EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Key words : Cholestasis, Liver transplant, Low-level γ-glutamyl transferase, Neonatal cholestasis

Neonatal physiological jaundice is a benign clinical condition that typically resolves within the first 2 weeks after birth.¹ However, neonatal cholestasis (NC) is always a pathological clinical condition present in affected newborn infants with unconjugated hyperbilirubinemia, indicative of impairment in hepatobiliary production or bile excretion. The incidence of NC is observed to be approximately 1 in 2500 live births.²

In the etiology of NC, abnormalities in the development of the bile ducts, defects in transporter proteins responsible for bile formation, ciliary dysfunction, and disorders in bile acid synthesis play significant roles.³ Observation of serum γ-glutamyl transferase (GGT) activity is essential for proper etiological evaluation of NC. High activity of GGT coincident with NC suggests extrahepatic biliary tract involvement, whereas typical or low GGT activity with NC generally indicates a genetic disorder. Genetic variants are identified in approximately 75% to 80% of patients.⁴

In patients with cholestasis, disruption of bile circulation may result in abnormalities in nutrition, detoxification, and cholesterol metabolism. However, the most serious complication of cholestasis in the long-term is the effect of bile acids to act as detergents and thereby cause severe liver damage. This deleterious effect of bile acids is the most common indication for liver transplant (LT) in patients with cholestasis.⁵

The progressive familial intrahepatic cholestasis (PFIC) group of genetic disorders is an important cause of NC. Recently, Maddirevula and colleagues identified a sequence variant of the ubiquitin-specific peptidase 53 (USP53) gene in children with low-GGT cholestatic liver disease.⁶ This newly identified disorder has been tentatively classified as PFIC type 7. Here, we present 2 siblings with a novel sequence variant of the USP53 gene who underwent LT on follow-up.

A 14-month-old boy was referred to our hospital for transplant to treat acute-on-chronic liver failure. He was the first-born child of parents who were related within the third degree of consanguinity. His prenatal and natal period had been unremarkable, and he had been assigned an ongoing follow-up regimen for cholestasis since birth. The results of his routine newborn hearing screening test were unremarkable. His growth and development were within reference ranges, and physical examination revealed jaundice, hepatomegaly, and splenomegaly. Laboratory results showed hypertransaminasemia, conjugated hyperbilirubinemia, increased international normalized ratio value, and GGT level within reference range (Table 1). Viral, toxic, metabolic, and autoimmune forms of liver disease were excluded. Pediatric end-stage liver disease score was 40. Ultrasonography of the liver revealed homogeneous hepatomegaly. The patient underwent LT, and explant liver biopsy revealed micronodular cirrhosis and giant cell transformation of hepatocytes. No pathological sequence variants were detected in the genetic tests conducted for PFIC type 1 and PFIC type 2. After LT, the patient was regularly monitored without any complication.

Six years later, his sister was admitted to our clinic with cholestasis at the age of 5 months. Like her brother, a physical examination of the sister revealed jaundice and hepatomegaly, and laboratory studies revealed conjugated hyperbilirubinemia, with typical healthy levels of GGT (Table 1). Liver ultrasonography revealed hepatosplenomegaly and grade 1 hepatosteatosis. We proceeded without a liver biopsy for the sister due to the similarity of her presentation to that of her brother, which raised suspicion of a genetic disorder. Whole exome sequencing was performed for the 2 siblings and revealed novel homozygous sequence variants (NCBI Reference Sequence NM_019050.2) c.454C>T (p.His152Tyr) in the USP53 gene in both patients. By Sanger sequencing, we confirmed this variant in both patients; also, it was the heterozygous form in parents and the wild-type form in the unaffected sibling (Figure 1). The sister was monitored with oral ursodeoxycholic acid (20 mg/kg/d) and fat-soluble vitamins (vitamin K, 1 mg/kg, 3 times per day; vitamins A and D, 1500 U per day; vitamin E, 10 mg/kg/d) until LT was performed at 14 months of age to treat acute-on-chronic liver failure in the same course of diagnosis and treatment that had been followed for her brother.

At the last follow-up, both siblings exhibited healthy psychomotor development and good condition after transplant. Both patients had no hearing impairment at the time of this publication.

Progressive familial intrahepatic cholestasis is the name assigned to a group of diseases with variable clinical severity characterized by impairment of bile excretion and production from hepatocytes, which begins in early childhood and may progress to end-stage liver failure.⁷ A number of gene sequence variants have been associated with PFIC through molecular genetic testing. So far, 11 subgroups of PFIC have been identified in the Online Mendelian Inheritance in Man database. The form of PFIC associated with USP53 gene sequence variants has been tentatively classified as PFIC type 7. In the extant literature, patients with USP53 gene sequence variants have generally exhibited mild cholestasis that responded to treatment. The 2 siblings we present here are very important, because this is the first report of LT to treat acute-on-chronic liver failure.

