Anti‐kelch‐like 12 and anti‐hexokinase 1: novel autoantibodies in primary biliary cirrhosis

Abstract

Background Using high-density human recombinant protein microarrays, we identified two potential biomarkers, kelch-like 12 (KLHL12) and hexokinase-1 (HK1), in primary biliary cirrhosis (PBC). The objective of this study was to determine the diagnostic value of anti-KLHL12/HK1 autoantibodies in PBC. Aims Initial discovery used sera from 22 patients with PBC and 62 non-PBC controls. KLHL12 and HK1 proteins were then analyzed for immunoglobulin reactivity by immunoblot and enzyme-linked immunosorbent assay (ELISA) in two independent cohorts of PBC and disease/healthy control patients. Methods Serum samples from 100 patients with PBC and 165 non-PBC disease controls were analyzed by immunoblot and samples from 366 patients with PBC, 174 disease controls, and 80 healthy donors were tested by ELISA. Results Anti-KLHL12 and anti-HK1 antibodies were each detected more frequently in PBC compared with non-PBC disease controls (p < 0.001). Not only are both markers highly specific for PBC (≥ 95%), but they also yielded higher sensitivity than anti-gp210 and anti-sp100 antibodies. Combining anti-HK1 and anti-KLHL12 with available markers (MIT3, gp210 and sp100) increased the diagnostic sensitivity for PBC. Most importantly, anti-KLHL12 and anti-HK1 antibodies were present in 10~35% of AMA-negative PBC patients and adding these two biomarkers to conventional PBC assays dramatically improved the serological sensitivity in AMA-negative PBC from 55% to 75% in immunoblot and 48.3% to 68.5% in ELISA. Conclusions The addition of tests for highly specific anti-KLHL12 and anti-HK1 antibodies to AMA and ANA serological assays significantly improves efficacy in the clinical detection and diagnosis of PBC, especially for AMA-negative subjects.

A contemporary perspective on the molecular characteristics of mitochondrial autoantigens and diagnosis in primary biliary cholangitis

“…Interestingly, another study using an independent cohort of serum samples from patients with PBC and controls validated the specificity of anti-KLHL12 antibodies and anti-HK1 antibodies in both AMA-positive and AMA-negative PBC patients. In addition, antibodies to KLHL12 and anti-HK1 have higher sensitivity than anti-gp210 and anti-sp100 [67]. …”

“…AMA is a serological marker in PBC that can be detected years before any clinical symptoms of PBC. Although AMA is not associated with the severity of PBC, combination of AMA and discovery of other biomarkers in PBC [67,101-104] will increase the sensitivity and specificity in the diagnosis, disease prediction and prognosis of PBC. Finally, increasing knowledge on antigen specificity of AMA to lipoylated and non-lipoylated forms of the mitochondrial autoantigens, lipoic acid, xenobiotics and isotypes of the Ig molecule [52,58,101,105], may lead to discovery of AMA subpopulations which can be of indicative value for disease progression and prognosis.…”

Recent advances in the diagnosis and treatment of primary biliary cholangitis

“…By screening the PBC-focused microarrays with PBC patients, 6 proteins were identified as new PBC autoantigens in the presence of high specificities and sensitivities, covering hexokinase-1 (HK 1, and isoforms I and II), Kelch-like protein 7 (KLHL7), KLHL12, zinc finger, BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1[109]. In addition, both anti-KLHL12 and anti-HK1 antibodies with higher specificity and sensitivity were more likely to be detected in PBC in comparison with controls without PBC ( P < 0.001)[110]. Anti-HK1 in combination with anti-KLHL12 in the presence of usable signs ( i.e ., MIT3, gp210 and sp100), improved the sensitivity of PBC diagnosis[110].…”

“…In addition, both anti-KLHL12 and anti-HK1 antibodies with higher specificity and sensitivity were more likely to be detected in PBC in comparison with controls without PBC ( P < 0.001)[110]. Anti-HK1 in combination with anti-KLHL12 in the presence of usable signs ( i.e ., MIT3, gp210 and sp100), improved the sensitivity of PBC diagnosis[110]. Importantly, both anti-KLHL12 and anti-HK1 autoantibodies had been detected in 10% to 35% of AMA-negative patients with PBC, and increasing both biomarkers in routine PBC tests significantly increased the sensitivity in AMA-negative patients with PBC from 55% to 75% by means of immunoblot and from 48.3% to 68.5% with the ELISA method[110].…”

“…Anti-HK1 in combination with anti-KLHL12 in the presence of usable signs ( i.e ., MIT3, gp210 and sp100), improved the sensitivity of PBC diagnosis[110]. Importantly, both anti-KLHL12 and anti-HK1 autoantibodies had been detected in 10% to 35% of AMA-negative patients with PBC, and increasing both biomarkers in routine PBC tests significantly increased the sensitivity in AMA-negative patients with PBC from 55% to 75% by means of immunoblot and from 48.3% to 68.5% with the ELISA method[110]. Supplementing both anti-KLHL12 and anti-HK1 autoantibodies with highly specific assays for AMAs and ANA serological tests observably enhanced the serological surveillance effect and PBC diagnosis, particularly for AMA-negative patients[110].…”