MYO5B mutations cause cholestasis with normal serum gamma‐glutamyl transferase activity in children without microvillous inclusion disease

HepatologyVolume 65, Issue 1 p. 164-173 Autoimmune, Cholestatic and Biliary DiseaseFree Access MYO5B mutations cause cholestasis with normal serum gamma-glutamyl transferase activity in children without microvillous inclusion disease Emmanuel Gonzales, Emmanuel Gonzales Pediatric Hepatology and Pediatric Liver Transplantation Unit and National Reference Centre for Rare Pediatric Liver Diseases, Bicêtre University Hospital, University of Paris-Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, France INSERM, UMR-S1174, Hepatinov, University of Paris-Sud, Orsay, FranceSearch for more papers by this authorSarah A. Taylor, Sarah A. Taylor Department of Pediatrics, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, ILSearch for more papers by this authorAnne Davit-Spraul, Anne Davit-Spraul Department of Biochemistry, Bicêtre University Hospital, University of Paris-Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, FranceSearch for more papers by this authorAlice Thébaut, Alice Thébaut Pediatric Hepatology and Pediatric Liver Transplantation Unit and National Reference Centre for Rare Pediatric Liver Diseases, Bicêtre University Hospital, University of Paris-Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, France INSERM, UMR-S1174, Hepatinov, University of Paris-Sud, Orsay, FranceSearch for more papers by this authorNadège Thomassin, Nadège Thomassin Department of Pediatrics, Grenoble University Hospital, Grenoble, FranceSearch for more papers by this authorCatherine Guettier, Catherine Guettier Department of Pathology, Bicêtre University Hospital, University of Paris-Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, FranceSearch for more papers by this authorPeter F. Whitington, Peter F. Whitington Department of Pediatrics, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, ILSearch for more papers by this authorEmmanuel Jacquemin, Corresponding Author Emmanuel Jacquemin [email protected] Pediatric Hepatology and Pediatric Liver Transplantation Unit and National Reference Centre for Rare Pediatric Liver Diseases, Bicêtre University Hospital, University of Paris-Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, France INSERM, UMR-S1174, Hepatinov, University of Paris-Sud, Orsay, France ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Emmanuel Jacquemin, M.D., Ph.D. Service d'Hépatologie et de transplantation hépatique pédiatriques Centre Hospitalier Universitaire de Bicêtre 78 rue du Général Leclerc 94275 Le Kremlin-Bicêtre Cedex, France E-mail: [email protected] Tel: +33-1-45-21-31-68Search for more papers by this author Emmanuel Gonzales, Emmanuel Gonzales Pediatric Hepatology and Pediatric Liver Transplantation Unit and National Reference Centre for Rare Pediatric Liver Diseases, Bicêtre University Hospital, University of Paris-Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, France INSERM, UMR-S1174, Hepatinov, University of Paris-Sud, Orsay, FranceSearch for more papers by this authorSarah A. Taylor, Sarah A. Taylor Department of Pediatrics, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, ILSearch for more papers by this authorAnne Davit-Spraul, Anne Davit-Spraul Department of Biochemistry, Bicêtre University Hospital, University of Paris-Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, FranceSearch for more papers by this authorAlice Thébaut, Alice Thébaut Pediatric Hepatology and Pediatric Liver Transplantation Unit and National Reference Centre for Rare Pediatric Liver Diseases, Bicêtre University Hospital, University of Paris-Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, France INSERM, UMR-S1174, Hepatinov, University of Paris-Sud, Orsay, FranceSearch for more papers by this authorNadège Thomassin, Nadège Thomassin Department of Pediatrics, Grenoble University Hospital, Grenoble, FranceSearch for more papers by this authorCatherine Guettier, Catherine Guettier Department of Pathology, Bicêtre University Hospital, University of Paris-Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, FranceSearch