Heterozygosity for the rs696217 SNP in the preproghrelin gene predicts weight loss after bariatric surgery in severely obese individuals
Short title: Preproghrelin and weight loss Manuscript type: type I
Abstract
Background Several patients encompass a scarce weight loss after Roux-en-Y gastric bypass (RYGB). As such event is not related to surgical complications, finding markers able to identify “well-responders” and to predict weight loss outcome is clinically relevant. Ghrelin regulates appetite and energy balance. Common single nucleotide polymorphisms (SNPs) in its encoding genes have been associated with body weight regulation. Other peptides involved in satiety modulation, like the CD40/CD40L complex, are less explored.
Methods One-hundred, otherwise healthy, obese subjects (age 45±11 yrs, 65 females, BMI 48.0±0.7 Kg/m2) were sequentially enrolled in 2014-2015 years. SNPs rs2241766 for adiponectin gene, rs490683 for ghrelin receptor, rs696217 and rs27647 for the preproghrelin/ghrelin gene and rs1126535 for the CD40L gene were determined on DNA extracted from circulating
lymphomonocytes. Patients were re-evaluated at 6 (n=100), 26 (n=91) and 52 (n=79) weeks after RYGB.
Results Subjects carrying the rs696217 T allele encompassed a significantly greater
reduction in BMI 52 weeks after surgery (GGvsGT: 30.5±1.1 vs 38.1±2.1%; p<0.001). Carrying the rs1126535 C allele in the CD40L gene was associated with a significantly lower BMI reduction at week 52 (TTvsCT: 33.2±1.1 vs 28.1±2.3%, p=0.049). rs490683 and rs27647 SNPs of ghrelin and rs2241766 for adiponectin gene did not show any difference between carriers and non-carriers of the mutant allele.
Conclusion Carrying a G to T substitution in rs696217 (preproghrelin gene) seems to mark a successful weight loss outcome; we also report for the first time that the rs1126535 C allele (CD40L gene) may predict a worse response to bariatric surgery.
Introduction
Morbid obesity had progressively increased in the last decades, reaching pandemic diffusion. Bariatric surgery guarantees a relevant weight loss, with a deeply positive impact in reducing comorbidities and improving the quality of life of these patients [1, 2]. However, the response to surgery is not always satisfactory, mainly for non-surgical causes, including inadequate behaviors after surgery procedures [3]. Therefore, considering costs and clinical efforts, managing these patients after the surgical procedures is a task, and searching for predictors of a successful and durable weight loss is a major issue. In this scenario, several hormones involved in regulating adipose tissue metabolism and appetite, major determinant of whatever successful program of weight loss, are preferential targets. Among these, adiponectin and ghrelin play important roles in modulating satiety and food intake. Adiponectin, a secretory product of adipocytes, is linked to improved insulin sensitivity and liver function [4, 5]; however, it can unfavorably affect satiety via a central mechanism (increased food intake and weight gain following activation of hypothalamic adiponectin receptors) [6]. Ghrelin (GRHL), secreted in the gastrointestinal tract, requires to bind to its growth hormone secretagogue receptor type 1a (GHSR) to become active. It stimulates growth hormone release from the pituitary, regulates appetite and food intake, gastrointestinal motility, gastric acid secretion, endocrine and exocrine pancreatic secretions, thus exerting a not trivial influence on glucose and lipid metabolism [7]. The multifaceted biological activity of these
hormones is, obviously, genetically regulated: relatively common single nucleotide polymorphisms (SNPs) in adiponectin gene have been linked to insulin resistance and metabolic syndrome [8, 9], while SNPs of ghrelin and its receptor genes are associated with obesity and eating disturbances [10, 11], and might mark an increased risk to develop diabetes [12].
Soluble CD40 ligand (sCD40L), a cytokine pertaining to the TNFα family, is involved in the pathological link between metabolic syndrome, subclinical inflammation and cardiovascular risk [13, 14]. Its role in the pathogenesis of obesity and related comorbidities seems intriguing [15], and
serum sCD40L levels tend to reduce after Roux-en-Y gastric bypass or vertical banded gastroplasty [16, 17]. Some CD40L gene polymorphisms may confer susceptibility to diseases whose
inflammatory component is predominant, like systemic lupus erythematosus [18], while common variants of the CD40L gene, X-linked, have been recently associated with tuberculosis in a Chinese population [19]; such polymorphisms have not been previously examined in relation to weight loss. Aim of the present study was to evaluate whether a panel of SNPs pertaining to genes involved in the appetite and hunger control might influence weight loss following a standard bariatric surgery procedure in morbidly obese, otherwise healthy individuals.
