Hepatitis E virus infection-triggered intrahepatic cholestasis: A case report

Abstract

BACKGROUND

Genetic disorders affecting hepatobiliary transporters can be triggered by various factors, resulting in marked cholestasis.

CASE SUMMARY

We report two patients who experienced a severe episode of intrahepatic cholestasis triggered by an acute hepatitis E virus infection. Following an extensive clinical examination that ruled out common causes of cholestatic liver damage, we conducted next-generation sequencing to determine the genetic profiles of the patients. The analysis revealed several known and unknown variants in genes associated with hepatobiliary transporters and bile salt regulation, including ATP8B1, ABCB11, ABCB4, MYO5B, and FXR. For a comprehensive understanding of the pathophysiology, we performed ClinVar analysis and utilized PolyPhen for bioinformatic prediction of functional impact. Both patients exhibited rapid symptom improvement and a decrease in hyperbilirubinemia when treated with either rifampicin or bezafibrate.

CONCLUSION

Our findings introduce hepatitis E viral infection as a novel trigger for intrahepatic cholestasis, and we categorize the significance of the various genetic variants based on the current state of research.

Key Words: Intrahepatic cholestasis; Hepatitis E; Next generation sequencing; ATP8B1; Case report

Core Tip: This case report highlights the role of genetic variations in hepatobiliary transporters in predisposing individuals to intrahepatic cholestasis and identifies hepatitis E virus (HEV) as a potential trigger. We present two cases of HEV-triggered intrahepatic cholestasis in patients with polymorphisms in ATP8B1 and other transporter-related genes. These findings suggest that these genetic variants may act as predisposing factors for cholestatic episodes, underscoring the need for further research to confirm these results in larger patient cohorts and through functional studies.

INTRODUCTION

Genetic variations in hepatobiliary transporters may predispose individuals to intrahepatic cholestasis, potentially leading to conditions such as benign recurrent intrahepatic cholestasis (BRIC) or progressive familial intrahepatic cholestasis (PFIC). BRIC is a heterogeneous group of rare cholestasis syndromes, which is caused by various triggers in susceptible patients carrying polymorphic variants of genes essential for bile formation[1,2]. The first episode usually occurs in early adulthood and is triggered by various factors, such as viral infections or, less frequently, certain drugs[3]. Affected individuals suffer from pronounced cholestasis with jaundice as a consequence. The most commonly affected genes are ATP8B1, ABCB11, and ABCB4, resulting in impaired function of the aminophospholipid translocase FIC1 and the transporters BSEP and MDR3[4-6].

Mutations in the same transporters can also lead to the more severe phenotype of PFIC, which typically manifests in childhood and causes profound cholestatic liver damage[6]. Patients with PFIC due to mutations in FIC1 or BSEP often show low gamma-glutamyltransferase (GGT) serum levels, whereas impaired function of MDR3 typically presents with elevated GGT levels[6].

While more than six subtypes of PFIC have been described, only mutations in ATPB8 (BRIC type 1) and ABCB11 (BRIC type 2) have been associated with BRIC[3,6]. The resulting clinical phenotype varies depending on the underlying polymorphisms and mutations as well as whether these alterations are heterozygous or homozygous. While certain pathogenic mutations can be related to certain severe clinical phenotypes, such genotype-phenotype correlations are less well-defined in milder cases, where the relationship between genetic variants and clinical manifestation remains ambiguous.

There are numerous triggers for intrahepatic cholestasis, and one potential cause is an infection with the hepatitis E virus (HEV). This zoonotic infection is known to elicit jaundice and nonspecific symptoms such as fatigue, nausea, and itching. Besides elevated liver enzymes, the presence of positive IgM antibodies, quickly followed by IgG antibodies, confirms the diagnosis. Nucleic acid testing for HEV RNA in blood and stool exhibits the highest sensitivity, detecting viremia 3–6 weeks after infection[7].

Herein, we describe two phenotypes of clinical intrahepatic cholestasis triggered by HEV infection in patients with various homozygous and heterozygous variants.

CASE PRESENTATION

Chief complaints

We report on two male patients, aged 39 and 58 years, who presented to our clinics in Erlangen and Aachen, Germany. Both had a normal body weight and initially presented to their general practitioner or to a peripheral hospital with fever, fatigue, jaundice, nausea, and loss of appetite.

History of present illness

Symptoms had started 2–4 weeks before referral to the respective university hospital.