for more papers by this authorPeter F. Whitington, Peter F. Whitington Department of Pediatrics, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, ILSearch for more papers by this authorEmmanuel Jacquemin, Corresponding Author Emmanuel Jacquemin [email protected] Pediatric Hepatology and Pediatric Liver Transplantation Unit and National Reference Centre for Rare Pediatric Liver Diseases, Bicêtre University Hospital, University of Paris-Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, France INSERM, UMR-S1174, Hepatinov, University of Paris-Sud, Orsay, France ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Emmanuel Jacquemin, M.D., Ph.D. Service d'Hépatologie et de transplantation hépatique pédiatriques Centre Hospitalier Universitaire de Bicêtre 78 rue du Général Leclerc 94275 Le Kremlin-Bicêtre Cedex, France E-mail: [email protected] Tel: +33-1-45-21-31-68Search for more papers by this author First published: 17 August 2016 https://doi.org/10.1002/hep.28779Citations: 94 Potential conflict of interest: Nothing to report. Supported in part by Association Maladies Foie Enfants (Malakoff, France), Monaco Liver Disorder (Monaco), and Foundation Rumsey-Cartier (Geneva, Switzerland). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Some patients with microvillus inclusion disease due to myosin 5B (MYO5B) mutations may develop cholestasis characterized by a progressive familial intrahepatic cholestasis-like phenotype with normal serum gamma-glutamyl transferase activity. So far MYO5B deficiency has not been reported in patients with such a cholestasis phenotype in the absence of intestinal disease. Using a new-generation sequencing approach, we identified MYO5B mutations in five patients with progressive familial intrahepatic cholestasis-like phenotype with normal serum gamma-glutamyl transferase activity without intestinal disease. Conclusion: These data show that MYO5B deficiency may lead to isolated cholestasis and that MYO5B should be considered as an additional progressive familial intrahepatic cholestasis gene. (Hepatology 2017;65:164-173). Abbreviations ARC arthrogryposis, renal dysfunction, and cholestasis (syndrome) BSEP bile salt export pump GGT gamma-glutamyltransferase MDR3 multidrug resistance protein 3 MVID microvillous inclusion disease MYO5B myosin 5B PFIC progressive familial intrahepatic cholestasis TJP2 tight junction protein-2 UDCA ursodeoxycholic acid ULN upper limit of normal Myosin 5B gene (MYO5B) mutations cause microvillus inclusion disease (MVID), a congenital disorder of the enterocyte leading to intractable diarrhea.1-4 MYO5B interacts with rab11, and this interaction is essential for proper functioning of polarized epithelial cells, including hepatocytes.5-9 Progressive familial intrahepatic cholestasis (PFIC) comprises several genetically determined disorders of bile formation, all of which are characterized by chronic cholestasis. PFIC can be grouped according to the serum gamma-glutamyl transferase (GGT) activity.10 Normal or relatively low (i.e., <100 IU/L) levels of GGT despite severe cholestasis characterize normal/low-GGT PFIC. To date, mutations in three genes have been described to cause normal/low-GGT PFIC: ATP8B1, encoding the aminophospholipid flippase FIC-111, 12; ABCB11, encoding bile salt export pump (BSEP)13, 14; and TJP2, encoding tight junction protein-2.15 It has been noted that some patients with MVID develop cholestasis that appears to be unrelated to bowel disease and/or therapy such as total parenteral nutrition required for their support.16, 17 In these patients it has been proposed that cholestasis resulted from an impairment of MYO5B/RAB11A interaction, altering the targeting of the BSEP to the canalicular membrane of hepatocytes.8, 16 These patients were noted to have normal serum GGT activity. The MYO5B genotypes were not detailed.16 To date, MYO5B deficiency has not been reported in patients with a normal-GGT PFIC phenotype in the absence of clinical bowel disease. Using a new-generation sequencing approach, we identified MYO5B mutations in five patients with normal-GGT PFIC phenotype in whom other causative