Publication: Hepatit B virüsü ile enfekte hastalarda genotip belirlenmesi
Abstract
Hepatit B virüsünün genomik yapısının araştırılmasında ileri moleküler biyolojik yöntemler kullanılarak önemli adımlar atılmıştır. HBV infeksiyonunun ciddi seyirli olması, kronikleşmesi ve tedavideki güçlükler infeksiyonun süregenliğinde konağın dışında virüsün genom yapısındaki bazı değişikliklerle de ilişkili olduğunu akla getirmektedir. Hepatit B virüsü genomları moleküler düzeyde incelendiğinde, genomlar arasında en az %8 veya daha fazla farklılık olanlar kendi aralarında gruplandırılarak günümüzde A'dan G'ye 7 genotip tanımlanmıştır. Bu genotipler farklı coğrafi dağılım ve klinik tablolara eşlik etmektedirler. Bu çalışma, kronik HBV infeksiyonlarında PZR-RFLP yöntemiyle HBV genotiplerinin belirlenmesi ve hastalığın klinik seyriyle karşılaştırılması amacıyla, Marmara Üniversitesi Tıp Fakültesi Gastroenteroloji Bilim Dalı işbirliği ile Marmara Üniversitesi Gastroenteroloji Enstitüsü'nde (M.Ü.G.E.) planlanmış ve enstitünün moleküler biyoloji laboratuvarında yapılmıştır. Bu amaçla çalışmaya, M.Ü.G.E.'nde Digene Kiti ile rutin olarak serumdan bakılan HBV DNA miktarlarından sonuçları 5 pg/ ml üzerinde pozitif olan 52 hasta alındı. HBV DNA, genotip tayini için NaOH ve HCl yöntemi ile serumdan ekstrakte edilip PZR ile çoğaltıldıktan sonra Tsp 5091 ve Hinf I restriksiyon enzimleri ile kesildi. RFLP yöntemi ile elde edilen sonuçları doğrulamak ve nükleotid farklılıklarını göstermek amacıyla 13 hastanın da otomatik DNA dizi analizleri, Ankara Üniversitesi Hepatoloji Enstitüsü'nde ABI 310 Genetic Analyser sistemi ile yapıldı. Çalışmada 52 kronik B hepatitli olgunun 24'ü çocuk ve 28'i ise erişkin hastalardan oluşmaktaydı. Hastaların tamamında HBV genotipinin genotip D olduğu saptandı. Aynı zamanda hastalarda bu genotipe ait literatürde belirtilen altı paternden üçü tespit edildi. D2 paterni %83 oranla baskın olarak saptandı. Sonuç olarak; ülkemizde hepatit B virüsü ile enfekte hastalarda D2 paternli genotip D dominant HBV genotipidir. Determination of Hepatitis B Genotypes in Patients with HBV Infection
Important steps have been achieved in the investigation of hepatitis B virus' genomic structure by the use of advanced molecular biological techniques. Genomic variation is an important subject since there are frequent relapsers or nonresponders among treated patients and the disease course can be quite severe. When HBV genomes were studied at molecular level, seven genomic groups called genotypes of HBV were defined based on a divergence of 8% or more of the complete genome and were designated from A to G. These genotypes were from different geographic regions and associated with different clinical outcomes. This study has been carried out at Marmara University Gastroenterology Institute (M.U.G.I.) in collaboration with Marmara University School of Medicine, Department of Gastroenterology in patients with chronic HBV infection with the aim of determining HBV genotypes by PCR-RFLP method and to compare the results with the patients clinical outcome. Fifty-two patients with an HBV DNA level over 5 pg/ ml as determined by Digene Kit were entered into the trial. To determine genotypes, HBV DNA was extracted from the serum by the NaOH and HCl method and amplified by PCR and then restricted by Tsp 5091 and Hinf I enzymes. Thirteen of patients DNA samples were sequenced automatically by ABI 310 system in Ankara University Hepatology Institute with the aim of confirming the results of RFLP method and to determine the nucleotide differences. In this study, 24 out of 52 patients with chronic hepatitis B were children and 28 were adults. Genotype D was found in all of the patients. Among 6 patterns of genotype D reported in the literature 3 were present in our series and D2 pattern was found to be the most frequent (83%). As a result, genotype D with pattern D2 is the dominant genotype in patients infected with HBV in our country.
Important steps have been achieved in the investigation of hepatitis B virus' genomic structure by the use of advanced molecular biological techniques. Genomic variation is an important subject since there are frequent relapsers or nonresponders among treated patients and the disease course can be quite severe. When HBV genomes were studied at molecular level, seven genomic groups called genotypes of HBV were defined based on a divergence of 8% or more of the complete genome and were designated from A to G. These genotypes were from different geographic regions and associated with different clinical outcomes. This study has been carried out at Marmara University Gastroenterology Institute (M.U.G.I.) in collaboration with Marmara University School of Medicine, Department of Gastroenterology in patients with chronic HBV infection with the aim of determining HBV genotypes by PCR-RFLP method and to compare the results with the patients clinical outcome. Fifty-two patients with an HBV DNA level over 5 pg/ ml as determined by Digene Kit were entered into the trial. To determine genotypes, HBV DNA was extracted from the serum by the NaOH and HCl method and amplified by PCR and then restricted by Tsp 5091 and Hinf I enzymes. Thirteen of patients DNA samples were sequenced automatically by ABI 310 system in Ankara University Hepatology Institute with the aim of confirming the results of RFLP method and to determine the nucleotide differences. In this study, 24 out of 52 patients with chronic hepatitis B were children and 28 were adults. Genotype D was found in all of the patients. Among 6 patterns of genotype D reported in the literature 3 were present in our series and D2 pattern was found to be the most frequent (83%). As a result, genotype D with pattern D2 is the dominant genotype in patients infected with HBV in our country.