[Frontiers in Bioscience 17, 206-220, January 1, 2012]

Genetic determinants of acquired cholestasis: A systems biology approach

Silvia Sookoian1, Carlos Jose Pirola2

1Department of Clinical and Molecular Hepatology, Institute of Medical Research A Lanari-IDIM, University of Buenos Aires-National Council of Scientific and Technological Research (CONICET), Ciudad Autonoma de Buenos Aires, Argentina, 2Department of Molecular Genetics and Biology of the Complex Diseases, Institute of Medical Research A Lanari-IDIM, University of Buenos Aires-National Council of Scientific and Technological Research (CONICET), Ciudad Autonoma de Buenos Aires, Argentina

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Systems approach and mechanistic and therapeutic insights
3.1 .Evidence of multifactorial genetic basis
3.2 .Systems Biology-based approaches and gene-regulatory networks in the etiology of PSC, PBC, and ICP
3.3 .Identifying potential candidate genes for disease susceptibility based on the strategy of gene prioritization
4. Perspective
5. Acknowledgments
6. References

1. ABSTRACT

Cholestatic liver diseases encompass a complex spectrum of intrahepatic and cholangiocellular cholestasis, whose etiologies include genetic and environmental components. This review focuses on the role of the genetic component of three adult cholestatic diseases, namely, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), and intrahepatic cholestasis of pregnancy (ICP). In particular, we integrate genomic, molecular, and physiological data to understand the putative interplay between the underlying genetic mechanisms involved in the susceptibility of these diseases. This approach is based on the hypothesis that a more integrative knowledge of the genetic determinants of cholestatic diseases may have a strong impact on the development of improved therapies. We also propose the strategy of gene prioritization to identity potential candidate genes for disease susceptibility, and show some examples of "leading genes of human cholestatic pathways". Finally, based on the hypothesis that common physiologic processes and molecular networks may influence the risk of adult cholestatic diseases, we used a candidate gene prioritization application based on the use of a protein-protein interaction network as part of the 'interactome'.

2. INTRODUCTION

Human cholestatic liver diseases cover a complex range of intrahepatic and cholangiocellular cholestasis. The etiologies of these diseases include genetic and environmental components, and the triggering-associated factors are diverse, including infections, toxins, and autoimmune processes, among others. Here we examine the role of the genetic component of three adult cholestatic diseases: primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), and intrahepatic cholestasis of pregnancy (ICP). Genomic, molecular, and physiological data are integrated to give us an understanding of the putative interplay between the underlying genetic mechanisms involved in the susceptibility of these clinical pictures. This particular approach is based on the hypothesis that a more integrative knowledge of the genetic determinants of cholestatic diseases may have a strong impact on the development of improved therapies.

While cholangiopathies such as PBC and PSC are progressive diseases, the first one characterized by portal inflammation and immune-mediated destruction of the intrahepatic bile ducts and the second one by inflammation, destruction, and fibrosis of intrahepatic and extrahepatic bile duct, ICP is a relatively benign disorder, at least for the mother. Despite such dramatic clinical and histopathological differences, there are some biological similarities that suggest the involvement of common biological pathways among them (Table 1) (1-3). Hence, our goal is to infer gene networks that capture common physiological pathways among the three diseases and predict new candidate genes potentially associated with them.

3. SYSTEMS BIOLOGY APPROACH, AND MECHANISTIC AND THERAPEUTIC INSIGHTS

3.1. Evidence of multifactorial genetic basis

As shown in Table 1, the three cholestatic diseases with which we will deal here show evidence of heritability and familial clustering. In addition, DNA variation in either candidate genes or signals identified by genome-wide association studies (GWAS) has been regarded as susceptibility or modifier variants of these disorders. Nevertheless, like in other complex diseases, the associated gene variants have small or moderate effects on disease risk. This picture is in part explained by the nature of the complex disorder itself and the strong impact of environmental factors working in concert with the genetic influence. In this regard, the influence of epigenetic factors, such as DNA methylation and histone modifications, remain totally unexplored, but it is very likely that they play a major role in the disease etiology.

In addition, while these diseases have worldwide distribution, their incidence is strongly variable according to the different ethnic groups, suggesting the involvement of population-specific genetic risk factors. This is supported by the observation that some gene variants are associated with disease susceptibility in some but not in all populations. For example, gene variants of cytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152CTLA-4) were associated with PBC in populations from United Kingdom (4), United States (5), and China (6), but the findings were not replicated in the Italian population (7), in a combined study from the United Kingdom and northern Italy (8), or in the Brazilian population (9). Similarly, a 32-base pair deletion in the chemokine receptor 5 gene (CCR5-D32), expressed on T-cells and macrophages, shows conflicting results about a putative association with susceptibility to PSC. In Australian (10) and Belgian populations (11), but not in Scandinavian ones (12), the variant was associated either with susceptibility or progression of the disease.

Interestingly, even in the presence of different mechanisms for the induction of cholestasis in PSC, PBC, and ICP, some genes are reported as being associated with all of them (Figure 1). The most remarkable example is ABCB4 (also known as MDR3), a member of the superfamily of ATP-binding cassette (ABC) transporters. ABCB4 is a liver-specific membrane transporter of phosphatidylcholine - a major component of the bile, from the inner to the outer leaflet of the canalicular membrane (13); the biliary phospholipid excretion is a critical mechanism to protect plasma membranes of cholangiocytes from bile salts.

Mutations of this gene lead to progressive familial intrahepatic cholestasis type 3; the gene is also regarded as PFIC3. As mentioned before, gene variants of ABCB4 were associated with ICP (14), and also were regarded as modifiers of disease progression in PBC or predictors of survival to liver transplantation in PSC (15). Interestingly, another family member of the ABC transporter superfamily, MRP2 (ABCC2), was found to be associated with ICP (16).

Besides the relevant data about the function of ABCB4, it is also important to note from a clinical point of view that most patients with ABCB4 deficiency have a favorable outcome with ursodeoxycholic acid (UDCA) therapy (17). Hence, ABCB4 has been proposed as good candidate for a targeted pharmacologic approach (17).

Moreover, ABCB4 mutations also represent a genetic factor involved in the presence of cholesterol gallstone disease in adults (18), a condition also associated with the three cholestatic diseases. Additional evidence from human and animal studies supporting the role of ABCB4 as a "cholestatic-candidate gene" is shown in Table 2.

