Exome sequencing identifies mutations in CCDC114 as a cause of primary ciliary dyskinesia

Abstract

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ~60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.

Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Cross-section Analysis of Ciliary Ultrastructure by Electron Microscopy and Segregation Analysis of CCDC114 (A) Normal ciliary ultrastructure from healthy subject (left) and absence of outer dynein arms (right) from UNC family 62 with biallelic mutations in CCDC114. (B–E) Pedigrees with mutations segregating in trans, consistent with autosomal-recessive mode of inheritance.

Figure 2

Genomic Organization of CCDC114 and Location of PCD-Causing Mutations (A) The transcript of CCDC114 consists of 3,215 bp and encodes 670 amino acid protein. Filled box designates exons “E,” horizontal lines designate intron “IVS,” and location of 5′ UTR, 3′ UTR, and start and stop codons are shown. Introns/exons are not drawn to scale. The positions of all identified CCDC114 mutations are indicated. (B, D, and E) Heterozygous sequences in affected (bottom) and corresponding wild-type (top) sequences in controls. (C) Homozygous (bottom) and heterozygous (middle) sequences in affected and the corresponding wild-type sequences (top) in control. Base sequence, amino acid sequence, and codon numbers are shown. Intron-exon junctions are shown with the red dotted lines.

Figure 3

Expression Analysis via Reverse-Transcriptase PCR (A) Expression pattern of CCDC114 in a panel of normal human tissues. (B) Expression pattern of CCDC114 (top) during ciliogenesis in human airway cells. RNA was isolated from human airway epithelia cells at different times after plating on collagen-coated culture inserts at an air/liquid interface. Under these conditions the cells initially grow as a single monolayer of undifferentiated “basal-like” cells. After 10–14 days, ciliated cells begin to appear, increasing over the next 2–3 weeks before plateauing at approximately 60%–80% of the culture. For both expression analyses, DNAI1 (middle) was used as a known ciliary gene control and cyclophilin (bottom) as a positive RNA control.