HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59:B227-B232 (2004)
© 2004 The Gerontological Society of America

Genome-Wide Scan for a Healthy Aging Phenotype Provides Support for a Locus Near D4S1564 Promoting Healthy Aging

Terry Reed1,, Danielle M. Dick1, Sean K. Uniacke2, Tatiana Foroud1 and William C. Nichols2

1 Department of Medical & Molecular Genetics, Indiana University School of Medicine, Indianapolis.
2 Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Ohio.

Address correspondence to Dr. Terry Reed, Department of Medical & Molecular Genetics, IB 130, 975 West Walnut St., Indianapolis, IN 46202-5251. E-mail: treed{at}iupui.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Living to a late age without suffering any major health problems is a genetically influenced trait. To identify the genes contributing to this important phenotype, a 10 cM genome screen was performed in 95 pairs of male fraternal twins concordant for healthy aging. Individuals meeting these criteria were defined as those attaining the age of 70 free of cardiovascular disease (coronary surgery, diabetes, heart attack, and stroke) and prostate cancer. Six chromosomal regions were identified with logarithm of odds (LOD) scores greater than 1.2 (p <.01). A region on chromosome 4 at marker D4S1564 produced a LOD score of 1.67; this was the same marker previously linked to extreme longevity segregating as an autosomal dominant trait in centenarian families. Our results provide independent evidence that a locus on the long arm of chromosome 4 is associated with better physical aging and/or longevity.

MOST genetic studies involved with aging have focused on identifying genes contributing to particular diseases. More recently, it has been recognized that it is also valuable to examine genetic factors related to disease-free or healthy aging (1,2). Utilizing twins from the National Academy of Sciences–National Research Council (NAS-NRC) twin panel, we have demonstrated that healthy physical aging is under a significant degree of genetic influence, with a heritability over 50% (3). Our definition of healthy aging focused principally on freedom from cardiovascular disease, and has received considerable support in the more recent literature. Brand and colleagues (4) reported that parental age at death was a significant predictor of coronary heart disease death in the Framingham offspring study and concluded that familial similarities for age at death may be mediated through shared coronary heart disease risk factors. Frederiksen and colleagues (5) reported that increased parental life was associated with a reduction in odds ratio for their children to have diabetes, ischemic heart disease, heart failure, stroke, and hypertension. We have found that better midlife lipid levels and blood pressures were associated with increased parental longevity in the National Heart, Lung, and Blood Institute twin study (6). Centenarian siblings and offspring, besides having increased longevity, have been shown to have better health and better cardiovascular risk factor profiles (7–10).

Siblings who are concordant for survival to a late age, free of specified chronic diseases, can be recruited and used to search for genes involved in healthy aging. By recruiting fraternal (dizygotic [DZ]) twin-pairs from the NAS-NRC twin panel, we have also matched for sex and perhaps less environmental variability than comparisons between other pairs of relatives. In this article, we report the results of a genome-wide screen and fine mapping studies designed to identify genes contributing to healthy aging in this sample of twins.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Participants
The NAS-NRC veteran twin registry is a nationwide registry of male twin births from 1917 and 1927 that was created by matching birth certificate information to Veteran Administration service records. The creation of the registry is described in more detail elsewhere (11,12). For the phenotype of healthy aging employed in this study, responses were used from a health history questionnaire (Q8) mailed in the fall of 1998. Questionnaires were received from 6109 of 8848 (69%) eligible respondents with a mean age of 74.3 years (range 69–82 years; only 1 respondent was aged 69 years). The definition of healthy aging from the Q8 responses was reaching the age of 70 and answering "No" to all of the following questions: [1] Has a doctor ever told you that you had a heart attack? [2] Have you ever had coronary bypass surgery or angioplasty? [3] Have you ever been told by a doctor that you had a stroke? [4] Has a doctor ever told you that you have diabetes? [5] Have you ever been diagnosed with prostate cancer? Prostate cancer was included in the healthy aging definition because it was felt to be a relatively common cancer that can have lengthy survival in men. Most other cancers, if the participant even completed Q8, would probably result in death or the twin being too ill to participate at the time of recruitment for the current linkage study. These traits were selected to define an individual who has successfully aged into his 70s free from most of the major physical health problems at Q8 (3). Kappa statistics on a subset of the twins with medical record review gave excellent agreement with the responses to the above health questions validating the Q8 responses (3).

