GSE202440
An ancient founder mutation located between ROBO1 and ROBO2 is responsible for increased microtia risk in Amerindigenous populations (RNA-Seq)
Microtia is a congenital malformation that encompasses mild hypoplasia to complete loss of the external ear, or pinna. Although contribution of genetic variation and environmental factors to microtia remains elusive, Amerindigenous populations have the highest reported incidence. Here, using both transmission disequilibrium tests (TDT) and association studies in microtia trios (parents and affected child) and microtia cohorts enrolled in Latin America, we map a ~10 kb microtia locus (OR 4.7; p=6.78e-18) to the intergenic region between Roundabout 1 (ROBO1) and Roundabout 2 (ROBO2) (chr3: 78546526-78555137).  While alleles at the microtia locus significantly increase the risk of microtia, their penetrance is low (<1%).  We demonstrate that the microtia locus contains a polymorphic complex repeat element that is expanded in affected individuals.  The locus is located near a chromatin loop region that regulates ROBO1  and ROBO2 expression in induced pluripotent stem cell-derived neural crest cells. Furthermore, we use single nuclear RNA sequencing (snRNAseq) to demonstrate ROBO1 and ROBO2 expression in both fibroblasts and chondrocytes of the mature human pinna. Because the microtia allele is enriched in Amerindigenous populations and shared by some East Asian subjects with craniofacial malformations, we propose both populations share a mutation that arose in a common ancestor prior to the ancient migration of Eurasian populations into the Americas, and that the high incidence of microtia among Amerindigenous populations reflects the population bottleneck that occurred during the migration out of Eurasia. Overall design: Comparative gene expression profiling analysis of RNA-seq data for undifferentiated iPSC and iPSC-derived neural crest cells (NCC) with 40 and 100kb deletions of an intergenic region between ROBO1 and ROBO2 genes