History of past illness

While Patient 1 had no medical history, Patient 2 had already experienced an episode of jaundice in his early twenties.

Personal and family history

Neither patient had a family history of liver disease or jaundice, and both denied alcohol consumption or illicit drug use.

Physical examination

Both patients exhibited pronounced jaundice along with pruritus. There were no signs of decompensation, such as ascites, encephalopathy, or gastrointestinal bleeding.

Laboratory examinations

Both patients initially exhibited markedly elevated transaminases and significant hyperbilirubinemia but only slightly elevated gamma-glutamyltransferase (Figure 1A). Diagnostic assessments revealed evidence of acute hepatitis E, supported by the presence of positive anti-HEV-IgM in both patients. Additionally, Patient 1 exhibited positive nucleic acid amplification testing for HEV RNA detection in the blood.

Figure 1 Longitudinal course of liver tests and genetic variants. A: Clinical course of Patient 1 and Patient 2 showing bilirubin levels (black line) and gamma-glutamyltransferase (GGT) levels (gray line) over time (in days). B: Genetic variants determined by next-generation sequencing and genotyping. Variants are annotated with corresponding minor allele frequencies (MAFs), obtained from ensembl.org, and linked to previously published references (Supplementary Table 1). GGT, gamma-glutamyl transferase; MAF, minor allele frequency; ABCB4: ATP-binding cassette 4; ABCB11: ATP-binding cassette, sub-family B member 11; ATPB1: ATPase phospholipid transporting 8B1; MYO5B: Myosin VB; FXR, farnesoid X receptor; n/a: Not available.

Imaging examinations

Abdominal ultrasonography in both patients revealed a liver of normal size with a regular homogeneous parenchymal structure and no focal lesions. Magnetic resonance cholangiography in Patient 1 demonstrated a normal intrahepatic and extrahepatic bile duct system.

Further diagnostic and histological examination

Due to progressive hyperbilirubinemia with marked pruritus and loss of appetite associated with weight loss, both patients were referred to tertiary care centers at university hospitals. Investigations excluded obstructive cholestasis, autoimmune hepatitis, primary biliary cholangitis or primary sclerosing cholangitis, alpha-1-antitrypsin deficiency, hemochromatosis, Wilson's disease, and other acute viral infections. Histology of liver biopsies demonstrated panacinar cholestasis with mild fibrosis. BSEP expression was preserved in both cases. More than 4 weeks after the onset of symptoms, neither the HEV ORF2 (capsid) protein in liver tissue[8,9] nor HEV-RNA in the blood could be detected, providing no evidence of chronic HEV infection.

Next generation sequencing

Genomic DNA was isolated using standard procedures applying membrane-based QIAamp DNA extraction protocol (Qiagen, Hilden, Germany). For library preparation, the Illumina DNA prep with Enrichment (formerly Nextera Flex for Enrichment) was used according to the manufacturer’s instructions (Illumina San Diego, USA). The custom panel designed in cooperation with Illumina included the exons of ABCB4, ABCB11, ABCC2, ALG8, ATP8B1, GANAB, MYO5B, PRKCSH, PRSS1, SEC61B, and SEC63. Direct paired-end NGS sequencing (2 × 150 bp) was performed on an Illumina MiniSeq platform. Results were analyzed using the JSI SeqNext software (JSI, Ettenheim, Germany), and the detected genetic variants were further checked using the gnomAD (gnomad.broadinstitute.org)

Genotyping

The predisposing cholestatic variant NR1H4 (FXR) c.-1G>T (rs56163822, C__25598386_10, Thermo Fisher Scientific) was genotyped using allelic discrimination on a TaqMan 7500 Fast PCR System. Quality control was maintained by including both negative controls and DNA samples with established genotypes as internal standards. Fluorescence signals were analyzed using allelic discrimination software 7500 v.2.3.

Genetic analyses

Subsequently, we performed genotyping for various genes as well as panel sequencing from the blood (Figure 1B). In both patients, genetic variants in ATP8B1, ABCB4, ABCB11, and MYO5B could be identified, and Patient 2 also had a heterogenous pathogenic mutation in NR1H4.

FINAL DIAGNOSIS

Based on the clinical presentation, laboratory findings, and exclusion of other causes, a diagnosis of HEV-triggered intrahepatic cholestasis was established.

TREATMENT

Patient 1 was subsequently treated with rifampicin 150 mg and Patient 2, with bezafibrate 400 mg until normalization of bilirubin.

