An ABCB11 variant registry and novel knockin mouse model of PFIC2 based on the clinically relevant ABCB11 E297G variant

Abstract

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a rare pediatric cholestatic liver disease caused by genetic deficiency in the bile salt export pump (BSEP, ABCB11). BSEP is an ATP-binding cassette transporter and the primary regulator of hepatic bile acid efflux. Loss of BSEP function in PFIC2 leads to cholestasis and intrahepatic accumulation of bile acids, the native toxicity of which drives progressive liver injury, in a manner that correlates with ABCB11 genotype. Here, to support ongoing PFIC2 research, we present two novel translational tools, 1) a codified evidence-based catalog of published disease relevant ABCB11 mutations and 2) a knockin mouse model of the PFIC2-associated missense variant E297G. Using a combination of AI-based indexing of the literature and manual review, we identified 476 nonbenign ABCB11 variants in published patients with cholestatic disease, of which 240 were associated with PFIC2. Additionally, we present phenotypic validation of a novel knockin mouse model of the cholestasis-associated ABCB11 E297G variant. Bsep^E297G homozygous mice recapitulate the core molecular and pathophysiological aspects of PFIC2, including perturbed Bsep processing and membrane trafficking, cholestasis, and hepatotoxicity. Moreover, and consistent with clinical data, pharmacological ileal bile acid transporter inhibition improved the cholestatic phenotype of Bsep^E297G mice through increased fecal bile acid excretion. Together, these tools can support clinical and translational efforts to advance understanding and treatment of PFIC2.

Keywords: Abcb11; BSEP; PFIC2; bile acid; cholestasis; missense.

Fig. 1

Frequency, position, and associated phenotypes of ABCB11 missense variants. A: Breakdown by effect type of nonbenign (by ACMG criteria), published ABCB11 variants that have been observed in a patient with cholestatic disease. B: number of nonbenign, published ABCB11 missense variants associated with progressive familial intrahepatic cholestasis type 2 (PFIC2), an unspecified/nonsyndromic form of cholestasis, benign recurrent intrahepatic cholestasis type 2 (BRIC2), and/or intrahepatic cholestasis of pregnancy (ICP). C: nonbenign, published ABCB11 missense variants observed in PFIC2 patients mapped by protein position and number of published, unrelated probands harboring the variant. ACMG, American College of Medical Genetics.

Fig. 2

Generation of PFIC2 mouse model by introduction of pathogenic BSEP human missense variant into mouse Bsep. A: Sequence of mouse Bsep edited from WT nucleotides (red) to the E297G allele nucleotides containing both the E to G conversion and introduction of BsmI restriction site (green). B: Genotyping gel of F1 HET animals indicating PCR product band and BmsI digest band (top). Sequencing chromatograms demonstrating heterozygosity of the edited nucleotides. BSEP, bile salt export pump; HET, heterozygote; PFIC2, progressive familial intrahepatic cholestasis type 2.

Fig. 3

BSEP protein processing is defective in Bsep^E297G mice. A: Graphic depiction of BSEP protein maturation and sequential glycosylation in the ER and Golgi apparatus as it traffics to the plasma membrane. B: Western blot of liver lysate from female (top) and male (bottom) WT, HET, and HOM animals indicating the presence of B- and C-band. C: Bsep mRNA qPCR analysis from WT, HET, and HOM female mice. D: Western blot of liver lysates from WT, HET, HOM animals subjected to PNGase to generate A- and B-band. E: Immunohistochemistry for Bsep in frozen liver cryo-sections from WT, HET, and HOM mice. BSEP, bile salt export pump; ER, endoplasmic reticulum; HET, heterozygote; HOM, homozygous; qPCR, quantitative real-time PCR.

Fig. 4

BSEP^E297G mice demonstrate evidence of cholestasis and liver injury. A and B: Body weight of female and male WT, HET, and HOM mice from 3 weeks to 12 weeks of age. C and D: Serum BA levels in female and male mice from 6 to 12 weeks of age. E and F: Liver BA levels in female and male mice at 12 weeks of age. G and H: Biliary BA levels in female and male mice at 12 weeks of age. I and J: Serum ALT levels in female and male mice from 6 to 12 weeks of age. K and L: Serum alkaline phosphatase levels in female and male mice from 6 to 12 weeks of age. M and N Liver index in female and male mice at 12 weeks of age. O: H&E staining in male and female WT and HOM at 6 weeks of age (n = 2/group). Black arrows: cytoplasmic inclusions, black arrowheads: lipid-type vacuoles, open arrows: single-cell hepatocyte necrosis/apoptosis, white∗: infiltrating lymphocytes and macrophages, PV: portal vein, ∗: bile duct. ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 Fischer’s least significant difference for WT versus HOM, #P < 0.01 Mixed effects model for Time x Genotype. $$$ P < 0.001 Mixed effects model for Genotype. ALT, alanine aminotransferase; BA, bile salt; BSEP, bile salt export pump; HET, heterozygote; HOM, homozygous.

Fig. 5

Hepatobiliary bile homeostasis is disrupted in Bsep^E297G mice. Composition of bile from the gallbladder of female 12-week-old WT and HOM Bsep^E297G mice. A: Bile phospholipids levels and (B) cholesterol levels. BA composition of WT and HOM Bsep^E297G mice for (C) serum, (D) liver, and (E) bile. ∗∗P < 0.01, ∗∗∗∗P < 0.0001 unpaired two-tailed t test. BA, bile salt; BSEP, bile salt export pump; HET, heterozygote; HOM, homozygous.

Fig. 6

Transcriptomic and pathway analysis of Bsep^E297G mice identified altered gene expression profile consistent with a counter-regulatory response to cholestasis. A: Changes in primary BA synthesis pathways in WT and HOM mice. B: Changes in canalicular transporter gene expression in WT and HOM mice. C: Changes in expression of sinusoidal BA efflux genes. D: Changes in expression of sinusoidal BA import genes. ∗P < 0.05, ∗∗P < 0.001, and ∗∗∗∗P < 0.0001 with Benjamini–Hochberg correction. BA, bile salt; BSEP, bile salt export pump; HOM, homozygous.

Fig. 7

Administration of clinical IBAT inhibitor decreases BA levels in Bsep^E297G mice. Fourteen-day treatment of 10- to-14-week-old female HOM Bserp^E297G mice with A2450. A: Total BA concentration of feces. B: Total BA concentration of serum. C: Total BA concentration of liver. D: Total BA concentration of bile. BA composition from (E) serum, (F) liver, and (G) bile. H: Serum ALT levels. I: Serum alkaline phosphatase levels. J: Liver index (liver weight to body weight). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001 one-way ANOVA or Kruskal–Wallis test.ALT, alanine aminotransferase; BA, bile salt; BSEP, bile salt export pump; HOM, homozygous; IBAT, ileal bile acid transporter.