New light on anion exchangers in primary biliary cirrhosis

Genetic factors of susceptibility and of severity in primary biliary cirrhosisJournal of HepatologyVol. 49Issue 6PreviewIn primary biliary cirrhosis (PBC), pathogenesis is influenced by genetic factors that remain poorly elucidated up to now. We investigated the impact of sequence diversity in candidate genes involved in immunity (CTLA-4 and TNFα), in bile formation (10 hepatobiliary transporter genes) and in the adaptative response to cholestasis (three nuclear receptor genes) on the susceptibility and severity of PBC. Full-Text PDF Primary biliary cirrhosis [[1]Ahrens E.H. Payne M.A. Kunkel H.G. Eisenmenger W.J. Blondheim S.H. Primary biliary cirrhosis.Medicine. 1950; 29: 299-364Crossref PubMed Scopus (272) Google Scholar] (PBC) is an enigmatic liver disease of predominantly middle-aged women that may slowly progress to cirrhosis and liver failure. Patients are often asymptomatic at the time of diagnosis, but may present with disabling fatigue, recurrent pruritus and progressive jaundice. A diagnosis of PBC is made “with confidence” [[2]Heathcote E.J. Management of primary biliary cirrhosis. The American Association for the Study of Liver Diseases practice guidelines.Hepatology. 2000; 31: 1005-1013Crossref PubMed Scopus (391) Google Scholar] when biochemical markers of cholestasis (alkaline phosphatase, γ-glutamyltransferase) are elevated in the presence of serum AMA (⩾1:40); compatible histological findings confirm the diagnosis and allow staging before therapeutic intervention. Unravelling the pathogenesis of PBC remains one of the major challenges in hepatology. PBC is regarded as a model autoimmune disease which develops from interaction between environmental factors and inherited genetic predisposition [[3]Gershwin M.E. Mackay I.R. The causes of primary biliary cirrhosis: convenient and inconvenient truths.Hepatology. 2008; 47: 737-745Crossref PubMed Scopus (246) Google Scholar]. A concordance rate of about 60% for monozygotic twins (as opposed to 0% for dizygotic twins) [[4]Invernizzi P. Selmi C. Mackay I.R. Podda M. Gershwin M.E. From bases to basis: linking genetics to causation in primary biliary cirrhosis.Clin Gastroenterol Hepatol. 2005; 3: 401-410Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar] and familial clustering in 1–6% in comparison to a prevalence of 0.02–0.04% in the general population in northern Europe and the U.S. [5Metcalf J. James O. The geoepidemiology of primary biliary cirrhosis.Semin Liver Dis. 1997; 17: 13-22Crossref PubMed Scopus (80) Google Scholar, 6Kim W.R. Lindor K.D. Locke 3rd, G.R. Therneau T.M. Homburger H.A. Batts K.P. et al.Epidemiology and natural history of primary biliary cirrhosis in a US community.Gastroenterology. 2000; 119: 1631-1636Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar] mirror the impact of genetic factors on development of PBC. It remains still open to speculation whether the female preponderance (gender ratio 9:1) reflects a potential X chromosome-linked major locus of susceptibility: genes involved in immune responsiveness or sex hormone homeostasis are located on the X chromosome. There could also be a protective role of Y-linked genes, haploinsufficiency to specific X-linked genes due to enhanced X monosomy in PBC [[7]Invernizzi P. Miozzo M. Battezzati P.M. Bianchi I. Grati F.R. Simoni G. et al.Frequency of monosomy X in women with primary biliary cirrhosis.Lancet. 2004; 363: 533-535Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar], or just a gender-specific exposure to environmental triggers like cosmetics [[8]Rieger R. Leung P.S. Jeddeloh M.R. Kurth M.J. Nantz M.H. Lam K.S. et al.Identification of 2-nonynoic acid, a cosmetic component, as a potential trigger of primary biliary cirrhosis.J Autoimmun. 2006; 27: 7-16Crossref PubMed Scopus (149) Google Scholar] or nail polishers [[9]Gershwin M.E. Selmi C. Worman H.J. Gold E.B. Watnik M. Utts J. et al.Risk factors and comorbidities in primary biliary cirrhosis: a controlled interview-based study of 1032 patients.Hepatology. 2005; 42: 1194-1202Crossref PubMed Scopus (481) Google Scholar]. Genetic linkage studies in affected families as well as association studies in large cohorts of unrelated patients may disclose genetic variants conferring susceptibility or influencing progression and severity of a disease. In PBC, no linkage studies in affected families are available. Association studies in the past targeted candidate genes encoding immune modulatory proteins and genes encoding proteins involved in bile formation. The results were disappointing. Gene loci reported to be associated with PBC showed either a weak association or were not replicated [[4]Invernizzi P. Selmi C. Mackay I.R. Podda M. Gershwin M.E. From bases to basis: linking genetics to causation in primary biliary cirrhosis.Clin Gastroenterol Hepatol. 