Genetics of maximally attained lung function: A role
for leptin?
Bram van den Borst
a,*
,i, Nicole Y.P. Souren
b,c,d,i, Ruth J.F. Loos
e,f,
Aime
´e D.C. Paulussen
g, Catherine Derom
h, Annemie M.W.J. Schols
a,
Maurice P. Zeegers
b,da
Department of Respiratory Medicine, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centerþ, Maastricht, The Netherlands
b
Department of Complex Genetics, Cluster of Genetics and Cell Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centerþ, Maastricht, The Netherlands
c
Department of Genetics/Epigenetics, FR8.3 Life Sciences, Saarland University, Saarbru¨cken, Germany
dUnit of Urologic and Genetic Epidemiology, Department of Public Health and Epidemiology, University of Birmingham,
Birmingham, UK
e
Medical Research Council Epidemiology Unit, Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, UK
fDepartment of Biomedical Kinesiology, Faculty of Kinesiology and Rehabilitation Sciences,
University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium
gDepartment of Clinical Genetics, Maastricht University Medical Centerþ, Maastricht, The Netherlands h
Department of Human Genetics, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium Received 14 February 2011; accepted 1 August 2011
Available online 4 November 2011
KEYWORDS Genetics; Heritability; Twin study; Leptin; Lung function Summary
Objectives: To estimate the heritabilities of maximally attained lung function in young adult twins, and to examine whether circulating leptin, leptin (LEP) and leptin receptor (LEPR) gene polymorphisms are associated with maximally attained lung function.
Methods: Measures on forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC)
were available of 578 twins recruited from the East Flanders Prospective Twin Survey (165 monozygotic (MZ) and 73 dizygotic (DZ) complete pairs and 102 single twins). Twin model fitting and (genetic) association analyses were performed.
Results: Intra-pair correlations of FEV1and FVC did not differ significantly between MZ
mono-chorionic and MZ dimono-chorionic pairs. Heritability estimates of FEV1and FVC were 69% and 63%,
respectively. The A allele of the LEP 19G>A SNP was significantly associated with a lower FEV1
(pAdditiveZ 0.01) and FVC (pDominantZ 0.047), while the LEPR K109R and Q223R SNPs showed no
associations. Accounting for body mass index, serum leptin was negatively associated with FVC
* Corresponding author. Tel.:þ31 43 3881327.
E-mail address:[email protected](B. van den Borst).
iThese authors contributed equally to this manuscript.
a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / r m e d
0954-6111/$ - see front matterª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2011.08.001
(pZ 0.02) in men, but not in women.
Conclusions: More than 60% of variation in maximally attained FEV1and FVC is explained by
genetic factors. Moreover, these results suggest that leptin may be important in the determi-nation of maximally attainable lung function.
ª 2011 Elsevier Ltd. All rights reserved.
Introduction
A lower maximally attained lung function is considered a risk factor for the development of Chronic Obstructive Pulmonary Disease (COPD).1,2 The level of maximally attained lung function is determined by both environ-mental and genetic factors and to get a better under-standing of the pathogenesis of COPD, much research is currently focused on the genetic component.3e5To unravel the genetics of lung function, it is of great interest to assess the size of the genetic component contributing to lung function parameters. Heritability is a commonly used measure to quantify to what extent genetic factors explain the total variance of a trait.6Heritabilities can in principle be estimated from all kinds of related individuals, but twin studies allow the variation to be split into genetic, shared environmental and unique environmental components, thus offering one of the most valid estimations.7 Twin studies reporting the genetic influences on lung function are mainly performed in adult populations (age > 40 y).8e13Considering the physiological course of lung function during life, which is characterized by a peak around the age of twenty-five,14it is of interest to perform such studies in young adults.
