Molecular Diagnostics of Primary Biliary Cirrhosis

Review Molecular diagnostics of primary biliary cirrhosis

Eirini I Rigopoulou & George N Dalekos† † University of Thessaly, Department of Medicine, Academic Liver Unit and Research Lab of 1. Introduction Internal Medicine, Medical School, Papakiriazi 22 Street, 41222 Larissa, Greece and 2. Antimitochondrial antibodies Institute of Biomedical Research and Technology, Research Group of Investigational Medicine, 3. Conclusion Centre for Research and Technology-Thessaly (CE.RE.TE.TH), Larissa, Greece

4. Expert opinion Background : Primary biliary cirrhosis (PBC) is an autoimmune liver disease of unknown etiology characterized by the presence of antimitochondrial antibodies (AMA) in 90 – 95% of patients. AMA are directed against members of 2-oxo-acid dehydrogenase complex, including mainly the E2 subunit of pyruvate dehydrogenase, the E2 subunit of branched chain 2-oxo-acid dehydrogenase complex and the E2 subunit of the oxoglutarate dehydrogenase complex. Apart from AMA, PBC is characterized by the presence of PBC-specific antinuclear antibodies (ANA). The molecular targets of these PBC-specific ANA have been characterized as gp210, lamin B receptor, nucleoporin 62, sp100 and promyelocytic leukemia proteins. Objective : To discuss the molecular diagnostics of PBC in the context of AMA and PBC-specific ANA detection by the use of conventional and ‘new’ novel technologies. Methods : Critical analysis of all published data regarding PBC serology between 1985 and 2007 was performed in order to suggest a diagnostic algorithm for the serological diagnosis of PBC. Results/conclusions : AMA are first detected by indirect immunofluorescence (IIF) on frozen sections of rat liver, kidney and stomach substrates. However, because IIF is time-consuming, labor-intensive and observer-dependent, molecular-based assays such as immunoblot and enzyme-linked immunosorbent assays have been developed with high sensitivity and specificity. Similarly, molecular-based assays have also been developed for the detection of PBC-specific ANA. The latter investigation seems to be of outmost importance because these autoantibodies can be used as a positive tool in the diagnosis of AMA-negative PBC while at the same time identifying a subgroup of PBC patients with more advanced disease. New test systems for the detection of PBC-specific antibodies based on the xMultiple Analyte Profiling Luminex methodology seems to be the future in molecular diagnostics of PBC as it was expected first to decrease the cost and second to speed up an accurate serological profile, although they may decrease further the proportion of AMA-negative PBC cases.

Keywords: antigp210 antibodies , antimitochondrial antibodies , antinuclear antibodies , anti-PDC-E2 antibodies , antisp100 antibodies , autoimmune cholestatic liver diseases , primary biliary diagnosis

Expert Opin. Med. Diagn. (2008) 2(6):621-634

1. Introduction

Primary biliary cirrhosis (PBC) is an autoimmune cholestatic liver disease of unknown etiology that primarily affects women [1,2] . Recent data suggest that this predominance of women among patients with PBC is related to a higher incidence of X chromosome monosomy in lymphoid cells [3,4]. The peak incidence of the disease occurs in the fifth decade of life, and it does not affect children and is uncommon in people < 25 years of age. Continuing destruction and loss of

A. B.

Cristae Matrix Inner membrane

Outer membrane

Intermembrane space

M2 antigens

PDC-E2

BCOADC-E2 OGDC-E2

Figure 1 . Schematic presentation of the mitochondrion structure and the major mitochondrial antigens. A. Structure of a mitochondrion. B. Components of the 2-oxo-acid dehydrogenase multi-enzyme complexes are located in the inner mitochondrial membrane. C. The major mitochondrial antigens are the E2 subunits of the pyruvate dehydrogenase complex (PDC-E2), the branched chain 2-oxo-acid dehydrogenase complex (BCOADC-E2) and the oxoglutarate dehydrogenase complex (OGDC-E2). small intrahepatic bile ducts, progressive fibrosis and the studies have shown the presence of PBC-specific antinuclear eventual development of cirrhosis and liver failure characterize antibodies (ANA), which are of diagnostic and clinical relevance the disease [1,2]. The course of the disease, however, is variable because they can be used as a positive tool in the diagnosis and an early diagnosis is desirable to identify individuals of AMA-negative PBC, while at the same time identifying a with rapidly progressing disease, initiate adequate therapeutic subgroup of patients with more advanced liver disease [5-10] . measures and evaluate the necessity of liver transplantation. In this review, after an historical note, the molecular Serologically, PBC is characterized by the presence of circu- diagnostics of PBC is discussed in the context of AMA and lating autoantibodies directed against mitochondrial antigens PBC-specific ANA detection by using conventional and (AMA) of the inner mitochondrial membrane [1,2,5,6] . As ‘new’ novel technologies. these autoantibodies are found in 90 – 95% of patients with PBC, they have been considered as the key diagnostic feature 2. Antimitochondrial antibodies of the disease. The diagnosis of PBC is based at present on three criteria: the presence of detectable AMA, elevation of 2.1 Historical overview cholestatic enzymes (most commonly alkaline phosphatase) The presence of AMA in PBC patients was first described for more than 6 months, and histological findings in the by Ian Mackay in 1958 [11]. In 1967, the target antigen of liver that are compatible with the presence of the disease. A AMA was found to be located within the inner mitochondrial probable diagnosis requires the presence of two of these membrane and termed M2 (Figure 1A, B) [12] , while in 1985, three criteria, and a definite diagnosis requires all three, but further analysis of M2 antigens led to their subdivision into some hepatologists believe that a liver biopsy is not needed, separate antigen fractions with molecular targets between although liver biopsy may allow the stage of the disease to be 36 and 74 kDa [13-15] . It was not until 1987 that analytical determined and provides a baseline for evaluating progression and molecular techniques led to the identification of of the disease and the response to treatment schedules [1,2] . 2-oxo-acid dehydrogenase complex (2-OADC) as the major Of note, as many as 5 – 10% of patients have no detectable autoantigen of PBC-related AMA [16]. Actually, using an AMA, but their disease appears to be identical to that in patients original expression vector, Gershwin and colleagues with AMA antibodies. In these cases, recent continuing identified for the first time the cDNA encoding the 70-kDa

