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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:355-362 (2006)
© 2006 The Gerontological Society of America

Chromosome 4q25, Microsomal Transfer Protein Gene, and Human Longevity: Novel Data and a Meta-Analysis of Association Studies

Marian Beekman, Gerard Jan Blauw, Jeanine J. Houwing-Duistermaat, Bernd W. Brandt, Rudi G. J. Westendorp and P. Eline Slagboom

1 Molecular Epidemiology, 2 Gerontology and Geriatrics, Department of General Internal Medicine, and 3 Medical Statistics, Leiden University Medical Center, The Netherlands.

Address correspondence to Marian Beekman, PhD, Leiden University Medical Center, Section of Molecular Epidemiology, P.O. Box 9503, 2300 RA Leiden, The Netherlands. E-mail: M.Beekman{at}LUMC.nl


   Abstract
Top
 Abstract
Participants and Methods
 Results
 Discussion
 References
 
Recently, chromosome 4q25 was linked to exceptional human longevity, and a haplotype of the positional candidate microsomal transfer protein (MTP) gene was associated to the phenotype in U.S. Caucasians. We investigated whether linkage to 4q25 could be detected in 164 nonagenarian sibships of the Leiden Longevity Study. Additionally, we compared the MTP –493G/T and Q95H allele and haplotype frequencies in the Leiden Longevity Study (379 nonagenarians, 525 of their offspring, and 251 partners of their offspring) and in the Leiden 85-Plus Study (655 octogenarians and 244 young controls). The latter study population was followed for at least 7 years, providing the opportunity to perform also prospective analyses using the longitudinal data. We found neither evidence for linkage at 4q25 nor association of the MTP locus with longevity in nonagenarian individuals. Meta-analyses of all previous studies implied that the association in U.S. Caucasians may have its source in admixture of the U.S. control population rather than in the genetic effect of the locus on exceptional longevity.

NONAGENARIANS and centenarians, who represent the extreme tail of the distribution of human life span, have a history of aging slowly, and either markedly delay or escape age-associated diseases (1,2). Influencing the human life span and reaching old age in good health remains a wish for many people. Human variation in life span is influenced by three main factors: (i) unique environmental factors (e.g., an individual's diet or hazardous line of work); (ii) familial environmental factors (e.g., familial socioeconomic status); and (iii) genetic factors (e.g., genetic susceptibility for cancer). Environmental factors are hard to study in humans. Current genetic research of longevity aims to identify the genes involved in the ability to become long-lived from the perspective that such may indicate the major relevant pathways of human life-span regulation. Evidence for the inheritance of longevity was provided by the finding that siblings of centenarians have a much higher chance of reaching the age of 100 years than do siblings of controls from the same birth cohort (3). Furthermore, the study of the comprehensive population-based genealogy database of the Icelandic population shows that a clear familial component is present in longevity (4). This hereditary component could represent shared environmental factors as well as shared genetic factors. From twin studies, it was estimated that approximately 25% of the variation in life span is contributable to genetic variation (5,6). However, the genetic basis of the inherited component of longevity still remains to be elucidated.

The search for genes having a major influence on longevity could be started by performing a genome scan in long-lived sibling pairs. In this approach, no assumptions are made with respect to the inheritance pattern of the trait and no control group for the nonagenarian participants is required. Puca and colleagues (7) measured 400 microsatellite markers with 10-cM spacing in 308 individuals belonging to 137 sibships, of which the oldest sibling was at least 98 years old and other siblings were older than either 91 years (if male) or 95 years (if female). Chromosome 4q25 was found to be linked with the exceptional longevity phenotype, and after fine mapping of this region, a maximum Logarithm of odds (LOD) score of 3.65 was obtained at a 12-cM region containing approximately 50 genes. To investigate which of the 50 genes in the linkage region might be responsible for the remarkable linkage result, the whole region was saturated with single-nucleotide polymorphisms (SNPs) (8) to test for association with longevity. A two-SNP haplotype in the microsomal transfer protein (MTP) gene was found to be associated with longevity, marked by the SNPs rs2866164 and Q95H. Variation in this gene has previously been associated with plasma levels of low-density lipoprotein (LDL) cholesterol (9–12), lipoprotein particle size (13), obesity (14), and insulin levels (14). However, contradictory results have also been published (15–17). It is possible that the MTP gene is involved in cardiovascular risk by influencing lipid metabolism, and thus may contribute to longevity.