Methods
Subjects - One-hundred severely obese individuals were consecutively recruited between January 2014 and September 2015 among those referring to the obesity outpatient clinic of our university hospital. Exclusion criteria were age above 65 years, presence of type 2 diabetes (excluded by an oral glucose tolerance test, OGTT, performed one week before the study day), hypertension or systemic inflammatory diseases.
After 12 hours fasting, subjects underwent a clinical examination; blood pressure and vital
parameters were recorded, height and weight (in light clothing using a digital scale with an accuracy of 0.1 kg) were measured, calculating BMI. Blood samples were collected from an antecubital vein to extract genomic DNA and to determine plasma levels of glucose, cholesterol, triglycerides, HDL cholesterol, creatinine, uric acid, TSH, PTH and vitamin status (vitamin B12, folate, vitamin D). All the participants underwent gastric bypass within three month from the study day. Weight was checked the day of the surgery procedure and 6, 26 and 52 weeks after surgery; at week 52, determination of biochemical variables was also repeated. Weight change was calculated as total weight loss percent (TWL%) (weight change/baseline or operation-day weight) × 100.
Analytical determinations - Plasma glucose was measured by the hexokinase method; cholesterol and triglycerides by an enzymatic colorimetric assay; uric acid and creatinine by the uricase/PAP method and a modification of the Jaffé method, respectively.
Serum TSH, PTH and 25(OH)vitamin D were measured by immunoradiometric assays (DiaSorin, Saluggia, Italy, Nichols Institute Diagnostics, San Juan Capistrano, CA, USA and DiaSorin Inc., Stillwater, MN, USA, respectively). Fasting serum levels of total ghrelin, adiponectin and sCD40L were determined by ELISA kits (ghrelin: EMD Millipore Corporation, Billerica, MA, USA; adiponectin and sCD40L: R&D Systems, Minneapolis, MN, US). The sensitivity cutoff was 50 pg/ml for ghrelin, 0.891 ng/ml for adiponectin and 10.1 pg/ml for sCD40L, respectively.
DNA extraction was performed using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). Allelic discrimination of genes was performed using an Eco Real-Time System (Illumina Inc., San Diego, CA, USA) according to the standard protocol and with validated TaqMan® SNP genotyping assays (Applied Biosystems Carlsbad, CA, USA). PCR reactions were carried out according to the manufacturer’s protocol. The following polymorphisms were determined: rs2241766 (+45T>G) for adiponectin gene (ADIPOQ), rs490683 (GHSR) and rs696217 and rs27647 for the ghrelin gene (GHRL), and rs1126535 (CD40L), for the CD40L gene.
SNPs selection - The SNPs included in our study were selected on the basis of the following reasons: i) they are involved in weight control; ii) no interplay role has been previously
demonstrated among them; and iii) they modulate gene expression of hormones that regulate food intake, or determine an impaired activity of their receptors. In particular, rs696217 is a
nonsynonimous polymorphism that alters the aminoacid sequence of preproghrelin, resulting in an impairment of mRNA or in the splicing of the preprohormone [20]. The association with weight control and obesity has been reported for ghrelin receptor SNP rs490683[21] and ghrelin SNP rs27647 [10]. Concerning the adiponectin SNP we have selected, it might be a ‘genetic marker’ of body weight, other than insulin resistance and type 2 diabetes [22]. Finally, we explored rs1126535, indeed not commonly related to obesity, but whose variations affect CD40L, a potential marker of obesity-inflammatory profile.
Statistical analysis - Data are given as mean±SEM or median [interquartile range]. Differences between before and after bariatric surgery were analyzed by Wilcoxon signed rank test.
All SNPs were analyzed for deviation from the Hardy-Weinberg Equilibrium (HWE) through the comparison between the observed allelic distribution and those expected from HWE by Χ2 test. Clinical parameters among the genotypes were evaluated with a one-way analysis of variance (ANOVA) or multivariate analysis of variance (MANOVA) test for continuous variables. Statistics were performed using JMP7.0; because of studying five different SNPs, a Bonferroni’s correction
for multiple comparisons was applied, and the cut-off p value for considering a result to be statistically significant was set at 0.05/5=0.01.
The statistical power of the analysis was evaluated by means of the statistical package ClinCalc (http://clincalc.com/Stats/SampleSize.aspx).
