disease, UGT1A1, association study, polymorphism bilirubin, coronary artery disease, UGT1A1, association study, polymorphism

Low levels of the antioxidative serum bilirubin are associated with vascular aging and an increased risk for coronary artery disease (CAD). UGT1A1 is the major gene influencing bilirubin concentrations. Therefore, we investigated an association of bilirubin levels and two polymorphisms in the promoter of UGT1A1 polymorphism and T-3279G] in 477 patients with premature, familial CAD and 619 age-and sex-matched controls. Bilirubin concentrations were significantly lower in cases than in controls (0.62±0.36 vs. 0.76±0.41 mg/dl for men, p=1.2x10 -10 ; and 0.42±0.29 vs. 0.55±0.23 mg/dl, p=1.9x10 -9 for women). Both polymorphisms showed a strong association with bilirubin levels with higher levels for homozygote carriers of the minor allele. These associations were most pronounced in male controls and patients (p=5.9x10 -26 and p=3.4x10 -16 , respectively, for the -53(TA-repeat) polymorphism). Logistic regression analysis revealed low bilirubin levels but not the UGT1A1 polymorphisms to be significantly associated with CAD: OR (95%C) 0.90 (0.86-0.94), p=2.6x10 -6 for men and 0.77 (0.68-0.87), p=3.2x10 -5 for women, respectively for each 0.1 mg/dl increase of bilirubin. These results indicate that it is rather decreased bilirubin levels in general than the changes in the genetic variation of this gene that increase the risk for CAD.

Introduction

Several studies have shown that bilirubin, a major product of heme catabolism, has a potent antioxidative capacity [START_REF] Stocker | Antioxidant activity of albumin-bound bilirubin[END_REF][START_REF] Neuzil | Free and albumin-bound bilirubin are efficient coantioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation[END_REF]. Animal experiments in congenitally hyperbilirubinemic Gunn rats with a defective uridinediphosphate-glucuronosyltransferase compared to wildtype rats demonstrated virtually no neointimal proliferation after intimal injury caused by balloon angioplasty.

When wildtype rats were pretreated with the bilirubin precursor biliverdin before injury, neointimal formation was also significantly reduced. Cultured vascular smooth muscle cells from rats showed a significantly reduced proliferation when treated with bilirubin (Öllinger et al., 2005). These properties and bilirubin's ability to scavenge peroxyl radicals indicates a protective role of bilirubin against vascular aging and associated atherosclerotic diseases [START_REF] Schwertner | Gilbert syndrome, UGT1A1*28 allele, and cardiovascular disease risk: possible protective effects and therapeutic applications of bilirubin[END_REF]. This is in line with epidemiological studies reporting low serum bilirubin levels to be associated with an increased risk for coronary artery disease (CAD) or other atherosclerosis outcomes [START_REF] Schwertner | Association of low serum concentration of bilirubin with increased risk of coronary artery disease[END_REF][START_REF] Hopkins | Higher serum bilirubin is associated with decreased risk for early familial coronary artery disease[END_REF] Two recent genome-wide linkage studies suggested UGT1A1, the gene encoding for uridine-diphosphate-glucuronosyltransferase, as a major locus for serum bilirubin levels [START_REF] Kronenberg | A genome scan for loci influencing anti-atherogenic serum bilirubin levels[END_REF][START_REF] Lin | Evidence for a gene influencing serum bilirubin on chromosome 2q telomere: a genomewide scan in the Framingham study[END_REF]. The gene product of UGT1A1 catalyzes the conjugation of serum bilirubin to facilitate its removal by bilary excretion. Polymorphisms in this gene result in a decreased capacity to glucuronidate bilirubin, as observed in Gilbert syndrome and some forms of perinatal jaundice [START_REF] Clarke | Genetic defects of the UDP-glucuronosyltransferase-1 (UGT1) gene that cause familial non-haemolytic unconjugated hyperbilirubinaemias[END_REF][START_REF] Maruo | Association of neonatal hyperbilirubinemia with bilirubin UDP-glucuronosyltransferase polymorphism[END_REF]. A common polymorphism in the promoter of UGT1A1 with five to nine TA-repeats in the TATA box at position -53 was reported to show an inverse association with the transcriptional activity [START_REF] Bosma | The genetic basis of the reduced expression of bilirubin UDPglucuronosyltransferase 1 in Gilbert's syndrome[END_REF]. So far five different alleles with (TA) 5-9 -repeats have been described with (TA) 6 and (TA) 7 repeats as the main alleles with a frequency of up to 60% and 40%, respectively, in Caucasian populations [START_REF] Beutler | Racial variability in the UDPglucuronosyltransferase 1 (UGT1A1) promoter: a balanced polymorphism for regulation of bilirubin metabolism[END_REF]. Functional studies demonstrated that carriers of an allele with (TA) 7 -repeats show a lower transcriptional efficiency and a decrease of uridine-diphosphate-glucuronosyltransferase activity of about 70% compared to those with less than 7 repeats [START_REF] Bosma | The genetic basis of the reduced expression of bilirubin UDPglucuronosyltransferase 1 in Gilbert's syndrome[END_REF]. This reduced enzyme activity causes increased unconjugated bilirubin concentrations in