Because the 4q25 locus was also suggestively linked to healthy aging in a small study of 70-year-old U.S. male veterans (18), it can be questioned whether this locus is of importance only for attaining the most exceptional longevity. We therefore performed linkage analysis in the chromosome 4q25 linkage region in 379 nonagenarian participants belonging to 164 sibships from the Leiden Longevity Study (19). Recently, we have reported excessive survival in three generations of relatives of these nonagenarian sibling pairs (19), demonstrating that our selection criteria result in individuals carrying familial longevity effects. Additionally, we performed association analysis of the two-SNP haplotype of the MTP gene in the nonagenarian sib pairs, the offspring of these sib pairs (assuming that they are predisposed to be long-lived), and the partners of the offspring (assuming that they represent the general population). Furthermore, we investigated the first and second cohorts of the prospective population-based Leiden 85-Plus Study (20,21) for association with the two-SNP haplotype of the MTP gene. Association of the two MTP SNPs were tested cross-sectionally, by comparing allele frequencies in the octogenarians with those in a young control group from the same Leiden area, as well as prospectively, by testing (using the follow-up data) the effects of the MTP genotypes on survival after the age of 85 (22). Finally, we performed a meta-analysis of all known data on the MTP T-Q haplotype and longevity to estimate the odds ratio of this putative risk haplotype on survival in Caucasian populations.


   PARTICIPANTS AND METHODS
Top
 Abstract
 Participants and Methods
Results
 Discussion
 References
 
Participants: Leiden Longevity Study
The individuals investigated in the present study participate in the ongoing Leiden Longevity Study (19). Families participating in the Leiden Longevity Study have at least two siblings meeting four inclusion criteria: (i) men are at least 89 years old and women are at least 91 years old, (ii) participants have at least one living brother or sister who fulfils the first criterion and is willing to participate, (iii) the nonagenarian sibship has an identical mother and father, and (iv) the parents of the nonagenarian sibship are Dutch and Caucasian.

The offspring of nonagenarian sib pairs, representing individuals susceptible to longevity, are also enrolled. In addition, their partners were included as the age-matched and environment-matched control group for the offspring cases and as the young control group for the nonagenarian sib pairs. It was previously found that the parents, the siblings, as well as the offspring of the nonagenarian sib pairs show lower mortality ratios as compared to the general population (19). In this Leiden Longevity Study we thus include families enriched for longevity.

In accordance with the Declaration of Helsinki, written informed consent was obtained from all participants prior to entering the study. Good clinical practice guidelines were maintained. The study protocol was approved by the medical ethical committee of the Leiden University Medical Center before the start of the study.

For the present study, data on families enrolled between the start of the study (September, 2002) and January 2004 were collected. We had available 379 nonagenarian participants belonging to 164 sibships (mean age = 93.3 ± 3.1 standard deviation [SD] years), 525 offspring of the nonagenarian sib pairs belonging to 238 sibships (mean age = 60.6 ± 6.8 SD years), and 251 of their partners (mean age = 59.6 ± 7.3 SD years).

Participants: Leiden 85-Plus Study
As a confirmation study for the cross-sectional association analysis, we investigated the population-based Leiden 85-Plus Study that consists of two cohorts of inhabitants of Leiden, The Netherlands (20–22). In total, 655 participants (mean age = 87.5 ± 3.1 SD years) born in the city of Leiden were compared to 244 control participants (mean age = 31.0 ± 5.6 SD years), with parents and grandparents from the same geographic region.

