Whole genome sequencing enhances molecular diagnosis of primary ciliary dyskinesia

Abstract

Background: Primary ciliary dyskinesia (PCD) is a genetic disorder affecting motile cilia. Most cases are inherited recessively, due to variants in >50 genes that result in abnormal or absent motile cilia. This leads to chronic upper and lower airway disease, subfertility, and laterality defects. Given overlapping clinical features and genetic heterogeneity, diagnosis can be difficult and often occurs late. Of those tested an estimated 30% of genetically screened PCD patients still lack a molecular diagnosis. A molecular diagnosis allows for appropriate clinical management including prediction of phenotypic features correlated to genotype. Here, we aimed to identify how readily a genetic diagnosis could be made using whole genome sequencing (WGS) to facilitate identification of pathogenic variants in known genes as well as novel PCD candidate genes.

Methods: WGS was used to screen for pathogenic variants in eight patients with PCD.

Results: 7/8 cases had homozygous or biallelic variants in DNAH5, DNAAF4 or DNAH11 classified as pathogenic or likely pathogenic. Three identified variants were deletions, ranging from 3 to 13 kb, for which WGS identified precise breakpoints, permitting confirmation by Sanger sequencing. WGS yielded identification of a de novo variant in a novel PCD gene TUBB4B.

Conclusion: Here, WGS uplifted genetic diagnosis of PCD by identifying structural variants and novel modes of inheritance in new candidate genes. WGS could be an important component of the PCD diagnostic toolkit, increasing molecular diagnostic yield from current (70%) levels, and enhancing our understanding of fundamental biology of motile cilia and variants in the noncoding genome.

Keywords: ciliopathies; molecular diagnosis; primary ciliary dyskinesia; rare respiratory disease; whole genome sequencing.

Figure 1

Ultrastructure analysis supports genetic diagnosis for outer arm dynein variants in PCD. (Left) DNAH5 variants disrupt outer dynein arms (red arrowhead = disrupted, black arrowheads normal) across axonemes of nasal brush samples from cases HG‐001, HG‐002, and HG‐004. (Right) In contrast, DNAH11 variants do not uniformly disrupt outer dynein arms (black arrowheads) in cilia from nasal brush of case HG‐007. Disruption of both inner and outer dynein arms (red arrowheads) is observed in DNAAF4 variants, as shown for HG‐005. Scale bars = 100 nm.

Figure 2

Structural and evolutionary analysis of DNAH11 missense mutations. (A) Structure of the human cytoplasmic dynein‐1 dimer (PDB ID: 5NUG), with the location of the C‐terminal domain colored beige, and the equivalent sites of the DNAH11 mutations highlighted in red. (B) Homology model of the DNAH11 C‐terminal domain with the sites of the missense mutations shown in red, along with the ΔΔG values calculated with FoldX. (C) Multiple sequence alignment of human dynein proteins around the region where the missense mutations occur.