Mutational analysis of the mitochondrial tRNALeu(UUR) gene in Tunisian patients with mitochondrial diseases

Biochemical and Biophysical Research Communications 355 (2007) 1031–1037 www.elsevier.com/locate/ybbrc

Mutational analysis of the mitochondrial tRNALeu(UUR) gene in Tunisian patients with mitochondrial diseases Emna Mkaouar-Rebai a,*, Abdelaziz Tlili a, Saber Masmoudi a, Neila Belguith a, Ilhem Charfeddine b, Mouna Mnif d, Chahnez Triki c, Faiza Fakhfakh a a

Laboratoire de Ge´ne´tique Mole´culaire Humaine, Faculte´ de Me´decine, 3029 Sfax, Tunisia b Service d’O.R.L., C.H.U. Habib Bourguiba de Sfax, Tunisia c Service de Neurologie, C.H.U. Habib Bourguiba de Sfax, Tunisia d Service d’endocrinologie, C.H.U. Habib Bourguiba de Sfax, Tunisia Received 13 February 2007 Available online 26 February 2007

Abstract The mitochondrial tRNALeu(UUR) gene (MTTL) is a hot spot for pathogenic mutations that are associated with mitochondrial diseases with various clinical features. Among these mutations, the A3243G mutation was associated with various types of mitochondrial multisystem disorders, such as MIDD, MELAS, MERRF, PEO, hypertrophic cardiomyopathy, and a subtype of Leigh syndrome. We screened 128 Tunisian patients for the A3243G mutation in the mitochondrial tRNALeu(UUR) gene. This screening was carried out using PCR-RFLP with the restriction endonuclease ApaI. None of the 128 patients or the 100 controls tested were found to carry the mitochondrial A3243G mutation in the tRNALeu(UUR) gene in homoplasmic or heteroplasmic form. After direct sequencing of the entire mitochondrial tRNALeu(UUR) gene and a part of the mitochondrial NADH dehydrogenase 1, we found neither mutations nor polymorphisms in the MTTL1 gene in the tested patients and controls, and we confirmed the absence of the A3243G mutation in this gene. We also found a T3396C transition in the ND1 gene in one family with NSHL which was absent in the other patients and in 100 controls. Neither polymorphisms nor other mutations were found in the mitochondrial tRNALeu(UUR) gene in the tested patients.  2007 Elsevier Inc. All rights reserved. Keywords: A3243G mutation; tRNALeu(UUR) gene; Mitochondrial ND1 gene; Diabetes; Hearing loss; MIDD; T3396C

Mitochondrial diseases are clinically a very heterogeneous group of disorder ranging from single organ involvement to a severe multisystem diseases [1]. The diagnosis and classification of these disorders are difficult because the same genetic cause can be associated with different clinical syndromes. Mutations in mitochondrial DNA (mtDNA) are involved in the pathogenesis of several clinical syndroms. Point mutations in mitochondrial tRNA genes are particularly frequent since over 100 pathogenic mtDNA mutations were described in various tRNA genes (www.mitomap.org). *

Corresponding author. Fax: +216 74 46 14 03. E-mail addresses: [email protected] (E. MkaouarRebai), [email protected] (F. Fakhfakh). 0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.02.083

The mitochondrial tRNALeu(UUR) gene is a hot spot for pathogenic mutations that are associated with mitochondrial diseases with various clinical features. In fact, until today, 21 different point mutations in this gene have been reported (www.mitomap.org; Fig. 1). Among these mutations in the mitochondrial tRNALeu(UUR) gene, the A3243G mutation was associated with various types of mitochondrial multisystem disorders, such as mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) [2–4]. Mitochondrial inherited diabetes and deafness (MIDD) [5], Myoclonus epilepsy with ragged red fibres (MERRF) [6], maternally inherited progressive external ophthalmoplegia (PEO) [7], hypertrophic cardiomyopathy [8], and Leigh syndrome [4,8–10].

