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vol. 7

Clinical research
Investigation of tRNALys/Leu and ATPase 6/8 gene mutations in Iranian ataxia telangiectasia patients

Massoud Houshmand
Sadaf Kasraie
Solmaz Etemad Ahari
Mostafa Moin
Mohammadali Bahar
Akram Zamani

Arch Med Sci 2011; 7, 3: 523-527
Online publish date: 2011/07/11
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Ataxia telangiectasia (AT) is a rare, neurodegenerative, autosomal recessive inherited disease that affects a number of different organs in the body and causes severe disability during childhood. About 20% of patients with AT develop cancer, particularly acute lymphocytic leukaemia or lymphoma. Many of these patients have been reported to have an impaired immune system, making them susceptible to recurrent respiratory infections. Ataxia telangiectasia is caused by mutations to the ATM (ataxia telangiectasia mutated) gene, which has been identified, sequenced and is located primarily on chromosome 11q22-23. Ataxia telangiectasia patients have shown more than 400 distinct ATM mutations, of which 85% are null mutations [1, 2]. The ATM protein is a member of the phosphatidylinositol 3-kinase-like family of serine/threonine protein kinases (PIKK) [3, 4]. Ataxia telangiectasia mutated protein is required for radiation-induced apoptosis and acts before mitochondrial collapse [5]. This protein is also involved in cell cycle control, intracellular protein transport, and the DNA damage response. Apoptosis is induced either through the death receptor pathway of apoptosis, or the mitochondrial pathway of apoptosis. This protein also plays a role in normal development and activity of body systems such as the nervous and immune system, which obviously indicates that any problem in the functions of the ATM protein could lead to immune system related diseases.

Mitochondria are present in almost all mammalian cells, and are responsible for energy generation. Despite the fact that the mitochondrial genome is very small, mitochondrial DNA mutations have an important role in genetic diseases [6, 7]. Mitochondrial DNA (mtDNA) mutations can result in both maternally inherited and sporadic diseases. The mitochondrial genome is 16.6-kb double-stranded circular DNA encoding 37 genes, including 13 proteins, 22 transfer RNAs, and 2 ribosomal RNAs. A number of mutations in the mitochondrial (mt) tRNA genes have been reported to be associated with human mitochondrial diseases.

It has been reported that the ATM protein is involved in regulation of cell check points and mutations leading to apoptosis, leaving us to hypothesize that mitochondrial DNA could play a role in AT patients who develop cancer, especially in the apoptotic pathway [8]. Many exhaustive screening approaches have been applied to investigate the mutations in mtDNA causing various human diseases. Many scientists have reported that point mutations in tRNALys/Leu genes and ATPase 6 and 8 genes are the leading causes of mitochondrial disorders [9]. To elucidate the molecular and functional role of mtDNA in patients with AT, it is required to establish that the mutations to mitochondrial (mt) tRNA could lead to the mitochondrial pathway of apoptosis in AT patients.

In this study we performed screening of mutations to the tRNALys/Leu genes and ATPase 6 and 8 genes in Iranian patients with AT who were referred to our centre. Primers for mitochondrial tRNALys/Leu and ATPase 6 genes were used on the DNA extracted from the blood samples of these patients and amplified by PCR. Automated DNA sequencing of the amplified DNA led to detection of possible DNA mutations in the mitochondrial (mt) tRNALys/Leu and ATPase 6 genes. Our additional analysis suggested that the presence of these mutations could be one of the causes of mitochondrial dysfunction in these AT patients leading to cancer.

Material and methods

Diagnosis of ataxia telangiectasia patients

All the patients were clinically defined as having AT by a neurologist according to generally accepted diagnosis criteria of AT [10]; all of the patients had gait ataxia, oculocutaneous telangiectasias, apraxia of eye movement or immunological defects that include immunoglobulin deficiencies (particularly IgA and IgE), high serum -fetoprotein concentration and lymphopenia. If the diagnosis was uncertain, molecular genetic tests for ATM mutations were performed to confirm the diagnosis [2].

