TTC12 Loss-of-Function Mutations Cause Primary Ciliary Dyskinesia and Unveil Distinct Dynein Assembly Mechanisms in Motile Cilia Versus Flagella

Abstract

Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.

The Role of Genetics and Oxidative Stress in the Etiology of Male Infertility—A Unifying Hypothesis?

“…A classic example of this condition is Kartegener syndrome ( Figure 1C ), which is associated with a complete lack of sperm motility due to the absence of dynein arms, in association with chronic sinusitis, bronchiectasis and, in 50% of cases, situs inversus caused by an inability of the embryonic cilia to shift the heart to the left hand side. The condition is inherited in an autosomal recessive manner as a result of biallelic homozygous or compound heterozygous mutations in several candidate genes including coiled-coil domain containing 40 ( CCDC40 ), dynein axonemal heavy chain 1, 5, and 11 ( DNAH1, DNAH5, DNAH7 DNAH11 ) dynein axonemal intermediate chain 1 ( DNAI1 ), leucine rich repeat containing 6 ( LRRC6 ), Zinc finger MYND-type containing 10 ( ZMYND10 ) armadillo repeat containing 4 ( ARMC4 ) and tetratricopeptide repeat domain 12 ( TTC12 ) ( 29 – 33 ). Overall, more than 40 genes have been implicated in this heterogenous disease to date and the list of genes involved is expanding rapidly in concert with improvements in our understanding of ciliary structure and function.…”

Primary ciliary dyskinesia: a major player in a bigger game

“…Despite rapid advances bringing new diagnostic tools from bench to bedside [ 22 , 23 ], gaps in our knowledge remain. A greater understanding of the underlying disease mechanisms and genetics, where next-generation sequencing has advanced gene discovery with loss-of-function mutations in more than 50 respiratory ciliopathy genes now reported [ 24 ]. Increased knowledge of cilia genes and observed functional defects in a wider array of ciliopathy conditions is challenging perceptions of the clinical phenotype, where overlapping features between motile and nonmotile ciliopathies are emerging.…”

“…The advance in genome sequencing has had significant impact on the diagnosis of PCD. To date, mutations in at least 50 genes have been identified as pathogenic in PCD [ 24 , 42 , 43 ]. Biallelic recessive, hemizygous X-linked and, more recently, heterozygous de novo dominant [ 19 ] pathogenic mutations in known ciliopathy genes are accepted as diagnostic, but around 20–25% of cases cannot be confirmed using genetic testing.…”

“…The clinical impact of mutations affecting motile cilia results from disruption of the normal mucociliary escalator with mutation specific phenotypic variations. Genotypic–phenotypic correlations have shown some mutations are not associated with laterality defects, others more likely to affect fertility, have a higher prevalence of hydrocephalus or affect the severity of pulmonary disease [ 21 , 24 , 44 – 46 ].…”

The Male Is Significantly Implicated as the Cause of Unexplained Infertility

“…70 Mutations in a large number of genes are associated with this syndrome including ZMYND10 (zinc finger MYND-type containing 10), 71 ARMC4 (armadillo repeat containing 4), 72 and TTC12 (tetratricopeptide repeat domain 12). 73 It is important to note that while ciliary dyskinesias have the potential to affect many ciliary systems in the body, mutations can also occur in sperm-specific genes affecting flagellar formation and function, generating isolated asthenozoospermia. In this context, many axonemal genes have been identified to cause sperm-specific motility phenotypes, termed "multiple morphological abnormalities of the sperm flagella (MMAF)."…”