Progressive Familial Intrahepatic Cholestasis: A Study in Children From a Liver Transplant Center in India

Abstract

Background/Aims

This study aimed to delineate the clinical profile of children diagnosed with progressive familial intrahepatic cholestasis (PFIC).

Methods

This study was a retrospective analysis of case records of children in the tertiary care hospital, with the diagnosis of PFIC from January 2017 to January 2020. The diagnosis was made using clinical and laboratory parameters and with genetic testing when available. Medical and surgical management was according to the departmental protocol. Liver transplant was offered to children with end-stage liver disease, intractable pruritus, or severe growth failure.

Result

There were 13 identified PFIC cases (familial intrahepatic cholestasis 1 [FIC1] deficiency-4, bile salt export pump (BSEP) deficiency-3, tight junction protein [TJP2] deficiency 3, multidrug-resistant protein 3 [MDR3] deficiency 2 and farnesoid X receptor deficiency-1). PFIC subtypes 1, 2, and 5 presented in infancy, whereas MDR3 presented in childhood. TJP2 deficiency had varied age of presentation from infancy to adolescence. Jaundice with or without pruritus was present in most cases. Genetic testing was carried out in 10 children, of which five had a homozygous mutation, three had a compound heterozygous mutation, and two had a heterozygous mutation. Three children (FIC1-2 and TJP2-1) underwent biliary diversion, of which clinical improvement was seen in two. Six children underwent liver transplantation, which was successful in four.

Conclusion

Byler's disease was the most common subtype. A clinicopathologic correlation with molecular diagnosis leads to early diagnosis and management. Liver transplantation provides good outcomes in children with end-stage liver disease.

What is known

• •Progressive familial intrahepatic cholestasis is a group of genetic disorders with varying clinical presentation ranging from progressive neonatal cholestasis to end-stage liver disease in adolescents.
• •Management includes medical and surgical interventions (biliary diversion) and liver transplantation.

What is new?

• •Next-generation sequencing may play an important role in the early identification of PFIC subtypes with cryptogenic cholestasis or cirrhosis, especially in developing countries with limited resources.
• •Living related donor transplant offers a good outcome in advanced liver disease in PFIC.

Progressive familial intrahepatic cholestasis (PFIC) is a family of a heterogeneous group of rare autosomal recessive liver disorders. It represents major defects in bile acids handling, presenting as intrahepatic cholestasis in infancy or early childhood and resulting in end-stage liver disease (ESLD), and death without liver transplantation in infancy to adulthood.¹

Recently, genetic advances have led to expansion of disease characterization into more subtypes of PFIC, apart from traditionally known variants—FIC1 deficiency (Byler disease, ATP8B1), bile salt export pump (BSEP) deficiency (ABCB11), and MDR3 deficiency (ABCB4). It is now understood that PFIC has been ever-growing family of diseases with different responses to medical and surgical modalities. The disease is classified on the basis of genetic defect involved in bile transport (ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, and MYO5B). The TJP2 deficiency is caused by the mutation in the TJP2 gene on chromosome 9q12, and farnesoid X receptor (FXR) deficiency is caused by mutation in the NR1H4 gene on chromosome 12q. MYO5B mutation leads to deficiency of MYO5B (PFIC6) in the absence of microvillous inclusion disease.¹

The clinical features of PFIC include cholestasis with pruritus and growth failure. Complications include portal hypertension (PH), liver failure, cirrhosis and hepatocellular carcinoma, and extrahepatic manifestations in FIC1 deficiency. The diagnosis of PFIC has been based on a combination of clinical and laboratory or biochemical approaches, but more recently, genetic testing has become a useful modality.¹ The medical management involves ursodeoxycholic acid (UDCA), fat-soluble vitamin supplementation, bile acid sequestrants, and symptomatic treatment for pruritus. Invasive surgical procedures are used to reduce bile acid concentration. Liver transplant (LT) is indicated in ESLD, growth failure, and intractable pruritus.¹

Methods

A retrospective review of 13 unrelated children and adolescents aged <18 years, diagnosed as PFIC between January 2017 and January 2020, was carried out. This study was conducted in the Department of Pediatric Gastroenterology and Hepatology at Indraprastha Apollo Hospital, New Delhi. The study was approved by the Institutional Review Board.

