Background

Progressive familial intrahepatic cholestasis (PFIC) is an ever-growing group of autosomal recessive liver disorders caused by defects in genes associated with bile secretion, bile salt and lipid transporters and regulators [1, 2]. Disease-causing genes of PFIC were gradually revealed, including ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, and MYO5B (named PFIC1 to 6) [3,4,5,6]. NR1H4 gene (OMIM *603,826), located on 12q23.1, encodes the farnesoid X receptor (FXR), a bile acid (BA)-activated transcription factor, and plays an essential role in BA homeostasis [7]. Biallelic pathogenic variants in NR1H4 were first identified in low-γ-glutamyl transferase (GGT) cholestasis patients and termed as PFIC5 in 2016 4. So far, only 10 patients with PFIC5 from six unrelated families have been reported, usually presenting as rapidly progressive liver failure, vitamin K independent coagulopathy, high alpha-fetoprotein (AFP) and ultimately required a liver transplant (LT) to save lives [4, 8,9,10]. To assess phenotypic spectrum and clinical outcomes in NR1H4-related PFIC, we studied our patients in detail and reviewed previously reported patients in the literature.

Methods

Subjects

Our patients were all Chinese children referred to the Center for Pediatric Liver Disease, Children’s Hospital of Fudan University from February 2016 to March 2023. Genetic testing, either whole-exome sequencing or liver panel sequencing, was performed in patients after excluding other causes of liver diseases (including infection, drug exposure, autoimmune hepatitis, and biliary atresia) [11]. Cytomegalovirus infection was not excluded due to its high prevalence in Chinese infants. When other known inherited liver disorders were excluded, patients with two or biallelic NR1H4 pathogenic/likely pathogenic/uncertain significance (P/LP/US) variants were collected. P/LP/US variants were classified according to the American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP) criteria [12].

Genetic analyses and in silico assessment of NR1H4 variants

We confirmed these variants by Sanger sequencing and confirmed parental origins when available. Assessment of variant pathogenicity were performed with seven in silico tools including MutationTaster (http://www.mutationtaster.org/), Sorting Intolerant From Tolerant (SIFT, http://sift.jcvi.org), Rare Exome Variant Ensemble Learner (REVEL, https://labworm.com/tool/revel), MutPred (http://mutpred.mutdb.org/), and Protein Variation Effect Analyzer (PROVEAN, http://provean.jcvi.org/index.php). Two programs, SpliceAI (https://spliceailookup.broadinstitute.org/) and varSEAK (https://varseak.bio/), were used to evaluate the effect of variants on mRNA splicing. Default settings were used for all in silico tools.

Literature review

A comprehensive literature review of the current literature was performed on March 2023 by searching PubMed (https://pubmed.ncbi.nlm.nih.gov/), CNKI (https://www.cnki.net/), and Wan fang (https://www.wanfangdata.com.cn/) databases using the keywords “NR1H4 variants, progressive familial intrahepatic cholestasis 5, and PFIC5”.

Results

Identification of biallelic NR1H4 variants in 3 new patients and in silico assessment

We identified 5 unique NR1H4 variants from 3 unrelated Chinese patients with low GGT intrahepatic cholestasis from our cohort. Among these, there were 3 missense variants, one nonsense variant, and one canonical splicing variant. All variants have not been previously reported in medical literatures (See Table 1). Three out of 5 variants were absent in gnomAD (c.505T > A/p. (Cys169Ser), c.1235T > C/p. (Leu412Pro), and c.1066 + 1G > A/p.?). The other two were present in gnomAD, with a population frequency of 0/1/251,458 (number of homozygotes/allele count/allele number) for the c.688 C > T/p. (Arg230Ter) variant and 0/1/31,410 (number of homozygotes/allele count/allele number) for the c.527G > A/p. (Arg176Gln) variant. MutationTaster predicted that the nonsense variant (c.688 C > T/p. (Arg230Ter)) may lead to nonsense-mediated mRNA decay (NMD). SpliceAI and varSEAK predicted that the canonical splicing variant (c.1066 + 1G > A/p.?) lead to loss of donor splice site, most likely leading to protein truncation. Three missense variants were predicted to be pathogenic by five pathogenicity prediction tools, and have no effect on pre-mRNA splicing. According to the ACMG/AMP criteria, all of the variants were classified as P/LP/US (Table 1).