gene mutations had not been found. These data show that MYO5B deficiency may lead to isolated cholestasis and that MYO5B should be considered as an additional PFIC gene. Patients and Methods PATIENT 1 This Caucasian girl was born healthy at 41 weeks of gestation to unrelated parents with a birth weight of 3,080 g. Familial medical history was negative for chronic bowel or liver disease. Antenatal ultrasounds were normal. She presented at the age of 14 months with jaundice, severe pruritus, discolored stools, and moderate hepatomegaly. Abdominal ultrasound confirmed an enlarged liver and showed a normal biliary tree. Serum clinical laboratory tests showed cholestasis (Table 1) with normal GGT activity, normal transaminase levels, and normal prothrombin time. No liver biopsy was performed. Ursodeoxycholic acid (UDCA) therapy was started at a dose of 600 mg/m2 body surface area/day together with rifampicin at a dose of 12.5 mg/kg body weight/day. At 18 months of age serum bilirubin levels were normal, but pruritus, hepatomegaly, and increased levels of serum bile acids (228 μmol/L) persisted. Thereafter and until the age of 3.5 years this girl experienced recurrent bouts of cholestasis while on UDCA therapy, with fluctuations of the severity of pruritus, serum transaminase levels (2 × upper limit of normal [ULN]), and bile acid levels (163 μmol/L) and persistence of a slight hepatomegaly. Serum albumin and cholesterol levels normalized. Besides cholestasis, the child developed neurological symptomatology of unknown cause characterized by language development delay and a pyramidal syndrome. Serum vitamin E levels were normal. She experienced no significant persistent intestinal symptomatology, and height and weight were normal for age. Table 1. Blood Findings at First Evaluation in 5 Children With MYO5B Mutations and Cholestasis With Normal Serum GGT Activity Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Age at presentation 14 months 12 months 7 months 10 months 15 months Total/conjugated bilirubin (<17 μmol/L) 89/78 158/93 79/53 70/ND 46/31 Bile salts (<15 μmol/L) 286 380 253 256 NDa GGT (<45 IU/L) 7 10 11 9 10 Alanine aminotransferase (<35 IU/L) 31 170 136 71 55 Aspartate aminotransferase (<40 IU/L) 57 202 207 64 51 Alpha-fetoprotein (<7 IU/mL) 5 2 ND 1 ND Prothrombin timeb (>70%) 100 100 100 100 100 Albumin (>35 g/L) 30 40 32 ND 45 Total cholesterol (3.2-5.5 mmol/L) 6.7 4.3 3.5 3.4 ND Normal values are given in parentheses. a Urine bile acid analysis (performed by fast atom bombardment mass spectrometry) demonstrated large quantities of normal primary bile acids indicative of severe cholestasis and not indicative of a bile acid synthetic defect. b Values after intravenous injection of vitamin K. Abbreviation: ND, not determined. PATIENT 2 This boy was an abandoned/adopted child of Cambodian origin. Family history was unknown. The adoptive parents reported that he developed pruritus at 2 months of age, and at 12 months he presented with jaundice, severe pruritus, discolored stools, and firm hepatomegaly. Abdominal ultrasound confirmed an enlarged liver and showed a normal biliary tree. Serum clinical laboratory tests showed cholestasis (Table 1) with normal GGT activity, modestly elevated transaminase levels, and normal prothrombin time. Liver histology showed lobular and portal fibrosis, numerous multinucleate giant hepatocytes, hepatocellular and canalicular cholestasis, and absence of bile duct proliferation (Fig. 1). UDCA therapy was started at a dose of 600 mg/m2 body surface area/day together with rifampicin at a dose of 10 mg/kg body weight/day. At 16 months of age, serum bilirubin levels were normal, and pruritus, hepatomegaly, serum transaminase levels (2-3 × ULN), and bile acid levels (98 μmol/L) had improved. Thereafter and until the age of 13.5 years this child experienced recurrent bouts of cholestasis while on UDCA therapy, with fluctuations of the severity of pruritus, serum bilirubin (5 × ULN), transaminase levels (2-3 × ULN), and bile acid levels (11-463 μmol/L) and persistence of moderate hepatomegaly. He experienced no significant persistent intestinal symptomatology, and