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which is located next to the mitochondrial tRNALeu(UUR) gene. This transition was found in only one family with a nonsyndromic hearing loss and was absent in the other patients and in 100 normal individuals. No polymorphisms or other mutations were found in the mitochondrial tRNALeu(UUR) gene. Patients and methods Patients

Fig. 1. Secondary structure of the mitochondrial tRNALeu(UUR) gene with illustrated point mutations.

Cellular studies investigating the A3243G mutant revealed deficiencies in RNA processing [11], aminoacylation [12], post-transcriptional tRNA modification [13], and translation [14]. Patients harbouring the A3243G mutation present an impaired oxidative phosphorylation with a deficient activity of respiratory chain complexes [15]. The prevalence of the mitochondrial A3243G mutation may be as high as 1–1.5% in certain diabetic populations [16]. However, the frequency of this mutation varies considerably in Asian patients: from 0.2% in Korea [17] to 1% in unselected diabetic patients in Japan [18,19], to 4.1% in diabetic Japanese patients [20]. In the common population of Finland, the prevalence of the mitochondrial A3243G mutation was found to be 16.3% in the adult population after a clinical selection for the MELAS and diabetes clinical features, and was thus one of the most common contributors to neurodegenerative disorders [21]. In the population of two British diabetes hospitals, the prevalence of the A3243G mutation was 0.13% [22]. Up to now, the prevalence of the mitochondrial A3243G mutation in the Tunisian population have been unknown since no data have been published yet. In order to determine the prevalence of the mitochondrial A3243G mutation in the mitochondrial tRNALeu(UUR) gene in Tunisian patients with type 2 diabetes, with nonsyndromic hearing loss and with other mitochondrial diseases, one-hundred and twenty-eight Tunisian patients with mitochondrial disorders were screened for the A3243G mutation by PCR-RFLP with ApaI restriction enzyme. None of the 128 patients or the 100 controls tested were found to carry this mutation. We also explored the entire mitochondrial tRNALeu(UUR) gene and a part of the mitochondrial NADH dehydrogenase 1 (ND1) by direct sequencing in order to find mitochondrial variants in these genes, and we revealed the presence of a transition in the mitochondrial ND1 gene,

A total of 128 Tunisian patients were tested in this study including 70 patients with nonsyndromic hearing loss, 30 diabetic subjects, and 28 patients belonging to 28 unrelated Tunisian families having clinical presentation highly suggestive of mitochondrial diseases. All the latter 28 patients showed two or more of the following clinical features: psychomotor regression, hearing loss, diabetes, myopathy, cardiomyopathy, leucodystrophy, hypotonia, hyperlacticacidemia, cortical atrophy. . . (Table 1). In addition, 100 Tunisian normal individuals were tested as controls. Methods DNA extraction. After getting informed consent from all the participating family’s members, total DNA was extracted from peripheral blood using standard procedures [23]. Screening of the mitochondrial A3243G mutation in the tRNALeu(UUR) gene. The mitochondrial A3243G mutation was analysed by PCR-RFLP. PCR amplification of a 528 bp fragment containing the entire mitochondrial tRNALeu(UUR) gene was performed using the following primers: 5 0 TCTAGAGTCCATATCAACAA-3 0 (nt 2953–2972) and 5 0 -TTTGGTGA AGAGTTTTATGG-3 0 (nt 3480–3461) in a total volume of 50 lL, containing 200 ng of genomic DNA, 6 pmol of each primer, 500 lM of each dNTP, 2 mM MgCl2, 1· PCR buffer, and 2 U of Taq DNA polymerase. The conditions for the PCR were: 95 C for 5 min, followed by 35 cycles at 94 C for 1 min, 53 C for 1 min, 72 C for 1 min, and a final extension at 72 C for 5 min in a thermal cycler (GenAmp PCR System 9700; Applied Biosystem). After PCR amplification, 23 ll of PCR product were digested with 10 U of the restriction endonuclease ApaI (BioLabs), separated through 2% agarose gel and visualised with ethidium bromide in UV. The A3243G mutation created a restriction ApaI site and the digestion resulted in two fragments of 234 and 294 bp, whereas in the absence of this mutation, we observed a 528 bp fragment corresponding to the PCR product. Mutational analysis of the mitochondrial tRNALeu(UUR) gene. The 528 bp PCR product amplified previously contains the entire mitochondrial tRNALeu(UUR) gene (MTTL1) corresponding to the mitochondrial genome at position nt 3230–3304 and a part of the mitochondrial NADH dehydrogenase 1 gene (ND1). After PCR amplification, each fragment was purified using NucleoSpin (MACHEREY-NAGEL) and subsequently analysed by direct sequencing in an ABI PRISM 3100-Avant automated DNA sequencer using the BigDye Terminator Cycle Sequencing reaction kit v1.1. The resultant sequences were compared with the update Cambridge sequence (GenBank Accession No. NC_001807) [24].