For the prospective association analyses, we investigated the follow-up data available for all participants of the Leiden 85-Plus Study. For cohort '87, participants aged 85 years or older were enrolled between 1987 and 1989 and were followed for mortality until May 2001. For cohort '97, participants who were exactly 85 years old were enrolled between 1997 and 1999 and were followed for mortality until April 2004. In cohort '87, almost 14 years after the start of the study, 704 participants (98%) had died. In cohort '97, almost 7 years after the start of the study, 308 participants (51%) had died. For the participants in both cohorts, the cause of death was registered and subdivided into death by cardiovascular disease, by cancer, by infectious disease, or by other causes. From the combined cohorts, a total of 1245 participants who originated from the Leiden area were genotyped for the prospective association analysis.

The Leiden 85-Plus Study was approved by the medical ethical committee of the Leiden University Medical Centre. Written informed consent was obtained from all participants after the nature and possible consequences of the studies were explained.

Methods
DNA isolation.-- From all participants in the Leiden Longevity Study, blood was drawn in a 10-ml coagulation Vacutainer (BD Alphen a/d Rijn), 8-ml and 4.5-ml EDTA Vacutainers, an 8-ml citrate Vacutainer, and a 2.5-ml PAXgene tube (PreAnalytiX, Hombrechtikon). Serum, EDTA, and citrate plasma were stored at –80°C, and EDTA and citrate buffy coats at –20°C. DNA was isolated from the EDTA buffy coats. From generation 2, the whole buffy coat was isolated using a QIAamp DNA Blood Maxi Kit (Qiagen, Venlo) according to the manufacturer's protocol. DNA isolation from generation 3 was outsourced to BaseClear (Leiden, The Netherlands), where 200 µl of the buffy coat was isolated using the Chemagic DNA Blood 100 Kit (Chemagen, Baesweiler) and carried out according to the manufacturer's protocol on a Magnetic Separation Module I (Chemagen), equipped with a 96-rod head. All DNA concentrations were determined using OD260 measurement. In the Leiden 85-Plus Study, DNA was available for 1245 participants of the combined cohorts.

Genotyping.-- The 379 nonagenarian participants belonging to 164 sibships were genotyped for the same six microsatellite markers at which linkage was initially detected by Puca and colleagues (7): D4S2964, D4S1534, D4S414, D4S1572, D4S406, and D4S402 with an average intermarker spacing of 7.22 cM. They observed the highest LOD score between D4S1572 and D4S406. These markers were taken from the Human Linkage Set v2.5 MD10 and HD5, amplified using AB9700 polymerase chain reaction (PCR) machines (Applied Biosystems, Foster City), and measured using an ABI Prism DNA Analyzer 3700 (Applied Biosystems). Genotyping was performed according to the manufacturer protocols, except that the amount of primer pairs for the markers was reduced up to 5-fold and duplex PCRs were designed if possible to reduce costs, time, expense, and amount of genomic DNA used. Genemapper 3.0 (Applied Biosystems) was used for allele calling using the LIZ500(–250) size standard (Applied Biosystems). For the purpose of quality control, {approx}5% of samples were genotyped twice and the results compared. The comparisons indicated that these markers could be genotyped reliably. Five Mendelian errors (0.2% of all genotypings) were detected using PedStats and no unlikely double recombinants using error detection in Merlin (both programs freely available at http://www.sph.umich.edu/csg/abecasis/software.html). The errors were removed using the PEDWIPE (23) command in Merlin. The location of the markers was taken from the Marshfield integrated genetic map (http://research.marshfieldclinic.org/genetics/) using Kosambi cM.