Table 1 NR1H4 variants and the results of in silico pathogenicity prediction
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Clinical features and outcome of 3 new patients with NR1H4-related cholestasis

All 3 male patients born at full-term with normal weight to non-consanguineous parents following uneventful pregnancies. The first child in family II died of unexplained liver disorders at the age of 7 months, whereas the other families did not have positive family history. All of them presented with jaundice during the first few days after birth. Normal growth and development were observed in patient 1(P1), and failure-to-thrive occurred in patients 2 (P2) and 3 (P3). Our patients have similar clinical features resembling previously reported patients, including low-GGT cholestasis, rapidly progressive liver failure/decompensated cirrhosis, vitamin K independent coagulopathy, and markedly elevated AFP levels (Table 2). Initial, pre-transplantation, or pre-death laboratory testing results were shown in Table 3. In addition, our patients had recurrent severe pneumonia, splenomegaly, and elevated urinary microalbumin. Two patients (P1 and P3) had hepatomegaly and hydrocele.

Table 2 Clinical features and outcomes of our 3 patients with NR1H4-related cholestasis
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Table 3 Initial and pre-transplantation/pre-death laboratory testing of our 3 patients with NR1H4-related cholestasis
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The patient P1 suffered massive ascites from decompensated cirrhosis prior to liver transplantation (LT). He had a positive plasma Epstein-Barr virus DNA from 2 months post-LT until the last visit. He also underwent a single surgery due to small bowel obstruction and right-sided diaphragmatic hernia one year after LT. The patient P3 presented with hyperammonemia due to acute liver failure (ALF) and his magnetic resonance of the brain showed minor abnormalities such as widened extracerebral space and cavum septum pellucidum.

Only one patient (P1) was treated by LT at the age of 4 months, and the other two patients died of infection at 3 months of age. Up to the latest assessment, P1 had normal liver function at one-year post-transplant.

Clinical and genetic characterization of 13 NR1H4-related PFIC patients

A total of 13 patients from 10 unrelated families were collected, with 3 new and 10 reported patients (Supplementary Table 1) [4, 8,9,10]. Eight were males, 4 were females, and one patient’s gender was unknown. All patients were delivered at full term without maternal or fetal complications. The age of onset ranged from the neonatal period to 17 months, with the median age of onset being 7 days after birth. Twelve out of 13 patients showed neonatal jaundice in the early neonatal period (7 patients, 0–6 days) and late neonatal period (5 patients, 7–28 days), respectively. Only one patient was admitted to hospital due to jaundice and abdominal distention at the age of 17 months. Reported clinical manifestations include: cholestasis (13/13, 100%), persistently elevated AFP (11/11, 100%), coagulopathy (11/11, 100%), hypoglycemia (9/13, 69%), splenomegaly (7/13, 54%), hyperammonemia (7/13, 54%), failure to thrive (8/13, 62%), and hepatomegaly (6/13, 46%). Six of 13 patients received LT at a median age of 6.2 months (range 2–20). Only one patient died of acute infection at one year after LT. Five out of 6 patients with LT are still alive, with a median age of 6 years (range 1.3–10). Other 7 patients without LT died at a median age of 5 months (range 1.2-8). The causes of death included ALF, multiple organ dysfunction syndrome (MODS), sepsis, and others (Supplementary Table 1).

Eight patients had homozygous genotype and 5 patients had compound heterozygous genotype. In total, 13 different variants were detected, including 4 nonsense variants, 4 missense variants, 2 frameshift variants, one splice site variant, one in-frame insertion variant, and one large DNA fragment deletion variant. Overall, there were five variants (41.6%) in exon 5, two variants (16.7%) in exon 4, one variant (8.3%) in exon 6, one variant (8.3%) in exon 8, one variant (8.3%) in exon 9, one variant (8.3%) in intron 9 and one variant (8.3%) in exon 11 (Figure 1).

