The genetic background of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy and its autoimmune disease components.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disorder characterized by an immune-mediated destruction of endocrine tissues, chronic candidiasis, and additional ectodermal disorders. In contrast to many other autoimmune diseases, APECED is associated with mutations of a single gene, designated autoimmune regulator (AIRE). To date, 45 different mutations of the AIRE gene have been identified and are distributed throughout the entire coding region. Several of the AIRE mutations predict the transcription and translation of a truncated protein, which may be nonfunctional. In contrast to the genetic background of APECED, in all of the autoimmune conditions typically associated with APECED the conclusive role of a single genetic locus capable of providing insight into the etiology of the disease has not been identified. Here we provide an overview of the current clinical and genetic features of APECED in comparison to the genetic background of the frequently associated disease components of APECED.

Increased incidence of anti-LKM autoantibodies in a consecutive cohort of hepatitis C patients from central Greece.

OBJECTIVES: In Greece, there are insufficient data regarding the presence of non-organ and liver-related autoantibodies in hepatitis C patients. This study in a consecutive cohort of 39 such patients from central Greece investigates (1) the prevalence of non-organ and liver-related autoantibodies, and (2) the reactivity of anti-liver-kidney microsomal type 1 antibodies (in the case of positivity with at least one of the methods used) against their molecularly defined antigens. DESIGN: All serum samples were tested by standard and molecular assays for the presence of anti-nuclear antibodies, smooth muscle antibodies, anti-liver-kidney microsomal type 1 antibodies, antibodies against parietal cells, anti-CYP2A6, anti-CYP1A2 and anti-CYP2D6 autoantibodies. METHODS: Indirect immunofluorescence, competitive enzyme-linked immunosorbent assays, immunoblotting and novel radioligand assays based on immunoprecipitation of [35S]-methionine labelled recombinant CYP2A6, CYP1A2 and CYP2D6 His-taq fusion proteins produced by in vitro transcription/translation were used. RESULTS: Seven out of 39 patients (17.9%) tested positive for smooth muscle antibodies, 2/39 (5.1%) tested positive for anti-nuclear antibodies, 1/39 (2.5%) tested positive for parietal cell antibodies, and 4/39 (10.3%) were found to be anti-liver-kidney microsomal positive (with at least one of the methods used). All sera were negative for anti-CYP2A6 and anti-CYP1A2 autoantibodies. Three out of four anti-liver-kidney microsomal positive samples had the typical liver-kidney microsomal staining pattern shown by indirect immunofluorescence. However, none tested positive for anti-CYP2D6 autoantibodies using the competitive CYP2D6 enzyme-linked immunosorbent assay, the specific CYP2D6 radioligand assay, and western blot using either human microsomes or recombinant CYP2D6. The fourth patient tested negative for anti-liver-kidney autoantibodies by either indirect immunofluorescence or the competitive enzyme-linked immunosorbent assay, but was repeatedly positive for anti-CYP2D6 autoantibodies by the sensitive and specific radioligand assay. Western blot experiments using human microsomes in this patient serum revealed two bands of 50 kDa and 55 kDa that documented as anti-CYP2D6 and anti-uridine triphosphate glucuronosyltransferase autoantibodies when recombinant CYP2D6 and recombinant uridine triphosphate glucuronosyltransferase autoantigens were used for immunoblot, respectively. CONCLUSIONS: A relatively high incidence of anti-liver-kidney microsomal autoantibodies (10.3%) was found in a consecutive sample of Greek patients with hepatitis C. The expanded panel of assays, however, failed to document CYP2D6 as the target autoantigen of anti-liver-kidney microsomal autoantibodies in most patients. We report for the first time the detection of parietal cell antibodies and both anti-CYP2D6 (anti-liver-kidney microsomal type 1) and anti-uridine triphosphate glucuronosyltransferase (anti-liver-kidney microsomal type 3) autoantibodies in patients who were hepatitis C positive/hepatitis D negative. Further studies are needed to confirm our findings and to determine whether these preliminary results have a clinical importance or not.

Cytochrome P450 2A6: a new hepatic autoantigen in patients with chronic hepatitis C virus infection.

