Open Access

Comprehensive analysis of mitochondrial DNA variants, mitochondrial DNA copy number and oxidative damage in psoriatic arthritis

  • Authors:
    • Materah Salem Alwehaidah
    • Manhel Alsabbagh
    • Ghada Al-Kafaji
  • View Affiliations

  • Published online on: September 26, 2023
  • Article Number: 85
  • Copyright: © Alwehaidah et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Growing evidence suggests that abnormalities in mitochondrial DNA (mtDNA) are involved in the pathogenesis of various inflammatory and immuno‑mediated diseases. The present study analysed the entire mitochondrial genome by next‑generation sequencing (NGS) in 23 patients with psoriatic arthritis (PsA) and 20 healthy controls to identify PsA‑related variants. Changes in mtDNA copy number (mtDNAcn) were also evaluated by quantitative polymerase chain reaction (qPCR) and mtDNA oxidative damage was measured using an 8‑hydroxy‑2'‑deoxyguanosine assay. NGS analysis revealed a total of 435 variants including 187 in patients with PsA only and 122 in controls only. Additionally, 126 common variants were found, of which 2 variants differed significantly in their frequencies among patients and controls (P<0.05), and may be associated with susceptibility to PsA. A total of 33 missense variants in mtDNA‑encoded genes for complexes I, III, IV and V were identified only in patients with PsA. Of them, 25 variants were predicted to be deleterious by affecting the functions and structures of encoded proteins, and 13 variants were predicted to affect protein's stability. mtDNAcn analysis revealed decreased mtDNA content in patients with PsA compared with controls (P=0.0001) but the decrease in mtDNAcn was not correlated with patients' age or inflammatory biomarkers (P>0.05). Moreover, a higher level of oxidative damage was observed in patients with PsA compared with controls (P=0.03). The results of the present comprehensive analysis of mtDNA in PsA revealed that certain mtDNA variants may be implicated in the predisposition/pathogenesis of PsA, highlighting the importance of NGS in the identification of mtDNA variants in PsA. The current results also demonstrated that decreased mtDNAcn in PsA may be a consequence of increased oxidative stress. These data provide valuable insights into the contribution of mtDNA defects to the pathogenesis of PsA. Additional studies in larger cohorts are needed to elucidate the role of mtDNA defects in PsA.


Psoriatic arthritis (PsA) is a heterogeneous chronic immune-mediated disease characterized by musculoskeletal inflammation. Numerous patients develop PsA on the background of psoriasis, a skin condition of scaly erythematous plaques that commonly affects the extensor surfaces of the elbows and knees, and other parts of the body (1-3). The onset of PsA often occurs between the age of 30 and 50 years but may arise at any point throughout a patient's lifetime. The clinical manifestations of PsA vary greatly between patients and range from relatively mild to severe disease, and disease flares can alternate with periods of remission (4). Due to the shared similarities in the clinical presentation of PsA and other arthritic diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), PsA is frequently undiagnosed and/or misdiagnosed (5). However, six clinical domains are involved in PsA including peripheral arthritis, enthesitis, dactylitis, psoriasis, psoriatic nail disease and axial disease (6).

Although the aetiology of PsA is not fully understood, genetics, epigenetics and environmental factors contribute to abnormal immune responses and disease expression (7). At the genetics level, both human leukocyte antigen (HLA) and non-HLA genes have been associated with PsA (7). Moreover, 33-50% of patients with PsA have at least one first-degree relatives who are also affected by psoriasis or PsA (8). Previous studies have shown that mitochondrial dysfunction contributes significantly to the pathogenesis of PsA by modulating innate immunity via redox-sensitive inflammatory pathways (9,10). Oxidative stress can disrupt redox signalling and cause molecular damage, which impacts angiogenesis, inflammation and immune cell function (11). Mitochondria produce most of the cellular energy through the process of oxidative phosphorylation (OXPHOS) and are also the major site of reactive oxygen species (ROS). Besides their central role in cellular metabolism, mitochondria also participate in other important cellular processes such as innate immune, inflammatory and stress responses (12). Mitochondria have their own genome called mitochondrial DNA (mtDNA), which is a double-stranded molecule encoding 37 genes. In total, 13 of the mtDNA genes are involved in the OXPHOS and the remaining genes are essential in assembling amino acids into functional proteins (13). mtDNA presents in multiple copies (1,000-10,000 copies) per cell, resulting in both homoplasmic and heteroplasmic mtDNA variants. Moreover, the mtDNA copy number (mtDNAcn) is regulated in a tissue-specific manner (14) and correlates positively with the number of mitochondria, and thus is considered an indicator of mitochondrial function (15). Numerous factors make mtDNA particularly vulnerable to ROS and oxidative damage, including its proximity to the site of the electron transport chain (ETC), absence of protective histones and inadequate DNA repair capacity (16). Oxidative stress is an important source of mitochondrial genomic instability and can induce mtDNA variations and copy number changes, which may lead to abnormalities in mitochondrial function (17,18). Both mtDNA variants and copy number alterations have been implicated in human aging and various pathological conditions including mitochondrial disorders, cancer, and neurodegenerative diseases (19,20).

Harty et al (21) evaluated the total mtDNA mutational load in PsA/RA using a mitochondrial random capture assay, which revealed a significant increase in the frequency of mtDNA variants in synovial tissue from patients with RA and PsA compared with controls. However, mitochondrial random capture assay has limitations such as low sensitivity and inability to detect heteroplasmy, which is an important characteristic of numerous mtDNA-related diseases. Previously, next-generation sequencing (NGS) has emerged as a robust technique for screening the mitochondrial genome. It enables comprehensive analysis of the entire mitochondrial genome for the detection of common and rare mtDNA variants, mtDNA disease-associated variants, and accurate measurement of heteroplasmy (22). Since no previous studies have analysed the entire mitochondrial genome or evaluated changes in mtDNAcn and oxidative stress in PsA, the present study aimed to investigate mtDNA variants related to PsA and/or associated with the risk of PsA via NGS. The present study also aimed to examine changes in mtDNAcn as well as evaluate mtDNA oxidative damage in patients with PsA and healthy controls.

