Introduction: Systemic lupus erythematosus (SLE) is a common autoimmune disease with unknown etiology. Recently, a growing number of evidence suggested that mitochondrial dysfunctions played active roles in the pathogenesis of SLE, but its detailed mechanism remains largely undetermined. The aim of this study was to analyze the frequencies of mitochondrial tRNA (mt-tRNA) variants in Chinese individuals with SLE. Methods: We carried out a mutational screening of mt-tRNA variants in a cohort of 200 patients with SLE and 200 control subjects by PCR-Sanger sequencing. The potential pathogenicity of mt-tRNA variants was evaluated by phylogenetic conservation and haplogroup analyses. In addition, trans-mitochondrial cybrid cell lines were established, and mitochondrial functions including ATP, reactive oxygen species (ROS), mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential (MMP), superoxide dismutase (SOD), and mt-RNA transcription were analyzed in cybrids with and without these putative pathogenic mt-tRNA variants. Results: We identified five possible pathogenic variants: tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, and tRNAThr A15924G that only found in SLE patients but were absent in controls. Interestingly, these variants were located at extremely conserved nucleotides of the corresponding tRNAs and may alter tRNAs’ structure and function. Furthermore, cells carrying these tRNA variants had much lower levels of ATP, mtDNA copy number, MMP, and SOD than controls; by contrast, the levels of ROS increased significantly (p < 0.05 for all). Furthermore, a significant reduction in mt-ND1, ND2, ND3, ND5, and A6 mRNA expression was observed in cells with these mt-tRNA variants, while compared with controls. Thus, failures in tRNA metabolism caused by these variants would impair mitochondrial translation and subsequently lead to mitochondrial dysfunction that was involved in the progression and pathogenesis of SLE. Conclusions: Our study suggested that mt-tRNA variants were important causes for SLE, and screening for mt-tRNA pathogenic variants was recommended for early detection and prevention for this disorder.

Systemic lupus erythematosus (SLE) is an autoimmune-mediated connective tissue disease characterized by the production of multiple autoantibodies and multisystem damage, primarily affecting women of childbearing age [1]. Its pathogenesis is complex and is partially explained by the interactions between genetic [2], epigenetic [3], environment [4], and mitochondrial dysfunction [5]. These factors lead to innate and adaptive immune disorders with failure of autoantigen tolerance and autoreactive lymphocyte activation, which induces injury through autoantibodies, immune complexes, and chronic inflammation [6]. Although great improvement has been achieved in SLE survival, the morbidity from both the disease and the medications makes the prognosis still far from satisfactory.

Cellular bioactivity cannot be accomplished without energy, and mitochondria are the critical organelle in initiating and maintaining cellular energy metabolism. By generating ATP through oxidative phosphorylation (OXPHOS), mitochondria provide energy for multiple cell activities and determine the cell fate like activation and differentiation [7]. Human mitochondrial DNA (mtDNA) is a 16,569-bp closed circular molecule encoding 13 proteins involved in the electron transport chain, two rRNAs, and 22 tRNAs. Because of the high levels of reactive oxygen species (ROS), a lack of protective histones, and limited DNA restoration capacity, mtDNA is more prone to DNA mutations than nuclear DNA [8]. Within the mitochondrial genome, tRNA variants are common and have the potential to cause structural or functional changes in the mutated tRNA molecules. Such tRNA variants can in turn lead to altered RNA processing or nucleotide modification, in addition to disrupting normal tRNA metabolism [9], thereby potentially resulting in mitochondrial dysfunction that can in turn drive SLE development. However, to date, no studies were reported on the associations between mitochondrial tRNA (mt-tRNA) variants and SLE.

In the present study, we analyzed the frequencies of mt-tRNA variants in a cohort of 200 Chinese patients with SLE and 200 healthy controls, sequence analysis of 22 mt-tRNA genes revealed the presence of 43 genetic variants, to further assess the potential pathogenicity of mt-tRNA variants, phylogenetic conservation and haplogroups were carried out, and this analysis led us to identify five possibly pathogenic variants. We also generated cybrid cell lines which were derived from these mt-tRNA variants and evaluated mitochondrial functions to assess their pathogenicity.

Patient Recruitments

Between January 2019 and January 2022, the blood specimens of 200 Chinese patients (mean age: 45.3 years; range, 20 to 79 years) with SLE were obtained from the Department of Rheumatism and Immunology, the First Affiliated Hospital of Wannan Medical College for this cross-sectional study. Furthermore, a total of 200 control subjects (mean age: 41.2 years, range, 23 to 60) without any autoimmune diseases were recruited. All patients met the SLE classification criteria revised by the American College of Rheumatology in 1997 [10]. The study was in compliance with the Declaration of Helsinki. Informed consent, blood samples, and clinical evaluations were obtained from all participants under protocols approved by the Ethics Committees of the First Affiliated Hospital of Wannan Medical College (No. 201905). In addition, we designed a general information questionnaire to collect some relevant data about these patients and controls, including the gender, ethnicity, age, disease duration, comorbidities, medical history, and the drug history of SLE.

Mutational Analysis of mt-tRNA Genes

The DNA was extracted from 2 mL peripheral blood using a QIAamp DNA Blood Minikit (Qiagen Chins Co., Ltd, China). The DNA concentrations >1.0 ng/µL were employed for the next experiments.

To screen mt-tRNA variants, subjects’ DNA (200 SLE patients and 200 controls) fragments spanning 22 mt-tRNA genes were amplified by PCR using the primers as described previously [11]. For the subjects carrying the putative pathogenic variants in the tRNA genes, fragments spanning the remaining regions of mitochondrial genome were PCR-amplified and sequenced to define the mtDNA haplogroups [12]. Bidirectional sequencing in both directions was conducted in order to confirm amplicon sequences, after which sequences were compared with the most recent Cambridge consensus sequence (GenBank accession No. NC_012920.1) [13]. Direct PCR product was subsequently analyzed by direct sequencing in an ABI 3700 automated DNA sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using the Big Dye Terminator Cycle Sequencing Reaction Kit (Thermo Fisher Scientific, Inc).

