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ARTICLE
Evidence of genetic enrichment for exceptional
survival using a family approach: the Leiden
Longevity Study
Manja Schoenmaker
1
, Anton JM de Craen*
,1
, Paul HEM de Meijer
2
, Marian Beekman
3
,
Gerard J Blauw
1
, P Eline Slagboom
3
and Rudi GJ Westendorp
1
1
Department of Gerontology and Geriatrics, Leiden University Medical Center (LUMC), Leiden, The Netherlands;
2
Department of General Internal Medicine, Leiden University Medical Center (LUMC), Leiden, The Netherlands;
3
Section of Molecular Epidemiology, Leiden University Medical Center (LUMC), Leiden, The Netherlands
We conducted a sib pair study in very old subjects for the purpose of mapping longevity loci. In the
present analysis, we explore whether our recruitment strategy has resulted in a population enriched
for a heritable component for exceptional longevity. Our study includes families with at least two long-
living siblings (men aged 89 years or above; women aged 91 years or above). Data were collected on
date of birth and, if applicable, date of death of parents, brothers and sisters, offspring, and spouses
of the long-living participants. Standardised mortality ratios (SMRs) compared with the general Dutch
population, were calculated. The SMR for all siblings of the long-living participants was 0.66 (95%
CI 0.60– 0.73). A similar survival benefit was also observed in the parents (SMR ¼0.76, 95% CI 0.66– 0.87)
and in the offspring of the long-living subjects (SMR ¼0.65, 95% CI 0.51–0.80). The SMR of the spouses
of the long-living subjects was 0.95 (95% CI 0.82 – 1.12). The familial clustering of extended survival is
unlikely to be caused by ascertainment bias, because in all analyses the long-living participants
were excluded. Moreover, it is also unlikely to be caused by environmental factors, because the spouses
of the long-living participants had a mortality risk comparable with the general Dutch population, whereas
they share the same environment. We conclude that our sample is genetically enriched for extreme
survival.
European Journal of Human Genetics (2006) 14, 79– 84. doi:10.1038/sj.ejhg.5201508; published online 26 October 2005
Keywords: longevity; epidemiology; genetics
Introduction
Each species has its own characteristic lifespan.
1
However,
within a species, individual lifespans vary widely.
This variation is in part the result of an individual ability
to avoid or cope with internal and external damage,
which has a strong genetic basis.
2
For example, single
point mutations in the more than 17 000 genes of
Caenorhabditis elegans can lower the rate of aging and
lengthen lifespan up to nearly five times as long as the
wild-type worms.
3
In mice, a single point mutation in the
p66shc gene delays the rate of aging and extends average
lifespan by about 30%.
4
These experimental data suggest
that the majority of age-related changes are under
coordinated genetic control.
5
By definition, experimental research into aging has been
performed in model organisms. However, several observa-
tional studies in humans have also explored the genetic
component in susceptibility to death. During the last
Received 13 April 2005; revised 6 July 2005; accepted 16 September 2005;
published online 26 October 2005
*Correspondence: Dr AJM de Craen, Department of Gerontology and
Geriatrics, C-2-R Leiden University Medical Centre, PO Box 9600, 2300 RC
Leiden, The Netherlands. Tel: þ31 71 526 6640; Fax: þ31 71 524 8159;
E-mail: craen@lumc.nl
European Journal of Human Genetics (2006) 14, 79 – 84
&
2006 Nature Publishing Group All rights reserved 1018-4813/06
$30.00
www.nature.com/ejhg
decade, a number of twin studies have shown that
approximately 25% in the variation of human lifespan
is explained by genetic factors.
6,7
The remainder of
the variation has to be explained by private environmental
factors and gene – environment interaction. More-
over, recent studies have demonstrated a clustering of
extreme longevity within families.
8,9
Puca et al
10
reported
on the localisation of a longevity locus on chromosome 4
after a genome-wide scan in 308 individuals belonging to
137 long-living sibships. Additionally, Reed et al
11
identi-
fied the same region as Puca on chromosome 4 in 95
healthy male twin pairs with a mean age of 74 years.
Hence, it is likely that genetic factors play an important
role in human longevity, but the exact pathways remain to
be elucidated.
The aim of the Leiden Longevity study is to locate
genetic loci explaining the interindividual differences in
human longevity using an affected sib pair design. There-
fore, we collected a large series of sib pairs aged 90 years
and over. By collecting sib pairs instead of long-living
singletons, we expected an enrichment of genetic factors
contributing to longevity in this population, whereas the
likelihood of having reached a long life because of
exceptional environmental conditions or chance would
be lower. Here we report whether our study of long-living
sib pairs has indeed resulted in a population genetically
enriched for longevity.