Materials and methods

Study subjects

A total of 43 subjects including 23 patients with PsA and 20 healthy controls were enrolled in the present study. Patients with PsA were recruited from the out-patient clinic at the Department of Rheumatology of Mubarak Hospital, (City of Kuwait, State of Kuwait). The patients fulfilled the classification criteria for PsA (CASPAR). Patients with other inflammatory or autoimmune diseases were excluded from study (23). Clinicopathological characteristics of patients (including sex and age distribution) are presented in Table I.

Table I

Demographic and clinical data of patients with psoriatic arthritis and controls.

Table I

Demographic and clinical data of patients with psoriatic arthritis and controls.

Number of subjects2320 
Sex  0.6
     Male, n (%)11(48)8(50) 
     Female, n (%)12(52)12(50) 
Age, years (mean ± SD)39±330±1.30.01
C-Reactive protein, mg/dl5±0.90.1±0.050.02
Rheumatoid factor, U/ml21±20.4±0.02<0.001
Erythrocyte sedimentation28±92.6±10.003
rate, mm/h   
     Topical treatment4  
     Systemic treatment19  

Healthy control individuals without inflammatory dermatoses or autoimmune diseases were recruited from the Central Blood Bank, State of Kuwait. Basic clinical characteristics and laboratory data were obtained from the medical and electronic records of patients and controls. Written informed consent was obtained from all participants under protocols approved by Kuwait University and Ministry of Health (City of Kuwait, State of Kuwait) (approval no. 2018/496).

Extraction of genomic DNA

Blood samples (5 ml) were collected in EDTA tubes from the participants and centrifuged at 4˚Cn 1,000 x g for 15 min to separate the buffy coat which was subjected to genomic DNA extraction using QIA amp DNA Mini kit (cat. no. 51304; Qiagen GmbΗ) according to the manufacturer's instructions as previously described (24). Briefly, a mixture of 200 µl buffy coat, 20 µl protease and 200 µl lysis buffer was incubated at 56˚C for 10 min and then centrifuged at 20,000 x g for 1 min at 4˚C. Next, absolute ethanol (200 µl) was added and centrifuged at 6,000 x g for 1 min followed by washing with 500 µl washing buffer. The mixture was then centrifuged at room temperature at 6,000 x g for 1 min and then at 20,000 x g for 3 min. Genomic DNA was eluted with 200 µl elution buffer after incubation at room temperature for 1 min and centrifugation at 6,000 x g for 1 min at room temperature. The DNA samples were quantified and assessed for purity using a NanoDrop ND-1000 ultraviolet-visible light spectrophotometer (Thermo Fisher Scientific, Inc.).

Mitochondrial genome sequencing

The entire mitochondrial genome was sequenced using the S5™XL NGS system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, as previously described (25). After library preparation and purification, the raw data were automatically transferred from the Ion Torrent S5 XL sequencer to the Torrent Suite software version 5.0 (Applied Biosystems; Thermo Fisher Scientific, Inc.), which allowed the conversion of the raw voltage semiconductor sequencing data into DNA base calls. For identification of variants, the Ion Torrent Variant Caller plug-in, Ion Reporter software version 5.2. and Torrent Variant Caller version 5.2 (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used. For alignment, the Revised Cambridge Reference Sequence of the Human mtDNA (NC_012920.1) was applied as a reference mitochondrial sequence (26). The average throughput of the Ion 520 chip was 3.5 Mb. The sequence data sets were registered in the Sequence Read Archive repository (reference no. PRJNA 928743).

Bioinformatics analysis

The impact of nonsynonymous mtDNA variants on protein function and structure was determined using three in-silico prediction tools used: i) Combined Annotation Dependent Depletion (CADD): Incorporates multiple annotations including conservation and functional information into one tool and categorizes variants as benign or deleterious using a machine learning approach. Variants with scores ≥20 were predicted to be deleterious (27); ii) CONsensus DELeteriousness (Condell): Integrates the output of five algorithms including Pfam E-value (Logre), MAPP, Mutation Assessor, Polyphen2, and SIFT to assess the outcome of nonsynonymous single nucleotide variants (SNVs) on protein function. Variants with scores ≥0.5 were predicted to be deleterious (28); and iii) Protein Variation Effect Analyzer (PROVEAN) predicts the impact of an amino acid substitution or indel on protein function. PROVEAN performance is comparable to SIFT or PolyPhen-2 and it can process a large number of protein variants. Variants with scores ≤-2.5 were predicted to be deleterious, while those with scores >-2.5 were considered neutral (29).

Analysis of protein stability

Analysis of the impact of nonsynonymous mtDNA variants on protein stability was conducted using Site-Directed Mutator (SDM), which is a statistical potential energy function that uses environment-specific amino acid substitution frequencies within the family of homologous proteins of known (3-D) structures to calculate a stability score, which is analogous to the free energy difference between the wild-type and mutant protein (30). A change in the Gibbs free energy for protein stability is expressed as ΔΔG (30).

Determination of relative mtDNAcn

Quantitative polymerase chain reaction (qPCR) was used to determine the mtDNAcn relative to nuclear DNA (nDNA). Mitochondrial NADH dehydrogenase subunit 2 (ND2) was used as a target gene for the amplification of mtDNA with the following primers: forward, 5'-CAC AGA AGC TGC CAT CAA GTA-3' and reverse, 5'-CCG GAG AGT ATA TTG TTG AAG AG-3'; while nuclear b2-microglobulin (β2M) was used as a reference gene for the amplification of nDNA with the following primers: forward, 5'-CCA GCA GAG AAT GGA AAG TCA A-3' and reverse, 5'-TCT CTC TCC ATT CTT CAG TAA GTC AAC T-3'. The PCR mixture contained 10 ng genomic DNA, 1X SYBR1 Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), forward and reverse primers (50 nM each), and nuclease-free water to a final volume of 10 µl. PCR was performed in a 7900HT real-time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the following thermocycling conditions: Initial denaturation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 10 sec, 60˚C for 30 sec and 72˚C for 30 sec. The experiments were performed in duplicate and non-template control was included in each run. Relative quantitation of mtDNAcn was performed using the 2-ΔΔCq method (31) by obtaining the Cq values of the ND2 and β2M genes, and then the ΔCq (Cq ND2-Cq β2M) values for cases and controls were calculated.

