Genetic factors contributing to human primary ciliary dyskinesia and male infertility.

Primary ciliary dyskinesia (PCD) is an autosomal-recessive disorder resulting from the loss of normal ciliary function. Symptoms include neonatal respiratory distress, chronic sinusitis, bronchiectasis, situs inversus, and infertility. However, only 15 PCD-associated genes have been identified to cause male infertility to date. Owing to the genetic heterogeneity of PCD, comprehensive molecular genetic testing is not considered the standard of care. Here, we provide an update of the progress on the identification of genetic factors related to PCD associated with male infertility, summarizing the underlying molecular mechanisms, and discuss the clinical implications of these findings. Further research in this field will impact the diagnostic strategy for male infertility, enabling clinicians to provide patients with informed genetic counseling, and help to adopt the best course of treatment for developing directly targeted personalized medicine.

INTRODUCTION

Primary ciliary dyskinesia (PCD) (Mendelian Inheritance in Man, MIM 242650) is a rare genetic disorder affecting approximately one in 20 000 individuals worldwide, which is multisystemic and caused by motility defects in the cilia and flagella.12 Specifically, ineffective cilia movement results in limited mucociliary clearance in the upper and lower respiratory tract, leading to rhinitis, sinusitis, rhinorrhea, chronic cough, recurrent respiratory infections, infertility, and ultimately, scarring of the lungs in the form of bronchiectasis.3 PCD is associated with situs inversus in 50% of the patients, which is termed Kartagener syndrome (KS; MIM 244400).4

In humans, motile cilia have a common microtubule-based ultrastructure (axoneme), comprising nine peripheral microtubule doublets surrounding a central microtubule pair,5 which are linked to a variety of microtubule-associated proteins. These proteins include the inner dynein arm (IDA) and outer dynein arm (ODA) motor complexes, which project from the peripheral microtubule doublets; the radial spokes, which provide a radial scaffold between the central pair and peripheral microtubules to facilitate signal transduction from the center out to the dynein arms to govern ciliary beats and waveforms;6 and nexin–dynein regulatory complexes, which attach between adjacent peripheral doublets to facilitate IDA attachment and regulate dynein activity.7

Male infertility has often been described as part of the clinical symptoms of PCD, but this particular aspect of the PCD physiopathology has not been systematically explored and is generally poorly described in scientific reports. The sperm of infertile male PCD patients is usually immotile and presents various ultrastructural defects of the flagella, such as missing dynein arms, microtubular translocations, and lack of radial spokes. There has been recent research progress on PCD, contributing to the identification and characterization of the numerous proteins required for adequate axonemal molecular structure and assembly. In this review, we focus on the progress made in identifying the genes related to PCD that are directly or potentially associated with male infertility, provide an update of the genetic etiology, summarizing the proposed underlying molecular mechanisms, and discuss the clinical implications arising from these findings.

COILED-COIL DOMAIN-CONTAINING 39 (CCCD39)

FAP59, the Chlamydomonas ortholog of human CCDC39 (MIM 613798), which is located on chromosomal region 3q26.33 and consists of twenty exons, was predicted to be essential for motile ciliary function, as no orthologs have been found in nonciliated organisms (“CiliaCut”) or in Caenorhabditis elegans (“MotileCut”).8

Merveille et al.9 found that a substantial proportion of human PCD cases with axonemal disorganization and abnormal ciliary beating is associated with loss-of-function mutations in CCDC39. Furthermore, functional analyses indicated that CCDC39 localizes to the ciliary axonemes and is essential for the assembly of IDA and the dynein regulatory complex.

Antony et al.10 sequenced the CCDC39 and CCDC40 genes in 54 “radial spoke defect” families, as these are the two genes identified so far to cause this defect. These findings highlighted a key role of both genes in the development of PCD with axonemal disorganization and IDA loss. IDA defects account for about 16% of all PCD cases, and were found in 14% up to 29% of all PCD cases in other cohorts.

DYNEIN, AXONEMAL, ASSEMBLY FACTOR 1–3 (DNAAF1–3)

The most frequent defects in PCD involve ODAs and IDAs, the large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Duquesnoy et al.11 investigated whether PCD might result from mutations in the human gene LRRC50 (also named DNAAF1; MIM 611088), which is located at chromosome 16q24.1 and encodes a member of the superfamily of leucine-rich repeat (LRR)-containing proteins. Functional analyses performed in Chlamydomonas reinhardtii and in another flagellated protist, Trypanosoma brucei, support a key role for DNAAF1 in the cytoplasmic preassembly of the dynein arms. Furthermore, Loges et al.12 demonstrated that DNAAF1 deficiency disrupts the assembly of distal and proximal dynein, axonemal, heavy chain 5 (DNAH5)- and dynein, axonemal, intermediate chain 1 (DNAI2)-containing ODA complexes, as well as dynein, axonemal, light chain 1 (DNALI1)-containing IDA complexes, resulting in immotile cilia.