All DZ twin-pairs concordant for the definition of healthy aging, and a small percentage of concordant pairs listed in the registry with unknown zygosity, were identified for recruitment into the linkage study. It has been estimated that zygosity is correctly determined for at least 95% of twin-pairs assigned a zygosity in the NAS-NRC panel (11). Approximately 80% of pairs assigned a zygosity were classified solely on their own assessment from a zygosity questionnaire mailed in 1965 (13). Beginning in February of 2001, 347 twin-pairs were mailed a letter inviting them to participate in the linkage study to search for genes related to healthy aging. Pairs agreeing to participate were sent a blood kit to take to their doctor or health facility for blood to be drawn. Instructions for collecting the sample and returning it via overnight express delivery were included with the packet. Eliminating pairs who were lost to contact or where one or both of the co-twins had died, approximately 40% of pairs that were reached agreed to participate. Of the 123 concordant pairs initially agreeing to participate, blood samples were received from 110 pairs (89% of those agreeing to participate). Some pairs of unknown zygosity (conflicting responses on the zygosity questionnaire) were determined to be monozygotic (MZ) twins during the analysis of genetic markers for the linkage study, and the expected approximate 5% of DZ pairs also were found on genome-wide scanning (see below) to be identical. The linkage results presented in this manuscript are for 95 complete DZ pairs concordant for the healthy phenotype. The average age for these pairs was 73.9 years at the time they completed Q8 and 76.3 years when the blood sample was obtained. Participants signed inform consent forms approved by the Indiana University, Purdue University at Indianapolis Institutional Review Board.

Genotyping
A genome screen was completed using 400 dinucleotide markers from the ABI Prism Linkage Mapping Set (Applied Biosystems, Foster City, CA) with an average heterozygosity of 79% and an average intermarker spacing of 8.6 centimorgans (cM) or map units. Briefly, 30 ng of genomic DNA was polymerase chain reaction (PCR)-amplified using each individual marker in a 10 µl reaction. After PCR, the PCR products were pooled using equal amounts of each PCR reaction. One µl of this multiplexed mix was added to 10 µl formamide containing the GeneScan-400HD ROX-size standard (Applied Biosystems). Genotypes were determined using the ABI 3700 DNA Analyzer (Applied Biosystems) and GeneScan 3.5, Genotyper 3.6, and GeneMapper 1.1 software (14).

Marker genotypic data were used to verify the twin relationships using the computer program PREST (15). Pairs originally identified as dizygotic were eliminated from the linkage analyses if there was significantly higher sharing of alleles identical by descent (IBD) than would be expected for full siblings, suggesting that they were MZ twins. In one instance, a pair shared a number of alleles IBD that was intermediate between that expected for DZ and MZ twins. Repeated genotyping of the same sample led to similar results. A second sample was obtained and the results verified the pair was MZ. Another DZ pair shared no alleles IBD; a second blood sample confirmed the pair was in fact unrelated.

Linkage Methods
Multipoint nonparametric linkage analysis was performed for our definition of healthy aging using the maximum likelihood method implemented in the computer program Mapmaker/SIBS (16). This program analyzes the extent of allele sharing among sibling pairs. Since each sibling receives 1 allele from each parent, siblings can share 0, 1, or 2 alleles IBD at any given autosomal locus. Under the null hypothesis of no linkage, 25% of siblings should share 0 alleles IBD, 50% of siblings should share 1 allele IBD, and 25% of siblings should share 2 alleles IBD. Affected sibling pair analyses analyze the extent to which there is deviation from the expected proportion of.25:.50:.25 alleles shared IBD. Since the siblings are selected to be concordant for the trait of interest, if a marker is near a gene influencing that trait, then a greater percentage of sibling pairs should share 2 alleles IBD at that marker, with a corresponding decrease in the percentage of pairs who share 0 alleles IBD.

