GHSR rs2232165 Gen Polimorfizminin Genotiplendirilmesi için PCR-RFLP Yönteminin Optimizasyonu ve Türk Erkeklerinde Alel Frekansının Belirlenmesi

Year 2022, , 1245 - 1255, 28.12.2022

Selin Özkan Kotiloğlu , Dilek Kaya Akyüzlü , Gizem Düzgün , Mustafa Danışman , Ece Ağtaş Ertan , İnci Özgür İlhan

https://doi.org/10.35414/akufemubid.1137753

Abstract

Çağımızın ciddi sorunlardan biri olan alkol bağımlılığı, genetik, çevresel, kültürel, gelişimsel ve nörobiyolojik faktörlerin etkisiyle ortaya çıkmaktadır. Son yıllarda yeme davranışı nörobiyolojisi ile bağımlılık yapan maddelere aşerme davranışının nörobiyolojisinin benzer özelliklere sahip olduğunun keşfedilmesi araştırmacıları, alkol bağımlılığı ile grelin gibi besin alımında önemli olan hormonlar arasındaki ilişkiyi araştırmaya sevk etmiştir. Alkol kullanım bozukluğunda ve alkole aşermede grelin sisteminin önemi mevcut çalışmalar tarafından ortaya konulmasına karşın bu ilişkinin biyolojik mekanizmasının aydınlatılması için daha çok çalışmaya ihtiyaç vardır. Bu nedenle, bu çalışmada, alkol kullanım sorunu olan Türk erkeklerinde, grelin reseptörünü kodlayan GHSR genindeki rs2232165 polimorfizminin alkol kullanım sorunu ile ilişkisi araştırılmıştır. Çalışmaya alkol kullanım bozukluğu tanısı konmuş 72 erkek birey ile herhangi bir madde bağımlılığı olmayan 82 sağlıklı erkek dahil edilmiştir. GHSR rs2232165 gen polimorfizmi, ilk defa bu çalışmada optimize edilen PCR-RFLP yöntemi ile genotiplendirilmiştir. GHSR rs2232165 polimorfizmi için alel frekansları alkol kullanım sorunu olan grupta (n:144); C aleli için 0,99 (n:142), T aleli için 0,01 (n:2) olarak hesaplanmıştır. Karşılaştırma grubunda ise (n:164), C aleli frekansı 0,98 (n:161), T aleli frekansı 0,02 (n:3) olarak belirlenmiştir. İki grup arasında, T alel frekansı açısından istatistiksel olarak anlamlı bir fark bulunmamıştır (p>0,05). Gen polimorfizmleri, çevresel faktörlerden etkilenmediği için Türk erkeklerindeki minör alel frekansını (MAF) belirlemek için iki gruptaki bireyler birleştirilmiş (n=154) ve Türk erkeklerinde GHSR rs2232165 polimorfizminin MAF değeri 0,02 olarak belirlenmiştir. Bu çalışmada, çağımızda alkol kullanım bozukluğu kadar önemli olan obezite etiyolojisinde de rol oynayan GHSR rs2232165 polimorfizminin Türk erkeklerinde alel frekansı ile ilgili ilk veriler toplanmıştır.

Keywords

Alkol Kullanım Bozukluğu, Grelin, Gen Polimorfizmi, GHSR rs2232165, PCR-RFLP

Supporting Institution

Ankara Üniversitesi

Project Number

20B0217001

Thanks

Bu çalışma Ankara Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü tarafından 20B0217001 No.’lu proje kapsamında desteklenmiştir.