Kintoun (KTU, previously termed C14 or f104, also named DNAFF2; MIM 612517) is located on chromosome 14q21.3 and consists of three exons, encoding cDNAs of 2511 bp or 2367 bp (an in-frame splice variant lacking exon 2). This gene was first identified in a medaka mutant, and was found to be mutated in PCD patients from two affected families.13 In the absence of DNAFF2, both ODAs and IDAs are missing or defective in the axoneme, leading to general loss of motility. Biochemical and immunohistochemical studies have shown that KTU/PF13 is one of the long-sought after proteins involved in the preassembly of dynein arm complexes in the cytoplasm before being loaded for intraflagellar transport to the ciliary compartment.14

The gene DNAAF3 (MIM 614566) on human chromosome 19q13 (previously designated C19 or f51) encodes a 588-amino acid protein (GenBank NP_849159).15 Lunt et al.16 showed that PF22/DNAAF3 is essential for the preassembly of dyneins into complexes prior to their transport into cilia. Loss-of-function mutations were identified in the human DNAAF3 gene in patients from families with situs inversus, causing defects in the assembly of the inner and outer dynein arms. Moreover, DNAAF3 loss prevents correct assembly of the inner and outer dynein arms, abolishing the motility of respiratory cilia, and giving rise to classical PCD associated with defective left–right organ asymmetry and male infertility.17

DNAH5

DNAH5 (MIM 603335) was identified based on homozygosity mapping and a candidate gene approach.18 This 79-exon gene (with one alternative first exon) encodes a heavy-chain protein that localizes to the outer dynein arm and is the homolog of the dynein c-heavy chain of Chlamydomonas reinhardtii.

A PCD-related locus was identified at chromosome 5p15.2, which contains DNAH5, the human ortholog of the Chlamydomonas ODA γ-heavy chain gene.19 Recessive mutations of DNAH5 result in nonfunctional DNAH5 proteins,20 and affected patients have dysmotile respiratory cilia with ODA defects. Mutations in DNAI1, encoding an ODA intermediate chain orthologous to Chlamydomonas IC78, also cause ODA defects in patients with PCD.21

In the normal ciliated airway epithelium, DNAH5 and DNAH9 show a specific regional distribution along the ciliary axoneme, indicating the existence of at least two distinct ODA types. Cilia with complete axonemal DNAH5 deficiency were immotile, whereas cilia with distal DNAH5 deficiency showed residual motility. In addition, the observation of the absence of DNAH5 within the respiratory ciliary compartment but a normal DNAH5 distribution within the sperm flagellum in a patient with DNAH5 mutations raised the possibility that the two organelle types are assembled via distinct mechanisms.22

DNAI1

The first gene in which mutations were found to be associated with PCD was DNAI1 (MIM 604366).23 DNAI1 is an axonemal dynein intermediate chain gene that was isolated from Chlamydomonas reinhardtii, a unicellular alga with two flagella showing an axonemal structure similar to that of human respiratory cilia and sperm tails.24 DNAI1 is localized on chromosome 9p13-p21 and is composed of twenty exons encoding a 699-amino acid protein.

Guichard et al.25 demonstrated a link between ciliary function and situs determination, given that heterozygosity for a compound mutation in DNAI1 results in PCD with situs solitus or situs inversus (KS). Zariwala et al.26 found that mutations in DNAI1 cause PCD with ODA defects, and are likely the genetic origin of clinical disease in some PCD patients with ultrastructural defects in the ODA.

DNAI2

DNAI2 (MIM 605483), the human ortholog of Chlamydomonas intermediate ODA chain IC69/IC2, which is located on chromosome 17q25, comprises 14 exons extending over a 39-kb genomic distance.272829

Applying a positional and functional candidate gene approach, Loges et al.30 identified homozygous loss-of-function DNAI2 mutations (IVS11þ1G>A) in four individuals from a family with PCD and ODA defects. DNAI2 and DNAH5 mutations affect the assembly of proximal and distal ODA complexes, whereas DNAI1 mutations mainly disrupt the assembly of proximal ODA complexes. Mutations in an ODA intermediate dynein chain are reported in 2%–13% of all PCD patients with defined ODA defects.

