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Invited Commentary

Progressive Familial Intrahepatic Cholestasis: Is It Time to Transition to Genetic Cholestasis?

Squires, Robert H.; Monga, Satdarshan Pal

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Journal of Pediatric Gastroenterology and Nutrition: May 2021 - Volume 72 - Issue 5 - p 641-643
doi: 10.1097/MPG.0000000000003111
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See “Progressive Familial Intrahepatic Cholestasis Associated With USP53 Gene Mutation in a Brazilian Child.” by Porta et al on page 674. and See “Cholestasis Due to USP53 Deficiency” by Bull et al on page 667.

In 1969, Clayton et al described seven individuals, born between 1947 and 1967, with features of infantile-onset cholestasis who developed hepatomegaly, steatorrhea, pruritus, poor weight gain and growth which became considerably more stunted after 6 months of age, coagulopathy corrected by parenteral vitamin K, and other features of fat-soluble vitamin deficiency (eg, rickets). Of the seven, five died between 17 months and 8 years of age. All were Amish, bore the surname Byler, and traced their ancestry to Jacob Byler (1799–1867) and his wife Nancy Kaufman. Clayton et al (1) deemed this fatal familial intrahepatic cholestasis in an Amish kindred to be Byler disease. Whitington et al (2) later characterized 33 patients with clinical features indistinguishable from Byler disease, but only two with known instances of consanguinity and none with an apparent link to the Byler lineage. Histologic features in 28 patients were also similar to those described by Clayton (3). In the absence of precise genetic or biological markers to distinguish or align Byler's patients from non-Byler patients (also known as, Byler syndrome), the authors preferred the more general term of progressive familial intrahepatic cholestasis (PFIC) to describe what could by a genetically diverse population with similar clinical phenotypes (2). Descriptive pediatric hepatology related to cholestatic diseases was in full swing.

As if on cue, Houwen et al screened the human genome for linkage disequilibrium in three distantly related subjects with benign recurrent intrahepatic cholestasis (BRIC) and mapped the BRIC gene to chromosome 18 (4). Carlton et al then mapped a locus in two distantly related patients with Old Order Amish Byler disease to a similar region of chromosome 18q21–q22 (5). However, as Whitington predicted, Strautnieks et al reported a Middle Eastern cohort with a phenotype similar to Byler disease but the gene locus did not map to chromosome 18q21–q22 (6), rather to a new gene locus on chromosome 2q24 was identified and designated PFIC2(7). Locus heterogeneity between patients with Byler disease and Byler syndrome was also confirmed by Bull et al (8).

Confirmation that mutations in the familial intrahepatic cholestasis gene (FIC1) (9) were associated with Byler disease (PFIC1) whereas mutations in the gene encoding the bile salt export pump (BSEP) were associated with PFIC2 (10) led to further discovery of genetic causes of pediatric cholestatic liver disease. Over the next 25 years, at least 60 genes have been identified associated with a myriad of intracellular and structural processes including mutations in proteins associated with cytoskeleton defects including mutations in the ubiquitin-specific protease 53 (USP53) gene (11). Mutations in USP53, with associated low-gamma glutamyl transferase (GGT) cholestasis in infants, children, and young adults, are the subject of two manuscripts in this issue of the Journal of Pediatric Gastroenterology and Nutrition.

The case report with literature review from Porta et al (12) describes a Brazilian child born to non-consanguineous parents and paternal family history of “intrahepatic cholestasis and pruritus” presenting at 10 days of life with normal GGT cholestasis, elevated serum aminotransferase levels, pruritus, and liver biopsy at 5 months of age revealing mild fibrosis and giant cell hepatitis. A novel heterozygous mutation in the USP53 gene was identified. Treatment with ursodiol and rifampicin was associated with normalization of serum aminotransferase levels, normal weight gain and growth and minimal pruritus at 18 months of age.

The report by Bull et al (13) screened local and international repositories and gathered seven additional patients with homozygous USP53 mutations, six with deletion mutations that included three with an additional deletion of MYOZ2 (a gene associated with cardiomyopathy), and one missense mutation. While data were not complete for all individuals, they tended to have mild or intermittent cholestasis and pruritus responsive to medication and those biopsied (N = 4) had evidence of fibrosis. Findings on electron microscopy in three subjects revealed apparently intact tight junctions, although two patients had disparate non-specific findings with one having granular bile with peri-canalicular filamentous actin and the other showing a desmosome detached from the plasma membrane with a normal canaliculus. Strikingly, the age of onset ranged from infancy to adolescence, although it was the adolescent that had the missense mutation that could be associated with less severe disease. Long-term follow-up of 2–13 years in subjects presenting with or reported to have had cholestasis beginning <6 months of age suggests an excellent outcome for most subjects with normal or near-normal total bilirubin, alanine aminotransferase, and total bile acids.