Results
Six weeks after bariatric surgery full data were recorded for the entire cohort, while data were missing for 9 and 21 individuals at 26 and 52 weeks, respectively. Table 1 refers to the comparison of basal vs 52 weeks after surgery clinical and biochemical parameters, for all the subjects that were available at week 52. After surgery, the subjects exhibited a 32% BMI reduction; metabolic
parameters like fasting plasma glucose, lipid profile and uric acid improved significantly. In Suppl Table 1 clinical characteristic were reported for study group at baseline.
SNPs in the GRHL (rs696217; rs27647) and GRSH (rs490683) genes were analyzed, together with two SPNs in the adiponectin and CD40L genes (rs2241766 and rs1126535, respectively). As shown in Suppl Table 2, none of these genotypes significantly deviated from HWE distribution. Patients with rs696217 (G>T) variant, despite a similar BMI at week 6 and 26, exhibited
significantly higher weight loss 52 weeks after surgery (p = 0.002) (Table 2). Moreover, given the number of G-G cases (63) and G-T (16), a post-hoc power analysis revealed that this comparison had >95% power to detect differences of TWL% between genotypes. In addition, this association was still present in a multivariate model, after controlling for gender, age, basal BMI, basal fasting plasma glucose and serum ghrelin concentrations (p=0.007). Conversely, heterozygous T allele carriers in CD40L SNP (rs1126535) demonstrated a worse response one year after intervention, comparing with homozygous subjects (p=0.049), although no difference in BMI at intermediate follow-up visits was observed. The post-hoc analysis reached 71% power to detect differences in TWL% between genotypes. Moreover, when multivariate analysis was ran, rs1126535 genotype was no more significantly related to sustained weight loss, neither the statistical significance survived after correction for multiple comparisons.When genotypes distribution was evaluated according to biochemical parameters, such as fasting plasma glucose or lipid profile, there was no difference between homozygous and heterozygous for rs696217 (modulator of preproghrelin activity). With regard to rs1126535 (functional polymorphism affecting CD40L gene), there was a
significant association, even if marginal, with total cholesterol (p=0.049); again, the association did not survive after correcting for multiple testing.
Table 3 shows basal circulating levels of cytokines of interest in the whole study group and
according to SNPs. Serum ghrelin, sCD40L and adiponectin concentrations were not influenced by genotype distribution of the polymorphisms. In the whole study population, serum ghrelin
concentrations were negatively and modestly related with basal BMI (r= -0.019, p=0.039) and BMI at 52 weeks (r= -0.027, p=0.011). A similar correlation, although not significant at the follow-up, was reported for adiponectin concentrations (r=-0.221, p=0.034 at baseline; r=-0.219, p=0.063 at 52 weeks). No association was observed between serum sCD40L levels and either basal or post-surgery BMI.
When combining genotypes for rs696217 and rs112653, the most successful weight loss was obtained by combining favorable genotypes (G-T and T-T: TWL% 38.8±2.2%) while patients harboring both the unfavorable genotypes showed the worst profile (G-G and C-T: 27.3±2.3%). ANOVA analysis showed a significant difference among genotypes (p=0.006), due to the difference between G-T/T-T vs G-G/C-T groups as shown in Figure 1. In representing the combined
genotypes, we also reported the homozygous mutants although very few (2 subjects); furthermore statistically significance without this group is maintained (p=0.003).
Carrying both favorable genotypes determined a significantly higher weight loss than carrying T-T rs112653 (TWL%: 38.8±2.3% for G-T/T-T and 33.2±1.1% for T-T, p=0.03); on the contrary, carrying both favorable genotypes did not add any further weight change than G-T genotype by itself (TWL%: 38.8±1.5% for G-T/T-T and 38.1±1.4% for G-T, p=ns).
Discussion
The present study aimed at evaluating whether polymorphisms of genes encoding for major regulators of food intake and hunger may impact on weight loss induced by bariatric surgery. In our cohort we found that i) patients heterozygous for the SNP rs696217 in preproghrelin region exhibited the most relevant weight loss 52 weeks following surgery; such association remained significant after adjusting for all measured confounders, including age, gender or basal BMI; ii) heterozygous carriers for the rs1126535 in CD40L showed the worst response to RYGB; iii) preproghrelin variant could exert an additive effect on CD40L polymorphism and iiii) no association with weight loss was revealed by genotype distribution in all the other SNPs.