Determination of mtDNA oxidative damage

8-Hydroxyl 2'-deoxyguanosine (8-OHdG) is one of the most common markers of oxidative DNA lesions and is widely applied for the measurement of oxidative DNA damage (32,33). In the detection of oxidative damage with 8-OHdG, the marker does not deform the DNA structure but causes inhibition of Taq DNA polymerase during PCR. Therefore, the presence of 8-OHdG in a certain region of mtDNA can be digested by formamidopyrimidine [fapy]-DNA glycosylase (FPG) which breaks the DNA fragment at the lesion site and reduces further amplification of this particular region. In the present study, 8-OHdG assay was conducted to measure mtDNA oxidative damage by qPCR. A total of 100 ng DNA was incubated for 1 h at 37˚C in a 10 µl of reaction mixture containing 1 U FPG enzyme (New England Bio Labs, Inc), 1X NEB buffer and 0.1 mg/ml bovine serum albumin (New England Bio Labs, Inc). Next, the digested DNA was amplified by PCR under the same aforementioned cycling protocol and conditions. DNA damage was measured as ΔCq (ΔCq=Cq treated-Cq untreated). The presence of oxidative damage in DNA after treatment of FPG reduces the PCR efficiency and increases the Ct value.

Statistical analysis

Statistical analysis of data was performed using SPSS software (version 23; IBM Corp.). The normal distribution of data was first evaluated using the Kolmogorov-Smirnov test. Accordingly, the comparisons of variables between patients and controls were conducted using the χ2 test for categorical variables and the Mann-Whitney test for normally distributed variables. The Fisher's exact test was used to assess differences in the frequency of variants between patients and controls and the odds ratio (OR) and 95% confidence interval (CI) were reported. P≤0.05 was considered to indicate a statistically significant difference.


Characteristics of the study subjects

The demographic and clinical data of patients with PsA (n=23) and healthy control individuals (n=20) are presented in Table I. The sex ratio (male: female) was 11:12 for patients and 8:12 for controls. There was no significant difference in sex distribution between PsA and controls (P=0.6), but there was a significant difference in mean age between the two groups (P=0.01). The clinical inflammatory marker C-reactive protein (CRP) (0-0.8 mg/dl), rheumatoid factor (RF) (0-20 U/ml), and erythrocyte sedimentation rate (ESR) (0-20 mm/hr) were all higher than the normal range in patients with PsA. The mean value of CRP, RF and ESR were significantly higher in PsA patients compared with controls (P<0.05). Patients were under the following medications: Topical treatment with corticosteroid cream (n=4), or systemic treatment (n=19) including Methotrexate (n=4), Adalimumab (n=8), Etanercept (n=3), Secukinumab (n=2) and Ixekizumab (n=2).

mtDNA sequence variants identification

A total of 435 mtDNA sequence variants were identified in 43 samples. Among them, 187 (43%) variants were exclusive for patients with PsA, and 122 (28%) variants were found in control individuals only (Fig. 1). In both patients and controls, the highest number of variants was observed in the D-loop region, while the lowest number of variants was observed in the tRNAs genes. In protein-coding genes of patients with PsA, there were 152 variants including 33 nonsynonymous and 92 synonymous silent variants. By contrast, in protein-coding genes of control individuals, there were 76 variants, including 18 non-synonymous and 58 synonymous silent variants.

Particularly, a higher number of variants were observed in the MT-ND4, MT-ND5 and MT-CYB genes. A higher number of variants in the MT-ND4, and MT-CYB genes was also observed in patients than in controls. Moreover, the majority of mtDNA variants in patients and controls were homoplasmy accounting for 183 and 118, respectively, compared with the small number of heteroplasmy variants (n=4) in each group. Details of the identified variants in patients or controls as well as their localization in different mtDNA regions are shown in Table II. Notably, of non-synonymous variants in controls only, 4640C>A in the MT-ND2 gene was reported as a pathogenic variant causing Leber optic atrophy according to the Mito Map ( and ClinVar ( databases.

Table II

Details of mtDNA variants exclusive for patients with psoriatic arthritis or controls.

Table II

Details of mtDNA variants exclusive for patients with psoriatic arthritis or controls.