Methods
Families can participate in the Leiden Longevity Study
if at least two siblings are long-living. There are four
inclusion criteria for the long-living subjects: (1) men
must be aged 89 years or above and women must be
aged 91 years or above; (2) subjects must have at least one
living brother or one living sister who fulfils the first
criterion and is willing to participate; (3) the sib pairs
have an identical mother and father; (4) the parents
of the sibship are Dutch and Caucasian. The minimum
age of 89 years for men and 91 years for women was
chosen because the age of the participants needed to be
extreme enough to be considered long-lived. In 2001, less
than 0.5% of the Dutch population fulfilled these
sex-specific criteria. We have not increased these age
criteria any further because we then would not be able
to recruit a series of 500 long-living sib pairs from a source
population of about 16 million people. In accordance
with the Declaration of Helsinki, we obtained informed
consent from all participants prior to their entering the
study. Good clinical practice guidelines were maintained.
The study protocol was approved by the ethical committee
of the Leiden University Medical Center before the start of
the study.
In total, 500 sibships were collected to enable a genome-
wide scan for longevity. Long-living cousins are also asked
to participate. In addition, the offspring of the long-living
sib pairs and their partners were enrolled to enable
future association studies. In these studies, the offspring
of long-living subjects, who are assumed to have a
higher susceptibility to become long-lived, will be
the cases, and their partners, who are representatives of
the general population, will be the controls. One of the
advantages of using partners of offspring of long-living
subjects as the control population is that they are likely to
have the same age, socio-economic, and geographical
background.
Data collection
All long-living subjects were visited at home, preferably in
the company of a next-of-kin. During the visit the
pedigrees were completed (see below) and a number of
questionnaires were administered. Cognitive performance
is assessed with the Mini-Mental State Examination
(MMSE).
12
We defined severe cognitive impairment as an
MMSE-score below 19 points. In case of cognitive impair-
ment, informed consent and assessment on competence
and actual performances in activities of daily living (ADL)
were obtained from a guardian. Disabilities in ADL are
measured with the Barthel ADL Index
13
and a modified
Fillenbaum questionnaire.
14
The sum score of the Barthel
Index ranges from 0 (unable to perform any activity) to 20
(competent in all activities). The Fillenbaum questionnaire
has a sum score that ranges from 0 (unable to perform any
activity) to 14 (competent in all activities). The Cantril
ladder,
15
a visual analogue scale on perceived quality of life
varying from 1 to 10 points, is administered to assess well-
being.
Each participating family provides us with the genealo-
gical information regarding the parents (ie P1), all siblings
(ie F1), and the offspring (ie F2) of the long-living
participants. With this genealogical data, three-generation
pedigrees were constructed (see Figure 1). Whenever
possible, this information was verified by passport, or by
birth or marriage certificate. Moreover, all data were also
verified with the personal record cards of the deceased
family members in the national population registry located
at the Central Bureau of Genealogy in The Hague, The
Netherlands.
From the long-living subjects, their offspring, the
partners of their offspring, and the long-living cousins, a
venous blood sample was drawn for isolation of DNA, RNA,
serum, and plasma. In case a participant prefered a mouth
swab instead of a blood sample, a mouth swab was
collected for DNA-extraction only. Moreover, place of birth
of the parents of the partners of the offspring was recorded,
to facilitate classification of partners as being ‘Caucasian’.
Statistical methods
The statistical analysis proceeded in various stages.
First, the mortality of various generations was compared
Genetic enrichment for exceptional survival
M Schoenmaker et al
80
European Journal of Human Genetics
with the general population using standardised mortality
ratios (SMRs). The SMR is the ratio of the observed
number of deaths in the study relative to the expected
number of deaths in the general population, adjusted
for sex and calendar period. A description of the
method has been described by others.
16,17
Second, the
mortality between different groups was directly compared
using Cox regression analysis. Level of significance was set
at 0.05.
Results
Recruitment to the Leiden Longevity Study started in July
2002. The number and age distribution of all family
members of the first 100 families included in the study
are listed in Table 1. The characteristics described are
derived from generations P1, F1, and F2. The proportion of
deceased subjects increases with an earlier year of birth
(P1-F1-F2). The proportion of deceased males in gen-
erations F1 and F2 is higher than the proportion of
deceased females. There were two living male spouses of
long-living females, while there were 21 living female
spouses of long-living males.
son1
(F2)
daughter 2
(F2)
partner son
(F2)
partner daughter 2
(F2)
spouse subject 2
(F2)
spouse subject 1
(F1)
daughter3
(F2)
partner daughter 3
(F2)
sibling
(F1)
sibling
(F1)
subject 2
(F1)
subjects 1
(F1)
mother
(P1)
father
(P1)
Figure 1 Representative pedigree. The closed symbols represent the long-lived individuals; the arrows point at the ascertained long-living subjects.
