Whole-exome sequencing identifies novel mutation in intrahepatic cholestasis of pregnancy: A case report and literature review

Abstract

Objective

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disease characterized by pruritus and elevated total bile acid (TBA) levels. The most serious impact of ICP is sudden unexplained intrauterine fetal death, especially when an associated TBA ≥ 100 µmol/L is confirmed.

Methods

We report a case of a 27-year-old female patient with early-onset severe refractory ICP. Whole-exome sequencing and mutation analyses were performed to obtain genetic data on the patient and her mother. Sanger sequencing was performed to screen the mutation site. Computer-based algorithms were applied to predict the pathogenesis of the identified mutation. Subsequently, we conducted a literature review to characterize the pathological features and perinatal management of severe refractory ICP, especially ICP with genetic susceptibility.

Results

A heterozygous mutation in the ABCD3 gene: c.130C > T/p.Pro44Ser was detected in this patient. Through the analysis of pathogenicity prediction software, the mutations were disease-causing. This is the first report to identify the novel p.Pro44Ser mutations of ABCD3 gene in ICP patients.

Conclusions

Our report provides new insights into the genetic architecture of ICP involving ABCD3 variants. Early-onset severe refractory ICP is rare and mutations in bile acid metabolism genes might accentuate the phenotype. Emphasized perinatal management and screening for potential pathogenicity sites of variants that drive specific recognition of ICP is necessary.

Keywords

intrahepatic cholestasis of pregnancy - bile acids - ABCD3 - ursodeoxycholic acid - mutation

Introduction

Intrahepatic cholestasis of pregnancy (ICP) is the most common pregnancy-related liver disease and ICP is diagnosed based on a peak nonfasting total bile acid (TBA) concentration ≥19 μmol/L and/or fasting TBA concentration ≥10 μmol/L and itchy skin with a normal appearance; it usually occurs in the third trimester [1]. ICP affects 0.1–2% of pregnant women and is associated with adverse pregnancy outcomes, and elevated TBA is the main culprit. The most serious cause of ICP is sudden unexplained fetal death in utero, which is positively correlated with TBA ≥ 100 μmol/ L [2]. As no superior alternatives are currently available, ursodeoxycholic acid (UDCA) remains the first-line treatment for ICP. UDCA reduces maternal itch and diminishes bile acid levels, but controversy remains over whether UDCA is effective in treating ICP and ameliorates adverse perinatal outcomes [1] [3]. The etiology of ICP is complex and has significant genetic susceptibility. Recent studies have systematically characterized common sequence variations in liver-enriched genes and regulatory elements contributing to ICP susceptibility [4]. These patients tend toward severe refractory ICP, which is unresponsive even if they receive standardized treatment, posing challenges for perinatal management.

Here, we present a clinical case of severe refractory ICP, whole exome sequencing (WES) identified four variants, with a variant of the ABCD3 gene being identified as potentially linked to ICP development. This marks the first documentation of an ABCD3 gene variant in ICP. Additionally, we provide new insights into the perinatal management of severe refractory ICP.

Case presentation

The 27-year-old pregnant female patient was of Asian Chinese Han descent, gravida 1, para 0, conceived through natural conception. At 13⁺³ weeks of gestation, elevated total bile acid (TBA) levels were recognized for the first time but the patient did not complain of skin pruritus. Viral hepatitis (A–E), cytomegalovirus, and Epstein-Barr virus infection status were excluded. There were no signs of cholecystitis, bile duct dilatation, terminal bile duct biliary sludge formation, bile duct stones, or portal hypertension on abdominal ultrasound. She denied a history of any other medical condition and alcohol consumption, illicit drug abuse, or hepatotoxic medications. She was diagnosed with severe intrahepatic cholestasis of pregnancy.

She underwent ursodeoxycholic acid (UDCA) treatment at a dosage of 750 µg per day from 13⁺³ weeks of gestation. Because TBA continued rising to 50.8 µmol/L after two weeks, UDCA was adjusted to 1 mg per day. One month later, the TBA level was 32 µmol/L. Since then, S-adenosylmethionine (SAMe) was added at a dose of 1 g per day. Over-standard TBA levels were observed repeatedly during pregnancy and even peaked at 69.6 µmol/L at 33⁺⁵ weeks of gestation. Then she was administered UDCA (1 mg per day) in combination with SAMe (1 g per day) again until admission.