Leptin exerts its biological function by binding to the leptin receptor which is present in many tissues, including lung epithelium.15Leptin is primarily produced by adipo-cytes, but is also expressed in alveolar macrophages and pulmonary epithelium.15,16 Whereas leptin was initially identified as a satiety signal regulating food intake and energy expenditure, it is now regarded as a pluripotent protein that is implicated in a wide range of body func-tions, including the inflammatory response and angiogen-esis.17,18 For example, leptin has been shown to induce cytokine production of monocytes/macrophages, aid in the activation and chemotaxis of neutrophils and increases the cytotoxicity of natural killer cells.18 In addition to these actions of leptin, evidence from animal studies suggests that leptin is also involved in the regulation of embryonic lung growth and maturation.19,20Leptin and its
receptor are expressed in fetal type 2 pneumocytes and stimulation of these cells with leptin results in an increased surfactant-related protein production.20,21 In mice, antenatal administration of leptin results in increased pulmonary cellularity of the newborn, indicative of improved lung development.20 Leptin-deficient mice are characterized by impaired alveolar formation and lower lung volumes at birth. Notably, postnatal leptin replacement in these mice increased the alveolar count and lung volumes.19These experimental data congruently suggest that proper functioning of both leptin and its receptor is necessary for normal lung growth and maturation.
In COPD, leptin has attracted growing interest with respect to its potential role in the pulmonary and systemic inflammatory response.22 Upon an acute exacerbation serum leptin levels have been reported to be increased which is in analogy to pro-inflammatory cytokines.23 Bron-chial expression of leptin was found to be increased in COPD and lung epithelium contains an active leptin receptor.16 Recently, single nucleotide polymorphisms (SNPs) in the leptin receptor (LEPR) gene were associated with a decline in forced expiratory volume in 1 s (FEV1) in
COPD patients, indicating that LEPR polymorphisms might be related to pulmonary disease risk.24
In the present study, we investigated to what extent genetic factors explain the variation in maximally attained lung function parameters determined in young adulthood (18e34 years), by estimating the heritabilities of maximally attained FEV1and forced vital capacity (FVC) in young adult
twins. Subsequently, we analysed whether common SNPs in the LEP (19G>A) gene and its receptor (K109R and Q223R) explain some of the genetic variation observed for the lung function measures. In addition, we examined whether serum leptin levels are associated with measures of lung function.
Methods
Participants and phenotypic measurements
Twins were recruited from the population-based East Flanders Prospective Twin Survey (EFPTS), which started in 1964 and has been recording all multiple births in the Belgian Province of East Flanders until the present.25
Between February 1997 and April 2000, 424 twins visited the research center in Leuven (Belgium), which started in the morning after an overnight fast. Participants were measured barefoot and lightly clothed. Standing height (cm) was measured to the nearest 0.1 cm with a Harpenden fixed stadiometer (Holtain, Crosswell, UK). Body mass (kg) was measured on a balance scale (SECA, Hamburg, Germany) to the nearest 0.1 kg. Body mass index (BMI) was calculated as body mass divided by the square of height (kg/m2). Fat-free mass was measured using a bioelectrical impedance analyser (BIA310; Biodynamics, Seattle, WA, USA). Fat mass (kg) was calculated by subtracting the value for fat-free mass from the total body mass. From the body fat measurements, fat mass index and fat-free mass index were calculated as fat (or fat-free) mass divided by the square of height (kg/m2). Plasma leptin concentrations
were measured using an immunoradiometric assay in a coated tube (Diagnostic Systems Laboratories, Webster, TX, USA). Lung function was measured with Spirobank (Medical International Research, Waukesha, WI, USA)
according to international guidelines.26 At least three
reproducible tests had to be obtained, and measured volumes had to be within a 5% range. The highest obtained values were used. After excluding those participants whose lung function test did not meet the criteria for accep-tance,26578 individuals were included in the present study,
including 165 monozygotic (MZ) and 73 dizygotic (DZ) complete twin pairs and 102 single twins (47 MZ, 55 DZ). Birth weights were obtained from obstetric records, and gestational age reported by the obstetrician was calculated as the number of completed weeks of pregnancy based on the last menstrual period. Zygosity was determined using sequential analysis based on sex, fetal membranes, umbil-ical cord blood groups, placental alkaline phosphatase and DNA marker analysis. The Ethics Committee of the Faculty of Medicine of the Katholieke Universiteit Leuven approved the project and all participants gave informed consent.