Structural Analysis

Stem and loop structures were defined based on published human mt-tRNA secondary structures, with tertiary structure interactions for these tRNA molecules being determined by referring to the relevant literature [14].

Phylogenetic Analysis

We carried out a phylogenetic conservation analysis for the identified mt-tRNA variants, as described previously [15]. The conservation index (CI) was then calculated by comparing the human nucleotide variants with the other 14 vertebrates. The CI ≥75% was regarded to have functional significance [16].

Cell Lines and Culture Conditions

Immortalized lymphoblastoid cell lines were generated by transformation with the Epstein-Barr virus, as described elsewhere [17]. Fourteen cell lines were derived from 7 SLE patients (SLE-100, SLE-005, SLE-103, SLE-094, SLE-149, SLE-044, and SLE-065) harboring putative pathogenic mt-tRNA variants (tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, and tRNAThr A15924G), as well as seven genetically unrelated control individuals (C-001, C-008, C-088, C-105, C-115, C-120, and C-121). All cell lines were grown in Dulbecco’s Modified Eagle Medium, containing 4.5 mg of glucose, 0.11 mg pyruvate/mL, and 50 mg of uridine/mL (Gibco), supplemented with 10% fetal bovine serum (Gibco).

ATP Analysis

The mitochondrial ATP levels in control and mutant cell lines were measured using the CellTiter-Glo® Luminescent Cell Viability kit (Promega, Wisconsin, USA), following the manufacturer’s instructions [18].

Assessment of ROS Production

The ROS levels of the SLE patients carrying these mt-tRNA variants and controls without these variants were analyzed according to the method described in a previous study [19]. Cells were incubated with the fluorescent probe (5 × 106 mol/L) 2′,7′-dichlorodihydrofluorescein diacetate for 30 min. The ROS production was assessed by fluorescence plate reader.

Analysis of mtDNA Copy Number

The mtDNA content was analyzed by using quantitative real-time PCR using SYBR Green DNA intercalating dye on an Applied Biosystems StepOnePlus Real-Time PCR system (Thermo Scientific, Wilmington, DE, USA), according to the protocol described previously [20]. The primer sequence for amplification of the β-globin gene was as follows: forward – 5′-GAA​GAG​CCA​AGG​ACA​GGT​AC-3′; reverse – 5′-CAA​CTT​CAT​CCA​CGT​TCA​CC-3′, while the primer for genetic amplification of mt-ND1 gene was forward: 5′-AAC​ATA​CCC​ATG​GCC​AAC​CT-3′; reverse: 5′-AGC​GAA​GGG​TTG​TAG​TAG​CCC-3′.

Analysis of Mitochondrial Membrane Potential

Mitochondrial membrane potential (MMP) in mutant and control cell lines was evaluated using JC-1 dye (Life Technology, California, USA), according to the protocol as previously described [21].

Evaluation of Superoxide Dismutase Concentration

To determine the effects of mt-tRNA variants on oxidative stress, the concentrations of superoxide dismutase (SOD) in mutant and control cell lines were analyzed using colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) [22].

mt-RNA Transcription Analysis

The total RNA was isolated from 14 cybrids using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). In brief, 500 ng of RNA was used with a reverse transcription kit (Takara, Kusatsu, Shiga, Japan). Then, fluorogenic SYBR Green (Bio-Rad, Hercules, CA, USA) was used for qPCR, following the protocol suggested previously [23]. The primers for the qPCR amplification are listed in Table 1.

Table 1.

Oligonucleotide primers for mtDNA transcription

LocusStartingEndingLength, bpForward (5′-3′)Reverse (5′-3′)
ND1 3307 4262 956 CCC​ATG​GCC​AAC​CTC​CTA​CTC​CTC AGC​CCG​TAG​GGG​CCT​ACA​ACG 
ND2 4470 5511 1,042 AAC​CCT​CGT​TCC​ACA​GAA​GCT GGA​TTA​TGG​ATG​CGG​TTG​CT 
ND3 10059 10404 346 ACG​AGT​GCG​GCT​TCG​ACC​CT TCA​CTC​ATA​GGC​CAG​ACT​TAG​GGC​T 
ND4(L) 10760 12137 1,377 CCC​ACT​CCC​TCT​TAG​CCA​ATA​TT TAGGCCCACCGCTGCTT 
ND5 12337 14148 1,812 TGCTCCGGGTCCATCATC TGA​GTA​GTC​CTC​CTA​TTT​TTC​GAA​TAT​CT 
ND6 14149 14673 525 GCC​CCC​GCA​CCA​ATA​GGA​TCC​TCC​C CCT​GAG​GCA​TGG​GGG​TCA​GGG​GT 
CO1 5904 7445 1,542 GCC​CCC​GAT​ATG​GCG​TTT​CCC​CGC​A GGG​GTC​TCC​TCC​TCC​GGC​GGG​GTC​G 
CO2 7586 8269 684 ACC​AGG​CGA​CCT​GCG​ACT​CCT ACC​CCC​GGT​CGT​GTA​GCG​GT 
CO3 9207 9990 784 CCC​CCA​ACA​GGC​ATC​ACC​CCG​C ATG​CCA​GTA​TCA​GGC​GGC​GGC 
A8 8366 8572 207 CCC​ACC​ATA​ATT​ACC​CCC​ATA​CT GGT​AGG​TGG​TAG​TTT​GTG​TTT​AAT​ATT​TTT​AG 
A6 8527 9207 681 TTA​TGA​GCG​GGC​ACA​GTG​ATT GAA​GTG​GGC​TAG​GGC​ATT​TTT 
CytB 14747 15887 1,141 CCC​ACC​CTC​ACA​CGA​TTC​TTT​A TTG​CTA​GGG​CTG​CAA​TAA​TGA​A 
LocusStartingEndingLength, bpForward (5′-3′)Reverse (5′-3′)
ND1 3307 4262 956 CCC​ATG​GCC​AAC​CTC​CTA​CTC​CTC AGC​CCG​TAG​GGG​CCT​ACA​ACG 
ND2 4470 5511 1,042 AAC​CCT​CGT​TCC​ACA​GAA​GCT GGA​TTA​TGG​ATG​CGG​TTG​CT 
ND3 10059 10404 346 ACG​AGT​GCG​GCT​TCG​ACC​CT TCA​CTC​ATA​GGC​CAG​ACT​TAG​GGC​T 
ND4(L) 10760 12137 1,377 CCC​ACT​CCC​TCT​TAG​CCA​ATA​TT TAGGCCCACCGCTGCTT 
ND5 12337 14148 1,812 TGCTCCGGGTCCATCATC TGA​GTA​GTC​CTC​CTA​TTT​TTC​GAA​TAT​CT 
ND6 14149 14673 525 GCC​CCC​GCA​CCA​ATA​GGA​TCC​TCC​C CCT​GAG​GCA​TGG​GGG​TCA​GGG​GT 
CO1 5904 7445 1,542 GCC​CCC​GAT​ATG​GCG​TTT​CCC​CGC​A GGG​GTC​TCC​TCC​TCC​GGC​GGG​GTC​G 
CO2 7586 8269 684 ACC​AGG​CGA​CCT​GCG​ACT​CCT ACC​CCC​GGT​CGT​GTA​GCG​GT 
CO3 9207 9990 784 CCC​CCA​ACA​GGC​ATC​ACC​CCG​C ATG​CCA​GTA​TCA​GGC​GGC​GGC 
A8 8366 8572 207 CCC​ACC​ATA​ATT​ACC​CCC​ATA​CT GGT​AGG​TGG​TAG​TTT​GTG​TTT​AAT​ATT​TTT​AG 
A6 8527 9207 681 TTA​TGA​GCG​GGC​ACA​GTG​ATT GAA​GTG​GGC​TAG​GGC​ATT​TTT 
CytB 14747 15887 1,141 CCC​ACC​CTC​ACA​CGA​TTC​TTT​A TTG​CTA​GGG​CTG​CAA​TAA​TGA​A 