height and weight were normal for age. Figure 1Open in figure viewerPowerPoint Liver histology and BSEP and MDR3 immunostaining studies in two children (patients 2 [D,E] and 3 [A,F,G]) with MYO5B mutations and cholestasis with normal serum GGT activity. (A) Presence of giant hepatocytes (arrowhead) and of canalicular cholestasis (arrow). Hematoxylin and eosin, ×200. (B) Normal pattern of canalicular expression. Normal human liver, BSEP immunostaining, ×400. (C) Normal pattern of canalicular expression. Normal human liver, MDR3 immunostaining, ×400. (D,F) Presence of an abnormal pattern of expression, with thickened canalicular staining and a granular and patchy pattern in the subcanalicular area. BSEP immunostaining, ×400. (E,G) Presence of an abnormal pattern of expression, with thickened canalicular staining and a granular and patchy pattern in the subcanalicular area. MDR3 immunostaining, ×400. PATIENT 3 This Caucasian boy was born healthy at 39 weeks of gestation to unrelated parents with a birth weight of 3,220 g. Familial medical history was negative for chronic bowel or liver disease. Antenatal ultrasounds were normal. At the age of 7 months, he presented with jaundice, severe pruritus, discolored stools, and moderate hepatomegaly. Abdominal ultrasonography confirmed an enlarged liver and showed a normal biliary tree. Serum clinical laboratory tests showed cholestasis (Table 1) with normal GGT activity, moderately elevated transaminase levels, and normal prothrombin time. UDCA therapy was started at a dose of 600 mg/m2 body surface area/day together with rifampicin at a dose of 19 mg/kg body weight/day. At 20 months of age serum bilirubin levels were normal, and serum transaminase levels (1.5 × ULN) and bile acid levels (182 μmol/L) had improved; but pruritus and hepatomegaly persisted, and splenomegaly developed. Liver histology showed lobular and portal fibrosis, some multinucleate giant hepatocytes, hepatocellular and canalicular cholestasis, and absence of bile duct proliferation. Thereafter and until the age of 4.5 years this boy experienced recurrent bouts of cholestasis while on UDCA therapy, with severe pruritus and fluctuation of serum bilirubin (4 × ULN), transaminase levels (2-3 × ULN), and bile acid levels (50-282 μmol/L) and persistence of hepatomegaly and splenomegaly. The child experienced some bouts of severe acute diarrhea before the age of 3 years. Duodenal histology was normal (data not shown), and this intestinal symptomatology disappeared and did not recur thereafter. Weight was normal for age, but the child had short stature (<−2 standard deviations). PATIENT 4 A North African boy, Moroccan in origin, was born healthy at term to unrelated parents. The family history was negative for any chronic liver or bowel disease. He presented at the age of 10 months with jaundice, discolored stools, pruritus, and moderate hepatomegaly. Abdominal ultrasonography confirmed an enlarged liver and showed a normal biliary tree. Serum clinical laboratory tests showed cholestasis (Table 1) with normal GGT activity, mildly elevated transaminase levels, and normal prothrombin time. No liver biopsy was performed. UDCA therapy was started at a dose of 600 mg/m2 body surface area/day together with rifampicin at a dose of 10 mg/kg body weight/day. Serum bilirubin, transaminase levels, and bile acid levels normalized and pruritus disappeared. UDCA therapy was stopped at age 3.5 years. Pruritus recurred, and UDCA therapy was started again. Thereafter and until the age of 5 years this boy experienced recurrent bouts of cholestasis while receiving UDCA therapy, with fluctuations of the severity of pruritus and of serum bile acid levels (279-415 μmol/L) and moderate hepatomegaly. He experienced no significant persistent intestinal symptomatology, and height and weight were normal for age. PATIENT 5 This Caucasian boy was born healthy at term to unrelated parents. The family history was negative for any chronic liver or bowel disease. He presented at 15 months of age with pruritus and acholic stools, at which time liver and abdominal ultrasonography showed a normal liver and biliary