Patients and Methods

Patients and Controls

This case-control study is based on 1096 individuals of European ancestry from the same geographical region of Utah who are known to have little racial admixture.

The 477 patients (365 men and 112 women) with premature, familial CAD had survived a myocardial infarction, percutaneous transluminal angioplasty, or coronary artery bypass grafting before age 55 for men or age 65 for women. To minimize artifactual effects of the acute coronary syndrome on biochemical measurements, patients were seen at least 6 months after their acute event. Each of the CAD cases was from a family in which at least one additional first-degree relative had early CAD by this definition. The control group is based on 833 individuals representing a general population sample from the same geographical region as described recently [START_REF] Schoenborn | The ATGL gene is associated with free fatty acids, triglycerides and type 2 diabetes[END_REF]. From this control group we selected 397 men and 222 women who represented a similar age distribution as the respective gender-specific patient groups and which can be considered as an approximate 1:1 matching for men and 1:2 matching for women (patients:controls). Ethical approval by local committee and written informed consent was obtained from each participant.

Bilirubin measurement

All biochemical measurements were made in overnight fasting venous samples.

Total serum bilirubin was measured by a thin film adaptation of a diazonium salt, colorimetric method using the Vitros analyzer (Ortho-Clinical Diagnostics, Inc.

Rochester NY 14650).

Genotyping

The UGT1A1 -53(TA-repeat) promoter polymorphism was analyzed by the ABI 3130xl sequencing system exactly as described recently [START_REF] Lin | Association between UGT1A1*28 allele, bilirubin levels, and coronary heart disease in the Framingham Heart Study[END_REF]. The -3279T>G polymorphism (rs4124874) was determined by use of a 5'-Nuclease Assay and the ABI Prism® 7900HT system. Additionally 10 % of the samples were genotyped in duplicate for quality control. Genotyping was performed within the Genotyping Unit of the Gene Discovery Core Facility at the Innsbruck Medical University, Austria.

Statistical analysis

Results

Baseline characteristics

The demographic and laboratory parameters of the 477 CAD patients and the 619 controls are presented in Table 1. All analyses were stratified by gender. In both genders cases showed higher concentrations of total cholesterol, LDL-cholesterol, triglycerides, glucose and serum creatinine and lower levels for HDL-cholesterol and albumin concentrations as expected. A higher frequency of current smokers and exsmokers was found among the cases. Serum bilirubin levels were about 20% lower in CAD patients compared to controls of both genders. Independent of case status, females exhibited lower bilirubin concentrations compared to males (p<0.001).

Genotype characteristics

Lewontin's D' of the -53(TA) and T-3279G locus was almost unity (D'=0.9969) with a correlation coefficient of r=0.77. There were no statistical significant differences in the genotype distribution when comparing men and women or when comparing cases and controls. The minor allele frequencies for the -53(TA-repeat) and the T-3279G polymorphism ranged between 30.7% and 31.9% and between 42.5 and 49.1%, respectively, in the four subgroups, male and female controls or patients (Table 2).

Bilirubin concentrations depend on UGT1A1 polymorphisms (analysis stratified by case-control status)

We observed strong associations between both polymorphisms and bilirubin levels in the case control-stratified analysis (Table 2). Homozygote carriers of the minor allele of the -53(TA-repeat) polymorphism showed the highest bilirubin concentrations. Heterozygote individuals showed bilirubin levels closer to the levels of homozygote wildtypes. These associations were very strong in the recessive model in male controls (p=5.9x10 -26 ), male CAD patients (p=3.4x10 -16 ) and female CAD patients (p=0.00001), but were of borderline significance in female controls (p=0.09). Similar observations but with less strong effect sizes were observed for the T-3279G polymorphism (Table 2).

