Multiple sclerosis (MS) is a neurological chronic inflammatory disorder affecting the white matter of the central nervous system (CNS) that involves immune-mediated mechanisms. The disease is characterized by demyelination and axonal loss as a result of myelin sheath damage by the body's own immune system, which affects the ability of nerve cells in the brain and spinal cord to effectively communicate with each other (1,2). However, the exact immunopathogenic mechanisms responsible for the initiation and progression of the disease remain unknown. Clinically, MS presents with a range of signs and symptoms that appear as episodic or progressive neurological impairments, such as numbness and tingling, blurry vision, mobility and balance issues, muscle weakness and tightness, bladder and bowel dysfunction, decreased memory and fatigue, as well as other conditions, such as depression (3,4). These symptoms present in each of the 4 types of MS that are as follows: i) Relapsing-remitting MS (RRMS), which is the most common type and occurs in 85-90% of patients; ii) secondary progressive MS (SPMS), which occurs in 70-80% of patients with RRMS within 10-15 years; iii) primary progressive MS (PPMS), which occurs in 15% of cases; and iv) progressive relapsing MS (PRMS), the least common form which occurs in 5% of patients (5). MS affects approximately 2.5 million individuals worldwide and commonly appears in young adults with a mean age of 32 years (6). Moreover, MS usually occurs 2-3 times more frequently in females than in males (7). Higher rates of MS have been reported in Europe, Southern Canada, Northern United States, New Zealand and Southeast Australia (8,9). Studies from the Arab Gulf countries, including Kuwait, Saudi Arabia and United Arab Emirates have also demonstrated noticeable increases in the incidence and prevalence of MS (10,11).

Although the cause of MS remains unclear, combinations of genetic and environmental factors may contribute to the etiopathogenicity of the disease (12-14). Recently, MS has become increasingly viewed as a neurodegenerative disorder, in which mitochondrial dysfunction occurs early in the pathogenic process and plays an important role in axonal degeneration and demyelination, as well as in disease progression (15-17). Therefore, it has been suggested that targeting mitochondrial pathways along with neuroprotection and immunomodulation may provide a novel approach for the treatment of MS (16-18).

The mitochondria are the main site of energy production in the cell and are also the major source of reactive oxygen species (ROS). Mammalian mitochondria have their own genome [mitochondrial DNA (mtDNA)], a single, circular double-stranded molecule of 16,569 base pairs. mtDNA encodes 2 rRNAs and 22 tRNAs, as well as 13 polypeptides that are all subunits of complexes of the respiratory chain, located in the inner mitochondrial membrane, that drives oxidative energy metabolism. While subunits of complex II are entirely encoded by mtDNA, subunits of complexes I, III, IV and V are encoded by either mtDNA or nuclear DNA (nDNA) (19). Specifically, mtDNA encodes 7 subunits of complex I (NADH dehydrogenase), 1 subunit of complex III (ubiquinol-cytochrome c oxidoreductase), 3 subunits of complex IV (cytochrome c oxidase) and 2 subunits of complex V (20). It has been demonstrated that mtDNA has a higher mutation rate than the nuclear genome. A number of factors contribute to the increased rate of mutations in mtDNA, such as the close proximity of mtDNA to the site of ROS production, the lack of protective histones and the low efficiency of DNA repair pathways (21,22). The loss of mitochondrial genomic integrity can lead to a progressive decline in mitochondrial function (23,24) and, eventually, to a reduction in energy within the cell, which has been implicated in a number of neuroinflammatory and neurodegenerative diseases (25). The role of defects in mtDNA in MS stems from the observation that a number of patients with Leber hereditary optic neuropathy (LHON), a maternally inherited mitochondrial disease caused by mutations in the ND1, ND4, ND4L and ND6 genes of complex I, develop neurological features, including inflammatory demyelinating disorders compatible with a diagnosis of MS (26,27). Studies on mtDNA in patients with MS from different ethnic backgrounds have identified variations within complex I genes to be associated with the pathogenicity of MS. A specific variant in the ND2 gene of complex I has been reported to play a role in the susceptibility to MS in Caucasians (28). Other variants in different genes of complex I have been shown to be associated with the risk of developing MS in a Filipino population (29). These observations suggest that although the disease etiology is common between populations, genetic variants can be population-specific. Therefore, in the current study, we carried out genetic analysis of mtDNA-encoded complex I genes to identify specific mutations in Saudi patients with MS. The identification of ethnicity-related mtDNA variations in MS may lead to the development of novel approaches for personalized treatment.