The diagnosis of PFIC in infants and children with jaundice and/or pruritus and cryptogenic cirrhosis was made on the basis of histopathology and/or genetic analysis. A liver biopsy was done in 11 of 13 children; the diagnosis of PFIC was made on clinical and biochemical grounds and confirmed using a genetic test. We did not perform tissue immunostaining at our center. Genetic analysis with next-generation sequencing (NGS) was used for 11 patients after taking appropriate consent.

Upper gastrointestinal (GI) endoscopy was done in cases with clinical evidence of PH. The medical management involved nutritional care, multivitamin supplementation and treatment of co-existing fat-soluble vitamin deficiencies. Pruritus was managed symptomatically with antihistaminics and UDCA with or without bile acid sequestrants; additional drugs such as rifampicin and ondansetron were added in unresponsive cases.

Surgical intervention (biliary diversion [BD]) was offered to children with intractable pruritus unresponsive to maximal medical treatment. Living donor liver transplant (LRLT) was performed in children with ESLD, uncontrolled variceal bleeding, intractable pruritus, or severe growth failure. Postsurgery/post-LT children were monitored for clinical improvement using laboratory and radiological parameters. The explant specimens in children who underwent LT were examined histologically. Graft dysfunction in post-LRLT period was investigated using radiology and/or liver biopsy.

Result

Thirteen children (boys, n = 8) with PFIC were identified. FIC1 deficiency was the most common subtype (n = 4), followed by BSEP and TJP2 deficiency subtypes (n = 3 each), and MDR3 disease (n = 2). There was a single case of NR1H4 mutation (PFIC5). Seven children presented to our center for undiagnosed cholestasis, whereas the remaining were referred to our institution for LT. The mean age of presentation was 47.79 months (3–180 months). The median age of onset of symptoms was 3 months (range 3–6 months) for the FIC1 deficiency, 1 month for BSEP disease (range 1–2 months) and 3 months for TJP2 mutation (range 1–48 months). Two children with MDR3 deficiency had onset of symptoms at 5 years and 9 years, whereas the child with FXR deficiency (NR1H4) developed symptoms in the first week of life. The median time lag from the onset of symptoms to diagnosis was 10 months (2.5–132 months).

The initial presentation of cholestasis in the form of jaundice (91.66%) was present in all the cases except in a child with TJP2 who presented with severe pruritus. Itching on presentation was present in 11 of 13 children (except in children with MDR3 and FXR deficiencies; 84.61%). On presentation, hepatomegaly was present in 10 cases, whereas splenomegaly was present in nine cases.

Extrahepatic manifestation in the form of recurrent diarrhea since infancy was present in two of four children with Byler's disease. Two of three children with BSEP deficiency presented with the decompensated liver disease before the first year of life; one of them received LT, whereas the other died of sepsis awaiting LT. Of the two children with MDR3 disease, one presented as cryptogenic cirrhosis with decompensation (acute variceal bleeding) at 11 years, whereas the other had pruritus, jaundice, and growth failure with decompensation (ascites) at 10 years of age. Pruritus was predominantly present in all three cases of TJP2, but the clinical presentation was varied. Two of these three children showed features of decompensation (ascites and growth faltering) before infancy, whereas the third case presented with cryptogenic cirrhosis in adolescence. He developed variceal bleed and is on medical management and endoscopic treatment of esophageal varices. The only child with NR1H4 mutation presented with uncorrected coagulopathy and low gamma-glutamyl transferase (GGT) cholestasis, succumbing at 9 months of age to ESLD awaiting LT.

We did not find any significant antenatal history of cholestasis in mothers of any of our cases. Third-degree consanguinity was noted in 7 of 13 cases (53.84%) and second degree in one case. Only one child with BSEP deficiency was born preterm (34 weeks), weighing 1.6 kg, whereas all others were born at term with an average weight of over 2.7 kg.