Non-coding region
LocusVariantType of variantAmino acid changers ID Homoplasmy/HeteroplasmyVariantVariant typeAffected amino acidrs ID Homoplasmy/Heteroplasmy
D-loopm.57T>TCInsertion rs21245905Heteroplasmym.143G>ASubstitution rs375589100Homoplasmy
D-loopm.72A>GSubstitution rs869183622Heteroplasmym.178A>GSubstitution Not providedHomoplasmy
D-loopm.93A>GSubstitution rs369034419Homoplasmym.182C>TSubstitution rs41473347Homoplasmy
D-loopm.153A>GSubstitution rs370716192Homoplasmym.185 G>TSubstitution rs879015046Homoplasmy
D-loopm.189A>GSubstitution rs371543232Homoplasmym.199T>CSubstitution rs72619362Homoplasmy
D-loopm.200A>GSubstitution rs372099630Homoplasmym.250 T>CSubstitution Not providedHomoplasmy
D-loopm.207G>ASubstitution rs369669319Homoplasmym.285C>TSubstitution rs201801609Homoplasmy
D-loopm.215A>GSubstitution rs879219259Homoplasmym.357A>GSubstitution rs28678375Homoplasmy
D-loopm.217T>CSubstitution rs41531144Homoplasmym.385A>GSubstitution rs201801609Homoplasmy
D-loopm.235A>GSubstitution rs3937037Homoplasmym.497C>TSubstitution rs28660704Homoplasmy
D-loopm.236T>CSubstitution rs375896687Homoplasmym.16093T>CSubstitution rs2853511Heteroplasmy
D-loopm.340C>TSubstitution rs117394573Homoplasmym.16150C>TSubstitution rs879004379Homoplasmy
D-loopm.508A/GSubstitution rs113683159Homoplasmym.16163 A>GSubstitution rs41479950Homoplasmy
D-loopm.524 C>CACInsertion Not providedHeteroplasmym.16185C>TSubstitution rs1556424787Homoplasmy
D-loopm.16041A>GSubstitution rs369904200Homoplasmym.16186C>TSubstitution rs879166752Homoplasmy
D-loopm.16051A>GSubstitution rs117565943Homoplasmym.16224T>CSubstitution rs386420031Homoplasmy
D-loopm.16067C>TSubstitution rs1556424732Homoplasmym.16232C>ASubstitution rs1603225749Homoplasmy
D-loopm.16111C>TSubstitution rs35315169Homoplasmym.16249T>CSubstitution rs372301309Homoplasmy
D-loopm.16124T>CSubstitution rs386829272Homoplasmym.16264C>GSubstitution rs878922147Homoplasmy
D-loopm.16148C>TSubstitution rs201893071Homoplasmym.16288T>CSubstitution rs386829301Homoplasmy
D-loopm.16178T>CSubstitution rs1603225705Homoplasmym.16293A>GSubstitution rs878890610Homoplasmy
D-loop m.16179CA-AAA>CAA§Deletion rs371240719Heteroplasmym.16296C>TSubstitution rs879138789Homoplasmy
D-loopm.16183A>TSubstitution rs28671493Homoplasmym.16318A>TSubstitution rs879067317Homoplasmy
D-loopm.16192C>TSubstitution rs879025248Homoplasmym.16319G>ASubstitution rs35105996Homoplasmy
D-loopm.16201C>TSubstitution Not providedHomoplasmym.16343A>GSubstitution rs374065731Homoplasmy
D-loopm.16214C>TSubstitution rs368055283Homoplasmym.16380C>TSubstitution rs878952395Homoplasmy
D-loopm.16217T>CSubstitution rs35134837Homoplasmym.16381T>CSubstitution rs1556424876Homoplasmy
D-loopm.16219A>GSubstitution rs878960666Homoplasmym.16526G>ASubstitution rs386829315Homoplasmy
D-loopm.16230A>GSubstitution rs2853514Homoplasmy     
D-loopm.16234C>TSubstitution rs368259300Homoplasmy     
D-loopm.16289A>GSubstitution rs1603225781Homoplasmy     
D-loopm.16290C>TSubstitution rs386828866Homoplasmy     
D-loopm.16295C>TSubstitution rs878874012Homoplasmy     
D-loopm.16300A>GSubstitution rs879082592Homoplasmy     
D-loopm.16301C>TSubstitution rs879194775Homoplasmy     
D-loopm.16304T>CSubstitution rs386829305Homoplasmy     
D-loopm.16320C>TSubstitution rs62581338Homoplasmy     
D-loopm.16324T>CSubstitution rs1556424863Homoplasmy     
D-loopm.16399A>GSubstitution rs139001869Homoplasmy     
rRNA genes
MT-RNR1 (12sRNA)m.961T>CSubstitution rs3888511Homoplasmym.954C>TSubstitution Not providedHomoplasmy
MT-RNR1 (12sRNA)m.1008A>GSubstitution rs727504505Homoplasmym.980T>CSubstitution rs397515731Homoplasmy
MT-RNR1 (12sRNA)m.1048C>TSubstitution rs2000974Homoplasmym.1018G>ASubstitution rs2856982Homoplasmy
MT-RNR1 (12sRNA)m.1442G>ASubstitution rs28358573Homoplasmym.1189T>CSubstitution rs28358571Homoplasmy
MT-RNR1 (12sRNA)m.1598G>ASubstitution rs3135027Homoplasmy     
MT-RNR2 (16sRNA)m.2028G>ASubstitution rs2124591154Homoplasmym.1733C>TSubstitution rs878868960Homoplasmy
MT-RNR2 (16sRNA)m.2245A>GSubstitution rs3020600Homoplasmym.1738T>CSubstitution rs28358574Homoplasmy
MT-RNR2 (16sRNA)m.2259C>TSubstitution rs201336470Homoplasmym.2218C>TSubstitution rs200813159Homoplasmy
MT-RNR2 (16sRNA)m.2283C>TSubstitution rs200131896Homoplasmym.2380C>TSubstitution rs1556422622Homoplasmy
MT-RNR2 (16sRNA)m.2484C>TSubstitution rs2124591301Homoplasmym.2768A>GSubstitution rs3895615Homoplasmy
MT-RNR2 (16sRNA)m.2626T>CSubstitution rs879158835Homoplasmym.2772C>TSubstitution rs200221487Homoplasmy
MT-RNR2 (16sRNA)m.3221A>GSubstitution rs1556422691Homoplasmym.2833A>GSubstitution rs3928312Homoplasmy
MT-RNR2 (16sRNA)     m.3158A>ATInsertion rs1556422679Homoplasmy
MT-RNR2 (16sRNA)     m.3221A>GSubstitution rs1556422691Homoplasmy
tRNA genes
MT-TV (tRNA)m.1676A>GSubstitution rs1603218612Homoplasmy     
MT-TL1 (tRNA)m.3387T>CSubstitution rs1569483877Homoplasmym.3277G>ASubstitution rs386828902Homoplasmy
MT-TQ (tRNA)m.4454T>CSubstitution rs11510098Homoplasmy     
MT-TA (tRNA)m.5603C>TSubstitution rs369496446Homoplasmym.5655T>CSubstitution rs1556423019Homoplasmy
MT-TS1 (tRNA)m.7476C>TSubstitution rs201950015Homoplasmym.7474G>CSubstitution rs2068703713Heteroplasmy
MT-TS1 (tRNA)m.7570A>GSubstitution rs1556423311Homoplasmy     
MT-TD (tRNA)     m.7581T>CSubstitution rs201582552Homoplasmy
MT-TK (tRNA)m.8292G>ASubstitution rs1556423422Homoplasmy     
MT-TG (tRNA)m.10042A>GSubstitution rs1603222643Homoplasmym.10034T>CSubstitution rs41347846Homoplasmy
MT-TH (tRNA)m.12171A>GSubstitution rs1603223589Homoplasmy     
MT-TH(tRNA)m.12175T>CSubstitution rs1057520099Homoplasmy     
MT-TT (tRNA)m.15907A>GSubstitution rs41383248Homoplasmy     
MT-ND1m.4188A>GSilentp.Leu294=Not providedHomoplasmym.3741C>TSilentp.Thr145=rs878907222Homoplasmy
MT-ND2m.4695T>CMissensep.Phe76Leurs1556422885Homoplasmym.4774T>TGInsertion Not providedHeteroplasmy
MT-ND2m.5090T>CSilentp.IIe207=Not providedHomoplasmym.5120A>GSilentp.Leu217=rs1603219790Homoplasmy
MT-ND2m.5390A>GSilentp. Met307=rs41333444Homoplasmym.5393T>CSilentp.Ser308=rs28357987Homoplasmy
MT-CO1m.6257G>ASilentp.Val118=rs2856983Homoplasmym.6521C>TSilentp.IIe 206=Not providedHomoplasmy
MT-CO2     m.8155G>ASilentp.Gly190=rs374052533Homoplasmy
MT-CO3m.9776C>TSilentp.Asp190=Not providedHomoplasmy     
MT-ND3m.10184C>TSilentp.Asp42=Not providedHomoplasmym.10238T>CSilentp.IIe60=rs193302927Homoplasmy
MT-ND4Lm.10628C>TSilentp. Ser53=Not providedHomoplasmy     
MT-ND6m.14494T>CSilentp.Leu60=rs879250748Homoplasmym.14364G>ASilentp Leu104=rs879086798Homoplasmy

