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A polymorphic variant of the insulin-like growth factor 1 (IGF-1) receptor correlates with male longevity in the Italian population: a genetic study and evaluation of circulating IGF-1 from the "Treviso Longeva (TRELONG)" study
© Alban et al; licensee BioMed Central Ltd. 2009
Received: 01 October 2008
Accepted: 21 May 2009
Published: 21 May 2009
An attenuation of the insulin-like growth factor 1 (IGF-1) signaling has been associated with elongation of the lifespan in simple metazoan organisms and in rodents. In humans, IGF-1 level has an age-related modulation with a lower concentration in the elderly, depending on hormonal and genetic factors affecting the IGF-1 receptor gene (IGF-1R).
In an elderly population from North-eastern Italy (n = 668 subjects, age range 70–106 years) we investigated the IGF-1R polymorphism G3174A (rs2229765) and the plasma concentration of free IGF-1. Frequency distributions were compared using χ2-test "Goodness of Fit" test, and means were compared by one-way analysis of variance (ANOVA); multiple regression analysis was performed using JMP7 for SAS software (SAS Institute, USA). The limit of significance for genetic and biochemical comparison was set at α = 0.05.
Males showed an age-related increase in the A-allele of rs2229765 and a change in the plasma level of IGF-1, which dropped significantly after 85 years of age (85+ group). In the male 85+ group, A/A homozygous subjects had the lowest plasma IGF-1 level. We found no clear correlation between rs2229765 genotype and IGF-1 in the females.
These findings confirm the importance of the rs2229765 minor allele as a genetic predisposing factor for longevity in Italy where a sex-specific pattern for IGF-1 attenuation with ageing was found.
Human longevity is the result of a complex interaction between genetics and environment (including lifestyle) and the contribution of each factor is difficult to quantify. Genetic components account for from 20 to 30% of lifespan, according to twin-based population studies [1, 2].
In model organisms like Caenorhabditis elegans, Drosophila and the mouse, several genes involved in different basic cell pathways (stress response, DNA repair, microbial immunity and inflammation, metabolism and calorie restriction) have been shown to be related to longevity [3–5]. Genes belonging to evolution-conserved pathways, like the insulin/insulin like-growth factor 1 (IGF-1) network, are of particular interest in view of their key roles in cell physiology [6, 7].
Insulin/IGF-1 signaling (IIS) triggers intracellular downstream transcription factors (for instance, belonging to the FOXO family) as the final step in a signal transduction pathway mediated by the homologous membrane receptors of insulin and IGF-1 . Down-regulation of IIS has been associated with increased longevity, probably as a consequence of the decrease in cell growth and reduction in oxidative metabolism [9–11]. In humans the level of circulating IGF-1 is low in the elderly, but is higher in childhood than adulthood, probably as a consequence of different hormonal regulation. IGF-1 expression is sensitive to the growth hormone (GH) concentration and is also influenced by sex hormones [12–14].
Genetic variations in genes belonging to the IIS pathway have been explored in relation to longevity, dementia, metabolic diseases and cancer [15–18]. In the Italian population, a synonymous polymorphism (rs2229765) on the IGF-1 receptor gene (IGF-1R) [GenBank:NM_000875] consisting of a G to A transition at nucleotide 3174 leading to the amino acid change Glu->Glu at position 1043 (E1043E) [GenBank:NP_000876] was evaluated and the A-allele showed a positive association with aging and a negative influence on circulating levels of IGF-1 . The same polymorphism has also been reported as predisposing factor for ischemic stroke in China .
We have collected a sample of elderly people (n = 668) from Treviso, Northeastern Italy, from 70 to 106 years old, homogeneous for ethnicity and geographical origin, and we have assessed their rs2229765 genotype and free IGF-1 plasma level.
Population sample recruitment
A comprehensive description of the study design has been reported . Briefly, starting from a list of about 14,000 Treviso inhabitants over 70 years of age, a population sample was built up comprising three classes (70–79, 80–89, 90 years and over). The list from which the sample has been drawn comes from the population register. The final sample size was of 668 independent subjects (311 males and 357 females). The participants were evaluated from the biologic, clinical and socio-economic point of view, with a blood sample collection and a structured interview. The study protocol presenting the inclusion criteria, collecting procedure and questionnaire to be administered to all people involved in the project, was submitted to and approved by the Ethical Committee of the National Institute on Research and Care of the Elderly (INRCA, Italy). This protocol includes an informed, written consent to be obtained from the participant, or from a legally responsible relative in case of mentally impaired subjects, for clinical and genetic studies.
