Hereditary cholestasis in childhood and infancy with normal serum gamma‐glutamyltransferase (GGT) activity is linked to several genes. Many patients, however, remain genetically undiagnosed. Defects in myosin VB (MYO5B; encoded by MYO5B) cause microvillus inclusion disease (MVID; MIM251850) with recurrent watery diarrhea. Cholestasis, reported as an atypical presentation in MVID, has been considered a side effect of parenteral alimentation. Here, however, we report on 10 patients who experienced cholestasis associated with biallelic, or suspected biallelic, mutations in MYO5B and who had neither recurrent diarrhea nor received parenteral alimentation. Seven of them are from two study cohorts, together comprising 31 undiagnosed low‐GGT cholestasis patients; 3 are sporadic. Cholestasis in 2 patients was progressive, in 3 recurrent, in 2 transient, and in 3 uncategorized because of insufficient follow‐up. Liver biopsy specimens revealed giant‐cell change of hepatocytes and intralobular cholestasis with abnormal distribution of bile salt export pump (BSEP) at canaliculi, as well as coarse granular dislocation of MYO5B. Mass spectrometry of plasma demonstrated increased total bile acids, primary bile acids, and conjugated bile acids, with decreased free bile acids, similar to changes in BSEP‐deficient patients. Literature review revealed that patients with biallelic mutations predicted to eliminate MYO5B expression were more frequent in typical MVID than in isolated‐cholestasis patients (11 of 38 vs. 0 of 13). Conclusion: MYO5B deficiency may underlie 20% of previously undiagnosed low‐GGT cholestasis. MYO5B deficiency appears to impair targeting of BSEP to the canalicular membrane with hampered bile acid excretion, resulting in a spectrum of cholestasis without diarrhea. (Hepatology 2017;65:1655‐1669).
Background and Aims Progressive familial intrahepatic cholestasis (PFIC) 6 has been associated with missense but not biallelic nonsense or frameshift mutations in MYO5B , encoding the motor protein myosin Vb (myoVb). This genotype‐phenotype correlation and the mechanism through which MYO5B mutations give rise to PFIC are not understood. The aim of this study was to determine whether the loss of myoVb or expression of patient‐specific myoVb mutants can be causally related to defects in canalicular protein localization and, if so, through which mechanism. Approach and Results We demonstrate that the cholestasis‐associated substitution of the proline at amino acid position 600 in the myoVb protein to a leucine (P660L) caused the intracellular accumulation of bile canalicular proteins in vesicular compartments. Remarkably, the knockout of MYO5B in vitro and in vivo produced no canalicular localization defects. In contrast, the expression of myoVb mutants consisting of only the tail domain phenocopied the effects of the Myo5b‐P660L mutation. Using additional myoVb and rab11a mutants, we demonstrate that motor domain‐deficient myoVb inhibited the formation of specialized apical recycling endosomes and that its disrupting effect on the localization of canalicular proteins was dependent on its interaction with active rab11a and occurred at the trans ‐Golgi Network/recycling endosome interface. Conclusions Our results reveal a mechanism through which MYO5B motor domain mutations can cause the mislocalization of canalicular proteins in hepatocytes which, unexpectedly, does not involve myoVb loss‐of‐function but, as we propose, a rab11a‐mediated gain‐of‐toxic function. The results explain why biallelic MYO5B mutations that affect the motor domain but not those that eliminate myoVb expression are associated with PFIC6.
Background and AimsLarge indels are commonly identified in patients but are not detectable by routine Sanger sequencing and panel sequencing. We specially designed a multi-gene panel that could simultaneously test known large indels in addition to ordinary variants, and reported the diagnostic yield in patients with intrahepatic cholestasis.MethodsThe panel contains 61 genes associated with cholestasis and 25 known recurrent large indels. The amplicon library was sequenced on Ion PGM system. Sequencing data were analyzed using a routine data analysis protocol and an internal program encoded for large indels test simultaneously. The validation phase was performed using 54 patients with known genetic diagnosis, including 5 with large insertions. At implement phase, 141 patients with intrahepatic cholestasis were evaluated.ResultsAt validation phase, 99.6% of the variations identified by Sanger sequencing could be detected by panel sequencing. Following the routine protocol, 99.8% of false positives could be filtered and 98.8% of retained variations were true positives. Large insertions in the 5 patients with known genetic diagnosis could be correctly detected using the internal program. At implementation phase, 96.9% of the retained variations, following the routine protocol, were confirmed to be true. Twenty-nine patients received a potential genetic diagnosis when panel sequencing data were analyzed using the routine protocol. Two additional patients, who were found to harbor large insertions in SLC25A13, obtained a potential genetic diagnosis when sequencing data were further analyzed using the internal program. A total of 31 (22.0%) patients obtained a potential genetic diagnosis. Nine different genetic disorders were diagnosed, and citrin deficiency was the commonest.ConclusionSpecially designed multi-gene panel can correctly detect large indels simultaneously. By using it, we assigned a potential genetic diagnosis to 22.0% of patients with intrahepatic cholestasis.