2005; 3: 401-410Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar]. In the present issue of this Journal, Poupon et al. [[10]Poupon R. Ping C. Chrétien Y. Corpechot C. Chazouillères O. Simon T. et al.Genetic factors of susceptibility and of severity in primary biliary cirrhosis.J Hepatol. 2008; 49: 1038-1045Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] made a new effort to gain further insight into contributing genetic factors in the development and progression of PBC. Several aspects of this work make it unconventional and a stimulus to further investigations. The authors selected 15 candidate genes: two related to immunity encoding cytotoxic T lymphocyte antigen 4 (CTLA-4) and tumor necrosis factor α (TNFα); ten related to bile formation encoding hepatobiliary transporters; and three related to adaptive response to cholestasis encoding nuclear receptors. They included a cohort of 258 PBC patients in comparison to two independent control groups of 286 and 269 healthy volunteers, respectively. By sequencing all exons, flanking intron sequences and the 5′ and 3′ untranslated regions in 32 individuals, they identified 42 haplotype-tagging single nucleotide polymorphisms (htSNPs), of which 12 were novel at the time of detection. The screening strategy allowed the detection of htSNPs of >5% frequency covering 96% of the genetic variance. In a first case-control analysis, only htSNPs in CTLA-4 and TNFα showed differences in distribution between PBC and controls suggesting a potential role of CTLA-4 and TNFα variants in the pathogenesis of PBC. In contrast, htSNPs of the 12 transporter genes as well as the 3 nuclear receptor genes under study were equally distributed. These findings were not unexpected as CTLA-4 [[11]Agarwal K. Czaja A.J. Jones D.E. Donaldson P.T. Cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms and susceptibility to type 1 autoimmune hepatitis.Hepatology. 2000; 31: 49-53Crossref PubMed Scopus (220) Google Scholar] and TNFα polymorphisms [[12]Donaldson P.T. TNF gene polymorphisms in primary biliary cirrhosis: a critical appraisal.J Hepatol. 1999; 31: 366-368Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar] affect various immune-mediated disorders whereas htSNPs of selected transporters had been studied in the past without major impact [13Pauli-Magnus C. Kerb R. Fattinger K. Lang T. Anwald B. Kullak-Ublick G.A. et al.BSEP and MDR3 haplotype structure in healthy Caucasians, primary biliary cirrhosis and primary sclerosing cholangitis.Hepatology. 2004; 39: 779-791Crossref PubMed Scopus (158) Google Scholar, 14Trauner M. Fickert P. Wagner M. MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes.Semin Liver Dis. 2007; 27: 77-98Crossref PubMed Scopus (190) Google Scholar]. In a second approach, Poupon et al. [[10]Poupon R. Ping C. Chrétien Y. Corpechot C. Chazouillères O. Simon T. et al.Genetic factors of susceptibility and of severity in primary biliary cirrhosis.J Hepatol. 2008; 49: 1038-1045Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] tested a potential association of the identified gene polymorphisms with disease severity at the time of diagnosis. Although the association of htSNPs in the multidrug resistance 1 gene, ABCB1/MDR1, and the pregnane X receptor gene, NR1L2/PXR, with more severe disease did not hold after stringent correction for multiple testing, this may provide an interesting lead for replicative studies in the future. The somewhat hidden jewel in this work is confined in the progression profiles of a subgroup of 147 patients with PBC treated with ursodeoxycholic acid (UDCA). Progression was defined as hyperbilirubinemia (>2 mg/dl), evolution towards cirrhosis, and referral to liver transplantation. A Cox regression analysis revealed serum bilirubin, alkaline phosphatase, albumin, and the SLC4A2/AE2 variant rs2303932 as potential independent prognostic factors for disease progression in PBC patients. While the former three factors have been documented as surrogate markers of prognosis in PBC in the past, the finding of an AE2 variant as a strong and independent prognostic factor for disease progression in PBC under UDCA therapy deserves particular attention. This finding suggests that cholangiocyte dysfunction may contribute to or aggravate the immune response against these cells. Thus, if cholangiocytes undergo apoptosis at higher than normal rates, this might lead to enhanced presentation of antigenic epitopes like pyruvate dehydrogenase complex-E2 (PDC-E2: the mitochondrial antigenic polypeptide structure against which T cell responses as well as AMA are directed in PBC) and/or accelerated destruction of bile ducts. Consequently, genetic variants that give rise to reduced cholangiocyte function may accelerate disease progression. The AE2 gene encodes a chloride/bicarbonate exchanger that is involved in intracellular pH homeostasis and cell volume regulation [[15]Alper S.L. Molecular physiology of SLC4 anion exchangers.Exp Physiol. 