Introduction

Primary biliary cholangitis (PBC) is a chronic autoimmune genetic disorder characterized by the occurrence of serum anti-mitochondrial antibodies (AMA), lymphocytic infiltration of the portal tract, progressive intrahepatic destruction of the interlobular bile ducts, which eventually leads to cirrhosis of the liver1. An increasing prevalence of PBC has been observed worldwide, presumably due to improved diagnosis, better care and survival of PBC patients, as well as enhanced awareness and knowledge of the disorder among clinicians2. The etiology and pathogenesis of PBC are poorly understood. It is possible that exposure to environmental factors triggers the disease process in genetically susceptible individuals3.

AMA, a disease-specific autoantibody found in 90% of PBC patients, is the characteristic serological hallmark of PBC4. The target antigens are members of the 2-oxo-acid dehydrogenase complex (2-OADC), including pyruvate dehydrogenase complex-E2 (PDC-E2), the branched chain 2-oxo-acid dehydrogenase complex (BCOADC-E2), and the 2-oxo-glutaric acid dehydrogenase complex (OGDC-E2). Each of the 2-OADC members has distinct antigenicity and shows no cross-reactivity5. The dominant epitopes contain a lysine-lipoyl acid domain, that can be recognized by both B and T cells and is subject to xenobiotic modifications6. It is unknown whether the presence of AMAs targeted to different antigens is predictive or prognostic for a particular clinical and biological profile of PBC. The clinical significance and prevalence of AMAs other than PDC-E2 have not been analyzed among PBC patients in the Chinese population.

In this study, we examined the prevalence and clinical significance of three different AMAs using enzyme-linked immunosorbent assay (ELISA) and investigated the association between these autoantibodies and the clinical and biochemical features in a large cohort of Chinese PBC patients.

MethodsSubjects

This study was conducted on patient samples collected from the member hospitals of the Jiangsu Provincial PBC Collaboration Group (JSPPCG) as part of a genome-wide association study7. PBC patients were recruited according to the guidelines of the Declaration of Helsinki (2008). The diagnosis of PBC was based on the following criteria: AMA positive (PDC-E2, BCOADC-E2, or OGDC-E2); AMA negative, anti-sp100 or -gp210 positive, with elevated alkaline phosphatase; or antibody negative, but with histological evidence and elevated alkaline phosphatase. Since a liver biopsy was not conducted for most patients, those who tested negative for AMA, sp100, and gp210 antibodies and did not undergo biopsy were excluded.

Biochemical test records for 780 of the 1096 PBC patients in this study were included at the time of disease onset (supplementary table 1). Of these 780 patients, 666 were female (85.38%) and 114 were male (14.62%). The median age of the patients was 55.9 years. Patients who had received ursodeoxycholic acid (UDCA) at a daily dose of 13 to 15 mg/kg of body weight and had at least 1 year of follow-up were selected to evaluate their response to UDCA. The biochemical response to treatment was evaluated according to the Barcelona (BA) and Paris I (PA) criteria8.

Cloning and expression of PDC-E2, BCOADC-E2 and OGDC-E2

For plasmid construction, homologous recombination/ET cloning was used to insert gene into the expression vectors9. pET28a with 6×His tag was used to clone human with the following primers (PDC-E2. FP: 5’-CAGCCATCATCATCATCATCA CGGATCCAGTCTTCCCCCGCATCAGAA, PDC-E2.RP: 5’-TGCTCGAGTGCGGCCGCAA GCTTCATTACAACAACATAGTGATAG; BCOADC-E2. FP: 5’-CATCATCATCATCATCACGGAT CCGGACAGGTTGTTCAGTTCAA-3’; BCOADC-E2.RP: 5’-GTGGTGGTGGTGGTGCTCG AGTTCAATCTAGTAGCATAAAAGCTGG-3’; OGDC-E2. FP: 5’-ACTTTAAGAAGGAGATATA CCATGGATGACTTGGTTACAGTC-3’; OGDC-E2.RP: 5’-GTGGTGGTGGTGGTGGTGCTC GAGAAGATCCAGGAGGAGGACTCTG-3’).

RNA from human liver cells (HepG2) served as the template for cDNA preparation by reverse transcribed-polymerase chain reaction (RT-PCR) using the oligos (dT) primer. PCR was performed using the high-fidelity LA Taq polymerase (TaKaRa, China). The DNA sequence of the constructed plasmid was verified by DNA sequencing analysis. Plasmids were transformed into BL21 competent cells. The transformed cells were grown at 37°C overnight in Luria-Bertani medium containing 50 µg/mL ampicillin and induced with 1mM isopropylthiogalactoside (IPTG) overnight at 25°C. PDC-E2 was purified from under native conditions by Ni-NTA agarose (QIAGEN, Germany) with elution buffer (50 mM NaHPO, 300 mM NaCl, 250 mM imidazole, pH 8.0). The eluted proteins were loaded to the HiLoad 26/600 Superdex 75 PG column (GE Healthcare Bio-Sciences AB, Sweden) for the final purification of PDC-E2, BCOADC-E2 and OGDC-E2.