She was admitted in pregnancy week 35⁺⁴ with a TBA of 31 umol/L. Lung maturation for the fetus was initiated (dexamethasone 6 mg im q12h). A healthy female baby was delivered by planned cesarean section at 36⁺¹ weeks of gestation. The newborn was 47 cm in length and 2710 g in weight, with an Apgar score of 10–10–10. UDCA was used continuously after cesarean section until the TBA was 21.1μmol/L on the second day after surgery. We advised her to continue taking oral SAMe for an additional week after discharge and check her TBA regularly. At 23⁺⁴ weeks postpartum, her TBA was elevated to 97.7 µmol/L and we advised her to seek advice from a specialist in gastroenterology. The details of the biochemical indicators and medication regimens used during pregnancy and postpartum are shown in [Fig. 1].

Fig. 1 Fluctuations in the concentrations of biochemical indicators and drug therapy during pregnancy. UDCA: ursodeoxycholic acid, SAMe: S-adenosyl-methionine, Tid: three times a day; Qid: four times a day; Bid: twice a day; ALT: alanine aminotransferase; AST: aspartate aminotransferase; TBA: total serum bile acid; ALP: alkaline phosphatase.

At the one-year follow-up, the patient’s daughter was in line with her peers in terms of both physical growth and developmental characteristics. Written informed consent was obtained from the patient, and supplementary data and images were used for this case report only.

Given that the patient’s clinical characteristics did not match those of a typical ICP and that the efficacy of the drugs was poor, we intended to conduct a genetic analysis of the patient together with her first-degree family members. Unfortunately, gene mutation analysis could not be performed on her father because of his refusal. Whole-exome sequencing (WES) was utilized to detect the variants. Novel variants from WES analyses were confirmed using Sanger sequencing. Detailed WES sequencing and mutation screening methods are provided in the Supplementary Material .

We initially detected variants in seven genes in the proband. Compared with those in the protestorʼs mother, who reported no relevant family history of ICP, we suspected that the four separate variants might be genetic risk variants: RPGRIP1 Like (RPGRIP1 L), ABCD3, Keratin 7 (KRT7), and Dolichyl-phosphate mannosyltransferase subunit 3 (DPM3). Compared with the reference sequences NM_001122674 and NM_002858, the proband’s sequence analysis revealed a heterozygous variant of the ABCD3 gene in exon 2 at codon 130, resulting in a substitution of proline (ACC) for serine (ATC). The genetic test revealed a heterozygous missense variant (c.208C>G/p.Pro70Ala) of the DPM3 gene on exon 1 at codon 208 of chromosome 1 compared with the reference sequence NM_018973. Another heterozygous missense variant is (c.491C>T/p. Ala164Val) of the KRT7 gene on exon 2 at codon 491 of chromosome 12 was identified. In addition, a deletion mutant allele was detected in RPGRIP1L (deletion c. 1419_1421TTT), resulting in a protein deficiency.

Computer-based algorithms, including SIFT, PolyPehn-2, and MutationTaster were used to predict the associations of genetic mutations with disease risk. The predicted result indicated that p.P44S on ABCD3 was disease-causing in MutationTaster and that p.P70A on DPM3 is probably damaging according to Polyphen-2. The full details for the variants of both the patient and her mother are described in [Fig. 2] and [Table 1].

Fig. 2 Sanger sequencing map of the proband and her mother. A deletion mutant was detected in RPGRIP1L (deletion c. 1419_1421TTT), resulting in protein deficiency. Missense heterozygous variants in ABCD3(p.P44S) and KRT7(p.A164V) were detected in our ICP patients. A new SNP was also detected in the DPM3 gene (p.P40A). More details on coding DNA (c.) and protein (p.) levels are shown in the figure. Ref seq: reference sequence at the nucleotide level.