DYSLEXIA SUSCEPTIBILITY 1 CANDIDATE 1 (DYX1C1)

DYX1C1 (MIM 608706) was initially identified as a candidate dyslexia gene due to a single-balanced translocation t(2;15)(q11;q21) that coincidentally segregates with dyslexia in a family, and was confirmed in subsequent single nucleotide polymorphism association studies.31 DYX1C1 is located on chromosome 15q21.3 and comprises ten exons (translation starts at exon 2) encompassing 77.93 kb of genomic DNA. Follow-up gene association studies have provided both positive323334 and negative353637 support for the association with dyslexia. Molecular and cellular analyses of DYX1C1 have indicated potential functional roles with chaperonins,3839 estrogen receptor trafficking,40 and neuronal migration.4142

Tarkar et al.43 found that deletion of Dyx1c1 exons 2–4 in mice caused a phenotype resembling PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans revealed disruptions of the ODA and IDA. DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA/IDA assembly factor DNAAF2/KTU.

HEAT REPEAT-CONTAINING 2 (HEATR2)

HEATR2 (MIM 614864), located on chromosome 7p22.3, encodes a member of the family of ten other uncharacterized HEAT repeat-containing proteins in humans. Preliminary analyses have shown that HEATR2 gene and protein sequences are highly conserved, and that HEATR2 is enriched in organisms with motile cilia and flagella.44

Horani et al.45 demonstrated that airway epithelial cells isolated from PCD-affected individuals showed markedly reduced HEATR2 levels, absence of dynein arms, and loss of ciliary beating. Moreover, immunohistochemistry studies in human airway epithelial cells showed that HEATR2 was localized to the cytoplasm and not to the cilia, which suggests a role for this protein in either dynein arm transport or assembly.

HYDIN, AXONEMAL CENTRAL PAIR APPARATUS PROTEIN (HYDIN)

Homozygous-recessive Hydin mutations are lethal in the 1st week of life in hy3 mice, due to hydrocephalus from abnormal ependymal ciliary motility.46474849

With homozygosity mapping, Olbrich et al.50 identified a PCD-associated locus at the chromosomal region 16q21-q23, which contains HYDIN (MIM 610812). However, a nearly identical 360-kb paralogous segment (HYDIN2) in the chromosomal region 1q21.1 complicated the mutational analysis. Electron microscopy tomography of HYDIN mutant respiratory cilia showed results consistent with the effects of loss-of-function mutations, in that the C2b projection of the central pair apparatus was lacking; similar findings were reported in Hydin-deficient Chlamydomonas and mice. High-speed video microscopy demonstrated markedly reduced beating amplitudes of the respiratory cilia and stiff sperm flagella.

LRR-CONTAINING 6 (LRRC6)

LRRC6 (MIM 614930) is expressed in the flagella of Chlamydomonas reinhardtii and in human cilia.51 In mice, Lrrc6 transcripts were found to be expressed in tissues that have flagella or motile cilia.52 In addition, a putative PCD-related locus containing LRRC6 was identified in a genome-wide linkage study performed in familial cases of PCD.53 Most importantly, the phenotypic features of several animal models with mutations in LRRC6 orthologs are consistent with ciliary defects. In zebrafish, seahorse mutants display a curved body with pronephric cysts,54 which was associated with left–right abnormalities in half of the cases.55 In Drosophila, a null allele of the LRRC6 ortholog called tilB was found to be responsible for the ciliary dysfunction of sensory neurons of the auditory organ and male sterility.56 In humans, LRRC6, which is located on chromosome 8q24.22, consists of 12 exons, and the only predicted transcript (RefSeq accession number NM_012472.3) encodes a 466-amino acid residue protein with five N-terminal LRR motifs. The LRRs of LRRC6 contain the consensus sequence LxxLxLxxNxIxxIxxLxzx Lxx (“z” indicates frequent deletions), which defines the SDS22-like subfamily of LRR-containing proteins.57 Each LRR is a beta-strand-turn/alpha-helix structure, and together, these motifs are known to form a solenoid (repeated structural units that form a continuous superhelix).58 LRRC6 also contains an LRR-cap that shields the solenoid, a coiled-coil (CC) domain, a polylysine motif, and a C-terminal alpha-crystallin p23-like domain.

Kott et al.59 demonstrated that in spite of the structural and functional similarities between LRRC6 and DNAAF1, another LRR-containing protein was associated with the same PCD phenotype, the two proteins are not redundant. Therefore, the evolutionarily conserved LRRC6 emerges as an additional player in IDA assembly, a process that is essential for proper axoneme building and that appears to be much more complex than previously thought.

Horani et al.60 revealed a single novel mutation in LRRC6 in PCD patients, which fits the model of autosomal-recessive genetic transmission, leading to a change of a highly conserved aminoacid from aspartic acid to histidine (Asp146His). These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking, and its mutation leads to PCD with laterality defects.