The total variation of bilirubin levels explained by the two polymorphisms combined was 15.4% in cases, and 13.6% in controls (Table 3). The analysis of the variation of bilirubin levels explained by the two single polymorphisms separately revealed that the T-3279G polymorphism explained less of the bilirubin variation than the -53(TA-repeat) polymorphism.

Bilirubin concentrations depend on CAD case-control status (analysis stratified by genotype groups)

When analyzing the genotype-specific differences in bilirubin levels between patients and controls, highly significantly lower bilirubin levels were observed among CAD patients compared to controls when focusing on subjects homozygous for the major allele (6/6 or T/T) or heterozygous (6/7 or T/G) (see most right column of Table 2 with p values ranging from 0.0004 to 1.0x10 -7 ). For subjects homozygous of the minor allele (7/7 or G/G), we observed a trend towards lower bilirubin levels in men (p=0.06 and 0.07 for the -53(TA-repeat) and the T-3279G polymorphism, respectively), but no difference in women (p=0.15 and 0.32, respectively).

Haplotype analysis

In concordance with the single polymorphism analysis, individuals carrying two copies of the major allele of both polymorphisms had the lowest bilirubin levels and haplotypes based on the minor allele at both SNPs were associated with the highest bilirubin levels (data not shown).

Association of bilirubin concentrations with CAD outcome, but no association of the polymorphisms with CAD outcome

After adjustment for age (model 1) and other risk factors (models 2 and 3), we observed a significant decrease in CAD risk per 0.1 mg/dl increase in serum bilirubin concentrations of about 6-10% in men and for about 25% in women (Table 4). No significant change in CAD risk depending directly on the genotype groups of the two polymorphisms was observed.

Discussion

Confirmed protective properties of bilirubin for CAD

The role of bilirubin in the pathogenesis of cardiovascular disease is still not entirely understood. The fact that it is antioxidative and cytoprotective suggests antiatherogenic properties. As demonstrated, bilirubin has a comparable antioxidative There are several explanations which might have contributed that we did not find an association of the UGT1A1 polymorphisms with CAD and some of them were discussed in detail recently by Schwertner and Vítek [START_REF] Schwertner | Gilbert syndrome, UGT1A1*28 allele, and cardiovascular disease risk: possible protective effects and therapeutic applications of bilirubin[END_REF]. The first one is the case-control design of our study. Due to the fact that all included cases in our study had survived a CAD event for at least six months, we might speculate on a survival bias considering that about half of the incident cases do not survive the first half year of a CAD event and that the further survival after the first event is influenced by genetic factors [START_REF] Marin | A pharmacogenetic effect of factor XIII valine 34 leucine polymorphism on fibrinolytic therapy for acute myocardial infarction[END_REF]. Given this hypothesis, the fact that we still detected the association of bilirubin levels with CAD might be explained by the strengths of this association and the situation that the UGT1A1 polymorphisms explain only about 10-15% of the bilirubin variance (Table 3). An additional and more likely explanation could be the existence of other factors that increase CAD risk and decrease bilirubin levels and are responsible for the ~85-90% of the variance in bilirubin not explained by the two UGT1A1 polymorphisms. The markedly lower bilirubin levels in CAD patients compared with controls within the genotype groups suggest such factors. These factors could have been confounding the gene-CAD association in our study. Assuming that bilirubin production is most likely affected by heme oxygenase, a further reason for the association of low bilirubin levels and an increased risk for CAD and might be the role of heme oxygenase-1 (HO-1) polymorphisms in the development of CAD [START_REF] Morita | Heme oxygenase and atherosclerosis[END_REF][START_REF] Exner | The role of heme oxygenase-1 promoter polymorphisms in human disease[END_REF][START_REF] Vitek | The heme catabolic pathway and its protective effects on oxidative stress-mediated diseases[END_REF]. Another possible explanation is reverse causation, meaning that the CAD status influences bilirubin levels rather than bilirubin levels modifying CAD risk. However, prospective studies rather indicate a truly causative effect of bilirubin on CAD with decreased levels long before the CAD event [START_REF] Djousse | Total serum bilirubin and risk of cardiovascular disease in the Framingham offspring study[END_REF][START_REF] Breimer | Serum bilirubin and risk of ischemic heart disease in middle-aged British men[END_REF][START_REF] Lin | Association between UGT1A1*28 allele, bilirubin levels, and coronary heart disease in the Framingham Heart Study[END_REF][START_REF] Troughton | Bilirubin and coronary heart disease risk in the Prospective Epidemiological Study of Myocardial Infarction (PRIME)[END_REF]. Given that the causative effect of bilirubin levels on CAD risk is true, the most likely explanation for not finding an UGT1A1 association with CAD is a lack of power. If increased levels cause a decreased risk of CAD of 10-25% per unit bilirubin, we have sufficient power to find this association. However, since the polymorphism shows various expressivity within each genotype group, the polymorphism explains only 10-15% of the variation in bilirubin levels. Therefore, our ability to detect this polymorphism-CAD association drops substantially if we consider also the fact that the frequency of the -53(TA) 7/7 genotype in CAD patients was expected to be half of the controls in case of a power of 80%.