Analyses were performed with dominance variance free to vary, allowing for the possibility that some alleles may not simply act in an additive fashion. This analysis provides for a more sensitive test for putative genes acting in a recessive fashion than does the analysis with dominance variance fixed at zero (17), ensuring maximum power to detect loci contributing to aging. In some behavior genetic studies of age-related phenotypes, the best-fitting model included nonadditive genetic variance (18,19). This is consistent with previous family studies that showed lower correlations in parent–child longevity than between siblings (19,20). Dominant genetic effects would only be reflected in comparisons among siblings but not in parent–child correlations.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Table 1 presents all chromosomal regions in the genome scan yielding logarithm of odds (lod) scores >=1.2; this corresponds to a pointwise p value of.01. While these regions do not reach statistical significance at the genome-wide level (16), this study had a relatively small sample size and was designed to identify chromosomal regions of interest. Lod scores are a measure of significance rather than effect size; therefore, they are necessarily influenced by sample size. Thus, we also report the IBD sharing to provide an indication of the extent of genetic effects at each marker.


View this table:
[in this window]
[in a new window]
  		Table 1. Chromosomal Regions Yielding Lod Scores > 1.2 (p <.01).

 
Chromosome 1 yielded a maximum lod score of 1.31 at 3 cM near the marker D1S468. Chromosome 3 yielded a peak lod score of 1.71 at the marker D3S1297. A second peak also emerged on chromosome 3; a lod score of 1.38 was obtained at 119 cM near the marker D3S1278. Chromosome 4 had a lod score of 1.37 at 96 cM at the marker D4S414. Chromosome 6 yielded a peak lod score of 1.41 at 5 cM at the marker D6S309. Chromosome 16 yielded broad evidence of linkage across the chromosome, with a peak lod score of 1.70 at 49 cM near the marker D16S3136. Finally, the X chromosome yielded a lod score of 1.60 at 160 cM at the marker DXS8043.

We followed-up regions containing multiple adjacent markers exceeding a lod score of 1.0 by genotyping additional markers in the region of linkage. The lod score on chromosome 4 remained constant with a denser marker map (Figure 1). However, the maximum lod score moved distally to the marker D4S1564. The lod score at D4S1564 using single-point linkage analysis was 1.67 (p =.003). At the marker D4S1564, the percentage of sibs sharing 0, 1, and 2 alleles IBD was 0.16, 0.43, 0.41, as compared to the expected proportions of 0.25, 0.50, 0.25, respectively. Thus, there was a significant increase in allele sharing at this marker among siblings concordant for the phenotype of healthy aging. The evidence for linkage was somewhat decreased on chromosome 16 with the addition of more markers. Additionally, the region of linkage remained quite broad; genotyping of additional markers did not narrow the potential region of interest (Figure 2). Denser marker genotyping in the region of linkage on the X chromosome increased the evidence for linkage (Figure 3). A maximum lod score of 1.98 (p =.001) was obtained at 156 cM near the marker DXS8106 with IDB 0 (0.32) and IDB 1 (0.68).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 1. Lod (logarithm of odds) score graph for chromosome 4 after additional markers were added to the region of linkage. The highest lod score was at the marker D4S1564

 


View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. Lod (logarithm of odds) score graph for chromosome 16 after additional markers were added to the region of linkage

 


View larger version (18K):
[in this window]
[in a new window]
  		Figure 3. Lod (logarithm of odds) score graph for the X chromosome after additional markers were added to the region of linkage. The highest lod score was at the marker DXS8106