Results Screening of the mitochondrial A3243G mutation in the tRNALeu(UUR) gene We screened 128 Tunisian patients for the A3243G mutation in the mitochondrial tRNALeu(UUR) gene. After PCR amplification, the PCR product was digested with

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Table 1 Clinical features of the 28 Tunisian patients with mitochondrial diseases Patients

Year of birth

Clinical phenotype

Group 1: Leigh syndrome Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11

1982 2003 Unknown Unknown Unknown 1996 1982 1967 2000 Unknown 1986

Leigh Leigh Leigh Leigh Leigh Leigh Leigh Leigh Leigh Leigh Leigh

Group 2: MIDD Patient 12 Patient 13 Patient 14 Patient 15

1958 1993 1959 Unknown

Diabetes mellitus + deafness maternally inherited Diabetes + hearing loss Diabetes + hearing loss + maculopathy Diabetes + hearing loss + hypothyroidy

Group 3: Leucodystrophy Patient 16 Patient 17 Patient 18

2003 1985 Unknown

Leucodystrophy + psychomotor retardation Leucodystrophy + seizures Leucodystrophy + hearing loss + hypotonia

Group 4: MELAS Patient 19

2004

Myoclonic encephalopathy + hearing loss

Group 5: Metabolic encephalopathy Patient 20 Patient 21

1992 Unknown

Metabolic encephalopathy + seizures + learning disabilities Encephalomyopathy + leucodystrophy

Group 6: Cardiomyopathy Patient 22

Unknown

Cardiomyopathy

Group 7: Syndromic hearing loss Patient 23 Patient 24 Patient 25 Patient 26

1997 2001 2000 ND

Congenital deafness + psychomotor regression Hearing loss + muscular weakness Hearing loss + seizures Hearing loss + leucodystrophy

Group 8: Others Patient 27 Patient 28

Unknown Unknown

Psychomotor regression + atrophy + mental retardation Psychomotor regression + metabolic dysfunction

syndrome + optic atrophy syndrome syndrome syndrome syndrome syndrome + staturoponderal retardation syndrome + optic atrophy + muscular weakness syndrome syndrome syndrome syndrome

MIDD, maternally inherited diabetes and deafness; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes.

the restriction endonuclease ApaI (Biolabs). The A3243G mutation created a restriction ApaI site and the digestion resulted in two fragments of 234 and 294 bp, whereas in the absence of this mutation, we observed a 528 bp fragment corresponding to the PCR product. After PCR amplification and a subsequent digestion, none of the 128 patients or the 100 controls were found to carry the A3243G mutation in the mitochondrial tRNALeu(UUR) gene in the homoplasmic or in the heteroplasmic form (Fig. 2). Mutational analysis of the mitochondrial tRNALeu(UUR) gene After direct sequencing of the entire PCR product amplified previously which contains the entire mitochondrial tRNALeu(UUR) gene and a part of the mitochondrial NADH