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Table 1 . Major mitochondrial antigens in primary 2.3 Non-PBC-speciﬁ c AMA biliary cirrhosis. Although AMA against members of the 2-OADC family are highly specific for PBC diagnosis or at least suggest that the Mitochondrial antigens Molecular person is at substantial risk of having PBC over the next weight (kDa) 5 – 10 years, several AMA exist that occur in several E2 subunit of pyruvate dehydrogenase 74 non-PBC disorders. Based on differentially centrifuged complex (PDC-E2) mitochondria, a system containing nine antigen subfractions, E2 subunit of branched chain 2-oxo-acid 51 namely M1 – M9, has been proposed with M2 containing dehydrogenase complex (BCOADC-E2) the 2-OADC antigens specific for PBC [21]. However, E2 subunit of the oxoglutarate dehydrogenase 48 a prognostic and diagnostic significance of M-based complex (OGDC-E2) classification of AMA remains speculative as apart from M2 AMA, the other anti-M antibodies have been found E3 binding protein (E3BP) of PDC* 55 in diverse pathological conditions, namely, syphilis, acute E1a subunit of pyruvate dehydrogenase 41 myocarditis, drug-induced hepatitis and autoimmune complex (PDC-E1a)* rheumatic diseases [6,8,22] . E1b subunit of pyruvate dehydrogenase 36 complex (PDC-E1b)* 2.4 Detection of AMA

* Reactivity to these autoantigens is rarely found in patients who do not have 2.4.1 Indirect immunoﬂ uorescence reactivity against PDC-E2. Originally, AMA were detected by indirect immunofluorescence (IIF) on frozen sections of rat liver, kidney and mitochondrial antigen that led to the identification of the stomach, and/or immobilized HEp2 cells are considered to E2 subunit of pyruvate dehydrogenase (PDC-E2). Since be the technique of choice for routine screening [5,6,8,23] . then the same group has identified the other mitochondrial As AMA in the serum are formed by all three main immuno- autoantigens of AMA as two other components of the globulin subtypes, that is, IgG, IgA and IgM, an antihuman 2-OADC. These AMA directed against members of the polyvalent immunoglobulin IgA, IgG, IgM should be used 2-OADC represent those previously defined as anti-M2 AMA, as secondary antibody in these assays. The typical pattern of are highly PBC-specific and can be differentiated from non- AMA by IIF is characterized by staining of the distal and specific AMA using molecularly defined seroimmunological proximal tubuli on rat kidney cryostat sections, whereas on methods [6,8]. The association of these PBC-specific AMA HEp2 cells a granular cytoplasmic pattern is usually present with the disease is so striking that practically all individuals (interesting note: proximal staining only is indicative of liver positive for AMA and with no clinical liver involvement will, kidney microsomal antibodies; anti-LKM; Figure 2 ) [24,25] . sooner or later, go on to develop overt disease [17] . For this reason, preparation of kidney substrate requires careful orientation of the sectioning and should first be cut 2.2 Target autoantigens of PBC-speciﬁ c AMA sagitally and then in cubes that contain both medulla and Sera from PBC patients react mainly against members of cortex. The sections (including liver, kidney and stomach) 2-OADC, including PDC-E2, the E2 subunit of branched are used without further fixation or can be stored at -20° C chain 2-oxo-acid dehydrogenase complex (BCOADC-E2), for 4 – 8 weeks [26] . Equivalent sections that are commercially the E2 subunit of the oxoglutarate dehydrogenase complex available are of variable quality as, to lengthen expiry date, (OGDC-E2), the dihydrolipoamide dehydrogenase (E3)-binding they are treated with fixatives, which result in enhanced protein (E3BP) and the E1a and E1b subunits of pyruvate background staining that usually limits interpretation of dehydrogenase complex (PDC-E1a and PDC-E1b) ( Table 1 , fluorescence patterns. Figure 1C ) [18] . Most commonly, AMA react against the PDC-E2 Of interest, a recent study investigated the pattern of and also show reactivity against the BCOADC-E2, the IgG subclass-specific AMA detected by IIF on rodent liver, OGDC-E2, or both. Several studies using oligopeptides or kidney and stomach tissue substrate using affinity-purified recombinant proteins have shown that the dominant epitopes IgG subclass monospecific antisera as revealing reagents [27] . recognized by AMA are located within the lipoyl domain of This study demonstrated that AMA are not restricted to a the three molecules and are conformation-dependent [18-20] . specific IgG subclass and, furthermore, IgG3 AMA-positive Of note, ∼ 10% of patients with PBC react only to PBC patients had a histologically more advanced disease and BCOADC-E2 and/or OGDC-E2, indicating that in clinical were more frequently cirrhotic compared with those who practice testing for AMA by using only the PDC-E2 as the were negative, whereas there was a positive correlation primary substrate would result in low sensitivity of the index between AMA IgG3 titre and the Mayo risk score, possibly test. AMA directed against the other components of 2-OADC reflecting the peculiar ability of this IgG subclass to engage are of minor diagnostic importance as reactivity to these mediators of damage [27] . If these results are confirmed on antigens is rarely found in patients who do not have a larger number of patients and in other laboratories, the reactivity against PDC-E2 [2,5,6,8,10,18] . possibility of testing simultaneously for conventional AMA

