Low Phospholipid‐Associated Cholelithiasis in a Mother–Son Pair Associated With a Novel ABCB4 Frameshift Variant

ABSTRACT

Low phospholipid‐associated cholelithiasis (LPAC) syndrome is a rare inherited disorder caused by ABCB4 mutations with heterogeneous manifestations. We describe two LPAC cases in a mother–son pair, detailing their clinical presentations, diagnostic evaluations, and management. A novel ABCB4 frameshift variant (c.715_716insTT; p.Ser239PhefsTer9) was identified, expanding the genetic spectrum of LPAC syndrome.

Key Clinical Message

This report describes a mother–son pair with low phospholipid‐associated cholelithiasis (LPAC) syndrome caused by a novel ABCB4 gene variant (c.715_716insTT). Early genetic testing enables timely diagnosis and ursodeoxycholic acid treatment, which can slow disease progression and prevent hepatobiliary complications in affected families.

1. Introduction

Low phospholipid‐associated cholelithiasis (LPAC) syndrome, also known as gallbladder disease 1 (GBD1), is a cholestatic liver disorder caused by mutations in the ABCB4 gene on chromosome 7q21. Notably, these mutations can lead to partial functional impairment of multidrug resistance protein 3 (MDR3) and may be inherited in either an autosomal dominant or autosomal recessive pattern [1]. The resulting reduction in MDR3 activity leads to cholesterol supersaturation and crystallization in bile ducts. This process damages biliary epithelial cells and promotes chronic biliary inflammation. Ultimately, these pathological changes lead to cholestasis, resulting in liver function impairment, cholelithiasis, acute and chronic biliary complications (including cholecystitis, cholangitis, and pancreatitis), liver fibrosis, cirrhosis, and an increased risk of hepatobiliary tumors [2].

In 2001, Rosmorduc et al. first described the association between ABCB4 gene mutations and symptomatic cholelithiasis in adults [3]. However, the overall prevalence of LPAC syndrome remains poorly defined, with an estimated 1% of patients with symptomatic cholelithiasis meeting the diagnostic criteria for LPAC syndrome [4]. A formal diagnosis for LPAC requires fulfillment of at least two of the following three criteria: onset of symptoms before 40 years of age, recurrence of biliary symptoms after cholecystectomy, and ultrasonographic evidence of intrahepatic hyperechoic foci, sludge, or microlithiasis [3, 4]. This case report describes two cases of LPAC syndrome in a mother–son pair, detailing their clinical presentations, diagnostic evaluations, and management.

2. Case History and Examination

A 49‐year‐old woman presented with recurrent abdominal distension and pain for more than 7 years, with a recent onset of fever lasting 2 weeks. Her surgical history included a cholecystectomy performed more than 20 years earlier and endoscopic retrograde cholangiopancreatography (ERCP) with endoscopic sphincterotomy (EST) for stone extraction 7 years ago to treat hepatolithiasis and choledocholithiasis. She denied any history of hepatotoxic drug use or schistosomiasis infection. On admission, pertinent laboratory findings were as follows: albumin (ALB), 29.73 g/L; total bilirubin (TBIL), 151.74 μmol/L; direct bilirubin (DBIL), 133.88 μmol/L; alanine aminotransferase (ALT), 121.4 U/L; aspartate aminotransferase (AST), 272.2 U/L; total bile acids (TBA), 193.52 μmol/L; alkaline phosphatase (ALP), 1196.9 U/L; and gamma‐glutamyl transferase (GGT), 472.5 U/L. Serological testing was negative for viral hepatitis and autoimmune liver diseases, and ceruloplasmin levels were within the normal range. Abdominal magnetic resonance imaging (MRI) and magnetic resonance cholangiopancreatography (MRCP) demonstrated dilatation of both the intrahepatic and extrahepatic bile ducts with numerous calculi, accompanied by features of cholangitis and pericholangitis. Additional findings included the absence of the gallbladder (post‐cholecystectomy), liver cirrhosis, splenomegaly, and a small amount of ascites.