The clinical vitamin D deficiency was seen in 6 of 13 children. Two children with FIC1 deficiency presented with severe form of vitamin D deficiency, that is, hypocalcemic tetany and pathological femoral fracture. A case with TJP2 mutation presented with severe deficiency of fat-soluble vitamins (A, D, and K); he developed an intracranial hemorrhage in the form of subdural hematoma after trivial trauma that necessitated surgical drainage at the age of 2 months. He also had a unilateral corneal ulcer at the age of 6 months that responded to vitamin A supplementation.

Significant liver function and hemogram of all the patients is given in Table 1. All the cases with FIC1, BSEP, and FXR deficiencies and two of three TJP2 mutations had normal GGT cholestasis; both the children with MDR3 deficiency had high GGT cholestasis. Liver biopsy was evaluated in 11 of 13 children; common features are reported in Table 2.

Table 1.

Significant Laboratory Parameters of Children With PFIC.

Parameters	FIC1 deficiency (n = 4)				BESP deficiency (n = 3)			MDR3 (n = 2)		TJP2 deficiency (n = 3)			FXR deficiency
	1	2	3	4	1	2	3	1	2	1	2	3	1
Total bilirubin (mg/dL)	6	7.2	2.8	4.5	26.9	17.3	9	25.3	6.6	15.1	2.5	16.2	24.1
DB (mg/dL)	4.6	5.9	2.5	4	17.4	13	6.2	18.4	5.4	11.1	1.7	12	16.1
AST (IU/L)	96	62	97	71	254	286	283	155	163	918	49	272	247
ALT (IU/L)	91	35	122	37	82	319	140	109	83	410	60	245	112
ALP (IU/L)	650	613	608	1681	466	270	587	216	529	490	342	612	434
GGT (IU/L)	17	13	38	11	44	37	34	140	330	75	34	245	34
ALB (g/dL)	4.1	4.0	4.2	4.5	3.2	3.9	3.9	1.9	3.4	3.9	3.5	3.6	3.0
INR	0.9	0.9	1.2	1	2.5	1.5	1.9	2.0	1.7	1.4	1.39	1.1	2
Hemoglobin (g/dL)	10	10.3	11.3	11.6	10	7.1	9.4	9.0	12.5	9.2	12.5	11.5	8.7
TLC per cmm	13.2	9.5	18.3	12.6	10.9	25.2	11.3	5.0	3.95	18.8	4.43	14.2	6090
PC (×103) per cmm	373	484	400	519	81	268	262	66	45	335	57	225	133
Vit D (ng/mL)	–	–	<3	–	–	11.4	–	9.7	16	–	–	9.49	–

DB, direct bilirubin; AST, aspartate transaminase; ALT, alanine transaminase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transferase; ALB, albumin; INR, International Normalized Ratio; TLC, total leucocyte count; PC, platelet count; Vit D, vitamin D.

Table 2.

Liver Biopsy Features of 11 Children With PFIC.

Liver biopsy Important features	FIC1 deficiency( n = 4)	BSEP deficiency( n = 3)	MDR3( n = 2)	TJP2 deficiency( n = 2)
Cholestasis	4 (panlobular canalicular)	3 Intrahepatocytic and canalicular 2	2	Intrahepatic 1
Inflammation	0	Focal lobular (2) Mild 1 Significant lymphocytic 1 Rosetting and ballooning 1	0	Moderate lymphocytic in portal 1 Chronic portal inflammatory cell with interface activity 1
Giant cell transformation	0	3	0	0
Bile ductular proliferation	0	0	1	0
Paucity of bile ducts	3	0	1	0
Fibrosis Ishak score	0	F3-1 F0-2	F1 F5	F5-1 F4-1
Lobular architecture	Maintained (4)	Disarray (3)		Disrupted 2

Cryptogenic cirrhosis was seen in three children referred for LT evaluation—2 children were found to have TJP2 mutation, whereas one had an ABCB4 mutation (MDR3 deficiency). Two of these cases underwent LRLT, and the other is awaiting LT.

