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Hepatology. Author manuscript; available in PMC 2017 May 01. Published in final edited form as: Hepatology. 2016 May ; 63(5): 1421–1423. doi:10.1002/hep.28408.
MDR3 mutation analysis: A step closer to precision medicine Jorge A Bezerra Division of Gastroenterology, Hepatology and Nutrition and the Liver Care Center, Cincinnati Children’s Hospital Medical Center Department of Pediatrics, University of Cincinnati College of Medicine Jorge A Bezerra: [email protected]
Author Manuscript Author Manuscript
Among the promises brought about by the genomic revolution is the greater understanding of how genes cause human diseases. In principle, major sequence variations in genes regulating critical biochemical processes result in severe clinical manifestations. In contrast, minor variants have milder phenotypes or are linked to disease susceptibility. The promise and the principle hold true for liver genes. In fact, heritability and biochemical defects had long been proposed for disorders like Wilson’s disease and alpha-1-antitrypsin deficiency prior to their genetic discoveries. Following several years of work with standard capillary sequencing technologies and complementary assays, investigators made 1998 a referenceyear for the genetics of inherited syndromes of intrahepatic cholestasis, with the identification of disease-causing mutations in the genes JAG1 (for the Alagille disease), ATP8B1 (for the progressive familial intrahepatic cholestasis type 1 = PFIC1), ABCB11 (for PFIC2), ABCB4 (for PFIC3), and AKR1D1 (for bile acid synthetic defect due to a deficiency in the enzyme 3-oxo-5- beta(β)-steroid 4-dehydrogenase [reviewed in reference (1)]. This was the start of a new era in which clinical syndromes would be defined molecularly (2), aided in the clinical setting by the availability of high-throughput technologies that made genetic screens a reality as part of diagnostic algorithms for patients with idiopathic liver disease (https://www.genetests.org).
Author Manuscript
Often, sequencing of cholestasis-related genes yields several nucleotide variants of unknown significance, thus identifying the need for “post-genomic era” studies to precisely define the impact of these variants to clinical phenotypes. In this issue of Hepatology, the article by Delaunay et al. does just that: A post-genomic study investigating how sequence variants in the ABCB4 gene (encoding the multi-drug resistance protein type 3 = MDR3) provide insight into how mutations impact the function of the encoded protein (3). MDR3 is responsible for the canalicular transport of phosphatidylcholine, a key bile constituent that protects the bile duct epithelium from the detergent properties of bile acids and prevents precipitation of cholesterol. Defective transport linked to ABCB4/MDR3 mutations manifest as at least four syndromes: intrahepatic cholestasis (mild or severe), low phospholipid-associated cholelithiasis, fibrosing cholangiopathy, and intrahepatic cholestasis of pregnancy. Although reports suggest phenotype-genotype relationships, very few studies directly demonstrate the biological and functional impact of missense variants, the most common sequence abnormalities in patients with MDR3 deficiency. These variants are found along the entire
Bezerra
Page 2
Author Manuscript
gene length, and laboratories use algorithms to classify variants into mutation or benign polymorphisms, taking into account typical mutation-causing changes (nonsense and frame shifting deletions and insertions), the likelihood that nucleotide changes (missense variants) may impair the function of encoded proteins, the conservation of nucleotide sequences across species, the prevalence of variants in the normal population, etc. While valid for many variants, the accuracy of prediction algorithms would be enhanced by the direct testing of biological activity/function of the variants in complementary biological assays.