DNA isolation and genotyping
As previously described,27 genomic DNA was initially
extracted from available placental tissue collected at birth and/or twins were contacted and whole blood or mouth swabs were taken for DNA extraction. The DNA mini kit (Qiagen, Venlo, the Netherlands) was used for placental and mouth swab DNA extraction, the Wizard kit (Promega, Leiden, the Netherlands) was used for blood DNA extrac-tion, both according to manufacturer’s instructions.
Only putative functional polymorphisms were analysed (functionality has not been proven yet), including the LEPR K109R SNP (rs1137100) in exon 4, the LEPR Q223R SNP (rs1137101) in exon 6 and the LEP 19G>A SNP (rs2167270) located in the untranslated exon 1 (in LEP no common nonsynonymous SNPs have been identified). The SNPs were genotyped using pyrosequencing technology (Pyrose-quencing AB, Uppsala, Sweden) in the Genome Center Maastricht of Maastricht University Medical Center. PCR conditions and primer sequences have been previously described in detail.27Pyrosequencing was performed for
sequence determination and allele designation in a Biot-age PSQ HS 96A System according to the manufacturer’s instructions. Prior to genotyping, 20 samples (including all 3 possible genotypes) were sequenced directly to validate the assays. No discrepancies were observed. The geno-typing success rate for LEP 19G>A, LEPR K109R SNP and LEPR Q223R SNP was 95%, 98% and 96%, respectively. Ac2
-test (1 df ) was used to check for Hardy-Weinberg proportions among genotype frequencies of the SNPs, using only one randomly selected twin per pair (see Souren et al.27 for genotype/allele frequencies). All genotype frequencies were in accordance with Hardy-Weinberg equilibrium (p> 0.20). Since not all participants of whom data on long function parameters were available were willing to provide DNA, genotypes were available of maximal 440 individuals.
General statistical analysis
Differences in means between men and women and MZ and DZ twins were calculated using the PROC MIXED method implemented in the SAS package (version 9.2, SAS Institute).
A random intercept model was used, where the intercept of each twin pair was modelled as a function of the population intercept plus a unique contribution of the pair. Further-more, the variance-covariance structure of the random intercept was allowed to differ between MZ and DZ pairs. Variables with a skewed distribution were transformed to a natural logarithmic scale when performing statistical tests. For the lung function parameters, the effect of the potential covariates sex, age, height, birth weight and smoking status (current, former and never smokers) were checked using the PROC MIXED method. A covariate was considered significant if p< 0.10 (seeTable 2). Intra-pair correlation coefficients were calculated according to chorionicity and zygosity status, after adjustment for significant covariates. To examine whether chorion-type of MZ twins biases the heri-tability estimates, linear regression analysis was carried out to test whether twin correlations differed between MZ monochorionic (MC) and MZ dichorionic (DC) twins.
Heritability analysis
Twin methodology makes use of the fact that MZ twins are genetically identical, whereas DZ twins share on average 50% of their segregating genes. Assuming that MZ and DZ twins share their common environment to the same extent, a higher within-pair concordance rate in MZ twins than in DZ twins reflects genetic influences. To estimate the genetic and environmental components of variance of the traits, twin model fitting of raw data was implemented using the statistical package Mx.28Scripts were downloaded from the GenomEUtwin Mx-script library (http://www.psy.vu.nl/ mxbib/). Univariate twin analyses were performed, where the phenotypic variance can be decomposed into additive genetic (A, additive effects of genes on multiple loci), non-additive genetic (D, interactions between alleles at the same locus (dominance) or on different loci (epistasis)), common environmental (C, environmental effects shared by twins reared in the same family) and unique environmental effects (E, non-shared environmental effects unique to the individual). MZ twins are assumed to share the same A and D genetic variance; DZ pairs are assumed to share half of the A variance and a quarter of the D variance. The C variance is assumed to be the same for both MZ and DZ twin pairs. The broad-sense heritability (H2), which estimates the extent to which variation of a trait in a population can be explained by genetic variation, is defined as the proportion of genetic variance to total phenotypic variance. As non-additive genetic (D) and shared environmental effects (C) cannot be identified simultaneously in data from twins reared together, ACE and ADE models were fitted separately. The significance of variance components A, C or D in the model was tested by dropping these parameters and comparing the fit of the models.