Under the hypothesis that the UGT1A1 polymorphisms' changes decreased CAD directly, we would expect an equally strong or even stronger association of the polymorphism with CAD than the association of bilirubin levels with CAD. As this was not the case, we can conclude that it is low bilirubin levels in general that seem to increase CAD risk than just the UGT1A1 polymorphisms. Interestingly, the recent genome-wide association study by the Wellcome Trust Case Control Consortium did not find a significant association of the UGT1A1 gene region with CAD (The Wellcome Trust Case Control Consortium 2007).

Gender differences

Hunt and colleagues [START_REF] Hunt | Association of plasma bilirubin with coronary heart disease and segregation of bilirubin as a major gene trait: The NHLBI Family Heart Study[END_REF]) reported a borderline significant difference in serum bilirubin levels between CHD cases and controls in males (p=0.08) but no differences in females. Similar results were found by Endler et al. [START_REF] Endler | Is low serum bilirubin an independent risk factor for coronary artery disease in men but not in women?[END_REF]. Our study found an association of low bilirubin levels with CAD in both genders, which was even stronger in females: logistic regression analysis revealed a decreasing risk for CAD of about 10% and 25% for men and women, respectively, with each increase of bilirubin of 0.1 mg/dl. Similarly, Erdogan et al. [START_REF] Erdogan | Low serum bilirubin levels are independently and inversely related to impaired flow-mediated vasodilation and increased carotid intima-media thickness in both men and women[END_REF] found an association in both, men and women. Reasons for these contrasting results might be the small number of women with the investigated outcomes and therefore low power or major age differences between women cases and controls. Interestingly, the association of the two investigated polymorphisms with bilirubin levels was much smaller in female controls than in male controls but still very strong in both genders of cases (Table 2).

Limitations of the Study

We did not adjust our findings for liver enzymes since these parameters were not available in the entire group. However, when we performed a sensitivity analysis in those patients and controls with alkaline (ALP), aspartate transaminase (AST), or gammaglutamyltransferase (GGT) available, we observed similar results as described above (data not shown).

A stratified analysis of subjects with hyperbilirubinemia above 1 mg/dL was not suitable since the frequency of this state was only 4.3% and 12.5% in females and males, respectively.

We did not investigate further polymorphisms in the region of UGT1A1. Hong et al. reported besides the TA repeat polymorphism 14 other SNPs and demonstrated that conditional on the TA repeat polymorphism no other variant was shown to be significantly associated with bilirubin in both genotype and haplotype analyses [START_REF] Hong | UDP-Glucuronosyltransferase 1A1 Gene Polymorphisms and Total Bilirubin Levels in an Ethnically Diverse Cohort of Women[END_REF]. According to this thorough study, we have already tagged the common haplotypes with the two polymorphisms investigated in our study. If there is indeed an additional variant which contributes to bilirubin levels independent from the TA repeat polymorphism, it can be expected that the effect is quite small which would require a large sample size.

