Identification of a Novel Cause of X-linked Heterotaxy. M. Tariq1, A. E. Cast1, J. W. Belmont2, S. M. Ware1,3 1) Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 2) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 3) Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.

   Heterotaxy is characterized by severe cardiovascular malformations resulting from abnormalities of the proper specification of left-right (LR) asymmetry during embryonic development with a prevalence of ~1 in 10,000 newborns. As a result of abnormal LR development, an unusual spatial arrangement of thoracic and/or abdominal organs may occur. The high relative risk of heterotaxy within families highlights its genetic basis. However, heterotaxy is challenging for traditional genetic analyses because of clinical and genetic heterogeneity, variable expressivity, and non-penetrance. Although the genetic basis is most frequently multifactorial, Mendelian inheritance does occur, and X-linked inheritance resulting from mutations in the transcription factor ZIC3 was initially described in 1997. However, some X-linked pedigrees do not contain mutations in coding regions of ZIC3 despite being closely linked to the ZIC3 locus. In this study we recruited 4 X-linked heterotaxy pedigrees, previously found to be negative for ZIC3 mutations, and investigated genetic causation using whole-exome or custom X-exome sequencing and segregation analysis of DNA variants. In one pedigree with 4 affected males including two with asplenia, gut malrotation, L-transposition of the great arteries, and abdominal situs inversus, a rare missense hemizygous variant in GPR101 (p.Val409Met) was identified, predicted to be damaging for protein function by bioinformatic tools. This variant falls in the sixth transmembrane domain (TM1-7) of GPR101, an orphan G-protein coupled receptor with no known ligand. This candidate, which is located just 534 Kb upstream of ZIC3, was further investigated by expression analyses in mouse, including RT-PCR and whole mount in-situ hybridization, with results suggesting a possible role in LR pattering and early heart development. Morpholino-induced knockdown of Gpr101 in Xenopus laevis resulted in heterotaxy with randomization of heart and gut looping, suggesting Gpr101 is a novel gene involved in embryonic asymmetric laterality. This study identifies GPR101 as a novel X-linked genetic cause of heterotaxy and provides opportunity to elucidate new mechanisms controlling embryonic LR asymmetry.

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