Analysis of tRNALys/Leu genes and ATPase genes

Mutation screening of tRNALys/Leu genes and also ATPase6 genes was performed in 20 patients who were diagnosed based on the typical clinical features of ataxia telangiectasia. Peripheral blood samples were obtained and the DNA was purified using a Diatom DNA Extraction Kit (Genefanavaran, Tehran).

Two pairs of primers were used to amplify the targeted genes. The primers and the PCR conditions were replicated from previous studies [11, 12]. The nucleotide sequences were directly determined by automated sequencing in a 3700 ABI machine using forward primers (Macrogene Seoul, Korea). The obtained mtDNA sequences were aligned with a multiple sequence alignment interface CLUSTAL_X with comparison to Mitomap references (http: /www.gen.emory.edu/mitomap/ mitoseq.html).


The sequence analysis of samples showed the following mutations: A8774G, A8982C and C8684T in the mt-ATPase6 gene. Each mutation was found in one patient out of 20. These mutations in the MT-ATPase6 gene result in amino acid replacements. In 6 patients, an mtDNA polymorphism without changing the amino acid was identified. The findings showed six known polymorphisms in the mt-ATP6 gene. All of the variations were homoplasmic based on the sequencing results. Nucleotide changes in mitochondrial genes found in our patients are shown in Table I [18-26]. Nucleotide changes in mitochondrial tRNALeu were not found in the studied patients.


A prominent characteristic of mtDNA diseases is that tissues with a high energy demand, such as muscle and brain, are mostly affected [7]. Based on other investigations on Iranian AT patients, mtDNA deletions have been observed in about 54% of the patients. The discovered deletions remove or impair some structural genes including ATPase 6 and 8, COIII, ND3, ND4L, ND4, ND5, ND6, Cytb and eight tRNA genes. Such deletions in AT patients may result in multiple respiratory chain deficiencies [13]. The human mtDNA encodes 13 polypeptides that are essential for the mitochondrial energy generating system, oxidative phosphorylation (OXPHOS), as well as the tRNA genes which are necessary for their expression; thus, any mutation in the mtDNA coding region will alter mitochondrial energy production. Series of enzyme complexes are present in the mitochondria inner membrane which are known collectively to constitute the electron transport chain which consists of five complexes. Complex V (ATP synthase) is composed of 14 subunits. The protons expelled from other complexes create an electrochemical gradient for ATP production. Human mtDNA encodes 2 subunits (ATPases 6 and 8) of complex V subunits [14]. So any mutation in these genes may also disorganize the mitochondrial energy production processes. Defective energy metabolism resulting from loss of ATP production is certainly a common attribute of most OXPHOS diseases [6]. For example, point mutations in the ATP synthase subunit 6 have been observed in some disease including neuropathy ataxia retinitis pigmentosa (NARP) and Leigh syndromes [15].

Mitochondria use OXPHOS to generate most of the cellular ATP and as a toxic product they produce most of the endogenous reactive oxygen species (ROS). The ROS can damage the OXPHOS enzymes and mtDNA in turn, eroding mitochondrial function. In the presence of mtDNA mutations which may be caused by ROS during aerobic metabolism, sensitive cells such as skeletal muscles are deprived of ATP (due to the defective respiratory functions of mitochondria). Apoptosis is induced either through the death receptor pathway of apoptosis, or the mitochondrial pathway of apoptosis. When mitochondrial energy production gets too low and/or mitochondrial ROS damage becomes too high, similar to the condition happening in AT patients, the mitochondrial permeability transition pore (mtPTP) is activated and the cell is removed by apoptosis, thus resulting in more profound oxidative damage [16].