3.2. Systems biology-based approaches and gene-regulatory networks in the etiology of PSC, PBC, and ICP

A second explanation for the relatively small effect attributed to the associated gene variants in the three cholestatic disorders analyzed here is that neither the common SNPs nor the rare mutations identified so far as putative susceptibility genes are necessarily the "causative agents" by themselves but they act as networks of genes that interact among each other. Then, it seems very unlikely that the complex gene-gene and gene-environment interactions that characterize PSC, PBC, and ICP can be easily explained or even modeled in a transgenic model of disease. Hence, system biology-based approaches based on the combinations of certain data, e.g., expression array analysis, the measurement of transcript levels, the relationships between transcript levels and clinical traits, etc., might be able to address such complex interactions.

Systems biology introduces a new and integrative concept of the pathogenesis of human disorders, and suggests the presence of common physiologic processes and molecular networks influencing the risk of a disease. The significance of systems biology-based strategy and a "proof-of-concept" about the potential application of this novel strategy was previously shown (19, 20). Here, we show a model of this concept for the explanation of the genetic determinants of the three cholestatic diseases.

An example of this approach is the prediction of integrated associations and gene networks, derived from several resources such as a collection of PubMed abstracts, high-throughput proteomic screening, and microarray profiling. Figure 2 shows a putative gene network for the ABCB4 gene. Interestingly, ABCB4 interacts closely with four genes that may explain its pleiotropic effects, namely, ABCB1, ABCB7, HAX1, and NR1H4 (FXR).

The observation on the impact of the putative gene network of ABCB4 on the pathogenesis of the above-mentioned cholestatic disorders fits also with the association between gene variants of the nuclear bile acid receptor NR1H4 (FXR) and susceptibility to ICP (21, 22), PSC (23), and gallstone disease (24) (Figure 2).

The second remarkable gene reported as a susceptibility gene of the three diseases is the nuclear receptor subfamily 1, group I, member 2 (NR1I2, PXR, SXR), a nuclear receptor that binds and is activated by a variety of endogenous and xenobiotic compounds. PXR is also involved in the detoxification of cholestatic bile acids, and in the regulation of the expression of genes involved in the biosynthesis transport and metabolism of bile acids. PXR activates the transcription of multiple genes involved in the metabolism and secretion of potentially harmful, exogenous and endogenous compounds. Interestingly, PXR is activated by pregnanes and by the toxic bile acid lithocholic acid. In addition, PXR binds to a response element in the promoters of the CYP3A4 and ABCB1/MDR1 genes. Functional gene variants in PXR were not only associated with susceptibility to ICP (25), but also appear to modify disease course in PSC (26). Finally, endogenous and exogenous PXR activators are used to treat pruritus in chronic inflammatory liver diseases such as PBC (27). A gene network for PXR (NR1I2, SXR) based on data of PubMed abstracts, high-throughput proteomic screening, and microarray profiling is shown in Figure 3. A gene that encodes a UDP-glucuronosyltransferase (UGT1A1, also known as bilirubin UDP-glucuronosyltransferase isozyme 1) is predicted to be co-expressed and also up-regulated by PXR; UGT1A1 transforms small lipophilic molecules, such as steroids, bilirubin, hormones, and drugs, into water-soluble, excretable metabolites. UGT1A1 plays a major role in controlling serum levels of bilirubin by contributing to its conjugation with glucuronic acid, a key detoxification step of this potentially neurotoxic compound.

3.3. Identifying potential candidate genes for disease susceptibility based on the strategy of gene prioritization

While the phenotypic manifestations of the adult cholestatic diseases are readily characterized, the underling genetic component is far more difficult to understand. Some progress, however, has been made for the understanding of PSC and PBC since the era of the GWAS. For instance, a GWAS on PSC showed strong HLA-associations near the HLA-B at chromosome 6p21, and also with a subset of genes involved in bile homeostasis and inflammatory pathways (a genetic region surrounding the GPC6 locus) (28). Similarly, common genetic variants at the HLA class II, IL12A, and IL12RB2 loci were reported as significantly associated with PBC from a genome-wide association analysis including DNA samples from 2072 Canadian and U.S. subjects (29).

Despite the thorough scanning of thousands of markers across the complete human genome during the post-genome era, only a small proportion of the genetic component of the adult cholestatic diseases, namely, PSC, PBC, and ICP, can be explained by either common gene variants or gene mutations. However, this observation is not restricted to human cholestatic diseases; on the contrary, it is a common finding in the genetic of complex diseases. In addition, GWAS generate large sets of potential candidate genes, with the identification of the most likely disease-related ones being extremely difficult, in part owing to the need for multiple testing adjustments to avoid false positives but at the cost of imposing a really high statistical cutoff.

Some strategies have been proposed to improve the identification of susceptibility genes based on computational tools for prioritization of candidate genes, by assuming that the majority of causative genes are functionally closely related from the functional point of view. This approach has the advantage of not only facilitating the identification of a disease-associated gene, but it may also help to elucidate the disease biology.

To accomplish this purpose, we used three different strategies. First, we looked for sets of functionally related genes in combinations of loci associated with ICP, PBC, and PSC, on the basis of all the available data about either candidate gene association studies or GWAS, when available for each disease. Following this premise, we used the bioinformatic tool Prioritizer, available at http://pcdoeglas.med.rug.nl/prioritizer/, in which the basic premise for ranking candidate genes is positional candidate gene prioritization. Briefly, this application uses a Bayesian approach to generate a gene network based upon data from Gene Ontology, KEGG, BIND, HPRD, and Reactome, a dataset that contains approximately 70,000 predicted protein-protein interactions, 3,000 predicted human protein-protein interactions and co-expression data, derived from approximately 10,000 human microarray experiments stored within the Gene Expression Omnibus and the Stanford Microarray Database (30). As a result, this interaction network ranks the best positional candidates genes on the basis of their interactions, assuming that the causative ones for any disorder will be involved in only a few different biological pathways (30).