2006; 91: 153-161Crossref PubMed Scopus (195) Google Scholar]. AE2 gives rise to several isoforms of the transporter by alternative transcription and is ubiquitously expressed in tissues of the body. In most epithelia it is present in the basolateral membrane, but, strikingly, in hepatocytes and cholangiocytes it was reported to be present in the apical membrane, which raises the possibility that it is involved in bicarbonate excretion into bile. Prieto, Medina and coworkers reported a decade ago that AE2 expression [[16]Medina J.F. Martinez A. Vazquez J.J. Prieto J. Decreased anion exchanger 2 immunoreactivity in the liver of patients with primary biliary cirrhosis.Hepatology. 1997; 25: 12-17Crossref PubMed Google Scholar] as well as biliary bicarbonate secretion [[17]Prieto J. Garcia N. Marti-Climent J.M. Penuelas I. Richter J.A. Medina J.F. Assessment of biliary bicarbonate secretion in humans by positron emission tomography.Gastroenterology. 1999; 117: 167-172Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar] are impaired in PBC. This does not appear to be a genetic defect as UDCA treatment led to a partial recovery of AE2 expression [[16]Medina J.F. Martinez A. Vazquez J.J. Prieto J. Decreased anion exchanger 2 immunoreactivity in the liver of patients with primary biliary cirrhosis.Hepatology. 1997; 25: 12-17Crossref PubMed Google Scholar] and biliary bicarbonate secretion [[17]Prieto J. Garcia N. Marti-Climent J.M. Penuelas I. Richter J.A. Medina J.F. Assessment of biliary bicarbonate secretion in humans by positron emission tomography.Gastroenterology. 1999; 117: 167-172Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar]. However, secondary reduction in AE2 expression (e.g. due to local inflammation) might accelerate cholangiocyte dysfunction with consequently a faster destruction of bile ducts in PBC. It could be assumed that genetic variants that are associated with decreased AE2 function have similar detrimental effects under pathological conditions. It is important to note, that knockout mice with a disruption of most isoforms of Ae2 develop symptoms of PBC [[18]Salas J.T. Banales J.M. Sarvide S. Recalde S. Ferrer A. Uriarte I. et al.Ae2a, b-deficient mice develop antimitochondrial antibodies and other features resembling primary biliary cirrhosis.Gastroenterology. 2008; 134: 1482-1493Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar]. Strikingly, the variant haplotype of AE2 reported in the study by Poupon et al. [[10]Poupon R. Ping C. Chrétien Y. Corpechot C. Chazouillères O. Simon T. et al.Genetic factors of susceptibility and of severity in primary biliary cirrhosis.J Hepatol. 2008; 49: 1038-1045Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] negatively correlated with PBC disease progression suggesting that this haplotype may improve rather than impair cholangiocyte function. Alternatively, this AE2 variant could influence the antigenic response by an altered lymphocyte pH homeostasis which would influence T-cell balance. Indeed, Salas et al. observed a decreased number of T regulatory cells (Tregs) in mice with a disrupted Ae2 gene [[18]Salas J.T. Banales J.M. Sarvide S. Recalde S. Ferrer A. Uriarte I. et al.Ae2a, b-deficient mice develop antimitochondrial antibodies and other features resembling primary biliary cirrhosis.Gastroenterology. 2008; 134: 1482-1493Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar]. We have recently shown that the absence of Ae2 in fibroblasts [[19]Mardones P. Medina J.F. Elferink R.P. Activation of cyclic AMP Signaling in Ae2-deficient mouse fibroblasts.J Biol Chem. 2008; 283: 12146-12153Crossref PubMed Scopus (20) Google Scholar] and lymphocytes [[18]Salas J.T. Banales J.M. Sarvide S. Recalde S. Ferrer A. Uriarte I. et al.Ae2a, b-deficient mice develop antimitochondrial antibodies and other features resembling primary biliary cirrhosis.Gastroenterology. 