Neuropeptide Y Leu7Pro polymorphism is not associated with risk of developing obesity in Pakistani population

Accepted Manuscript Neuropeptide Y Leu7Pro polymorphism is not associated with risk of developing obesity in Pakistani population

Pasha Ghazal, Wishal Khan PII: DOI: Reference:

S2214-5400(18)30027-6 doi:10.1016/j.mgene.2018.02.013 MGENE 412

To appear in:

Meta Gene

Received date: Revised date: Accepted date:

12 December 2017 14 February 2018 24 February 2018

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ACCEPTED MANUSCRIPT Neuropeptide Y Leu7Pro polymorphism is not associated with risk of developing obesity in Pakistani population NPY Leu7Pro gene variant in Pakistani Population

Pasha Ghazal *, Wishal Khan

Department of Biosciences, COMSATS institute of information technology, Islamabad,

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Pakistan.

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Corresponding author: Dr.Pasha Ghazal, PhD Assistant professor, Department of Biosciences, COMSATS, institute of information technology, Islamabad, Pakistan

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[email protected]

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Abstract:

Background/Objectives: The role of Neuropeptide Y in the hypothalamic control of energy balance is well established. NPY is a well-known, orexigenic peptide. Apart

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from increasing food intake and reducing state of satiety, NPY stimulates fat angiogenesis, proliferation and differentiation of new adipocytes, resulting in development of abdominal obesity and metabolic syndrome-like conditions. Although there is extensive evidence of the key role of NPY in energy regulation in pre-clinical studies, evidence in humans is limited. However, single nucleotide polymorphism in the signal peptide of NPY Leu7Pro, which causes an amino acid change from leucine

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(7) to proline (7), has been found to increase susceptibility to obesity in various populations. Therefore, we sought to genotype screen NPY Leu7Pro allele in

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overweight, obese and normal Pakistani individuals.

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Method: A total of 364 randomly selected participants were genotyped screen for the NPY Leu7Pro using PCR-RFLP followed by Sanger sequencing for conformation of

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the results. The participants were divided into normal, overweight and obese

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categories based on the BMI.

Results: Of the 364 samples screened, only one sample showed the heterozygous TC

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genotype. All the other samples showed homozygous TT (normal) genotype. No CC genotype was observed in both the overweight, obese and control groups.

Conclusion: The NPY SNP Leu7Pro is found to be extremely rare in Pakistani population and does not contribute towards risk of developing obesity.

Keywords: NPY, Leu7Pro, obesity, Pakistan

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ACCEPTED MANUSCRIPT Introduction:

The problem of obesity has turned into a global pandemic and has fast penetrated into developing countries like Pakistan, where, 13.6% of population is affected with it.

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The health risks associated with obesity increases with increase in BMI, therefore, identification of genetic risk markers is imperative for timely and effective therapeutic

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interventions. Peptidergic systems have emerged as key regulators of energy homeostasis with high abundance in the hypothalamus and brainstem regions.

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Therefore, aberrations at single nucleotide level in neuropeptides genes are being envisaged as critical risk markers to assess predisposition towards development of obesity.

Neuropeptide Y has a well-established role in regulation of feeding behaviors as

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evidenced by both mammalian and non-mammalian based studies. NPY has potent orexigenic effects and hence, increased central NPY-ergic tone mediates obsogenic effects by increasing food intakes and delaying satiety, consequently, increasing over

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all calorie intakes. At cellular levels, NPY stimulates fat angiogenesis, proliferation and differentiation of new adipocytes, resulting in development of abdominal obesity and metabolic syndrome-like conditions. Importantly, gene knockout models of NPY

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demonstrate reductions in abdominal fat 3. Given the role of NPY system in regulation of energy homeostasis, Leu7Pro polymorphism (rs16139) in NPY gene has been

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strongly linked with higher risk of developing obesity and associated co- morbidities in European cohorts.

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The NPY rs16139 is a functional mutation that leads to a

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synonymous change from leucine to proline in the amino acid sequence of the propeptide.18 Studies show that in mutant allele carriers, this change results in higher

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levels of peptide secretion. Where as, basal NPY levels are lower in heterozygous carriers of this SNP at rest but plasma NPY levels increases in response to sympathetic stimulation.19, 20 However, only two

association studies have investigated the link between obesity

and Leu7Pro gene variant in south east-asian populations.

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Therefore, we sought to

genotype screen NPY Leu7Pro allele in Pakistani overweight and obese vs. lean subjects.