In addition, 126 (28.9%) mtDNA variants were common for both patients with PsA and controls (Appendix I). The frequency of two common variants differed significantly between the two groups. The substitution variant m.152T>C in the D-loop region was found in 26% of patients and 55% of controls (OR=0.3, 95% CI=0.1-0.5, P=0.02), whereas the silent variant m.15301G>A in the MT-CYB gene was found in 30% of patients and 10% of controls (OR=3.8, 95% CI=1-8, P=0.04). The remaining variants showed no significant differences in their prevalence among patients or controls (P>0.05).

Variants with amino acid substitutions in patients with PsA and their impact on protein function and protein stability

Out of 187 (43%) mtDNA variants detected only in patients with PsA, 33 missense variants in mtDNA-encoding genes of complexes I, III, IV, and V resulted in amino acid substitutions.

Bioinformatics analysis using CADD, Condel, and PROVEAN was conducted to determine the potential impact of 33 missense variants that detected in PsA patients, on protein function and structure. The analysis predicted 25 variants to be deleterious by at least one bioinformatics tool (Table III). The highest impact on protein function and structure (by all three in-silico algorithms) was predicted for 2 variants, namely the m.11172A>G variant (referred to as rs2853489 polymorphism) in the MT-ND4 gene and the m.15257G>A variant (referred to as rs41518645 polymorphism) in the MT-CYB gene. The allelic frequencies of these variants in the gnome AD database were 0.0007. and 0.01, respectively.

Table III

mtDNA variants with amino acid substitutions in patients with psoriatic arthritis and their impact on protein function and structure.

Table III

mtDNA variants with amino acid substitutions in patients with psoriatic arthritis and their impact on protein function and structure.

GeneVariantVariant typeAmino acid changers IDMAFCADD/scoreCondel/scorePROVEAN/score
MT-CO1m.6546C>TMissensep.Leu215Phers16032205310.0002Neutral/6.11 Deleterious/0.84Neutral/-0.45
MT-ATP8m.8554A>GMissensep.Ile10Valrs16032215830.0001Neutral/7.68 Deleterious/0.54Neutral/0.18
MT-ATP6m.8618T>CMissensep.Ile31Thrrs283588850.009Neutral/5.55 Deleterious/0.5Neutral/2
MT-ATP6m.8705T>CMissensep.Met60Thrrs8789594040.0043Neutral/0.5 Deleterious/0.75Neutral/0.32
MT-ATP6m.8860A>GMissensep.Thr112Alars20010310.99Neutral/6.13 Deleterious/0.81 Deleterious/-3.9
MT-ATP6m.9007A>GMissensep.Thr161Alars16032219730.0058Deleterious/23Neutral/0.06 Deleterious/-3.5
MT-ATP6m.9103T>CMissensep.Thr161Alars16032220770.00045Neutral/9.74Deleterious/1 Deleterious/-2.5
MT-CO3m.9336A>GMissensep.Met44Leurs284747790.0004Neutral/0.01 Deleterious/0.68Neutral/-0.32
MT-CO3m.9948G>AMissensep.Val248Ilers15564237470.0012Neutral/9.11 Deleterious/0.65Neutral/-0.84
MT-ND3m.10143G>AMissensep.Gly29Serrs2021314190.0015Neutral/5.51 Deleterious/0.96Neutral/1.9
MT-ND4m.11016G>AMissensep.Ser86Thrrs285949040.004Neutral/0.11 Deleterious/0.68Neutral/0.21
MT-ND4m.11172A>GMissensep.Asn138Serrs28534890.0007Deleterious/20 Deleterious/0.69 Deleterious/-3.5
MT-ND5m.13759G>AMissensep.Ala475Thrrs3864200240.0143Neutral/0.69 Deleterious/0.73Neutral /1.6
MT-ND5m.13813G>AMissensep.Val493Ilers15564243320.0013Neutral/6.86 Deleterious/0.74Neutral/-0.37
MT-ND5m.13886T>CMissensep.Leu517Prors283591820.0018Neutral/9.6 Deleterious/0.6Neutral/0.23
MT-ND5m.13967 C>TMissensep.Thr544Metrs3868291970.0011Neutral/1.23 Deleterious/0.61Neutral/0.85
MT-ND5m.14053 A>GMissensep.Thr573Alars2001348390.002Neutral/0.17 Deleterious/0.76Neutral/1.8
MT-ND6m.14562 C>TMissensep.Val38Ilers16032247910.0002Neutral/1.7 Deleterious/0.8Neutral/0.06
MT-ND6m.14634 T>CMissensep.Met14Valrs16032248160.0011Neutral/12Neutral/0.17Neutral/0.001
MT-CYBm.14862 C>TMissensep. Ala39Valrs16032249330.0007Neutral/17.5 Deleterious/0.65Neutral/1.4
MT-CYBm.15218 A>GMissensep.Thr158Alars28535060.03Neutral/13.3Neutral/0.29Neutral/-1.6
MT-CYBm.15257 G>AMissensep.Asp171Asnrs415186450.01 Deleterious/23.5 Deleterious/0.67 Deleterious/-3.5
MT-CYBm.15314 G>AMissensep.Ala190Thrrs5272361760.002Neutral/16.4 Deleterious/0.61Neutral/-1.6
MT-CYBm.15431 G>AMissensep.Ala229Thrrs1933029930.002 Deleterious/24.4 Deleterious/0.61Neutral/-0.22
MT-CYBm.15468 C>TMissensep.Thr241Metrs16032253010.0004 Deleterious/20.8Neutral/0.41Neutral/-0.81
MT-CYBm.15735 C>TMissensep.Ala330Valrs16032254460.0001Deleterious/22 Deleterious/0.78Neutral/-1.7