Optimization of PCR-RFLP Method for Genotyping of GHSR rs2232165 Gene Polymorphism and Determination of Allele Frequency in Turkish men

Abstract

Alcohol addiction, which is one of the crucial problems of the current era, arises with the effects of genetic, environmental, culturel, developmental and neurobiological factors. In recent years, the discovery that the neurobiology of eating behavior and the neurobiology of craving for addictive substances have similar characteristics has prompted researchers to investigate the relationship between alcohol addiction and hormones important in food intake such as ghrelin. Even though the importance of the ghrelin system in alcohol use disorder and alcohol craving has been stated by existing studies , more studies are needed to enlighten the biological mechanism of this relationship. Therefore, in this study the relationship between rs2232165 polymorphism in GHSR gene encoding ghrelin receptor and alcohol use disorder in Turkish men with alcohol use problem. Seventy-two individuals diagnosed with alcohol use disorder and 82 healthy men without any substance addiction were included in this study. The GHSR rs2232165 gene polymorphism was genotyped by PCR-RFLP which was optimized for the first time in this study. The allele frequency of the GHSR rs2232165 gene polymorphism was determined in the group with alcohol use problem (n:144) as 0,99 (n:142) for C allele and 0,01 (n:2) for T allele. The frequency of the C allele was 0,98 (n:161) and T allele was 0,02 (n:3) in the healthy group. There was no statistically significant difference between two groups in terms of T allele frequency. Since gene polymorphisms are not affected by environmental factors, individuals in two groups were combined (n=154) in order to determine minor allele frequency (MAF) in Turkish males, and the MAF value of GHSR rs2232165 polymorphism in Turkish males was determined as 0,02. In this study, the first data on the allele frequency of GHSR rs2232165 polymorphism, which plays a role in the etiology of obesity, which is as important as alcohol use disorder in the current era, were collected in Turkish males.

