Mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganization and absent inner dynein arms

Abstract

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed "radial spoke defect." We sequenced CCDC39 and CCDC40 in 54 "radial spoke defect" families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice, and frameshift predicting early protein truncation, which suggests this defect is caused by "null" alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganization and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as "IDA and microtubular disorganisation defect," rather than "radial spoke defect."

Figure 1

Mutation segregation and ultrastructural defects in selected CCDC39 and CCDC40 patients. A, B: PCD family pedigrees from the UCL-ICH cohort with autosomal recessive inheritance of CCDC39 and CCDC40 mutations. Affected individuals are indicated by black symbols, consanguineous marriages by a double horizontal line. Segregation patterns for the rest of the cohort are shown in Supp. Figure S1. C: (a) Normal 9+2 ciliary ultrastructure shown; ODA, outer dynein arm; IDA, inner dynein arm; N-DRC, nexin-dynein regulatory complex. Representative transmission electron micrographs of cells from nasal brush biopsy of CCDC40 patient 114 II:1 show (b) consistent loss of inner dynein arms (lightening strikes), typical translocation of peripheral outer doublet microtubules (white arrow), accentric microtubular central pairs (white arrow head). In addition both extra central microtubules (c, black arrow) and total absence of the central microtubular pair (d, black arrowhead) were occasionally seen. (d) Similar findings were observed in a fallopian tube biopsy of the same patient. Scale bar, 200 nm.

Figure 2

Location of mutations in CCDC39 and CCDC40. A, B: Location of CCDC39 and CCDC40 mutations identified in this study indicated below the gene and protein images in red; mutations identified in two previous studies indicated above in black ((Merveille et al., 2011; Becker-Heck et al., 2011; Nakhleh et al., 2012). Several predicted protein domains are indicated by rectangles: coiled coil domains (green, amino acid numbers shown), Structural Maintenance of Chromosomes domains (SMC, blue) and domains with similarity to yeast BRE1 histone ubiquitylation protein (BRE1, purple).

Figure 3

Localisation of axonemal components in CCDC40 patient respiratory cells. A: Representative immunofluorescent images of respiratory cells localise CCDC39 protein (green) along the length of the ciliary axonemes in control cells. B: Complete absence of CCDC39 from axonemes from a patient 114 II:1 carrying CCDC40 mutations. Dual staining with anti-acetylated alpha tubulin (axonemes) and anti-gamma tubulin (basal bodies) was used to stain the cilia (red). C, D: RSPH4A (green) localises along the length of axonemes at similar levels in cells from both control and patient 114 II:1 carrying CCDC40 mutations. DNA in nuclei was stained using DAPI. DIC indicates differential interference contrast microscopy images.