Methodology

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ACCEPTED MANUSCRIPT Study population This is a case-control, multi-centre study of obese and respective controls in adults. A total of 364 (189 males and 175) female subjects participated in this study. All the participants recruited; between August 2016- June 2017 from various cities of Pakistan, were of Pakistani-asian heritage and aged between 37 to 40 years. We ascertained relatively homogeneous genetic background of our study population in

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order, to preclude factor of population stratification affecting our study results. Participants were classified according to their BMI values; 126 subjects were over weight and 138 were obese and 100 subjects were with normal BMI. At the time of sample collection obesity was assessed on the basis of body mass index (BMI) as weight (kg) in kilograms divided by height in meters square (m2). A single examiner at each hospital made anthropometric measurements. The participants were barefoot

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and in light clothes when the measurements were taken. Body weight (Kg) was measured using a calibrated standard weighing balance. Height (cm) was measured using a precision stadiometer. BMI, defined as weight (Kg) divided by the square of

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height (m2), was calculated. Waist circumference was measured by applying an inelastic tape horizontally midway between the lowest rib margin and the iliac crest of

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the subject at the end of a gentle expiration.

Blood samples were drawn via the antecubital vein after the patient had fasted overnight.

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In accordance with WHO defined criteria, patients with BMI greater or equal to 30kg/m2 were classed as obese; those with BMI values between 25-29.9 kg/m2 were

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considered overweight where as individuals with a BMI less than 24 kg/m2 were considered control individuals. The study inclusion criteria were Pakistani heritage

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and absence of congenital metabolic disorders. The exclusion criteria were nonPakistani heritage and the presence of congenital metabolic diseases (e.g., diabetes or hyperlipidemia), under nutrition, polycystic ovary disease, cancer or thyroid problems and the use of medication that alters BP, glucose or lipid metabolism. The study protocol was in adherence to the Helsinki Declaration of 1975 as revised in 2000 and the research study was accepted and approved by the Ethics Review Board of the Department of Biosciences, COMSATS Institute of Information Technology Islamabad. The participants of the study were briefed about the aims and objectives of the study. Demographic information and blood samples for biochemical and genetic analysis were collected from all study participants only after the signed informed 4

ACCEPTED MANUSCRIPT consents of the participants.

Biochemical tests Biochemical tests for random blood glucose level and lipid profile (cholesterol, HDL, LDL and triglycerides levels) were performed for all the three weight groups: obese, overweight and control subjects to ascertain obesity related phenotypes in a university

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affiliated diagnostic laboratory, using automated enzymatic analyzers. Genotyping

Genotyping of the Leu7Pro polymorphism was performed using the PCR-RFLP method as described earlier 9. Briefly, genomic DNA was extracted using kit method

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(Qiagen, USA ) and was amplified for rs16139 in a 25 l total reaction volume (Axygen, Union City, CA, USA). Amplification of the gene was done using thermal (Thermo

Electron

Corporation)

CCCGTCCGTTGAGCCTTCTG

3’

using

and

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cycler

NPY

NPY

RS16 RS16

Forward Reverse

5’5’-

CGGTCCCGCGGTCCC-3’ primer set under the following conditions: 95° C for 5

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minutes, followed by 34 cycles of 95° C for 1 min (denaturation), 58.4° C 1 minute (annealing), 72° C for 1 minute (extension), and final extension for 5 minutes at

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72°C.The PCR product of 238bp was analyzed on 2% agarose gel electrophoresis. Gel electrophoresis was carried out for 45 min at 95 volts and 400 mA followed by

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visualization under UV light using the trans-illuminator. Digestion of the amplicon was done by type II restriction endonuclease strain of thermophilic Bacillus circulans

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(BsiEI) (Fermentas, Lithuania), which cuts at 5′-CGRY↓CG-3′restriction site. The reaction mixture was incubated at 60 °C for 4 hours and then at 80 °C for 20 minutes. Digested products were then electrophoresed in 2.5% gel and visualized in UV illuminator (Alpha Imager Mini Bucher Biotech, Basel, Switzerland). The PCR product, which do not contain a BsiEI restriction enzyme site results in a 238 bp band; the heterozygotes results in 3 band 238, 190 and 48 bp; and the Pro7/Pro7 homozygotes yields bands of 190 and 48 bp. Sanger sequencing PCR-RFLP results were later confirmed with Sanger sequencing using Big Dye Terminator Cycle Sequencing Ready Reaction Kit and the ABI PRISM 3730 DNA 5

ACCEPTED MANUSCRIPT analyzer (Applied Biosystems, Foster City, CA, USA).

Statistical analyses: Data from anthropometric measurements and biochemical tests were analyzed using one-way analysis of variance ANOVA. Where as, Chi-Square test was performed where appropriate, to analyze genotype and allele frequencies . Statistical significance

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level was set at p< 0.05.

