Leukocyte mitochondrial DNA alteration in systemic lupus erythematosus and its relevance to the susceptibility to lupus nephritis

Abstract

The role of mitochondrial DNA (mtDNA) alterations in the pathophysiology of systemic lupus erythematosus (SLE) remains unclear. We investigated sequence variations in the D310 region and copy number change of mtDNA in 85 SLE patients and 45 normal subjects. Leukocyte DNA and RNA were extracted from leukocytes of the peripheral venous blood. The D310 sequence variations and copy number of mtDNA, and mRNA expression levels of mtDNA-encoded genes in leukocytes were determined by quantitative real-time polymerase chain reaction (Q-PCR) and PCR-based direct sequencing, respectively. We found that leukocyte mtDNA in SLE patients exhibited higher frequency of D310 heteroplasmy (69.4% vs. 48.9%, p = 0.022) and more D310 variants (2.2 vs. 1.7, p = 0.014) than those found in controls. Among normal controls and patients with low, medium or high SLE disease activity index (SLEDAI), an ever-increasing frequency of D310 heteroplasmy was observed (p = 0.021). Leukocyte mtDNA copy number tended to be low in patients of high SLEDAI group (p = 0.068), especially in those harboring mtDNA with D310 heteroplasmy (p = 0.020). Moreover, the mtDNA copy number was positively correlated with the mRNA level of mtDNA-encoded ND1 (NADH dehydrogenase subunit 1) (p = 0.041) and ATPase 6 (ATP synthase subunit 6) (p = 0.030) genes. Patients with more D310 variants were more susceptible to lupus nephritis (p = 0.035). Taken together, our findings suggest that decrease in the mtDNA copy number and increase in D310 heteroplasmy of mtDNA are related to the development and progression of SLE, and that the patients harboring more D310 variants of mtDNA are more susceptible to lupus nephritis.

Keywords: D310 sequence variation; copy number; heteroplasmy; mitochondrial DNA; systemic lupus erythematosus.

Figure 1

Representative cases to demonstrate the D310 sequence variations. T (thymine) is shown in red, A (adenine) in green, C (cytosine) in blue and G (guanine) in black during sequencing. The Arabic number above the red arrow denotes the number of C before the indicated T peak. (A) In patient S5, leukocyte mtDNA harbored 1 kind of D310 variant, C₇TC₆. As a result, S5 was classified as D310 homoplasmy with C₇TC₆ being the dominant variant; (B) In patient S10, leukocyte mtDNA harbored 2 kinds of D310 variants, C₈TC₆ and C₉TC₆ in order, with C₈TC₆ being the dominant one. As a result, S10 was classified as D310 heteroplasmy with C₈TC₆ as the dominant variant; (C) In patient S15, leukocyte mtDNA harbored 4 kinds of D310 variants, C₉TC₆, C₁₀TC₆, C₈TC₆ and C₁₁TC₆ in order, with C₉TC₆ being the dominant one. As a result, S15 was classified as D310 heteroplasmy with C₉TC₆ as the dominant variant. Clinically, patient S15 developed lupus nephritis.

Figure 2

(A) The distribution of mean mtDNA copy number in the leukocytes of normal controls and SLE patients with low, medium and high SLE Disease Activity Index (DAI) scores. The mean mtDNA copy number in the leukocytes of the group with low SLEDAI score (0.243 ± 0.147) was higher than that of controls (0.193 ± 0.065) (p = 0.049) and the group with a high SLEDAI score (0.179 ± 0.077, p = 0.068); (B) In SLE patients (n = 59) with D310 heteroplasmy in mtDNA of the leukocytes, we found a negative correlation between the mtDNA copy number and SLEDAI score (p = 0.020, Pearson’s correlation coefficient γ_pcc = −0.268); (C) Among the 44 analyzed SLE patients, a positive correlation was found between mtDNA copy numbers and the mRNA expression levels of mtDNA-encoded ND1 gene; (D) Among the 44 analyzed SLE patients, a positive correlation was found between mtDNA copy numbers and the mRNA expression levels of mtDNA-encoded ATPase 6 gene.

Figure 2

(A) The distribution of mean mtDNA copy number in the leukocytes of normal controls and SLE patients with low, medium and high SLE Disease Activity Index (DAI) scores. The mean mtDNA copy number in the leukocytes of the group with low SLEDAI score (0.243 ± 0.147) was higher than that of controls (0.193 ± 0.065) (p = 0.049) and the group with a high SLEDAI score (0.179 ± 0.077, p = 0.068); (B) In SLE patients (n = 59) with D310 heteroplasmy in mtDNA of the leukocytes, we found a negative correlation between the mtDNA copy number and SLEDAI score (p = 0.020, Pearson’s correlation coefficient γ_pcc = −0.268); (C) Among the 44 analyzed SLE patients, a positive correlation was found between mtDNA copy numbers and the mRNA expression levels of mtDNA-encoded ND1 gene; (D) Among the 44 analyzed SLE patients, a positive correlation was found between mtDNA copy numbers and the mRNA expression levels of mtDNA-encoded ATPase 6 gene.