P1, F1 and F2 represent the three generations included in the study.
Table 1 Characteristics of the first 100 families
Males Females
n Age n Age
Parents of long-living subjects (P1)
Deceased 100 78 (68– 87) 98
a
80 (67– 88)
Total sibships
b
(F1)
Alive 106 90 (84– 93) 228 92 (89 – 95)
Deceased 238 75 (47– 84) 152 84 (61 – 90)
Spouses of long-living subjects
c
(F1)
Alive 2 88 (86– 89) 21 86 (83– 89)
Deceased 119 76 (68– 84) 36 81 (78– 86)
Offspring of long-living subjects (F2)
Alive 351 59 (54– 64) 371 59 (55 – 65)
Deceased 51 50 (26– 59) 31 33 (6– 56)
Partners of offspring of long-living subjects
d
(F2)
Alive 93 62 (57– 66) 101 57 (52 – 61)
Age displayed as median (interquartile range). P1, F1 and F2 represent
the three generations included in the study (see also Figure 1).
a
Two mothers had unknown dates of birth.
b
Total sibship includes the long-lived subjects and all the siblings.
c
Calculations include only the first spouse.
d
Calculations include only the partners who participated in the study.
Genetic enrichment for exceptional survival
M Schoenmaker et al
81
European Journal of Human Genetics
The median MMSE score of the long-living
subjects, indicating global cognitive functioning, was 25
(interquartile range (IQR) 21 – 27). Severe cognitive impair-
ment (MMSE o19) was found in 14% of the long-living
subjects. The median Barthel and Fillenbaum scores,
measuring disability, were 18 (IQR 15– 20) and 8 (IQR
4– 11). Of the long-living subjects, 26% had a Barthel score
of 20, indicating they are competent to perform all ADL
independently. The proportion of participants who were
able to perform all seven items of the Fillenbaum
independently was 5%. The median score on the Cantril
ladder, a test to measure well-being, was 8 points out of 10
(IQR 7– 8).
Table 2 lists the mortality characteristics of the
various generations in the included families, expressed
as SMR. An SMR below unity indicates that the mortality
in the included sample is lower than in the general
population, adjusted for sex, age distribution, and calendar
time. The SMR for all 221 long-living participants and
their 503 siblings was 0.31 (95% CI 0.28 – 0.34). When
we excluded the 221 long-living participants, the SMR
was 0.66 (95% CI 0.60 – 0.73) for the 503 remaining
brothers and sisters. The survival benefit is also present
in the parents (SMR ¼0.76, 95% CI 0.66 – 0.87) and
in the offspring of the long-living participants (SMR ¼0.65,
95% CI 0.51 – 0.80). Moreover, the spouses of the
long-living participants, who have a similar socio-
economic background as the long-living participants
themselves, have a mortality pattern similar to
that of the general population (SMR 0.95, 95% CI
0.82– 1.12).
In a direct comparison, the relative mortality risk
of the brothers and sisters of the long-living participants
versus the spouses of the long-living participants was
0.65 (sex-adjusted hazard ratio 0.65, 95% CI 0.54–0.80).
The relative mortality risk between the siblings and
the spouses was 0.66 (95% CI 0.52 – 0.83) for the men
and 0.65 (95% CI 0.44 – 0.94) for the women.
Discussion
Using the first 100 families of the Leiden Longevity Study,
we found a consistent survival benefit in the parents, the
siblings, and the offspring of the long-living sib pairs. This
clearly indicates that we have succeeded in generating a
study sample that is enriched for longevity. As the benefit
was present in all family members of the long-living sib
pairs but not in their partners, we think it is likely that the
survival benefit is not attributable to ascertainment bias or
to environmental factors but is caused by heritable factors.
We have two arguments to infer that the survival benefit
is caused by heritable factors. First, there is a clear survival
advantage in all three generations of the included families.
The parents (P1), siblings (F1), and offspring (F2) of the
included long-living subjects all have a comparable
survival benefit. The fact that family members beyond
the participating long-living sib pairs have a lower
mortality risk makes ascertainment bias unlikely. Second,
in line with expectation, a direct comparison of the
survival of the siblings of the long-living participants with
the spouses of the long-living participants showed a
survival benefit of 0.65. We think that the adult environ-
mental conditions of the siblings of the long-living
participants are likely to be similar to the adult environ-
mental conditions of the spouses of the long-living
participants. Moreover, although it is possible that
the early life conditions of the two groups are different,
we consider it unlikely that familial nongenetic preadult
environmental factors explain the observed survival
benefit of 0.65.