2008; 134: 1482-1493Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar] leads to a sustained increase in intracellular pH. Interestingly, this cytosolic alkalinization induces higher cAMP levels in fibroblasts [[19]Mardones P. Medina J.F. Elferink R.P. Activation of cyclic AMP Signaling in Ae2-deficient mouse fibroblasts.J Biol Chem. 2008; 283: 12146-12153Crossref PubMed Scopus (20) Google Scholar], a phenomenon that is most likely driven by bicarbonate-stimulated soluble adenylate cyclase [[20]Zippin J.H. Levin L.R. Buck J. CO(2)/HCO(3)(−)-responsive soluble adenylyl cyclase as a putative metabolic sensor.Trends Endocrinol Metab. 2001; 12: 366-370Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]. Concomitantly, these “alkalinized cells” with increased cAMP levels have a sustained induction of inducible cAMP early repressor (ICER). ICER is the only member of the cAMP response element binding and modulator (CREB/CREM) family of transcription factors that acts as a direct repressor. ICER may play a crucial role in peripheral conversion of responder T cells into Tregs by inhibition of TGF-β mediated induction of Foxp3 expression [[21]Bodor J. Fehervari Z. Diamond B. Sakaguchi S. ICER/CREM-mediated transcriptional attenuation of IL-2 and its role in suppression by regulatory T cells.Eur J Immunol. 2007; 37: 884-895Crossref PubMed Scopus (51) Google Scholar]. Hence, disturbance of intracellular pH homeostasis may have important consequences for T cell differentiation. It remains to be demonstrated whether soluble adenylate cyclase is expressed to any significant extent in T cells, and whether impaired function of AE2 in T cells leads to chronically elevated cAMP levels and consequent induction of ICER. The functional nature of the human variant AE2 gene highlighted by Poupon et al. [[10]Poupon R. Ping C. Chrétien Y. Corpechot C. Chazouillères O. Simon T. et al.Genetic factors of susceptibility and of severity in primary biliary cirrhosis.J Hepatol. 2008; 49: 1038-1045Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] in the present issue remains to be elucidated, but the potential for targeted therapeutic interventions is intriguing [[22]Arenas F. Hervias I. Uriz M. Joplin R. Prieto J. Medina J.F. Combination of ursodeoxycholic acid and glucocorticoids upregulates the AE2 alternate promoter in human liver cells.J Clin Invest. 2008; 118: 695-709PubMed Google Scholar]. Apart from these considerations on pathophysiological aspects of PBC, we would like to add some general remarks with regard to genetic association studies. These are often plagued by lack of reproducibility caused by false-negative and, more frequently, false-positive associations. False-positive associations can be caused by hidden stratification within the case or control group, by statistical fluctuation, or by the inappropriate use of p-values of 0.05 as a threshold criterion for success. Newton-Cheh and Hirschhorn [[23]Newton-Cheh C. Hirschhorn J.N. Genetic association studies of complex traits: design and analysis issues.Mutat Res. 2005; 573: 54-69Crossref PubMed Scopus (235) Google Scholar] argued that association studies should utilize prior probability estimates on basis of the type of association that is studied. This involves estimation of the number of candidate genes involved in a certain disease, the odds ratio of an involved gene and whether a gene represents a major or minor determinant. They calculated that in the best case scenario (a top candidate gene in a polygenic disease with an odds ratio lower than 1.5) one should use a p < 0.003 rather than 0.05. On the basis of this approach, they also calculated that when a p < 0.05 is used in such a study with 200 cases and 200 controls, the chance of finding a true-positive association is 1.7%. If a p < 0.001 is applied this chance increases to 26%. When the same study involves 2000 cases and 2000 controls, the chance of true positivity increases to 4.9% (with p < 0.05%) and 60% (with p < 0.001). These calculations demonstrate that conclusions from small association studies have to be interpreted cautiously. In summary, the data provided by Poupon et al. [[10]Poupon R. Ping C. Chrétien Y. Corpechot C. Chazouillères O. Simon T. et al.Genetic factors of susceptibility and of severity in primary biliary cirrhosis.J Hepatol. 2008; 49: 1038-1045Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] are intriguing with regard to a potential role of ABCB1, PXR and, in particular, AE2 gene variants in the development and progression of PBC. However, confirmation of these data in large and independent cohorts of PBC patients is warranted. Statistical advice by Prof. Thomas Gasser is gratefully acknowledged.