Upper GI endoscopy was performed in cases with clinical or radiological evidence of PH (n = 9); six children showed mild esophageal varices with or without portal gastropathy. Three children (MDR3: 2 cases and TJP2: 1 case) presented with hematemesis; all grade 3 varices with a red color sign requiring endoscopic variceal ligation sessions.

We evaluated targeted gene sequencing of 11 children and found a homozygous mutation in five children and compound heterozygous mutation in three cases (Table 3). In a child with a clinical picture of BSEP deficiency, we found a single heterozygous missense mutation in the ABCB11 gene. A child with cryptogenic cirrhosis had heterozygous mutations in the TJP2 gene and tRNA mitochondrial 2-thiouridylase (TRMU) gene. Of interest, a child with a homozygous mutation in ATP8B1 gene also had an additional variant of a heterozygous mutation in TJP2 gene.

Table 3.

Genetic Tests of 11 Children With PFIC.

Parameters	Case 1	Case2	Case 3	Case 4	Case 5	Case 6	Case 7	Case 8	Case 9	Case 10	Case 11
Gene	ATP8B1, TJP2	ATP8B1	ATP8B1	ABCB11	ABCB11	ABCB11	ABCB4	TJP2	TJP2	TJP2, TRMU	NR1HA
Classification	Homozygous, heterozygous	Homozygous	Homozygous	Heterozygous	Homoygous	Heterozygous	Heterozygous	Homozygous	Heterozygous	Hetero, heterozygous	Homozygous
Mutation	c.1933-1G>T, intron 17; c.932G>A, exon 5	c.2198G>A	1804C > T	c.2693G>A, exon 22; c.1156 T > A, exon11	c.477+5G>C	c.3959 T>A, Exon 28	c.1529 A > G, Exon 13 c.370G>C, Exon 6	c.2380delA, exon 17	c.2465 T > C, exon 17 CNV deletion, exon 1-16	c.296C > T, exon 4 c.993C > G, exon 9	c.557G>A, Exon 3, missense
Significance	Pathogenic, VOUS	VOUS	VOUS	Pathogenic (both)	Pathogenic	VOUS	VOUS	Pathogenic	VOUS	VOUS	Pathogenic

TJP2, tight junction protein 2; TRMU, tRNA mitochondrial 2-thiouridylase; VOUS, variation of uncertain significance.

Surgical intervention by BD was done in three children (FIC1: two cases and TJP2: one case) as a measure for intractable pruritus despite maximal medical management; partial external BD (PEBD) was done in a child with TJP2 mutation (genetic diagnosis was available later), and partial internal BD (PIBD) was performed in both the children with FIC1 deficiency. In the child with TJP2 mutation, BD was done despite liver biopsy suggesting stage 5 fibrosis. All the children reported an improvement in pruritus transiently only. The child with a TJP2 mutation ultimately required LRLT after 13 months of BD in view of intractable pruritus.

Table 4 shows the clinical data of patients who underwent LRLT. Of 13 cases, six children (four boys) underwent living related donor transplantation at our institution (FIC1: 1 and BSEP: 1 and MDR3 and TJP2: two cases each). Either parent was the liver graft donor in five children and a maternal aunt in one case. The age at LT, ranged from 19 to 190 months, was early for BSEP and TJP2 diseases. Pediatric ESLD scores ranged from 5.1 to 22.9, and the model of ESLD score of an adolescent was 32. We encountered two deaths in immediate postoperative period; a child with FIC1 suffered hyperacute graft rejection and succumbed in the first week post-LT. The other adolescent with MDR3 had liver dysfunction with hyperbilirubinemia and normal synthetic function in the post-LT period. A post-LT liver biopsy showed bland cholestasis without any features of inflammation or cellular rejection. He succumbed because of sepsis at 3 months post-LT. A child with TJP2 mutation suffered hypoxic ischemic injury with seizures requiring antiseizure medications. Other children did not have any post-LT complications and showed resolution of original symptoms and normal graft function till the latest period of follow-up. On explant tissue analysis of LT recipients, micronodular cirrhosis with intrahepatocytic bile accumulation and canalicular bile plugs was seen in all. There was no evidence of malignancy in any patients (Supplemental Table I).