Results:

The panel in this study consisted of 364 subjects in total, out of which 138 were obese, 126 were overweight, and 100 were normal individuals (Table 1). Overall, 48% (n=175) of the subjects were females and 52% (n=189) were males. The mean

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age of the subjects was around 35 years, and the mean height and weight were 66 inches and 84 kg, respectively. The subjects were grouped into different categories based on their BMI values. The anthropometric measurements (height, weight, BMI),

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and majority of the clinical markers (glucose, cholesterol, triglycerides levels) were significantly higher in the obese group than in overweight and normal-BMI

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group. In contrast, HDL levels were lower in obese individuals in comparison to overweight and normal BMI group.

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All the samples screened showed the homozygous TT (normal) genotype except only one sample which had a heterozygous CT genotype, whereas no CC genotype was

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observed in both the control and the obese subjects (Figure. 1) Out of 138 obese individuals, 137 were homozygous for the wild type genotype TT, only one sample

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was heterozygous CT, there were no homozygous genotype CC. Out of the 100 normal individuals, all were homozygous TT, and there were no heterozygous CT and homozygous CC genotypes. All of 126 overweight subjects were homozygous for the wild type genotype TT, while no heterozygous TC, and homozygous CC genotype was found in this group (see Table. 2).

Discussion:

The present study reports for the first time that Leu7Pro allele is extremely rare in Pakistani population. All 364 samples showed Leu (7)/Leu (7) (TT) genotype of the 6

ACCEPTED MANUSCRIPT NPY gene. Only one sample showed Leu (7)/Pro (7) (TC) allele, whereas no Pro7/Pro7 (CC) allele was found in normal and obese or over weight subjects of pure Pakistani-Asian ethnicity.

This data corroborates the accumulating evidence that Leu7Pro allele is rare in people of pure Asian heritage. Previously, Leu7Pro allele has been reported to be completely 10

and Japanese

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population whereas in Chinese

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population it is

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absent in Korean

found to be extremely rare, only one individual out of 304 study participants showed CT genotype, whereas, all the other participants had TT genotype. This is similar to our observations. In contrast, the frequency of this allele is reported to be quite high in European populations ranging from 7% in Dutch people

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to 14% in Finns 9. The

presence of this allele has also been reported in some African tribes, Yoruba and Esan

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in Ibadan, Nigeria (www.internationalgenome.org) and at lower frequencies in Moroccans and Ethiopians. 13

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A review of recent reports on presence of Leu7Pro in different ethnicities reveals that, in south east -asian populations, a relatively high frequency of this allele has only

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been reported in those populations, which have an ethnicity overlap with either Caucasians or with Africans. In the Persian population, the genotype frequency of Leu7Pro has been reported to be 5.9%.

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This high frequency of Leu7Pro is

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comparable to that observed in Caucasian populations and stems from their mixed

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Persian-Caucasian ethnicity.

Whereas, in Indian Asians, over all low allele frequency of this SNP has been

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reported in northern Indians with no association established with obesity. 7, 8 However, two small tribes Kota and Siddi have relatively high frequency of the Pro 7 allele. Interestingly, Siddi is an African immigrant tribe, where as Kota is a small isolated population. The observed, relatively high frequency of Pro7 alleles in these populations can be largely ascribed to the phenomenon of genetic drift and founder effect. 15 Over all these observations might suggest that (Leu7Pro) genetic variant of NPY gene might have origins in northern Europe and could have spread to other parts of the world in different proportions depending on their geographical distance with northern Europe, the Leu7Pro allele shows a decreasing frequency from north to south.16, 17 7

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The NPY rs16139 is a functional mutation that leads to a synonymous change from leucine to proline in the amino acid sequence of the propeptide.18 Studies show that in mutant allele carriers, this change results in higher levels of peptide secretion. Where as, basal NPY levels are lower in heterozygous carriers of this SNP at rest but plasma

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NPY levels increases in response to sympathetic stimulation.19,20

A review of literature suggests that NPY SNP rs16139 has modest effect on weight gain and BMI and might not cause extreme obesity. In Asian populations, very few studies have investigated the role of NPY SNP rs 16139 in causing obesity. Bhushan et al., 2010

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and Bhaskar et al., 2011

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failed to find any significant association

between this SNP and risk of developing obesity in obese Indian Asians. In our study,

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this SNP has been found to be extremely rare in Pakistani population and is not associated with the risk of developing obesity. Where as, in a large association study, among 1842 European Americans and 1031 polish participants, no replicable 21

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association was found between 26 SNPs in NPY gene and risk of developing obesity .Intriguingly, in some European populations, rs16139 C allele has been reported to

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increase BMI in non-obese individuals 6. Yeung et al., 2011,22 reported a rather small combined weight gain of approximately 5 kg over 20–30 yr in non-obese carriers of this SNP. These findings suggest the presence of some other genetic factors, which

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might override the effects of this SNP in extreme obesity.