To test whether genetic and environmental factors influence a trait to the same degree in men and women, we compared a quantitative heterogeneity model (variance components free to differ across sexes) with a homogeneity model (variance components constrained to be equal for both sexes). In addition, we verified whether the distribu-tion of a trait differed among men and women by testing a scalar model, which assumes that the female variance
components are common multiples of the male variance components. In this model the variance components were constrained to be equal for both sexes, but total variances were allowed to differ between men and women.29
Variance components were estimated adjusted for signif-icant covariates by modelling the covariates as definition variables in the means model. The difference in fit between the nested models was evaluated with the likelihood ratioc2 -test, which uses the difference between thee2log likelihood of the full and the restricted model. This difference is distributed asc2. The df of the test were calculated as the difference in df between the models. When thec2was not
statistically significant (p < 0.05), the most parsimonious model was selected, i.e. the model with the best fit given the number of df. When comparing the fit of non-nested models (e.g. ACE with ADE), the model with the lowest Akaike’s information criterion (AIC) was preferred.
(Genetic) association analyses
The relation between maximally attained lung function and serum leptin levels was analysed, unadjusted and adjusted
for body composition in men and women separately, using the PROC MIXED method as described above. Associations between the LEP and LEPR SNPs and the lung function parameters were also analysed using the PROC MIXED method, adjusted for significant covariates. Models assuming an additive, dominant and recessive mode of inheritance were tested and an association was considered significant if the p-value <0.05. The genetic association study has 80% power to detect a difference between two means with an effect size (Cohen’s d) of approximately 0.28, assuming a significance threshold of 0.05 and a group-size ratio (e.g. nKK:nxR) of 1:2. Effect sizes of 0.20,
0.50 and 0.80 are defined as small, medium and large, respectively.30
Results
General statistical analysis
Compared to women, men were taller and heavier, had a higher birth weight, fat-free mass index, higher FEV1and
FVC, but lower fat mass index and leptin levels (p< 0.05) Table 1 Phenotypic characteristics of the twin sample according to sex and zygosity status.
Trait Sex p Zygosity p
Men Women MZ DZ n 272 306 377 201 Gestational age (wks) 37.0 2.3 37.1 2.5 0.59a 36.9 2.4 37.4 2.5 0.009a Birth weight (g) 2581 467 2490 491 0.02 2492 485 2610 467 0.02 Age (yrs)a 24.8 4.4 25.1 4.7 0.50a 24.6 4.4 25.7 4.7 0.007a Height (cm) 178.6 6.2 165.5 6.1 <0.0001 171.3 9.1 172.4 8.7 0.31 Weight (kg) 70.1 9.3 60.5 9.8 <0.0001 64.5 10.7 66.0 10.6 0.18 BMI (kg/m2)b 21.7 1.1 21.9 1.2 0.90 21.7 1.1 21.9 1.1 0.46
Fat mass indexb 3.6 1.5 6.1 1.3 <0.0001 4.7 1.6 4.8 1.5 0.65 Fat-free mass index 18.1 1.6 15.7 1.7 <0.0001 16.7 1.9 16.9 2.1 0.34 FEV1(L) 4.6 0.7 3.3 0.5 <0.0001 3.9 0.9 4.0 0.9 0.30
FVC (L) 5.6 0.8 3.9 0.6 <0.0001 4.6 1.1 4.7 1.0 0.52 Leptin (ng/ml)b 1.5 2.9 11.7 2.1 <0.0001 4.4 3.9 4.3 3.9 0.93
BMIZ body mass index, FEV1Z forced expiratory volume in the first second, FVC Z forced vital capacity. Data are expressed as
mean SD unless stated otherwise.
a p-value calculated using standard linear regression, because convergence criteria could not be met using a random intercept model. b Transformed to natural logarithmic scale and for these variables the geometric mean SD is presented.
Table 2 Intra-pair correlations of maximally attained lung function parameters and variance components estimates (95% confidence interval) of the best fitting models expressed in percentages; adjusted for significant covariates.