Fig. 1
figure 1

Schematic presentation of the farnesoid X receptor (FXR) protein and locations of 12 unique single or multiple nucleotides variants identified in our cohort and literature. The exon 5 was depicted in red, and other exons of the NR1H4 gene are depicted in blue; FXR protein domains are shown in green

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Liver pathological characteristics in NR1H4-related PFIC patients

Liver pathology was documented in 7 of 13 NR1H4-related PFIC [8, 9]. Characteristic pathological features included cholestasis, steatosis, micro-nodular cirrhosis, hepatocellular ballooning, fibrous tissue proliferation, fibrosis, inflammatory cell infiltration, and proliferation of bile ducts. Immunohistochemical stainings of both bile salt export pump (BESP) and FXR proteins were absent, and the multi-drug resistance protein 3 (MDR3) expression was decreased or normal in all 7 patients.

Discussion

It was first discovered 7 years ago that the NR1H4 was responsible for PFIC [4]. Previous studies showed that the NR1H4-associated PFIC had early-onset and rapid disease progression with high mortality [4, 8]. As only few patients have been reported, current understanding of PFIC5 caused by NR1H4 defect is limited. Therefore, we performed a retrospective analysis to obtain a better understanding of clinical phenotype and outcomes of PFIC5 caused by NR1H4 in our center and literature.

The FXR, encoded by NR1H4 gene, as the master regulator of BA homeostasis, regulates BA homeostasis, biliary BA secretion, and intestinal re-absorption [13,14,15]. Compared with other PFIC patients, the PFIC5 patients caused by NR1H4 defect had significantly worse prognosis due to more rapid progression [8, 16, 17]. All patients without LT died and survival with native liver has not been observed. Three new patients in our cohort exhibited similar clinical characteristics as published case [7, 8]. The liver phenotypes of all reported patients were extremely similar and presented as low GGT neonatal cholestasis with rapid progression to ALF (with/without related complications such as hypoglycemia, hyperammonemia, coagulopathy, hepatosplenomegaly, hydrothorax, and ascites) [8, 9].

While NR1H4 gene is predominantly expressed in liver and intestine, it also presents in kidney, spleen, heart, gallbladder, pancreas, adrenal glands, bone marrow, and other tissues [18,19,20,21]. We further summarized the extra-hepatic phenotypes of 13 patients. Of those, the failure to thrive was the most common finding. Other extrahepatic manifestations were also described, such as atrial septal defect, butterfly vertebra, decreased bone mineral density, intestinal obstruction, diaphragmatic hernia, inguinal hernia, and iris coloboma. Although NR1H4 is highly expressed in the intestine, recurrent or severe diarrhea has not been observed in patients with NR1H4-related disorder [8,9,10].

Notably, pharmacological therapy is typically not effective for NR1H4 disease [8]. Those patients without LT died in early infancy, and the common causes of death included ALF, MODS, and severe infection [8]. Therefore, LT may be the only curative option. Fortunately, 5 out of 6 patients are still alive after LT without serious postoperative complications, and with good clinical outcome during the follow-up. Only one patient died of acute infections one month after the transplant [9], this suggests that minimizing the risk of infection is the key to reduce morbidity and mortality associated with LT for PFIC5 patients [22, 23].

All enrolled patients had poor prognosis with native liver. So, we were not able to analyze the relationship between genotypes and phenotype. However, we observed that 41.6% of all reported variants are located on the exon 5 of NR1H4 gene. Exon5 encodes a highly conserved DNA binding domain of FXR by binding to specific DNA sequences called hormone response elements, thereby possibly regulating other gene expression [24, 25]. More cases and further studies are needed to confirm whether exon 5 is a susceptible or hotspot region for NR1H4 gene mutation.

Conclusions

NR1H4-related PFIC is characterized by severe neonatal cholestasis, rapid progression to liver failure, and early death. LT might be the only lifesaving therapy that can lead to cure. At present, no severe complications of LT related to NR1H4 gene were observed, but long-term outcome of LT still needs to be validated in more patients.