Whole-exome sequencing reveals LRP5 mutations and canonical Wnt signaling associated with hepatic cystogenesis

Abstract

Polycystic livers are seen in the rare inherited disorder isolated polycystic liver disease (PCLD) and are recognized as the most common extrarenal manifestation in autosomal dominant polycystic kidney disease. Hepatic cystogenesis is characterized by progressive proliferation of cholangiocytes, ultimately causing hepatomegaly. Genetically, polycystic liver disease is a heterogeneous disorder with incomplete penetrance and caused by mutations in PRKCSH, SEC63, PKD1, or PKD2. Genome-wide SNP typing and Sanger sequencing revealed no pathogenic variants in hitherto genes in an extended PCLD family. We performed whole-exome sequencing of DNA samples from two members. A heterozygous variant c.3562C > T located at a highly conserved amino acid position (p.R1188W) in the low density lipoprotein receptor-related protein 5 (LRP5) gene segregated with the disease (logarithm of odds score, 4.62) but was not observed in more than 1,000 unaffected individuals. Screening of LRP5 in a PCLD cohort identified three additional mutations in three unrelated families with polycystic livers (p.V454M, p.R1529S, and p.D1551N), again all undetected in controls. All variants were predicted to be damaging with profound structural effects on LRP5 protein domains. Liver cyst tissue and normal hepatic tissue samples from patients and controls showed abundant LRP5 expression by immunohistochemistry. Functional activity analyses indicated that mutant LRP5 led to reduced wingless signal activation. In conclusion, we demonstrate that germ-line LRP5 missense mutations are associated with hepatic cystogenesis. The findings presented in this study link the pathophysiology of PCLD to deregulation of the canonical wingless signaling pathway.

Keywords: gene identification; β-catenin pathway.

Fig. 1.

Identification of LRP5 variants p.R1188W in an extended Dutch PCLD-1 family (A) and three additional LRP5 variants in three unrelated PCLD families. Generations are denoted with Roman numerals, and individuals are numbered in a counterclockwise way. Squares indicate male sex, and circles indicate female sex. Solid symbols denote affected individuals, and open symbols are individuals without or unknown for PCLD. A slash indicates that the individual is deceased. (B) Simplified pedigree from PCLD-1 family with the clinical features by abdominal CT scanning and ultrasound of the liver of both probands (III/18 and II/18) in which whole-exome sequencing was performed (green arrow). (C) LRP5 is located at chromosome 11q13.2, and the sequence electropherogram shows heterozygous germ-line mutations. (D) All LRP5 missense mutations were located at highly evolutionary conserved amino acid regions with ortholog proteins from human to frog.

Fig. 2.

Functional and structural analyses of LRP5 variants in polycystic liver disease. (A) Immunohistochemistry of liver cyst tissue from proband III/18 of PCLD-1 family with LRP5 mutation c.3562C > T (p.R1188W). The cyst lining cholangiocytes present positive staining for LRP5 compared with the negative control next. (B) PCLD patient with a PRKCSH c.1341–2A > G mutation shows similar staining of liver cyst tissue and expression of LRP5 compared with A. (C) Localization of LRP5 protein was analyzed by immunofluorescence microscopy. CHO cells were transfected with constructs expressing LRP5_WT, (D) LRP5_R1188W, or LRP5_D1551N and compared with negative controls. No differences in LRP5 localization between all constructs was detected. (E) Presentation of the human LRP5 protein and homology modeling of the β-propeller domains highlighting the amino acid changes to emphasize the impact on the configuration and surrounding protein domains. (F) Homology modeling of the LRP5 domains and detailed view of the amino acid changes shows extracellular LRP5 mutation (*indicated) p.R1188W located at the sixth blade of the fourth β-propeller domain, and LRP5 mutation p.V454M at the third blade of the second β-propeller domain. Both intracellular mutations p.R1529S and p.D1551N are located between PPPSP motifs A and B and PPPSP motifs B and C, respectively. (G) CHO cells were transiently transfected with empty, WT, or mutant LRP5 vector. Whole cell lysate was analyzed by Western blotting using the V5 antibody and anti–β-actin. LRP5 protein expression levels (normalized to β-actin) are similar between LRP5_WT and both mutant constructs LRP5_R1188W and LRP5_D1551N. (H) Canonical Wnt signaling activity was analyzed by firefly luciferase activity and normalized to renilla luciferase activity with (in gray) or without addition of 250 ng/mL Wnt3a. All LRP5 constructs showed a significant increase in Wnt signaling activity compared with the empty vector (P < 0.0001). Both LRP5 mutants showed a decreased Wnt3a-induced signal activity (*P < 0.001; **P < 0.0001). Activity of LRP5_D1551N without Wnt3a was significantly decreased compared with LRP5_WT.