Statistical Analysis

The SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis. Student’s t test was used to calculate the p values between the control and SLE group; p < 0.05 was regarded as having statistical significance.

Study Subjects

This study recruited 200 SLE patients and 200 control subjects from the First Affiliated Hospital of Wannan Medical College. The mean age of SLE patients was 45.3 (range: 20 to 79 years); of these patients, 180 were women (90%). Furthermore, 152 patients expressed skin involvement, 144 patients had arthritis, 65 subjects had oral ulcerations, 68 patients developed serositis, and 38 patients had CNS involvements. Moreover, 95 subjects received acetylcysteine, 130 patients were treated with sirolimus, 120 patients received glucocorticoid, and 59 subjects were supplied with vitamin D. Details about the study subjects’ demographics and disease characteristics are given in Table 2.

Table 2.

Demographics and disease characteristics of the study subjects

Parameter (unit)SLE (n = 200)Controls (n = 200)
Age 
 <30 years 50 48 
 30∼50 years 130 126 
 >50 years 20 26 
Mean age, years 45.3 41.2 
Mean age at onset, years 35.2 
Female, n (%) 180 (90) 170 (85) 
Ethnicity, n Yellow (200) Yellow (200) 
Disease duration 
 <5 years 55 
 5∼10 years 117 
 >10 years 28 
SLE history, n (%) 
 Skin involvement 152 (76) 
 Arthritis 144 (72) 
 Oral ulcerations 65 (32.5) 
 Serositis 68 (34) 
 CNS involvement 38 (19) 
 Kidney involvement 29 (14.5) 
Comorbidities, n (%) 
 Cardiovascular disease 118 (59) 
 Serious infections 28 (14) 
 Coexistence with antiphospholipid syndrome 67 (33.5) 
 Anti-dsDNA antibodies (+) 138 (69) 
 Lupus encephalopathy 35 (17.5) 
 Photosensitivity 32 (16) 
 Raynaud phenomenon 10 (5) 
 Alopecia 90 (45) 
Treatment, n (%) 
 Acetylcysteine 95 (47.5) 
 Sirolimus 130 (65) 
 Glucocorticoid 120 (60) 
Current vitamin D supplementation 59 (29.5) 
Parameter (unit)SLE (n = 200)Controls (n = 200)
Age 
 <30 years 50 48 
 30∼50 years 130 126 
 >50 years 20 26 
Mean age, years 45.3 41.2 
Mean age at onset, years 35.2 
Female, n (%) 180 (90) 170 (85) 
Ethnicity, n Yellow (200) Yellow (200) 
Disease duration 
 <5 years 55 
 5∼10 years 117 
 >10 years 28 
SLE history, n (%) 
 Skin involvement 152 (76) 
 Arthritis 144 (72) 
 Oral ulcerations 65 (32.5) 
 Serositis 68 (34) 
 CNS involvement 38 (19) 
 Kidney involvement 29 (14.5) 
Comorbidities, n (%) 
 Cardiovascular disease 118 (59) 
 Serious infections 28 (14) 
 Coexistence with antiphospholipid syndrome 67 (33.5) 
 Anti-dsDNA antibodies (+) 138 (69) 
 Lupus encephalopathy 35 (17.5) 
 Photosensitivity 32 (16) 
 Raynaud phenomenon 10 (5) 
 Alopecia 90 (45) 
Treatment, n (%) 
 Acetylcysteine 95 (47.5) 
 Sirolimus 130 (65) 
 Glucocorticoid 120 (60) 
Current vitamin D supplementation 59 (29.5) 

Screening for mt-tRNA Variants

We used PCR-Sanger sequencing analysis to detect mt-tRNA variants in SLE and control subjects. As shown in Table 3, we identified 43 different nucleotide variants in our studied individuals. The distribution of these variants was as follows: two variants in tRNAPhe, two variants in tRNAVal, two variants in tRNALeu(UUR), one variant in tRNAIle, three variants in tRNAGln, two variants in tRNAMet, two variants in tRNAAla, three variants in tRNACys, one deletion in tRNATyr, two variants in tRNASer(UCN), two variants in tRNAAsp, two variants in tRNALys, four variants in tRNAGly, two variants in tRNAArg, three variants in tRNAHis, one variant in tRNASer(AGY), two variants in tRNALeu(CUN), one variant in tRNAGlu, five variants in tRNAThr, and one variant in tRNAPro, respectively.

