EVALUATION OF MITOCHONDRIAL DNA MUTATIONS IN SIX FAMILIES BY RESEQUENCING ARRAY

Öz

Objective: Human mitochondrial DNA is a circular, double stranded molecule which is inherited through maternal lineage. Point mutations in tRNA, rRNA or protein coding genes and structural rearrangements such as partial deletions or duplications can cause mitochondrial disorders. The prevalence of mitochondrial diseases is estimated to be 1/5000 worldwide. For the analysis of mtDNA mutations, Sanger sequencing, Southern blot, long and quantitative PCR, Resequencing Array and next-generation sequencing methods can be used. In this study, we analysed whole mitochondrial genomes of six children (along with their mothers) who were admitted to Gazi University Hospital with symptoms suggestive of mitochondrial disease. Materials and Methods: After the extraction of genomic DNA from six children and their mothers, mtDNA resequencing with the analysis of obtained data was performed. In order to determine whether one of the mutations found in Patient 4 was homoplasmic or heteroplasmic, PCR and RFLP techniques were also used. Results: Among six patients included in this study group, none of the variants detected could be attributed to any mitochondrial diseases, except the pathogenic mutation detected in Patient 4. The m.3460 G>A mutation detected in Patient 4 was located in the MT-ND1 gene that was known to be responsible for LHON. This mutation detected in Patient 4 was also detected both in his mother and sister with homoplasmic state. The lack of clinical findings in his mother and sister was thought to be due to decreased penetrance of the disease in females and modifying genes in nuclear genome. Conclusion: Screening of mtDNA using resequencing method could provide fast, effective and more reliable results in the diagnosis of mitochondrial diseases. Also, currently, the NGS technology analysis of nuclear DNA along with mtDNA will provide more reliable results in diagnosis of mitochondrial diseases, thus allowing more accurate genotype-phenotype correlation.

Anahtar Kelimeler

• Mitochondrial DNA
• mutation
• Array

MİTOKONDRİYAL DNA MUTASYONLARININ TEKRAR DİZİLEME ARRAY YÖNTEMİ İLE ALTI AİLEDE DEĞERLENDİRİLMESİ

Öz

Amaç: İnsan mitokondriyal DNA’sı, maternal kalıtılan dairesel, çift sarmallı bir moleküldür. tRNA, rRNA veya protein kodlayan genlerdeki nokta mutasyonları ve kısmi delesyonlar veya duplikasyonlar gibi yapısal yeniden düzenlemeler mitokondriyal bozukluklara neden olabilir. Mitokondriyal hastalıkların dünya genelinde prevalansının 1/5000 olduğu tahmin edilmektedir. MtDNA mutasyonlarının analizi ile ilgili olarak Sanger dizileme, Southern blot, kantitatif PCR, tekrar dizileme ve yeni nesil dizileme yöntemleri kullanılabilir. Gereç ve Yöntem: Bu çalışmada, mitokondriyal hastalığı düşündüren semptomları olan ve Gazi Üniversitesi Hastanesi’ne başvuran 6 çocuk ve annesinden genomik DNA’nın elde edilmesinin ardından mtDNA yeniden dizileme yöntemi ile elde edilen verilerin analizi yapıldı. Dördüncü hastada bulunan mutasyonlardan birinin homoplazmik veya heteroplazmik olduğunu belirlemek için PCR-RFLP tekniği kullanıldı. Bulgular: Çalışma grubumuza dahil edilen altı hastadan 4. hastada saptanan patojenik mutasyon dışında, saptanan değişikliklerin hiçbiri hastalarımızdaki mitokondriyal hastalık ile ilişkilendirilmemiştir. Dördüncü hastada saptanan m.3460 G>A mutasyonu: MT-ND1 geninde lokalize olup LHON’dan sorumludur. Dördüncü hastada saptanan bu mutasyon, hastanın anne ve kız kardeşinde de homoplazmik olarak saptandı. Anne ve kız kardeşinde klinik bulgu olmamasının, kadınlarda hastalığın penetransının azalmasına ve nükleer genomdaki genlerin modifiye edilmesine bağlı olduğu düşünüldü. Sonuç: mtDNA’nın yeniden dizileme yöntemiyle taranması mitokondriyal hastalıkların tanısında hızlı, etkili ve daha güvenilir sonuçlar sağlayabilir. Ayrıca, şu anda NGS teknolojisi ile nükleer DNA’nın mtDNA ile birlikte analiz edilmesi mitokondriyal hastalıkların tanısında daha güvenilir sonuçlar verecek ve böylece daha doğru genotip-fenotip korelasyonuna izin verecektir.

Anahtar Kelimeler

• Mitokondriyal DNA
• mutasyon
• array

Kaynakça

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