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

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\u27s 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

<i>dnah6</i> morpholino knockdown in zebrafish embryo cause laterality defects.

<p><b>(A,B)</b><i>dnah6</i> MO injected embryos exhibited curly tail and cardiac edema phenotype at 48 hours post fertilization (hpf). <b>(C,D)</b> <i>dnah6</i> MO injected embryos at 48hpf exhibited heart (C) and gut (D) looping defects, including failure to loop (middle panels) and reversal looping (right panels). <b>(E)</b> In <i>situ</i> hybridization analysis revealed abnormal right sided (R), bilateral (B), and absent (Ab) <i>spaw</i> expression after <i>dnah6</i> MO knockdown. <b>(F,G)</b> Heart/gut looping defects were observed in <i>dnah6 MO</i>-injected embryos, including normal (N), right sided (R), and straight (St) heart/gut looping phenotypes. <b>(H).</b> KV cilia were shorter (middle) in <i>dnah6</i> AUG-MO but not <i>dnah6</i> spl-MO injected embryos (p-value = 0.0017). <b>(I)</b>. High-speed videomicroscopy showed reduction in KV motile cilia of <i>dnah6</i> MO injected embryos (p = 1.6x10^-4 for 7.5 ng and p = 1.1x10^-5 for 10 ng <i>dnah6</i> MO; two-tailed Student’s t-test). <b>(J)</b> Little bead movement (color tracing) was observed in <i>dnah6</i> MO injected embryos. <b>(K)</b> KV flow was absent or reduced with <i>dnah6</i> knockdown (Chi-square test, p-value = 0.0192).</p

<i>DNAH6</i> and PCD gene mutations identified in heterotaxy patients.

<p><i>DNAH6</i> and PCD gene mutations identified in heterotaxy patients.</p

Heterotaxy patient cohorts analyzed by targeted and whole exome sequencing analyses.

<p><b>(A)</b> 25 heterotaxy (HTX) patients from Children’s National Medical Center were sequence analyzed with targeted ciliome sequencing (yellow) to identify cilia-related mutations. This includes 13 HTX patients with airway ciliary dysfunction (CD) and 12 patients with normal airway cilia function (without CD). This analysis identified <i>DNAH6</i> as the only candidate gene with mutations found exclusively in patients with CD (n = 2). <b>(B)</b> Whole-exome sequencing (pink) was conducted in 23 HTX patients from Cincinnati Children’s Hospital, with 1 patient identified with a novel homozygous <i>DNAH6</i> mutation. <b>(C)</b> <i>DNAH6</i> amplicon resequencing (blue) was conducted on 72 HTX patients from Tokyo Women’s Medical University and 54 patients from Children’s Hospital of Philadelphia. Of these 126 patients, 6 were found to have heterozygous <i>DNAH6</i> mutation. The latter 6 patients were further analyzed by whole exome sequencing (pink). In 5 of these patients, additional heterozygous mutations were found in other PCD genes including, 4 mutations in <i>DNAH5</i>.</p

<i>Dnah6</i> genetically interacts with <i>Dnai1</i> and <i>Dnah5</i> to cause heterotaxy and PCD.

<p><b>(A,B)</b> Embryos injected with subthreshold dose of <i>dnah6</i> and <i>dnai1</i> MO show increased heart looping defects compared with Ctrl MO injections (n = 177, p-value = 3.8x10^-8), or single injection of either <i>dnai1</i> (p = 9.29x10^-9) or <i>dnah6</i> (p = 2.04x10^-8) MO at the same MO dose (A). Similar results were observed with subthreshold <i>dnah5/dnah6</i> double MO knockdown (n = 82; p = 1.74x10^-5, Bonferroni corrected)<b>. (C,D)</b> Reciliating mouse airway epithelia from wildtype (+/+) and heterozygous (+/-) <i>Dnai1</i> knockout (C) or <i>Dnah5</i> mutant (D) mice show robust ciliation and ciliary motion (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s017" target="_blank">S5 Movie</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s018" target="_blank">S6 Movie</a>). 30nM <i>Dnah6</i> siRNA had no effect on ciliation or cilia motility in wildtype airway epithelia, but in heterozygous <i>Dnai1 or Dnah5</i> mutant airway, ciliation was reduced and ciliary motion was dyskinetic (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s017" target="_blank">S5 Movie</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s018" target="_blank">S6 Movie</a>). With 50nM siRNA, little or no cilia was seen in wildtype and heterozygous <i>Dnai1</i> or <i>Dnah5</i> mutant mouse airway.</p

Aberrant TGF-β signaling reduces T regulatory cells in ICAM-1-deficient mice, increasing the inflammatory response to Mycobacterium tuberculosis

Foxp3+ T regulatory cells are required to prevent autoimmune disease, but also prevent clearance of some chronic infections. While natural T regulatory cells are produced in the thymus, TGF-β1 signaling combined with T-cell receptor signaling induces the expression of Foxp3 in CD4+ T cells in the periphery. We found that ICAM-1−/− mice have fewer T regulatory cells in the periphery than WT controls, due to a role for ICAM-1 in induction of Foxp3 expression in response to TGF-β1. Further investigation revealed a functional deficiency in the TGF-β1-induced translocation of phosphorylated Smad3 from the cytoplasmic compartment to the nucleus in ICAM-1-deficient mice. This impairment in the TGF-β1 signaling pathway is most likely responsible for the decrease in T regulatory cell induction in the absence of ICAM-1. We hypothesized that in the presence of an inflammatory response, reduced production of inducible T regulatory cells would be evident in ICAM-1−/− mice. Indeed, following Mycobacterium tuberculosis infection, ICAM-1−/− mice had a pronounced reduction in T regulatory cells in the lungs compared with control mice. Consequently, the effector T-cell response and inflammation were greater in the lungs of ICAM-1−/− mice, resulting in morbidity due to overwhelming pathology