Table 3.

Mt-tRNA variants in 200 patients with SLE and 200 controls

GenesPositionReplacementCI (%)aHomoplasmy/heteroplasmyNumbering in tRNAWatson-Crick base pairingbLocation in tRNA200 SLE patients, n (%)200 controls, n (%)
Pathogenic/likely pathogenic variants 
tRNAVal 1606 G to A 98.0 Heteroplasmy C-G↓ Acceptor arm 1 (0.5) 
tRNALeu(UUR) 3243 A to G 100 Heteroplasmy 14  D-arm 2 (1) 
tRNAIle 4295 A to G 100 Heteroplasmy 39  Anticodon stem 1 (0.5) 
tRNAGly 9997 T to C 100 Homoplasmy A-U↓ Acceptor arm 1 (0.5) 
tRNAThr 15924 A to G 86.5 Homoplasmy 38 A-U↓ Anticodon stem 2 (1) 
Other Variants 
tRNAPhe 596 InsC 26.9 Homoplasmy 20  D-arm 1 (0.5) 1 (0.5) 
633 A to G 0.25 Homoplasmy 57  TψC loop 2 (1) 1 (0.5) 
tRNAVal/16S rRNA 1704 A to T 73.1 Homoplasmy 73  Acceptor arm 1 (0.5) 
tRNALeu(UUR) 3290 T to C 23.1 Homoplasmy 59  TψC loop 1 (0.5) 3 (1.5) 
tRNAGln 4392 G to A 88.4 Homoplasmy  D-arm 2 (1) 2 (1) 
4394 C to T 36.5 Homoplasmy A-U↓ Acceptor arm 4 (2) 3 (1.5) 
4395 T to C 82.7 Homoplasmy C-G↑ Acceptor arm 1 (0.5) 1 (0.5) 
tRNAMet 4453 A to G 71.2 Homoplasmy 52  TψC loop 0 (0) 1 (0.5) 
4454 T to C 59.6 Homoplasmy 53  TψC loop 0 (0) 1 (0.5) 
tRNAAla 5601 C to T 59.0 Homoplasmy 59  TψC loop 2 (1) 4 (2) 
5603 C to T 77.0 Homoplasmy 57  TψC loop 1 (0.5) 
tRNACys 5773 G to A 24.0 Homoplasmy 61  TψC loop 1 (0.5) 
5821 G to A 65.0 Homoplasmy  Acceptor arm 2 (1) 2 (1) 
5823 A to G 29.0 Homoplasmy C-G↑ D-loop 1 (0.5) 1 (0.5) 
tRNATyr 5878 delT 64.0 Homoplasmy 14  D-loop 2 (1) 
tRNASer(UCN) 7476 G to A 63.5 Homoplasmy 43  Anticodon stem 1 (0.5) 
7492 C to T 68.0 Homoplasmy 27 U-A↑ Anticodon stem 2 (1) 2 (1) 
tRNAAsp 7521 G to A 13.5 Homoplasmy U-A↑ Acceptor arm 1 (0.5) 
7543 A to G 63.5 Homoplasmy 29 U-A↓ Anticodon stem 1 (0.5) 
tRNALys 8334 G to A 92.3 Homoplasmy 45  Variable region 2 (1) 
8343 A to G 41.0 Homoplasmy 54  TψC loop 1 (0.5) 2 (1) 
tRNAGly 10007 T to C 49.0 Homoplasmy 19  D-arm 2 (1) 
10031 T to C 51.0 Homoplasmy 44  Variable region 1 (0.5) 
10042 A to G 80.7 Homoplasmy 55  TψC loop 2 (1) 
tRNAArg 10410 T to C 12.0 Homoplasmy  Acceptor arm 1 (0.5) 2 (1) 
10454 T to C 56.0 Homoplasmy 55  TψC loop 1 (0.5) 3 (1.5) 
tRNAHis 12153 C to T 59.0 Homoplasmy 16  D-arm 1 (0.5) 
12189 T to C 36.5 Homoplasmy 38  TψC loop 1 (0.5) 2 (1) 
12200 A to G 88.4 Homoplasmy 49  Acceptor arm 1 (0.5) 1 (0.5) 
tRNASer(AGY) 12245 T to C 42.3 Homoplasmy 39  TψC loop 2 (1) 
tRNALeu(CUN) 12280 A to G 59.0 Homoplasmy 42  D-arm 1 (0.5) 
12331 A to G 94.2 Homoplasmy 68  Acceptor arm 1 (0.5) 
tRNAGlu 14693 A to G 100 Homoplasmy 54  TψC loop 1 (0.5) 2 (1) 
tRNAThr 15889 T to C 41.0 Homoplasmy U-A↓ Acceptor arm 1 (0.5) 
15900 T to C 73.0 Homoplasmy 13  D-arm 2 (1) 
15907 A to G 65.4 Homoplasmy 20  D-arm 1 (0.5) 1 (0.5) 
15941 T to C 69.0 Homoplasmy 60  TψC loop 1 (0.5) 2 (1) 
tRNAPro 16017 A to G 34.6 Homoplasmy U-A↓ Acceptor arm 2 (1) 
GenesPositionReplacementCI (%)aHomoplasmy/heteroplasmyNumbering in tRNAWatson-Crick base pairingbLocation in tRNA200 SLE patients, n (%)200 controls, n (%)
Pathogenic/likely pathogenic variants 
tRNAVal 1606 G to A 98.0 Heteroplasmy C-G↓ Acceptor arm 1 (0.5) 
tRNALeu(UUR) 3243 A to G 100 Heteroplasmy 14  D-arm 2 (1) 
tRNAIle 4295 A to G 100 Heteroplasmy 39  Anticodon stem 1 (0.5) 
tRNAGly 9997 T to C 100 Homoplasmy A-U↓ Acceptor arm 1 (0.5) 
tRNAThr 15924 A to G 86.5 Homoplasmy 38 A-U↓ Anticodon stem 2 (1) 
Other Variants 
tRNAPhe 596 InsC 26.9 Homoplasmy 20  D-arm 1 (0.5) 1 (0.5) 
633 A to G 0.25 Homoplasmy 57  TψC loop 2 (1) 1 (0.5) 
tRNAVal/16S rRNA 1704 A to T 73.1 Homoplasmy 73  Acceptor arm 1 (0.5) 
tRNALeu(UUR) 3290 T to C 23.1 Homoplasmy 59  TψC loop 1 (0.5) 3 (1.5) 
tRNAGln 4392 G to A 88.4 Homoplasmy  D-arm 2 (1) 2 (1) 
4394 C to T 36.5 Homoplasmy A-U↓ Acceptor arm 4 (2) 3 (1.5) 
4395 T to C 82.7 Homoplasmy C-G↑ Acceptor arm 1 (0.5) 1 (0.5) 
tRNAMet 4453 A to G 71.2 Homoplasmy 52  TψC loop 0 (0) 1 (0.5) 
4454 T to C 59.6 Homoplasmy 53  TψC loop 0 (0) 1 (0.5) 
tRNAAla 5601 C to T 59.0 Homoplasmy 59  TψC loop 2 (1) 4 (2) 
5603 C to T 77.0 Homoplasmy 57  TψC loop 1 (0.5) 
tRNACys 5773 G to A 24.0 Homoplasmy 61  TψC loop 1 (0.5) 
5821 G to A 65.0 Homoplasmy  Acceptor arm 2 (1) 2 (1) 
5823 A to G 29.0 Homoplasmy C-G↑ D-loop 1 (0.5) 1 (0.5) 
tRNATyr 5878 delT 64.0 Homoplasmy 14  D-loop 2 (1) 
tRNASer(UCN) 7476 G to A 63.5 Homoplasmy 43  Anticodon stem 1 (0.5) 
7492 C to T 68.0 Homoplasmy 27 U-A↑ Anticodon stem 2 (1) 2 (1) 
tRNAAsp 7521 G to A 13.5 Homoplasmy U-A↑ Acceptor arm 1 (0.5) 
7543 A to G 63.5 Homoplasmy 29 U-A↓ Anticodon stem 1 (0.5) 
tRNALys 8334 G to A 92.3 Homoplasmy 45  Variable region 2 (1) 
8343 A to G 41.0 Homoplasmy 54  TψC loop 1 (0.5) 2 (1) 
tRNAGly 10007 T to C 49.0 Homoplasmy 19  D-arm 2 (1) 
10031 T to C 51.0 Homoplasmy 44  Variable region 1 (0.5) 
10042 A to G 80.7 Homoplasmy 55  TψC loop 2 (1) 
tRNAArg 10410 T to C 12.0 Homoplasmy  Acceptor arm 1 (0.5) 2 (1) 
10454 T to C 56.0 Homoplasmy 55  TψC loop 1 (0.5) 3 (1.5) 
tRNAHis 12153 C to T 59.0 Homoplasmy 16  D-arm 1 (0.5) 
12189 T to C 36.5 Homoplasmy 38  TψC loop 1 (0.5) 2 (1) 
12200 A to G 88.4 Homoplasmy 49  Acceptor arm 1 (0.5) 1 (0.5) 
tRNASer(AGY) 12245 T to C 42.3 Homoplasmy 39  TψC loop 2 (1) 
tRNALeu(CUN) 12280 A to G 59.0 Homoplasmy 42  D-arm 1 (0.5) 
12331 A to G 94.2 Homoplasmy 68  Acceptor arm 1 (0.5) 
tRNAGlu 14693 A to G 100 Homoplasmy 54  TψC loop 1 (0.5) 2 (1) 
tRNAThr 15889 T to C 41.0 Homoplasmy U-A↓ Acceptor arm 1 (0.5) 
15900 T to C 73.0 Homoplasmy 13  D-arm 2 (1) 
15907 A to G 65.4 Homoplasmy 20  D-arm 1 (0.5) 1 (0.5) 
15941 T to C 69.0 Homoplasmy 60  TψC loop 1 (0.5) 2 (1) 
tRNAPro 16017 A to G 34.6 Homoplasmy U-A↓ Acceptor arm 2 (1) 