Author Manuscript Author Manuscript
In the study by Delaunay et al., the investigators used complementary assays to evaluate the biological consequences of 12 variants in ABCB4 in 9 children with the progressive form of intrahepatic cholestasis, explored pharmacological approaches to restore/improve the function of mutant proteins, and proposed a functional classification of gene variants (3). Using site-directed mutagenesis, they expressed mutant MDR3 proteins in cell lines, assessed their pattern/localization of expression, and subjected them to biological assays. Functionally, all but 4 mutants produced abnormal secretion of phosphatidylcholine. Among these 4 mutants, T424A and N510S had normal pattern of canalicular expression but reduced protein stability, while T175A and R652G had no detectable defect (and were classified as polymorphisms). Notably, mutations that resulted in defective maturation of the protein (I541F, L556R, and Q855L), as evidenced by the retention in the endoplasmic reticulum, had improved canalicular localization in the presence of cyclosporins. Using the available information, the authors classified variants into Classes I–V according to severity (nonsense and frame shifting deletions for Class I), impact on maturation, activity or stability of the protein (Classes II, III and IV, respectively), or no detectable defect (Class V). Interestingly, the T175A mutant placed in Class V was initially proposed to be “deleterious” to the encoded protein based on predictive algorithms. In agreement with the authors’ choice to place this variant into Class V, the presence of T175A did not change the canalicular expression of the protein and allowed for adequate phosphatidylcholine secretion.
Author Manuscript
Despite the scientific merits and potential clinical relevance of these studies, the experimental and classification strategies would have been strengthened by the inclusion of more variants and broader/combinatorial pharmacological screen. Further, the number of subjects in the study was relatively small, thus limiting the analysis of the relationship between genotype, biological function and clinical phenotype. Without the inclusion of a greater number of patients, the authors’ claim that “the most severe phenotypes appreciated by the duration of transplant-free survival were caused by ABCB4 variants that were markedly retained in the endosplasmic reticulum…” may not be easily defended because children carrying mutants that had apparently proper canalicular expression (S346I, G954S, and T424A) also required liver transplantation. Last, the experimental approach did not include an analysis of biologically significant mutants in the heterozygous state, a particularly intriguing scenario based on the previous report of ABCB4 heterozygosity in adults with idiopathic cholestasis and liver fibrosis (4). These limitations do not minimize the contribution of the report by Delaunay et al. to the field, which join other proof-of-principle studies directly examining the biological and clinical relevance of variants in ABCB4 and other genes related to inherited cholestasis (5– Hepatology. Author manuscript; available in PMC 2017 May 01.
Bezerra
Page 3
Author Manuscript Author Manuscript
7). While individually sporadic, these syndromes are important diagnostic considerations when evaluating children and adults with idiopathic liver disease. Among them, the Alagille syndrome results from mutations in JAG1 or NOTCH2, genes implicated in the regulation of cell fate determination (8). Mutations in other genes encoding canalicular transporters, such as ATP8B1 (encoding the familial intrahepatic cholestasis-1 = FIC1 protein; responsible for PFIC1) and ABCB11 (the bile salt export protein = BSEP; for PFIC2) cause chronic cholestasis. In 2014, mutations in the TJP2 gene identified a new group of children with chronic cholestasis, with a proposed mechanism of cholestasis linked to a defective interhepatocyte junction produced by the abnormal localization of CLAUDIN1 caused by the mutant TJP2 (9). Like for ABCB4/MDR3, sequence variants for these genes are scattered along exons, intron-exon boundaries, and regulatory regions. Thus, a greater insight into the relevance of these variants will benefit from the type of investigation reported by Delaunay et al. The expression of MDR3 mutants (or mutants for JAG1, FIC1, BSEP and TJP2) in cells that allow for easier quantification of function, perhaps in a high-throughput assay, will enable the rapid screen of pharmacologic agents to identify new drugs that restore cellular function by targeting the mutant protein. The ability to carry these types of experiments and validate the findings in clinical trials will greatly increase the usefulness of mutation screening as part of diagnostic algorithms for patients with chronic cholestasis. It is then that “precision hepatology” will become a reality by enabling the selection of treatments that meet the biological requirements dictated by the patient’s genetic makeup.