The 379 nonagenarian participants, 525 of their offspring, and 251 of their partners from the Leiden Longevity Study, as well as the 1245 octogenarian participants from the Leiden 85-Plus Study and the 244 young Leiden controls, were genotyped for two SNPs in the MTP gene of which the two-SNP-haplotype associated with longevity (8) consisted. This haplotype was formed by the SNPs rs2866164/rs1800591 and Q95H, where the genotypes of the MTP promoter SNPs rs2866164 and rs1800591 are perfectly correlated in the U.S. cases and controls as well as in the French cases and controls. Hence, these two SNPs provide exactly the same genetic information. Although Geesaman and colleagues (8) reported on the rs2866164, we genotyped rs1800591 (alias –493G/T in the MTP gene) beside the SNP Q95H, the semiconservative mutation in exon 3 of the MTP gene. The minor allele G of rs28166164 described in the article by Geesaman and colleagues (8) corresponds with the minor allele T of –493G/T described in this article.

Genotyping of the –493G/T SNP was performed using Taqman Assay by Design (Applied Biosystems). Assay by Design was used as recommended, except for the following modifications. A qPCR core kit was used (Eurogentec, Maastricht) with one third of the recommended amount of assay mix. PCRs were performed using an AB9700 (Applied Biosystems), and post-PCR fluorescence measurements were carried out on an ABI7900 (Applied Biosystems). For genotyping, cluster plots were made of the fluorescent labels using SDS software (v2.2; Applied Biosystems). Spots falling outside a cluster were set to missing genotypes. As quality control, 10% of samples were genotyped in duplicate; no inconsistencies were observed.

Genotyping of the Q95H SNP was performed using the MassARRAY platform according to protocols of the manufacturer (Sequenom, San Diego). Briefly, after PCR, a primer extension reaction was performed to introduce mass differences between alleles and, after removing salts by adding a resin, {approx}15 nl of the product was spotted onto a target chip with 384 patches containing matrix. Mass differences were detected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), and genotypes were assigned real-time using Typer 3.0 software (Sequenom). As quality control, cluster plots were made of the low- and high-mass allele signals as detected using mass spectrometry. Calls falling outside a cluster and no-calls falling inside a cluster were checked manually. In addition, 10% of samples were genotyped in duplicate and no inconsistencies were observed.

Statistical Analysis
Linkage.-- Nonparametric linkage analysis ALL Pairs was performed as implemented in MERLIN-0.10.2 (23) on 379 nonagenarians belonging to 164 sibships.

Cross-sectional association analysis.-- For association analysis with the two MTP gene SNPs, z-tests (adjusted for the covariance between siblings in the case of the Leiden Longevity Study) were performed to compare allele frequencies in different age categories. In the Leiden Longevity Study, the 379 nonagenarian sib pairs were compared to the 251 unrelated partners of the offspring as a younger control group. The 525 offspring of the nonagenarians harboring potentially future long-lived cases were compared to their 251 partners as an age-matched and environment-matched control group. In the Leiden 85-Plus Study, the 655 octogenarians born in the city of Leiden were compared to 244 young Leiden controls with parents and grandparents from the same geographic region.

Haplotype frequencies were estimated using THESIAS (freely available at http://ecgene.net/genecanvas/modules/mydownloads/), which is designed for haplotype-based association analysis in unrelated individuals (24,25). These frequencies were also compared between the groups of the Leiden Longevity Study and the Leiden 85-Plus Study as described above, except that in the Leiden Longevity study only one individual of each sibship was analyzed to meet the requirement of analyzing unrelated individuals.

Prospective association analysis.-- For prospective association analyses in the Leiden 85-Plus Study, Kaplan–Meier estimates of the survival curves for the various genotype groups were calculated. A sex-adjusted hazard ratio was estimated using a Cox proportional hazards model in all 1245 octogenarians from the Leiden area. For these analyses, we took delayed entry into account using STATA/SE 8 (Cosinus Computing, Waalwijk).