tree. Serum clinical laboratory tests showed normal GGT activity, mildly elevated transaminase and bilirubin levels, and normal prothrombin time (Table 1). Urine bile acid analysis (performed by fast atom bombardment mass spectrometry) demonstrated large quantities of normal primary bile acids indicative of severe cholestasis and not indicative of a bile acid synthetic defect. Liver histology showed features of cholestasis, mild architectural changes, giant cell formation, normal portal bile ducts with periportal ductular metaplasia of hepatocytes, and no significant fibrosis. UDCA was started at a dose of 20 mg/kg body weight/day together with hydroxyzine, but pruritus persisted with poor weight gain. At 18 months of age, the patient underwent surgical partial external biliary diversion.18, 19 Within 1 month after surgery, pruritus markedly improved. UDCA was discontinued, he was maintained on fat-soluble vitamin supplementation, and weight growth improved. At age 3 years, the serum bile acid level, checked for the first time, was 4.5 μmol/L (see patient 5 in Jericho et al.20). Immunohistochemistry performed on a prior liver biopsy demonstrated canalicular expression of BSEP with a patchy and granular subcanalicular staining being noted20 (data not shown). He has remained free of cholestatic symptoms up to the present (age of 7 years). The patient had no intestinal symptomatology unless some bouts of constipation. GENETIC AND IMMUNOHISTOCHEMICAL STUDIES In all patients, ATP8B1 and ABCB11, the main genes involved in normal-GGT PFIC (PFIC1 and 2) were sequenced as described.21 Because no deleterious mutations were identified in these genes, DNA of patients was further studied by sequencing of a panel of genes involved in genetic cholestasis by new-generation sequencing (Illumina MISEQ) (Supporting Table S1) or by whole-exome analysis (Illumina Hiseq2500 system). MYO5B mutations identified were confirmed by Sanger sequencing, as described.1 In silico analysis (Sift, Mutation Taster, Polyphen2) of identified mutants was performed. Written informed consent was obtained from each family, according to the guidelines of each local ethical committee. Liver immunostaining studies were performed in patients 2 and 3, as described, and analyzed the hepatocellular distribution of the following proteins: BSEP, multidrug resistance protein 3 (MDR3), RAB11A, and MYO5B (Figs. 1 and 2).16, 21 Figure 2Open in figure viewerPowerPoint MYO5B and RAB11A immunostaining studies in one child (Patient 3) with MYO5B mutations and cholestasis with normal serum GGT activity. (A) MYO5B staining was localized at the cell membrane without a specific domain of localization (arrows) and more faintly in the cytoplasm of hepatocytes. Normal human liver, MYO5B immunostaining, ×400. (B) Normal pattern of canalicular expression (arrow). Normal human liver, RAB11A immunostaining, ×400. (C) MYO5B staining was abnormal and showed an intense and granular pattern in the cytoplasm of hepatocytes (arrow) but not at the plasma membrane level. MYO5B immunostaining, ×400. (D) RAB11A staining was abnormal with a loss of or weak canalicular expression (arrow) and a granular cytoplasmic pattern (star). RAB11A immunostaining, ×400. (E,F) Absence of MYO5B or RAB11A staining. Negative control for MYO5B or RAB11A immunostaining, ×200. Results IDENTIFICATION OF MYO5B MUTATIONS Sequencing of a panel of genes involved in genetic cholestasis (including RAB11A) in patients 1-4 by new-generation sequencing (Illumina MISEQ) and in patient 5 by whole-exome analysis (Illumina Hiseq2500 system) revealed new mutations in the MYO5B gene (Table 2). Patients 1, 2, 4, and 5 were found to be compound heterozygous for MYO5B gene mutations, whereas patient 3 was homozygous for a single mutation. The parents of patients 1, 3, and 5 were shown to each carry a single copy of one mutation present in the child. The mother of patient 4 carries a splice mutation and a missense mutation and the father, a missense mutation. Parental allelic distribution of mutations was not studied in patient 2 because parental DNA was not available (abandoned/adopted child). No mutations in