Table 1 Overview of the prioritized variants identified in the proband.

Gene (OMIM)	Chrs	Nucleotide	Protein consequence	Zygosity	Function	Allele frequency			Function prediction
						1000g-all	EXAC-all	gnomAD-all	SIFT	polyPhen-2	Mutation-Taster
ABCD3 (616278)	1	c.130C>T	p.Pro44Ser	Heterozygous	Missense	–	0.0000165	0.0000159275	Ta	Bb	Dc
LYST (214500)	1	c.8624G>A	p.Arg2875Hismat	Heterozygous	Missense	0.000399361	0.0002	0.000227101	Dd	D	D
DPM3 (608093)	1	c.208C>G	p.Pro70Ala p.Pro40Ala	Heterozygous	Missense	–	–	–	T	De	D
KRT7 (N/A)	12	c.491C>T	p.Ala164Val	Heterozygous	Missense	–	0.00002492	0.0000199173	T	B	Nf
PIEZO1 (N/A)	16	c.5636A>G	p.Glu1879Glymat	Heterozygous	Missense	0.00159744	0.0006	0.000744397	T	B	N
RPGRIP1L (216360)	16	c.1419_1421del	p.Lys473del	Heterozygous	Inframe deletion	–	0.0002	0.000134556	–	–	–
TMEM165	4	c.208–15->T	–	Heterozygous	Splice region variant	–	0.0064	0.00264685	–	–	–

OMIM: phenotype MIM number in Online Mendelian Inheritance in Man database; a: T means tolerated in SIFT; b: B means benign in Polyphen-2; c: D means disease-causing in MutationTaster; d: D means deleterious in SIFT; e: D means probably damaging in Polyphen-2; f: N means polymorphism in MutationTaster; mat: mutation is inherited maternally.

Discussion and Conclusions

ABCD3 variants associated with ICP

We report a case of early refractory ICP associated with unsatisfactory TBA decline after combination treatment with UDCA and SAMe. To further explore the etiology of the disease, we conducted WES and the predicted result suggested that p.P44S on ABCD3 and p.P70A on DPM3 might be pathogenic variants. Screen candidate genes based on gene function, OMIM, HPO, HGMD, and MGI database annotations, combined with GO and KEGG information. We focus on compound heterozygous ABCD3 sequence variations, which may be rare causes associated with ICP.

Functional bile-acid transporters are the core regulators of the bile acid cycle and homeostasis. Mutations affecting proteins can disrupt the synthesis and secretion of bile acids by hepatocytes [5].

Many studies have focused on expanding the genetic details that contribute to ICP, as mentioned above, especially the ATP-bound cassette transporter (ABC) family. It is a group of membrane-bound proteins that transport a variety of substrates both extracellularly and intracellularly, playing a key role in the secretion of bile into canaliculi [6].

The human ABCD3 gene is located on chromosome 1 and encodes ATP-binding cassette subfamily D member 3 isoform X2. ABCD3 transports branched fatty acids and C27-bile acid intermediates to peroxisomes, participating in a crucial step in bile acid biosynthesis [7]. However, variants in ABCD3 have not been identified in patients with ICP. Hence, this is the first study to present an ICP patient with a heterozygous variant in the ABCD3 gene. Gene sequencing studies have shown that serine at amino acid position 44 is highly conserved, and computer-based algorithms predict that p.P44S is harmful; however, the site has not been previously reported. The presence of refractory cholestasis in our case report suggested a possible genetic susceptibility. However, this cannot be defined as evidence of causation, and further characterization on the protein basis of structure and function is needed.

ICP and hereditary cholestasis

The synthesis and metabolism of bile acids is a delicate process that requires every link to work properly. High circulating estrogen levels during pregnancy may increase the risk of ICP and the underlying liver disease can be diagnosed as ICP because of the special period of pregnancy. Hormonal changes may obscure a genetic predisposition to cholestasis, and some cases of unexplained cholestasis during pregnancy involve genetic variation.