Since point mutations of mitochondrial tRNA genes are the hot spots of mitochondrial disorders, in this research the sequence of mitochondrial tRNALys/Leu genes and ATPase genes of 20 AT patients were analysed by PCR and automated sequencing methods. The results showed a significant level of mtDNA variations in AT patients. In one case a nucleotide variation (C8684T) resulting in amino acid change from Thr to Ile was observed in the MT-ATPase6 gene. Thr is categorized in polar amino acids, but Ile contains non-polar properties. This alteration may strongly affect the tertiary structure of protein that will impair its interaction with ATP.

Moreover, two samples in this study showed novel point mutations in the MT-ATP6 gene causing amino acid replacement. The first one was A8982C mutation in an affected patient which changes Gln to His. Gln has neutral properties but His is a basic amino acid. Therefore, it could deeply influence the structure of the subunit of respiratory complex V and the activity of this complex as a result. Regarding the second sample, we found a point mutation at position 8774 in the MT-ATP6 gene that alters Asn to Ser, where both of the amino acids have similar properties. Neither of these variations have been reported previously.

In the present research, approximately 60% of our patients had mtDNA mutations. Among the 12 mitochondrial mutations in this study, four of them (~30%) were missense mutations. Three of the patients (25%) in this study had mutations in the non-coding region of the MT-NC7 gene. All of the mutations in the coding region belonged to the mt-ATP6 gene with the exception of one variation in the mt-ATP8 gene.

Recent studies proposed involvement of mitochondrial pathway mediated apoptosis in the development of cancers in this group of patients and in immunopathogenesis of AT. In fact, apoptosis in AT could be via the mitochondrial pathway, which would offer a basis for establishment of mtDNA-targeted therapeutic interventions in AT [8].

Ataxia telangiectasia mutated is the gene mutated in ataxia telangiectasia and numerous different mutations in the ATM gene have been identified in classical AT and in some patients with variant forms of AT [17-26]. It was explained by Eaton et al. (2007) that their results show a novel function for ATM in mtDNA maintenance. This study hypothesizes that since ATM influences the de novo pathway for dNTP synthesis which is required for mtDNA replication and repair, and also since AT cells have higher amounts of ROS, the loss of ATM will affect the mtDNA copy number. Finally they offered novel roles for ATM in mitochondrial homeostasis.

Based on our results, there was probably a second effect of mtDNA mutations in AT disease and mtDNA plays a main role in establishment of AT. Perhaps mtDNA mutations are responsible for the decline of mitochondrial function in AT patients. In conclusion, we suggest more analysis to unravel the precise connection between ATM, mtDNA mutations and AT disease.


This work was supported by a grant (with research project No. 183) from the National Institute of Genetic Engineering and Biotechnology. We would like to thank all patients and their families whose collaboration and understanding allowed us to do this work, as well as their permission to publish it.


 1. Becker-Catania SG, Cheng G, Hwang MJ, et al. Ataxia-telangiectasia: phenotype/genotype studies of ATM protein expression, mutations, and radiosensitivity. Mol Genet Metab 2000; 70: 122-33.  

2. Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003; 3: 155-68.  

3. Goodarzi AA, Block WD, Lees-Miller SP. The role of ATM and ATR in DNA damage-induced cell cycle control. Prog Cell Cycle Res 2003; 5: 393-411.  

4. Vit JP, Moustacchi E, Rosselli F. ATM protein is required for radiation-induced apoptosis and acts before mitochondrial collapse. Int J Radiat Biol 2000; 76: 841-51.  

5. Taylor RW, Turnbull M. Mitochondrial DNA mutations in human disease. Nat Rev Genet 2005; 6: 389-402.  

6. Isaian A, Bogdanova NV, Houshmand M, et al. BAK, BAX and NBK/BIK proapoptotic gene alterations in Iranian patients with ataxia talangiectasia. J Clin Immunol 2010; 30: 132-7.  

7. Houshmand M. Mitochondrial DNA mutations, pathogenicity and inheritance. Iran J Biotechnol 2003; 1: 1-18.  

8. Sedgwick RP, Boder E. Ataxia talangiectasia. In: Handbook of clinical neurobiology. Elsevier 1991; 60: 347-423.  

9. Safaei S, Houshmand M, Banoei MM, et al. Mitochondrial tRNA Leu/Lys and ATPase 6/8 gene variations in spinocerebellar ataxias. Neurodegen Dis 2008; 6: 16-22.