Meta analysis.-- The meta-analysis was carried out on the MTP T-Q haplotype estimating random and fixed effect using the "meta" library in the free software R version 2.1.1 (26,27) in a total of 4915 long-lived cases older than 85 years and 3002 Caucasian controls from the United States first tier (190 cases and 190 controls) and second tier (190 cases and 190 controls), United States proactively matched (488 cases and 462 controls) (8), French [564 cases and 564 controls; (8)], German [1039 cases and 550 controls; (28)], Danish [1625 cases and 551 controls; (29)], the Leiden Longevity Study [164 cases and 251 controls; this article), and the Leiden 85-Plus Study (655 cases and 244 controls; this article). For fixed effects, the inverse variance weighting was used for pooling and the DerSimonian–Laird estimator was used in the random-effects model.


   RESULTS
Top
 Abstract
 Participants and Methods
 Results
Discussion
 References
 
Linkage Analysis at Chromosome 4q25
In total, 379 nonagenarian participants belonging to 164 sibships of the Leiden Longevity Study were genotyped for the same set of markers at 4q25 with which Puca and colleagues (7) initially detected linkage with the longevity phenotype. We found no evidence for linkage at this chromosome 4 region in the Leiden Longevity Study (Figure 1). Markedly, in the 164 nonagenarian sibships the LOD score is for the whole region even little less than zero (Table 1), suggesting support against linkage. Also, when repeating the linkage analyses on the more stringent phenotype as investigated by Puca and colleagues, in which one sibling is at least 98 years old (21 sibships), we observed no significant effect of this 4q25 locus on longevity (Table 1).


Figure 01
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  		Figure 1. Nonparametric linkage analysis of chromosome 4q25 in 164 nonagenarian sibships of the Leiden Longevity Study (—) and the 137 nonagenarian sibships described by Puca and colleagues (7) (- - -). *Indicates markers from the initial marker set used by Puca and colleagues with an average spacing of 7.22 cM, resulting in a logarithm of odds (LOD) score of 2.67, and markers genotyped in the Leiden Longevity Study

 

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  		Table 1. Nonparametric LOD Scores Obtained in 164 Nonagenarian Sibships of the Leiden Longevity Study in the Chromosome 4 Linkage Region of Puca and Colleagues (7) and in 21 Sibships in Which at Least One Sibling Has Reached the Age of 98 Years.

 
Cross-Sectional Association Analyses
To test for association of the MTP gene with longevity, the two SNPs forming the previously associated haplotype, i.e., –493G/T and Q95H, were genotyped in the nonagenarian sib pairs of the Leiden Longevity Study, their offspring, and the partners of the offspring (Table 2). In addition, the octogenarians of the Leiden 85-Plus Study born in the city of Leiden were compared to the young Leiden control group (Table 2). The genotype distributions of all groups were in Hardy–Weinberg equilibrium.


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  		Table 2. Demographic Statistics of the Leiden Longevity Study and the Leiden 85-Plus Study.

 
Table 3 shows the frequencies of the –493 T and 95 Q alleles (the alleles of the previously associated haplotype) of the MTP gene polymorphisms in all study groups. The allele frequencies did not differ between the case and control groups in the Leiden Longevity Study or in the Leiden 85-Plus Study (p >.32). Also, no association of these polymorphisms was found in sex-specific analyses (data not shown).


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  		Table 3. Frequencies (%) of the –493 T and 95 Q Microsomal Transfer Protein (MTP) Alleles and Their Haplotype in the Leiden Longevity Study and the Leiden 85-Plus Study.

 
To investigate whether an MTP haplotype is associated with longevity, the MTP haplotype frequencies are estimated in the age groups of the Leiden Longevity Study and the Leiden 85-Plus Study (Table 3). The two MTP polymorphisms are in complete linkage disequilibrium (D' = 1.0) and the haplotype –493G/95H is extremely rare. Because the THESIAS program was designed for haplotype-based association analysis in unrelated individuals, we investigated just one sibling per sibship to perform association analysis of the MTP haplotype in the Leiden Longevity Study. We found no association of an MTP haplotype with longevity in either of our Leiden Studies (p >.46) (Table 3).

