Considering the genetic etiology of pediatric cholestasis in the literature, the diagnostic yield of genetic tests varies between 25% and 50% [18]. This rate has been increasing with the discovery of novel genes associated with cholestasis in recent years, after the prevalence of genetic testing has increased. Next generation sequencing technologies are a promising tool for discovering new genes related to pediatric cholestasis. For example; in a study on pediatric cholestatic liver disease in 2019, it was suggested that 5 novel loci and candidate genes may play a role in the etiology of the disease. These genes are KIF12, PPM1F, USP53, LSR, and WDR83OS [7].
Biallelic variants in USP53 have recently been reported in cholestasis phenotype. Up to now, a total of 29 cases from 22 families have been reported in 6 articles in the literature showing that the USP53 gene is associated with cholestasis and/or some additional phenotypes [5, 7, 18-21]. Detailed clinical, laboratory, and genetic findings of all cases reported in the literature and our own case (totally 30 cases) are summarized in Table-2. A total of 18 different variants have been reported in these patients. Of these variants, 5 were frameshift, 5 were nonsense, 4 were misspelled, 2 were insertions, and 1 were gross deletion. Also, one family had large copy number variation (CNV) involving exon 1 of the USP53 gene. When the patients are clinically evaluated; three of reported USP53 cases had additional hearing loss. In mice, studies have shown that USP53 co-locates with TJP2 and contributes to tight junction structures, at least in the ear [8]. Pathogenic variants detected in the TJP2 gene have been shown to be associated with hypercholanemia, normal or low GGT intrahepatic cholestasis, hepatocellular carcinoma as well as deafness [5, 8, 22]. However, we recommend that cases with variants in USP53 gene should be monitored for hearing loss and that aminoglycoside antibiotics should not be used in these patients because of the additional damage they may cause. In one patient, speech and developmental delay is accompanied. Also, he has delayed gross motor skills. The USP53 variant in this patient was the c.951delT(p.Phe317fs). In the same article, 4 siblings from another family had the same variant but these patients had no speech and developmental delay [18]. Although no other genetic variant was reported in the WES result for the additional cause of mental and motor delay in this patient, the absence of developmental delay in all other cases in the literature suggests a possible secondary genetic etiology. On the other hand, as the number of cases identified in the USP53 gene increases, we will have clearer information about the genotype-phenotype correlation.
It has been reported in studies that patients with USP53-related cholestasis generally respond to medical treatment and liver transplantation was performed in only one case among the cases reported in the literature so far. In this case, intractable itching that did not respond to medical treatment was reported as an indication for transplantation. It was also stated that the case may not have had a chance to be treated with rifampicin 20 years ago. In the same article, it was reported that intractable itching during BRIC episodes was also reported in 2 other adult patients and even led to depression and suicidal attempts in patients [18].
Due to intractable itching that did not respond to antihistamines, our patient was also started on rifampicin. At the 3 months of follow-up, ALT, AST and bilirubin levels returned to normal and the complaint of itching decreased dramatically. Although early fibrosis has been described, cholestasis is transient in most patients with variants in the USP53 gene [20]. Our patient also had porto-portal, porto-central and centrilobular fibrosis as a result of liver biopsy performed at the age of 9 months. We think that patients should be followed closely in terms of fibrosis.
USP53, a member of the Ubiquitin Specific Proteases involved in the deconjugation of ubiquitin and ubiquitin-like protein adducts, is a cysteine protease with complex organizational structures and different functional properties [23]. All known USPs contain three basic domains called Cys-box, QQD-box and Her-box important for their catalytic activity [24-26]. The generated model demonstrated the successful formation of the conserved catalytic triad in all other known USPs (Figure 5a). Asp108, located near the catalytic triad of Cys41, His301, Asp319, may be responsible for substrate stability. Apart from this core catalytic domain, the N-terminal and C-terminal domains of the protein may be important in substrate specificity and cellular localization. Although conserved Rossman fold-like structures in the N-terminal region reveal an irregular formation, they may provide data on the secondary functions of USP53 (tight junction, interaction with multiple substrates, and additional regulatory roles) (Figure 5b). The protein sequence of USP53, which terminates early with mutation, brought about the loss of protein structure including functional properties. The functional deficiency of USP53 will also disrupt the metabolic and regulatory processes with which it interacts. The elucidation of the molecular basis of cholestasis will be possible with a detailed analysis of the USP53 interaction map (Figure 6). The relationship between USP53 and Crk-like protein (CRKL) in the interaction network created by considering the experimental data is remarkable. CRKL acts as a control interface that mediates intracellular signal transduction in many processes including transcriptional repression, apoptotic mechanisms, ensuring membrane integrity, and substance transport. Chloride intracellular channel protein 4 co-expressed with CRKL maintains basolateral membrane polarity, induces endothelial cell proliferation, and creates selective ion channels [27-29]. Programmed cell death protein 6 is a calcium sensor protein involved in processes such as endoplasmic reticulum-golgi vesicular transport and membrane repair. Activation with calcium triggers the formation of 3 different hydrophobic pockets that allow interaction with different protein groups, thus enabling translocation [30-32]. Tyrosine-protein kinase ABL1 co-expressed with CRKL plays a key role in many vital cellular processes such as cytoskeletal remodeling, cell motility, cell adhesion, receptor endocytosis, autophagy, apoptosis, and response to DNA damage [33-35]. Another, Rap guanine nucleotide exchange factor-1 co-expressed with CRKL is important in establishing basal endothelial barrier function [36]. SAMD4B, which represses the translations of activator protein-1, tumor protein-53, and cyclin-dependent kinase inhibitor 1A, and E3- ubiquitin-protein ligase CBL, which acts as a negative regulator of many signaling pathways, are genes that interact directly or indirectly with USP53 [37].
As we tried to explain above, USP53 has critical roles in many important processes for the cell, especially substance transport, membrane permeability, membrane stability, apoptotic processes, repair, regulation at the transcription/translation level. Loss of UPS53 function due to c.238-1G>C variation may cause disruption or deterioration in these processes. The disruption that will occur in the mechanisms that regulate the transport and passage of various biological components and the deterioration in the mechanisms that will control these malfunctions will reveal a process that results in the passage of bile into the systemic circulation, as in cholestasis. In particular, weaknesses that affect membrane stability, cell adhesion, and the stability of tight junctions can result in leakage of bile salts into the plasma. The C-terminal domain of USP53 interacts with the TJP1 and TJP2 heterodimer associated with cholestasis risk [38, 39]. Tight junction protein 2 (TJP2) belongs to a family of membrane-associated guanylate cyclase homologs involved in the organization of tight junctions. Tight junctions in hepatocytes are important in separating bile from plasma and the canalicular membrane area from the sinusoidal membrane area. TJP2 is hypothesized to affect the tight junction structure by binding to claudins and occluding. It has been suggested that weakness in tight junctions may result in leakage of bile salts into the plasma and high serum bile salt concentrations [22, 40, 41]. The c.238-1G>C variant abolishes the USP53-TJP2 interaction, which can result in a weakening of tight junctions and leakage of bile salts into the plasma.