Cheng Cui, Hisato Yagi, Elizabeth Goldmuntz, Yoshiyuki Furutani, Dong Min Lee, Toshio Nakanishi, Richard Francis, Cecilia W. Lo, Stephen M. King, Manush Saydmohammed, Muhammad Tariq, Rama Rao Damerla, You Li, Iman Sami, Ezenwa O. Onuoha, Stephanie M. Ware, Maliha Zahid, Gregory J. Pazour, Michael Tsang, Linda Leatherbury, and Gregory M. Hendricks
Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer’s vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease model with broad relevance for further interrogating the genetic etiology of human ciliopathies., Author Summary Heterotaxy is a birth defect involving randomization of left-right body axis. Its genetic etiology is still poorly understood, but recent studies suggest mutations in genes causing primary ciliary dyskinesia (PCD), a sinopulmonary disease, also can cause heterotaxy. Moreover, heterotaxy patients can show airway cilia dysfunction reminiscent of PCD. The link between these two seemingly disparate diseases reflects the common requirement for motile cilia in both left-right patterning and airway mucus clearance. Sequencing analysis of heterotaxy patients together with experimental modeling identified DNAH6 as a novel gene that can cause both heterotaxy and PCD. We further showed DNAH6 can interact with other PCD genes to mediate a more complex oligogenic etiology of disease. Thus experimental modeling with double gene knockdown showed digenic interactions of DNAH6 with DNAH5 or DNAI1 could disrupt motile cilia function in the respiratory epithelia and also cause heterotaxy in zebrafish embryos. These findings provide the first experimental evidence indicating oligogenic interactions can contribute to the complex genetics of heterotaxy.