Targeted disruption of Tbc1d20 with zinc-finger nucleases causes cataracts and testicular abnormalities in mice

Loss-of-function mutations in TBC1D20 cause Warburg Micro syndrome 4 (WARBM4), which is an autosomal recessive syndromic disorder characterized by eye, brain, and genital abnormalities. Blind sterile (bs) mice carry a Tbc1d20-null mutation and exhibit cataracts and testicular phenotypes similar to those observed in WARBM4 patients. In addition to TBC1D20, mutations in RAB3GAP1, RAB3GAP2 and RAB18 cause WARBM1-3 respectively. However, regardless of which gene harbors the causative mutation, all individuals affected with WARBM exhibit indistinguishable clinical presentations. In contrast, bs, Rab3gap1 (-/-) , and Rab18 (-/-) mice exhibit distinct phenotypes; this phenotypic variability of WARBM mice was previously attributed to potential compensatory mechanisms. Rab3gap1 (-/-) and Rab18 (-/-) mice were genetically engineered using standard approaches, whereas the Tbc1d20 mutation in the bs mice arose spontaneously. There is the possibility that another unidentified mutation within the bs linkage disequilibrium may be contributing to the bs phenotypes and thus contributing to the phenotypic variability in WARBM mice. The goal of this study was to establish the phenotypic consequences in mice caused by the disruption of the Tbc1d20 gene.

Our findings show that the disruption of Tbc1d20 in mice results in cataracts and aberrant acrosomal formation, thus establishing bs and Tbc1d20 (ZFN/ZFN) as allelic variants. Although the WARBM molecular disease etiology remains unclear, both the bs and Tbc1d20 (ZFN/ZFN) mice are excellent model organisms for future studies to establish TBC1D20-mediated molecular and cellular functions.

The zinc finger nuclease (ZFN) mediated genomic editing generated a Tbc1d20 c.[418_426del] deletion encoding a putative TBC1D20-ZFN protein with an in-frame p.[H140_Y143del] deletion within the highly conserved TBC domain. The evaluation of Tbc1d20 (ZFN/ZFN) eyes identified severe cataracts and thickened pupillary sphincter muscle. Tbc1d20 (ZFN/ZFN) males are infertile and the analysis of the seminiferous tubules identified disrupted acrosomal development. The compound heterozygote Tbc1d20 (ZFN/bs) mice, generated from an allelic bs/+ X Tbc1d20 (ZFN/+) cross, exhibited cataracts and aberrant acrosomal development indicating a failure to complement. Conclusions: Our findings show that the disruption of Tbc1d20 in mice results in cataracts and aberrant acrosomal formation, thus establishing bs and Tbc1d20 (ZFN/ZFN) as allelic variants. Although the WARBM molecular disease etiology remains unclear, both the bs and Tbc1d20 (ZFN/ZFN) mice are excellent model organisms for future studies to establish TBC1D20-mediated molecular and cellular functions.