Gestasyonel Diabetes Mellitus'lu hastalarda irisin (FNDC5) gen polimorfizmlerinin araştırılması
Investigation of irisin (FNDC5) gene polymorphisms in patients with gestational diabetes mellitus
Yazar: HALİL CAŞKA
Danışman: PROF. DR. HALİT AKBAŞ
Harran Üniversitesi / Sağlık Bilimleri Enstitüsü / Tıbbi Biyoloji Ana Bilim Dalı
Derece: Yüksek Lisans, 2020
Konu: Tıbbi Biyoloji = Medical Biology
Dizin: Diabet-gebelik sırasında = Diabetes-gestational ; Diabetes mellitus = Diabetes mellitus ; Genler = Genes ; Polimorfizm-genetik = Polymorphism-genetic ; İrisin = Irisin

Özet:
Amaç: Bu çalışmada İrisin (FNDC5) genindeki iki polimorfizmin rs726344 (G>A) ve rs16835198 (G>T) serum irisin düzeyi üzerindeki etkisi ve Gestasyonel Diabetes Mellitus (GDM) ile ilişkisinin araştırılması amaçlanmıştır.
Yöntem: Bu çalışma için; Harran Üniversitesi Tıp Fakültesi Kadın Hastalıkları ve Doğum Anabilim Dalı polikliniklerine başvuran, ADA (American Diabetes Association) kriterlerine göre, Gestasyonel Diabetes Mellitus tanısı alan 110 hasta ve benzer yaş grubunda olan 123 sağlıklı birey kontrol grubu olarak çalışmaya dahil edilmiştir. Çalışma grubundaki bireylerden rutin laboratuvar tetkikleri amacıyla hemogram tüplerine (EDTA'lı) alınmış olan kan örneklerinden spin kolon yöntemi ile Genomic DNA izolasyon kiti kullanılarak DNA izolasyonu yapılmıştır. Her bireydeki FNDC5 genine ait polimorfizmlerin [c.499+250 C>T (rs726344) ve g.33326681G>T (rs16835198)] moleküler analizi, Real-Time PCR yöntemi kullanılarak gerçekleştirilmiştir. FNDC5 geni üzerinde seçilen iki çift primer (forward ve reverse), ve iki çift prob sekans dizilerine göre sentezlettirilerek Real-Time PCR'da genomik DNA'nın çoğaltılmasında ve genin söz konusu polimorfizmler bakımından genotiplendirilmesinde kullanılmıştır.
Bulgular: Bu araştırmada FNDC5 geni c.499+250 C>T (rs726344) polimorfizminin GDM'li hasta ve kontrol grubundaki dağılımı GDM'li hasta grubunda homozigot GG genotipi 97 bireyde (%88), heterozigot GA genotipi 13 bireyde (%12) tespit edilmiş olup homozigot AA genotipine ise hiçbir bireyde rastlanmamıştır. Kontrol grubu genotipler açısından incelendiğinde, homozigot GG genotipi 108 bireyde (%88), heterozigot GA genotipi 14 bireyde (%11) ve homozigot AA genotipi ise 1 bireyde (%1) tespit edilmştir. Yapılan analizler sonucunda FNDC5 geni c.499+250 C>T (rs726344) polimorfizminin GDM'li hasta ve kontrol grubundaki genotip dağılımında istatiksel olarak anlamlı bir fark saptanmamıştır (p>0.05). FNDC5 geni g.33326681G>T (rs16835198) polimorfizminin GDM'li hasta ve kontrol grubundaki genotip dağılımına bakıldığında homozigot GG genotipinin 64 bireyde ve %58 oranında, kontrol grubunda ise 70 bireyde ve %57 oranında olduğu saptanmıştır. Heterozigot GT genotipinin hasta grubunda 34 bireyde %31 oranında, kontrol grubunda ise 43 bireyde %35 oranında olduğu tespit edilmiştir. Homozigot TT genotipinin hasta grubunda 12 bireyde %11 oranında, kontrol grubunda ise 10 bireyde %8 oranında olduğu görülmüştür. FNDC5 geni g.33326681 G>T (rs16835198) polimorfizmi bakımından da hasta ve kontrol gruplarındaki genotip dağılımları karşılaştırıldığında istatiksel olarak anlamlı bir fark saptanmıştır (p>0.05).