Expert Opin. Med. Diagn. (2008) 2(6) 623 Molecular diagnostics of primary biliary cirrhosis

A. B.

C. D.

Figure 2 . A, B. Antimitochondrial antibodies react to the proximal and distal tubules of the rat kidney ( B). In these cases there is also reactivity to the parietal cells of the rat stomach (A ). C. Typical staining of antimitochondrial antibodies on immobilized HEp-2 cells in a female patient with primary biliary cirrhosis. Few nuclear dots are also shown in the nucleus (reactivity against sp100 was conﬁ rmed by molecular-based assays). D. Antibodies against liver-kidney microsomes type 1 (anti-LKM-1) from a patient with autoimmune hepatitis type 2 react to the proximal tubules of the rat kidney. The absence of reactivity against the distal tubules of the rat kidney and parietal cells of the rat stomach (see also A, B ) can distinguish anti-LKM-1 autoantibodies from antimitochondrial antibodies. and for IgG3-AMA could be considered because double include reactivity against OGDC-E2 [6,8] . However, as noted positive patients are likely to have a more severe disease and above, commercially available first generation AMA ELISAs potentially a worse prognosis. (using only the PDC-E2 derived usually from bovine tissues as the primary substrate) would result in low sensitivity of 2.4.2 Enzyme-linked immunosorbent assays the test [24,28] . It should be kept in mind that, for practical and diagnostic More recently, a new M2 enhanced performance ELISA reasons [17], the main aim in AMA determination is to (MIT3), which utilizes the recombinant MIT3 antigen [24,30] detect AMA that are PBC-specific and to exclude AMA of has been made available. This recombinant antigen contains low diagnostic relevance for the disease. The fact that IIF is the three immunodominant epitopes of AMA, namely time-consuming, labor-intensive and observer-dependent has PDC-E2, BCOADC-E2 and OGDC-E2, in one molecule. led to the development of molecular-based assays such as Interestingly and more importantly, several studies have immunoblot and ELISAs, with the latter appearing very shown that this ELISA was able to unmask the presence attractive as they are objective, rapid, semiautomated and of AMA in 30 – 50% of AMA-negative samples by IIF, can be applied for AMA detection at the same time in a suggesting a superiority in sensitivity compared with IIF large number of samples. Therefore, when positive AMA assays [24,30-34]. As a result, these findings may suggest the are detected by IIF, further analysis is needed by using use of the MIT3-based assays as first-line investigation molecularly defined antigen preparations. The detection for AMA detection, particularly when the laboratories are of antibodies against PDC-E2 and BCOADC-E2 can be unfamiliar with the use and interpretation of IIF patterns performed by ELISAs using recombinant antigens or of AMA. In addition, MIT3-based ELISA appears to reference sera [24,28,29]. If both are negative, testing should be more rapid and cost-effective than the IIF, which is