Table 4.

Clinical Profile of Children With PFIC Who Underwent Living Related Liver Transplant.

No	G	Age (months)	PFIC	PELD/MELD#	Donor	Outcome	Complication
1	M	54	1	5.1	Mother	Died	Hyperacute graft rejection
2	F	23	2	22.9	Aunt	Alive	Nil
3	M	130	3	17.2	Mother	Alive	Nil
4	M	31	4	11.9	Mother	Alive	HIE with seizures
5	F	19	4	17.4	Mother	Alive	Nil
6	M	190	3	32#	Father	Died	Bland cholestasis, sepsis

G gender; M, male; F, female; PELD, pediatric end-stage liver disease; MELD, model of end-stage liver disease; HIE, hypoxic ischemic encephalopathy.

^#Age more than 12 years.

Discussion

The literature on PFIC from India is scarce; the largest series of 25 children has been published earlier from northern India.² In this study, we have described relatively newer subtypes of PFIC using clinical exome sequencing and our center's experience with the PFIC group of illnesses. The clinical spectrum of PFIC ranges from mild disease to severe ESLD, requiring liver transplantation.¹ The higher number of children with ESLD in our study may be because of referral bias because our center is a tertiary care hospital with transplant facility.

The median age of onset for FIC1 and BSEP deficiency was 3 months and 2 months, respectively. This is in line with recent studies,^2,3 which represents the onset of disease in early infancy. On the other hand, both the cases of PFIC3 presented in the first decade, at 5 years and 9 years respectively. The main function of TJP2 is to prevent back diffusion of bile salts from canaliculi to the blood circulation.^3,4 In this study, two of the three cases with TJP2 presented in infancy, whereas one presented in childhood. This is similar to a previous series of cases with TJP2 disease.⁵ A child with homozygous NR1H4 mutation (FXR deficiency) presented early with progressive jaundice and vitamin K–independent coagulopathy. This gene encodes FXR, which regulates BSEP, and also induces fibroblast growth factor 19 to inhibit the synthesis of bile acids. The biochemistry showed markedly high bilirubin levels, transaminitis, normal GGT level, and uncorrectable coagulopathy. This presentation was similar to that noted by Gomez-Ospina et al.⁶ This is the first case of FXR deficiency to be reported from India.

Similar to a previous study, term delivery with normal birth weight was seen in most cases. The median delay in diagnosis of the disease was 8 months (2.5–132 months).² The lack of awareness among general pediatricians about PFIC may be the reason behind this. FIC1, BSEP, and TJP2 deficiencies are characterized by normal to low GGT with direct hyperbilirubinemia, whereas MDR3 is associated with high GGT cholestasis. These observations were consistent in our study.^7,8

Rickets due to cholestasis was seen in 50% of our cases. A child, with TJP2 mutation, had a subdural hematoma after trivial trauma in early infancy. Haney et al. reported a case of PFIC2 with an intracranial bleed secondary to vitamin K deficiency mimicking abusive head injury.⁹ Thus, nutritional deficiencies in children with cholestatic liver disease, especially PFIC, need utmost attention.

Histopathological findings in FIC1 deficiency showed bland cholestasis with maintained lobular architecture, whereas in BSEP deficiency, typical features included intrahepatic cholestasis with giant cell formation and lobular disarray. In MDR3 disease, bile ductular proliferation was prominent along with portal fibrosis suggestive of advanced disease.⁷ Both the children with TJP2 mutation showed disrupted lobular architecture and significant portal fibrosis; intrahepatic cholestasis was noted in one specimen only.¹⁰