Blood sampling and rs2229765 genotyping
Blood samples (about 30 ml) were collected by venipuncture; one part was used to isolate leukocytes for gDNA extraction, and the other was centrifuged at 2000 rpm for 10 minutes at 4°C (with sodium EDTA as anticoagulant) to separate the plasma fraction, which was then divided into aliquots and stored at -80°C until required. From the final population of 668 subjects, consent for blood collection was obtained from 590 Treviso inhabitants and, in all, 587 plasma samples were successfully prepared and stored for further analysis.
Genomic DNA was extracted from leukocytes using a semi-automated nucleic acid extractor (AB6100, Applera, USA) and stored at 4°C. To assess the rs2229765 genotype, a gDNA aliquot (about 50 ng) was amplified by polymerase chain reaction (PCR) using the following primers: forward 5'-tcttctccagtgtacgttcc-3', reverse 5'-ggaactttctctttaccacatg-3'. The resulting PCR product was digested by MnlI (New England Biolabs Inc, UK). The rs2229765 genotype was examined after loading the corresponding enzymatic digestions on a capillary electrophoresis unit with standard reference markers for instrumental lining-up (Agilent Technologies, USA).
From the available 590 gDNA samples, 510 samples gave unambiguous digestion result and were considered for subsequent analysis.
Plasma IGF-1 assay
Plasma IGF-1 was assayed by a specific sandwich-type enzyme-linked immunosorbent assay (ELISA) (Diagnostic System Laboratories, Inc, USA), according to the manufacturer's instructions. The kit sensitivity was 10 ng/ml and the intra-assay %CV was <10%. From the assayed 587 plasma samples, 564 gave a IGF-1 value above the kit sensitivity limit and were considered for further analysis.
Frequency distributions were compared using χ2-test "Goodness of Fit" test (Preacher, K. J. Calculation for the chi-square test: An interactive calculation tool for chi-square tests of goodness of fit and independence [Computer software]. http://www.quantpsy.org) and means were compared by one-way analysis of variance (ANOVA) followed by Tukey's or Dunnett's post-hoc test. Analyses were done using StatView program ver. 5.0. Multiple regression analysis was performed using JMP7 for SAS software (SAS Institute, USA). The limit of significance for genetic and biochemical comparison was set at α = 0.05.
Age-dependent distribution of rs2229765
Summary of the main demographic and clinical data of the TRELONG study.
Age bracket (no. of people)
No. of people (male:female)
Mean ± SD
84.0 ± 8.0
235.0 ± 75.0
6.43 ± 1.70
214.0 ± 44.0
136.0 ± 37.0
56.0 ± 15.0
1125 ± 412
Body mass index (BMI)
24.8 ± 4.1
105 ± 33
2.3 ± 1.6
5.8 ± 2.0
We first assessed the genotypic distribution of rs2229765 in our sample in five-year groups. No significant difference came to light (data not shown).
rs2229765 genotypic and allelic frequencies according to age and sex.
Genotype count (%)
Allele count (%)
(no. of people)
ap = 0.03
b p = 0.02
c p = 0.04
d p = 0.02
e p = 0.02
Plasma IGF-1 concentration and relation between IGF-1 level and rs2229765 genotype
Multivariate linear model using plasma IGF-1 levels as dependent variable.
sex (×) age**
Age-related IGF-1 plasma concentrations.
No. of people
IGF-1 [ng/mL] (mean ± SD)
70 – 75
192 ± 80
76 – 80
197 ± 89
81 – 85
184 ± 77
86 – 90
159 ± 68**
91 – 95
171 ± 76
96 – 100
146 ± 51**
117 ± 87
70 – 79
199 ± 88
80 – 89
174 ± 70 #
90 – 99
168 ± 75 #
132 ± 63
Sex-related decrease of IGF-1.
(no. of people)
plasma IGF-1 [ng/mL]
(mean ± SD)
Males (n = 264)
213 ± 88
204 ± 76
200 ± 81
157 ± 65 **
159 ± 74 **
212 ± 83
189 ± 76
158 ± 72 §
Females (n = 300)
175 ± 70
189 ± 104
164 ± 64
161 ± 70
170 ± 73
185 ± 91
159 ± 59
173 ± 76
142 ± 86
Total subjects (n = 564)
M (n = 264)
188 ± 80
F (n = 300)
172 ± 77#
Human longevity is influenced by genetic and environmental factors. The present study addressed the question whether the genetic variability due to rs2229765 polymorphism of the IGF-1R plays a role in human longevity. We also sought a correlation between these genetic data and the circulating level of IGF-1, an important molecule in the general cell metabolism, whose reduction was found to be beneficial for longevity in model organisms [5–7].