The USP53 gene is located on chromosome 14q24.3 and encodes a member of the deubiquitinating enzyme family.^8,9 The USP53 protein is known to be expressed in the liver, brain, kidney, and inner ear.^6,10 Sequence variation in the USP53 gene has been identified in animal models and has been reported to cause deafness, and the disorder in tight junction skeletal models plays a role in its mechanism.¹¹ The USP53 protein is a component of the tight junction complex and interacts with tight junction protein 1 (TJP1) and TJP2.¹⁰ Tight junction proteins are cytoplasmic proteins and link intrinsic membrane TJP to the cytoskeleton.¹² In this connection, normal organization is ensured in the apical membranes, and the epithelial structure is protected through cell-to-cell interaction and apical-to-basal polarization.^13,14 It has been reported that disruption of the tight junction structure will cause a defect in the separation of bile from the plasma and the canalicular area from the sinusoidal area and thereby cause bile leakage into the plasma.¹³ Clinical and pathological results of sequence variation in the USP53 gene are similar to results from studies of the TJP2 gene sequence variant (PFIC type 4) and the ATPase phospholipid carrier 8B1 (ATP8B1) gene sequence variant (PFIC type 1), and this similarity may be due to a common damage mechanism.¹⁵ In all 3 diseases (PFIC type 4, type 1, and type 7), the primary damage occurs in membrane symmetry, and these gene sequence variants are thought to cause a secondary loss in each other’s functions.^15,16

In the literature, there was a report of 3 patients in 1 family presented with hearing loss and GGT normal cholestasis.⁶ Late-onset hearing loss in patients is frequently reported; therefore, it is recommended that such patients should be monitored for hearing dysfunction.⁶ In our patients, hearing impairment was not detected, but annual hearing tests were included in the ongoing follow-up regimen. In another study,³ homozygous sequence variants in the USP53 gene were identified in 7 patients, and it was reported that mild and intermittent cholestasis responded to medications in all patients. All 7 patients exhibited fibrosis on liver biopsy, and all 7 eventually developed splenomegaly. However, none of these patients required transplant during follow-up. Although the age of clinical onset varies in the literature, patients with moderate cholestasis who present with intermittent jaundice and itching that are responsive to treatment have been reported. Both of our patients had been followed up with severe cholestasis that was unresponsive to treatment since the neonatal period. Liver findings of the patients worsened during follow-up and required LT in the early period. A study in the literature reported on a patient who required LT.¹⁵ Alhebbi and colleagues reported the case of a 6-year-old patient who received a transplant to treat persistent itching that was unresponsive to treatment and worsening his quality of life.¹⁵ Interestingly, the itching could not be treated with rifampicin in this patient. With regard to the favorable response of patients to rifampin in the literature, such reports suggest that rifampin treatment could have been successful without the need for LT.

Biopsy findings from patients with USP53 gene sequence variants are variable. Lobular cholestasis, giant cells, mild inflammatory cell infiltration, and fibrosis have been reported on liver biopsy from these patients.¹⁷ We performed a liver biopsy for the elder brother in our study, and giant cell formation was observed, which is consistent with the literature. We proceeded without a biopsy for the sister because her clinical presentation was surprisingly similar to symptoms that we observed in her brother. As the prevalence and comprehensiveness of genetic examinations improve over time in patients with cholestasis coincident with typical healthy levels of GGT activity, it is likely that new gene sequence variants will be discovered in these patients.

Most sequence variants reported in the USP53 gene are truncating variants, whereas missense variants are reported less frequently. The missense homozygous c.454C>T (p.His152Tyr) sequence variant detected in our patients has not been previously described. This variant is located at a highly conserved residue associated with the zinc-binding site in USP catalytic cores,¹⁸ as previously reported in patients with missense variants.^3,18 It is also predicted to be pathogenic by in silico tools (Meta Recurrent Neural Network score, 0.9633; BayesDel addAF score, 0.2736; Rare Exome Variant Ensemble Learner score, 0709). In addition, considering its homozygosity, intrafamilial segregation, and clinical consistency, this variant was thought to be responsible for the findings of the patients and was interpreted as likely pathogenic according to the American College of Medical Genetics and Genomics standards.¹⁹ The patients with different clinical severity have been identified in the same family for USP53 sequence variants.⁶ For this reason, it seems difficult to establish a genotype-phenotype correlation with presently available data. Therefore, with each detected novel gene sequence variant, we obtain new clinical information about these diseases, which are rare in the literature.

Sequence variants of the USP53 gene result in cholestasis with typical healthy GGT activity or cholestasis with low GGT activity. However, this novel gene sequence variant can lead to severe cholestasis that is unresponsive to treatment and requires LT. Particularly in patients with a family history of similar disease or affected siblings without a genetic diagnosis and/or consanguineous parents, reanalysis is crucial as it offers the chance to diagnose the condition and provide prenatal counseling to the parents of these patients.

Volume: 22 Issue: 10 October 2024 - Supplement - 5

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