ALG9 Mutation Carriers Develop Kidney and Liver Cysts

Abstract

Background: Mutations in PKD1 or PKD2 cause typical autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic kidney disease. Dominantly inherited polycystic kidney and liver diseases on the ADPKD spectrum are also caused by mutations in at least six other genes required for protein biogenesis in the endoplasmic reticulum, the loss of which results in defective production of the PKD1 gene product, the membrane protein polycystin-1 (PC1).

Methods: We used whole-exome sequencing in a cohort of 122 patients with genetically unresolved clinical diagnosis of ADPKD or polycystic liver disease to identify a candidate gene, ALG9, and in vitro cell-based assays of PC1 protein maturation to functionally validate it. For further validation, we identified carriers of ALG9 loss-of-function mutations and noncarrier matched controls in a large exome-sequenced population-based cohort and evaluated the occurrence of polycystic phenotypes in both groups.

Results: Two patients in the clinically defined cohort had rare loss-of-function variants in ALG9, which encodes a protein required for addition of specific mannose molecules to the assembling N-glycan precursors in the endoplasmic reticulum lumen. In vitro assays showed that inactivation of Alg9 results in impaired maturation and defective glycosylation of PC1. Seven of the eight (88%) cases selected from the population-based cohort based on ALG9 mutation carrier state who had abdominal imaging after age 50; seven (88%) had at least four kidney cysts, compared with none in matched controls without ALG9 mutations.

Conclusions: ALG9 is a novel disease gene in the genetically heterogeneous ADPKD spectrum. This study supports the utility of phenotype characterization in genetically-defined cohorts to validate novel disease genes, and provide much-needed genotype-phenotype correlations.

Keywords: ADPKD; chronic renal disease; cystic kidney; human genetics; kidney stones; polycystic kidney disease.

Graphical abstract

Figure 1.

Alg9 loss causes abnormal biogenesis of PC1. (A) Immunoblots of cell lysate with anti-HA (left panel) and anti-LRR PC1–N-terminal antibody (7e12) (right panel) show quantitative decrease in PC1–C-terminal fragment (PC1-CTF) and the mature EndoH-resistant fraction of PC1–N-terminal fragment (PC1-NTR), as well as faster migration of PC1 full-length (PC1-FL) and the immature EndoH-sensitive PC1–N-terminal fragment (PC1-NTS) in Alg9^−/− cells compared with controls. Re-expression of human ALG9-GFP in Alg9^−/− cells rescues both the quantitative and migration differences (third lane). (B) Cell lysate was treated with EndoH and blotted with anti-HA. The migration difference of PC1-FL between control and Alg9^−/− cell lysates is eliminated after EndoH treatment, showing that there is altered glycosylation of PC1 in the absence of Alg9. (C and D) Anti-HA immunoblots of cell lysate from Alg9^−/− cells with or without stable re-expression of human ALG9-GFP with either wild-type (ALG9) or the indicated missense variants. The known pathogenic missense mutation, p.Y287C, and the experimental missense variant, p.R370K, do not rescue the PC1 phenotype in Alg9^−/− cells. (D) The other experimental missense variants rescue the PC1 phenotypes. This bioassay identifies p.R370K as a deleterious missense mutation and p.A232P, p.A280V, pN315S, and p.R517L as benign variants. (E) Imaging for YU202 with ALG9-p.R370K. Serial CT scan sections show multiple small kidney cysts (arrows) many of which are likely hemorrhagic/proteinaceous (left panel) and innumerable liver cysts (right panel).

Figure 2.

Cystic kidney phenotypes in carriers of ALG9 heterozygous loss-of-function variants are of varying severity. (A) Ultrasound long-axis views from anteroposterior sweep, and (B and C) axial CT scan sections. Quantification of lesions in all cases is presented in Table 2.