Trait MZ DZ Model a2(H2) c2 d2(H2) e2 S Significant
covariatesa All MC DC All
n 165 93 72 73
FEV1 0.74 0.73 0.75 0.18 DE 69 (61e75) 31 (25e39) M> W Sex, height and
birth weight FVC 0.68 0.65 0.71 0.38 AE 63 (54e70) 37 (30e46) M> W Sex, height,
age and birth weight
a2 Z additive genetic variance, c2 Z common environmental variance, d2 Z non-additive genetic variance, DC Z dichorionic,
e2Z unique environmental variance, FEV
1Z forced expiratory volume in the first second, FVC Z forced vital capacity, H2Z
broad-sense heritability, MZ men, MC Z monochorionic, n Z number of pairs, S Z scalar effect, W Z women.
(Table 1). MZ twins had a lower birth weight, shorter gestational age and were younger than DZ twins (p< 0.05). In addition, FEV1 and FVC did not differ between current
(nZ 172), former (n Z 39) and never (n Z 366) smokers (FEV1 current, former and never smokers (mean SD):
3.9 0.9, 3.8 0.8 and 3.9 0.9 L, respectively, p Z 0.56; FVC current, former and never smokers: 4.7 1.1, 4.6 1.2 and 4.6 1.1 L, respectively, p Z 0.64).
Intra-pair correlations of FEV1 and FVC were higher in
MZ-DC compared to MZ-MC twins, but did not differ signif-icantly (p> 0.05) (Table 2). For FEV1, the DZ correlations
were less than half of the MZ correlations, suggesting that non-additive genetic effects might be important. The intra-pair correlations for the FVC were in agreement with a model containing additive genetic and unique environ-mental influences (Table 2).
Heritability analysis
For FEV1, the best fitting model was a DE model containing
a non-additive genetic and unique environmental compo-nent (Table 2). For FVC, the variation was best explained by an AE model containing an additive genetic and unique environmental component (Table 2). The broad-sense heritability estimates (encompassing both additive and non-additive genetic effects) of FEV1and FVC were 69 and
63%, respectively (Table 2).
For both FEV1 and FVC, scalar sex differences were
observed, indicating that although variance components are equal across sexes, the total variances differ. Total variances of FEV1and FVC were larger in men compared to
women.
(Genetic) association analysis
The A allele of the LEP 19G>A SNP was associated with a lower FEV1 and a lower FVC (p < 0.05). For FEV1 the
model assuming an additive mode of inheritance was most
significant (pZ 0.01), while for FVC the dominant model had the lowest p-value (pZ 0.047). The LEPR K109R and Q223R SNPs showed no association (p> 0.25) with the lung function parameters (Table 3). As reported previously in this cohort, the LEP and LEPR SNPs showed no association with serum leptin levels.27
In men, serum leptin levels were negatively associated with FVC (pZ 0.02), but only after adjusting for BMI (Table S1). No significant associations between lung function parameters and serum leptin levels were observed in women (Table S1).
Discussion
In this study, we assessed the size of the genetic component contributing to maximally attained lung function by esti-mating the heritabilities of FEV1 and FVC in young adult
twins recruited from the EFPTS. In our sample, 69% and 63% of the total variation in maximally attained FEV1 and FVC
was explained by genetic factors, respectively. One study in a younger twin sample (mean age 19 years) failed to iden-tify significant heritabilities for FEV1 and FVC,31 which
might be due to their relatively small sample size (74 twin pairs). Conversely, twin studies in older samples revealed heritability estimates for FEV1 and FVC between 32 and
91%.8e13While it has been shown that the genetic influence on lung function is modified by smoking status,11,12we did not observe a significant effect of cigarette exposure. This is probably the result of the relatively low pack years exposure in our young sample.
Low birth weight, a proxy for a poor prenatal environ-ment, has been associated with reduced lung function in adulthood.32,33 Compared to MZ-DC and DZ twins, MZ-MC twins experience a more adverse prenatal environment, which is translated in a lower birth weight and higher perinatal mortality and morbidity.25Since the classical twin design assumes equal prenatal environment for MC, MZ-DC and DZ twins, this design might be an unreliable method
Table 3 Associations between SNPs in the LEP and LEPR gene and maximally attained lung function; adjusted for significant covariates.
