Guyem KOLBAŞI DEMİRCİOĞLU, Sezen GÜNTEKİN ERGÜN, Kıvılcım GÜCÜYENER, Ferda E. PERÇİN, Mehmet Ali ERGUN

MİTOKONDRİYAL DNA MUTASYONLARININ TEKRAR DİZİLEME ARRAY YÖNTEMİ İLE ALTI AİLEDE DEĞERLENDİRİLMESİ

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

EVALUATION OF MITOCHONDRIAL DNA MUTATIONS IN SIX FAMILIES BY RESEQUENCING ARRAY

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.

Keywords:

Mitochondrial DNA, mutation, Array,

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1. Schapira AHV. Mitochondrial disease. Lancet 2006;368(9529):70-82. [CrossRef] google scholar
2. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, editors. Molecular biology of the cell. 5th ed. New York: Garland Science; 2008. [CrossRef] google scholar
3. Tuppen HA, Blakely EL, Turnbull, Taylor RW. Mitochondrial DNA mutations and human disease. Biochim Biophys Acta 2010;1797(2):113-28. [CrossRef] google scholar
4. Arning L, Haghikia A, Taherzadeh-Fard E, Saft C, Andrich J, Pula B, et al. Mitochondrial haplogroup H correlates with ATP levels and age at onset in Huntington disease. J Mol Med (Berl) 2010;88(4):431-6. [CrossRef] google scholar
5. Andreu AL, DiMauro S. Current classification of mitochondrial disorders. J Neurol 2003;250:1403-6. [CrossRef] google scholar
6. Buajitti E, Rosella LC, Zabzuni E, Young LT, Andreazza AC. Prevalence and health care costs of mitochondrial disease in Ontario, Canada: A population-based cohort study. PLoS ONE 2022;17(4): e0265744. [CrossRef] google scholar
7. Jakupciak JP, Maragh S, Markowitz ME, Greenberg AK, Hoque MO, Maitra A, et al. Performance of mitochondrial DNA mutations detecting early stage cancer. BMC Cancer 2008;8:285. [CrossRef] google scholar
8. Legati A, Zanetti N, Nasca A, Peron C, Lamperti C, Lamantea E, et al. Current and new next-generation sequencing approaches to study mitochondrial DNA. J Mol Diagn 2021;23(6):732-41. [CrossRef] google scholar
9. Dhorne-Pollet S, Barrey E, Pollet N. A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants. BMC Genomics 2020;21(1):785. [CrossRef] google scholar
10. Wolf NI, Smeitink JA. Mitochondrial disorders: a proposal for consensus diagnostic criteria in infants and children. Neurology 2002;59(9):1402-5. [CrossRef] google scholar
11. Morava E, van den Heuvel L, Hol F, de Vries MC, Hogeveen M, Rodenburg RJ, et al. Neurology 2006;67:1823-6. [CrossRef] google scholar
12. Shokrzadeh M, Mohammadpour A. Evaluation of a modified salt-out method for DNA extraction from whole blood lymphocytes: A simple and economical method for gene polymorphism. Pharm Biomed Res 2018;4(2):28. [CrossRef] google scholar
13. Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 1999;23(2):147. [CrossRef] google scholar
14. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17(5):405-24. [CrossRef] google scholar
15. McCormick EM, Lott MT, Dulik MC, Shen L, Attimonelli M, Vitale O. Specifications of the ACMG/AMP standards and guidelines for mitochondrial DNA variant interpretation. Hum Mutat 2020;41(12):2028-57. [CrossRef] google scholar
16. Mustafa MF, Fakurazi S, Abdullah MA, Maniam S. Pathogenic Mitochondria DNA Mutations: Current detection tools and interventions. Genes (Basel) 2020;11(2):192. [CrossRef] google scholar
17. Rusecka J, Kaliszewska M, Bartnik E, Tonska K. Nuclear genes involved in mitochondrial diseases caused by instability of mitochondrial DNA. J Appl Genet 2018;59(1):43-57. [CrossRef] google scholar
18. Vasta V, Ng SB, Turner EH, Shendure J, Hahn SH. Next generation sequence analysis for mitochondrial disorders. Genome Medicine 2009;1:100. [CrossRef] google scholar
19. Hartmann A, Thieme M, Nanduri LK, Stempfl T, Moehle C, Kivisild T, et al. Validation of microarray-based resequencing of 93 worldwide mitochondrial genomes. Hum Mutat 2009;30:115-22. [CrossRef] google scholar
20. Wikman FP, Lu ML, Thykjaer T, Olesen SH, Andersen LD, Cordon-Cardo C, et al. Evaluation of the performance of a p53 sequencing microarray chip using 140 previously sequenced bladder tumor samples. Clin Chem 2000;46(10):1555-61. [CrossRef] google scholar
21. Sigurdsson S, Hedman M, Sistonen P, Sajantila A, Syvänen AC. A microarray system for genotyping 150 single nucleotide polymorphisms in the coding region of human mitochondrial DNA. Genomics 2006;87(4):534-42. [CrossRef] google scholar
22. Riordan-Eva P, Harding AE. Leber’s hereditary optic neuropathy: the clinical relevance of different mitochondrial DNA mutations. J Med Genet 1995;32(2):81-7. [CrossRef] google scholar
23. Sharkawi E, Oleszczuk JD, Holder GE, Raina J. Clinical and electrophysiological recovery in Leber hereditary optic neuropathy with G3460A mutation. Doc Ophthalmol 2012;125(1):71-4. [CrossRef] google scholar
24. Hudson G, Keers S, Yu Wai Man P, Griffiths P, Huoponen K, Savontaus ML, et al. Identification of an X-Chromosomal locus and haplotype modulating the phenotype of a mitochondrial DNA disorder. Am J Hum Genet 2005;77(6):1086-91. [CrossRef] google scholar
25. Newman NJ. From genotype to phenotype in Leber hereditary optic neuropathy: still more questions than answers. J Neuroophthalmol 2002;22(4):257-61. [CrossRef] google scholar