In total, 5 other variants were predicted to be deleterious by two in-silico algorithms tools: 3 variants in the MT-ATP6 gene namely m.8860A>G (referred to as rs2001031 polymorphism), m.9007A>G (referred to as rs1603221973 polymorphism) and m.9103T>C (referred as rs1603222077 polymorphism); and 2 variants in the MT-CYB gene namely m.15431G>A (referred to as rs193302993 polymorphism) and m.15735C>T (referred to as rs1603225446 polymorphism). With the exception of the MT-ATP6 variant m.8860A>G with an allelic frequency of 0.99, all other variants had allelic frequencies of <0.01 in the gnome AD database (

In addition, 18 other variants were predicted to be deleterious by one of the in-silico algorithms tools. These included 2 variants in the MT-CO1 gene, 1 variant in the MT-ATP8 gene, 2 variants in the MT-ATP6 gene, 2 variants in the MT-CO3, 1 variant in the MT-ND3 gene, 1 variant in the MT-ND4 gene, 5 variants in the MT-ND5 gene, 1 variant in the MT-ND6 gene and 3 variants in the MT-CYB gene. The majority of these variants also had low allelic frequencies (<0.01) in the gnome AD database.

In subsequent analysis, the deleterious mtDNA variants were further evaluated for their effect on protein stability using SDM. In SMD, a stability score is calculated using environment-specific amino acid substitution frequencies within the family of homologous proteins of known 3-D structures (30). All variants in mtDNA-encoded genes of complexes I, III and IV were examined at the level of protein stability. However, the effect of variants in the MT-ATP6 and MT-ATP8 genes of complex V could not be demonstrated as the human 3-D structures were not available for complex V proteins.

As shown in Table IV, SDM predicted 19 destabilizing variants in mtDNA-encoded genes of complexes I, III and IV. Of them, 13 variants were predicted to decrease the stability of encoded proteins, including 1 variant in the MT-CO1 gene (m.6546C>T), 2 variants in the MT-CO3 gene (m.9336A>G and m.9948G>A), 1 variant in the MT-ND3 gene (m.10143G>A), 2 variants in the MT-ND4 gene (m.11016G>A and m.11172A>G), 3 variants in the MT-ND5 gene (m.13759G>A, m.13813G>A and m.13886T>C), and 4 variants in the MT-CYB gene (m.14862C>T, m.15314G>A, m.15431G>A, and m.15735C>T). Furthermore, 6 variants were found to increase the stability of encoded proteins: 1 variant in the MT-CO1 gene (m.6366G>A), 2 variants in the MT-ND5 gene (m.13967C>T and m.14053A>G), 1 variant in the MT-ND6 gene (m.14562C>T) and 2 variants in the MT-CYB gene (m.15257G>A and m.15468C>T).

Table IV

Deleterious mtDNA variants in patients with psoriatic arthritis and their impact on protein stability.

Table IV

Deleterious mtDNA variants in patients with psoriatic arthritis and their impact on protein stability.

GeneVariantVariant typeAmino acid changers IDDDGStability outcome
Relative leukocyte mtDNAcn

Using qPCR, the relative mtDNAcn was determined in the leukocytes of patients with PsA (n=23) and healthy controls (n=20). The results showed a 3.44-fold reduction in mtDNAcn in patients with PsA compared with controls. The mean ± SD mtDNAcn was 93.3±10 in patients vs. 321±29 in controls (P=0.0001) (Fig. 2).

mtDNA oxidative damage

mtDNA oxidative damage was assessed using qPCR by measuring the 8-OHdG content in patients with PsA and controls. The ΔCq value of the difference between the Cq value of DNA samples treated with FPG and the Cq value of DNA samples without FPG treatment was calculated. The results showed a 2-fold increase in the level of mtDNA oxidative damage in patients with PsA compared with controls. The ΔCq value was 0.98±0.29 in patients vs. 0.49±0.3 in controls (P=0.03) (Fig. 3).


PsA is a chronic inflammatory disease, which presents in a significant number of individuals with psoriasis (1-3). PsA is widely regarded as a multi-factorial disease with underlying autoimmune mechanisms including infiltration of plasma cells and mononuclear cells that are observed in both psoriatic plaque and PsA articular space (7). Mitochondrial dysfunction plays an important role in the pathogenesis of PsA by modulating innate immunity via redox-sensitive inflammatory pathways or directly through activation of the inflammatory response (9,10). An imbalance in oxidant-antioxidant mitochondrial system results in oxidative stress, which can induce mtDNA variations and copy number changes leading to mitochondrial functional impairment (17,18). Currently, there is limited knowledge on whether abnormalities in mtDNA a possible factor could be involved in PsA.

The present study sequenced the entire mitochondrial genome using NGS, a high-throughput and sensitive method with the ability to detect any variants (22), investigated changes in mtDNAcn and evaluated mtDNA oxidative damage in patients with PsA and healthy controls.