aCI, conservation index.

bClassic Watson-Crick base pairing: created (↑) or abolished (↓).

Evaluation of mt-tRNA Gene Variants

These variants on the tRNA genes were further evaluated for the pathogenicity using the following criteria: (1) evolutional conservation: CI ≥ 75%, proposed by Ruiz-Pesini and Wallace [16]; (2) present <1% in controls; (3) potential structural and functional alterations [24]; (4) impair mitochondrial functions. First, we performed the phylogenetic analysis of these variants by comparing human mt-tRNA sequences with corresponding tRNAs of other 14 vertebrate, as shown in Table 3. the CIs among these nucleotides ranged from 0.25% to 100%. Of these, there were 12 variants whose CIs ≥ 75%. However, the CIs of other 31 variants were <75%. In addition, these variants were then evaluated by examining the allelic frequency in our cohort of 200 Han Chinese controls. Consequently, we classified five variants (tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, and tRNAThr A15924G) that met these criteria. Furthermore, the G1606A variant occurred in one out of 200 SLE patients (0.5%), the A3243G variant was found in 2 patients with SLE (1%), the A4295G variant was detected in one subject with SLE (%), the T9997C variant was identified in 1 out of 200 patients (1%), and the A15924G variant was identified in 2 patients with SLE (1%) (Fig. 1).

Fig. 1.

Cloverleaf structures of SLE-associated mt-tRNAVal, tRNALeu(UUR), tRNAIle, tRNAGly, and tRNAThr; arrows indicated the positions of mt-tRNA variants.

Fig. 1.