Using the Prioritizer program we investigated the putative biological relationships among all the genetic loci that have been reported until now in association with ICP, PBC, and PSC; Prioritizer input (training sets) for the three diseases is shown in Figures 4, 5, and 6, respectively. The application of Prioritizer to the three adult cholestatic diseases revealed a complex biological network containing several genes operating in interesting related pathways. For example, for genes related to ICP, Prioritizer analysis revealed a putative gene network associated with ABCC2 composed of NFKb (nuclear factor of kappa light polypeptide gene enhancer in B-cells 2), a transcription factor involved in many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis, and apoptosis, and HIF1a (hypoxia inducible factor 1a ), a transcription factor found in mammalian cells cultured under reduced oxygen tension that plays an essential role in cellular and systemic homeostatic responses to hypoxia. Hypoxic liver injury, even at molecular level, was not related before to the pathogenesis of ICP. Nevertheless, severe hypoxic injury was reported in a variant of hepatic sickle cell crisis associated with intrahepatic cholestasis, which has severe jaundice and high transaminase levels (31). Thus, one may speculate that some level of insufficient hepatic perfusion in women suffering from ICP may occur.

In the case of PBC, both CASP8 (caspase 8, apoptosis-related cysteine peptidase), involved in the programmed cell death induced by Fas and various apoptotic stimuli, and CD28, a T-cell-specific surface glycoprotein, composed the CTLA4-associated gene network. Finally, for the PSC-associated genes, the Prioritizer analysis revealed a putative gene network associated with HLA-DRB2 composed of PSMB8 (proteasome subunit, beta type, 8 that plays critical roles in major histocompatibility complex class I antigen processing) and BRD2 (bromodomain containing 2, a protein serine/threonine kinase having distinct roles in initiating apoptosis; the dotted distribution pattern in nucleus may be a morphologic marker of cell apoptosis).

The second approach we used was gene prioritization through genomic data fusion by the ENDEAVOUR program (32). ENDEAVOUR is a software application for the computational prioritization of candidate genes underlying biological processes or diseases, based on their similarity to known genes involved in a disease (32). The hypothesis underlying the gene prioritization procedure by ENDEAVOUR is that test genes (candidates) are ranked based on their similarity with a set of known training genes. Briefly, ENDEAVOUR makes use of statistics to compute a ranking of test genes according to their similarity to the training genes. In a subsequent step, these rankings are integrated into a single ranking by making use of order statistics. The ranking of the test genes is built by ENDEAVOUR after integrating the data into a mathematical model based on the similarity of the test genes with the training genes. Vector-based data are scored by the Pearson correlation between a test profile and the training average, whereas attribute-based data are scored by Fisher's omnibus analysis on statistically over-represented training attributes (32). We used the same list of training genes as we had used for prioritization by the Prioritizer program (all the genes already known to be involved in the genetic susceptibility of ICP, PSC, and PBC).

Based on the description above, Table 3 lists the first 50 prioritized genes out of 23.712 from the whole human genome with a significant association with the ICP training set. Similar analyses are shown for PSC and PBC in Table 4 and Table 5, respectively. Interestingly, gene prioritization by ENDEAVOUR showed that some gene families might be regarded as involved in the genetic susceptibility to the adult cholestatic diseases. For instance, the Cytochrome P450 (CYP) superfamily, represented by almost 13 genes, was prioritized with high significance levels for ICP. As already known, the function of most CYP enzymes is to catalyze the oxidation of organic substances, including metabolic intermediates such as lipids and steroidal hormones, as well as xenobiotic substances such as drugs and other toxic chemicals. There are just few studies in the Chinese population that have evaluated the association between gene variants of the CYP enzymes and the obstetric cholestasis (33, 34), so that replication in other populations deserves to be performed.

In the case of PSC, the family of chemokine receptors was significantly over-represented, suggesting that they may play a significant role in the etiology of this disorder. An interesting hypothesis about pathogenetic similarities between PSC and arteriosclerosis was previously formulated, suggesting that PSC represents "arteriosclerosis of the bile duct" initiated by toxic biliary lipids (35). In this scenario, adhesion molecules like ICAMs, and chemokine receptors may explain the pathobiology of the bile ductular lesions.

The signal transducer and activator of transcription (STAT) genes deserve particular attention regarding their putative role in the genetic susceptibility of PBC. STAT proteins are involved in several functions, including the maintenance of immune tolerance, tumor surveillance, and apoptosis. There is no evidence for the association between gene variants in any STAT genes and PBC as yet, and hence additional studies are warranted to investigate these candidate genes.

A final and remarkable observation involves a group of genes prioritized for the three disorders that overlap each other with significant p values, namely, genes belonging to the nuclear receptors family (Figure 7). ENDEAVOUR prioritized several susceptibility loci that reveal a molecular link between the three disorders. Hence, the complex network shared by ICP, PSC, and PBS includes the thyroid hormone receptor alpha (THRA), liver X receptor a and b (LXRA and LXRB), constitutive androstane receptor (CAR), retinoic acid receptor (RARB), hepatocyte nuclear factor 4a (HNF4A), and RAC3. None of them were considered before as candidate genes, while their role in the bile acid homeostasis and cholestatic liver injury is unquestionable (36). The complex transcriptional and post-transcriptional regulations exerted by these nuclear receptors make them promising therapeutic targets for cholestatic liver diseases (37). List of references about candidate gene association studies is shown in Table 6.

Based on the hypothesis that common physiologic processes and molecular networks may influence the risk of adult cholestatic diseases, we proposed as a final approach for candidate gene prioritization: the use of a protein-protein interaction network (PPIN) or 'interactome'. For this purpose, we used the open web bioinformatic resource ToppGene Suite (http://toppgene.cchmc.org) to detect functional enrichment of our gene list based on Transcriptome, Proteome, Regulome (TFBS and miRNA), Ontologies (GO, Pathway), Phenotype (human disease and mouse phenotype), Pharmacome (Drug-Gene associations), and literature co-citation (38). Briefly, the protein-protein interaction network-based disease candidate gene prioritization uses social and Web networks analysis algorithms (extended versions of the PageRank and HITS algorithms, and the K-Step Markov method) (38).

