Genetic mutations in podocyte genes can increase susceptibility to focal segmental glomerulosclerosis (FSGS), but only a small proportion of patients have been identified who have a clear genetic basis underlying their disease. A new study from Haiyang Yu and colleagues describes the development of a pipeline to detect FSGS susceptibility genes. Nine FSGS candidate genes were identified by next-generation sequencing and three were validated in vivo using a novel high-throughput method.

Credit: Lara Crow/NPG

The researchers performed DNA sequencing of 2,500 genes that are highly and/or specifically expressed in podocytes, in 214 unrelated individuals of Northern European ancestry with biopsy-confirmed sporadic or familial FSGS. They found missense variants in WNK4, KANK1, and ARHGEF17 by single variant analyses. XYLT1, KAT2B, BPTF, DLG5, WNK4, and GC1 were identified as potential FSGS susceptibility genes by rare variant analyses. Enrichment was also found in two known FSGS susceptibility genes: APOL1 and COL4A4.

Next, a high-throughput method was designed to validate the candidate genes. An embryonic stem (ES) cell line was generated from FSGS-susceptible Cd2ap+/−; Synpo+/− mice, which was engineered to express a podocyte-specific doxycycline (DOX)-inducible transactivator. WNK4, KANK1, ARHGEF17, DLG5, and KAT2B were knocked down in the ES cells using shRNAs targeted specifically to the Hprt locus. KANK2 was also targeted by shRNA, as the precise mouse orthologue for KANK1 is unknown. Upon verification of shRNA targeting in vitro, mice were generated by laser-assisted microinjection of the ES cells into eight-cell embryos. Consequently, the mice were almost 100% derived from the shRNA-targeted ES cells without the need for further breeding.

proteinuria was identified in the Wnk4, Arhgef17, and Kank2 mice

Proteinuria was assessed in the animals 4–8 weeks after treatment with DOX. Substantial proteinuria was identified in the Wnk4, Arhgef17, and Kank2 mice by 8 weeks, but not in the Kank1 mice until 12 weeks; podocyte foot process effacement was also observed. No effect on renal function was detected in the Dlg5 animals.

The researchers conclude that their data support “a broader role for genetic susceptibility of both sporadic and familial cases of FSGS”. They propose that their methodology allows for rapid evaluation of candidate disease genes, and that this pipeline could be applied to other common or rare diseases.