Fig. 2. Screening of the A3243G mutation by PCR-RFLP: A 528 bp PCR fragment is digested with ApaI. The wild-type PCR product not contains the ApaI restriction site, whereas PCR product containing the A3243G mutation is cleaved into two fragments of 234 and 294 bp in length. Lane 1, undigested PCR fragment; lanes 2 and 3, digestion in normal individuals; lane 4, pBR322 DNA/AluI Marker; lanes 5 and 6, digestion in patients.

dehydrogenase 1, we found no mutations or polymorphisms in the MTTL1 gene in the tested patients and controls, and

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proband’s mother who had a normal hearing. However, it was absent in the father and in the paternal grand-mother who were normal (Fig. 4). Discussion

Fig. 3. Sequence chromatograms from normal individual (A) and affected patient (B) with the T3396C transition in the mitochondrial ND1 gene.

we confirmed the absence of the A3243G mutation in this gene. However, we detected a T to C transition at the nucleotide 3396 of the mitochondrial genome located in the mitochondrial ND1 gene (NADH dehydrogenase 1) in a patient with a nonsyndromic hearing loss (Fig. 3). This transition was absent in the other patients and in 100 normal individuals tested. The analysis of the other members of the proband’s family revealed the presence of the T3396C transition and its inheritance in a maternal pattern. Indeed, this substitution was detected in the proband’s sister and in her aunt’s mother who were deaf and it was also found in the

Clinical features were very heterogeneous in patients harbouring the common pathogenic A3243G point mutation in the mitochondrial tRNALeu(UUR) gene. In fact, this mutation was associated with various types of mitochondrial multisystem disorders, such as MELAS, MERRF, PEO, MIDD, hypertrophic cardiomyopathy, and Leigh syndrome. In the present study, we performed a mutational screening of the mitochondrial tRNALeu(UUR) gene and a part of the mitochondrial ND1 gene by direct sequencing in 128 Tunisian patients including 70 patients with nonsyndromic hearing loss, 30 diabetic subjects, and 28 patients belonging to 28 unrelated families and presenting with mitochondrial diseases. We also tested 100 normal individuals as controls. Neither the PCR-RFLP analysis, nor sequencing detected the A3243G mutation in the mitochondrial tRNALeu(UUR) gene neither in the homoplasmic nor in the heteroplasmic form in the 70 deaf patients, in the 30 diabetic subjects, and in the 28 patients with mitochondrial diseases.

Fig. 4. Pedigree of the Tunisian family presenting the T3396C transition in the mitochondrial ND1 gene. The proband is indicated by an arrow. Asterisks indicate the individuals from whom DNA samples were obtained and tested. Generations are indicated on the left in Roman numerals and the numbers under the individuals represent identification numbers for each generation. Hearing impaired individuals are indicated by filled symbols.