624 Expert Opin. Med. Diagn. (2008) 2(6) Rigopoulou & Dalekos time-consuming, and the cost per well is double compared Nevertheless, from the diagnostic point of view, it should with MIT3-based ELISA [34]. However, it should be noted be noted that 2 – 3% of PBC patients might have only that, at least in our hands, there is no ‘gold standard’ assay AMA of the IgA specific isotype, indicating the need for IgA (with 100% sensitivity and 100% specificity) for the AMA determination, at least in PBC cases with negative detection of AMA at present, as in a recent study 18 out of IgG AMA results [31,34] . The serial determinations of AMA 103 PBC sera showed a discrepancy in results between IIF titers by IIF or ELISA proved of no benefit in clinical and the MIT3-based assays [34]. A possible explanation practice, as they do not correlate with histopathological, for these discrepancies could be the fact that kit-based biochemical or clinical features of the disease in most of the commercial assays may have been validated only in-house, published studies [46,47], whereas a recent study with very such that the performance characteristics would not long period of follow-up (7 – 28 years) also showed that necessarily be available to the end users [26] . isotype-specific IgG, IgM and IgA AMA titers against each Ideally, the detection of AMA by ELISAs should be of PDC-E2, BCOADC-E2 and OGDC-E2 antigens did not performed by investigating all three main specific isotypes, change significantly over time [48] . namely, IgG, IgM and IgA AMA. Among them, IgA AMA Alternatively to ELISAs, the use of enzyme inhibition have been suggested to play a critical role in bile duct injury assays could be another option for AMA detection as it is as these autoantibodies can be detected in bile, saliva and well known that AMA in PBC sera have the capacity to urine samples of PBC patients [18] . Secretory IgA is the inactivate rapidly the catalytic function of 2-OADC predominant immunoglobulin of the mucosal immune in vitro [49]. The enzyme inhibition assay is non-subjective system and is the major protein in bile. Nishio et al. [35] compared with IIF, and seems to be more rapid and simpler and Floreani et al. [36] suggested that IgA AMA might have than ELISAs and immunoblot, carrying a very high specificity. a pathogenetic role because during its transport through However, its sensitivity is lower as at present this assay does biliary epithelial cells into the bile, IgA AMA may bind to not detect the inhibitory activity of PBC sera to 2-OADC its target antigen(s) in biliary epithelial cells, and this may enzymes other than PDC. be followed by dysfunction and finally destruction of biliary epithelial cells. Further evidence of the possible importance 2.4.3 Immunoblotting of IgA AMA in PBC comes from clinical observations [37] Preparations of mitochondrial fractions of human and/or rat and experiments by Malmborg et al. [38] as well as by liver or bovine heart have been used as sources of antigens Gershwin’s group, which showed recently that IgA but for AMA detection by immunoblot testing [29,32] . Whole not IgG AMA induce caspase activation in Madine-Darby extract from human or rat liver is an alternative, but the canine kidney cells transfected with the human polymeric former preparations are considered more specific for AMA Ig receptor [39] . detection. Western blot analysis can detect both the presence Although the hypothesis of IgA-driven pathogenesis and antigen-specificity of AMA, as the indicative 74 kDa in PBC is very attractive [40], it is still uncertain and (PDC-E2), 51 kDa (BCOADC-E2) and 48 kDa (OGDC-E2) the results on the clinical significance of IgA AMA in bands can be visualized [5,6,8,50,51] . As in IIF and ELISA PBC largely conflict. In more detail, Kisand et al. [41] assays, an antihuman polyvalent immunoglobulin IgA, IgG, reported that the levels of IgA AMA by ELISA correlated IgM should be used as secondary antibody for the detection negatively with several histopathological parameters as of all three main specific isotypes of AMA, namely IgG, fibrosis and inflammatory infiltrate, and Nakajama et al. [42] IgM and IgA AMA. Using this second antibody, a recent also reported that IgA AMA were more frequently study has shown that immunoblotting has higher sensitivity detected in the early stage of PBC. On the contrary, and specificity for the detection of AMA compared with Tanaka et al. [43] and Masuda et al. [44] have reported IIF on rat tissues [32]. However, in clinical practice an an association of IgA AMA with advanced histological antihuman monospecific IgG is used as secondary antibody stage of the disease and subsequently with advanced in most laboratories. An alternative to mitochondrial histopathological lesions of the liver, although the latter particles for AMA detection by immunoblotting could be investigators, in a more recent study, failed to show the use of Escherichia coli-expressed recombinant antigens, any association between IgA AMA and markers of but in this case it has been shown that there is a weak histopathological progression of PBC [45] . Finally, crossreactivity with E. coli PDC-E2 in non-PBC sera at Gabeta et al. [34] failed to show any association between lower (1:10, 1:20 and 1:40) dilutions that it is not due to IgA AMA and the histological or clinical stage of PBC, specific AMA positivity [51,52] . although there was a positive trend with the Mayo risk score Finally, a recent study by Rigopoulou et al. [53] of the patients. Therefore, a prospective study employing a using modern, computer-assisted imaging technology large number of PBC patients at early stages with detectable provides new information regarding the quantitative IgA AMA is required to conclude safely whether these relationship of AMA detected by IIF and the fine specificity antibodies contribute to the injury of biliary epithelial of AMA responses determined by immunoblot. Actually, cells and progression of PBC. it has been demonstrated that the AMA titers by IIF