other genes known to lead to normal-GGT PFIC (i.e., ATP8B1, ABCB11, HSD3B7, AKR1D1, BAAT, BACL, VPS33B, VIPAS39, TJP2, and NR1H4) were found.12, 13, 15, 22-25 The MYO5B gene mutations were confirmed by Sanger sequencing. None of the mutations are reported in the single-nucleotide polymorphism database and exome variant server, nor have they been reported in the literature in patients with isolated MVID or MVID with cholestasis. In silico analysis (Sift, Mutation Taster, Polyphen2) predicted a deleterious effect of the mutants (Table 2). Table 2. MYO5B Mutations in 5 Children With Cholestasis and Normal Serum GGT Activity Exon/Intron Location MYO5B Mutations Homozygous/heterozygous Myosin 5b Domain Predicted Effect on RNA and/or Protein Patient 1 Exon 3 c.274C>T (p.Arg92Cys) Compound heterozygous Head, motor domain Nonconservative substitution, highly evolutionarily conserved S = 0, MT = DC, PP2 = 1 Exon 19 c.2395C>T (p.Arg799Trp) IQ domains, EF-hand binding site Nonconservative substitution, evolutionarily conserved S = 0, MT = DC, PP2 = 1 Patient 2 Exon 12 c.1499T>C (p.Ile500Thr) Heterozygous mutationsa Head, motor domain Nonconservative substitution, highly evolutionarily conserved S = 0, MT = DC, PP2 = 0.999 Exon 16 c.1925T>C (p.Leu642Pro) Head, motor domain Nonconservative substitution, evolutionarily conserved S = 0, MT = DC, PP2 = 0.251 Patient 3 Exon 4 c.356A>G (p.Tyr119Cys) Homozygous Head, motor domain Nonconservative substitution, highly evolutionarily conserved S = 0, MT = DC, PP2 = 1 Patient 4 Exon 10 c.1135C>T (p.Arg379Cys) Compound heterozygousb Head, motor domain Nonconservative substitution, highly evolutionarily conserved S = 0, MT = DC, PP2 = 1 Intron 15 c.1906-2A>G (splicing, Phe636Leufs*2) Head, motor domain Exon 16 skipping (98-bp deletion), prematurely terminated protein, RNA decay Exon 20 c.2470C>T (p.Arg824Cys) IQ domains, EF-hand binding site Nonconservative substitution, highly evolutionarily conserved S = 0, MT = DC, PP2 = 1 Patient 5 Intron 14 c.1753-1G>T (splicing) Compound Head-like Exon 15 skipping, in-frame 153-bp/51-amino acid deletion Exon 19 c.2395C>T (p.Arg799Trp) heterozygous IQ domains, EF-hand binding site Nonconservative substitution, evolutionarily conserved S = 0, MT = DC, PP2 = 1 Prediction effects and protein data are deduced from Sift, Mutation Taster, Polyphen2 programs and from GenBank RefSeq-file accession number NG_012925.1 for the human MYO5B gene.3, 4 The nonsynonymous MYO5B variants are predicted to be deleterious, according to the following methods of analysis: S, SIFT (Sorting Intolerant From Tolerant): values range from 0 to 1. Values close to 0 are less tolerated, whereas those near 1 are better-tolerated substitutions. PP2, PolyPhen 2: predicts impact of an amino acid substitution on the structure and function of a human protein using straightforward physical and comparative considerations. Values range from 0 to 1. Values close to 0 are better-tolerated substitutions considered as benign, whereas those near 1 are predicted to be damaging. MT, MutationTaster: evaluates disease-causing potential of sequence alterations: DC, disease-causing mutation (P = 1). a Parental allelic distribution was not studied because parental DNAs were not available. According to patient phenotype, a compound heterozygous status is likely. b The mother carries the Arg379Cys mutation and the splice mutation and the father, the Arg824Cys mutation. LIVER IMMUNOHISTOCHEMICAL STUDIES In normal human liver, BSEP, MDR3, or RAB11A staining was localized to the canalicular membrane of hepatocytes showing a linear, thin, and intense staining. MYO5B staining was localized at the cell membrane without a specific domain of localization and more faintly in the cytoplasm of hepatocytes. In patient livers, we observed an abnormal pattern for BSEP and MDR3, with thickened canalicular staining and a granular and patchy pattern in the subcanalicular area (Fig. 1). MYO5B immunoreactivity was abnormal in the two cases studied and showed intense and granular staining in the cytoplasm of hepatocytes but not at the plasma membrane level. RAB11A immunoreactivity was also abnormal