BackgroundUnderlying causes in Chinese children with recurrent acute liver failure (RALF), including liver crises less than full acute liver failure, are incompletely understood. We sought to address this by searching for genes mutated in such children.MethodsFive unrelated Chinese boys presenting between 2012 and 2015 with RALF of unexplained etiology were studied. Results of whole exome sequencing were screened for mutations in candidate genes. Mutations were verified in patients and their family members by Sanger sequencing. All 5 boys underwent liver biopsy.Results NBAS was the only candidate gene mutated in more than one patient (biallelic mutations, 3 of 5 patients; 5 separate mutations). All NBAS mutations were novel and predictedly pathogenic (frameshift insertion mutation c.6611_6612insCA, missense mutations c.2407G > A and c.3596G > A, nonsense mutation c.586C > T, and splicing-site mutation c.5389 + 1G > T). Of these mutations, 3 lay in distal (C-terminal) regions of NBAS, a novel distribution.Unlike the 2 patients without NBAS mutations, the 3 patients with confirmed NBAS mutations all suffered from a febrile illness before each episode of liver crisis (fever-related RALF), with markedly elevated alanine aminotransferase and aspartate aminotransferase activities 24-72 h after elevation of body temperature, succeeded by severe coagulopathy and mild to moderate jaundice.ConclusionsAs in other countries, so too in China; NBAS disease is a major cause of fever-related RALF in children. The mutation spectrum of NBAS in Chinese children seems different from that described in other populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12876-017-0636-3) contains supplementary material, which is available to authorized users.
Profiling of plasma bile acids has provided insights into cholestasis alleviation and may be useful for the clinical management of cholestatic diseases.
BackgroundBranchio-oto-renal (BOR) syndrome is a dominant autosomal disorder characterized by phenotypes such as hearing loss, branchial fistulae, preauricular pits, and renal abnormalities. EYA1, the human homolog of the Drosophila “eye absent” gene on chromosome 8q13.3, is recognized as one of the most important genes associated with BOR syndrome.MethodsThe proposita in this study was a 5-year-old Chinese girl with hearing loss, bilateral otitis media with effusion, microtia, facial hypoplasia, palatoschisis, and bilateral branchial cleft fistulae. The girl’s family members, except two who were deceased, agreed to undergo clinical examination. We collected blood samples from 10 family members, including six who were affected by the syndrome. Genomic DNA was extracted and subjected to Sanger sequencing. A minigene assay was performed to confirm whether splicing signals were altered. In addition, we performed western blotting to determine alterations in protein levels of the wild-type and mutant gene.ResultsClinical tests showed that some of the family members met the criteria for BOR syndrome. The affected members harbored a novel heterozygous nonsense variation in exon 11 of EYA1, whereas no unaffected member carried the mutation at this position. Functional experiments did not detect abnormal splicing at the RNA level; however, western blotting showed that the mutated protein was truncated.ConclusionsThis study reports a novel mutation associated with BOR syndrome in a Chinese family. We highlight the usefulness of genetic testing in the diagnosis of BOR syndrome. Thus, we believe that this report would benefit clinicians in this field.Electronic supplementary materialThe online version of this article (10.1186/s12881-018-0653-2) contains supplementary material, which is available to authorized users.
Myosin 5B-Associated Cholestatic Liver Disease Cholestatic liver disease (CLD) is characterized by an increase in the serum concentrations of compounds that are normally excreted with bile, such as bile acids and bilirubin. (1) CLD clinically manifest with cholestasis, jaundice, and pruritis. Diagnosis involves evaluation of the patient's clinical manifestations, exclusion of common causes of childhood cholestasis, and analyses of blood biochemistry and liver histology. (1) One blood marker that aids in the differential diagnosis of liver diseases is gamma-glutamyltransferase (GGT), a liver enzyme which is typically elevated in serum upon liver damage. The best known CLDs characterized by low/normal GGT are the benign recurrent and progressive forms of familial intrahepatic cholestasis (BRIC/PFIC), which are caused by mutations in the adenosine triphosphatase phospholipid transporting 8B1 (ATP8B1) gene encoding the ATP8B1 protein (BRIC/PFIC1) or in the adenosine triphosphate-binding cassette family B member 11 (ABCB11) gene encoding the canalicular bile salt export pump (BSEP) (BRIC/PFIC2). (2,3) In hepatocytes, ATP8B1 and BSEP are localized to the apical bile canalicular domain, where they contribute to biliary secretion. When mutated as in PFIC1/2 patients, the respective proteins are less expressed or mislocalized and/or display impaired activity, leading to defective biliary secretion and, consequently, cholestasis. (2,3) Since 2017 four independent reports have identified in total 30 different myosin 5B (MYO5B) mutations in 22 patients with non-microvillus inclusion disease (MVID) who were diagnosed with intermittent, recurrent, or progressive cholestasis presenting with jaundice, pruritus, hepatomegaly, and low/normal serum levels of GGT (Fig. 1; Supporting Table S1). These patients tested negative for mutations in other PFIC-associated genes. (4-7) MYO5B mutations may account for approximately 20% of pediatric patients with idiopathic low-GGT intrahepatic cholestasis. (8)
To assess the spectrum of pediatric clinical phenotypes in TJP2 disease, we reviewed records of our seven patients in whom intrahepatic cholestasis was associated with biallelic TJP2 variants (13; 12 novel) and correlated clinical manifestations with mutation type. The effect of a splicing variant was analyzed with a minigene assay. The effects of three missense variants were analyzed with protein expression in vitro. Our patients had both remitting and persistent cholestasis. Three exhibited growth retardation. Six responded to treatment with cholestyramine, ursodeoxycholic acid, or both. Two had cholecystolithiasis. None required liver transplantation or developed hepatocellular or cholangiocellular malignancy. None manifested extrahepatic disease not attributable to effects of cholestasis. The variant c.2180-5T>G resulted in exon 15 skipping with in-frame deletion of 32 amino acid residues in TJP2. The three missense variants decreased but did not abolish TJP2 expression. Patients with truncating or canonical splice-site variants had clinically more severe disease. TJP2 disease in children includes a full clinical spectrum of severity, with mild or intermittent forms as well as the severe and minimal forms hitherto described. Biallelic TJP2 variants must be considered in children with clinically intermittent or resolved intrahepatic cholestasis.
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