Expert Opin. Med. Diagn. (2008) 2(6) 625 Molecular diagnostics of primary biliary cirrhosis correlate to the number and magnitude of immunofixed that the presence of ANA reactivities, especially those 2-OADC bands in PBC sera [53]. In addition, this study regarded as disease-specific, is associated with disease severity offers fresh evidence to suggest that although PDC-E2 is the and, in contrast to AMA, considered as a marker of poor predominant AMA target, it is not always responsible for prognosis [5,9,53]. In addition, the presence of PBC-specific most of the reactivity seen by IIF, suggesting for one more ANA has become an important clinical tool for the diagnosis time that assays aimed at the maximum performance for the of PBC, particularly in AMA-negative patients [5,7,9,10] . detection of AMA may need to take into account the whole Most PBC sera are heterogeneous and have auto- spectrum of the mitochondrial antigens [53] . antibodies with different ANA specificities. PBC sera display primarily rim-like, nuclear dot, speckled, homogeneous 2.4.4 New assays – techniques and centromere staining patterns. In such cases overlapping A new ELISA (PBC screen) using a mixture of recombinant staining patterns can be expected and IIF cannot distinguish fusion protein, which includes the immunodominant portions between various kinds of circulating autoantibodies. In of the three major mitochondrial antigens, that is, PDC-E2, a large cohort of 492 PBC patients, 72% were ANA BCOADC-E2 and OGDC-E2, along with sp100 and gp210 positive by IIF on HEp-2 cell substrate, with more than nuclear antigens that are the major molecular targets of 1 staining pattern in 12.8% of cases; antibodies against PBC-specific antinuclear antibodies (ANA; vide infra), has been nuclear pore complexes (19%), nuclear bodies (20%) and developed for the potential screening of PBC-autoantibody centromeres (13%) were most prevalent [57] and comparable serology in suspected PBC. In a small study, the PBC to previous studies [56,61,62] . screen ELISA based on an antigenic mixture of the major mitochondrial and nuclear antigenic targets performs as well 2.5.1 ANA not speciﬁ c for PBC as the ELISAs based on the individual antigens [54]. However, These ANA include anticentromere antibodies (ACA) these findings need to be confirmed and validated on a large and extractable nuclear antigens (anti-ENA), which recognize number of PBC patients and pathological controls to address a wide spectrum of molecular targets such as nuclear whether screening for PBC-autoantibody serology could be ribonucleoproteins (nRNP), ribosomal phosphoproteins used instead of a combination of individual ELISAs. (PO) and cellular enzymes such as DNA topoisomerase I The persistence of some AMA-negative PBC patients with (Scl-70) and histidyl-tRNA synthetase (Jo-1). all conventional methods raises the question whether these Initially, counterimmunoelectophoresis (CIE) on thymic patients represent a subgroup with distinct clinical features and spleen extracts and IIF on HEp-2 cells have been used or simply have antibody titers and specificities that are for the determination of anti-ENA and ACA, respectively [63] . undetectable with the available technology [5]. Recently, the The identification of antigen specificities within each anti- group of Gershwin at Davis, California, has tried to address ENA reactivity (nRNP, Sm, SSB/La, SSA/Ro 60 and 52) has this question by developing a new sensitive bead assay for facilitated the development of different and more sensitive AMA detection using the Luminex principle [55] . These assays such as ELISAs and immunoblot. In a recent study, investigators, using individual bead assays with the three anti-ENA detected by immunoblot were present in 30% of major mitochondrial antigens PDC-E2, BCOADC-E2 and PBC patients, the most frequent anti-ENA reactivity being OGDC-E2, demonstrated the detection of AMA in 100% anti-SSA/Ro-52kD (28%), which was associated with advanced of PBC patients known to be AMA-positive, but also in and active disease [64] . The CIE, however, remains the 20% of the rigorously defined AMA-negative patients, technique with the higher specificity compared with ELISAs suggesting the potential for automated AMA detection with and immunoblot, but its relative complexity makes this rapid, accurate and reliable assaying [55] . In addition, this assay difficult for screening in routine laboratories. assay highlights further the diminished number of ‘truly’ ACA antibodies have been reported in ∼ 20 – 30% of AMA-negative PBC patients, but again, as noted previously, PBC patients (ranging between 11 and 60%) [56,62,64-68] . an external validation in a large number of PBC patients and Such enormous variation in the prevalence of ACA can pathological controls is needed before this test is considered be attributed to differences in methodology (IIF versus as a highly specific diagnostic test for PBC. ELISA) [65], disease stage [67,68] and ethnic/geographic regions [67,68] . Whether the presence of non-PBC-specific 2.5 Antinuclear antibodies in PBC ANA and particularly that of ACA is of any clinical Although the serologic hallmark for the diagnosis of PBC is significance remains to be determined, as available data the presence of AMA, identified in up to 95% of patients, are conflicting [57,65-67] . ANA are also detected in up to 72% of patients [9,56-60] . The use of HEp2 cell line as a substrate for IIF has allowed the 2.5.2 PBC-speciﬁ c ANA identification of ANA specificities not observed on tissue The PBC-specific ANAs give two distinct immunofluoresence sections and thus increase the sensitivity of ANA detection. patterns: the perinuclear (rim-like) and the multiple nuclear ANA are distinguished between those that are not specific dot (MND) patterns (Table 2 ; Figure 2C). ANA-positive sera and those specific for the disease. Growing evidence suggests giving a rim-like/membranous pattern by IIF react with