Enzyme-linked immunosorbent assay (ELISA) for autoantibody analysis

Gel-purified proteins (antigens) (1 µg/mL) were suspended in 50 mM carbonate buffer (pH 9.6) and coated onto microtiter plates (Thermo-Fisher, USA) overnight at 4°C for enzyme-linked immunosorbent assay (ELISA). After blocking with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), the plates were incubated with sera (1:2000 dilution) for 2 hours at room temperature and then washed five times with PBS containing 0.1% Tween-20 (PBST). Peroxidase-conjugated anti-human immunoglobulin (IgG, IgM, and IgA) antibody (Millipore, Temecula, USA) was added to the plates (1:30,000 dilution, 100 µL/well) and then incubated for 2 hours at room temperature. After washing six times with PBST, 3,3′,5,5′-tetramethylbenzidine (TMB) was added as the substrate, and 2 M sulfuric acid was added as the stop solution. The optical density (OD) was measured using an ELISA plate reader (Bio-Rad, USA) at 450 nm. Reactivity against AMAs was also assessed using commercially available ELISA kits (Shanghai Kexin Biotech Co., Shanghai, China).

Statistical analysis

All statistical analyses were performed using SPSS version 19.0 for Windows (SPSS, Inc., Chicago, IL). Values were expressed as the mean ± SD. A P value less than 0.05 was considered statistically significant for all tests. The Mann-Whitney U-test was used to compare quantitative variables between two groups, and the χ² test was used to compare categorical variables.

ResultsHigher Prevalence of AMA to BCOADC-E2 in Chinese PBC Patients

The overall prevalence of AMA to PDC-E2, BCOADC-E2, and OGDC-E2 is summarized in Table 1. In this study, 1081 patients were immunoreactive to at least one of the three 2-OADC components. Anti-PDC-E2 was the most frequently detected AMA, while anti-OGDC-E2 was the least prevalent among the three 2-OADC components. Notably, the prevalence of anti-BCOADC-E2 was 84.21% in Chinese PBC patients, which was much higher than the prevalence observed in Caucasian PBC patients (64.32%)10. Approximately one-third of patients were simultaneously reactive to PDC-E2, BCOADC-E2, and OGDC-E2.

Table 2

Biochemical characteristic of PBC patients according to the presence of M2-AMA

Clinical Features

Anti-PDC-E2

Anti-BCOADC-E2

Anti-OGDC-E2

Positive

(673)

Negative

(107)

P-value

Positive

(657)

Negative

(123)

P-value

Positive

(316)

Negative

(464)

P-value

Age

56.0 ± 11.5

55.4 ± 10.4

0.461

56.0 ± 11.1

55.0 ± 12.7

0.484

56.5 ± 11.3

55.5 ± 11.3

0.286

Sex (F/M)

571/102

95/12

0.284

554/103

112/11

0.052

262/54

404/60

0.107

ALT (IU/L)

121.6 ± 217.1

140.4 ± 165.5

0.039

120. 2 ± 211.4

145.9 ± 207.2

0.172

105.2 ± 128.2

137.1 ± 251.4

0.156

AST (IU/L)

120.0 ± 184.0

123.2 ± 144.0

0.890

114.9 ± 152.4

150.1 ± 280.6

0.795

110.3 ± 112.6

127.3 ± 212.6

0.891

GGT (U/L)

357.0 ± 308.7

387.0 ± 317.6

0.443

370.0 ± 314.4

312.5 ± 280.5

0.022

369.7 ± 337.5

355.2 ± 289.9

0.799

ALP (U/L)

339.1 ± 223.4

319.0 ± 197.4

0.285

343.9 ± 221.5

296.2 ± 208.0

0.003*

347.5 ± 231.6

328.8 ± 211.7

0.220

TB (μmol/)

44.6 ± 591

50.5 ± 94.5

0.234

47.1 ± 66.8

36.2 ± 54.2

＜0.001*

42.5 ± 57.8

47.3 ± 69.6

0.919

DB (μmol/)

28.5 ± 45.9

32.1 ± 75.7

0.072

30.5 ± 52.9

20.8 ± 38.0

＜0.001*

27.3 ± 46.2

30.2 ± 54.0

0.835

TP (g/L)

73.7 ± 9.7

73.4 ± 12.3

0.760

73.7 ± 10.3

73.5 ± 8.7

0.634

74.7 ± 9.7

72.9 ± 10.3

0.007*

ALB (g/L)

37.7 ± 7.3

39.4 ± 6.6

0.018

37.8 ± 7.2

38.7 ± 6.8

0.223