Sonuç: Araştırma neticesinde elde edilen bulgular, FNDC5 geni c.499+250 C>T (rs726344) ve g.33326681 G>T (rs16835198) polimorfizmlerinin gestasyonel diabetes mellitus ile ilişkili olmadığı sonucuna varılmıştır.
Anahtar Kelimeler: FNDC5, İrisin, Polimorfizm, GDM, Gen

Abstract:
Objective: Gestational Diabetes Mellitus (GDM) is defined as a disease of glucose intolerance, which is first identified in pregnancy. Transmembrane protein fibronectin type III domain-containing protein 5 (FNDC5) that is encoded by the FNDC5 gene has been demonstrated to be cleaved and released as a novel hormone-like myokine irisin. FNDC5 is expressed from, heart, brain, liver, skeletal muscle, ovary, spleen, kidney, lung, pancreas, thymus, prostate, small intestine, large intestine and adipose tissues.  İrisin is a protein with 112 amino acids and a molecular weight of 12 kDa. Irisin belongs to the class of adipomyokines since it acts both in adipose and muscle tissue (adipokine and myokine) and is a thermogenic protein that promotes energy expenditure by WAT browning. Irisin plays an essential role in several functions associated with some important diseases. In the myokine group, Irisin peptide, released from muscle cells, acting as a messenger between muscle and adipose tissue, has been known to play an important role in insulin resistance and energy metabolism regulation. In this study, it was aimed to investigate the effects of two polymorphisms c.499+250 C>T (rs726344) and g.33326681 G>T (rs16835198) in the Irisin (FNDC5) gene on serum Irisin level and their relationship with Gestational Diabetes Mellitus (GDM).
Methods: The study included 233 pregnanat subjects who applied to Harran University Medical Faculty Obstetrics and Gynecology Outpatient Clinic. The study population was divided into two groups; the first one is 110 subjects of the GDM group and the second one is 123 subjects of the control (Non-GDM subjects) group. 110 subjects of the first group, who diagnosed with GDM at the 24-28 gestational period, were chosen as the patient group according to the criteria of ADA (American Diabetes Association). DNA isolation was performed by using Genomic DNA isolation kit with spin column method from blood samples taken from hemogram tubes (with EDTA) for routine laboratory tests from subjects in the study group. Molecular analysis of polymorphisms c.499+250 C>T (rs726344) and g.33326681 G>T (rs16835198) of the FNDC5 gene of each subject was performed by using the Real-Time PCR.
Two pairs of primers (forward and reverse) and two pairs of probes selected on the FNDC5 gene were synthesized according to sequence order and used to replicate genomic DNA in Real-Time PCR and the polymorphisms were genotyped by amplification curves gained from the RT-PCR results.
Results: In this study, distribution of FNDC5 gene G> A polymorphism in GDM patients and control group was determined as homozygous GG (88%) in 97 subjects  and heterozygous GA genotype in 13 subjects (12%), and homozygous AA genotype was not found in any subject. In terms of genotype when the control group was examined, homozygous GG genotype was detected in 108 subjects (88%), heterozygous GA genotype in 14 subjects (11%), and homozygous AA genotype in 1 subject (1%). As a result of the analyzes made, statistically no significant difference was observed in the genotype distribution of FNDC5 gene c.499+250 C>T (rs726344) polymorphism in GDM patient and control groups (p>0.05). The genotype distribution of the FNDC5 gene g.33326681 G>T (rs16835198) polymorphism in GDM patients and the control group was observed as homozygous GG genotype in 64 subjects and 58%, in the control group 70 subjects and 57%. The heterozygous GT genotype was observed as 31% in 34 patients and 35% in 43 subjects in the control group. The homozygous TT genotype was observed as 11% in 12 subjects in the patient group and 8% in 10 subjects in the control group. In terms of FNDC5 gene g.33326681 G>T (rs16835198) polymorphism, when the genotype distributions in the patient and control groups were compared it was found that it was not statistically significant in terms of genotype distributions (p>0.05).
Conclusions: As a result of this research, it was concluded that FNDC5 gene c.499+250 C>T (rs726344) and g.33326681 G>T (rs16835198) polymorphisms are not related to gestational diabetes mellitus.
Keywords: FNDC5, Irisin, Polymorphism, GDM, Gene