626 Expert Opin. Med. Diagn. (2008) 2(6) Rigopoulou & Dalekos

Table 2 . PBC-speciﬁ c nuclear antigens. inner nuclear membranes are joined and consist of 80 – 100 proteins. Some of them are identified and characterized and gp210 classified further into three groups: a group of integral Nucleoporin p62 membrane proteins, a family of glycoproteins modified with Lamin B receptor O -linked N -acetylglucosamine and a family of nucleoporins sp100 not substituted by carbohydrates. An integral membrane protein named gp210, nucleoporin 62 and the lamin B Promyelocytic leukemia protein receptor (LBR) have been identified as targets of ANA reactivity present in patients with PBC (Table 2 ) [9] . supramolecular structures called nuclear pore complexes The presence of an ANA specific for nuclear membrane (NPCs), which mediate nucleocytoplasmic transport, by IIF in sera from patients with PBC was first reported in including the import into the nucleus of proteins and the 1985 [72]. This reactivity was present in 18 of 63 PBC sera export to the cytoplasm of ribosomal and messenger RNAs. (28.5%) and in only 1 out of 431 control sera (0.2%) [72] . Antibodies against NPCs label the nuclear periphery in Using immunoprecipitation and western blot, three characteristic manners: a punctuated pattern of the nuclear subsequent studies have shown that up to 27% of sera from surface with each dot representing a nuclear complex is PBC patients with a rim-like nuclear staining in IIF on observed when the microscopic focal plane is on the top of rat liver tissue sections contain antibodies to 200 kDa the spherical interphase nucleus and the largest diameter is polypeptide of nuclear envelope [73-75] . Courvalin et al. have out of focus; a punctuated discontinuous perinuclear rim is shown that the target antigen is a 210 kDa integral glyco- seen, representing only the nuclear pores, when the focal plane protein named gp210 [76], which consists of a 1783-amino is in the largest diameter of the interphase nucleus [60] . acid amino-terminal domain located in the perinuclear space, The MND pattern involves the staining of 3 – 20 dots a 20-amino acid transmembrane segment, and a 58-amino distributed throughout the nucleus, but sparing the nucleoli acid cytoplasmic carboxy-terminal tail. Further analysis ( Figure 2C). It is generated by autoantibodies directed against revealed that all patients positive for gp210 recognized a the sp100 and promyelocytic leukemia (PML) proteins stretch of 15 amino acids in the cytoplasmic carboxy- (Table 2): those proteins colocalize and the corresponding terminal tail [77] , and Wesierska-Gadek et al. have shown autoantibodies tend to occur together [69] . that a new epitope located within the large amino-terminal PBC-specific ANAs are found in up to 50% of patients [60] . glycosylated domain was recognized by 66% of antigp210- The identification of the molecular targets of PBC-specific positive sera [78] . Interestingly, carbohydrate moieties, ANAs has been another major progress in the diagnostic recognized by 33% of antigp210-positive sera, were an evaluation of patients with this disease and has allowed the essential part of this new epitope [78]. The prevalence of establishment of observer-independent and more sensitive antigp210 by ELISA has been reported to be between methods based on the use of recombinant antigens, such as 10.4 and 32.4% [67,68]. Such a variation in the frequency ELISA and immunoblot. Applying immunoblotting, of these antibodies in PBC patients is probably related Invernizzi et al. were able to find anti-NPC reactivity in to geographical/ethical differences (Mediterranean versus 22% out of 105 PBC sera previously considered ANA- Japanese population) and to protocol differences (commercially negative by IIF and in 35% of ANA-positive individuals [70] . available versus in-house ELISA), suggesting the need Interestingly, only a quarter of these PBC sera testing for studies on large PBC cohorts. Antigp210 are highly positive for both ANA by IIF and anti-NPCs gave a specific for PBC (> 99%) [73-76,78,79] and persist after liver definite rim-like pattern [70] . transplantation, although no association with disease The visualization of ANA can be hampered by the recurrence has been reported [80] . presence of AMA and other antinuclear specificities present Early studies based mostly on IIF detection of PBC- in the serum [71]. This has led previously to the belief that specific ANA reactivities could not establish an association PBC-specific ANAs are mostly confined to AMA-negative between the presence of such antibodies and a worse PBC patients. In a recent study, the use of antisera specific prognosis [73,75,81]. With the use of IgG subclass-specific for individual IgG isotypes has been proved to increase the antisera, it has been demonstrated that PBC-specific ANA diagnostic accuracy of the conventional IIF. Sixty-five per (giving both a rim-like and MND pattern), especially of the cent of patients tested were positive for MND and/or IgG3 isotype, were associated with more severe disease [59] . rim-like reactivity with the use of anti-IgG isotype-specific Subsequent, mostly retrospective studies have suggested antisera (IgG1-4), compared with 15% ANA positivity when that antibodies against NPC and especially gp210 might be antitotal IgG antiserum was applied [59] . surrogate markers for the progression of PBC to end stage hepatic failure [56,67,70,79,82,83] . Anti-NPC antibodies usually 2.5.2.1 ANA directed against the nuclear pore complex remain stable during the course of disease and their titer, in The NPCs are 125 kDa supramolecular structures embedded particular of gp210, can be an important tool to identify in the bilayer nuclear membrane at sites where outer and asymptomatic patients with unfavorable disease course [79,84] .

Expert Opin. Med. Diagn. (2008) 2(6) 627 Molecular diagnostics of primary biliary cirrhosis