Cloverleaf structures of SLE-associated mt-tRNAVal, tRNALeu(UUR), tRNAIle, tRNAGly, and tRNAThr; arrows indicated the positions of mt-tRNA variants.

Close modal

Clinical, Molecular, and Biochemical Features of Seven SLE Subjects Carrying Putative Pathogenic mt-tRNA Variants

Of 200 SLE patients enrolled in this study, seven (0.35%) were found to be carriers of the potential tRNA variants identified through this analysis (Table 3). As shown in Table 4, among 7 SLE patients, there were five females and two males, and 6 patients exhibited positive anti-dsDNA antibody. Furthermore, two subjects (SLE-103 and SLE-094) were treated with glucocorticoid, and 2 patients (SLE-005 and SLE-065) were administrated with sirolimus. Four patients had family history of SLE (SLE-005; SLE-094; SLE-044; and SLE-065). Most of these SLE subjects expressed abnormal complement C3 and C4 levels, while the other seven control subjects had normal anti-dsDNA antibody, complement C3 and C4.

Table 4.

Summary of clinical and molecular data for 7 SLE patients and seven controls used for mitochondrial functional analyses

ProbandsEthnicityWeight, kgAnti-dsDNA positiveComplement 3 (0.9–1.8), g/LComplement 4 (0.1–0.4), g/LFamily history of SLEFunctional mtDNA variantsDrug history
SLE-100 Asian 73 0.79 0.18 No tRNAVal G1606A None 
SLE-005 Asian 75 0.41 0.09 Yes tRNALeu(UUR) A3243G and ND1 T3398C Sirolimus 
SLE-103 Asian 69 − 0.69 0.06 No tRNALeu(UUR) A3243G Glucocorticoid 
SLE-094 Asian 70 0.82 0.10 Yes tRNAIle A4295G and ND4 G11696A Glucocorticoid 
SLE-149 Asian 66 0.46 0.08 No tRNAGly T9997C None 
SLE-044 Asian 75 0.88 0.1 Yes tRNAThr A15924G and 12S rRNA 961insC None 
SLE-065 Asian 70 1.23 0.25 Yes tRNAThr A15924G and CO1/tRNASer(UCN) G7444A Sirolimus 
C-001 Asian 62 − 0.98 0.33 No None None 
C-008 Asian 59 − 1.2 0.28 No None None 
C-088 Asian 61 − 1.05 0.37 No None None 
C-105 Asian 66 − 1.58 0.20 No None None 
C-115 Asian 60 − 1.19 0.26 No None None 
C-120 Asian 58 − 1.30 0.39 No None None 
C-121 Asian 55 − 1.03 0.30 No None None 
ProbandsEthnicityWeight, kgAnti-dsDNA positiveComplement 3 (0.9–1.8), g/LComplement 4 (0.1–0.4), g/LFamily history of SLEFunctional mtDNA variantsDrug history
SLE-100 Asian 73 0.79 0.18 No tRNAVal G1606A None 
SLE-005 Asian 75 0.41 0.09 Yes tRNALeu(UUR) A3243G and ND1 T3398C Sirolimus 
SLE-103 Asian 69 − 0.69 0.06 No tRNALeu(UUR) A3243G Glucocorticoid 
SLE-094 Asian 70 0.82 0.10 Yes tRNAIle A4295G and ND4 G11696A Glucocorticoid 
SLE-149 Asian 66 0.46 0.08 No tRNAGly T9997C None 
SLE-044 Asian 75 0.88 0.1 Yes tRNAThr A15924G and 12S rRNA 961insC None 
SLE-065 Asian 70 1.23 0.25 Yes tRNAThr A15924G and CO1/tRNASer(UCN) G7444A Sirolimus 
C-001 Asian 62 − 0.98 0.33 No None None 
C-008 Asian 59 − 1.2 0.28 No None None 
C-088 Asian 61 − 1.05 0.37 No None None 
C-105 Asian 66 − 1.58 0.20 No None None 
C-115 Asian 60 − 1.19 0.26 No None None 
C-120 Asian 58 − 1.30 0.39 No None None 
C-121 Asian 55 − 1.03 0.30 No None None 

To see the contributions of mitochondrial genetic background to the phenotypic expression of these pathogenic mt-tRNA variants, we subsequently screened the whole mtDNA variants in these patients with SLE. Consequently, four functional variants were identified to be coexisted with these potential pathogenic mt-tRNA variants. Among them, the ND1 G3635A coexisted with tRNAVal G1606A variant. In fact, the G3635A variant changed a highly conserved serine to asparagine at the 110th residue of the ND1 protein to asparagine, caused a respiration defect with complex I-linked substrates, and had been regarded to be a pathogenic variant for Leber’s hereditary optic neuropathy [25, 26]. Furthermore, the ND1 T3398C variant coexisted with tRNALeu(UUR) A3243G variant, the T to C transition at position 3398 resulted in the substitution of a highly conserved methionine for a threonine at amino acid position 31 of the ND1 gene, and previous biochemical studies showed that this variant caused a reduction of complex I activity, and impaired the OXPHOS activities [27]. In addition, the ND4 G11696A resulted in the substitution of an isoleucine for valine at amino acid position 312 and was regarded as a pathogenic variant for diabetes, deafness, and Leber’s hereditary optic neuropathy [28‒30]. While the 961insC localized at the C-cluster of the region between loops 21 and 22, there was a secondary variant for aminoglycoside-induced and non-syndromic hearing loss-associated A1555G variant in Han Chinese pedigrees [31]. Interestingly, the G7444A variant was adjacent to the site of 3′ end endonucleolytic processing of L-strand RNA precursor, spanning tRNASer(UCN) and ND6 mRNA [32]. The G7444A variant resulted in a read-through of the stop codon AGA of the CO1 message, thereby adding three amino acids (Lys-Gln-Lys) to the C-terminal of the polypeptide [32].

Reduced in ATP Production

Because mitochondria generated ATP via OXPHOS, defects in ATP synthesis were found to be an important cause for mitochondrial dysfunction. For this purpose, we constructed cybrid cells containing these SLE-associated putative pathogenic variants (tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, and tRNAThr A15924G), as well as seven control subjects lacking these variants. As shown in Figure 2a, we noticed that cells carrying these variants caused approximately 43% reductions in ATP production when compared with controls (p < 0.001).