To build the candidate gene list, we used the same training set of genes as those mentioned above for ICP, PBC, and PSC. By the ToppFun application that relies on gene annotations and sequence features, namely, GO: Molecular Function, GO: Biological Process, Mouse Phenotype, Pathways, Protein Interactions, Protein Domains, transcription factor-binding sites, miRNA-target genes, disease-gene associations, drug-gene interactions, and Gene Expression, we observed again that the common functional pathway shared by ICP, PBC, and PSC is the one enlisted as Nuclear Receptors in Lipid Metabolism and Toxicity (identification source: CGAP BioCarta, h_nuclearRsPathway, p value = 1.191E-11) (Figure 8). This pathway is notably ranked first for the three diseases, suggesting that nuclear receptors play a critical role in the physiopathology of adult cholestatic diseases, regardless of their etiology and clinical presentation. Interestingly, the ToppFun application also predicts drug-gene interactions based on data and annotations deposited in databases, such as The Comparative Toxicogenomics Database (CTD), Drugbank, Metador (Manually Annotated Targets and Drugs Online Resource), and Stitch (search tool for interactions of chemicals). For example, CTD is able to predict which genes/proteins interact with a chemical, and as shown in Table 7, based on the list of training genes we used for this analysis, ursodeoxycholic acid was predicted for ICP, PBC, and PSC. Hence, the rationale for its use is biologically plausible as supported by the clinical indication in the three diseases and successful treatment response. STITCH, a resource to explore known and predicted interactions of chemicals and proteins, predicted another chemical for ICP and PSC, 7alpha-hydroxy-4-cholesten-3-one, which was ranked with high significant p values. Previous reports showed that serum concentrations of 7alpha-hydroxy-4-cholesten-3-one (alpha-HC) may serve as a convenient marker for the semiquantitative assessment of bile acid synthesis (39).

As a final observation, the application predicted a miRNA: the hsa-miR-148a for ICP and PSC (p value = 1.637E-3); miRNAs are post-transcriptional regulators that bind to complementary sequences in the 3' UTRs of target mRNAs, usually resulting in gene silencing. This miRNA was recently regarded as a modulator the inflammation-associated cytokine interleukin-6 and oncogenesis in cholangiocarcinoma (40).

4. PERSPECTIVE

The genetic component of human adult cholestatic disorders such as ICP, PSC, and PBC is still under elucidation. Integrative knowledge in genetics by different approaches such as systems biology is becoming a powerful instrument to identify not only associated candidate genes but also common disease-associated molecular pathways. These liver disorders need a deeper analysis of DNA sequence variations and their interactions in concert with liver expression patterns and transcript abundance. Gene prioritization is gaining acceptance for modeling and performing integrative strategies, and some examples of "leading genes of human cholestatic pathways" were shown. Finally a comprehensive examination of each phenotype, analyzing its similarities and differences, may also impact on the design of more effective therapeutic strategies. A clear example of this is the fact that UDCA treatment is the most commonly used pharmacological intervention for ICP and PBC. Nevertheless, although clinical and biochemical response to this drug is remarkable for both disorders, the underlying physiopathological mechanism of ICP and PBC is, at least until now, regarded as absolutely different.

5. ACKNOWLEDGMENTS

Authors SS and CJP equally contributed to this article. Supported in part by Grants UBACYT M055 (Universidad de Buenos Aires),PICT 2006-124 and PICT 2008-1521 (Agencia Nacional de Promoción Científica y Tecnológica). SS and CJP are members of Consejo Nacional de Investigaciones Científicas y Tecnicas (CONICET). The authors have no conflict of interest to declare.

6. REFERENCES

1. M. M. Kaplan and M. E. Gershwin: Primary biliary cirrhosis. N Engl J Med 353, 1261-1273 (2005)
doi:10.1056/NEJMra043898
PMid:16177252

2. Y. M. Lee and M. M. Kaplan: Primary sclerosing cholangitis. N Engl J Med 332, 924-933 (1995)
doi:10.1056/NEJM199504063321406
PMid:7877651

3. B. Pathak, L. Sheibani and R. H. Lee: Cholestasis of pregnancy. Obstet Gynecol Clin North Am 37, 269-282 (2010)
doi:10.1016/j.ogc.2010.02.011
PMid:20685553

4. K. Agarwal, D. E. Jones, A. K. Daly, O. F. James, B. Vaidya, S. Pearce and M. F. Bassendine: CTLA-4 gene polymorphism confers susceptibility to primary biliary cirrhosis. J Hepatol 32, 538-541 (2000)
doi:10.1016/S0168-8278(00)80213-5

5. B. D. Juran, E. J. Atkinson, E. M. Schlicht, B. L. Fridley and K. N. Lazaridis: Primary biliary cirrhosis is associated with a genetic variant in the 3' flanking region of the CTLA4 gene. Gastroenterology 135, 1200-1206 (2008)
doi:10.1053/j.gastro.2008.06.077
PMid:18778710    PMCid:2613835

6. L. Y. Fan, X. Q. Tu, Q. B. Cheng, Y. Zhu, R. Feltens, T. Pfeiffer and R. Q. Zhong: Cytotoxic T lymphocyte associated antigen-4 gene polymorphisms confer susceptibility to primary biliary cirrhosis and autoimmune hepatitis in Chinese population. World J Gastroenterol 10, 3056-3059 (2004)
PMid:15378793

7. S. Oertelt, T. P. Kenny, C. Selmi, P. Invernizzi, M. Podda and M. E. Gershwin: SNP analysis of genes implicated in T cell proliferation in primary biliary cirrhosis. Clin Dev Immunol 12, 259-263 (2005)
doi:10.1080/17402520500317859
PMid:16584111    PMCid:2270736

8. P. Donaldson, S. Veeramani, A. Baragiotta, A. Floreani, C. Venturi, S. Pearce, V. Wilson, D. Jones, O. James, J. Taylor, J. Newton and M. Bassendine: Cytotoxic T-lymphocyte-associated antigen-4 single nucleotide polymorphisms and haplotypes in primary biliary cirrhosis. Clin Gastroenterol Hepatol 5, 755-760 (2007)
doi:10.1016/j.cgh.2007.02.012
PMid:17482523

9. P. L. Bittencourt, S. A. Palacios, E. L. Cancado, G. Porta, F. J. Carrilho, A. A. Laudanna, J. Kalil and A. C. Goldberg: Cytotoxic T lymphocyte antigen-4 gene polymorphisms do not confer susceptibility to autoimmune hepatitis types 1 and 2 in Brazil. Am J Gastroenterol 98, 1616-1620 (2003)
PMid:12873588