The vast majority of the studies on ghrelin preprotein variant investigated its role in the susceptibly of polygenic obesity/metabolic disturbance, with inhomogeneous results [23, 24]. Our data are in agreement with those derived from three different association studies, that suggested that G-T genotype (and also the rare T-T one) protect from fat accumulation, compared to G-G genotype [25]. Furthermore, Tang and co-investigators [26] showed that G-T patients affected by coronary artery disease had significantly lower BMI than G-G carriers. However, at the best of our
knowledge, this is the first study addressing the role of this polymorphism on weight loss in
subjects that underwent bariatric surgery; the only report dealing with weight loss shows no role of this SNP after a seven-week dietary intervention in 109 Brazilian obese young women [27]. We cannot exclude that this negative result could be ascribed to a much lower weight loss after this short intervention trial than after bariatric surgery, such as in our case.
The functional role of the rs696217 polymorphism has not been completely elucidated yet. Some authors speculated that the ghrelin variant rs696217 might alter mRNA stability or interfere with the splicing of the preprohormone affecting ghrelin secretion or activity [20]; matter of fact that,
according to previous reports [25, 28], we did not observe serum ghrelin variations according to G-T variant; this is in agreement with functional effects on ghrelin activity exerted by such
polymorphism. On the other hand, this polymorphism might impact on both central (imbalancing leptin and ghrelin at the level of hypoythalamic neurons) [25] and peripheral mechanisms
(increased fat oxidation, reduced insulin secretion and/or impaired insulin action) [29, 30]. We cannot confirm or deny such associations, because we did not administer any questionnaire
regarding appetite/eating behavior, neither performed dynamic test to evaluate metabolic pathways; however fasting plasma glucose, a rough indicator of the metabolic status of our study participants, did not vary after surgery in relation with rs696217 variants.
With respect of the rs1126535 polymorphism in CD40L, our data suggest, for the first time, a negative association between surgery-induced weight loss and heterozygous carriers, although this association did not remain significant after multivariate analysis or corrections for multiple testing and revealed a milder power in post-hoc analysis. The allelic distribution of this SNP in relation with metabolic diseases has not been previously explored, except for bone mineral density in women [31]; we could therefore speculate that carrying the C-T genotype is related to a more ‘inflammatory’ phenotype, that might be less prone in losing weight. Concerning circulating sCD40L levels, these might be increased in obese vs lean individuals [32, 33]; we confirm this observation in our study cohort.
A nice observation coming from our dataset is that, when a combined analysis was performed, an additive effect of the two above-discussed polymorphisms was found. Although it is evident that such effect is mainly driven by G-T polymorphism in preproghrelin SNP variant, an
interaction/interplay between these two polymorphisms could not be ruled out. Literature on the interaction between these two molecules is scanty: an anti-inflammatory/immunoregulatory effect of ghrelin on the complex CD40/CD40L, likely mediated through the GHSR-1a receptor, has been reported [34].
We did not find any association with the GHRL (rs27647) and adiponectin (rs2241766) variant and weight decrement induced by bariatric surgery; however, no previous studies were conducted with this regard.
Finally, concerning the role of GHSR variant (rs490683), Matzko et al [35] found that C-C homozygous patients showed the most weight loss following RYGB; however in our population this very rare variant was not sufficiently represented (only one subject).
Several limitations need to be highlighted in the present study: missing data for some key anthropometric and biochemical variables at follow-up; the relatively small sample size for a genetic study, that might underestimate the results; the observational nature; the lack of mechanistic studies able to interpret our results, also in the light of GHRL/CD40L functional interaction: as a consequence, several questions about the biology supporting our findings remain unanswered. The main strength is to describe, for the first time, the predicting role of G-T genotype in the rs696217 of preproghrelin gene, also reporting an interaction of different polymorphisms (i.e. T-T genotype in rs1126535 of CD40L gene), on the main outcome of bariatric surgery in a relatively healthy and homogenous population. These very preliminary observations require confirmation from large cohort intervention studies, with specific markers, based on functional pathways. Furthermore, elucidating the functional/molecular significance of these polymorphisms, in particular of the preproghrelin variant, could be helpful in understanding whether it is a ‘protective’ allele/marker, other than exerting an active role on weight control. In the context of a personalized approach, this kind of studies could provide further knowledge in the research of better predicting outcome markers of complex and expensive intervention of obesity, such as bariatric surgery.
Conflict of Interest Disclosure The Authors have nothing to disclose.
Ethical Statement The protocol was approved by the Ethics Committee of the University of Pisa (n°3463/2011)
Consent Statement All participants in this study read and signed an informed consent References
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Figure legend
Figure 1 Distribution of % of total weight loss percent (TWL%) at 52 weeks according to combined rs696217 and rs1126535 genotypes. The graph was obtained by means of analysis of
covariance on TWL% values. The number (n) of individuals with each combined genotype is indicated. * p value is from ANOVA analysis. Dots indicate means; bars indicate SEM.