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
This investigation was designed to identify chromosomal regions of interest that may contain genes related to healthy physical aging. Six chromosomal regions were identified that exceeded our initial linkage threshold (p <.01). One of these was in a region of particular interest with regard to a related aging phenotype, longevity. It has been demonstrated that longevity has moderate (0.20–0.30) heritability (18,19,21), and the families of centenarians have been extensively studied to examine the genetics of longevity. In families with extreme longevity segregating as an autosomal dominant trait, Puca and colleagues (22) found significant linkage with the marker D4S1564, suggesting that a gene or genes in this region exert an influence on the ability to achieve exceptional age. The locus in this region is referred to as longevity 1 (lgv1) (http://www3.ncbi.nlm.nih.gov/omim/). In our genome scan of healthy aging, we also find linkage to this region of chromosome 4. Genotyping additional markers increased the evidence of linkage at the marker D4S1564 at chromosome band 4q25, with a multipoint lod score of 1.28. Single-point analyses at D4S1564 yielded a maximum lod score of 1.67. The higher lod score yielded in the single-point analysis, as compared to the multipoint analysis, would suggest that the markers surrounding D4S1564 are giving less evidence of linkage, dampening the signal at D4S1564. Recently, Geesaman and colleagues (23) reported an association of a single nucleotide polymorphism (SNP) near D4S1564 with longevity. The SNP was located in a microsomal transfer protein thought to be a rate-limiting step in the production of lipoproteins. Increasingly, cardiovascular disease profiles are reported to be better in centenarians, their siblings, and their offspring (7–10). Coronary heart disease risk factor levels in adults are also associated with parental age at death (4–6). Although our healthy aging phenotype, primarily based on the absence of cardiovascular disease to at least age 70, and longevity per se are considerably different phenotypes, there possibly may be at least a few shared common determinants.

In genotyping additional markers on the X chromosome, the linkage result increased to a lod score of 1.98 at the marker DXS8106 at Xq27.3. There is evidence in the literature for a stronger association of offspring longevity with longevity of mothers than longevity of fathers. This was first mentioned by Pearl (24), whose data were later updated by Abbott and colleagues (25,26), who found that the strongest relationship was between mother and son and the weakest between father and daughter. Brand and colleagues (4) found that maternal age at death was more strongly associated with age at death in Framingham offspring than paternal age at death, and that age at death may be mediated primarily through coronary risk factors such as systolic blood pressure. We also reported (6) that the risk of early death was more strongly related to the longevity of the mother in adult twins. This association has more recently been suggested to be due to mitochondrial genes (27,28); however, these were not genotyped in our sample. Because males are hemizygous for X-linked genes, a recessive gene on the X chromosome promoting healthy aging would be more likely to be detected in male than female siblings, making our sample ideal to detect such an association.

Linkage analysis is an excellent means to identify chromosomal regions likely to harbor a gene contributing to a particular phenotype; however, this statistical approach does not yield great precision in the location of the susceptibility gene. In fact, simulations have shown that the maximum lod score underlying a true susceptibility gene may be 10 or more million base-pairs from the position of the contributing gene (29). As a result, there are a number of genes near our markers that might be related to healthy aging. Previous studies have suggested that genes related to longevity might fall into several broad categories. These include genes related to cellular maintenance and repair (30,31), genes with homologs that influence longevity in lower organisms (31), and genes identified as related to aging in human cell culture studies. The latter includes genes identified from complementation studies of immortalized human cell lines (32). Senescence is a dominant phenotype. In cell complementation experiments, one of the four complementation group genes was localized on chromosome 4q33-34.1 (33). Interestingly, this gene, MORF4 (mortality factor chromosome 4), is relatively close (about 1 map unit) from the minor peak at D4S1539 (Figure 1).