The complex genetic variations in bile synthesis or secretion metabolism present different phenotypes, such as benign recurrent intrahepatic cholestasis, ICP, drug-induced cholestasis, low phospholipid-associated cholelithiasis, and progressive familial intrahepatic cholestasis [8]. ICP is usually caused by complex variations of the same gene, penetrance, and environmental factors combined with risk factors involving different diseases that contribute to the specific phenotype observed.

In our patient, ABCD3 variants may have been the underlying cause of the observed phenotype. In some patients, UDCA is less effective at alleviating disease symptoms, usually because of its genetic diversity. Hence, we propose systematic genetic screening for early-onset or severe refractory individuals with ICP. Identifying key candidate genes and causative variants is extremely valuable because it contributes to the accurate diagnosis of cholestatic liver disease and enlarges the spectrum of pathogenic genes.

Medical intervention with severe refractory ICP

The core of managing women with ICP revolves around ameliorating symptoms and adverse perinatal outcomes. However, until now, no conclusive evidence has supported the most effective treatment for severe refractory ICP, and the efficacy of UDCA was limited in our case.

Rifampicin strongly induces CYP3A4 expression by activating the heater sensor progesterone X receptor, which increases the 6-α hydroxylation of bile salts, and the bile acids, which enhances the excretion of gluconic acid in urine and promotes the binding and excretion of bilirubin [9].

Recently, a prospective study [10] was designed to compare the use of rifampicin vs. UDCA in early-onset severe ICP women, which was defined as cholestasis identified earlier than 34 weeks of gestation and a serum TBA level > 40 μmol/L. Maternal pruritus was considered the primary outcome. However, at the time of submission of our report, the research has not yet been completed. However, for Dubin-Johnson Syndrome with a specific locus variant of the ATP binding cassette subfamily C member 2 (ABCC2) gene, the use of rifampicin or UDCA alone or in combination may be dangerous, potentially worsening both bilirubinemia and the serum bile acid concentration [11].

For patients suffering from both ICP and GDM, both glycemic control and alleviating cholestasis should be taken into account. There is evidence that bile acid pools may be increased in patients with type 2 diabetes mellitus. These changes may increase insulin resistance, disrupt glucose homeostasis, and contribute to the mechanism underlying the diabetes mellitus [12]. Although none of the oral antidiabetic medications is recommended for GDM, individual cases have reported the efficacy of metformin in the treatment of recurrent ICP [13], and a clinical trial (Identifier: NCT03056274, registered on ClinicalTrials.gov on December 22, 2016) is currently being conducted to compare the effects of metformin vs. UDCA on lowering liver enzymes and bile salts. Further larger-scale studies may reveal the superior treatment of early-onset severe refractory ICP.

Postpartum liver function surveillance is desirable for women with ICP. ICP is associated with increased liver abnormalities (ALT >25 U/L, ALP >140 U/L, and liver disease) after childbirth [14]. The potential for long-term adverse consequences of ICP, including hepatobiliary cancer and immune-mediated disease. Furthermore, a small increased risk of subsequent cardiovascular disease is prevalent, especially in women with ICP and concomitant preeclampsia [15]. For pregnant women with ICP, especially those with a genetic predisposition, regular follow-up helps them recognize the warning signs of liver disease first and avoid progression.

In conclusion, our study highlights the heterogeneity of the variations associated with bile acid metabolism dysfunction. Notably, compound heterozygous ABCD3 sequence variations may be rare causes associated with ICP. Screening for potential pathogenicity variants that drive specific recognition of ICP is necessary. Women who are diagnosed with severe refractory ICP should be offered genetic counseling. Once pathogenic variants are confirmed, the patient should be counseled about the long-term risk of liver abnormalities, and a referral to the hepatology team should be made.

Contributor’s Statement

Tingting Xu and Xiaodong Wang conceived the study, revised the manuscript and supervised the entire study, they contributed equally to this work.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgement

We extend our sincere gratitude to the physicians of the Department of Pediatric Healthcare, West China Second University Hospital for their invaluable assistance in providing the data on the children involved in this study.

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Publication History

Received: 19 September 2024

Accepted after revision: 31 October 2024

Article published online:
15 January 2025