Prospective Association Analyses
Using the follow-up data of the two combined cohorts of the Leiden 85-Plus Study (N = 1245) from the Leiden area, the effect of the genotypes of the two MTP SNPs on survival after the age of 85 years was tested. The proportional hazard ratios of the heterozygous carriers of the minor alleles of the –493G/T and Q95H SNPs compared to the homozygous wild-type carriers were 0.98 (confidence interval [CI], 0.85–1.13) and 1.13 (CI, 0.89–1.42), respectively. Furthermore, the proportional hazard ratios of the homozygous carriers of the minor alleles of the –493G/T and Q95H SNPs compared to the homozygous wild-type carriers were 0.94 (CI, 0.70-1.26) and 1.18 (CI, 0.29–4.74), respectively. Because these proportional hazard ratios do not differ from 1.0 and the survival curves of the different genotypes overlaid (Figure 2) for both polymorphisms, these MTP polymorphisms were associated with neither overall mortality risk nor specific causes of death (data not shown).


Figure 02
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  		Figure 2. Kaplan–Meier survival curves per genotype in the two combined cohorts of the Leiden 85-Plus Study for the –493G/T and Q95H microsomal transfer protein (MTP) gene single-nucleotide proteins (SNPs)

 
Meta-Analysis
Recently, several studies (8,28,29) reported on the putative effect of the MTP T-Q haplotype on longevity. In a meta-analysis of all published studies, we analyzed 4915 long-lived cases older than 85 years and 3002 younger controls. When a heterogeneity test was performed that included the first and second tier of the U.S. study and the French, German, Danish, Leiden Longevity, and Leiden 85-Plus Studies, significant heterogeneity was observed (p =.039). The pooled odds ratio with random effects was 0.93 (95% CI, 0.77–1.14). When the two tiers were replaced by the U.S. proactively matched cases and controls, no heterogeneity could be observed among the studies (p =.256) and the pooled odds ratio with fixed effects was the same as with random effects: 1.00 (95% CI, 0.89–1.13) (Figure 3).


Figure 03
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  		Figure 3. Meta-analysis of the effects of the microsomal transfer protein (MTP) gene T-Q haplotype on longevity. The size of the black square indicates the size of the study

 

   DISCUSSION
Top
 Abstract
 Participants and Methods
 Results
 Discussion
References
 
To investigate whether the previously identified locus on chromosome 4q25, involved in exceptional human longevity, also plays a role in becoming a nonagenarian, we performed linkage analysis in 379 individuals belonging to 164 nonagenarian sib pairs from the Leiden Longevity Study. We found no evidence for linkage of nonagenarian longevity in this chromosomal region, despite previous observations that 4q25 was linked to healthy aging in a small study of 70-year-old twins (18).

A major reason for the absence of linkage in our study may be that the power to detect linkage with longevity increases with the age of the individuals (30) and that the siblings in our study are approximately 5 years younger than those in the study by Puca and colleagues (7). A test for linkage in 13% of our participants who match the criteria of Puca and colleagues (7) did not reveal any linkage but is also simply underpowered to allow any conclusion. Possibly the 4q25 locus is confined to exceptional longevity and the familial effects on nonagenarian longevity in our study (19) are explained by other loci. Alternatively, the genetic determinants of longevity in the Dutch population may differ from those of the Boston study, despite the fact that both populations are of Caucasian descent.

Because association analysis is more sensitive than linkage analysis (31), we might have missed the linkage signal even in the presence of association. Therefore, we tested two polymorphisms in the MTP gene for association with longevity in the Leiden Longevity Study and the two cohorts of the prospective Leiden 85-Plus Study. This gene is located at chromosome 4q25, and a haplotype of these two SNPs has previously been identified as a marker for human life span (8) showing association in U.S. centenarians compared to U.S. controls. Our analyses, however, provided no evidence for association of –493G/T, Q95H, or their haplotypes with longevity. This may have been explained if this 4q25 locus is simply not involved in the nonagenarian longevity trait of the Leiden Longevity population. Also, recent studies (28,29) describe the absence of association between the two-SNP haplotype of the MTP gene and human longevity in a cross-sectional design.