10. R. Eri, J. R. Jonsson, N. Pandeya, D. M. Purdie, A. D. Clouston, N. Martin, D. Duffy, E. E. Powell, J. Fawcett, T. H. Florin and G. L. Radford-Smith: CCR5-Delta32 mutation is strongly associated with primary sclerosing cholangitis. Genes Immun 5, 444-450 (2004)
doi:10.1038/sj.gene.6364113
PMid:15215889

11. L. Henckaerts, J. Fevery, S. W. Van, C. Verslype, P. Yap, F. Nevens, T. Roskams, L. Libbrecht, P. Rutgeerts and S. Vermeire: CC-type chemokine receptor 5-Delta32 mutation protects against primary sclerosing cholangitis. Inflamm Bowel Dis 12, 272-277 (2006)
doi:10.1097/01.MIB.0000209790.21737.28
PMid:16633049

12. E. Melum, T. H. Karlsen, U. Broome, E. Thorsby, E. Schrumpf, K. M. Boberg and B. A. Lie: The 32-base pair deletion of the chemokine receptor 5 gene (CCR5-Delta32) is not associated with primary sclerosing cholangitis in 363 Scandinavian patients. Tissue Antigens 68, 78-81 (2006)
doi:10.1111/j.1399-0039.2006.00604.x
PMid:16774544

13. P. J. Meier and B. Stieger: Bile salt transporters. Annu Rev Physiol 64, 635-661 (2002)
doi:10.1146/annurev.physiol.64.082201.100300
PMid:11826283

14. P. H. Dixon and C. Williamson: The molecular genetics of intrahepatic cholestasis of pregnancy. Obstet Med 1, 65-71 (2008)
doi:10.1258/om.2008.080010

15. T. H. Karlsen and J. R. Hov: Genetics of cholestatic liver disease in 2010. Curr Opin Gastroenterol 26, 251-258 (2010)
doi:10.1097/MOG.0b013e328336807d
PMid:20042859

16. S. Sookoian, G. Castano, A. Burgueno, T. F. Gianotti and C. J. Pirola: Association of the multidrug-resistance-associated protein gene (ABCC2) variants with intrahepatic cholestasis of pregnancy. J Hepatol 48, 125-132 (2008)
doi:10.1016/j.jhep.2007.08.015
PMid:17997497

17. A. Davit-Spraul, E. Gonzales, C. Baussan and E. Jacquemin: The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects. Semin Liver Dis 30, 134-146 (2010)
doi:10.1055/s-0030-1253223
PMid:20422496

18. O. Rosmorduc, B. Hermelin and R. Poupon: MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Gastroenterology 120, 1459-1467 (2001)
doi:10.1053/gast.2001.23947
PMid:11313316

19. A. S. Azmi, Z. Wang, P. A. Philip, R. M. Mohammad and F. H. Sarkar: Proof of concept: network and systems biology approaches aid in the discovery of potent anticancer drug combinations. Mol Cancer Ther 9, 3137-3144 (2010)
doi:10.1158/1535-7163.MCT-10-0642
PMid:21041384

20. S. Sookoian and C. J. Pirola: Metabolic Syndrome: From the Genetics to the Pathophysiology. Curr Hypertens Rep (2010). Epub (DOI: 10.1007/s11906-010-0164-9).

21. S. W. Van Mil, A. Milona, P. H. Dixon, R. Mullenbach, V. L. Geenes, J. Chambers, V. Shevchuk, G. E. Moore, F. Lammert, A. G. Glantz, L. A. Mattsson, J. Whittaker, M. G. Parker, R. White and C. Williamson: Functional variants of the central bile acid sensor FXR identified in intrahepatic cholestasis of pregnancy. Gastroenterology 133, 507-516 (2007)
doi:10.1053/j.gastro.2007.05.015
PMid:17681172

22. V. Zimmer, R. Mullenbach, E. Simon, C. Bartz, S. Matern and F. Lammert: Combined functional variants of hepatobiliary transporters and FXR aggravate intrahepatic cholestasis of pregnancy. Liver Int 29, 1286-1288 (2009)
doi:10.1111/j.1478-3231.2009.02026.x
PMid:19490418

23. L. Henckaerts, M. Jaspers, S. W. Van, L. Vliegen, J. Fevery, H. Nuytten, T. Roskams, P. Rutgeerts, J. J. Cassiman, S. Vermeire and H. Cuppens: Cystic fibrosis transmembrane conductance regulator gene polymorphisms in patients with primary sclerosing cholangitis. J Hepatol 50, 150-157 (2009)
doi:10.1016/j.jhep.2008.07.033
PMid:18992954

24. P. Kovacs, R. Kress, J. Rocha, U. Kurtz, J. F. Miquel, F. Nervi, N. Mendez-Sanchez, M. Uribe, H. H. Bock, R. Schirin-Sokhan, M. Stumvoll, J. Mossner, F. Lammert and H. Wittenburg: Variation of the gene encoding the nuclear bile salt receptor FXR and gallstone susceptibility in mice and humans. J Hepatol 48, 116-124 (2008)
doi:10.1016/j.jhep.2007.07.027
PMid:17931734

25. G. Castano, A. Burgueno, G. T. Fernandez, C. J. Pirola and S. Sookoian: The influence of common gene variants of the xenobiotic receptor (PXR) in genetic susceptibility to intrahepatic cholestasis of pregnancy. Aliment Pharmacol Ther 31, 583-592 (2010)
doi:10.1111/j.1365-2036.2009.04210.x

26. T. H. Karlsen, B. A. Lie, F. K. Frey, E. Thorsby, U. Broome, E. Schrumpf and K. M. Boberg: Polymorphisms in the steroid and xenobiotic receptor gene influence survival in primary sclerosing cholangitis. Gastroenterology 131, 781-787 (2006)
doi:10.1053/j.gastro.2006.05.057
PMid:16952547

27. K. Wallace, D. E. Cowie, D. K. Konstantinou, S. J. Hill, T. E. Tjelle, A. Axon, M. Koruth, S. A. White, H. Carlsen, D. A. Mann and M. C. Wright: The PXR is a drug target for chronic inflammatory liver disease. J Steroid Biochem Mol Biol 120, 137-148 (2010)
doi:10.1016/j.jsbmb.2010.04.012
PMid:20416375    PMCid:2937210

