The Snps Of Mitochondrial DNA Displacement Loop Region And Mitochondrial DNA Copy Number Is Associated With The Risk Of Rheumatic Diseases

Rheumatic diseases encompass a collection of disorders primarily affecting joints,bones,muscles,blood vessels,and associated soft tissues or connective tissues.The majority of these diseases are autoimmune in nature,such as rheumatoid arthritis(RA),systemic lupus erythematosus(SLE),Sj?gren’s syndrome(SS),and idiopathic inflammatory myopathies(IIM).They often exhibit a subtle onset and a protracted disease course.While the exact pathogenesis of rheumatic diseases remains elusive,it is postulated that environmental and infectious factors contribute to immune dysfunction in genetically predisposed individuals.This dysfunction involves innate immunity,adaptive immunity,and non-immune factors,ultimately resulting in multi-organ damage.Diagnosis and treatment of these diseases are currently challenging.During cellular metabolism,mitochondria generate a considerable amount of reactive oxygen species(ROS),which can cause damage to DNA,proteins,and lipids.As the cell’s powerhouses and primary sites of aerobic respiration,mitochondria are particularly vulnerable.The mitochondrial DNA(mtDNA)is more susceptible to mutations than nuclear DNA due to its frequent exposure to ROS and other free radicals,lack of histone protection,and absence of a comprehensive repair system.Abundant mtDNA variation has been observed in rheumatic diseases,degenerative diseases,and tumors.The mitochondrial D-loop region,a non-coding region of mtDNA,comprises three hypervariable regions(HV-I,HV-II,HV-III).Most mtDNA mutations are concentrated in this area,with mutations potentially disrupting overall mitochondrial function,impacting respiratory chain function,and altering the organism’s immune status.The mtDNA copy number is strictly regulated to ensure mitochondria produce adequate energy and intracellular signals for maintaining normal cellular functions.The mtDNA copy number,reflecting gene-environment interactions between unidentified genetic variables and oxidative stress exposures,has emerged as a promising predictor of disease risk.This study aims to explore the relationships between D-loop region single nucleotide polymorphisms(SNPs),mtDNA copy number,and rheumatic diseases.Additionally,it will measure ROS and cytokine levels,investigate the interplay between genetic,immune,and non-immune factors in the development of rheumatic diseases,and elucidate the potential role of mtDNA in disease progression.This research will also screen for markers associated with rheumatic disease development,identify relevant cytokines,and provide a foundation for precision diagnosis and biologically targeted treatment of these disorders.Part One: The SNPs of mitochondrial DNA displacement loop region and mitochondrial DNA copy number is associated with risk of polymyositis and dermatomyositisObjective: The potential activation of muscle catabolism and autophagy pathways due to oxidative damage-induced mitochondrial dysfunction may contribute to muscle weakening in idiopathic inflammatory myopathies(IIM).Methods: This study included 86 patients with polymyositis/dermatomyositis(PM/DM)and 110 healthy controls.Genomic DNA was extracted from peripheral blood,and the target fragment of the mitochondrial DNA(mtDNA)D-loop region was amplified using the polymerase chain reaction(PCR)technique.The PCR products were then sequenced.Serum reactive oxygen species(ROS)levels were measured utilizing a high-sensitivity ROS detection kit.Cytokine levels were assessed using the Flow Fluorescence Immunomicrobeads Assay.Quantitative real-time fluorescence PCR(q RT-PCR)analysis was employed to determine the relative mtDNA copy number.Results:1.The average frequency of single nucleotide polymorphisms(SNPs)in each PM/DM patient was significantly higher compared to controls(8.779±1.912 vs.7.972±1.903,P=0.004).The D-loop region demonstrated a high frequency of SNP mutations in patients with DM(8.803±1.890 vs.7.792±1.903,P=0.004).2.Two SNPs(16304T/C,16519T/C)were identified as being associated with the risk of developing PM/DM,and alleles 16304C(P=0.047)and16519C(P=0.043)in the mitochondrial D-loop region were confirmed to be correlated with PM/DM development risk.3.The PM/DM risk-associated allele 16519 C was also linked to antinuclear antibody(ANA)positive status(C: 90.48% vs.T: 68.18%,P=0.011).4.ROS