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Prevalence of the primary LHON mutations in Northern Finland associated with bilateral optic atrophy and tobacco-alcohol amblyopia

2012; Wiley; Volume: 91; Issue: 7 Linguagem: Inglês

10.1111/j.1755-3768.2012.02506.x

1755-3768

Paula Korkiamäki, Marko Kervinen, Karoliina Karjalainen, Kari Majamaa, Johanna Uusimaa, Anne M. Remes,

Metabolism and Genetic Disorders

Purpose: Leber hereditary optic neuropathy (LHON) is regarded as the most common mitochondrial disease. We have previously reported comprehensive population-based epidemiological data on common mitochondrial DNA (mtDNA) mutations including m.3243A>G, m.8344A>G and large-scale mtDNA deletions in Northern Finland. Our aim was to investigate the prevalence of primary LHON mutations and mutations in the four mtDNA genes considered hot spots for LHON in the same population. Methods: The study population consisted of 42 adult patients with an aetiologically undefined bilateral optic atrophy. The major LHON mutations m.3460G>A, m.11778G>A and m.14484T>C were analysed by restriction fragment length polymorphism (RFLP), and MTND1, MTND6 and MTATP6 genes were sequenced. MTND5 gene was analysed by conformation-sensitive gel electrophoresis (CSGE). Results: No major LHON mutations were found in the population of the province of Northern Ostrobothnia giving the prevalence of these mutations 0–1.36:100 000 (95% CI). However, two main mutations were found elsewhere in Northern Finland, homoplasmic m.11778G>A from Kainuu and heteroplasmic m.3460G>A from Central Ostrobothnia. Furthermore, tobacco-alcohol amblyopia was diagnosed in five patients in the study population and one of them had m.11778G>A. Conclusion: The prevalence of the three major LHON mutations is lower in Northern Finland than elsewhere in Finland or in Western Europe. As LHON and tobacco-alcohol amblyopia have a similar phenotype, we recommend analysing the known LHON-associated mutations before setting tobacco-alcohol amblyopia diagnosis. Leber hereditary optic neuropathy (LHON) is maternally inherited mitochondrial eye disorder characterized by acute, painless, loss of vision mostly affecting young men (Nikoskelainen 1994). Typical age of onset is between 15 and 30 years. However, the symptoms can occur at any age between early childhood and seventh decade of life (Harding et al. 1995; Yu-Wai-Man et al. 2008). LHON is regarded as the most common mitochondrial disease #535000 OMIM. The prevalence of LHON is estimated to be as high as 3.2:100 000 in North East of England, 2.6:100 000 in Netherlands and 2:100 000 in Finland (Man et al. 2003; Spruijt et al. 2006; Puomila et al. 2007). In all, 90–95% of LHON patients have one of the three mtDNA point mutations in NADH dehydrogenase (ND) subunit genes m.3460G>A in MTND1, m.11778G>A in MTND4 or m.14484T>C in MTND6 gene (Wallace et al. 1988; Howell et al. 1991; Huoponen et al. 1991; Johns et al. 1992; Mackey & Howell 1992). About 30 mtDNA mutations associated with LHON have been reported, and several of those are located in the MTND1 and MTND6 genes that can be called as LHON hot spot genes (Chinnery et al. 2001; Valentino et al. 2004; Yu-Wai-Man et al. 2011). Few other mitochondrial genes also harbour LHON mutations. LHON mutation m.9101T>C in ATP synthase subunit 6 (MTATP6) has been found from a Finnish family (Lamminen et al. 1995). Incomplete penetrance of LHON and male predominance are still enigmas. Environmental factors, such as tobacco and alcohol, have been hypothesized to influence the penetrance. However, only few tobacco and alcohol abusers develop optic neuropathy that leads debate on individual susceptibility. It has been proposed that susceptibility may be due to LHON-associated mitochondrial mutations (Cullom et al. 1993). MtDNA haplogroups are known to affect mutation penetrance. It has previously been reported that population of Northern Finland differs markedly from other European populations in the frequency of haplogroup U, especially U5. The frequency of U5 is double in Finland in comparison with Europe as a whole, and it appears to be even higher in Northern Finland (Meinila et al. 2001). Previously, we have investigated the prevalence of mitochondrial diseases in the province of Northern Ostrobothnia. The prevalence of mitochondrial encephalopathy lactic acidosis and stroke-like episodes (MELAS) mutation m.3243A>G in adult population is 16.3:100 000 and 18.4:100 000 in children (Majamaa et al. 1998; Uusimaa et al. 2007). However, prevalences of large mitochondrial DNA deletions and m.8344A>G mutation associated with MERFF (myoclonus epilepsy and