Analysis of the entire mitochondrial genome revealed a total of 435 variants, distributed across all regions of the mtDNA. These included 187 (43%) variants exclusively found in patients with PsA, and 122 (28%) only present in control individuals. A higher number of variants were observed in the D-loop region in both patients and controls, whereas a higher number of variants in mtDNA-coding genes was found in patients compared with controls, particularly in the MT-ND4, MT-ND5, and MT-CYB genes. In addition, common mtDNA variants (126, 28.9%) were identified among patients and controls. The frequency of two specific variants differed significantly (P<0.05) between the two groups and may be linked with the susceptibility to PsA. Namely, the D-loop m.152T>C variant occurred in 26% of patients and 55% of controls (OR=0.3, 95% CI=0.1-0.5, P=0.02) and may confer a protective role against PsA. The D-loop (non-coding or control region) contains essential regulatory sequences for replication and transcription (34). Although the majority of harmful variants are removed by natural selection, some of these variants are introduced in certain populations and may influence the risk of developing certain disorders (35), whereas other variants such as m.152T>C may be selectively beneficial on some genetic backgrounds. Additionally, the silent m.15301G>A variant in the MT-CYB gene occurred in 30% of patients and 10% of controls (OR=3.8, 95% CI=1-8, P=0.04) and may be associated with the risk of developing PsA. Synonymous variants in protein-coding genes are generally considered to be silent with no effect on protein function. However, previous studies have shown that silent variants can significantly alter gene expression by affecting the stability and folding of mRNA (36,37), and can also influence the rate of translation and posttranslational modification of proteins (38).

mtDNA variants that cause changes in amino acid sequences of protein-coding genes have been implicated in the pathogenicity of numerous diseases such as rheumatoid arthritis (RA) (39). In the present study, analysis of mtDNA in patients with PsA only revealed 33 missense variants in mtDNA-encoded genes of the ETC. In total, 25 of these variants were predicted to have deleterious effects on encoded proteins by 1-3 bioinformatics tools (CADD, Condel and PROVEAN).

Specifically, 2 missense transition variants in MT-ND4 gene and MT-CYB gene were predicted to have the highest impact on protein function and structure by all three in silico algorithms tools. Moreover, 5 mtDNA variants were predicted to be deleterious by two in silico algorithms tools, including 3 variants in the MT-ATP6 gene and 2 variants in the MT-CYB gene. A total of 18 other variants in all mtDNA-encoded complexes of the ETC were predicted to be deleterious by one of the in silico algorithms tools. The majority of these variants are considered rare with low allelic frequencies <0.01 in the gnome AD database.

Protein function and stability are closely related to protein structure. Variants that cause amino acid alterations can markedly change protein structure (30,40). Particularly, the unique amino acid sequence of a protein is reflected in its folded structure, which is important to perform proper biological function. Changes in the hydration status of a protein also affect protein folding (41). Hydrophobic interactions are important for protein folding, and changes in hydrophobicity can lead to a collapse of the protein chain in an aqueous environment (42). The polarity of amino acids also promotes appropriate folding by interacting with the water solvent, thus affecting protein stability (41). All the identified deleterious missense variants in the current study produce amino acids that are different from the usual amino acids at that position and alter the function of proteins. For instance, the MT-ND4 m.11172A>G variant causes changes in amino acid from Asparagine to Serine (p.Asn138Ser), while the MT-CYB m.15257G>A variant causes changes in amino acid from Asparagine to Aspartic acid (p.Asp171Asn). Asparagine is a hydrophilic uncharged polar amino acid, whereas serine is a neutral uncharged polar amino acid, and aspartic acid is a hydrophilic negatively charged polar amino acid.

Previous studies have demonstrated that protein flexibility is a key factor for its catalytic activity (43), and increased stability of thermophilic proteins has been shown to be associated with loss of protein flexibility and reduced enzymatic activity at low temperatures (44,45). Furthermore, high stability of proteins leads to increased proteolytic resistance, which make them difficult to regulate, particularly during cell signalling (46). It has been also shown that variants causing increased protein stability can lead to protein malfunction in human diseases, such as the stabilizing homozygous variant S37A in patients with parathyroid adenomas (47) and the Parkinson disease-associated A30P stabilizing variant in human neuroblastoma cells (48).

Analysis of the impact of deleterious variants on protein stability in present study revealed 19 destabilizing variants in mtDNA-encoded genes of complexes I, III and IV. Of them, 13 variants in different mtDNA-encoded complexes of the ETC were predicted to decrease protein stability. Moreover, 6 variants were found to increase the stability of encoded proteins. These variants may affect the stabilizing interaction within folded proteins, leading to protein instability and malfunction.

The mitochondrial OXPHOS system comprises four multi-enzymatic respiratory complexes (namely I-IV) and ATP synthase and is embedded in the inner mitochondrial membrane. A total of 4 of these complexes (I, III, IV and V) are encoded by both nDNA and mtDNA genes. Complex I (NADH: ubiquinone oxidoreductase) is one of the main contributors to ROS production within the mitochondrial matrix, which is a major cause of cellular oxidative stress and is associated with neuromuscular diseases and aging (49-52). mtDNA-encoded genes of complex I are hotspots for pathological variants (19). Such variants affect complex I assembly and activity leading to complex I deficiency with increased ROS production (51-55). Particularly, variants in mitochondrial complex I genes have been previously reported to be associated with severe erosive arthritis and to be implicated in the pathogenesis of RA (39). Complex III (bc1 complex; ubiquinol cytochrome c reductase) has been also identified as a main producer of superoxide within the mitochondrial respiratory chain (56,57). Previous studies have revealed that deleterious variants in the MT-CYB gene caused isolated complex III deficiency, leading to a variety of human diseases such as cardiomyopathy, encephalomyopathy, and Leber hereditary optic neuropathy (58-61). Complex IV or cytochrome c oxidase (COX) is one of the major regulation sites for OXPHOS and deficiency in the activity of COX has been linked to a variety of diseases (62). ATP6 and ATP8 are mtDNA-encoded subunits of the ATP synthase of complex V, which utilizes the energy provided by the proton electrochemical gradient across the inner membrane during OXPHOS and synthesizes ATP from ADP (63). Variants in MT-ATP8 gene can disturb the stability and function of complex V, affecting ATP production (64). In a previous study by Du et al (39), a higher rate of mtDNA variants in complex V was found in patients with RA compared with controls, suggesting that these variants may be associated with susceptibility to RA. Variants in complex V were also linked to ROS production and apoptosis pathways, and associated with RA progression (39).