levels were considerably higher in PM/DM patients compared to controls(18477.756±13574.916 vs.14484.191±5703.097,P=0.012).5.The PM/DM risk-associated allele 16304 C was significantly correlated with lower IL-4 expression(P=0.021),while 16519 C exhibited a trend toward association with higher IL-2 expression.6.The mtDNA copy number was notably higher in PM/DM patients than in controls(0.602±0.457 vs.0.300±0.118,P<0.001).In subgroup analysis,elevated mtDNA copy number was also observed in patients with DM(0.566±0.417 vs.0.300±0.118,P<0.001),and PM patients(0.876±0.652 vs.0.300±0.118,P=0.021)demonstrated increased mtDNA copy number compared to the control group,respectively.Conclusions: The findings indicate that mitochondrial D-loop SNPs may serve as potential biomarkers for PM/DM risk,and SNPs associated with cytokine expression could be implicated in the development of PM/DM.Targeting related cytokines as a therapeutic strategy may hold potential.Additionally,mtDNA copy number-mediated mitochondrial dysfunction might precede the onset of PM/DM.Part Two: The SNPs of mitochondrial DNA displacement loop region and mitochondrial DNA copy number is associated with risk of Sj?gren’s syndrome in Female PatientsObjective: Mitochondrial dysfunction may induce an innate immune response,leading to cytokine release and the initiation of Sj?gren’s syndrome(SS)onset.This study evaluates the association between single nucleotide polymorphisms(SNPs)in the mitochondrial D-loop and mitochondrial DNA(mtDNA)copy number in female SS patients.Methods: The study included 89 female SS patients and 98 healthy controls.Genomic DNA was extracted from peripheral blood,and the target fragment of the mtDNA D-loop region was amplified using the polymerase chain reaction(PCR)technique.The PCR products were subsequently sequenced.The serum reactive oxygen species(ROS)level was detected using a highly sensitive ROS detection kit.Cytokine levels were assessed using the Flow Fluorescence Immunomicrobeads Assay.The relative mtDNA copy number was determined by quantitative real-time PCR(q RT-PCR).Results:1.The average frequency of SNPs in SS patients was significantly higher than that in controls(8.955±2.028 vs.7.898±1.987,P<0.001).2.A total of 4 SNPs(152T/C,16304T/C,16311T/C,and 16362T/C)were identified as being associated with the risk of SS.At the nucleotide sites of152,16304,16311,and 16362 in the D-loop,the frequencies of alleles 152C(P=0.040),16304C(P=0.045),16311C(P=0.045),and 16362C(P=0.028)were significantly higher in SS patients than in female controls.3.The SS risk-related allele 16311 C was associated with higher IL-2levels(P=0.010)at a statistically significant level,while 152 C was associated with lower IL-10 levels(P=0.058)at a borderline statistical level.4.The mtDNA copy number was notably higher in SS patients compared to controls(1.509±0.836 vs.1.221±0.506,P=0.006).Conclusions: The findings suggest that mitochondrial D-loop SNPs may serve as predictors for SS risk and could influence SS development by modulating cytokine expression.As a result,targeting relevant cytokines could represent a potential therapeutic strategy.The elevated mtDNA copy number observed in SS patients may contribute to salivary gland cell damage.Part Three: Risk related single nucleotide polymorphisms in mitochondrial D-loop region promotes the levels of reactive oxygen species in female systemic lupus erythematosusObjective: Single nucleotide polymorphisms(SNPs)in the D-loop region of mitochondrial DNA(mtDNA)were previously found to be associated with an increased risk of systemic lupus erythematosus(SLE).This study primarily focuses on the correlation between SLE risk-associated SNPs in the D-loop and oxidative stress status.Methods: This study enrolled a total of 81 female SLE patients and 102age-matched female controls.Plasma reactive oxygen species(ROS)levels were measured using a high-sensitivity Plasma Active Oxygen Detection Kit.Results: ROS levels in SLE patients were significantly higher than those in controls(655.025±49.748 vs.533.284±30.033,P=0.030).Additionally,the SLE susceptible SNP of 195 T was associated with increased ROS generation(P=0.048).Conclusions: In summary,the SLE risk-associated SNPs in the D-loop region may contribute to SLE initiation by increasing oxidative stress levels.Antioxidant therapy could represent a potential therapeutic approach for the treatment of SLE.