10. Kasraie S, Houshmand M, Banoei MM, et al. Investigation of tRNALys/Leu and ATPase 6 genes mutations in Huntingtons disease. Cell Mol Neurobiol 2008; 28: 933-8.

11. Houshmand M, Sanati MH, Hooshiar Kashani B, et al. Investigation of mitochondrial deletions and haplogroups in Iranian ataxia-telangiectasia patient. Iran J Biotechnol 2006; 4: 64-8.

12. Wiesner RJ, Ruegg JC, Morano I. Counting target molecules by exponential polymerase chain reaction: copy number of mitochondrial DNA in rat tissues. Biochem Biophys Res Commun 1992; 183: 553-9.

13. Scheffler IE. A century of mitochondrial research: achievements and perspectives. Mitochondrion 2000; 1: 3-31.

14. Kokoszka JE, Coskun P, Esposito LA, Wallace DC. Increased mitochondrial oxidative stress in the Sod2 (+/-) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis. Proc Natl Sci USA 2001; 98: 2278-83.

15. Concannon P, Gatti RA. Diversity of ATM gene mutations detected in patients with ataxia – telangiectasia. Hum Mutat 1997; 10: 100-7.

16. Eaton JS, Ping Lin Z, Sartorelli AC, Bonawitz DN, Shadel SG. Ataxia-talangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis. J Clin Investig 2007; 117: 2723-34.

17. Brown MD, Starikovskaya YB, Derbeneva O, et al. The role of mtDNA background in disease expression: a new primary LHON mutation associated with Western Eurasian haplogroup. Human Genetics 2002; 110: 130-8.

18. Povalko N, Zakharova E, Rudenskaia G, et al. A new sequence variant in mitochondrial DNA associated with high penetrance of Russian Leber hereditary optic neuropathy. Mitochondrion 2005; 5: 194-9.

19. Poetsch M, Wittig H, Krause D, Lignitz E. The impact of mtDNA analysis between positions nt8306 and nt9021 for forensic casework. Mitochondrion 2003; 3: 133-7.

20. Rieder MJ, Taylor SL, Tobe VO, Nickerson DA. Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome. Nucleic Acids Res 1998; 26: 967-73.

21. Spagnolo M, Tomelleri G, Vattemi G, Filosto M, Rizzuto N, Tonin P. A new mutation in the mitochondrial tRNA(Ala) gene in a patient with ophthalmoplegia and dysphagia. Neuromusc Dis 2001; 11: 481-4.

22. Thomas AW, Edwards A, Sherratt EJ, Majid A, Gagg J, Alcolado JC. Molecular scanning of candidate mitochondrial tRNA genes in type 2 (non-insulin dependent) diabetes mellitus. J Med Genet 1996; 33: 253-5.

23. Qian Y, Zhou X, Hu Y, et al. Clinical evaluation and mitochondrial DNA sequence analysis in three Chinese families with Leber's hereditary optic neuropathy. Biochem Biophys Res Commun 2005; 332: 614-21.

24. Tanaka M, Ozawa T. Strand asymmetry in human mitochondrial DNA mutations. Genomics 1994; 22: 327-35.

25. Ozawa T, Tanaka M, Sugiyama S, et al. Patients with idiopathic cardiomyopathy belong to the same mitochondrial DNA gene family of Parkinson's disease and mitochondrial encephalomyopathy. Biochem Biophys Res Commun 1991; 177: 518-25.

26. Ozawa T, Tanaka M, Ino H, et al. Distinct clustering of point mutations in mitochondrial DNA among patients with mitochondrial encephalomyopathies and with Parkinson's disease. Biochem Biophys Res Commun 1991; 176: 938-46.
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