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
1
c.130C>T
p.Pro44Ser
Heterozygous
Missense
–
0.0000165
0.0000159275
Ta
Bb
Dc
LYST
1
c.8624G>A
p.Arg2875Hismat
Heterozygous
Missense
0.000399361
0.0002
0.000227101
Dd
D
D
DPM3
1
c.208C>G
p.Pro70Ala
Heterozygous
Missense
–
–
–
T
De
D
KRT7
12
c.491C>T
p.Ala164Val
Heterozygous
Missense
–
0.00002492
0.0000199173
T
B
Nf
PIEZO1
16
c.5636A>G
p.Glu1879Glymat
Heterozygous
Missense
0.00159744
0.0006
0.000744397
T
B
N
RPGRIP1L
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.