Gene (SNP) Trait Genotype b (SE) pA pD pR
LEP 19G>A GG GA AA
(rs2167270) n 161 206 58
FEV1(L) 3.95 (3.86e4.03) 3.86 (3.79e3.94) 3.74 (3.60e3.88) 0.10(0.04) 0.011 0.039 0.033
FVC (L) 4.75 (4.64e4.86) 4.62 (4.52e4.72) 4.60 (4.43e4.76) 0.09(0.05) 0.064 0.047 0.392
LEPR K109R KK KR RR
(rs1137100) n 267 137 36
FEV1(L) 3.86 (3.79e3.92) 3.89 (3.80e3.98) 3.97 (3.79e4.15) 0.05 (0.04) 0.255 0.359 0.310
FVC (L) 4.65 (4.57e4.73) 4.67 (4.55e4.78) 4.72 (4.50e4.94) 0.03 (0.05) 0.584 0.697 0.572
LEPR Q223R QQ QR RR
(rs1137101) n 137 223 69
FEV1(L) 3.86 (3.77e3.96) 3.91 (3.84e3.99) 3.90 (3.76e4.04) 0.03 (0.04) 0.534 0.407 0.946
FVC (L) 4.71 (4.59e4.82) 4.67 (4.58e4.77) 4.65 (4.48e4.82) 0.03(0.05) 0.548 0.577 0.710
FEV1Z forced expiratory volume in the first second, FVC Z forced vital capacity. Data are expressed as least squares mean (95%
confidence interval (CI)), calculated using a 2 df F-test and adjusting for significant covariates. nZ total number of individuals. pAZ
p-value additive model, pDZ p-value dominant model, pRZ p-value recessive model. FEV1analyses were adjusted for sex, height and
to estimate heritabilities for lung function parameters. However, in contrast to previous twin studies that exam-ined the heritability of lung function, we were able to control for birth weight and chorion-type in our analyses. Although the intra-pair correlations for FEV1 and FVC
(controlled for birth weight) were slightly higher in MZ-DC compared to MZ-MC twins, they did not differ signifi-cantly. This suggests that the heritability estimates pre-sented here are not biased by chorion-type. Nevertheless, failure to detect a significant difference due to low power cannot be excluded.
We are the first to report a model containing non-additive genetic factors as the best fitting model for FEV1. However, Redline et al.
13
reported for FEV1 a DZ
correlation of less than one-half of the MZ correlation, which also indicates that non-additive genetic factors play a role. The presence of non-additive genetic effects implies that part of the variation is explained by within-locus interactions (dominance) and/or between-locus interac-tions (epistasis). In addition to the non-additive genetic component, one would expect that additive genetic factors also explain part of the total variation in FEV1. However,
the identification of a model containing both an additive genetic and a non-additive genetic component (ADE model) requires greater statistical power, which is a limitation of this heritability analysis. In addition, one should keep in mind that heritabilities are specific for a particular pop-ulation at a particular moment in time and cannot be generalized to other populations.34
This means for instance that if the environment becomes more heterogeneous, the environmental variance will increase and the heritability will reduce and vice versa.
Knowledge on which genetic variants contribute to the genetic components underlying FEV1and FVC may
eventu-ally aid in the development of more personalized diagnos-tics, treatment and prevention strategies. These genetic variants may either alone (e.g.a1-antitrypsin variant) or in combination with others a (e.g. a-nicotinic acetylcholine receptor 3/5 and hedgehog interacting protein poly-morphisms) predict lung disease.35,36 In this study, we
examined whether common SNPs in the LEP (19G>A) gene and its receptor (K109R and Q223R) account for some of the genetic variation observed for the lung function measures. Recently, Hansel et al.24 reported that the R allele of the LEPR K109R SNP was associated with an attenuation in annual FEV1decline in COPD patients. We did not observe
a significant association between the lung function measures and the LEPR K109R and/or Q223R SNP. However, in our sample R allele carriers of the LEPR K109R SNP had a slightly higher lung function compared to the K allele carriers, which is in agreement with the protective effect of the R allele reported by Hansel et al.24Since our sample size is similar to the sample size of Hansel et al.,24 the absence of a significant association cannot be attributed to lower statistical power. Nevertheless, Hansel et al.24 studied middle-aged smoking COPD patients and therefore our insignificant results might also indicate that the effect of the LEPR SNP is modified by smoking and/or disease status.
