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PLoS Genet. 2019 Nov 15;15(11):e1008467. doi: 10.1371/journal.pgen.1008467. eCollection 2019 Nov.

Gpr63 is a modifier of microcephaly in Ttc21b mouse mutants.

Author information

1
Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
2
Neuroscience Graduate Program, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.
3
Department of Anatomy and Embryology, Faculty of Medicine (Girl's Section), Al-Azhar University, Cairo, Egypt.
4
Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.
5
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
6
Shriner's Hospital for Children - Cincinnati, Cincinnati, Ohio, United States of America.

Abstract

The primary cilium is a signaling center critical for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background and mutants on an FVB/NJ (FVB) background develop a forebrain significantly smaller than mutants on a C57BL/6J (B6) background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified an orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP that is polymorphic between the FVB and B6 strains in Gpr63 and creates a missense mutation predicted to be deleterious in the FVB protein. We used CRISPR-Cas9 genome editing to create two lines of FVB congenic mice: one with the B6 sequence of Gpr63 and the other with a deletion allele leading to a truncation of the GPR63 C-terminal tail. We then demonstrated that Gpr63 can localize to the cilium in vitro. These alleles affect ciliary localization of GPR63 in vitro and genetically interact with Ttc21baln/aln as Gpr63;Ttc21b double mutants show unique phenotypes including spina bifida aperta and earlier embryonic lethality. This validated Gpr63 as a modifier of multiple Ttc21b neural phenotypes and strongly supports Gpr63 as a causal gene (i.e., a quantitative trait gene, QTG) within the Moaq1 QTL.

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