With respect to the scarcity of prognostic models, relevance in the diagnosis of PBC must be evaluated in particularly in early stages of PBC, anti-NPC testing larger cohorts of PBC patients. and in particular determination of gp210 by commercially The PML protein is a transformation and cell growth- available ELISA can be added to tests applied to the suppressing protein aberrantly expressed in promyelocytic initial evaluation and ‘classification’ of PBC patients. leukemia cells, which also gives an MND pattern by IIF. Nucleoporin 62, a 60-kDa component of NPCs, is the Sternsdorf et al. has found anti-PML antibodies in almost second most frequently recognized component of the nuclear all antisp100-positive PBC patients using an immuno- envelope. Initially, reactivity against nucleoporin 62 was assessed precipitation assay with radiolabeled PML and an IIF assay by affinity chromatography on wheat-germ agglutinin based on a cell line overexpressing PML [69]. Only very few (WGA)-Sepharose [85] . The use of human recombinant p62 patients with PBC or other autoimmune diseases contained protein has improved the detection rate of these antibodies anti-PML or anti-Sp100 antibodies exclusively. In contrast by immunoprecipitation and immunoblotting [86] . to Sp100, immunoreactivity of recombinant PML in immuno- A recent study by Wesierska-Gadek and co-workers blots was only weak and was directed to one region, arguing has shown that 30% of 127 PBC patients had anti-NPC against the development of an ELISA [69]. Züchner et al. reactivity with the use of immunoblotting – 23% against have found anti-PML antibodies in 19% of PBC patients, antigp210, 15% against antip62 and 8% manifested both most of them being sp100 positive; very few exhibit only reactivities [84] . Moreover, the prevalence of antip62 was anti-PML reactivity [93] . estimated to be 55% by immunoprecipitation by the same Sp100 and PML have been shown to be covalently linked group [87]. As such a big discrepancy exists in the prevalence to small ubiquitin-like molecules (SUMO), in particular of antip62 depending on the antigen and protocol used, SUMO-1 and SUMO-2/3. Banding of SUMO to a protein further studies are warranted to explore the true prevalence stabilizes that target protein and regulates its cellular of these antibodies in PBC. distribution and function. Recently, Janka et al. have Less frequently, patients with PBC have antibodies demonstrated that autoantigens against SUMO-2 and against the LBR. Even though the prevalence of these SUMO-1 are found in 42 and 15% of PBC sera giving a antibodies has been found to be very low (1.29%), they are MND pattern, whereas anti-SUMO reactivity was not considered disease specific [81,88] . Recently, the prevalence of observed in MND-negative sera. It is of interest that anti-LBR has been found to be higher (9%) in patients anti-SUMO reactivity was always detected in the presence from Japan [83] . of antisp100, proposing antigen spreading as a possible The presence of antibodies against other nuclear envelope mechanism for anti-SUMO generation [98] . These data components, such as the Tpr protein, the lamina-associated suggest that SUMO proteins might represent new polypeptides LAP1 and LAP2 and lamins have been independent PBC autoantigens, and this hypothesis needs described in patients with PBC but are not considered as to be validated in future studies. disease specific. 3. Conclusion 2.5.2.2 ANA giving the multiple nuclear dot pattern by IIF The presence of ANA producing a MND pattern was From the diagnostic and clinical points of view, PBC is first described in 1987 by Szostecki et al. [89] . This pattern considered to be a model of organ-specific autoimmunity was attributed to a 95 – 100 kDa protein named sp100, with the existence of a diagnostic test of high specificity for a finding confirmed in subsequent studies [90,91]. Using an the disease. In fact, AMA represents the only disease-specific ELISA encompassing a 142aa sp100 fragment, previously seroimmunological test in patients with autoimmune liver identified as the immunodominant epitope [92] , the diseases, although these autoantibodies do not seem to prevalence of antisp100 has been reported to be between contribute directly to disease aetiopathogenesis and cannot 21 and 34% [56,93-95]. Even though antisp100 is considered predict the outcome of the disease. The application of to be specific for the diagnosis of PBC, its prevalence molecularly defined antigens of AMA either in ELISA or in in non-PBC patients and especially those with immunoblot test systems has led to the accurate and reliable rheumatologic disorders varies [92,94,96,97] . The occurrence diagnosis of PBC cases and, furthermore, the decrease of the of anti-MND antibodies tested by IIF or by ELISA group of AMA-negative PBC. When positive AMA by IIF is against sp100 has been associated with an unfavorable detected, further analysis should include subclassification disease course in terms of significantly more severe bio- using molecularly defined antigen preparations. This chemical and histological disease, faster disease progression multistep analysis secures a rational and reliable diagnosis and worst outcome in seropositive PBC patients [59,93] , of PBC-specific AMA excluding those detected in other although this was not confirmed in all studies [67] . Antisp100 non-PBC disorders. Similar investigation using molecularly antibodies usually remain stable during the disease course [93] defined antigens should be done in suspected PBC in cases and persist after liver transplantation [80] . Considering the with negative AMA results by IIF. It is worth stating that potential prognostic significance of antisp100, its clinical assays aimed at the maximum performance for the detection