28. T. H. Karlsen, A. Franke, E. Melum, A. Kaser, J. R. Hov, T. Balschun, B. A. Lie, A. Bergquist, C. Schramm, T. J. Weismuller, D. Gotthardt, C. Rust, E. E. Philipp, T. Fritz, L. Henckaerts, R. K. Weersma, P. Stokkers, C. Y. Ponsioen, C. Wijmenga, M. Sterneck, M. Nothnagel, J. Hampe, A. Teufel, H. Runz, P. Rosenstiel, A. Stiehl, S. Vermeire, U. Beuers, M. P. Manns, E. Schrumpf, K. M. Boberg and S. Schreiber: Genome-wide association analysis in primary sclerosing cholangitis. Gastroenterology 138, 1102-1111 (2010)
doi:10.1053/j.gastro.2009.11.046
PMid:19944697

29. G. M. Hirschfield, X. Liu, C. Xu, Y. Lu, G. Xie, Y. Lu, X. Gu, E. J. Walker, K. Jing, B. D. Juran, A. L. Mason, R. P. Myers, K. M. Peltekian, C. N. Ghent, C. Coltescu, E. J. Atkinson, E. J. Heathcote, K. N. Lazaridis, C. I. Amos and K. A. Siminovitch: Primary biliary cirrhosis associated with HLA, IL12A, and IL12RB2 variants. N Engl J Med 360, 2544-2555 (2009)
doi:10.1056/NEJMoa0810440
PMid:19458352    PMCid:2857316

30. L. Franke, B. H. van, L. Fokkens, E. D. de Jong, M. Egmont-Petersen and C. Wijmenga: Reconstruction of a functional human gene network, with an application for prioritizing positional candidate genes. Am J Hum Genet 78, 1011-1025 (2006)
doi:10.1086/504300
PMid:16685651

31. S. Banerjee, C. Owen and S. Chopra: Sickle cell hepatopathy. Hepatology 33, 1021-1028 (2001)
doi:10.1053/jhep.2001.24114
PMid:11343226

32. S. Aerts, D. Lambrechts, S. Maity, L. P. Van, B. Coessens, S. F. De, L. C. Tranchevent, M. B. De, P. Marynen, B. Hassan, P. Carmeliet and Y. Moreau: Gene prioritization through genomic data fusion. Nat Biotechnol 24, 537-544 (2006)
doi:10.1038/nbt1203
PMid:16680138

33. X. Wang, L. Zhang, R. Q. Ou, J. Wang, S. Y. Liu, Q. Chen and P. C. Su: (Association between CYP1A1 gene polymorphism and intrahepatic cholestasis of pregnancy). Zhonghua Yi Xue Yi Chuan Xue Za Zhi 25, 70-72 (2008)
PMid:18247309

34. L. Zhang, S. Y. Liu, Q. Chen, Q. Y. Shi, H. Zou and L. Wu: (Association between polymorphisms of CYP17 and CYP3A4 genes and intrahepatic cholestasis of pregnancy in Chengdu). Sichuan Da Xue Xue Bao Yi Xue Ban 37, 551-3, 582 (2006)
PMid:16909599

35. P. Fickert, T. Moustafa and M. Trauner: Primary sclerosing cholangitis--the arteriosclerosis of the bile duct? Lipids Health Dis 6, 3 (2007)
doi:10.1186/1476-511X-6-3
PMid:17254334    PMCid:1796546

36. M. Arreseand S. J. Karpen: Nuclear receptors, inflammation, and liver disease: insights for cholestatic and fatty liver diseases. Clin Pharmacol Ther 87, 473-478 (2010)
doi:10.1038/clpt.2010.2

37. G. Zollnerand M. Trauner: Nuclear receptors as therapeutic targets in cholestatic liver diseases. Br J Pharmacol 156, 7-27 (2009)
doi:10.1111/j.1476-5381.2008.00030.x
PMid:19133988    PMCid:2697779

38. J. Chen, E. E. Bardes, B. J. Aronow and A. G. Jegga: ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res 37, W305-W311 (2009)
doi:10.1093/nar/gkp427
PMid:19465376    PMCid:2703978

39. G. Sauter, F. Berr, U. Beuers, S. Fischer and G. Paumgartner: Serum concentrations of 7alpha-hydroxy-4-cholesten-3-one reflect bile acid synthesis in humans. Hepatology 24, 123-126 (1996)
PMid:8707250

40. C. Braconi, N. Huang and T. Patel: MicroRNA-dependent regulation of DNA methyltransferase-1 and tumor suppressor gene expression by interleukin-6 in human malignant cholangiocytes. Hepatology 51, 881-890 (2010)
PMid:20146264

41. K. Said, H. Glaumann and A. Bergquist: Gallbladder disease in patients with primary sclerosing cholangitis. J Hepatol 48, 598-605 (2008)
PMid:18222013

42. D. J. Brandt, R. L. MacCarty, J. W. Charboneau, N. F. LaRusso, R. H. Wiesner and J. Ludwig: Gallbladder disease in patients with primary sclerosing cholangitis. AJR Am J Roentgenol 150, 571-574 (1988)
PMid:3277348

43. J. A. Summerfield, E. Elias, G. D. Hungerford, V. L. Nikapota, R. Dick and S. Sherlock: The biliary system in primary biliary cirrhosis. A study by endoscopic retrograde cholangiopancreatography. Gastroenterology 70, 240-243 (1976)
PMid:1248684

44. A. Ropponen, R. Sund, S. Riikonen, O. Ylikorkala and K. Aittomaki: Intrahepatic cholestasis of pregnancy as an indicator of liver and biliary diseases: a population-based study. Hepatology 43, 723-728 (2006)
doi:10.1002/hep.21111
PMid:16557542

45. Y. Popov, E. Patsenker, P. Fickert, M. Trauner and D. Schuppan: Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary fibrosis via massive dysregulation of pro- and antifibrogenic genes. J Hepatol 43, 1045-1054 (2005)
doi:10.1016/j.jhep.2005.06.025
PMid:16223543

46. K. E. Nakken, S. Nygard, T. K. Haaland, K. E. Berge, A. Odegaard, K. J. Labori and M. G. Raeder: Gene expression profiles reflect sclerosing cholangitis activity in abcb4 (-/-) mice. Scand J Gastroenterol 44, 211-218 (2009)
doi:10.1080/00365520802400867
PMid:18785065

