Whole-Exome Capture and Sequencing Identifies HEATR2 Mutation as a Cause of Primary Ciliary Dyskinesia Amjad Horani, Todd E. Druley, Maimoona A. Zariwala,

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Whole-Exome Capture and Sequencing Identifies HEATR2 Mutation as a Cause of Primary Ciliary Dyskinesia Amjad Horani, Todd E. Druley, Maimoona A. Zariwala, Anand C. Patel, Benjamin T. Levinson, Laura G. Van Arendonk, Katherine C. Thornton, Joe C. Giacalone, Alison J. Albee, Kate S. Wilson, Emily H. Turner, Deborah A. Nickerson, Jay Shendure, Philip V. Bayly, Margaret W. Leigh, Michael R. Knowles, Steven L. Brody, Susan K. Dutcher, Thomas W. Ferkol The American Journal of Human Genetics Volume 91, Issue 4, Pages 685-693 (October 2012) DOI: 10.1016/j.ajhg.2012.08.022 Copyright © 2012 The American Society of Human Genetics Terms and Conditions

Figure 1 Family Pedigree and Genetic Analysis (A) Pedigree of consanguineous kindred with related nuclear families in Amish communities from the midwestern United States. Solid symbols represent affected individuals; central dots represent heterozygous individuals. The following abbreviations are used: si, situs inversus totalis; sa, situs ambiguus. VIII-1 specifies the proband. Asterisks indicate subjects who underwent exome sequencing. (B) Nucleotide sequences in affected HEATR2 region confirming exome sequencing results. HEATR2 mutation, NM_017802.3 (HEATR2): c.2384T>C, resulting in T-to-C transition at base position Chr7:819734 (hg19/GRCh37), is shown on the chromatograms for normal and PCD-affected subjects. Arrow indicates base change. This region does not have any reported nonsynonymous SNP or repeat elements. The HEATR2 variant allele segregated perfectly in the cohort and was homozygous mutant in all nine affected individuals. (C) HEATR2 mutation leads to an amino acid change from leucine to proline, Leu795Pro, an amino acid conserved from humans to microalgae. Arrow indicates amino acid change. The American Journal of Human Genetics 2012 91, 685-693DOI: (10.1016/j.ajhg.2012.08.022) Copyright © 2012 The American Society of Human Genetics Terms and Conditions

Figure 2 Ultrastructural and Functional Defects in Silenced C. reinhardtii (A) Phylogenetic tree showing the relationship of HEATR2 among diverse organisms generated from iTOL v2.28 A BLAST search identified two to four proteins with similarity to HEATR2 in organisms that have motile cilia or flagella. In contrast, none of the 72 species that lack an orthologous HEATR2 have motile cilia or flagella, which suggests a conserved role for HEATR2-like proteins in the formation of motile cilia and flagella. The numeral on the right side of each species indicates the number of HEATR2-like proteins found in the relevant genome database. (B) Ultrastructural appearance of outer microtubule doublets and dynein arms from C. reinhardtii flagella isolated from wild-type and amiRNA transformants with reduced HEATR2 mRNA. Outer dynein arms were absent in the transformant. Blue and white arrows indicate inner and outer dynein arms, respectively. (C) Isolated axonemes from wild-type (WT) and HEATR2-silenced (htr2) strains were resolved by SDS-PAGE and stained with Coomassie blue. Protein molecular weights are shown on the right, and asterisks indicate bands with obvious differences between the preparations. The polypeptide at 250 kDa is probably a membrane protein that is often variable in axonemal preparations, whereas identities of polypeptides at 150 kDa are unknown. Immunoblots for structural proteins were normalized to α-tubulin, and values are indicated as fractions. DIC2 is a component of the outer dynein arms and is nearly absent in htr2 axonemes; however, DIC4 is a component of the inner dynein arms and is equivalent. CNT1 is a component of the inner dynein arms and is overrepresented in the axonemes from silenced strains, which may reflect their shorter length. (D) Flagellar beat frequencies in wild-type uniflagellate (uni1-2) cells (WT) and uniflagellate double mutant (uni1-2:htr2) cells (htr2) (p < 0.0001). (E) Swimming speeds of WT, htr2-silenced, and oda2 (oda, outer dynein arm mutant) cells (p < 0.0001). The American Journal of Human Genetics 2012 91, 685-693DOI: (10.1016/j.ajhg.2012.08.022) Copyright © 2012 The American Society of Human Genetics Terms and Conditions

Figure 3 HEATR2 Localization in Ciliated Airway Epithelial Cells (A and B) Photomicrographs of normal human lung section (scale bar = 100 μm) (A) and bronchial epithelium (B) following immunofluorescent staining for HEATR2, which reveals the cytoplasmic localization of the protein (HEATR2, red) only in ciliated cells (cilia marker, acetylated α-tubulin (α-tub), green; nuclei stained with DAPI, blue) (scale bar = 10 μm). (C and D) Immunofluorescent staining of nasal epithelial cells cultured at an air-liquid interface from a healthy subject (NL) showing the presence of HEATR2 (C) and an individual with PCD showing a reduction of HEATR2 levels (D) (scale bar = 5 μm). (E) Immunoblot analysis of tracheobronchial (TR) epithelial cells and nasal epithelia (NP) cells from a healthy subject and an individual with PCD (PCD) confirms the very low level of the mutant form of HEATR2. (F and G) Immunoflourescent staining in representative human airway epithelial cells transfected with nontargeted, scrambled shRNA sequences (NT-shRNA) (F) and HEATR2-specific shRNA sequences (G) for HEATR2 (red) and acetylated α-tubulin (green) (scale bar = 5 μm). (H) Immunoblot analyses of airway epithelial cells transfected with each of three different HEATR2-specific shRNA (1–3) or nontargeted shRNA (NT) sequences and nontransfected control cells (M). (I–K) Ultrastructural appearance of cilium from the proband (I), and cilia from airway epithelial cells following transfection with (J) nontargeted and (K) HEATR2-targeted shRNA sequence 1. Blue and red arrows indicate inner and outer dynein arms, respectively. The American Journal of Human Genetics 2012 91, 685-693DOI: (10.1016/j.ajhg.2012.08.022) Copyright © 2012 The American Society of Human Genetics Terms and Conditions

Figure 4 Expression and Localization of Specific Inner and Outer Dynein Arm Proteins in Ciliated Airway Epithelial Cells (A) Immunofluorescent staining of nasal epithelial cells cultured at an air-liquid interface from a healthy subject (NL) showing the presence of outer dynein arm protein DNAI1 (red) and a subject with PCD (PCD) showing absence of HEATR2 expression. (B) Expression of DNAH7 (red), an inner dynein arm protein, as seen in cultured nasal epithelial cells from an individual with PCD (PCD) and a healthy subject (NL). DNAH7 was expressed and localized to cilia in both normal and PCD airway epithelial cells. Cilia were identified by immunofluorescent staining with α-tubulin (green), and nuclei were stained with DAPI (blue). Scale bar = 5 μm. The American Journal of Human Genetics 2012 91, 685-693DOI: (10.1016/j.ajhg.2012.08.022) Copyright © 2012 The American Society of Human Genetics Terms and Conditions