In the current study, we carried out sequence analysis for the mtDNA-encoded complex I (NADH dehydrogenase) genes in 164 Saudi subjects, 60 patients with RRMS and 64 healthy controls, in order to identify mutations relevant to MS. We found several variants in different positions of the ND1, ND3, NDL4, ND4, ND5 and ND6 genes both in the patients with MS and the healthy individuals. However, no variants were found in the ND2 gene in the 2 groups. Some of the identified variants in the ND1, ND3, NDL4, ND4, ND5 and ND6 genes were commonly observed at high frequencies in the patients with MS and the healthy individuals. These included variants in the ND4 gene, namely m.11719G>A and m.11251A>G, and variants in the ND5 gene, namely m.12372G>A, m.13708G>A, m.12612G>A and m.12705C>T. Moreover, numerous variants were identified which were observed only in patients with MS, whereas none of the healthy subjects carried any of these variants. These were found to be located in different positions of the ND1, ND3, ND4L, ND4, ND5 and ND6 genes. Bioinformatics analysis revealed that the majority of these variants were synonymous and did not cause any amino acid changes, and were thus considered as simple polymorphisms. Importantly, the significant finding in the group of MS patients was the identification of 4 mutations in the ND4 gene that were revealed to encode missense mutations. Of these, m.11150G>A exhibited an alanine to threonine alteration, m.11519A>C a threonine to proline alteration, m.11523A>C a lysine to threonine alteration and m.11527C>T a histidine to leucine alteration. It was also noted that 1 patient (patient no. 48, Table V) carried the m.11150G>A mutation, whereas another patient (patient no. 44, Table V) carried the m.11519A>C, m.11523A>C and m.11527C>T mutations.

MS has traditionally been considered as an immune-mediated neurological inflammatory demyelinating disease of the central nervous system with axonal degeneration (1,2). Recent studies have suggested that mitochondrial dysfunction occurs early in MS and plays an important role in disease development and progression (15-17). Targeting mitochondrial pathways along with neuroprotection and immunomodulation are a potential therapeutic target in MS (16-18). The mitochondria are the most efficient producers of cellular energy in the form of adenosine ATP and are also the major source of ROS. The mitochondrial respiratory chain is located in the inner mitochondrial membrane and consists of 5 multimeric protein complexes I-V (19). While complex II subunits are entirely encoded by nuclear DNA (nDNA), the subunits of complexes I, III, IV and V are encoded by both nDNA and mtDNA. Complex I is the largest enzyme complex of the mitochondrial respiratory chain, which is responsible for electron transport and the generation of protons across the mitochondrial inner membrane to drive energy production (20). It is a multi-subunit complex consisting of 44 subunits, of which 7 subunits, including ND1, ND2, ND3, NDL4, ND4, ND5 and ND6 are encoded by the mitochondrial genome (20).

In this study, numerous synonymous variants were found in different genes of mtDNA-encoded complex I in the patients with MS and the healthy individuals, as well in patients with MS only. Synonymous mutations, which do not result in changes in amino acid sequences and are considered biologically silent, have been shown to directly affect gene expression and function through diverse mechanisms, and may thus be implicated in human diseases (34-36). The presence of rare codons may lead to a premature arrest of the translation process, hence resulting in a truncated form of a protein. Although in the present study, we did not analyze the function of the identified polymorphisms, studies in our laboratory are ongoing to investigate the effects of these variant on gene expression, where silent mutations can lead to the generation of rare codons.

A deficiency in complex I is the most frequently enzyme deficit in mitochondrial diseases (37) and degenerative diseases (38), as well as in other pathological conditions (39), and contributes to neurodegeneration in MS (15,40). Moreover, ROS generated by mitochondrial complex I are considered the main source of cellular oxidative stress (41), and impaired complex I activity mediated by mtDNA oxidative damage has been shown in chronic active plaques in MS (42) and is also implicated in axonal degeneration (15).

It has been demonstrated that ND genes of complex I encoded by mtDNA are hotspots for pathological mutations (43). These mutations in complex I cause a deficiency in NADH ubiquinone oxidoreductase enzyme activity and can lead to mitochondrial dysfunction with increased ROS production (37). In particular, mutations in the ND4, ND5 and ND6 genes have been shown to affect complex I assembly and activity, leading to complex I dysfunction (44-47). Previous studies have confirmed a possible implication of several variations in mtDNA-encoded complex I genes in the pathogenicity of MS in different populations. For example, specific variants in the ND2 gene of complex I have been strongly linked to MS in Caucasians (28). Several other variants in different genes of complex I have been shown to be risk factors in the pathogenicity of MS in a Filipino population (29). The study by Yu et al (48) demonstrated an association between the m.13708G>A variant in the ND5 gene and an increased risk of MS in European cohorts. However, Kellar-Wood et al (49) demonstrated that this base change at position 13708 of the mtDNA-encoded ND5 gene does not contribute to genetically determined susceptibility in typical MS patients. Moreover, in LHON, a rare maternally inherited mitochondrial disease with clinical features associated with an MS-like illness (26,27), the m.13708G>A variant in the ND5 gene has been reported as a secondary mutation when occurring in patients with LHON (50-52) and does not functionally impair mitochondrial oxidative metabolism in vivo or determine the deficit of energy metabolism in LHON (52). In this study, the m.13708G>A variant in the ND5 gene was observed at high frequencies in both patients with MS and healthy individuals. These inconsistencies between studies suggest that the contribution of specific mtDNA variants to disease risk and susceptibility varies among different ancestral and ethnic groups.

In the current study, 4 missense mutations, including m.11150G>A, m.11519A>C, m.11523A>C and m.11527C>T in the ND4 gene of complex I were found in 2 Saudi patients with MS. As indicated by functional analysis, the identified mutations, m.11150G>A, m.11519A>C and m.11523A, in this study were predicted to be deleterious and to directedly cause ND4 protein instability, which may render the protein non-functional. As mentioned above, 3 mutations (m.11519A>C, m.11523A>C and m.11527A>C) were observed in 1 patient (patient no. 44, Table V) and 2 of them (m.11519A>C and m.11523A>C) caused protein instability, whereas the m.11150G>A mutation was found in another patient (patient no. 48, Table V). Although the m.11527C>T mutation in patient no. 44 had no effect on protein function, it may cause a cumulative destabilizing effect, and may thus disrupt ND4 protein function. Indeed, when these 3 mutations in patient 44 were analyzed for their cumulative destabilizing effect, they were found to affect ND4 protein stability. ND4 subunit is part of a large complex, and these mutations, if not affecting ND4 stability directly, may perturb the protein-protein interaction responsible for maintaining the complex and may consequently affect complex I function. The missense mutations in the ND4 gene observed in Saudi patients with MS in the present study have not been reported in previous studies, at least to the best of our knowledge, and may therefore be considered novel. Since all previous mtDNA studies have revealed a number of variations in MS patients of different populations, such as Caucasians and Filipinos (28,29,48), the identified novel mutations in Saudi patients with MS in the present study could be ethnic-related and may be important to personalized treatment. This study also confirmed the implication of mtDNA mutations in the pathogenicity of MS and the newly identified mutations may serve as a reference for future studies on the mitochondrial genome in MS.

The polyploid nature of the mitochondrial genome with the presence of up to several thousand copies per cell gives rise to an important feature of mitochondrial genetics, homoplasmy and heteroplasmy. Homoplasmy is when all copies of mtDNA are identical, which may be normal or mutated. Heteroplasmy is when there is a mixture of normal and mutated mtDNA (43). The level of heteroplasmic mutations is important for both the clinical expression of the disease and for biochemical defects (53). In most mtDNA disorders, when the percentage of mutant mtDNA exceeds a certain threshold level, mitochondrial dysfunction becomes clinically apparent (54). Due to organ-specific energetic requirements, the proportion of mutant mtDNA in any cell or tissue may be extremely variable, giving rise to variable disease severity (54). It is generally accepted that high levels of heteroplasmy are associated with severe clinical presentations (55). On the other hand, lower levels of heteroplasmy have been commonly detected in maternally inherited diseases (56,57). Moreover, low-level heteroplasmic sequence changes are important features of the pathology of several diseases, including neurodegenerative diseases, which can lead to mtDNA damage and consequently may induce alterations of OXPHOS enzymes (58).

For a DNA alteration to be classified as a deleterious mutation, it should not be present in healthy individuals, and must occur in a structurally and functionally important region (53). While in our results no heteroplasmic mutations were indicated, the identified mutations in the ND4 gene of complex I, which is a key element in cellular energy production, were observed in only patients with MS and were not found in healthy individuals, suggesting that they are pathogenic mutations and may play an important role in the pathogenicity MS. Nevertheless, further studies are required to predict the energetic effects of amino acid changes caused by these mutations.

A limitation of the current study was the relatively low number of subjects, which affected the statistical differences in age and sex distribution between the patients and controls. Theoretically, the age and sex distribution between cases and controls in a high number of participants should be very similar (Gaussian distribution). Moreover, further studies are warranted with larger cohorts to analyze the possible association between these variants and the risk of and/or susceptibility to MS.

In conclusion, in this study, we identified a number of variants as synonymous mutations in both patients with MS and healthy individuals in the mtDNA-encoded ND1, ND3, NDL4, ND4, ND5 and ND6 genes of complex I. Some of these variants in the ND4 and ND5 genes were commonly observed at high frequencies in the investigated groups. We also identified numerous variants in patients with MS only, which were absent in healthy individuals. While the majority of these variants in patients with MS were synonymous, 4 variants in the ND4 gene were missense mutations and could lead to complex I dysfunction. This study, to the best of our knowledge, is the first to report novel mutations in mtDNA-encoded ND4 gene of complex I in Saudi patients with MS, which may prove to be of importance in personalized treatment.