RADIAL SPOKE HEAD HOMOLOGS

In humans, RSPH1 (MIM609314; RSPH: radial spoke head homologs) is located on chromosomal region 21q22.3 and consists of nine exons; the only predicted transcript (RefSeq accession number NM_080860.2) encodes a 309-aminoacid residue protein with five N-terminal membrane occupation and recognition of nexus (MORN) repeats,61 followed by a linker and a sixth MORN repeat. RSPH1 is the homolog of Chlamydomonas RSP1.

Kott et al.62 combined homozygosity mapping and whole-exome sequencing in a consanguineous individual with central complex defects, and identified a nonsense mutation in RSPH1. RSPH1 mutations appear to play an important role in the etiology of this PCD phenotype, which includes radial spoke defects, thereby unveiling the importance of RSPH1 in the proper building of the central complex and radial spokes in humans.

Daniels et al.63 described a novel splice-site mutation (c.921+3_6delAAGT) in RSPH4A (MIM 612647), which leads to a premature translation termination signal, in nine subjects with PCD (seven families). Loss-of-function of this mutation was confirmed with quantitative ciliary ultrastructural analysis, measurement of ciliary beat frequency and waveform, and transcript analysis. All nine individuals carrying the c.921+3_6delAAGT splice-site mutation in RSPH4A were Hispanic with ancestry tracing to Puerto Rico. This mutation is considered to be a founder mutation and a common cause of PCD without situs abnormalities in patients of Puerto Rican descent.

Castleman et al.64 identified mutations in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect of an RSPH4A mutation present in the United Kingdom-Pakistani pedigrees. Both RSPH9 (MIM 612648) and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 showed that gene expression was restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicates that radial spoke head proteins are important in maintaining normal movement in motile, “9 + 2”-structure cilia and flagella. This effect could be rescued by the reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in the function of these genes was not associated with defects in left–right axis determination in humans or zebrafish.

Onoufriadis et al.65 found that mutations in RSPH1, RSPH4A, and RSPH9, which all encode homologs of components of the “head” structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. Using high-resolution immunofluorescence, they identified loss of RSPH4A and RSPH9 associated with RSPH1-mutated cilia, suggesting that RSPH1 mutations may result in loss of the entire spoke head structure.

ZINC FINGER, MYND-TYPE-CONTAINING 10 (ZMYND10)

ZMYND10 (also known as BLU; MIM 607070), which is located on chromosome 3p21.3, encodes a protein containing a C-terminal myeloid, nervy, and DEAF-1 (MYND) domain. ZMYND10 is highly enriched in ciliated cells compared to nonciliated cells,66 but little is known about its function. ZMYND10 is known to act as a tumor suppressor, inhibiting the clonogenic growth of nasopharyngeal carcinoma cells, arresting the cell cycle at the G1 phase, downregulating c-Jun N-terminal kinase and cyclin D1 promoter activities, and inhibiting the phosphorylation of c-Jun.67

In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, ZMYND10 is exclusively expressed in cells with motile cilia, chordotonal sensory neurons, and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein that is altered in PCD.68 Moore et al.68 concluded that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly, and that its variants cause ciliary dysmotility and PCD with laterality defects. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, they found that 6/38 (16%) of the subjects carried biallelic mutations in ZMYND10.

Zariwala et al.69 identified mutations in ZMYND10 that result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models also support the association between ZMYND10 and human PCD, given that Zmynd10 knockdown in zebrafish caused ciliary paralysis, leading to cystic kidneys and otolith defects, and that knockdown in Xenopus interfered with ciliogenesis. Thus, a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

Summary and perspectives

A male infertility phenotype has often been described as one of the clinical symptoms of PCD, but this particular aspect of the physiopathology has not yet been systematically explored and is often only scarcely described in scientific reports (). PCD/KS severely affects the quality of life of patients, and male infertility resulting from the disease should receive more attention. The gaining popularity and progress in assisted reproductive technology will certainly benefit infertile couples affected by this disease. However, full understanding of the underlying molecular mechanisms of PCD/KS will require further studies. This is a very exciting time in the field of the genetics of infertility. We have so far witnessed only the tip of the iceberg, but we are confident that the rest will come to light in the foreseeable future.

AUTHOR CONTRIBUTIONS

LD, YWS, and ZYJ drafted the manuscript, and PL revised it critically for important intellectual content. All authors have read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Table 1

Primary ciliary dyskinesia gene mutations and their consequences for axonemal ultrastructure and sperm phenotype