Fig. 2.

a–e Analysis of mitochondrial functions in seven control and seven mutant cell lines derived from normal individuals (mean age: 48) and SLE patients (mean age: 52) carrying putative pathogenic mt-tRNA variants (tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, tRNAThr A15924G). Student’s t test was performed in two groups to measure the p values.

Fig. 2.

a–e Analysis of mitochondrial functions in seven control and seven mutant cell lines derived from normal individuals (mean age: 48) and SLE patients (mean age: 52) carrying putative pathogenic mt-tRNA variants (tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, tRNAThr A15924G). Student’s t test was performed in two groups to measure the p values.

Close modal

ROS Increased in Mutant Cell Lines

The levels of mitochondrial ROS from SLE patients carrying mt-tRNA variants and the controls without these variants were analyzed. As shown in Figure 2b, SLE patients with mt-tRNA variants exhibited approximately 49.2% increase in ROS production when comparing with controls (p < 0.001).

Decreased in mtDNA Copy Number

The mtDNA copy number was a relative measure of the cellular number of mitochondria. Damage to mtDNA was considered to be an alternate measure of mitochondrial dysfunction because it led to reduced cellular metabolic activity [33]. As shown in Figure 2c, SLE patients had a much lower level of mtDNA content as compared with controls (p < 0.001).

Diminished Levels of MMP in SLE Patients

Loss of MMP was implicated to have functional importance because it was the early event for apoptosis [34]. As shown in Figure 2d, we found that, compared with control subjects, mutant cell lines carrying these mt-tRNA variants had a much lower level of MMP (p = 0.0003).

The Levels of SOD Decreased in Mutant Cell Lines

SOD was a universal enzyme of organisms that lived in the presence of oxygen. SOD enzyme controlled the levels of ROS, thus limiting the potential toxicity of these molecules and governing broad aspects of cellular life [35]. As shown in Figure 2e, approximately 32.8% reductions in SOD levels were found in mutant cells, when compared with controls (p = 0.0012).

mt-tRNA Variants Affected RNA Transcription

To see whether these mt-tRNA variants affected mitochondrial functions, we evaluated the mt-RNA transcription in patient-derived cell lines and found that significantly lower levels of ND1, ND2, ND3, ND5, and A6 mRNA were present in the cell lines carrying these mt-tRNA variants when compared with the cells without these tRNA variants (Fig. 3). This result showed that mt-tRNA variants might, at least partially, block mt-RNA transcription.

Fig. 3.

Analysis of the relative mt-RNA transcription in control and mutant cells. All p values were calculated by using unpaired, two-tailed Student’s t test.

Fig. 3.

Analysis of the relative mt-RNA transcription in control and mutant cells. All p values were calculated by using unpaired, two-tailed Student’s t test.

Close modal

SLE is a prototype of systemic autoimmune disease involving almost every organ. At present, the pathogenesis of SLE remains unclear. Polygenic predisposition and complicated epigenetic regulations are the upstream factors to elicit its development. In addition to genetic and environmental factors, mitochondrial dysfunction can also be involved [36]. mtDNA is a multi-copy, circular, double-stranded DNA molecule that is essential for OXPHOS. In SLE, neutrophil mtDNA is highly accessible to be oxidized by ROS and released from mitochondria [37]. Cytosolic mtDNA further promotes type I interferon (IFN) production through the cGAS-STING pathway [38, 39]. Extracellular mtDNA activates plasmacytoid dendritic cells and initiates CD4+ T-cell activation, which is critical to SLE pathogenesis [40].

mt-tRNAs are essential components for the translation and transcription of the 13 protein-encoding genes and are complemented by a number of imported nuclear-encoded factors such as the aminoacyl-tRNA synthetases [41]. Among 22 mt-tRNAs, tRNAGlu, tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer(UCN), tRNAGln, and tRNAPro are resided at the cytosine-rich light (L) strand, and the remaining tRNAPhe, tRNAVal, tRNALeu(UUR), tRNALeu(CUN), tRNAIle, tRNAMet, tRNASer(AGY), tRNATrp, tRNAAsp, tRNALys, tRNAGly, tRNAArg, tRNAHis, and tRNAThr are located at the guanine-rich heavy (H) strand. mt-tRNA variants are being increasingly recognized as important causes of disease, with novel pathogenic changes being reported across all the mt-tRNAs. Such variants can result in transcriptional and translational defects and consequently mitochondrial respiratory chain dysfunction [42].

In the present study, by using PCR and Sanger sequencing, we identified 43 genetic polymorphisms in 200 SLE subjects and 200 controls. By focusing on variants that were evolutionarily conserved, not present in control subjects, and predicted to induce functional or structural changes in tRNA molecules, we were ultimately able to identify five possibly pathogenic variants: tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, and tRNAThr A15924G. In fact, the G to A substitution at nucleotide 1606 had been reported to cause sensorineural deafness, ataxia, myoclonus, seizures, and mental retardation [43]. The G1606A variant disrupted a G-C pair in the acceptor stem of the tRNAVal at the same position on the tRNA structure as reported on T7512C variant in the tRNASer(UCN) gene [44]. Furthermore, the heteroplasmic A3243G in tRNALeu(UUR) was one of the most common diabetes-associated pathogenic variants [45]. This variant also led to mitochondrial encephalomyopathy, lactic acidosis, and stroke-like symptoms [46]. Molecular analysis revealed that this variant reduced the steady-state level, aminoacylation, as well as codon recognition of tRNALeu(UUR) [47]. As a result, the A3243G variant caused the pretermination of transcription and expression impeding of normal rRNA, thus compromising mitochondrial protein and ATP synthesis [48], while the A4295G variant affected a highly conserved adenosine at position 37, 3′ adjacent to the tRNA’s anticodon. Furthermore, this variant reduced the 5′ end processing efficiency of tRNAIle precursors, catalyzed by RNase P, affected the tRNA steady-state level and aminoacylation, and led to mitochondrial dysfunction [49, 50]. The T to C transition at position 9997 abolished the U-A base pairing and affected tRNA structure and function. In fact, functional analysis of cybrid cell lines harboring the T9997C variant produced a significant reduction both in complex I and IV, as well as influenced the protein translation [51]. The homoplasmic A15924G variant occurred at the extremely conserved nucleotide of tRNAThr, which was the last base pair of the anticodon stem adjacent to the anticodon loop of this tRNA [52]. Interestingly, the A15924G variant abolished the Watson-Crick base pairing and may result in the failure in tRNA metabolism. Functional assessment of cell lines with this variant revealed a deficiency in complex IV activity as compared with controls suggesting a direct pathogenic role for mitochondrial diseases [53].