References
Author Manuscript Author Manuscript
1. Balistreri WF, Bezerra JA. Whatever happened to “neonatal hepatitis”? Clin Liver Dis. 2006; 10:27– 53. v. [PubMed: 16376793] 2. Bezerra JA, Balistreri WF. Intrahepatic cholestasis: order out of chaos. Gastroenterology. 1999; 117:1496–1498. [PubMed: 10579993] 3. Delaunay JL, Durand-Schneider AM, Dossier C, Falguieres T, Gautherot J, Anne DS, Ait-Slimane T, et al. A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3. Hepatology. 2015 4. Ziol M, Barbu V, Rosmorduc O, Frassati-Biaggi A, Barget N, Hermelin B, Scheffer GL, et al. ABCB4 heterozygous gene mutations associated with fibrosing cholestatic liver disease in adults. Gastroenterology. 2008; 135:131–141. [PubMed: 18482588] 5. Byrne JA, Strautnieks SS, Ihrke G, Pagani F, Knisely AS, Linton KJ, Mieli-Vergani G, et al. Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing. Hepatology. 2009; 49:553–567. [PubMed: 19101985] 6. Gordo-Gilart R, Andueza S, Hierro L, Martinez-Fernandez P, D’Agostino D, Jara P, Alvarez L. Functional analysis of ABCB4 mutations relates clinical outcomes of progressive familial intrahepatic cholestasis type 3 to the degree of MDR3 floppase activity. Gut. 2015; 64:147–155. [PubMed: 24594635] 7. Wang L, Dong H, Soroka CJ, Wei N, Boyer JL, Hochstrasser M. Degradation of the bile salt export pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II. Hepatology. 2008; 48:1558–1569. [PubMed: 18798335] 8. McDaniell R, Warthen DM, Sanchez-Lara PA, Pai A, Krantz ID, Piccoli DA, Spinner NB. NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway. Am J Hum Genet. 2006; 79:169–173. [PubMed: 16773578] 9. Sambrotta M, Strautnieks S, Papouli E, Rushton P, Clark BE, Parry DA, Logan CV, et al. Mutations in TJP2 cause progressive cholestatic liver disease. Nat Genet. 2014; 46:326–328. [PubMed: 24614073]
Hepatology. Author manuscript; available in PMC 2017 May 01.
Hepatology. Author manuscript; available in PMC 2017 May 01. Published in final edited form as: Hepatology. 2016 May ; 63(5): 1421–1423. doi:10.1002/hep.28408.
MDR3 mutation analysis: A step closer to precision medicine Jorge A Bezerra Division of Gastroenterology, Hepatology and Nutrition and the Liver Care Center, Cincinnati Children’s Hospital Medical Center Department of Pediatrics, University of Cincinnati College of Medicine Jorge A Bezerra: [email protected]
Author Manuscript Author Manuscript
Among the promises brought about by the genomic revolution is the greater understanding of how genes cause human diseases. In principle, major sequence variations in genes regulating critical biochemical processes result in severe clinical manifestations. In contrast, minor variants have milder phenotypes or are linked to disease susceptibility. The promise and the principle hold true for liver genes. In fact, heritability and biochemical defects had long been proposed for disorders like Wilson’s disease and alpha-1-antitrypsin deficiency prior to their genetic discoveries. Following several years of work with standard capillary sequencing technologies and complementary assays, investigators made 1998 a referenceyear for the genetics of inherited syndromes of intrahepatic cholestasis, with the identification of disease-causing mutations in the genes JAG1 (for the Alagille disease), ATP8B1 (for the progressive familial intrahepatic cholestasis type 1 = PFIC1), ABCB11 (for PFIC2), ABCB4 (for PFIC3), and AKR1D1 (for bile acid synthetic defect due to a deficiency in the enzyme 3-oxo-5- beta(β)-steroid 4-dehydrogenase [reviewed in reference (1)]. This was the start of a new era in which clinical syndromes would be defined molecularly (2), aided in the clinical setting by the availability of high-throughput technologies that made genetic screens a reality as part of diagnostic algorithms for patients with idiopathic liver disease (https://www.genetests.org).