Conclusion

In summary, the present case-control study showed a strong and consistent association of low serum bilirubin levels with CAD for both genders. Despite a clear association of the two analyzed UGT1A1 promoter polymorphisms with bilirubin concentrations, we could not detect a significant association of these polymorphisms with CAD indicating that it is rather decreased bilirubin levels in general than the changes in the gene product caused by the particular polymorphisms that increase the risk of CAD. a adjusted for age, gender, smoking status, and either of the two polymorphisms or the two polymorphisms combined; b same as a but without adjustment for any polymorphism; c The variation explained by the polymorphisms was calculated by the difference of r 2 between a full model a and the submodel b . Values given are the odds ratio (OR), 95% confidence intervals (CI), and the respective pvalue testing for deviance of the OR from unity.

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  ; Hunt et al., 2001; Novotny et al., 2003; Djousse et al., 2003; Endler et al., 2003; Schwertner et al., 2000; Breimer et al., 1995; Levinson 1997; Troughton et al., 2007; Hunt et al., 1996; Lin et al., 2006; Rantner et al., 2008). A study in healthy individuals grouped by low, intermediate and high serum bilirubin levels revealed that elevated bilirubin concentrations protect from coronary flow reserve impairment, coronary microvascular dysfunction, and possibly coronary atherosclerosis (Gullu et al., 2005).

  heterozygous and homozygous carriers of the (TA) 7 -repeats. A recent study in 11 Japanese and 12 Caucasian patients with Gilbert's syndrome reported homozygosity for both, the UGT1A1 -53(TA) 7 repeat allele and the G allele of the T-3279G (rs4124874) polymorphism. The T-3279G polymorphism is located in a phenobarbital-responsive enhancer element in the 5' region of the gene. The presence of the -3279G allele results in a lower transcriptional activity, a reduced conjugation of bilirubin and higher serum bilirubin concentrations (Maruo et al., 2004). Due to the contradictory results of association studies investigating the UGT1A1 TA-repeat polymorphism and atherosclerotic complications (Bosma et al., 2003; Lin et al., 2006; Gajdos et al., 2006; Rantner et al., 2008) we decided to investigate the association of this and another recently described promoter polymorphism with bilirubin levels as well as CAD in a large group of patients with premature familial CAD in comparison to a control group.

  To compare characteristics of patients and controls, we applied unpaired t-tests nonparametric Wilcoxon tests and Pearson's χ2-test. The genotype frequencies for cases and controls of both genders were in Hardy-Weinberg equilibrium for both polymorphisms. Linkage disequilibrium between the two polymorphisms was assessed by the correlation coefficient r and Lewontin's D'. Haplotypes based on the two SNPs were estimated via the expectation-maximization algorithm by the SAS® procedure PROC HAPLOTYPE (version 9.1, release 2004). Differences between bilirubin levels between the genotype and haplotype groups stratified for case-control status or between controls and CAD cases stratified for genotype groups were tested using linear regression adjusting for age. An additive (i.e. estimating the change of mean bilirubin levels per minor allele) and a recessive (i.e. change of mean bilirubin levels of subjects with two copies of the minor allele versus the rest) genetic model was applied. Percentage of bilirubin variance explained by classical factors and by the two polymorphisms was estimated by the r² (Field 2005). Bilirubin concentrations were analyzed on a transformed scale (ln(bilirubin+1)) in order to obtain a normally distributed variable. Logistic regression was applied to estimate the association of bilirubin levels and the polymorphisms with the change of odds for CAD. Statistical analysis was performed with SPSS for Windows 15 and SAS (version 9.1, release 2004).