The worldwide number of reported patients with cholestasis attributed to mutations in HSP53 is up to 18 and evidence suggests the gene should be included among those associated with cholestasis (14,15). Other than having low-GGT cholestasis with pruritus, a distinguishing clinical or histologic phenotype is elusive. Initial presentation of cholestasis occurs anywhere between infancy and adolescence, although it is not known if adults have screened for this mutation. Hearing loss occurs in a minority of patients. Hypocalcemia is an inconsistent finding. Cholestasis is generally mild although pruritus can be clinically important, but both may respond to rifampicin, ursodiol, and/or cholestyramine. While 1 patient received a liver transplant for intractable pruritus at age 6 years, all others are alive with their native liver, many with a resolution of cholestasis at the last testing point at which subjects were either on or sometimes off medication. Importantly, some patients may experience recurrent episodes of cholestasis months or years after the initial episode resolves or when medications are withdrawn. At the moment, we do not know to what extent this disorder is progressive. However, the presence of fibrosis, ultrasonographic evidence of splenomegaly, and a suggestion that cholestasis can be recurrent tell us patients should continue to be carefully monitored despite the initial resolution of cholestasis.

The association of USP53 mutations with disruption of junctional proteins were initially identified in studies related to progressive hearing loss. USP53 was found to co-localize with tight junction proteins (TJP) TJP1 and TJP2 in cochlear hair cells. Mutations in Usp53 in mice are associated with hair cell degeneration and progressive hearing loss suggesting that USP53 is important to the tight junction apparatus (16). In the liver, a complex structure that includes tight junctions, adherens junctions, desmosomes, and gap junctions form a critical barrier separating sinusoidal blood from canalicular bile known as the blood–bile barrier (17). Tight junction proteins include families of proteins critical to anchoring the cytoskeleton of adjoining cells. Mutations in TPJ2 are associated with progressive normal-GGT cholestasis often resulting in severe liver disease with many requiring liver transplantation (18). Likewise, when adherens junctional assembly in liver cells was disrupted in preclinical models through the combinatorial loss of β-catenin and γ-catenin (the latter compensating for β-catenin at adherens junctions by maintaining association with E-cadherin and eventually upholding cell-cell adhesion), mice displayed features of progressive intrahepatic cholestasis that associated with disruption of tight junctional assembly (19). These clinical and preclinical studies highlight the role of cell–cell junctions and in turn intercellular adhesion in maintaining the integrity of the blood–bile barrier (17). Disruption of these junctions could yield a progressive cholestatic disease through the presence of bile components in interstitial space, concomitant inflammation and associated fibrosis. Another consequence of loss of function of components of tight junction and adherens junctions on disease pathogenesis could be through altered cell polarity, which could lead to erroneous sorting and placement of transporters and polarized proteins (20). Such mislocalization could also impair normal and directional bile flow, affect biliary canaliculi structure and even bile composition, all of which could contribute to intrahepatic cholestasis.

Knowing the relevance of TJP and junctional integrity in general in the pathogenesis of cholestasis, it is tempting to speculate that USP53 is somehow interfering with junctional integrity and eventually leading to intrahepatic cholestasis. The exact mechanism by which USP53 might be regulating TJP function or junctional integrity, however, remains a dilemma and would require careful future studies. While USP53 does belong to the family of deubiquitinating enzymes (DUB), of which there are 83, only seven of these have been predicted to be inactive (21). In fact, USP53 lacks an essential histidine in the catalytic site and hence predicted to be catalytically inert. Indeed, Kazmierczak et al (16) showed an absence of activity in the DUB inhibitor assays. The authors speculated the mutant would bind to TJP and disrupt its normal turnover by interfering with the function of an active DUB on TJP, thus serving as a dominant-negative. Or, USP53 may be binding to ubiquitin modifiers like NEDD8 and others and thus interfere with their role in protein stabilization. A simple STRING Search ( for USP53 revealed interactions of this protein with several proteins including Alkaline ceramidase-1, -2, and -3, which can modulate levels of sphingosine-1-phosphate and ceramide and can cause apoptosis (22). In fact, bile acids are known to cause ERK1/2 activation and impact cell metabolism in a sphingosine-1-phosphate dependent manner (23). USP53 appears to interact with death associated protein kinase-2 and DNA-damage binding protein-2 and hence could play a role in causing disease through regulating cell death. Thus, the conundrum of regulation of TJP stability or junctional stability by USP53, if at all, will need future studies.

Over the last 50 years, beginning with Clayton (1), Watson (24), and Alagille (25,26), and continuing with an increasing number of observant pediatric gastroenterologists and hepatologists, the proportion of children with neonatal cholestasis due to “neonatal hepatitis” has fallen from 65% to 10–12%, while genetically associated diseases now account for approximately 60% when combining PFIC ∼25%, metabolic disease ∼25%, and alpha-1 antitrypsin deficiency ∼10%. As in the cases reported here, identifying a single gene is insufficient to account for phenotypic variability or for establishing mechanistic principals in many cases. A genetic diagnosis is becoming the new clinical descriptor, awaiting the further discovery of patient-specific circumstances that interact with the mutation or its gene product to configure the individual phenotype. Diagnostic classification of PFIC served its purpose to initially classify a novel clinical phenotype and may yet be useful to park those children without a known genetic or other cause for their progressive cholestasis. Moving to a genetic classification of cholestasis provides an opportunity to focus on the remarkable clinical variability manifested by mutations in a single gene that challenges us to ask Why? The answer to that question will likely identify opportunities for novel therapeutics.


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