Keywords

Alcohol Use Disorder, Ghrelin, Gene Polymorphism, GHSR rs2232165, PCR-RFLP

Project Number

20B0217001

References

• Ando, T., Komaki, G., Naruo, T., Okabe, K., Takii, M., Kawai, K., Konjiki, F., Takei, M., Oka, T., Takeuchi, K., Masuda, A., Ozaki, N., Suematsu, H., Denda, K., Kurokawa, N., Itakura, K., Yamaguchi C., Kono, M., Suzuki, T., Nakai, Y., Nishizono‐Maher, A., Koide, M., Murakami, K., Nagamine, K., Tomita, Y., Ookuma, K., Tomita, K., Tonai, E., Ooshima, A., et al., 2006. Possible role of preproghrelin gene polymorphisms in susceptibility to bulimia nervosa. American Journal Medical Genetics Part B Neuropsychiatric Genetics, 141, 929-934.
• Ando, T., Ichimaru, Y., Konjiki, F., Shoji, M., Komaki, G., 2007. Variations in the preproghrelin gene correlate with higher body mass index, fat mass, and body dissatisfaction in young Japanese women. The American Journal of Clinical Nutrition, 86, 25-32.
• Ariyasu, H., Takaya, K., Tagami, T., Ogawa, Y., Hosoda, K., Akamizu, T., Suda, M., Koh, T., Natsui, K., Toyooka, S., Shirakami, G., Usui, T., Shimatsu, A., Doi, K., Hosoda, H., Kojima, M., Kangawa, K., Nakao, K., 2001. Journal of Clinical Endocrinology and Metabolism, 86, 4753-4758.
• Aydin, S., 2006. Increased grelin levels in epileptic patients. Seizure, 15, 658.
• Aydin, S., 2007. A comparison of grelin, glucose, alpha-amylase and protein levels in saliva from diabetics. Journal of Biochemistry and Molecular Biology, 40, 29-35.
• Aydın, S., Özkan, Y., Çaylak, E., Aydın, S., 2006. Ghrelin and its biochemical functions. Türkiye Klinikleri, Journal of Medical Sciences, 26, 272-283.
• Baessler, A., Hasinoff, M.J., Fischer, M., Reinhard, W., Sonnenberg, G.E., Olivier, M., Erdmann, J., Schunkert, H., Doering, A., Jacob, H.J., Comuzzie, A.G., Kissebah, A.H., Kwitek, A.E., 2005. Genetic linkage and association of the growth hormone secretagogue receptor (ghrelin receptor) gene in human obesity. Diabetes, 54, 259-267.
• Coşkun, B., Çakmak, D., 2005. Alkol ve madde bağımlılarının grup psikoterapisinde psikodramanın kullanılması. Bağımlılık Dergisi, 6, 103-110.
• D’Addario, C., Shchetynsky, K., Pucci, M., Cifani, C., Gunnar, A., Vukojevic, V., Padyukov, L., Terenius, L., 2017. Genetic variation and epigenetic modification of the prodynorphin gene in peripheral blood cellsin alcoholism. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 76, 195-203.Gelder, M., Gath, D., Mayou, R., Cowen, P., 1995, The abuse of alcohol and drugs. Oxford Textbook of Psychiatry, Gelder M, Gath D, Mayou R, Cowen P (ed.) Oxford University Press, Oxford, 438-461.
• Emeç, H., Gülay, E., 2008. Alkol tüketimi ve sosyo-demografik değişkenlerin alkol tüketimi üzerine etkisi. Ekonomik Yaklaşım Dergisi, 19, 115-134.
• Feng, X., Wang, S., Duan, X., Li, C., Yan, Z., Feng, F., Yu, S., Wu, Y., Wang, W., 2016. An improved PCR-RFLP assay for the detection of a polymorphism rs2289487 of PLIN1 gene. Journal of Clinical Laboratory Analysis, 30, 986-989.
• Garcia, E.A., King, P., Sidhu, K., Ohgusu, H., Walley, A., Lecoeur, C., Gueorguiev, M., Khalaf, S., Davies, D:, Grossman, A.B., Kojima, M., Petersenn, S., Froguel, P., Korbonits, M., 2009. The role of ghrelin and ghrelin-receptor gene variants and promoter activity in type 2 diabetes. European Journal of Endocrinology, 161, 307-315.
• Görgün, S., Tiryaki, A., Topbaş, M., 2010. Üniversite öğrencilerinde madde kullanma biçimleri. Anadolu Psikiyatri Dergisi, 11, 305-12.
• Groschl, M., Uhr, M., Kraus, T., 2004. Evaluation of the comparability of commercial grelin assays. Clinical Chemistry, 50, 457-458.
• Ho, R.K.S., Fok, P.W.Y., Chan, H.C.H., 2021. Pattern and determinants of alcohol and tobacco co-use and its relationship with smoking cessation in Hong Kong. Tobacco Prevention and Cessation, 24, 21.
• Holder, H.D., 1998. Alcohol and the community: a systems approach to preventation. Cambridge University Press.