with a loss of or weak canalicular expression and granular cytoplasmic staining. Negative controls performed without primary antibody showed no MYO5B or RAB11A staining (Fig. 2). Discussion The novel data reported herein involve five children with normal-GGT chronic intrahepatic cholestasis related to MYO5B mutations but without clinically evident MVID. The clinical findings at presentation in these children closely resemble those of classical normal-GGT PFIC due to mutations in ATP8B1 (PFIC1, FIC1 disease) and ABCB11 (PFIC2, BSEP disease) with certain differences. All three diseases present primarily with cholestasis, manifest by jaundice and pruritus, and have similarly elevated serum bile salt concentrations. Our patients have somewhat less jaundice than typically observed in the other two. BSEP disease appears to have an earlier onset than FIC1 disease, and both are generally earlier than observed in our patients. The age at first symptoms was around 1 year in the patients reported here, while it was below 3 months of age in most patients with BSEP disease.21 BSEP disease generally has greater elevations of serum transaminases than FIC1,21, 26 and transaminases are somewhat greater than in our patients. It is difficult to distinguish between PFIC1 and PFIC2 on clinical grounds alone, and the clinical findings in our cases of MYO5B-cholestasis appear to be within the spectrum of either of these two well-described normal-GGT PFIC disorders.21 Recent work has shown that severe mutations in the farnesoid-X receptor gene NR1H4 produce low-GGT PFIC by way of failure to express BSEP.25 The affected patients had severe, early-onset progressive liver disease similar to that seen in severe ABCB11 mutations. Truncating mutations in the TJP2 gene have recently been identified in several cases of familial low-GGT cholestasis.15 The limited clinical data presented in this work suggest that the clinical presentation of TJP2 disease is also indistinguishable from PFIC1 and PFIC2. Thus, at presentation with an apparent case of isolated normal/low-GGT cholestasis, mutations in at least five genes should now be considered: ATP8B1, ABCB11, NR1H4, TJP2, and MYO5B. The genes whose mutations are responsible for low-GGT cholestasis are expressed in tissues other than liver with the exception of ABCB11, leading to the possibility of nonhepatic symptomatology. Patients with PFIC1 have variable degrees of chronic diarrhea from none to disabling and may develop severe hepatic steatosis after liver transplantation, presumably the effect of FIC1 deficiency in the bowel.21, 26, 27 Thus, patients with MYO5B mutations with dominant cholestasis and some diarrhea might well be clinically confused with PFIC1. For example, patient 3 in this series had several episodes of severe acute diarrhea, which may or may not have been related to MVID. The fact that the duodenal histology was normal suggests coincidence. The arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is another cause of normal-GGT cholestasis.22 The neurological aspects of ARC syndrome are often dominant and distinctive, but sometimes they are minimal. Of interest in this regard is the neurological symptomatology reported here in patient 1. It is not known if this could be attributed to a MYO5B defect, but neurological abnormalities have been reported in children with MVID and MYO5B mutations.17 Also, it has been shown that MYO5B/RAB protein interaction is important for axon development and regulation of dendritic arborization of neurons.28, 29 The neurological symptomatology in patient 1, which may or may not have been related to the MYO5B mutations, could possibly cause confusion with ARC syndrome. Histological features can also be confused with PFIC1, PFIC2, NR1H4-cholestasis, and ARC syndrome, showing numerous giant hepatocytes, fibrosis, and hepatocellular cholestasis. Immunohistochemical staining may permit differentiation of these conditions. Canalicular BSEP immunostaining is usually negative in PFIC230 and was negative in the published cases of NR1H4-cholestasis,25 whereas MDR3 staining is normal in PFIC2.21 In our patients immunostaining studi