We collected a homogeneous sample of people from Treviso (Italy), aged from 70 to 106 years (TRELONG Study) . The sample was of particular interest to highlight the specific features of extremely long-living individuals (over 85 years) in comparison to those with a normal lifespan (between 70 and 85 years).
The polymorphism we assessed is a synonymous amino acidic substitution (E1043E) in the IGF-1R protein, whose functional significance is unclear. Genetic data about the rs2229765 did not indicate any major differences when the population was grouped in five-year age brackets or split in the two groups (70–85) and 85+, but there was an increase of the A-allele in the males over 85, suggesting a sex-specific effect on male longevity. The significance of this increase was not robust, (p = 0.04, χ2-test) probably because of the sample size of 85+ males. The (70–85) male group had a lower A-allele frequency than (70–85) females. This might be interpreted as a sex-specific effect but might also hide a chance or recruitment bias. A confirmatory study in an independent male population sample from Veneto Region is required to better clarify the above described limitations of the study. Our finding about rs2229765 A-allele was partially in agreement with a previous study in the Italian population by Bonafè et al., which reported an increase of the rs2229765 A-allele in individuals over 85 years of age regardless of sex. This aspect can be explained considering that our study design was different, based on an elderly population sample, while Bonafè et al. considered a wider sample ranging from 20 to 100+ years . Consequently, their increase of the A-allele in all the people over 85 years old might have depended on a larger difference between male A+ carriers in the two groups and does not exclude a sex-specific pattern. A gender-specific influence on longevity is a well-known aspect that can be partially explained at genetic level. Other polymorphisms show a gender-specific pattern in relation to longevity as well [24–26].
The second part of our study investigated plasma IGF-1 concentration, and a possible correlation between rs2229765 and IGF-1 circulating levels. We found a linear relationship between plasma IGF-1 and age using a simple and a multivariate regression model, confirming that IGF-1 continues to fall through life, however long. A substantial gender-specific effect was embedded in the general population analysis, as men and women contributed differently to the IGF-1 reduction: female IGF-1 in the (70–85) group was lower than in males but it did not drop further over time; in contrast, male IGF-1 was significantly lower in the 85+ group than the (70–85) group. Consequently, the drop in IGF-1 over time in our population was mainly due to a specific effect in males. This agrees with a reported sex-specific variation of IGF-1 level in model organisms, where it is influenced by sex hormones [27–29]. Bonafé et al. also reported a linear reduction of circulating free IGF-1 in the Italian population , in accordance with our data that magnified the effect for the elderly and found previously unreported sex-specific difference.
We found no difference in the entire sample as regards a correlation between IGF-1 plasma level and rs2229765 genotype. However, men showed a genotype-specific age-related decrease of plasma IGF-1 that declined faster when the subject was homozygous for the A-allele. In females this association was less evident, however the A-allele carriers had a faster plasma IGF-1 decline over age than the G/G subjects. This might suggest that in females rs2229765 impacts on circulating IGF-1 level, but this is masked by other genetic or environmental factors. This sex-specific relation was confirmed when we split our population at 85 years and divided men from women, as we observed that in the 85+ males the reduction of mean IGF-1 was unequally distributed among the genotypic classes G/G, G/A and A/A, the last group having the lowest IGF-1 concentration (119 ± 50 ng/mL), and the G/G genotype the highest (185 ± 74 ng/mL) that was comparable to the mean IGF-1 value of (70–85) men (205 ± 81 ng/mL). However, this correlation was statistically weak (p = 0.048, Tukey's test) and a chance effect should be taken into account. Recently, a genetic screening in Ashkenazi Jewish centenarians reported gender-specific modulations of circulating IGF-1 that was associated to IGF-1R genetic variability (including rs2229765), confirming that also this synonymous genetic alterations in the human IGF-1R might alter IGF signaling pathway and human longevity .
This genetic and biochemical study in the Italian population confirmed and better detailed a genetic contribution to longevity coming from the A-allele of the rs2229765 polymorphism in the IGF-1R gene, that correlated with male longevity.
This study was supported by grants from the Veneto Region, the Treviso Municipality, Treviso Province and Cassamarca Foundation. We are grateful to all people who kindly agreed to participate in this study and to J.D. Baggott for manuscript editing. LP is recipient of a fellowship from "Golgi Cenci Foundation", Abbiategrasso, Milan, Italy.
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