Because the first cohort of the Leiden 85-Plus study was followed for 14 years and the second cohort for 7 years, we were also able to analyze the effect of the MTP SNPs prospectively. None of the genotypes of the –493G/T or Q95H MTP SNPs showed an effect on either overall mortality or on specific causes of death (such as cardiovascular disease, which would be the most likely cause of death when MTP would have an effect on mortality). This result is in accordance with the Danish longitudinal results (29).

To deal with the discrepancies reported in the literature, we performed a meta-analysis on all published data on the association of the MTP T-Q haplotype and longevity. The meta-analysis including the first and second tier of the U.S. samples showed significant heterogeneity. When, however, the U.S. proactively matched cases and controls were analyzed instead, no heterogeneity was detected among any of the studies. This finding may indicate that the U.S. first and second tier are confounded by population stratification. It should be noted that Geesaman and colleagues found an 18% frequency of the associated haplotype in the U.S. centenarian cases of the Boston study (8) and a 29% frequency in their young U.S. control group. In all published data (28,29) as well as in the French centenarians and controls (8), this haplotype frequency is approximately 19%. Also, the MTP allele frequencies found in the Framingham Offspring study, a Caucasian U.S. population, were similar to those in European Caucasians (32). Among all published MTP allele and haplotype frequencies (11–13,17,28,29,32,33), only the U.S. control groups show an overrepresentation of the associated alleles and haplotype.

It is, therefore, likely that the positive association found in the two U.S. tiers is due to population stratification in the control groups. The long-lived U.S. cases reflect the ethnic distribution of the United States near the beginning of the 20th century, whereas the U.S. control population reflects more recent generations (34). To minimize the problem of admixture, Geesaman and colleagues selected only the Caucasian individuals, and they proactively matched the controls to the long-lived cases using the genotypes of 60 random SNPs. However, moderate levels of stratification may remain undetected when a small number of SNPs (50–100) are investigated (35). This implies that, even after proactively matching, residual population stratification may still have been present in the U.S. control data set (36). Because our meta-analysis including the U.S. proactively matched sample showed that this putative MTP risk haplotype has no effect on human longevity in Caucasian populations, we conclude that it is more likely that the association found by Geesaman and colleagues has its source in the stratification of (especially) the control group of the U.S. population rather than in the genetic effect of the locus on exceptional longevity.

Conclusion
Considering the absence of linkage in our study, we find it unlikely that chromosome 4q25 harbors a gene explaining the familial effects on Dutch nonagenarian longevity. Furthermore, the current evidence does not support the MTP gene at chromosome 4q25 to be the major longevity gene explaining the previously detected linkage to exceptional longevity.


   Acknowledgments
Top
 Abstract
 Participants and Methods
 Results
 Discussion
 References
 
This study is supported by a grant from Innovation Oriented research Program IOP on Genomics (SenterNovem; IGE01014) and by the Centre for Medical Systems Biology (CMSB), a center of excellence approved by The Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research (NWO).

We appreciate the great effort of Michiel Leijnen and J. Kate van Duijn in genotyping the microsatellite markers and the MTP SNPs in samples of the Leiden Longevity Study and the two cohorts of the Leiden 85-Plus Study. We thank Bas Heijmans for his inspiring comments.


   Footnotes
Top
 Abstract
 Participants and Methods
 Results
 Discussion
 References
 
Decision Editor: James R. Smith, PhD

Received April 15, 2005

Accepted October 14, 2005


   References
Top
 Abstract
 Participants and Methods
 Results
 Discussion
 References
 

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