Abstract
ABSTRACTHuman bestrophin-1 (BEST1) is an integral membrane protein known to function as a Ca2+-activated and volume-regulated chloride channel. The majority of disease-associated mutations in BEST1 constitute missense mutations and were shown in vitro to lead to a reduction in mutant protein half-life causing Best disease (BD), a rare autosomal dominant macular dystrophy. To further delineate BEST1-associated pathology in vivo and to provide an animal model useful to explore experimental treatment efficacies, we have generated a knock-in mouse line (Best1Y227N). Heterozygous and homozygous mutants revealed no significant ocular abnormalities up to 2 years of age. In contrast, knock-in animals demonstrated a severe phenotype in the male reproductive tract. In heterozygous Best1Y227N males, Best1 protein was significantly reduced in testis and almost absent in homozygous mutant mice, although mRNA transcription of wild-type and knock-in allele is present and similar in quantity. Degradation of mutant Best1 protein in testis was associated with adverse effects on sperm motility and the capability to fertilize eggs. Based on these results, we conclude that mice carrying the Best1 Y227N mutation reveal a reproducible pathologic phenotype and thus provide a valuable in vivo tool to evaluate efficacy of drug therapies aimed at restoring Best1 protein stability and function.
Highlights
Bestrophin-1 (BEST1) was initially identified via positional cloning of the gene causing the autosomal dominant Best vitelliform macular dystrophy, known as Best disease (BD) (Marquardt et al, 1998; Petrukhin et al, 1998)
We report the generation and characterization of a gene-modified mouse line carrying the human BD-associated mutation Y227N in the endogenous murine Best1 gene. This gene mutation in the human homologue is well established to cause autosomal dominant BD, we show that the mouse fails to reveal any of the typical features of human BEST1-associated retinopathy but instead exhibits strong effects on protein stability in the testis and on sperm motility
The absence of an ocular/retinal phenotype in the Best1Y227N mouse, even in two genetically divergent backgrounds, may be explained by findings in this study and by others that normal as well as mutant Best1 protein is below immunohistochemical detection in the murine retinal pigment epithelium (RPE) which is in striking contrast to the human situation (Mullins et al, 2005)
Summary
Bestrophin-1 (BEST1) was initially identified via positional cloning of the gene causing the autosomal dominant Best vitelliform macular dystrophy, known as Best disease (BD) (Marquardt et al, 1998; Petrukhin et al, 1998). We and others showed that MDCKII BEST1transfected cell culture models are well suited to demonstrate mislocalization (Johnson et al, 2014, 2013; Milenkovic et al, 2011) and reduced protein stability (Uggenti et al, 2016; Milenkovic et al, 2018) of autosomal dominant as well as autosomal recessive BEST1 mutations These observations were further supported by studies in BD patient-derived human induced pluripotent stem cell (hiPSC) retinal pigment epithelium (RPE) (Milenkovic et al, 2015; Singh et al, 2015; Marmorstein et al, 2018). While this phenotype was associated with potential abnormalities in calcium homeostasis, a direct link of these findings with the mutant Best protein and the ocular phenotype has not been established so far
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