• Holst, B., Schwartz, T.W., 2006. Ghrelin receptor mutations-too little height and too much hunger. Journal of Clinical Investigations, 116, 637-641.
• Jorde, A., Bach, P., Witt, S.H., Becker, K., Reinhard, I., Vollstädt-Klein, S., Kirsch, M., Hermann, D., Charlet, K., Beck, A., Wimmer, L., Frank, J., Treutlein, J., Spanagel, R., Mann, K., Walter, H., Heinz, A., Rietschel, M., Kiefer, F., 2014. Genetic variation in the atrial natriuretic peptide transcription factor GATA4 modulates amygdala responsiveness in alcohol dependence. Biological Psychiatry, 75, 790-797.
• Kaiya, H., Darras, V.M., Kangawa, K., 2007. Grelin in Birds: Its structure, distribution and function. The Journal of Poultry Science, 44, 18.
• Karpyak, V.M., Winham, S.J., Biernacka, J.M., Cunningham, J.M., Lewis, K.A., Geske, J.R., Colby, C.L., Abulseoud, O.A., Hall-Flavin, D.K., Loukianova, L.L., Schneekloth, T.D., Frye, M.A., Heit, J.A., Mrazek, D.A., 2014. Association of GATA4 sequence variation with alcohol dependence. Addiction Biology, 19, 312-315.
• Kierson, J.A., Dimatteo, D.M., Locke, R.G., Mackley, A.B., Spear, M.L., 2006. Grelin and cholecystokinin in term and preterm human breast milk. Acta Paediatrica, 95, 991-995.
• Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Grelin is a growth hormone-releasing acylated peptide from stomach. Nature, 402, 656-659.
• Kojima, M., Kangawa, K., 2005. Grelin: structure and function. Physiological Reviews, 85, 495-522. Kojima, S., Nakahara, T., Nagai, N., Muranaga, T., Tanaka, M., Yasuhara, D., Masuda, A., Date, Y., Ueno, H., Nakazato, M., Naruo, T., 2005. Altered grelin and peptide YY responses to meals in bulimia nervosa. Clinical Endocrinology, 62, 74-78.
• Korbonits, M., Goldstone, A.P., Gueorguiev, M., Grossman, A.B., 2004. Grelin-a hormone with multiple functions. Frontiers in Neuroendocrinology, 25, 27-68.
• Landgren, S., Jerlhag, E., Zetterberg, H., Gonzalez-Quintela, A., Campos, J., Olofsson, U., Nilsson, S., Blennow, K., Engel, J.A., 2008. Association of pro-ghrelin and GHS-R1A gene polymorphisms and haplotypes with heavy alcohol use and body mass. Alcoholism: Clinical and Experimental Research, 32, 2054-2061.
• Landgren, S., Jerlhag, E., Hallman, J., Oreland, L., Lissner, L., Strandhagen, E., Thelle, D.S., Zetterberg, H., Blennow, K., Engel, J.A., 2010. Genetic variation of the ghrelin signaling system in females with severe alcohol dependence. Alcoholism: Clinical and Experimental Research, 34, 1519-1524.
• Landgren, S., Simms, J.A., Hyytiä, P., Engel, J.A., Bartlett, S.E., Jerlhag, E., 2012. Ghrelin receptor (GHS-R1A) antagonism suppresses both operant alcohol self-administration and high alcohol consumption in rats. Addiction Biology, 17, 86-94.
• Leggio, L., Ferrulli, A., Cardone, S., Nesci, A., Miceli, A., Malandrino, N., Capristo, E., Canestrelli, B., Monteleone, P., Kenna, G.A., Swift, R.M., Addolorato, G., 2012. Ghrelin system in alcohol-dependent subjects: role of plasma ghrelin levels in alcohol drinking and craving. Addiction Biology, 17, 452-64.
• McKee, K.K., Palyha, O.C., Feighner, S.D., Hreniuk, D.L., Tan, C.P., Phillips, M.S., Smith, R.G., Van der Ploeg, L.H.T., Howard, A.D., 1997. Molecular analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors. Molecular Endocrinology, 11, 415-423.
• Meza, E., Kranzler, H.R., 1996. Closing the gap between alcoholism research and practise: the case for pharmacotherapy. Psychiatric Services, 47, 917-920.
• Mignini, F., Napolioni, V., Codazzo, C., Carpi, F.M., Vitali, M., Romeo, M., Ceccanti, M., 2012. DRD2/ANKK1 TaqIA and SLC6A3 VNTR polymorphisms in alcohol dependence: association and gene-gene interaction study in a population of Central Italy. Neuroscience Letters, 522, 103-107.
• Miller, D.W., Harrison, J.L., Brown, Y.A., Doyle, U., Lindsay, A., Adam, C.L., Lea, R.G., 2005. Immunohistochemical evidence for an endocrine/paracrine role for ghrelin in the reproductive tissues of sheep. Reproductive Biology and Endocrinology, 3, 60-74.