4 . 2

 42 capacity as vitamin E[START_REF] Stocker | Antioxidant activity of albumin-bound bilirubin[END_REF] and alleviates the atherogenic stress of lipid peroxidation by preventing oxidative modification of LDL[START_REF] Neuzil | Free and albumin-bound bilirubin are efficient coantioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation[END_REF]. Thus, serum bilirubin concentration itself and/or factors that modify its equilibrium are responsible for the protective properties. The present study in 477 CAD patients and 619 healthy controls confirmed the results of several previous epidemiological studies (Schwertner et al., 1994; Hopkins et al., 1996; Hunt et al., 2001; Novotny et al., 2003; Djousse et al., 2003; Endler et al., 2003; Schwertner et al., 2000; Breimer et al., 1995; Levinson 1997; Troughton et al., 2007; Hunt et al., 1996; Lin et al., 2006) that low serum bilirubin levels were associated with increased risk for coronary events. Genetic variation within the UGT1A1 gene and CAD Two polymorphisms in the promoter region of the UGT1A1 gene, the -53(TArepeat) and the T-3279G polymorphism, were shown to be strongly associated with serum bilirubin levels (Bosma et al., 1995; Maruo et al., 2004). The presence of the -53(TA) 7 allele and the -3279G allele are known to have a reduced enzyme activity of the gene product and cause increased serum bilirubin concentrations (Bosma et al., 1995; Maruo et al., 2004). Our data clearly indicated a significant association of both polymorphisms with bilirubin levels, but no association of either of the polymorphisms with CAD status. Meantime four other studies (Bosma et al., 2003; Gajdos et al., 2006; Lin et al., 2006; Rantner et al., 2008) investigated the association between the -53(TA-repeat) polymorphism and CHD. First, the prospective population-based Rotterdam Study, a nested case-control study including 185 CHD cases and 255 controls, found no protective effect of the -53(TA) 7/7 genotype on CHD, although carriers of the -53(TA) 7/7 genotype showed significantly increased bilirubin concentrations. However, this study also did not observe an association between low serum bilirubin and increased risk of CHD, in clear contrast to the majority of the literature. Second, from the ECTIM Study including 366 male cases with myocardial infarction and 314 male controls, Gajdos and coworkers (Gajdos et al., 2006) obtained further evidence that the UGT1A1 TA-7 allele is not associated with a decreased risk for myocardial infarction. Bilirubin concentrations were not available in that study. Third, a recent study in patients with peripheral arterial disease (PAD) revealed similar results as the study at hand, i.e. a clear association between low bilirubin concentrations and PAD but no association between the UGT1A1 polymorphism and PAD. These three studies and the present study are in contrast to the results of the prospective Framingham Offspring Study, a 24-year follow-up study with 1780 participants (Lin et al., 2006) which found for the first time a significantly decreased risk of CVD and CHD for subjects with the -53(TA) 7/7 genotype. Individuals with this genotype had about a third of the risk for CVD and CHD than those with genotype 6/6 or 6/7.

Table 4 :

 4 Logistic regression analysis adjusted for age and stratified for gender investigating the association of bilirubin concentrations and the -53(TA-repeat) and the T-3279G polymorphisms (genotypes) on the change of odds for coronary artery disease (CAD).

Table 2 :

 2 Age-adjusted UGT1A1 genotype-specific bilirubin levels of controls and patients with coronary artery disease stratified by gender The mean bilirubin levels are the retransformed mean bilirubin levels computed on the ln(bilirubin+1) scale. b p values from linear regression adjusted for age to compare bilirubin levels between the genotype groups within controls or CAD cases. The additive genetic model calculates the p values for the changes of mean bilirubin levels per copy of the minor allele. The recessive genetic model calculates the p values for the changes of mean bilirubin levels of subjects with two copies of the minor allele versus subjects with only one or no copy of the minor allele. c p values from linear regression adjusted for age to compare bilirubin levels between controls and CAD cases with the same genotype group.

	Control group

a

Table 3 :

 3 Percentage of variation of bilirubin concentrations explained by classical factors and the -53(TA-repeat) and the T-3279G polymorphism (assuming a recessive genetic model).Values given are the r² values (as percentage) from linear regression with various

	adjustments.
		r 2 (%)	r 2 (%)	r 2 (%) explained by
	full model a	submodel b	polymorphisms c
	-53(TA-repeat) polymorphism
	Cases	24.1	8.7	15.4
	Controls	23.6	10.5	13.1
	SNP T-3279G
	Cases	17.9	8.7	9.2
	Controls	17.0	10.5	6.5
	-53(TA-repeat) polymorphism and SNP T-3279G combined
	Cases	24.1	8.7	15.4
	Controls	24.1	10.5	13.6

The Wellcome Trust Case Control Consortium, 2007. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447,

Acknowledgements

This study was supported by grants from the "Austrian Nationalbank" (Project 9331), the Austrian Heart Fund, the "Genomics of Lipid-associated Disorders -GOLD" of the GEN-AU Programme to Florian Kronenberg, by the Munich Center of Health Sciences and the German National Genome Research Net to the Ludwig-Maximilians-University (Iris M. Heid), and by grants from NIH (HL21088 and AG18734) to Steve Hunt.