Cybrid cell lines can incorporate human subject mitochondria and perpetuate its mtDNA-encoded components. Since the nuclear background of different cybrid lines can be kept constant, this technique allowed investigators to study the influence of mtDNA on cell function [54]. For this purpose, we generated seven cybrid cell lines harboring these variants and 7 control subjects without these variants. Although 4 SLE patients received sirolimus and glucocorticoid for regular treatment of this disease, and these anti-SLE drugs may affect mitochondrial functions [55, 56], we still found that mutant cells exhibited much more severe mitochondrial dysfunctions than control cells including lower levels of mtDNA content, ATP, MMP, and SOD, whereas ROS increased. In mitochondrial diseases, high mtDNA copy number mostly correlated with decreased disease severity, or even with incomplete disease penetrance [57, 58]. By contrast, decrease in mtDNA copy number was coupled with dysfunction in OXPHOS activity or impaired ROS homeostasis in mitochondria, eventually resulting in the manifestation of SLE progression [59]. In addition, a decrease in mtDNA copy number will induce the impairment of OXPHOS system, and subsequently reduce the ATP production in mutant cell lines, as in the case of tRNAHis T12201C variant [60]. Previous study indicated that mitochondrial hyperpolarization and ATP depletion were found in patients with SLE [61]. Indeed, the MMP reflected the pumping of hydrogen ions across the inner membrane during the process of electron transport and OXPHOS [62]. The defects in MMP may be due to strongly decreased efficiency of respiratory chain-mediated proto extrusion for the matrix [63]. Furthermore, SOD was an antioxidant enzyme that had the ability to break down the harmful oxygen molecular within the cell and changed it into less toxic products. The increased ROS production and decreased SOD levels in cells with these mt-tRNA variants indicated that an increased cellular oxidative stress was involved in SLE progression [64]. In SLE, mt-tRNA variants may lead to excessive production of ROS and oxidative stress, which in turn causes DNA damage and cell necrosis of local epithelial and endothelial cells, thereby contributing to the release of self-reactive T and B cells that drive the amplification of this inflammatory response [65], despite that exact pathophysiological role of oxidative stress in SLE was not completely understood. Moreover, Doherty et al. [66] showed that increased ROS production would enhance mitochondrial oxygen consumption and the accumulation of oxidative stress generating mitochondria in both human and mice with SLE [67, 68].

Mitochondrial haplogroups were collections of similar haplotypes defined by combinations of single-nucleotide polymorphisms in mtDNA inherited from a common ancestor. These haplogroups were formed as a result of the sequential accumulation of mutations through maternal lineages [69]. Recent experiment study suggested that mtDNA haplogroup affecting complex I and V genes played an active role in the development and progression of white patients with SLE; in particular, the A6 G9055A variant showed a strong correlation for this disease [70]. Our study revealed that mtDNA haplogroup N9b1-specific ND1 G3635A variant, haplogroup M9a-specific ND1 T3398C variant, D4j-specific ND4 G11696A variant, haplogroup F3b-specific 12S rRNA 961insC variant, and haplogroup D4b2b2-specific CO1/tRNASer(UCN) G7444A variant may enhance the expression of SLE-related mt-tRNA variants.

Next, the examination of the RNA level of mtDNA-encoded OXPHOS subunits revealed that the mRNA levels of ND1, ND2, ND3, ND5, and A6 were lower in cybrids carrying these mt-tRNA variants than in cybrids without these variants. This result indicated that defective mt-RNA translation caused by mt-tRNA variants may be responsible for multiple OXPHOS complex deficiencies in cybrid cells.

In summary, our study suggested that mitochondrial dysfunctions caused by mt-tRNA variants played important roles in SLE progression; in particular, tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, and tRNAThr A15924G variants that caused screening for SLE-related mt-tRNA variants was recommended. Thus, our study provided novel insight into the molecular pathogenesis of SLE that was manifested by mt-tRNA variants.

This study represented the first investigation between mt-tRNA variants and SLE among Chinese population. However, there were still several limitations. First, due to the limited sample size of our case-control study, we only found seven mt-tRNA variants associated with SLE. Second, since 4 patients had the history of using anti-SLE drugs, the impact of these drugs on mitochondrial functions may be overshadowed. Further investigation of additional potential functional mt-tRNA polymorphisms from multicenter study was needed to verify this conclusion.

All methods were carried out in accordance with the Declaration of Helsinki. The study received ethical approval from the Ethics Committee of the First Affiliated Hospital of Wannan Medical College (No. 201905). All participants in the study were informed and gave their written consent for anonymous and voluntary participation.

The authors have no conflicts of interest to declare.

There was no funding for this study.

D.X., F.Q., and Y.X. designed the study, H.X. and L.W. collected SLE and control samples, D.X. performed genetic analysis, F.Q. performed cellular experiments, D.X. and Y.X. wrote the manuscript, and all authors read and approved the final manuscript.

Additional Information

Dan Xuan and Fuyong Qiang contributed equally to this work.

Data cannot be shared publicly, because data from this study may contain potentially or sensitive patient information; furthermore, the Ethics Committee has not agreed to the public sharing of data as we do not have the participants’ permission to share their anonymous data. However, the data presented in this study are available on request from the corresponding author (Yonghui Xia, e-mail: [email protected]).

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