DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia.

You Li,Richard Francis,Stephanie M Ware,Toshio Nakanishi,Elizabeth Goldmuntz,Gregory J Pazour,Muhammad Tariq,Yoshiyuki Furutani,Maliha Zahid,Linda Leatherbury,Michael Tsang,Hisato Yagi,Ezenwa Obi Onuoha,Iman Sami,Cheng Cui,Stephen M King,Rama Rao Damerla,Manush Saydmohammed,Gregory M Hendricks ,Dong-Min Lee ,Cecilia Lo

doi:10.1371/journal.pgen.1005821

Abstract

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.

Highlights

Heterotaxy and primary ciliary dyskinesia (PCD, OMIM: 24440) are both rare heritable disorders with prevalence of approximately 1 in 10,000[1, 2]
We further showed DNAH6 can interact with other PCD genes to mediate a more complex oligogenic etiology of disease
These findings show DNAH6 is required for motile cilia function mediating airway clearance and left/right patterning, and this may involve oligogenic interactions that can contribute to the complex genetics of heterotaxy and PCD

Summary

Introduction

Heterotaxy and primary ciliary dyskinesia (PCD, OMIM: 24440) are both rare heritable disorders with prevalence of approximately 1 in 10,000[1, 2]. While heterotaxy patients exhibit defects in patterning of the left-right body axis, PCD patients suffer sinopulmonary disease due to airway mucus clearance defects caused by immotile or dyskinetic respiratory cilia in the airway These two seemingly disparate diseases may have overlapping genetic etiology, as PCD patients can exhibit laterality defects, comprising either of complete reversal of visceral organ situs (situs inversus) as in Kartagener’s syndrome or randomization of visceral organ situs as in heterotaxy. A link between PCD and heterotaxy is further supported by the recent finding of a high prevalence of airway ciliary dysfunction similar to that seen with PCD in heterotaxy patients with congenital heart disease (CHD) This is of clinical importance, as heterotaxy is highly associated with complex CHD and CHD/heterotaxy patients are known to have high postsurgical morbidity and mortality associated with more respiratory complications[3]. Insights into the genetic overlap between heterotaxy and PCD may have relevance for clinical management of this highly vulnerable CHD patient population

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