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In conclusion, no association was found between NPY Leu7Pro allele and obesity in

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Pakistani population.

These results and earlier observations might suggest that in south-east Asian populations, Leu7Pro allele does not influence changes in BMI and is not associated with obesity.

Acknowledgements This study was supported by Higher Education Commission (HEC) research grant  21-583 (HEC-SRGP to PG).

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Conflict of interest: The authors declare no conflict of interest.

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ACCEPTED MANUSCRIPT References 1. Ng M, Fleming T, Robinson M, Thomson B, Graetz N, Margono C, et al. Global, regional, and national prevalence of overweight and obesity in children and adults during 1980–2013: a systematic analysis for the Global Burden of Disease Study.

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2. Boughton CK, Murphy KG. Can neuropeptides treat obesity? A review of neuropeptides and their potential role in the treatment of obesity. Br J

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3. Kuo LE, Kitlinska JB, Tilan JU, Li L, Baker SB, Johnson MD et al.

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Neuropeptide Y acts directly in the periphery on fat tissue and mediates stress-

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induced obesity and metabolic syndrome. Nat Med. 2007; 13(7): 803-11

4. Bray MS, Boerwinkle E, Hanis CL. Sequence variation within the neuropeptide Y

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5.Van Rossum CTM, Pijl H, Adan R A H, Hoebee B, & Seidell J C. Polymorphisms

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in the NPY and AGRP genes and body fatness in Dutch adults. Int J Obes 2006; 30(10): 1522-1528.

6. Ding B, Kull B, Liu Z, Mottagui-Tabar S, Thonberg H, Gu H F, et al. Human neuropeptide Y signal peptide gain-of-function polymorphism is associated with increased body mass index: possible mode of function. Regul. Pept. 2005;127(1): 4553.

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ACCEPTED MANUSCRIPT 7.Bhaskar, L V K S, Thangaraj K, Pardhasaradhi G, Kumar KP, SinghL, RaoVR. Neuropeptide Y gene polymorphisms are not associated with obesity in a South Indian population. Eur J Clin Nutr 2010; 64(8): 868-872.

8.Bhushan B, Guleria R, Misra A, Luthra K, & Kumar G. Association of PPARγ2

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(Pro12Ala) and Neuropeptide Y (Leu7Pro) Gene Polymorphisms with Obstructive Sleep Apnea in Obese Asian Indians. Dis Markers 2011; 30(1): 31–38.

9. Karvonen M K, Pesonen U, Koulu M, Niskanen L, Laakso M, Rissanen A, et al.

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Association of a leucine (7)-to-proline (7) polymorphism in the signal peptide of neuropeptide Y with high serum cholesterol and LDL cholesterol levels. Nat Med

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1998; 4(12): 1434-1437.

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10. Ding B, Bertilsson L, Wahlestedt C. The single nucleotide polymorphism T1128C in the signal peptide of neuropeptide Y (NPY) was not identified in a Korean

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population. J Clin Pharm Ther 2002; 27(3): 211-212.

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11.Makino K, Kataoka Y, Hirakawa Y, Ikeda A, Yamauchi A. A leucine (7)‐to‐ proline (7) polymorphism in the signal peptide of neuropeptide Y was not identified in the Japanese population. J Clin Pharm Ther 2001; 26(3): 201-203.

12. Jia C, Liu Z, LiuT, Ning Y. The T1128C polymorphism of neuropeptide Y gene in a Chinese population. Arch. Med. Res. 2005; 36:175–177.

13. Lappalainen J, Kranzler HR, Malison R, Price LH, Van Dyck C, Rosenheck RA, 11

ACCEPTED MANUSCRIPT et al. A functional neuropeptide Y Leu7Propolymorphism associated with alcohol dependence in a large population sample from the United States. Arch Gen Psychiatry 2002; 59(9): 825–831

14. Masoudi-kazemabad A, Jamialahmadi K, Moohebati M, Mojarrad M, Dehghan-

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manshadi R, Mahdi M, Akhlaghi S. High frequency of Neuropeptide Y Leu7Pro polymorphism in an Iranian population and its association with coronary artery disease. Gene 2012; 496(1): 22–27.