In the present study, NGS was helpful in identifying PFIC in children with cryptogenic cirrhosis referred for liver transplantation. A recent review highlighted the use of NGS and whole exome sequencing (WES) in establishing etiologies, especially with the history of itching, of unexplained cholestasis caused by specific genetic pathways.¹¹ A child with clinical and laboratory features suggestive of BSEP disease was found to have one heterozygous missense mutation in the ABCB11 gene. This may be attributed to a pathogenic mutation outside sequenced areas. Mutations in other genes, epigenetic changes, or environmental factors may contribute to the severity of the phenotype.¹³ Similarly, a child with cryptogenic cirrhosis and pruritus, on NGS, showed heterozygous mutations in the TJP2 gene and TRMU gene. We suspect that these two mutations led to chronic liver disease or an unidentified second mutation in the TJP2 gene missed by NGS. Financial constraints limited the possibility of whole genomic sequencing or Sanger sequencing of targeted areas in the index case or parents.

Experience with PIBD in PFIC is evolving; a recent study showed it to be a safe procedure in children without cirrhosis.¹⁴ Squires et al. reported recurrent episodes of pruritus to be common after PEBD, which does not lead to the progression of liver fibrosis. This may be the reason for the transient resolution of pruritus in both the children with FIC1. Further follow-up is needed to ascertain the progress after BD. In the third child, PEBD was performed despite fibrosis because of several factors—the family had financial constraints and belonged to a resource-limited country. Lack of improvement after BD may be because of the original disease (TJP2 mutation) and the presence of liver fibrosis.

The indications for LT in PFIC include severe pruritus, significant growth retardation, liver cirrhosis, and liver failure.^7,15 It corrects the genetic defect and reverses many, if not all the effects of chronic liver disease. Graft survival and patient survival were 76.6% and 85.2%, respectively, with the longest reported follow-up interval being 19 years posttransplantation.¹⁶ A previous study reported inadequate outcomes in patients with FIC1 disease because of worsening of diarrhea, steatohepatitis, and fibrosis in LT recipients.¹⁷ In this study, the only child with PFIC1, whose donor was his mother, had a hyperacute graft rejection and ultimately died. We did not do a genetic mutation study in this patient, which could have ascertained the mutation-specific disease behavior in the posttransplant period.

The role of LT in PFIC2 is well documented with favorable outcomes, but the recurrence of disease secondary to anti-BSEP antibodies to donor liver is known.¹⁶ The only child with PFIC2, in this study, recovered well post-LT and is being followed up for over 12 months. In our series, two children with PFIC3 underwent LRLT, and we reported one death. Kaur et al. reported grade 1 acute graft rejection in a child with PFIC3 after LT.¹⁸ The survival of patients post-LT has been reported to be excellent. The other child is alive and well during 30 months of follow-up. There is scanty literature about LRLT in TJP2 disease. Both children in the present study had rapid progression to ESLD, thus requiring LT early. One child who had failed PEBD before LT developed hypoxic ischemic encephalopathy with seizure in the immediate postoperative period. Both children have reported adequate graft function till the last follow-up (6 months and 8 months). Other studies have reported successful LT in patients with TJP2; however, long-term follow-up is lacking.⁵

PFIC constitutes an important cause of liver disease in the pediatric population, and early diagnosis is vital for timely management. When available, NGS can help identify specific genetic mutations and defects involved in bile handling pathways and response to the treatment. Cost-effectiveness and clinical utility of NGS and WES in PFIC need to be evaluated in the Indian scenario. However, there are inherent limitations of these investigations, such as inadequate coverage of disease-causing mutations, which need adequate consideration. In centers where LRLT facility is available, an early transplant in children with ESLD can have better outcomes.

Data sharing statement

No additional data available.

Registration of study

Not applicable.

Credit authorship contribution statement

S.M. contributed to conception, data acquisition, analysis, and drafting of the article; Karunesh Kumar: Data analysis and interpretation; Ravi Bhardwaj: Data acquisition; Smita Malhotra: revision of manuscript; Neerav Goyal: Data interpretation and revision of manuscript; Anupam Sibal: Final approval before submission.

Conflicts of interest

The authors have none to declare.

Funding

None.

Appendix A. Supplementary data

The following is the Supplementary data to this article: