Mitochondria are the primary site of energy production via the process of oxidative phosphorylation (OXPHOS). This process involves the transfer of electrons from reduced nicotine adenine dinucleotide (NADH) or flavin adenine dinucleotide (FADH₂) to oxygen through highly conserved mitochondrial membrane-bound enzyme complexes (I-V) of the electron transport chain (ETC) to create ATP (1). Mitochondria are also an essential source of reactive oxygen species (ROS) generation as by-products of normal mitochondrial metabolism (2).

One of the mitochondria's unique features is that it contains its own genome (mtDNA), separate and distinct from the nuclear genome of the cell. Human mtDNA is a double-stranded and circular molecule of 16,569 bp and contains two regions (3). The coding region encompasses 37 genes, which encode 13 crucial protein subunits of the ETC, two ribosomal (r)RNAs, and 22 transfer (t)RNAs. The control or regulatory (D-loop) region consists of sites for replicating and transcribing of the mtDNA. Except for complex II subunits, which are entirely encoded by the nuclear DNA (nDNA), subunits of complex I, III, IV and V are encoded by both nDNA and mtDNA. Specifically, mtDNA codes for seven subunits (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6) of NADH-ubiquinone oxidoreductase of complex I, cytochrome b (CYTB) subunit of ubiquinol-cytochrome c oxidoreductase of complex III, three subunits (CO1, CO2 and CO3) of cytochrome c oxidase of complex IV and two subunits (ATPase 6 and 8) of ATP synthase of the complex.

The mtDNA is particularly susceptible to oxidative damage and has a high mutational rate due to its proximity to the site of ROS production, the lack of protective histones, and low DNA repair capacity (4,5). Since mtDNA encodes essential components of the ETC, these mutations can disrupt the mitochondria's ability to generate energy for the cell (6). Indeed, mtDNA mutations are linked with a wide range of human diseases (6).

Although primary mutations in the mtDNA have been observed in diseases of mitochondrial origin, secondary mutations and new variants are also involved in aging (7,8) and may underlie the predisposition of several common diseases, such as neurodegenerative, metabolic and inflammatory conditions (8-10). It is therefore useful to sequence the complete mitochondrial genome to explore disease-related variants in the mtDNA (11,12).

Psoriasis (Ps) is a chronic immune-mediated inflammatory skin disease characterized by hyperproliferative keratinocytes and the infiltration of the dermis by various immune cells (13,14). Ps affects ~3% of the population worldwide (15), and its incidence is also high in the Gulf countries, including Kuwait, where it affects around ~3% of people (16-18). Several studies have shown an association between Ps and metabolic syndrome (19-21), particularly type 2 diabetes (T2D), in which T2D was found to be twice as prevalent in patients with Ps (22). T2D is a progressive metabolic disease characterized by hyperglycaemia due to inadequate insulin secretion from the β-cells and insulin resistance. T2D is a leading cause of severe vascular complications, including cardiovascular disease (23,24), which is frequently observed in Ps patients (25,26).

Whilst the nature of the relationship between Ps and T2D remains ambiguous, both of these diseases are multifactorial, involving an interplay between genetic and environmental factors (27). Amongst the genetic factors that may explain the co-occurrence of Ps and T2D, variations in mtDNA have been suggested. In this context, studies have shown a potential role of mtDNA variants in the susceptibility or risk of T2D in different populations, including in Asian (28), European (29) and other populations (30,31). Similarly, the role of mtDNA variants in Ps has been observed in a European population (32). However, these studies have demonstrated ethnic diversity in the distribution or the implications of mtDNA variants in Ps and T2D.

To date, there are no studies that have investigated variations in the mtDNA in patients with Ps alone or in patients with Ps and T2D (Ps-T2D) in the Arab population, to the best of our knowledge. Therefore, this study aimed to sequence and compare whole mitochondrial genomes from Kuwaiti subjects with Ps, T2D, Ps-T2D and healthy controls to identify mtDNA variants in Arab individuals in Kuwait.

To the best of our knowledge, this is the first study to perform a comprehensive analysis of mitochondrial DNA (mtDNA) variants in Kuwaiti subjects with Ps, T2D and Ps-T2D, as well as in healthy controls. The average coverage depth was 24625.2X and the mean read length was 144 bp. However, the average total reads were 3,359,441, the frequency of reads was between 99.4-99.9%, and the coverage of reads was >100%. Whole mitochondrial genome sequencing revealed 36 novel non-synonymous and synonymous mutations and 51 sequence variations in the patient groups that were not detected in the controls. Additionally, several known sequence variations were seen in both patients and controls.

In general, a synonymous mutation is the substitution of a DNA base pair that does not result in a change in the amino acid sequence; in contrast, a non-synonymous mutation is the substitution of a DNA base pair that results in a single amino acid change in a given polypeptide. Non-synonymous mutations include a missense mutation (a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid), a nonsense mutation (a point mutation in a sequence of DNA that leads to the appearance of a stop codon, resulting in premature termination of translation and the production of a truncated protein), as well as insertion and deletion of one or more DNA base pairs.

Amongst the novel mutations identified in the patient groups, eight non-synonymous mutations resulted in amino acid changes and were detected primarily in the subunit genes of complexes I, III and V. These included missense mutations in the Ps group, primarily found in subunit genes of complex I, including G5262A in the ND2 gene and A12397G in the ND5 gene. Moreover, missense mutations were detected in the T2D group. These included the missense mutations C12084T and A11930G in the ND4 gene and G4959A in the ND2 gene, as well as the missense mutations C14751T in the CYB gene, and T8951C in the ATP6 gene. Additionally, the C15735T missense mutation in the CYB gene was found in the Ps-T2D group Moreover, 25 synonymous mutations were located in the coding and control regions in patient groups. Known variants previously reported as either missense or synonymous mutations were also identified. The majority of these were located in the coding region, and only a few were found in the control region.

Other known sequence variations were found in the patients groups and controls. Some of these variations were observed more frequently in all patient groups compared with the controls. Specifically, the frequencies of the G15301A variant in the CYB gene and the variant C150T in the D-loop region were significantly higher in all patient groups compared with the control. In contrast, the C12705T mutation in the ND5 gene and the A1438G in the 12S rRNA gene were found at significantly higher frequencies in the Ps and Ps-T2D groups compared with the control group. Moreover, other variants were found at higher frequencies in specific patient groups compared with the control group. Whilst most of these variants were synonymous, the A8701G variant in the ATPase6 gene that was found at a higher frequency in the Ps patients compared with the control group was identified as a missense mutation and resulted in an amino-acid substitution from threonine to alanine (Thr59Ala).

Mitochondria are the primary intracellular site of energy production, and mutations in the mitochondrial genome can affect mitochondrial function (6). In humans, the mtDNA encodes 13 protein subunits of the ETC, two rRNAs and 22 tRNAs, all of which are important for normal mitochondrial function (3). Mitochondria are also prone to damage from ROS, and several mutations of the mtDNA-encoded genes can enhance ROS production (37). Indeed, mitochondrial impairment as a result of mtDNA mutations have been observed in somatic tissues during normal aging (7,8), and have also been linked to several diseases, where oxidative stress serves a pivotal role in their development, such as in cancer and neurodegenerative diseases (6,7,9). Moreover, mitochondrial dysfunction serves a role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), as it affects hepatic lipid homeostasis and promotes ROS production and lipid peroxidation, and NAFLD has been linked to both T2D and psoriasis (38).

In the present study, the identified missense, and insertion mutations in the mtDNA genes were only observed in the patient groups. Although the identified mutations were homoplasmic, they showed changes in the amino acids of essential polypeptides complexes of the mitochondrial ETC, as well as in rRNAs and tRNAs, which are components of the mitochondrial gene expression system and the non-coding region. A thousand copies of the mitochondrial genome per cell gives rise to an essential feature of mitochondrial genetics: Homoplasmy and heteroplasmy. Homoplasmy is the presence of identical copies of mtDNA that may be normal or mutated. Heteroplasmy is the presence of a mixture of normal and mutated mtDNA. Whereas most deleterious mtDNA mutations are heteroplasmic in nature, not all are pathogenic, as some heteroplasmic mutations in the hypervariable D-loop region may be of little clinical significance (39). Moreover, some homoplasmic mutations have been reported to cause Leigh syndrome, a severe neurological disorder (40), or as secondary mutations that influence the disease severity of Leber's hereditary optic neuropathy (41). Secondary homoplasmic mutations may predispose an individual to specific symptoms of T2D, obesity and Alzheimer's disease from different ethnic groups (31,42).

The present study identified novel mutations that met at least 3 criteria classified as disease-causing mutations (6); they were present in structurally and functionally important regions of the mtDNA, resulted in changes in the amino acids, and were not found in healthy individuals. Therefore, these mutations may have detrimental effects on the structure and function of the ETC complexes. Notably, in the present study, most novel mutations were found in the NADH dehydrogenase subunit genes of complex I, the largest enzyme of the mitochondrial OXPHOS system, and the primary source of ROS in mitochondria (43). Altered complex I activity has been frequently observed in various pathologies such as mitochondrial disorders, cancer, neurodegenerative diseases and T2D (9,44,45).

The results of the present also showed several synonymous mutations in patient groups. Although mutations that do not result in amino changes are considered biologically silent, they have been implicated in human diseases through their direct effect on gene expression and function (46-48).

In addition to mtDNA pathogenic mutations, which are rare in a population, mtDNA polymorphisms have been linked with the susceptibility to or protection from various diseases. In this context, previous population-based studies have found an association between mtDNA variants with the susceptibility and risk of T2D (28-31), whereas a protective effect of mtDNA variants from Ps have also been identified (32).

The current study identified numerous reported mtDNA variations that are already present in the MITOMAP database, which were found more frequently in ≥1 group of patients compared with the controls. Although most of these were homoplasmic synonymous variants with no amino acid changes, they were reported in several disease conditions. The variants G15301A in the CYB gene of complex III and the variant C150T in the hypervariable segment of the D-loop region were found more frequently in all patient groups (Ps, T2D and Ps-T2D) compared with the controls. These variants have not been reported in any of the abovementioned diseases, but were previously reported in other conditions. The G15301A variant was described as a germline homoplasmic mtDNA mutation in 40% of Malaysian females with breast cancer (49), whereas the C150T variant was associated with the risk of cervical cancer and HPV infection (50). The T10410C variant in the tRNA^Arg gene and the G16390A variant in the D-loop region were found at increased frequencies in Ps-T2D patients compared with the controls. The T10410C variant was previously reported in children with Leigh syndrome (51), and the G16390A variant was found to be weakly associated with T2D in a Tunisian cohort (52). The variant A8701G in the ATPase 6 gene was found at a higher frequency in Ps patients compared with controls. This homoplasmic variant was previously reported in Japanese patients with T2D (53) and patients with mitochondrial maternally inherited diabetes and deafness (31). Numerous studies have shown a clear association between Ps and T2D, and patients with Ps are at increased risk of developing T2D (19-22). The presence of the G16390A variant in Ps-T2D patients and the A1438G variant in the Ps patients and their previous association with T2D suggest a possible role of these variants to predisposition of Ps and Ps-T2D.

In the present study, the variant A8701G, which occurred at a higher frequency in the Ps patients compared with the controls, was identified as a missense mutation and resulted in amino-acid substitution from threonine to alanine (Thr59Ala) in the ATPase6 subunit of complex V. This variant was previously associated with maternally inherited hypertension and cardiomyopathy in a Chinese pedigree of consanguineous marriage (54). Although the Ps patients triglyceride and total cholesterol levels were normal in the present study, Ps patients are at higher risk of developing cardiovascular diseases (25,26).

The present study has some limitations, including the relatively low number of subjects affecting the statistical power. Additionally, functional analysis should be performed to determine the potential biological significance of these mutations in the context of these diseases, which is lacking from the present study.

In conclusion, the present study is the first study to sequence and analyse the whole mitochondrial genome of Kuwaiti patients with Ps, T2D and Ps-T2D, and compared these with healthy controls. Novel mutations in patients that resulted in a change in the coded amino acid, which may be co-responsible in the determination of these diseases were identified. Additionally, known variants were detected in higher frequencies in the patient group compared with the controls, suggesting their role in predisposing patients to these diseases. These results warrant further functional analysis to determine the role of these variants in T2D, Ps and Ps-T2D.