ragged red fibres) were very low in the same population, being 1.6:100 000 and 0–1.5:100 000, respectively (Remes et al. 2003, 2005). LHON prevalence studies have mostly been performed in Western Europe. Puomila reported the LHON prevalence in Finland based on the known Finnish LHON families, which came mostly from Southern Finland. Thus, we report the prevalence of mtDNA mutations in the LHON hot spot genes MTND1, MTND5, MTND6 and MTATP6 based on the genetic screening of a cohort of patients with undefined optic atrophy. The study population consisted of over 18-year-old patients referred to Oulu University Hospital (OUH) because of an aetiologically undefined bilateral optic atrophy as described earlier (Remes et al. 2003). Patients with definite dominant optic atrophy, ischaemic brain damage, brain tumour or multiple sclerosis were excluded. The only neuro-opthalmologist practice in Northern Finland resides in OUH, and it serves for North or Central Ostrobothnia, Kainuu, and Lapland areas (Fig. 1). Therefore, we have extensive records from optic atrophy patients living in this area. Altogether, 42 patients were included in the study population, and the patients were examined in OUH during the years 1986–1998. The Ethics Committee of Medical Faculty of the University of Oulu approved the research protocol and guidelines of the Helsinki Declaration were followed. The patients of the cohort come from regions 1–4. Total DNA was purified from blood samples by QIAamp Blood kit (Qiagen, Hilden, Germany). The mtDNA haplogroups were determined by restriction digestion as described (Torroni et al. 1996; Finnila et al. 2001). Two mtDNA point mutations MELAS m.3243A>G and MERFF m.8344A>G together with large-scale mtDNA deletions have previously been excluded from the patients' DNA samples (Majamaa et al. 1998; Remes et al. 2003, 2005). The LHON primary mutations were detected by restriction fragment length polymorphism (RFLP) using BsaHI (New England Bio Labs) for m.3460G>A, Lwel (Fermentas) for m.11778G>A and DpnII (New England Bio Labs) for m.14484T>C mutations. The entire coding regions of MTND1, MTND6 and MTATP6 genes were sequenced using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The MTND5 gene was analysed by conformation-sensitive gel electrophoresis (CSGE) (Ganguly et al. 1993; Finnila et al. 2001). The fragments of patient samples and healthy control samples were amplified by PCR and mixed in pairs. The fragments differing in mobility in CSGE were sequenced. The MTND5 coding region 12 217–14 208 was analysed in eight fragments. Primers used for the amplification of the fragments for CSGE, and those used for subsequent sequencing have been reported previously (Finnilä et al. 2000). On the selected prevalence date (31 December 1998), there were 67728 over 18-year-old inhabitants in Kainuu area and 56385 in Central Ostrobothnia. The 95% confidence intervals (CI) were calculated based on Poisson distribution. Two patients with the major LHON mutations were detected in the population of Northern Finland (Table 1). Neither of them was found in Northern Ostrobothnia, suggesting the prevalence A was found in a male patient from Kainuu giving a minimum estimate for the prevalence 1.48:100 000 (95% CI 0.04–8.23:100 000). He lost his vision at the age of 36 years and was assumed to suffer tobacco-alcohol amblyopia. His brother also had poor vision since early adulthood, but the diagnosis of LHON had not been considered previously. The heteroplasmic m.3460G C that was expected according to previous studies (Puomila et al. 2007). Altogether 24 non-synonymous changes were detected in 42 patients (Tables 1 and 2). Patients of interest, clinical features and combinations of controversial mtDNA mutations are described in Table 3. A few cases showed typical LHON presentation or mitochondrial disease-associated phenotype without any primary LHON mutations. Instead, they carried combinations of several other non-synonymous mtDNA mutations. Patient 4 with typical LHON phenotype and family history with respect to LHON carried a combination of two MTND1 and one MTND5 gene mutations. Patient 3 carried a combination of three MTND1 and one MTND6 gene mutations. His amblyopia was thought to be a consequence of chronic carbon monoxide (CO) exposure during 2 years related to his work as a bus driver. In all, five of 42 optic atrophy cases that had been diagnosed to have tobacco-alcohol amblyopia. The pathogenic LHON mutation m.11778G>A was detected in one of them (patient 1). In addition, patient 7 had a homoplasmic non-synonymous m.14420C>T in MTND6 gene changing glycine to glutamine in a