Summary
Given the relatively small size of our sample, we cannot state definitively that there are genes related to healthy aging at each of the chromosomal regions in which we found a lod score exceeding our threshold of 1. These results should be interpreted cautiously and used as a guide for further exploration in future studies related to aging processes. Our results do provide further suggestion that there is a locus in the region of lgv1 (D4S1564) that is related to longevity and/or healthy aging. We plan to analyze the SNP reported to be associated with longevity in this narrow region of chromosome 4 and to study other marker haplotypes to further determine the extent of the genetic difference in individuals meeting our healthy physical aging phenotype.


   Acknowledgments
 
Supported by grant R01AG18736 (T.R.) and NRSA F32AA113358 (D.M.D.). Thanks to Shannon A. Rinehart for her technical help.


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received August 7, 2003

Accepted December 9, 2003


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Hadley EC, Rossi WK, Albert S. Genetic epidemiologic studies on age-specified traits. Am J Epidemiol.. 2000;152:1003-1008.[Abstract/Free Full Text]
  2. Collins FS, Green ED, Guttmacher AE, Guyer, MS. A vision for the future of genomics research. Nature.. 2003;422:835-847.[Medline]
  3. Reed T, Dick DM. Heritability and validity of healthy physical aging (wellness) in elderly male twins. Twin Res.. 2003;6:227-234.[Medline]
  4. Brand FN, Kiely DK, Kannel WB, Myers RH. Family patterns of coronary heart disease mortality: the Framingham Longevity Study. J Clin Epidemiol.. 1992;45:169-174.[Medline]
  5. Frederiksen H, McGue M, Jeune B, et al. Do children of long-lived parents age more successfully? Epidemiol.. 2002;13:334-339.[Medline]
  6. Reed T, Carmelli D, Robinson TS, Rinehart SA, Williams CJ. More favorable risk factor levels in male twins and mortality after 25 years of follow-up is related to longevity of their parents. J Gerontol Med Sci.. 2003;58A:M367-M371.
  7. Barzilai N, Gabriely H, Gabriely M, Iankowitz N, Sorkin JD. Offspring of centenarians have a favorable lipid profile. J Am Geriatr Soc.. 2001;49:76-79.[Medline]
  8. Perls TT, Wilmouth J, Levenson R, et al. Life-long sustained mortality advantage of siblings of centenarians. Proc Natl Acad Sci U S A.. 2002;99:8442-8447.[Abstract/Free Full Text]
  9. Terry DF, Wilcox M, McCormick MA, Lawler E, Perls TT. Cardiovascular advantages among the offspring of centenarians. J Gerontol Biol Sci Med Sci.. 2003;58A:425-431.
  10. Evert JLE, Perls T. Morbidity profiles of centenarians: survivors, delayers and escapers. J Gerontol Biol Sci Med Sci.. 2003;58A:232-237.
  11. Jablon S, Neel JV, Gershowitz H, Atkinson, GF. The NAS-NRC twin panel: methods of construction of the panel, zygosity diagnosis, and proposed use. Am J Hum Genet.. 1967;19:133-161.[Medline]
  12. Hrubec Z, Neal JV. The national academy of sciences-national research council twin registry: ten years of operation. Prog Clin Biol Res.. 1978;24B:153-172.
  13. Hrubec Z, Omenn GS. Evidence of genetic predisposition to alcoholic cirrhosis and psychosis: Twin concordances for alcoholism and its biological end points by zygosity among male veterans. Alcohol Clin Exper Res.. 1981;5:207-215.[Medline]
  14. Pankrantz N, Nichols WC, Uniacke S, et al. Genome screen to identify susceptibility genes for Parkinson disease in a sample without parkin mutations. Am J Hum Gen.. 2002;71:124-135.[Medline]
  15. McPeek MS, Sun L. Statistical tests for detection of misspecified relationships by use of genome-screen data. Am J Hum Gen.. 2000;66:1076-1094.[Medline]