Although the LEPR SNPs were not significantly associated with the lung function measures, the A allele of the LEP 19G>A SNP showed a significant association with a lower
FEV1and a lower FVC. To our knowledge, we are the first
examining the relation between a SNP in the LEP gene and lung function measures, thus additional studies in other populations are required to confirm this association. Since this SNP is located in the 50untranslated region, it might alter expression levels of LEP. Hager et al.37reported that
the A allele of the LEP 19G>A SNP was associated with increased serum leptin levels, but other studies failed to replicate this association.38,39Also in our twin sample, the LEP 19G>A SNP was not associated with serum leptin levels.27 Since adipose tissue is the main source of circu-lating leptin, lack of association with serum leptin levels indicates that the LEP 19G>A SNP has probably no func-tional consequences in adipocytes, which does not rule out functional relevance in other cells such as alveolar cells. To underline a differential pattern of systemic versus local leptin action, we recently showed an inverse association between serum leptin and sputum leptin in COPD.40If the LEP 19G>A SNP, or a SNP in linkage disequilibrium with the LEP 19G>A SNP, affects LEP expression locally in the developing lung, it may interfere with the regulation of normal lung growth. A feasible next approach to get more insight into a potential local effect of LEP SNPs could be to examine their relation with leptin expression in lung biopsies.
Eventually, we examined whether serum leptin levels were associated with measures of lung function. While in a large middle-aged US sample an independent relation between impaired FEV1and increased serum leptin levels
has been reported,41 in our sample serum leptin levels were not associated with FEV1. This might be due to the
small range of FEV1 in our study as in the US study data
from 2808 individuals were analysed, providing adequate statistical power for the identification of small effects. Nevertheless, in men, serum leptin levels were negatively associated with FVC, but only after adjusting for BMI. Because of the large number of tests performed, we want to emphasize that this association should be interpreted cautiously, because this might reflect a random association by chance. However, our results are supported by a recently published study of Hickson et al. (including 4679 AfricaneAmerican men and women), who observed an inverse association between serum leptin and FEV1and FVC
% predicted, independent of adiposity, which was more pronounced in men and absent in women with a normal body weight.42Finally, childhood lung infection and asthma were previously related to increased plasma leptin levels,43
and may be related to maximally attained lung function.2 However, because we did not observe a clear relation between plasma leptin and lung function, this was unlikely to confound our results.
In conclusion, this classical twin study reveals that in this young adult twin sample the total variation in maxi-mally attained FEV1and FVC is to a large extent explained
by genetic factors. Genetic association analyses with SNPs in the LEP and LEPR genes, revealed that the LEP 19G>A SNP was associated with a lower FEV1and a lower FVC. This
association needs to be confirmed in other populations and future studies should address whether the LEP 19G>A SNP has functional consequences and underlies a local patho-physiological mechanism related to impaired lung development.
Conflict of interest
None declared.
Acknowledgements
BB and NYPS wrote the first draft of this manuscript and contributed equally to this manuscript. RJFL, ADCP and CD were involved in data collection and reviewed the manu-script. AMWJS and MPZ supervised the study and reviewed the manuscript. BB and NYPS take the responsibility of the integrity of the work as a whole.
The EFPTS has been partly supported by grants from the Fund for Scientific Research Flanders and by Twins, Asso-ciation for Scientific Research in Multiple Births Belgium. NYP Souren was supported by an Alexander von Humboldt research fellowship. We are grateful to all the twins participating in this study and thank Lut De Zeure for excellent fieldwork.
Supplementary material
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.rmed. 2011.08.001.
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