628 Expert Opin. Med. Diagn. (2008) 2(6) Rigopoulou & Dalekos of AMA need to take into account at least the three account in an attempt to achieve the maximum performance immunodominant epitopes of AMA, namely PDC-E2, for AMA detection. BCOADC-E2 and OGDC-E2, and, ideally, the detection Non-observer-dependent assays, which will utilize a of all three main specific isotypes of AMA, namely mixture of recombinant mitochondrial antigens, that IgG, IgM and IgA. Investigation for the detection of is, PDC-E2, BCOADC-E2 and OGDC-E2, and sp100, PBC-specific ANA either by IIF on HEp2 cells or using p62 and gp210 nuclear antigens that are the main molecular molecular-based assays as immunoblot and ELISAs seems targets of PBC-specific ANA, could be used in the near to be of utmost importance because these autoantibodies future for PBC screening because extensive standardization can be used as a positive tool in the diagnosis of and validation of such assays is needed. AMA-negative PBC, while at the same time identifying During the last few years, the possibility of simultaneous a subgroup of PBC patients with more advanced disease. measurement of several correlated analytes (multiplex Based on recent technological advances (multiplex technologies) has overcome some of the limitations of technologies), further efforts are under way for the conventional methods and appears to be very interesting development of increasingly automated detection methods for analytical (sample volumes, reagents and low cost), of AMA and PBC-specific ANA associated with more managerial (association of markers measured at present by rapid and accurate assaying by decreasing the likelihood different methods) and pathophysiological reasons [99] . of human error in biological test performance. In this context, proteomics, the science that studies the expression, function and interaction of proteins on a large 4. Expert opinion scale, allows parallel analyses of hundreds of different antigens/antibodies in minimal quantities of biological In clinical practice, most of the routine laboratories are fluids, aiming to identify proteins or polypeptides encoded still using, for AMA detection, the IIF assay on frozen by messanger RNA transcripts, which are upregulated or sections of rat liver, kidney and stomach substrate. The downregulated in association with a particular disease [100] . use of immobilized HEp2 cells only for AMA detection Among the numerous systems developed in recent years, should be avoided owing to an increase in frequency of some microarray assays have been used to study the false-positive results. Ideally, it could be better for each gene expression and autoantibody profiles in autoimmune local laboratory to develop its own rodent tissue substrate diseases [101,102] . Of all these systems now available, planar sections because substrates that are commercially available and non-planar autoantigen arrays have attracted considerable are of variable quality, resulting sometimes in enhanced attention. Unfortunately, such studies have so far received background staining, which usually limits interpretation of limited attention in PBC [103] . However, a recently established fluorescence patterns. In addition, the use of an antitotal consortium (E-RARE consortium ‘Epinostics’) has been funded human Ig-fluorescein isothiocyanate conjugate (IgG, IgA, by the European Union since 2008 for 36 months to study IgM) as revealing reagent is preferred compared with the the diagnostics and pathophysiology of autoimmune liver use of single monospecific IgG antiserum. A positive diseases (including PBC) by using sophisticated technologies AMA result by in-house IIF performed in a well-known and such as immunoscope analysis/spectratyping, microarray qualified laboratory could be enough for PBC diagnosis in analysis, epitope peptide mapping and mass spectrometric the case of a typical PBC patient, that is, a middle-aged protein characterization. The final aim of this project is woman with cholestasis, elevated cholesterol levels and to determine how the findings could be used further for compatible liver histology. Unfortunately, in real life the achieving new and innovative diagnostic and therapeutic development of locally validated sections for IIF does applications in these diseases. not seem to be very feasible. The latter, along with the Until the final findings/conclusions of the above- problems that do exist between laboratory reporting and mentioned EU project come out for external evaluation, clinical interpretation of the serological tests, makes the the development of new test systems for the detection determination of AMA and PBC-specific ANA only by of PBC-specific antibodies based on the xMultiple Analyte the IIF assay inefficient for achieving a safe and accurate Profiling Luminex methodology seems to be of utmost diagnosis. Therefore, other assays based on molecularly importance. This is one of the newest and most promising defined antigens, such as ELISAs and immunoblot, laboratory technologies as in general it can determine up to should be used either for AMA or for PBC-specific 100 different antibody specificities of up to 96 samples in a ANA determinations. From the diagnostic point of view, single run, giving the opportunity for the biomedical the latter assays are of utmost importance, particularly scientist to perform up to 9600 (96 × 100) tests using when the laboratories are unfamiliar with the use and a single tray. It is promising that this multiplexing interpretation of IIF patterns of AMA. It should be technology offers significant reductions in cost (5 – 6 times stressed that for molecular-based assays at least the three lower) and up to 80 – 95% reduction in labor compared immunodominant epitopes of AMA, namely PDC-E2, with ELISAs and IIF (IIF has double the cost per well BCOADC-E2 and OGDC-E2, should be taken into compared with ELISAs) [33] , as this test system eliminates

Expert Opin. Med. Diagn. (2008) 2(6) 629 Molecular diagnostics of primary biliary cirrhosis the need to split samples as well as to purchase different The same, of course, could be an option for the immunoassay kits. molecular diagnostics of the other autoimmune liver As noted in this review, Gershwin’s group has developed diseases, namely, autoimmune hepatitis, primary sclerosing such a sensitive bead assay for AMA detection based on cholangitis and overlap syndromes, either separately or along the Luminex principle using the three major mitochondrial with the diagnostics of PBC, giving rise to a total ‘autoantigens [55]. This bead assay improved sensitivity associated immune liver assay panel’. Of course, before the use of these with spatial presentation, essential for conformational new diagnostic tools for routine screening, the tests should epitopes, allowing the detection of multiple antigens at once be validated in detail in large prospective multi-center and, furthermore, it incorporates the advantages of a fully studies by incorporating newly diagnosed PBC patients of automated procedure that minimizes operator variability different race and stage of the disease. If these tests proved and allows high standardization [55] . Of note, one future to be efficient and accurate in large studies, it could be perspective with using this assay is the inclusion in the then possible to explore further their use in population- bead panel not only of the three major mitochondrial based studies, which will be of utmost importance as they antigens, but also several minor mitochondrial (e.g., PDC-E1a, might shed light on the etiology, pathogenesis and the PDC-E1b and E3BP) and nuclear antigens (e.g., sp100, natural history of PBC. gp210, p62, PML and LBR) that have been considered PBC-specific, in the attempt to generate a comprehensive Declaration of interest assay for PBC. The development of such a panel could offer the definition of autoantibody profile with a single The authors have no conflict of interest to declare and no assay, simplifying and accelerating the diagnostics in PBC. fee has been received for preparation of the manuscript.

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