Author Manuscript
Often, sequencing of cholestasis-related genes yields several nucleotide variants of unknown significance, thus identifying the need for “post-genomic era” studies to precisely define the impact of these variants to clinical phenotypes. In this issue of Hepatology, the article by Delaunay et al. does just that: A post-genomic study investigating how sequence variants in the ABCB4 gene (encoding the multi-drug resistance protein type 3 = MDR3) provide insight into how mutations impact the function of the encoded protein (3). MDR3 is responsible for the canalicular transport of phosphatidylcholine, a key bile constituent that protects the bile duct epithelium from the detergent properties of bile acids and prevents precipitation of cholesterol. Defective transport linked to ABCB4/MDR3 mutations manifest as at least four syndromes: intrahepatic cholestasis (mild or severe), low phospholipid-associated cholelithiasis, fibrosing cholangiopathy, and intrahepatic cholestasis of pregnancy. Although reports suggest phenotype-genotype relationships, very few studies directly demonstrate the biological and functional impact of missense variants, the most common sequence abnormalities in patients with MDR3 deficiency. These variants are found along the entire
Bezerra
Page 2
Author Manuscript
gene length, and laboratories use algorithms to classify variants into mutation or benign polymorphisms, taking into account typical mutation-causing changes (nonsense and frame shifting deletions and insertions), the likelihood that nucleotide changes (missense variants) may impair the function of encoded proteins, the conservation of nucleotide sequences across species, the prevalence of variants in the normal population, etc. While valid for many variants, the accuracy of prediction algorithms would be enhanced by the direct testing of biological activity/function of the variants in complementary biological assays.
Author Manuscript Author Manuscript
In the study by Delaunay et al., the investigators used complementary assays to evaluate the biological consequences of 12 variants in ABCB4 in 9 children with the progressive form of intrahepatic cholestasis, explored pharmacological approaches to restore/improve the function of mutant proteins, and proposed a functional classification of gene variants (3). Using site-directed mutagenesis, they expressed mutant MDR3 proteins in cell lines, assessed their pattern/localization of expression, and subjected them to biological assays. Functionally, all but 4 mutants produced abnormal secretion of phosphatidylcholine. Among these 4 mutants, T424A and N510S had normal pattern of canalicular expression but reduced protein stability, while T175A and R652G had no detectable defect (and were classified as polymorphisms). Notably, mutations that resulted in defective maturation of the protein (I541F, L556R, and Q855L), as evidenced by the retention in the endoplasmic reticulum, had improved canalicular localization in the presence of cyclosporins. Using the available information, the authors classified variants into Classes I–V according to severity (nonsense and frame shifting deletions for Class I), impact on maturation, activity or stability of the protein (Classes II, III and IV, respectively), or no detectable defect (Class V). Interestingly, the T175A mutant placed in Class V was initially proposed to be “deleterious” to the encoded protein based on predictive algorithms. In agreement with the authors’ choice to place this variant into Class V, the presence of T175A did not change the canalicular expression of the protein and allowed for adequate phosphatidylcholine secretion.
Author Manuscript
Despite the scientific merits and potential clinical relevance of these studies, the experimental and classification strategies would have been strengthened by the inclusion of more variants and broader/combinatorial pharmacological screen. Further, the number of subjects in the study was relatively small, thus limiting the analysis of the relationship between genotype, biological function and clinical phenotype. Without the inclusion of a greater number of patients, the authors’ claim that “the most severe phenotypes appreciated by the duration of transplant-free survival were caused by ABCB4 variants that were markedly retained in the endosplasmic reticulum…” may not be easily defended because children carrying mutants that had apparently proper canalicular expression (S346I, G954S, and T424A) also required liver transplantation. Last, the experimental approach did not include an analysis of biologically significant mutants in the heterozygous state, a particularly intriguing scenario based on the previous report of ABCB4 heterozygosity in adults with idiopathic cholestasis and liver fibrosis (4). These limitations do not minimize the contribution of the report by Delaunay et al. to the field, which join other proof-of-principle studies directly examining the biological and clinical relevance of variants in ABCB4 and other genes related to inherited cholestasis (5– Hepatology. Author manuscript; available in PMC 2017 May 01.