47. O. Rosmorduc, B. Hermelin, P. Y. Boelle, R. Parc, J. Taboury and R. Poupon: ABCB4 gene mutation-associated cholelithiasis in adults. Gastroenterology 125, 452-459 (2003)
doi:10.1016/S0016-5085(03)00898-9

48. J. F. Lucena, J. I. Herrero, J. Quiroga, B. Sangro, J. Garcia-Foncillas, N. Zabalegui, J. Sola, M. Herraiz, J. F. Medina and J. Prieto: A multidrug resistance 3 gene mutation causing cholelithiasis, cholestasis of pregnancy, and adulthood biliary cirrhosis. Gastroenterology 124, 1037-1042 (2003)
doi:10.1053/gast.2003.50144
PMid:12671900

49. E. Jacquemin, J. M. De Vree, D. Cresteil, E. M. Sokal, E. Sturm, M. Dumont, G. L. Scheffer, M. Paul, M. Burdelski, P. J. Bosma, O. Bernard, M. Hadchouel and R. P. Elferink: The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood. Gastroenterology 120, 1448-1458 (2001)
doi:10.1053/gast.2001.23984
PMid:11313315

50. A. Floreani, I. Carderi, D. Paternoster, G. Soardo, F. Azzaroli, W. Esposito, A. Variola, A. M. Tommasi, D. Marchesoni, C. Braghin and G. Mazzella: Intrahepatic cholestasis of pregnancy: three novel MDR3 gene mutations. Aliment Pharmacol Ther 23, 1649-1653 (2006)
doi:10.1111/j.1365-2036.2006.02869.x

51. E. Jacquemin, D. Cresteil, S. Manouvrier, O. Boute and M. Hadchouel: Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy. Lancet 353, 210-211 (1999)
doi:10.1016/S0140-6736(05)77221-4

52. M. L. Eloranta, T. Hakli, M. Hiltunen, S. Helisalmi, K. Punnonen and S. Heinonen: Association of single nucleotide polymorphisms of the bile salt export pump gene with intrahepatic cholestasis of pregnancy. Scand J Gastroenterol 38, 648-652 (2003)
doi:10.1080/00365520310000807

53. R. Mullenbach, K. J. Linton, S. Wiltshire, N. Weerasekera, J. Chambers, E. Elias, C. F. Higgins, D. G. Johnston, M. I. McCarthy and C. Williamson: ABCB4 gene sequence variation in women with intrahepatic cholestasis of pregnancy. J Med Genet 40, e70 (2003)
doi:10.1136/jmg.40.5.e70
PMCid:1735459

54. M. L. Eloranta, J. T. Heiskanen, M. J. Hiltunen, A. J. Mannermaa, K. R. Punnonen and S. T. Heinonen: Multidrug resistance 3 gene mutation 1712delT and estrogen receptor alpha gene polymorphisms in Finnish women with obstetric cholestasis. Eur J Obstet Gynecol Reprod Biol 105, 132-135 (2002)
PMid:12381474

55. A. Floreani, I. Carderi, D. Paternoster, G. Soardo, F. Azzaroli, W. Esposito, A. Variola, A. M. Tommasi, D. Marchesoni, C. Braghin and G. Mazzella: Intrahepatic cholestasis of pregnancy: three novel MDR3 gene mutations. Aliment Pharmacol Ther 23, 1649-1653 (2006)
doi:10.1111/j.1365-2036.2006.02869.x

56. R. Mullenbach, A. Bennett, N. Tetlow, N. Patel, G. Hamilton, F. Cheng, J. Chambers, R. Howard, S. D. Taylor-Robinson and C. Williamson: ATP8B1 mutations in British cases with intrahepatic cholestasis of pregnancy. Gut 54, 829-834 (2005)
PMid:15888793    PMCid:1774530


57. J. G. Mella, E. Roschmann, J. C. Glasinovic, A. Alvarado, M. Scrivanti and B. A. Volk: Exploring the genetic role of the HLA-DPB1 locus in Chileans with intrahepatic cholestasis of pregnancy. J Hepatol 24, 320-323 (1996)
doi:10.1016/S0168-8278(96)80011-0

58. M. L. Eloranta, J. Heiskanen, M. Hiltunen, S. Helisalmi, A. Mannermaa and S. Heinonen: Apolipoprotein E alleles in women with intrahepatic cholestasis of pregnancy. Scand J Gastroenterol 35, 966-968 (2000)
doi:10.1080/003655200750023048
PMid:17934296

59. J. Laasanen, S. Helisalmi, S. Iivonen, M. L. Eloranta, M. Hiltunen and S. Heinonen: Gamma 2 actin gene (enteric type) polymorphism is not associated with obstetric cholestasis or preeclampsia. Fetal Diagn Ther 23, 36-40 (2008)
doi:10.1159/000109224
PMid:18930330

60. R. Poupon, C. Ping, Y. Chretien, C. Corpechot, O. Chazouilleres, T. Simon, S. C. Heath, F. Matsuda, R. E. Poupon, C. Housset and V. Barbu: Genetic factors of susceptibility and of severity in primary biliary cirrhosis. J Hepatol 49, 1038-1045 (2008)
doi:10.1016/j.jhep.2008.07.027
PMid:19003916    PMCid:2592501

61. P. Invernizzi, C. Selmi, F. Poli, S. Frison, A. Floreani, D. Alvaro, P. Almasio, F. Rosina, M. Marzioni, L. Fabris, L. Muratori, L. Qi, M. F. Seldin, M. E. Gershwin and M. Podda: Human leukocyte antigen polymorphisms in Italian primary biliary cirrhosis: a multicenter study of 664 patients and 1992 healthy controls. Hepatology 48, 1906-1912 (2008)
doi:10.1002/hep.22567
PMid:19458352    PMCid:2857316

62. G. M. Hirschfield, X. Liu, C. Xu, Y. Lu, G. Xie, Y. Lu, X. Gu, E. J. Walker, K. Jing, B. D. Juran, A. L. Mason, R. P. Myers, K. M. Peltekian, C. N. Ghent, C. Coltescu, E. J. Atkinson, E. J. Heathcote, K. N. Lazaridis, C. I. Amos and K. A. Siminovitch: Primary biliary cirrhosis associated with HLA, IL12A, and IL12RB2 variants. N Engl J Med 360, 2544-2555 (2009)
doi:10.1056/NEJMoa0810440
PMid:20639880