• Miyasaka, K., Hosoya, H., Sekime, A., Ohta, M., Amono, H., Matsushita, S., Suzuki, K., Higuchi, S., Funakoshi, A., 2006. Association of ghrelin receptor gene polymorphism with bulimia nervosa in a Japanese population. Journal of Neural Transmission, 113, 1279-1285.
• Monteleone, P., Tortorella, A., Castaldo, E.D.L., Filippo, C., Maj, M., 2007. The Leu72Met polymorphism of the ghrelin gene is significantly associated with binge eating disorder. Psychiatric Genetics, 17, 13-16.
• Otto, B., Cuntz, U., Fruehauf, E., Wawarta, R., Folwaczny, C., Riepl, R.L., Heiman, M.L., Lehnert, P., Fichter, M., Tschöp, M., 2001. Weight gain decreases elevated plasma ghrelin concentrations of patients with anorexia nervosa. European Journal of Endocrinology, 145, 669-673.
• Özen Bekar, E., 2014. Bağımlılık yapıcı madde kullanımı olan hemşireler ve hemşirelik hizmetleri yönetiminin yaklaşımı: bir literatür incelemesi. Sağlık ve Hemşirelik Yönetimi Dergisi, 1, 43-47.
• Öztürk, Y. E., Kırlıoğlu, M., Kıraç, R., 2015. Alkol ve madde bağımlılığında risk faktörleri. Selçuk Üniversitesi Sosyal Bilimler Meslek Yüksek Okulu Dergisi, 18, 97-118.
• Petersenn, S., Rasch, A.C., Penshorn, M., Beil, F.U., Schulte, H.M., 2001. Genomic structure and transcriptional regulation of the human growth hormone secretagogue receptor. Endocrinology, 142, 2649-2659.
• Schuckit, M.A., 1994. Low level of response to alcohol as a predictor of future alcoholism. American Journal of Psychiatry, 151, 184-189.
• Shiiya, T., Nakazato, M., Mizuta, M., Date, Y., Mondal, M.S., Tanaka, M., Nozoe, S-I., Hosoda, H., Kangawa, K., Matsukura, S., 2002. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. Journal of Clinical Endocrinology and Metabolism, 87, 240-244.
• Smith, R.G., Van der Ploeg, L.H.T., Howard, A.D., Feighner, S.D., Cheng, K., Hickey, G.J., Wyvratt, M.J., Fisher, M.H., Nargund, R.P., Patchett, A.A., 1997. Peptidomimetic regulation of growth hormone secretion. Endocrinology Reviews, 18, 621-645.
• Suchankova, P., Yan, J., Schwandt, M.L., Stangl, B.L., Jerlhag, E., Engel, J.A., Hodgkinson, C.A., Ramchandani, V.A., Leggio, L., 2017. The Leu72Met Polymorphism of the Prepro-ghrelin Gene is Associated With Alcohol Consumption and Subjective Responses to Alcohol: Preliminary Findings. Alcohol and Alcoholism. 52, 425-430.
• Tanrıkulu, A.C., Carman, K.B., 2009. Kars il merkezinde çeşitli üniversite öğrencileri arasında sigara kullanımı sıklığı ve risk faktörleri. Türk Toraks Dergisi, 10, 101-106.
• Tümer, A., 1989. Endokrinolojiye Giriş. Hacettepe Üniversitesi, Ankara, 1-4.
• Türkcan, A., 1998. Türkiye’de Madde Kullananların Profili: Hastane Verilerinin İncelenmesi. Düşünen Adam, 11, 56-64.
• Wang, H.J., Geller, F., Dempfle, A., Schauble, N., Friedel, S., Lichtner, P., 2004. Ghrelin receptor gene: identification of several sequence variants in extremely obese children and adolescents, healthy normal-weight and underweight students, and children with short normal stature. Journal of Clinical Endocrinology and Metabolism, 89, 157-162.
• Warnault, V., Darcq, E., Morisot, N., Phamluong, K., Wilbrecht, L., Massa, S.M., Longo, F.M., Ron, D., 2016. The BDNF Valine 68 to Methionine Polymorphism Increases Compulsive Alcohol Drinking in Mice That Is Reversed by Tropomyosin Receptor Kinase B Activation. Biological Psychiatry, 79, 463-473.
• Wren, A.M, Seal, L.J., Cohen, M.A., Brynes, A.E., Frost, G.S., Murphy, K.G., Dhillo, W.S., Ghatei, M.A., Bloom, S.R., 2001. Grelin enhances appetite and increases food intake in humans. Journal of Clinical Endocrinology and Metabolism, 86, 5992-5995.
• Yalçın, M., Eşsizoğlu, A., Akkoç, H., Yaşan, A., Gürgen, F., 2009. Dicle Üniversitesi öğrencilerinde madde kullanımını belirleyen risk faktörleri. Klinik Psikiyatri, 12, 125-133.
• Zhang, J.V., Ren, P.G., Avsian-Kretchmer, O., Luo, C.W., Rauch, R., Klein, C., Hsueh, A.J., 2005. Obestatin, a peptide encoded by the grelin gene, opposes grelin’s effects on food intake. Science, 310, 996-999.