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15.Bhaskar LV, Thangaraj K, Shah AM, Pardhasaradhi G, Praveen Kumar K, Reddy

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AG et al .Allelic variation in the NPY gene in 14 Indian populations. J Hum Genet. 2007; 52(7): 592-8

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16. Ding B. Distribution of the NPY 1128C allele frequency in different populations.

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J. Neural Transm 2003; 110:1199–1204.

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17. Pesonen U. NPY L7P polymorphism and metabolic diseases. Regul. Pept 2008;

18. Ding B, Kull B, Liu Z, Mottagui-Tabar S, Thonberg H, Gu HF, Brookes AJ, Grundemar L, Karlsson C, Hamsten A, Arner P, Ostenson CG, Efendic S, Monné M, von Heijne G, Eriksson P, Wahlestedt C. Human neuropeptide Y signal peptide gainof-function polymorphism is associated with increased body mass index: possible mode of function. Regul Pept; 2005, 127:45–53

19. Kallio J, Pesonen U, Jaakkola U, Karvonen MK, Helenius H, Koulu M. Changes in diurnal sympathoadrenal balance and pituitary hormone secretion in subjects with 12

ACCEPTED MANUSCRIPT Leu7Pro polymorphism in the prepro-neuropeptide Y. J Clin Endocrinol Metab; 2003.88:3278–3283

20.Kallio J, Pesonen U, Karvonen MK, Kojima M, Hosoda H, Kangawa K, Koulu M. Enhanced exercise-induced GH secretion in subjects with Pro7 substitution in the

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prepro-NPY. J Clin Endocrinol Metab; 2001,86:5348–5352

21. Campbell CD, Lyon HN, Nemesh J, Drake JA, Tuomi T, Gaudet D, Zhu X, Cooper RS, Ardlie KG, Groop LC, Hirschhorn JN. Association studies of BMI and type 2 diabetes in the neuropeptide Y pathway: a possible role for NPY2R as a candidate gene for type 2 diabetes in men. Diabetes; 2007, 56:1460–1467

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22. Yeung EH, Zhang C, Chen J, Bowers K, Hu F B, Kang G, Qi L. Polymorphisms in the Neuropeptide Y Gene and the Risk Of Obesity: Findings from Two Prospective

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ACCEPTED MANUSCRIPT Figure legends

Figure 1 PCR-RFLP analysis of Leu7Pro (rs16139) polymorphism in NPY gene in Pakistani population. The original product size is 238 bp and in case of heterozygous a band of

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238, 190 is formed after restriction.

Figure 2

Verification of the NPY rs16139 SNP by Sanger sequencing. Sequencing analysis

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showed the normal, overweight and obese patients to be homozygous for wild type TT genotype. Whereas, heterozygyous CT genotype is observed in one sample in the

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obese group.

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ACCEPTED MANUSCRIPT Abbreviation list

HDL: High density lipoproteins LDL: Low density lipoproteins

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Leu: Leucine Pro : Proline

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ACCEPTED MANUSCRIPT Normal

Overweight

Obese

(18-24.9)

(25-29.9)

(30-≥40)

100

126

138

53/47

65/61

71/67

N =364 Anthropometric charachteristics Sex (M/F) Age (years) Mean height (inches)

37.7 ±10.4 66.08±4.30

Mean weight (kg)

65.50±9.93

35.93 ±15.19 67.57±3.17

39.9± 8.8 65.76±4.03

80.43±8.76

96.19±9.67

p-value

>0.05 >0.05 <0.001**

22.05±0.44 28/26

27.32 ±1.32

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BMI (kg/m2) Waist circumference (inches) (M/F)

37/35

Biochemical tests

Cholestrol (mg/dl)

124.36±4.5

Triglycrides levels (mg/dl)

85.74±3.7

221.4±8.5

<0.0001*** <0.001***

228.4±11.5

<0.0001***

225.17± 3.8

201.17± 3.8

<0.0001***

167±3.6

185±2.5

<0.0001***

26.69 ± 1.904

24.8 ± 1.904

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104.84±4.3

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Glucose levels (mg/dl)

32.27±0.65 48/42

36.98 ± 1.206

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HDL levels (mg/dl)

<0.0001***

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Table 1 Anthropometric and biochemical parameters of the studied participants

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Genotype/Allele Controls Overweight Obese N=126

N=138

TT

100

126

137

TC

0

0

1

CC

0

0

0

Overweight

Obese 276

Allele frequency T

200

252

C

0

0

NS

NS

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N=100

P value

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Table 2: Genotypic distribution of NPY rs 16139 in normal controls, overweight and obese Pakistani subjects. P-values <0.05 were considered significant. NS: non significant

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Figure 1

Figure 2