non-conserved region. Previous epidemiological LHON data suggest that LHON is the most prevalent mitochondrial disease (Man et al. 2003; Spruijt et al. 2006; Puomila et al. 2007). We have comprehensive data on the epidemiology of common mtDNA mutations in Northern Ostrobothnia. There are extensive differences between prevalences of mtDNA mutations in Finland and also compared with the other European countries. The prevalence of the m.3243A>G MELAS mutation in adults is 16.3:100 000 and in children 18.4:100 000 thus raising it the most common mitochondrial disease in Northern Ostrobothnia, whereas MERRF m.8344A>G as well as primary LHON mutations are very rare (Majamaa et al. 1998; Remes et al. 2003; Uusimaa et al. 2007). We found several cases with combinations of two or more non-synonymous changes in MTND genes, which are associated with mitochondrial diseases and LHON. Patient 3 had mutations in both LHON hot spot genes MTND1 and MTND6. All of these changes m.3796A>G, m.3992C>T, m.4024A>G and m.14582A>G have been considered as polymorphisms (MITOMAP 2011). This patient also had chronic CO exposure, which can stress the oxidative phosphorylation. These polymorphisms are presumed to be neutral in effect, but there is accumulating evidence against this assumption and associations between mtDNA haplogroups, and several diseases have been reported (Herrnstadt & Howell 2004). Thus, the effect of these mutations needs to be studied further. Patient 4 with a typical LHON phenotype and belonging to haplogroup J, had homoplasmic non-synonymous m.4025C>T and m.4216 T>C in MTND1 and m.13708G>A in MTND5. The mutations m.4216T>C and m.13708G>A are considered as secondary LHON mutations (Chinnery et al. 1999). The change m.4216T>C has been shown to have an effect on the structural integrity of complex I in Eschericia coli bacterial model (Hinttala et al. 2010). The mutation m.4025C>T is relatively rare and considered to be a polymorphism, although it has been found in the Finnish LHON patients previously (Puomila et al. 2007; MITOMAP 2011). Interestingly, patient 4 with LHON-associated haplogroup J and prevalent heredity of the LHON phenotype in the family was found to be negative for primary LHON mutations. Patients 5 and 6 have LHON-like eye symptoms but both of them are untypically women. Their ND5 mutations are considered to be polymorphisms (MITOMAP 2011). Leber hereditary optic neuropathy and tobacco-alcohol amblyopia have a similar phenotype and are indistinguishable without genetic analyses. Our cohort included five tobacco-alcohol amblyopia cases, and one of them (patient 1) was found with a primary LHON mutation. Patient 7 diagnosed with tobacco-alcohol amblyopia belonged to haplogroup H and had homoplasmic non-synonymous m.14420C>T mutation in MTND6 gene. This was the only detected mutation in MTND1, MTND5, MTND6 and MTATP6 genes. This mutation has been reported twice. It was found from LHON patient carrying also m.14484T>C and presenting also with spondyloepiphyseal dysplasia. Mutation m.14420C>T was not found from 200 control samples, but patient's asymptomatic sister had both mutations (Howell et al. 2001). The effect of tobacco and alcohol on vision has been controversial in spite of numerous studies. Kirkman reported the largest epidemiological study so far investigating the role of smoking and alcohol exposure in the expression of visual loss in LHON. They found that smoking is associated with an increased rate of visual loss and that this relationship might even be dose responsive. They also identified a tendency towards increased visual failure with alcohol use, but only with heavy intake. Based on their results, the authors conclude that smoking has a consistent role in increasing disease penetrance in LHON (Kirkman et al. 2009a). In the present study, we report that the frequencies of mitochondrial diseases and mtDNA mutations vary regionally in Finland. The prevalence of primary LHON mutations is low in Northern Finland, whereas the MELAS m.3243A>G is the most common mitochondrial disease causing mutation in mtDNA in the same area. Based on the recent studies and our results, we suggest that at least the three LHON primary mutations should be investigated by molecular genetic analyses when considering tobacco-alcohol amblyopia diagnosis. We thank Ms Anja Heikkinen and Ms Pirjo Keränen for their excellent technical assistance. This work was supported financially in part by grants from the Finnish Medical Foundation (AMR and JU), the Arvo and Lea Ylppö Foundation (JU), the Foundation for Pediatric Research (JU) and by clinical EVO grants from Oulu University Hospital (AMR).