BACKGROUND/AIMS: Cytochromes P4502A6 (CYP2A6) and P4501A2 (CYP1A2) were described as hepatic autoantigens in the autoimmune polyglandular syndrome type-1 (APS-1). We evaluated the significance of anti-CYP2A6 and anti-CYP1A2 in several hepatic diseases in the absence of APS-1. METHODS: A radioligand assay (RLA) based on immunoprecipitation of [(35)S]-methionine-labeled CYP2A6 and CYP1A2 was used. Four hundred and thirty subjects with chronic viral hepatitis (n=185), autoimmune liver diseases (n=181), autoimmune rheumatic diseases (ARD, n=31) and healthy (n=33) were tested. RESULTS: Seven out of 366 patients with liver diseases were anti-CYP2A6 positive. Neither healthy nor ARD patients showed anti-CYP2A6. One out of 181 patients with autoimmune liver diseases tested anti-CYP2A6 positive. A significantly higher prevalence of anti-CYP2A6 (P<0.05) was detected with six out of seven patients positive in the viral hepatitis group. The latter were infected by flaviviruses (1 HGV/GBVC, 5 HCV). 4/5 HCV/anti-CYP2A6 positive sera were positive for anti-LKM-1 by immunofluorescence and for anti-CYP2D6 by RLA. None of the 430 sera recognized CYP1A2. CONCLUSIONS: For the first time CYP2A6 is reported as a hepatic autoantigen in patients with viral hepatitis caused by flaviviruses and in particular in HCV/anti-LKM-1 positive patients. Multicenter studies are needed in order to investigate the clinical importance of this novel finding. This study further supports that anti-CYP2A6 in the absence of flavivirus is rather limited to APS-1.

Characterization of a lipoyl domain-independent B-cell autoepitope on the human branched-chain acyltransferase in primary biliary cirrhosis and overlap syndrome with autoimmune hepatitis.

BACKGROUND AND AIMS: Antimitochondrial antibodies (AMA) which recognize pyruvate acetyltransferase (PDC-E2) represent a highly diagnostic feature of primary biliary cirrhosis (PBC). The analysis of immunofluorescence (IF)-AMA-positive sera in PBC patients indicates a conformational epitope located within the lipoyl binding domain of bovine branched-chain acyltransferase (BCKADC-E2) alone or in combination with AMA directed against PDC-E2 the significance of which is presently unclear. In the present study, immunoreactivities and disease associations of AMA against BCKADC-E2 were analyzed. B-cell autoepitopes on BCKADC-E2 were mapped by immunoprecipitation assay. METHODS: Sera of 96 IF-AMA-positive patients with serological evidence of anti-BCKADC-E2 alone (n = 26), anti-PDC-E2 alone (n = 15), and both anti-BCKADC-E2 and anti-PDC-E2 (n = 55) were analyzed by Western blot and ELISA in addition to an analysis of B cell autoepitopes on BCKADC-E2 by immunoprecipitation using in vitro translated, unmodified human proteins. Ninety-four patients without IF-AMA [blood donors (n = 30), rheumatoid arthritis (n = 40), autoimmune hepatitis (AIH)(n = 10) and primary sclerosing cholangitis (PSC) (n = 14) served as controls. RESULTS: Eighty of 81 (99%) sera positive for BCKADC-E2 recognized the full length, mature protein, while only 2/10 AIH sera and none of the other controls showed reactivity. Of the 68 PBC sera 58 (85%) recognized the N-terminus consisting of aa 1-144 representing the lipoyl domain. Surprisingly, C-terminal sequences (aa 143-421) were recognized by 46 out of 68 sera (68%). Three PBC sera reacted with the C-terminus only. Only 1/7 serum from patients with an "overlap syndrome of PBC and AIH" was reactive with C-terminal sequences. CONCLUSIONS: Our analysis of BCKADC-E2-positive PBC sera identified a novel B cell epitope on the C-terminal part of the human protein. Our data indicate that a distinct subset of AMA recognize sequence(s) on BCKADC-E2 which located outside of the lipoyl binding domain. The absence of immunoreactivity against C-terminal sequences may serve as a marker differentiating patients with PBC and overlap syndrome of PBC with AIH.