Bezerra
Page 3
Author Manuscript Author Manuscript
7). While individually sporadic, these syndromes are important diagnostic considerations when evaluating children and adults with idiopathic liver disease. Among them, the Alagille syndrome results from mutations in JAG1 or NOTCH2, genes implicated in the regulation of cell fate determination (8). Mutations in other genes encoding canalicular transporters, such as ATP8B1 (encoding the familial intrahepatic cholestasis-1 = FIC1 protein; responsible for PFIC1) and ABCB11 (the bile salt export protein = BSEP; for PFIC2) cause chronic cholestasis. In 2014, mutations in the TJP2 gene identified a new group of children with chronic cholestasis, with a proposed mechanism of cholestasis linked to a defective interhepatocyte junction produced by the abnormal localization of CLAUDIN1 caused by the mutant TJP2 (9). Like for ABCB4/MDR3, sequence variants for these genes are scattered along exons, intron-exon boundaries, and regulatory regions. Thus, a greater insight into the relevance of these variants will benefit from the type of investigation reported by Delaunay et al. The expression of MDR3 mutants (or mutants for JAG1, FIC1, BSEP and TJP2) in cells that allow for easier quantification of function, perhaps in a high-throughput assay, will enable the rapid screen of pharmacologic agents to identify new drugs that restore cellular function by targeting the mutant protein. The ability to carry these types of experiments and validate the findings in clinical trials will greatly increase the usefulness of mutation screening as part of diagnostic algorithms for patients with chronic cholestasis. It is then that “precision hepatology” will become a reality by enabling the selection of treatments that meet the biological requirements dictated by the patient’s genetic makeup.
References
Author Manuscript Author Manuscript
1. Balistreri WF, Bezerra JA. Whatever happened to “neonatal hepatitis”? Clin Liver Dis. 2006; 10:27– 53. v. [PubMed: 16376793] 2. Bezerra JA, Balistreri WF. Intrahepatic cholestasis: order out of chaos. Gastroenterology. 1999; 117:1496–1498. [PubMed: 10579993] 3. Delaunay JL, Durand-Schneider AM, Dossier C, Falguieres T, Gautherot J, Anne DS, Ait-Slimane T, et al. A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3. Hepatology. 2015 4. Ziol M, Barbu V, Rosmorduc O, Frassati-Biaggi A, Barget N, Hermelin B, Scheffer GL, et al. ABCB4 heterozygous gene mutations associated with fibrosing cholestatic liver disease in adults. Gastroenterology. 2008; 135:131–141. [PubMed: 18482588] 5. Byrne JA, Strautnieks SS, Ihrke G, Pagani F, Knisely AS, Linton KJ, Mieli-Vergani G, et al. Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing. Hepatology. 2009; 49:553–567. [PubMed: 19101985] 6. Gordo-Gilart R, Andueza S, Hierro L, Martinez-Fernandez P, D’Agostino D, Jara P, Alvarez L. Functional analysis of ABCB4 mutations relates clinical outcomes of progressive familial intrahepatic cholestasis type 3 to the degree of MDR3 floppase activity. Gut. 2015; 64:147–155. [PubMed: 24594635] 7. Wang L, Dong H, Soroka CJ, Wei N, Boyer JL, Hochstrasser M. Degradation of the bile salt export pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II. Hepatology. 2008; 48:1558–1569. [PubMed: 18798335] 8. McDaniell R, Warthen DM, Sanchez-Lara PA, Pai A, Krantz ID, Piccoli DA, Spinner NB. NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway. Am J Hum Genet. 2006; 79:169–173. [PubMed: 16773578] 9. Sambrotta M, Strautnieks S, Papouli E, Rushton P, Clark BE, Parry DA, Logan CV, et al. Mutations in TJP2 cause progressive cholestatic liver disease. Nat Genet. 2014; 46:326–328. [PubMed: 24614073]
Hepatology. Author manuscript; available in PMC 2017 May 01.