The proband's son, an 18‐year‐old male, was incidentally found to have abnormal liver function 6 months earlier, characterized by a GGT level exceeding 600 U/L. He was asymptomatic, reporting no abdominal pain, fever, or jaundice, and denied any history of hepatotoxic drug use or schistosomiasis infection. Laboratory findings were as follows: ALB, 39.04 g/L; TBIL, 20.6 μmol/L; DBIL, 6.13 μmol/L; ALT, 89.4 U/L; AST, 52.2 U/L; TBA, 88.44 μmol/L; ALP, 181.3 U/L; and GGT, 273.1 U/L. Serological testing excluded autoimmune liver disease, viral hepatitis, and Wilson disease. Liver biopsy findings (Figure 1) demonstrated small bile duct injury, liver fibrosis, and narrowing or occlusion of the interlobular portal veins.

FIGURE 1.

Pathological findings from the liver biopsy of the proband's son. (A, B) The portal tract shows mild lymphoplasmacytic infiltration, mainly composed of lymphocytes and plasma cells, within fibrotic stroma. The interlobular bile duct was absent, the portal venule was narrowed, and the hepatic arteriole was preserved. Lobular hepatocytes were swollen, without significant lobular inflammation or interface hepatitis (hematoxylin and eosin stain, ×200). (C) Periportal sinusoidal capillarization, indicating portal vein narrowing with compensatory predominance of arterial blood supply within the liver (CD34 immunohistochemistry, ×200). (D) Perisinusoidal fibrosis with portal‐to‐portal (P–P) bridging fibrosis (reticulin stain).

3. Methods (Differential Diagnosis, Investigations, and Treatment)

With common causes of liver disease excluded, the familial occurrence of similar cholestatic features, with or without gallstones, raised the suspicion of an inherited disorder. Genetic testing was therefore performed. Whole‐exome sequencing was performed for the proband's son using GenCap capture probes (mean depth 150.85×, > 99% of target regions covered at ≥ 10×). Reads were aligned to GRCh37/hg19, and variants were annotated according to American College of Medical Genetics and Genomics (ACMG) guidelines, using REVEL and SpliceAI. The genetic analysis revealed a heterozygous ABCB4 gene variant (c.715_716insTT; p.Ser239PhefsTer9) (Figure 2A) and two heterozygous UGT1A1 gene variants (c.‐41_‐40dup and c.‐3275T>G). For the proband, targeted bidirectional Sanger sequencing was performed to validate the identified variants in ABCB4 and UGT1A1. Maternal genetic analysis confirmed the presence of a heterozygous ABCB4 gene variant (c.715_716insTT) (Figure 2B) and a heterozygous UGT1A1 gene variant (c.‐3275T>G), as well as a homozygous UGT1A1 gene variant (c.‐41_‐40dup).

FIGURE 2.

Sanger sequencing chromatograms confirming the ABCB4 c.715_716insTT (p.Ser239PhefsTer9) variant in the mother–son pair. (A) Proband's son; (B) Proband (mother). Red arrows indicate the location of the heterozygous variant in both patients.

The proband underwent cholecystectomy before the age of 30. At 42 years old, she developed recurrent abdominal distension and pain, with imaging revealing multiple intra‐ and extrahepatic bile duct stones. Her symptoms persisted despite undergoing EST for stone removal. Laboratory evaluation showed a predominantly cholestatic liver injury pattern, with elevated DBIL, ALP, and GGT. Maternal genetic testing identified a heterozygous ABCB4 gene variant. Based on established diagnostic criteria [3, 4], she was diagnosed with definitive LPAC syndrome. Her son, although showing only mild liver injury with a predominantly cholestatic profile, did not fully meet the primary diagnostic criteria for LPAC. Nevertheless, liver biopsy revealed small bile duct injury, and genetic testing confirmed that he carried the same pathogenic ABCB4 variant as his mother (Figure 3). Consequently, a diagnosis of early‐stage LPAC was considered for him.

FIGURE 3.

Pedigree chart of the family.

Both patients were treated with ursodeoxycholic acid (UDCA). In the mother's case, no additional surgical or interventional procedures were performed.

4. Conclusions and Results (Outcome and Follow‐Up)

In the mother, after more than 3 months of UDCA therapy, follow‐up liver function tests showed marked improvement: ALB, 33.7 g/L; TBIL, 45.2 μmol/L; DBIL, 24.16 μmol/L; ALT, 44.2 U/L; AST, 131.5 U/L; TBA, 84.3 μmol/L; ALP, 428.0 U/L; and GGT, 211.0 U/L. These results indicate substantial improvement in liver function and significant alleviation of cholestasis.

5. Discussion

LPAC syndrome is an inherited disorder caused by mutations in the ABCB4 gene. Its characteristic features include symptom onset before age 40, recurrence of symptoms after cholecystectomy, and ultrasonographic evidence of intrahepatic hyperechoic foci, sludge, or microlithiasis. The diagnosis is typically established when at least two of the following three criteria are met [3, 4]. However, these criteria were derived from relatively limited case series, and not all individuals with LPAC syndrome meet them [4, 5]. Additional clinical clues may support the diagnosis, including a family history of gallstones in first‐degree relatives, intrahepatic cholestasis of pregnancy (ICP), responsiveness to UDCA therapy, an increased biliary cholesterol‐to‐phospholipid ratio, or identification of an ABCB4 gene mutation [1]. However, ABCB4 gene mutations demonstrate marked genetic heterogeneity, including nonsense and missense variants as well as partial deletions, with heterozygous missense variants being the most frequently identified [5]. Only approximately half of patients with a clinical diagnosis of LPAC syndrome harbor detectable ABCB4 mutations, and their absence does not exclude the diagnosis [4, 5]. The exact prevalence of LPAC syndrome remains uncertain; however, it has been estimated to account for approximately 1% of symptomatic cholelithiasis cases [4]. The male‐to‐female ratio is approximately 1:3 [6], with females presenting at a significantly younger age than males (mean age 29.1 vs. 38.7 years, respectively; p < 0.003). Truncating mutations are associated with an even earlier disease onset (mean age 26.5 vs. 33.9 years; p < 0.003) [5]. LPAC syndrome typically occurs in individuals with low‐to‐normal body weight, and nearly half of affected female patients experience concomitant ICP [5]. Conversely, classic cholelithiasis typically presents after the age of 50, is commonly associated with obesity and metabolic syndrome, and most often manifests as isolated cholecystolithiasis. Recurrence after cholecystectomy and concomitant ICP are uncommon. Histologically, LPAC syndrome lacks specific pathognomonic features; liver biopsy usually reveals changes consistent with biliary obstruction, including portal tract enlargement, ductular proliferation, focal capillary cholestasis, mild chronic portal inflammation, and, in some cases, early‐stage septal fibrosis [1].

The differential diagnosis of LPAC syndrome includes other ABCB4‐related disorders, such as progressive familial intrahepatic cholestasis type 3 (PFIC3), ICP, and drug‐induced liver injury (DILI) [7, 8]. PFIC3 is an autosomal recessive disorder typically caused by homozygous or compound heterozygous mutations that result in complete or near‐complete loss of MDR3 function. It usually presents between 1 month and 20.5 years of age, with approximately one‐third of affected patients developing progressive jaundice and cholestasis within the first year of life. Of note, the disease progresses rapidly to liver fibrosis and cirrhosis. In contrast to LPAC syndrome, gallstones are uncommon in PFIC3, and treatment with UDCA is often ineffective, frequently necessitating liver transplantation [9, 10]. Meanwhile, patients with LPAC syndrome exhibit only partial MDR3 dysfunction, resulting in milder clinical manifestations and a more favorable prognosis. This phenotypic spectrum suggests that the severity of ABCB4 mutations correlates with the degree of residual protein function. Both patients described in this report are heterozygous carriers with late disease onset and slow progression, findings that are inconsistent with PFIC3. On the other hand, ICP is a reversible cholestatic condition that typically occurs during the second or third trimester of pregnancy and is characterized by pruritus and elevated bile acid levels, with symptoms usually resolving after delivery. ICP is associated with increased risks of prematurity, fetal distress, and stillbirth. Although neither patient met the diagnostic criteria for ICP, the mother exhibited more severe liver injury than her son despite sharing the same pathogenic variant. This observation supports the hypothesis that elevated estrogen levels during pregnancy may suppress ABCB4 expression and precipitate cholestasis, thereby contributing to the female predominance observed in LPAC syndrome and the association of DILI with oral contraceptive use [11]. In addition to oral contraceptives, several other medications, including itraconazole, chlorpromazine, and imipramine, are known to suppress ABCB4 gene expression and inhibit biliary transporter function, potentially leading to cholestasis [12].

UDCA is the mainstay of treatment for LPAC syndrome. Its mechanisms of action include enhancement of MDR3 protein activity, reduction of intestinal reabsorption of endogenous bile acids, an increased proportion of hydrophilic bile acids, suppression of inflammatory cytokine release and oxygen free radical formation, and promotion of choleresis. Together, these effects facilitate litholysis, prevent new stone formation, alleviate cholangitis, and reduce cholestasis [13]. The standard dosage of UDCA is 7–10 mg/kg/day, which can be increased to a maximum of 20 mg/kg/day. Most patients respond favorably, with symptomatic and biochemical improvements typically observed within weeks, whereas radiological improvement may require several months [1]. Cholecystectomy does not cure LPAC syndrome and is therefore reserved for patients with symptomatic gallstones or acute cholecystitis. Interventional or surgical procedures should be considered in the presence of complications such as concomitant hepatobiliary stones, recurrent cholangitis, intrahepatic abscesses, or hepatobiliary tumors. Liver transplantation may be indicated for secondary biliary cirrhosis with end‐stage liver failure or for severe intrahepatic choledocholithiasis complicated by recurrent septicemia. Recurrence of the disease after transplantation is rare, as the transplanted liver provides functional biliary transporters.

In addition to the ABCB4 variant, both patients were found to carry variants in the UGT1A1 gene. However, these UGT1A1 variants were considered incidental findings. The clinical and biochemical features of our patients—predominantly cholestatic liver injury with elevated ALP, GGT, and DBIL, along with biliary stone formation—do not fall within the spectrum of UGT1A1‐related disorders, which typically present with unconjugated hyperbilirubinemia [14, 15] (e.g., Gilbert syndrome and Crigler‐Najjar syndrome).

The mother had a long‐standing history of liver disease, including a prior diagnosis of secondary biliary cirrhosis. Genetic testing, prompted by her son's presentation with abnormal liver function, identified a heterozygous frameshift variant in the ABCB4 gene (c.715_716insTT, p.Ser239PhefsTer9), thereby confirming the diagnosis of LPAC syndrome. According to the ACMG guidelines, this variant is classified as pathogenic, very strong (PVS1), as it is predicted to result in loss of gene function [16]. This particular variant has not been previously reported, and its pathogenicity is not yet annotated in the ClinVar database. The proband's son carries the same ABCB4 variant but presents with only mild liver injury and fibrosis. This phenotypic disparity may be related to his adolescent age and potential sex‐related protective factors. Nevertheless, without intervention, disease progression is likely, emphasizing the importance of early diagnosis and management. After more than 3 months of UDCA therapy, the mother's liver function and cholestasis showed significant improvement, indicating a favorable response to treatment, although radiological changes require long‐term follow‐up. Given the autosomal dominant inheritance pattern of heterozygous ABCB4 mutations, first‐degree relatives of affected individuals have a 50% risk of carrying the variant. Accordingly, screening of first‐degree relatives, including liver function tests, hepatic imaging, and genetic testing, is strongly recommended.

In summary, we described two cases of LPAC syndrome with differing clinical severities and identified a novel ABCB4 frameshift variant (c.715_716insTT; p.Ser239PhefsTer9). Although the co‐segregation of this variant with the disease phenotype supports its clinical relevance, definitive confirmation of pathogenicity requires functional studies and replication in larger cohorts. Increased clinical awareness of LPAC syndrome is warranted, and diagnosis should be considered in individuals presenting with early‐onset gallstones, recurrence of symptoms after cholecystectomy, unexplained cholestasis, or intrahepatic or extrahepatic bile duct stones, particularly when there is a family history of gallstones in first‐degree relatives. For confirmed cases, genetic testing and cascade screening of family members are essential for early detection and intervention, thereby preventing serious hepatobiliary complications.

Author Contributions

Hui‐Ming Yang: investigation, data curation, validation, visualization, writing – original draft. Jun Liu: investigation, data curation, validation, writing – review and editing. Zheng Zhang: conceptualization, resources, supervision, validation, project administration, writing – review and editing.

Funding

This work was supported by the Health Research Project of Hunan Provincial Health Commission (grant numbers: W20243223 and W20243248).

Ethics Statement

This case report was conducted in accordance with the ethical principles of the Declaration of Helsinki. All patient identifiers have been removed from the manuscript to protect privacy.

Consent

Written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this case report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The datasets generated and analyzed during this case report (including clinical records and imaging data) are not publicly available due to patient privacy and ethical restrictions. De‐identified data may be available from the corresponding author upon reasonable request, subject to approval by the institutional ethics committee.