Table 1 - Anthropometric and biochemical characteristics of study subjects.
Baseline 52 weeks after surgery p*
(n=79) (n=79)
Age (yrs) 45±11 ---
---BMI (Kg/m2) 47.4±0.7 32.1±0.6 <0.0001
Gender (M/F) 27/52
---Fasting plasma glucose (mg/dL) 88.3±2.0 82.4±1.5 0.035
Total cholesterol (mg/dL) 189.4±4.6 172.3±4.0 0.001 HDL-cholesterol (mg/dL) 46.7±1.8 55.1±1.7 0.0002 Triglycerides (mg/dL) 118.1±8.0 79.0±3.9 0.0001 Uric acid (mg/dL) 6.1±0.3 4.9±0.2 0.0003 Serum creatinine (mg/dL) 0.76±0.02 0.74±0.02 0.706 TSH (μU/mL) 1.53 [1.35] 1.68 [0.91] 0.332 PTH (pg/mL) 54.3 [33.7] 39.8 [22.1] 0.328 25(OH) Vitamin D (ng/mL) 15.5 [16.1] 20.6 [13.4] 0.242 Vitamin B12 (pg/mL) 393.0 [183.0] 289.0 [194.2] 0.0006 Folate (ng/mL) 4.8 [1.6] 7.4 [4.7] 0.179
Data are mean±SEM or median [interquartile range]; * for the comparison of Baseline vs 52 weeks after
Table 2 - Association among genotypes and total weight loss percent (TWL%) at 6, 26 and 52 weeks after surgery.
Data are mean±SEM; p° values are from ANOVA; p ¶ are from ANOVA adjusted for gender, age, basal weight, fasting plasma glucose (and also serum ghrelin and CD40L concentration in the case of rs696217 and rs1126535, respectively) SNP Genotype BMI (kg/m2) 0 week (n=100) TWL% 6 weeks (n=100) TWL% 26 weeks (n=91) TWL% 52 weeks (n=79) GHRL rs696217 G-G G-T p°; p ¶ 48.3±0.8 46.3±1.6 0.268; ---13.9±0.5 14.3±1.1 0.712; ---24.1±0.7 23.1±1.6 0.308; ---30.5±1.1 38.1±2.1 0.002; 0.007 GHSR rs490683 G-GC-G C-C p°; p ¶ 48.3±0.9 47.5±1.2 56.4±7.0 0.430; ---14.2±0.6 13.2±1.5 22.3±4.6 0.118; ---24.8±0.7 23.1±1.6 28.5±6.2 0.462; ---33.2±1.1 28.1±2.3 35.2±8.9 0.810; ---GHRL rs27647 T-T C-T C-C p°; p ¶ 48.0±1.1 47.5±1.0 51.8±2.3 0.233; ---14.4±0.7 13.9±0.7 12.7±1.6 0.605; ---24.5±1.1 24.6±0.9 24.0±2.3 0.976; ---30.3±1.7 33.2±1.3 33.6±3.6 1.040; ---CD40L rs1126535 T-T C-T p°; p ¶ 48.0±0.7 47.8±1.8 0.919; ---13.7±0.5 14.9±1.5 0.359; ---24.8±0.7 23.1±1.6 0.336; ---33.2±1.1 28.1±2.3 0.049; 0.188 ADIPOQ rs2241766 T-T G-T p°; p ¶ 48.7±0.9 46.7±1.2 0.180; ---14.2±0.6 13.6±0.8 0.436; ---24.5±0.8 24.4±1.1 0.917; ---32.0±1.3 32.3±1.6 0.908;
Genotype Ghrelin (pg/mL) Genotype CD40L (ng/mL) Genotype GHRL rs696217 Overall G-G G-T p° 471.4±27.3 469.0±30.0 484.1±68.3 0.840 CD40L rs1126535 Overall T-T C-T p ¶ 4.13 ±0.63 4.41±0.70 3.26±1.60 0.512 ADIPOQ rs2241766 Overall T-T C-T p*
Table 3 - Serum citokine concentrations among genotypes in rs696217, rs1126535 and rs2241766.
Data are as mean±SEM.
p° values are for G-G vs G-T comparison (ANOVA analysis); p ¶ and p* values are for T-T vs C-T comparison for CD40L and Adiponectin, respectively (ANOVA analysis).