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This mutation was described as the most prevalent mutation with a prevalence ranging from 0.1% to 10%. In fact, in the population of two British diabetes hospitals, the prevalence of the mitochondrial A3243G mutation was 0.13% [22]. This substitution was also reported in 0.2% of Korean diabetic patients [17], in 1% of Japanese patients [18], and in 6% of unselected groups of patients with diabetes mellitus type 2 [5] or type 1 [25]. In 1995, Odawara et al. found this same mutation in 3 out of 300 patients with noninsulin-dependent diabetes mellitus or impaired glucose tolerance, and in none of 94 insulin-dependent diabetes mellitus or 115 nondiabetic controls [19]. In the study of Yoshihiko Suzuki et al., the frequency of the 3243 mtDNA mutation was 4.1% of 271 Japanese patients with type 2 diabetes [20]. Besides, this mutation was found in 4 of 133 (3.0%) patients with early onset (640 years) diabetes who also had a positive maternal family history, and in one of 348 (0.3%) patients with late-onset (>40 years) diabetes and no family history [26]. Furthermore, the A3243G mutation in the mitochondrial tRNALeu(UUR) gene was also reported in patients with MIDD [27]. Indeed, it was detected in 4% of Australian patients with maternally inherited diabetes mellitus [28]. Lehto et al. described a prevalence of 2.6% for this mutation in Scandinavian families with early-onset diabetes and no further selection [29]. The 3243 mutation in the mitochondrial tRNALeu(UUR) gene was also detected in 25 out of 50 tested members of five French pedigrees. Mutation-positive family members presented variable clinical features, ranging from normal glucose tolerance to insulin-requiring diabetes [30]. In the same year, Yorifuji et al. found this mutation in a Japanese mother with diabetes mellitus and sensorineural hearing loss and in her son [31]. In addition, the A3243G mutation was also reported in patients with MELAS [15,21,32], with chronic progressive external ophthalmoplegia (CPEO) or with Kearns-Sayre syndrome (KSS) [27]. This mutation was also identified in 12/44 Japanese patients who showed the phenotype of Leigh syndrome, MELAS, and cardiomyopathy [4]. Sue et al. reported three unrelated children with the A3243G mutation who presented with severe psychomotor delay in early infancy. One patient’s clinical picture was more consistent with Leigh syndrome, ataxia, and bilateral striatal lesions on brain MRI. A second patient had generalised seizures refractory to treatment and bilateral occipital lesions on brain MRI. The third child had atypical retinal pigmentary changes, seizures, and cerebral atrophy on brain MRI [33]. Deschauer et al. detected the A3243G mutation in the heteroplasmic form in DNA from muscle and blood of a 61-year-old patient who at the age of 54 developed a myopathy with painful muscle stiffness as the predominant symptom. Additionally, a hearing impairment requiring a hearing aid for the left ear, numbness of the left arm and leg, and impaired glucose tolerance were present [34] and in 2001, Aggarwal et al. found the same mutation in a

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29-year-old woman with gestational diabetes, deafness, and Wolff–Parkinson–White (WPW) syndrome. The A3243G mitochondrial mutation was also reported in a child with cardiomyopathy who presented also severe failure to thrive, severely delayed gross motor milestones, and marked muscle weakness [35]. This mutation was also frequent in mitochondrial encephalomyopathies. In fact, Deschauer et al. found it in 16 patients with mitochondrial encephalomyopathies (10 index patients and 6 symptomatic relatives). Only six of these patients showed with stroke-like episodes and the classic criteria of MELAS syndrome. One had MELAS/MERRF overlap syndrome. Two patients presented with stroke-like episodes but did not meet the classic criteria of MELAS. Of the eight other patients, one had myopathy with hearing loss and diabetes mellitus, one had chronic progressive external ophthalmoplegia, one had diabetes mellitus with hearing loss, one had painful muscle stiffness with hearing loss, one had cardiomyopathy, one had diabetes mellitus, and two had hearing loss as predominant features. In 11 of the 16 patients, hearing impairment was obvious on clinical examination [36]. In addition, among 230 patients with sensorineural hearing loss in Otorhinolaryngology Clinics in Japan, Nagata et al. found four patients with the A3243G mutation (1.74%) [37]. Moreover, in our study, mutational analysis of the mitochondrial tRNALeu(UUR) gene by direct sequencing showed the absence of any polymorphisms or other mutations and confirmed the absence of the A3243G mutation in this gene in the 128 tested patients and controls. In addition, after direct sequencing of the entire PCR product amplified previously which contains the entire mitochondrial tRNALeu(UUR) gene and a part of the mitochondrial NADH dehydrogenase 1, we detected a T to C transition at the nucleotide 3396 of the mitochondrial genome in the mitochondrial ND1 gene in a patient with nonsyndromic hearing loss. This transition was absent in the other patients and in 100 normal individuals tested. The analysis of the other members of the proband’s family revealed the presence of the T3396C transition and its inheritance in a maternal pattern. In fact, this substitution was detected in the proband’s sister and in her aunt’s mother who were deaf and it was also found in the proband’s mother who had a normal hearing. However, the father and the paternal grand-mother who were normal did not present this mutation. No other clinical abnormalities were found in this family’s members. In addition, we previously screened the mitochondrial tRNASer(UCN) and the 12S rRNA genes and the nuclear GJB2, GJB3, and GJB6 genes in this family and we did not find any mutations associated with hearing loss [38]. Moreover, no mutations in other nuclear genes associated with deafness were found in this family’s members. Thus, we had to explore other mitochondrial genes or the entire mitochondrial genome to explain the relation between the T3396C transition and the deafness in this family.