63. X. Liu, P. Invernizzi, Y. Lu, R. Kosoy, Y. Lu, I. Bianchi, M. Podda, C. Xu, G. Xie, F. Macciardi, C. Selmi, S. Lupoli, R. Shigeta, M. Ransom, A. Lleo, A. T. Lee, A. L. Mason, R. P. Myers, K. M. Peltekian, C. N. Ghent, F. Bernuzzi, M. Zuin, F. Rosina, E. Borghesio, A. Floreani, R. Lazzari, G. Niro, A. Andriulli, L. Muratori, P. Muratori, P. L. Almasio, P. Andreone, M. Margotti, M. Brunetto, B. Coco, D. Alvaro, M. C. Bragazzi, F. Marra, A. Pisano, C. Rigamonti, M. Colombo, M. Marzioni, A. Benedetti, L. Fabris, M. Strazzabosco, P. Portincasa, V. O. Palmieri, C. Tiribelli, L. Croce, S. Bruno, S. Rossi, M. Vinci, C. Prisco, A. Mattalia, P. Toniutto, A. Picciotto, A. Galli, C. Ferrari, S. Colombo, G. Casella, L. Morini, N. Caporaso, A. Colli, G. Spinzi, R. Montanari, P. K. Gregersen, E. J. Heathcote, G. M. Hirschfield, K. A. Siminovitch, C. I. Amos, M. E. Gershwin and M. F. Seldin: Genome-wide meta-analyses identify three loci associated with primary biliary cirrhosis. Nat Genet 42, 658-660 (2010)
doi:10.1038/ng.627
PMid:20639879    PMCid:2929126

64. G. M. Hirschfield, X. Liu, Y. Han, I. P. Gorlov, Y. Lu, C. Xu, Y. Lu, W. Chen, B. D. Juran, C. Coltescu, A. L. Mason, P. Milkiewicz, R. P. Myers, J. A. Odin, V. A. Luketic, D. Speiciene, C. Vincent, C. Levy, P. K. Gregersen, J. Zhang, E. J. Heathcote, K. N. Lazaridis, C. I. Amos and K. A. Siminovitch: Variants at IRF5-TNPO3, 17q12-21 and MMEL1 are associated with primary biliary cirrhosis. Nat Genet 42, 655-657 (2010)
doi:10.1038/ng.631
PMid:18671305

65. Y. Ohishi, M. Nakamura, N. Iio, S. Higa, M. Inayoshi, Y. Aiba, A. Komori, K. Omagari, H. Ishibashi and K. Tsukamoto: Single-nucleotide polymorphism analysis of the multidrug resistance protein 3 gene for the detection of clinical progression in Japanese patients with primary biliary cirrhosis. Hepatology 48, 853-862 (2008)
doi:10.1002/hep.22382
PMid:19376604

66. A. Tanaka, S. Nezu, S. Uegaki, K. Kikuchi, A. Shibuya, H. Miyakawa, S. Takahashi, I. Bianchi, P. Zermiani, M. Podda, H. Ohira, P. Invernizzi and H. Takikawa: Vitamin D receptor polymorphisms are associated with increased susceptibility to primary biliary cirrhosis in Japanese and Italian populations. J Hepatol 50, 1202-1209 (2009)
doi:10.1016/j.jhep.2009.01.015

67. L. P. Lakatos, E. Bajnok, D. Hegedus, T. Toth, P. Lakatos and F. Szalay: Vitamin D receptor, oestrogen receptor-alpha gene and interleukin-1 receptor antagonist gene polymorphisms in Hungarian patients with primary biliary cirrhosis. Eur J Gastroenterol Hepatol 14, 733-740 (2002)
doi:10.1097/00042737-200207000-00004
PMid:17301827

68. T. H. Karlsen, K. M. Boberg, M. Vatn, A. Bergquist, J. Hampe, E. Schrumpf, E. Thorsby, S. Schreiber and B. A. Lie: Different HLA class II associations in ulcerative colitis patients with and without primary sclerosing cholangitis. Genes Immun 8, 275-278 (2007)
doi:10.1038/sj.gene.6364377
PMid:10372541

69. A. Spurkland, S. Saarinen, K. M. Boberg, S. Mitchell, U. Broome, L. Caballeria, E. Ciusani, R. Chapman, G. Ercilla, O. Fausa, I. Knutsen, A. Pares, F. Rosina, O. Olerup, E. Thorsby and E. Schrumpf: HLA class II haplotypes in primary sclerosing cholangitis patients from five European populations. Tissue Antigens 53, 459-469 (1999)
doi:10.1034/j.1399-0039.1999.530502.x
PMid:17257319

70. K. Wiencke, T. H. Karlsen, K. M. Boberg, E. Thorsby, E. Schrumpf, B. A. Lie and A. Spurkland: Primary sclerosing cholangitis is associated with extended HLA-DR3 and HLA-DR6 haplotypes. Tissue Antigens 69, 161-169 (2007)
doi:10.1111/j.1399-0039.2006.00738.x
PMid:15123348

71. X. Yang, S. N. Cullen, J. H. Li, R. W. Chapman and D. P. Jewell: Susceptibility to primary sclerosing cholangitis is associated with polymorphisms of intercellular adhesion molecule-1. J Hepatol 40, 375-379 (2004)
doi:10.1016/j.jhep.2003.11.009
PMid:17295178

72. M. Trauner, P. Fickert and M. Wagner: MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes. Semin Liver Dis 27, 77-98 (2007)
doi:10.1055/s-2006-960172
PMid:15888793    PMCid:1774530

Key Words: Cholestatic disorders, System Biology, Integrative Genetics, Gene Prioritization, Review

Send correspondence to: Silvia Sookoian, Instituto de Investigaciones Medicas A. Lanari, Av. Combatiente de Malvinas 3150, C1427ARO, Buenos Aires. Argentina, Tel: 54-11-4514 8701, Fax: 54-11-4523 8947, E-mail:ssookoian@lanari.fmed.uba.ar