In comparison with the general Dutch population, we
found a small, nonsignificant survival benefit of the
Table 2 Mortality of the parents, siblings, offspring and partners of the included sib pairs compared with the general Dutch
population
Standardised mortality ratio (95% CI)
n
Observed
deaths
Expected
deaths All Males Females
Parents of long-living subjects
a
(P1) 198
b
198 261 0.76 (0.66– 0.87) 0.81 (0.67 – 0.99) 0.70 (0.57 –0.85)
Total sibship (F1)
Including all participating long-living subjects 724 390 1194 0.31 (0.28 – 0.34) 0.43 (0.38 – 0.49) 0.24 (0.20– 0.28)
Excluding all participating long-living subjects 503 390 591 0.66 (0.60– 0.73) 0.71 (0.63 – 0.81) 0.59 (0.50 – 0.69)
Offspring of long-living subjects (F2) 804 82 117 0.65 (0.51 – 0.80) 0.68 (0.51 –0.89) 0.59 (0.42 – 0.84)
Partners of long-living subjects
c
(F1) 178 155 161 0.95 (0.82 –1.12) 1.00 (0.84 – 1.20) 0.82 (0.59– 1.14)
P1, F1 and F2 represent the three generations included in the study (see also Figure 1).
a
Person years were counted from the date of birth of the youngest long-living subject.
b
Two parents had unknown dates of birth.
c
Includes only the first spouse of long-living subjects; person years were counted from the date of marriage.
Genetic enrichment for exceptional survival
M Schoenmaker et al
82
European Journal of Human Genetics
partners of the long-living subjects. As high socio-econom-
ic background is associated with higher life expectancy,
18
our sample might have a small overrepresentation
of families with higher socio-economic background.
Hence, we think that the small, nonsignificant
survival benefit of the partners of the long-living subjects
is likely to be caused by environmental factors. More-
over, we could not calculate an SMR of the partners
of the offspring of the long-living participants because
we only included living partners. Therefore, any compar-
ison with the general population would have revealed a
survival benefit, which is clearly caused by this selective
inclusion.
The cognitive function of the long-living subjects is
comparable with other studies of octo- and nonagenar-
ians.
19
According to expectation, the number of subjects
who could independently do all ADL was higher than the
number of subjects who could perform all IADL indepen-
dently.
20
A median score of 8.0 for well-being is in the same
range as the elderly of 85 years and older.
21
The scores on
these tests indicate that our study sample is a good
representation of the general population of the oldest of
the Dutch elderly. Moreover, as the aim of our study is to
locate genetic loci explaining the interindividual differ-
ences in human longevity, we decided not to collect any
environmental risk factors associated with age-related
diseases or longevity. For, so far as an unintended selection
of a beneficial environment can explain for the survival
benefit, we emphasise that mortality in the offspring was
35% lower than expected whereas it was not different from
the general population in the spouses. These data strongly
suggest that the survival benefit is due to familial, genetic
factors.
Perls et al
22
demonstrated that siblings of centenarians
have a four-fold increased probability of surviving to the
age of 91 years (l
s
¼4). Kerber et al
23
calculated that
siblings of subjects achieving the 97th percentile of excess
longevity (for males this corresponded with an age of 95
years and for women an age of 97 years) had a l
s
of 2.30 to
achieve the 97th percentile. Normally, l
s
is the probability
for a long-living individual (not a sib pair) to have a long-
living sibling divided by the probability of this in the
general population. However, our study design does not
permit this calculation as we included sib pairs in our
study.
An important strength of our study is that the majority
of analyses were carried out in subjects after exclusion of
the long-living sib pairs. The parents, brothers and sisters,
and offspring of the long-living participants all had an
extended survival of about 0.70. The analysis where the sib
pairs were included showed a survival benefit of 0.31.
However, this is an overestimation of the survival benefit
because of ascertainment bias since it also includes the
long-living subjects. Hence, the true genetic enrichment of
the included long-living sib pairs is probably between these
two values. Another important strength is that all data
were verified by official documents and population-based
registries.
In conclusion, we observed familial clustering of ex-
tended survival in three generations in families included in
the Leiden Longevity Study. In this study design of
nonagenarian subjects, we distinguished shared environ-
mental influences and a significant genetic component
contributing to extended survival. Mapping of longevity
genes in this study allows for future linkage analysis. The
two generation inclusion further enables a combination of
linkage and association analysis.
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