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In the literature, the T3396C mutation in the mitochondrial ND1 gene was reported in only one patient with noninsulin-dependent diabetes mellitus by Thomas et al. in 1996 [39]. Thus, our study reports the second case with the mitochondrial T3396C mutation in the ND1 gene in a patient with nonsyndromic hearing loss. This mutation was transmitted through a maternal inheritance in the patient’s family members and was absent in the normal individuals. In conclusion, the mitochondrial A3243G mutation in the mitochondrial tRNALeu(UUR) gene is not a frequent cause of diabetes, hearing loss or other mitochondrial disorders in the Tunisian population. Further screening of enlarging study group is necessary to fully determine the prevalence of this mutation in our population. In addition, the search for other mitochondrial mutations in others genes should allow to determine the prevalence of MIDD and its specific molecular background in the Tunisian population. Acknowledgments We thank the patients and their families for their cooperation in the present study. This work was supported by The Ministry of Higher Education, Scientific Research and Technology in Tunisia. We extend our thanks to Mr. Jamil JAOUA, the coordinator of the English Unit at the Sfax Faculty of Science for having proofread this paper. References [1] J.H. Chae, H. Hwang, B.C. Lim, H.I. Cheong, Y.S. Hwang, K.J. Kim, Clinical features of A3243G mitochondrial tRNA mutation, Brain Dev. 26 (2004) 459–462. [2] Y. Goto, I. Nonaka, S. Horai, A mutation in the tRNA(Leu)(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies, Nature 13 (1990) 651. [3] T. Kanaumi, S. Hirose, Y. Goto, E. Naitou, A. Mitsudome, An infant with a mitochondrial A3243G mutation demonstrating the MELAS phenotype, Pediatr. Neurol. 34 (2006) 235–238. [4] I. Ueki, Y. Koga, N. Povalko, Y. Akita, J. Nishioka, S. Yatsuga, R. Fukiyama, T. Matsuishi, Mitochondrial tRNA gene mutations in patients having mitochondrial disease with lactic acidosis, Mitochondrion 6 (2006) 29–36. [5] J.M. Van den Ouweland, H.H. Lemkes, W. Ruitenbeek, L.A. Sandkuijl, M.F. de Vijlder, P.A. Struyvenberg, J.J. van de Kamp, J.A. Maassen, Mutation in mitochondrial tRNA(Leu)(UUR) gene in a large pedigree with maternally transmitted type II diabetes mellitus and deafness, Nat. Genet. 1 (1992) 368–371. [6] G.M. Fabrizi, E. Cardaioli, G.S. Grieco, T. Cavallaro, A. Malandrini, L. Manneschi, M.T. Dotti, A. Federico, G. Guazzi, The A to G transition at nt 3243 of the mitochondrial tRNALeu(UUR) may cause an MERRF syndrome, J. Neurol. Neurosurg. Psychiatry 61 (1996) 47–51. [7] C.T. Moraes, F. Ciacci, G. Silvestri, S. Shanske, M. Sciacco, M. Hirano, E.A. Schon, E. Bonilla, S. DiMauro, Atypical clinical presentations associated with the MELAS mutation at position 3243 of human mitochondrial DNA, Neuromuscul. Disord. 3 (1993) 43– 50. [8] L. Vilarinho, F.M. Santorelli, M.J. Rosas, C. Tavares, M. Melo-Pires, S. DiMauro, The mitochondrial A3243G mutation presenting as severe cardiomyopathy, J. Med. Genet. 34 (1997) 607–609.

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