Integrity of mtDNA which encodes essential proteins of the ETC subunits is mandatory for normal mitochondrial function (13). Improper function of ETC can enhance ROS production, which is implicated in psoriatic inflammation and PsA (9,65). Improper function of ETC can enhance ROS production, which is implicated in psoriatic inflammation and PsA (9,65). The deleterious mtDNA variants in patients with PsA identified in the present study were not detected in control individuals; were located in functionally/structurally important sites of mtDNA-encoded subunits of the ETC; and resulted in protein instability. Thus, these variants may be disease-related and could play a role in the pathogenicity of PsA.

There are several copies per cell of mtDNA, resulting in both homoplasmy (identical mtDNA) and heteroplasmy (mixture of mutated and wild-type mtDNA). In the present study, a higher rate of homoplasmic variants was detected compared with heteroplasmic variants. While most pathogenic mtDNA variants are heteroplasmic, the clinical expression of diseases is determined by the level of heteroplasmy. In this context, mitochondrial dysfunction becomes clinically apparent when the percentage of mutant mtDNA exceeds a certain threshold level. Instead, some mtDNA diseases such as Leber hereditary optic neuropathy are caused by homoplasmic variants (66). Pathogenic homoplasmic variants have been also reported in diseases such as Leigh syndrome (67) and multiple sclerosis (68), and may increase the risk of type 2 diabetes (69) and neurodegenerative diseases (70). Importantly, the phenotypic expression of homoplasmic variants can be tissue-specific, suggesting that incomplete penetrance and unidentified nuclear genetic and/or environmental factors are likely to contribute to the disease phenotype (66).

The present results also revealed a significant reduction in mtDNAcn in leukocytes of patients with PsA compared with healthy controls. mtDNAcn is an important indicator of mitochondrial biogenesis and function (15). Consequently, changes in mtDNA content could contribute to various pathological conditions (20). Low mtDNAcn was previously reported in numerous diseases such as OA (71). In our previous study, decreased mtDNAcn in patients with psoriasis was found compared with controls (24). Decreased mtDNAcn is associated with oxidative stress-induced mtDNA damage and poor oxidative capacity which can lead to a reduction in mitochondrial function and subsequent disruption of cellular functions which could affect several tissues (24,72).

The present study also found a higher level of mtDNA oxidative damage in patients with PsA compared with controls. It is well established that mtDNA is more susceptible to oxidative damage and has a higher mutational rate than nDNA (16). Oxidative damage to mtDNA can impair mitochondrial bioenergetics and causes defective mitochondrial ATP generation, which leads to further mitochondrial dysfunction and increased ROS production. Therefore, decreased mtDNAcn in patients with PsA may be a consequence of mtDNA oxidative damage.

In conclusion, the present study identified a number of unique variants in patients with PsA only or healthy controls only, as well as common variants in patients and controls, two of which may be associated with the susceptibility to PsA, and also identified various missense variants that were present only in patients with PsA and were predicted to be deleterious with important effect on the function and stability of encoded proteins. In addition, lower mtDNAcn and higher levels of mtDNA oxidative damage were found in patients with PsA compared with controls. Taken together, the present findings suggested that impaired mitochondrial function due to deleterious mtDNA variants, low mtDNAcn, and oxidative damage may be contributory factors in the pathogenesis of PsA. To the best of our knowledge, the present study is the first comprehensive analysis of mtDNA in PsA. However, the present study is limited by the small number of subjects enrolled; thus, additional large-scale studies are warranted to further elucidate the role of mtDNA defects in PsA. Moreover, the possible effect of nuclear genes and environmental factors on mitochondrial genetics in PsA should be considered in future studies. In addition, to provide an improved estimate of mtDNA damage, several regions within the mtDNA genome including the D-loop region should be targeted in future studies.

Supplementary Material

Common mtDNA variants among PsA patients and controls.


The authors would like to express their gratitude to Dr Betsy Sheena at research sector of Faculty of Sciences, Kuwait University (State of Kuwait) for the next generation sequencing data analysis and also the authors would like to acknowledge the next generation sequencing facility under the project GS01/02.


Funding: No funding was received.

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the Sequence Read Archive (SRA) repository with reference PRJNA 928743 (accession no.

Authors' contributions

MSA was the project administrator, was responsible for the conceptualization, methodology, investigation, acquisition of resources/funding and the data curation of the present study. MSA, GAK and MA implemented formal analysis. MSA and GAK confirm the authenticity of all raw data. MSA and GAK wrote the original draft, reviewed and edited the manuscript. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

The present study was conducted according to the guidelines of the Declaration of Helsinki and approved (approval no. 2018/496) by the Health Science Center Ethics Committee at Kuwait University and Health and Medical Research Committee in the Ministry of Health (City of Kuwait, State of Kuwait). Informed consent was obtained from all subjects involved in the study.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.



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Alwehaidah MS, Alsabbagh M and Al-Kafaji G: Comprehensive analysis of mitochondrial DNA variants, mitochondrial DNA copy number and oxidative damage in psoriatic arthritis. Biomed Rep 19: 85, 2023
Alwehaidah, M.S., Alsabbagh, M., & Al-Kafaji, G. (2023). Comprehensive analysis of mitochondrial DNA variants, mitochondrial DNA copy number and oxidative damage in psoriatic arthritis. Biomedical Reports, 19, 85.
Alwehaidah, M. S., Alsabbagh, M., Al-Kafaji, G."Comprehensive analysis of mitochondrial DNA variants, mitochondrial DNA copy number and oxidative damage in psoriatic arthritis". Biomedical Reports 19.5 (2023): 85.
Alwehaidah, M. S., Alsabbagh, M., Al-Kafaji, G."Comprehensive analysis of mitochondrial DNA variants, mitochondrial DNA copy number and oxidative damage in psoriatic arthritis". Biomedical Reports 19, no. 5 (2023): 85.