OUTCOME AND FOLLOW-UP

Both patients showed a rapid improvement, with a marked decline in hyperbilirubinemia and resolution of pruritus and other symptoms such as loss of appetite. During follow-up examinations, both patients remained asymptomatic, with normalization of bilirubin levels for more than a year (Figures 1 and 2).

Figure 2 Histology of liver biopsies. Representative histological staining of liver biopsy samples from both patients. The following stainings were performed: BSEP: Bile salt export pump; CK7: Cytokeratin 7; HE: Hematoxylin and eosin; MAS: Masson’s trichrome; MDR3: Multidrug resistance protein 3. Long scale bar: 100 µm; Short scale bar in inlay: 40 µm (BSEP and MDR3 staining).

DISCUSSION

In the search for possible susceptibility loci for cholestatic liver disease, several genes (ABCB4, ABCB11, ATP8B1, ALG8, GNAB, MYO5B, PRKCSH, SEC61B, and SEC63) of the patients were analyzed by next-generation sequencing (NGS; Figure 1B). We identified both known and novel homozygous and heterozygous variants. To enhance our understanding of the pathophysiology, we conducted a ClinVar analysis and performed a bioinformatic prediction using PolyPhen (Figure 1B), aligning the results with the current literature. All variants were classified as either benign or likely benign, with some lacking data. A comprehensive summary of the literature pertaining to the identified variants is presented in the Supplementary material. The phenotypes of liver injury associated with these variants vary widely, and a definitive correlation between the genotypes and the cholestatic episodes cannot be established at this time.

Nonetheless, we posit that ATP8B1 is a promising candidate gene contributing to cholestatic susceptibility in our patients. The rare variants rs319438 and rs222581 have previously been associated with intrahepatic cholestasis[5,8]. Supporting a potential role of ATP8B1 in Patient 2, it has been reported that mutations in ATP8B1 lead to decreased FXR1 activity[10]. This patient had a variant form of NR1H4 (Figure 1B). The farnesoid X receptor plays a central role in the regulation of bile acid metabolism, and clinically relevant severe mutations are now classified as PFIC type 5[11].

Moreover, the significantly more pronounced hyperbilirubinemia in Patient 2 compared to Patient 1 (Figure 1A) aligned with previous findings associating rs319438 and rs222581 of ATP8B1 with severe cholestatic liver disease[12,13]. Although a considerable number of other variants (Figure 1B) were found in our patients, we do not believe that they are a major cause of the cholestatic episodes. Functional variants of ABCB4 typically lead to elevated GGT levels[14], which were not observed in our patients. Variants in MYO5B typically lead to altered canalicular morphology and abnormal BSEP immunohistochemical staining, both of which were not observed in our patients. Based on the current literature, our sequencing information, and the immunohistochemical staining results, the two variants of ATP8B1 appear to be the most likely genetic factors predisposing these patients to HEV-triggered intrahepatic cholestasis.

Treatment

Treatment in the present cases was based on the pregnane X receptor agonist rifampicin and the peroxisome proliferator-activated receptor agonist bezafibrate. Rifampicin upregulates the expression of detoxification enzymes as well as hepatocellular export pumps including the apical transporter MRP2 and basolateral OSTb in hepatocytes[15,16]. Bezafibrate, through the activation of peroxisome proliferator-activated receptors, modulates lipid metabolism and anti-inflammatory responses[17]. The induction of these genes increases the elimination of bilirubin conjugates and bile salts and represents a therapeutic strategy in intrahepatic cholestasis. Furthermore, both drugs alleviate pruritus and are included in current treatment guidelines for cholestatic pruritus. Although their combined use may theoretically offer synergistic benefits, we advise caution due to the potential risk of hepatotoxicity with either agent. In contrast, ursodeoxycholic acid can be safely added as a baseline therapy for intrahepatic cholestasis.

CONCLUSION

After excluding common causes of cholestatic liver damage in the clinical workup, hereditary causes of cholestasis including disorders of hepatobiliary transporters represent an important differential diagnosis. In addition to known triggers, our case reports unravel HEV infection as a novel trigger for intrahepatic cholestasis. Based on our sequencing data, the ATP8B1 variants identified in both patients are likely susceptibility factors for this cholestatic phenotype. However, to confirm the pathogenic relevance of these variants, further evidence is required—either through the identification of additional patients carrying these genetic alterations or through functional studies demonstrating their biological impact.