  16. Kruglyak L, Lander ES. Complete multipoint sib-pair analysis of qualitative and quantitative traits. Am J Hum Genet.. 1995;57:439-454.[Medline]
  17. Holmans P. Asymptotic properties of affected-sib-pair linkage analysis. Am J Hum Genet.. 1993;52:362-374.[Medline]
  18. McGue M, Vaupel JW, Holm N, Harvald B. Longevity is moderately heritable in a sample of Danish twins born 1870-1880. J Gerontol.. 1993;48:237-244.
  19. Herskind AM, McGue M, Holm NV, Sorensen TI, Harvald B, Vaupel JW. The heritability of human longevity: a population-based study of 2872 Danish twin pairs born1870–1900. Hum Genet.. 1996;97:319-323.[Medline]
  20. Christensen K, Vaupel JW. Determinants of longevity: genetic, environmental and medical factors. J Intern Med.. 1996;240:333-341.[Medline]
  21. Ljungquist B, Berg S, Lanke J, McClearn GE, Pedersen NL. The effect of genetic factors for longevity: a comparison of identical and fraternal twins in the Swedish twin registry. J Gerontol Biol Sci.. 1998;53A:B441-B446.
  22. Puca AA, Daly MJ, Brewster SJ, et al. A genome-wide scan for linkage to human exceptional longevity identifies a locus on chromosome 4. Proc Natl Acad Sci U S A.. 2001;98:10505-10508.[Abstract/Free Full Text]
  23. Geesaman BJ, Benson E, Brewster SJ, et al. Haplotype-based identification of a microsomal transfer protein marker associated with the human lifespan. Proc Natl Acad Sci U S A.. 2003;100:14115-14120.[Abstract/Free Full Text]
  24. Pearl R. Studies on human longevity. IV. The inheritance of longevity, preliminary report. Hum Biol.. 1931;3:245-269.
  25. Abbott MH, Murphy EA, Bolling D, Abbey H. The familial component in longevity, a study of offspring of nonagenarians. II. Preliminary analysis of the completed study. Johns Hopkins Med J.. 1974;134:1-16.[Medline]
  26. Abbott MH, Abbey H, Bolling DR, Murphy, EA. The familial component in longevity—a study of offspring of nonagenarians: III. Intra-familial studies. Am J Med Genet.. 1978;2:109-120.
  27. Tanaka M, Gong J-S, Zhang J, Yoneda M, Yagi K. Mitochondrial genotype associated with longevity. Lancet.. 1998;351:185-186.[Medline]
  28. Niemi A-K, Hervonen A, Hurme M, Karhunen PJ, Jylha M, Majammaa K. Mitochondrial DNA polymorphisms associated with longevity in a Finnish population. Hum Genet.. 2003;112:29-33.[Medline]
  29. Roberts SB, MacLean CJ, Neale MC, Eaves LJ, Kendler KS. Replication of linkage studies of complex traits: an examination of variation in location estimates. Am J Hum Genet.. 1999;65:876-884.[Medline]
  30. Vijg J, Papaconstantinou J. Aging and longevity genes: strategies for identifying DNA sequences controlling life span. J Gerontol.. 1990;45:B179-B182.
  31. Schachter F, Cohen D, Kirkwood T. Prospects for the genetics of human longevity. Hum Genet.. 1993;91:519-526.[Medline]
  32. Pereira-Smith OM, Smith JR. Genetic analysis of indefinite division in human cells: identification of four complementation groups. Proc Natl Acad Sci U S A.. 1988;85:6042-6046.[Abstract/Free Full Text]
  33. Bertram MJ, Berube NG, Hang-Swanson X, et al. Identification of a gene that reverses immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes. Mol Cell Biol.. 1999;19:1479-1485.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Journals of Gerontology Series A: Biological Sciences and Medical SciencesHome page
D. Karasik, S. Demissie, L. A. Cupples, and D. P. Kiel
Disentangling the Genetic Determinants of Human Aging: Biological Age as an Alternative to the Use of Survival Measures
J. Gerontol. A Biol. Sci. Med. Sci., May 1, 2005; 60(5): 574 - 587.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS