Isolated polycystic liver disease genes define effectors of polycystin-1 function

Abstract

Dominantly inherited isolated polycystic liver disease (PCLD) consists of liver cysts that are radiologically and pathologically identical to those seen in autosomal dominant polycystic kidney disease, but without clinically relevant kidney cysts. The causative genes are known for fewer than 40% of PCLD index cases. Here, we have used whole exome sequencing in a discovery cohort of 102 unrelated patients who were excluded for mutations in the 2 most common PCLD genes, PRKCSH and SEC63, to identify heterozygous loss-of-function mutations in 3 additional genes, ALG8, GANAB, and SEC61B. Similarly to PRKCSH and SEC63, these genes encode proteins that are integral to the protein biogenesis pathway in the endoplasmic reticulum. We inactivated these candidate genes in cell line models to show that loss of function of each results in defective maturation and trafficking of polycystin-1, the central determinant of cyst pathogenesis. Despite acting in a common pathway, each PCLD gene product demonstrated distinct effects on polycystin-1 biogenesis. We also found enrichment on a genome-wide basis of heterozygous mutations in the autosomal recessive polycystic kidney disease gene PKHD1, indicating that adult PKHD1 carriers can present with clinical PCLD. These findings define genetic and biochemical modulators of polycystin-1 function and provide a more complete definition of the spectrum of dominant human polycystic diseases.

Figure 1. Quantile-quantile plots of observed P values versus an expected distribution of P values.

(A) Observed P values calculated using a binomial test by comparison of the observed burden of rare (MAF < 1 × 10^–3) loss-of-function variants in PCLD cases with the “expected” burden based on the gene transcript length (n = 102). (B) Observed burden in the European subset of discovery cohort (n = 92) compared with European controls (n = 3,274) using Fisher’s exact test.

Figure 2. Mutations in ALG8 cause abnormal biogenesis of PC1.

(A) YU313 has innumerable liver cysts larger than 1 cm (circle). (B) T-70 has innumerable small liver cysts (arrows). (C) W-YU363 has innumerable liver cysts, many larger than 1 cm (arrows, top panel), and 3–4 kidney cysts (arrows, bottom panel), while his 19-year-old daughter, YU364 (D), has the same ALG8 mutation but 8 kidney cysts (arrows, top and bottom panels) and no liver cysts. (E and F) Immunoblots of cell lysates with anti-HA (E) and anti-LRR PC1 N-terminal antibody (7e12) (F) show decreased PC1-CTF, PC1-FL, PC1-NTR, and PC1-NTS in Alg8^–/– cells. Re-expression of Alg8⁺ in Alg8^–/– cells rescues PC1 expression (F). The labels in red mark the migration of the respective PC1 fragments in the Alg8^–/– cell lysate. (G and H) Anti-HA immunoprecipitation of PC1-FL and PC1-CTF was treated with PNGase F or EndoH. (G) Altered migration of PC1-FL is due to hypoglycosylation in Alg8^–/– cells. PNGase F treatment shows equivalent deglycosylated PC1-FL migration. (H) The relative proportion of EndoH-resistant/EndoH-sensitive PC1-CTF is reduced in Alg8^–/– cells. In G and H, the 2 PC1-CTF bands in the PNGase F–treated lanes result from alternatively spliced forms present in rodents (66). (I) Immunofluorescence shows absence of detectable PC1 (anti-HA) in cilia of Alg8^–/– cells. (J) Alg8^–/– cells activate the IRE1α/XBP1 branch of UPR, demonstrated by presence of XBP1s by reverse transcription PCR (RT-PCR; top) and immunoblotting (bottom). (K) Anti-HA immunoblot shows that inactivation of both Alg8 and Xbp1 does not change PC1 hypoglycosylation or GPS cleavage compared with Alg8 knockout alone. HSP90 serves as loading control for cell lysates.

Figure 3. Mutations in GANAB cause PCLD.

(A) T-116 has innumerable large liver cysts (asterisks) with resultant hepatomegaly. (B) TOR6205 has innumerable small liver cysts (arrows). Kidney cysts are present in both cases (arrows). (C and D) Reduced protein expression and altered migration of PC1 fragments in Ganab^–/– cells. Immunoblot of cell lysates with anti–PC1-LRR N-terminal antibody (C, top panel) and anti-GANAB (C, GIIα; middle panel) demonstrates absence of PC1-NTR (asterisk) in Ganab^–/– cells. Anti-HA immunoblot (D) shows significant decrease in PC1-CTF. PC1-FL (C and D) and PC1-NTS (C) have slower migration in Ganab^–/– cells. The PC1 expression level and altered gel-migration pattern are rescued by re-expression of Ganab (C, Ganab^–/–;Ganab⁺). The re-expressed GIIα is larger than the native protein because of the inclusion of an epitope tag and an alternatively spliced exon 6 that is absent from the native protein in the mouse cell line. (E) PC2 and PC1-FL show higher molecular mass in Ganab^–/– cells that resolves to the same gel migration in WT cells following PNGase F treatment. *Nonspecific band. (F) Ganab^–/– cells only have EndoH-sensitive (S) and lack EndoH-resistant (R) PC1-CTF. PC1-CTF shows 2 bands due to alternative spice forms in rodents. (G) IRE1α/XBP1 branch of UPR is activated in Ganab^–/– cells as evidenced by increased XBP1s. Tunicamycin (Tun) treatment (2.5 μg/ml for 6 hours) serves as positive control for UPR activation. (H) Functional assay of GANAB missense variants. Fluorescence output from the glucosidase II fluorogenic substrate 4-methylumbelliferyl α-d-glucopyranoside (4-MUG) from HEK 293T cells cotransfected with GIIβ and the respective variants of GIIα. Background fluorescence from untransfected cell lysates is subtracted. HSP90 serves as loading control for cell lysates.

Figure 4. Mutations in Sec61b have profound effects on quantity and maturation of PC1.

(A) Liver imaging for YU356 showing numerous small liver cysts (arrows). (B) Immunoblots of cell lysates with C-terminal (anti-HA; left) and N-terminal (anti-LRR, 7e12; right) antibodies show that all PC1 fragments are markedly decreased to almost undetectable levels in Sec61b^–/– cells. PC1-NTR is absent even on long exposure. Immunoblot with anti-SEC61β antibody shows absence of SEC61β protein in Sec61b^–/– cells. WT, isogenic wild-type control cells. (C) Long-exposure immunoblot of cell lysates with anti-HA showing a minute amount of PC1-FL but complete absence of PC1-CTF. (D) Immunoblots of cell lysates with anti-HA showing complete rescue of PC1 phenotype with stable re-expression of Sec61b (Sec61b^–/–;Sec61b⁺). (E) Activation of the IRE1α/XBP1 branch of UPR as evidenced by increased levels of XBP1s in Sec61b^–/– cells. Tunicamycin (Tun) treatment (2.5 μg/ml for 6 hours) serves as positive control for UPR activation. HSP90 serves as loading control for all immunoblots.

Figure 5. Adult carrier parents for ARPKD can develop PCLD.

(A–F) Liver and kidney imaging for cases with heterozygous PKHD1 loss-of-function mutations. T-58 (A), TOR6216 (B), T-61 (C), T-59 (D), and TOR6321 (E) have innumerable small liver cysts (arrows). (F) T-74 has more than 10 liver cysts (arrows and asterisks), some greater than 1 cm in diameter (asterisks), and at least 1 kidney cyst larger than 1 cm (asterisk). (G) Immunoblots of cell lysates with the PC1 C-terminal epitope (anti-HA, left) and PC1 N-terminal anti-LRR antibody (7e12, right) using WT cells and 2 independent Pkhd1^–/– cell lines showing no significant difference in any PC1 fragment and no change in PC2 quantity. HSP90 serves as loading control. (H) Pkhd1^–/– cells show positive PC1 by anti-HA staining in cilia (green) marked by anti-ARL13B (red). Pkhd1^–/– (1) is shown, but this was found in Pkhd1^–/– (2) as well.

Figure 6. Schematic of the function of the PCLD genes in the ER protein biogenesis pathway.

The PCLD genes are numbered 1–5. Lipid-linked oligosaccharide precursors of N-linked glycans are initially assembled on dolichol on the cytoplasmic aspect of the ER membrane. These are flipped into the ER lumen, where ALG8 (1) catalyzes the addition of the second glucose residue. Nascent polypeptides undergo cotranslational translocation via the SEC61 translocation pore that is composed of α, β (2), and γ subunits and is associated with SEC62. SEC63 (3) and ERJ1 act in concert with the major ER HSP70 chaperone BiP to facilitate this translocation process. Oligosaccharyl transferase (OST) catalyzes the attachment of the glycan moiety to asparagine residues. Glucosidase I removes the outermost glucose before glucosidase II, composed of GIIα (4) and GIIβ (5) subunits, removes the second glucose. This step is necessary for the nascent peptide to enter the calnexin (CNX)/calreticulin (CRT) protein folding and quality control cycle. Glucosidase II (4, 5) subsequently removes the innermost glucose from the N-linked glycan, allowing for exit from the CNX/CRT cycle. If the protein has attained its properly folded conformation, it proceeds along the secretory pathway. Misfolded proteins are recognized and reglucosylated by UGGT, allowing for more time in the folding environment of the CNX/CRT cycle. Eventually, proteins that fail to fold properly undergo ER-associated degradation by retrotranslocation through the SEC61 translocon complex into the cytoplasmic compartment, where they are degraded by the proteasome.