New light on anion exchangers in primary biliary cirrhosis☆
Article Outline
Primary biliary cirrhosis [1] (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] 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]. A concordance rate of about 60% for monozygotic twins (as opposed to 0% for dizygotic twins) [4] 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. [5], [6] 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], or just a gender-specific exposure to environmental triggers like cosmetics [8] or nail polishers [9].
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].
In the present issue of this Journal, Poupon et al. [10] 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] and TNFα polymorphisms [12] affect various immune-mediated disorders whereas htSNPs of selected transporters had been studied in the past without major impact [13], [14].
In a second approach, Poupon et al. [10] 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]. 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] as well as biliary bicarbonate secretion [17] 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] and biliary bicarbonate secretion [17]. 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]. Strikingly, the variant haplotype of AE2 reported in the study by Poupon et al. [10] 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]. We have recently shown that the absence of Ae2 in fibroblasts [19] and lymphocytes [18] leads to a sustained increase in intracellular pH. Interestingly, this cytosolic alkalinization induces higher cAMP levels in fibroblasts [19], a phenomenon that is most likely driven by bicarbonate-stimulated soluble adenylate cyclase [20]. 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]. 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] in the present issue remains to be elucidated, but the potential for targeted therapeutic interventions is intriguing [22].
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] 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] 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.
Acknowledgement
Statistical advice by Prof. Thomas Gasser is gratefully acknowledged.
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☆ The authors declare that they do not have anything to disclose regarding funding from industries or conflict of interest with respect to this manuscript.
PII: S0168-8278(08)00621-1
doi:10.1016/j.jhep.2008.09.010
© 2008 European Association for the Study of the Liver. Published by Elsevier Inc. All rights reserved.
Refers to article:
- Genetic factors of susceptibility and of severity in primary biliary cirrhosis , 30 September 2008
