CFAP70 mutations lead to male infertility due to severe astheno-teratozoospermia. A case report

Julie Beurois1,†, Guillaume Martinez1,2,†, Caroline Cazin1,†,
Zine-Eddine Kherraf1,3, Amir Amiri-Yekta4, Nicolas Thierry-Mieg5, Marie Bidart6, Graciane Petre6, Véronique Satre1,2, Sophie Brouillet7, Aminata Touré8,9,10, Christophe Arnoult1, Pierre F. Ray1,3,‡, and Charles Coutton1,2,‡,*
1INSERM U1209, CNRS UMR 5309, Institute for Advanced Biosciences, Université Grenoble Alpes, 38000 Grenoble, France 2CHU Grenoble Alpes, UM de Génétique Chromosomique, Grenoble, France 3CHU de Grenoble, UM GI-DPI, Grenoble, F-38000, France
4Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
5CNRS, TIMC-IMAG, Université Grenoble Alpes, F-38000 Grenoble, France 6INSERM U1205, UFR Chimie Biologie, Université Grenoble Alpes, Grenoble, France 7Laboratoire d’AMP-CECOS, CHU Grenoble-Alpes, U1036 INSERM-UGA-CEA-CNRS, Université Grenoble Alpes, 38000 Grenoble, France 8INSERM U1016, Institut Cochin, Paris 75014, France. 9UMR 8104, Centre National de la Recherche Scientifique, Paris 75014, France 10Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, Paris 75014, France.

*Correspondence address. Laboratoire de Génétique Chromosomique, Hôpital Couple Enfant, CHU Grenoble Alpes, 38043 Grenoble Cedex 9, France. Tel: +33-4-76-76-54-82; E-mail: [email protected]
Submitted on April 9, 2019; resubmitted on July 14, 2019; editorial decision on July 18, 2019
Introduction
The past decade has been marked by the emergence of the next gener- ation sequencing (NGS), a fast-evolving technology which has allowed important advances in human genetics. This technical revolution has

. proved to be a highly powerful research and diagnostic tool in male
. infertility and has permitted a major step forward for the identification
. of genetic causes of male infertility (Krausz and Riera-Escamilla, 2018).
. In particular, NGS has contributed massively to deciphering the genetic
. causes of the multiple morphological abnormalities of the flagella

†The authors should be regarded as first authors respectively.
‡The authors should be regarded as last authors respectively.
© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: [email protected]

(MMAF phenotype), which is a subgroup of astheno-teratozoospermia characterised by the presence in the ejaculate of immotile spermatozoa with several flagellar defects including short, coiled or absent flagella and flagella of irregular caliber (Ray et al., 2017). To date, mutations in 14 genes have been found to be associated with MMAF (AK7, AKAP3, AKAP4, ARMC2, CEP135, CFAP43, CFAP44, CFAP65, CFAP69, DNAH1,
FSIP2, QRICH2, TTC21A and WDR66) (Supplementary Fig. S1) (Baccetti et al., 2005; Ben Khelifa et al., 2014; Sha et al., 2017a; Tang et al., 2017; Auguste et al., 2018; Coutton et al., 2018; Dong et al., 2018; Kherraf et al., 2018; Lorès et al., 2018; Martinez et al., 2018; Coutton et al., 2019; Liu et al., 2019; Shen et al., 2019). However, despite these plethoric findings, the etiology of MMAF remains unknown in more than 50% of the cases and the discovery of the remaining genes is a now a major challenge (Coutton et al., 2019).
Using different animal models and in particular flagellated protists such as Chlamydomonas or Trypanosoma, at least five of these known MMAF-related genes (DNAH1, CFAP43, CFAP44, WDR66 and CEP135)
have been already reported to encode different components of the axoneme, a central multi-protein complex which is highly conserved throughout evolution and shared by both the sperm flagellum and other motile cilia (Inaba, 2007; Satir and Christensen, 2008). Briefly,
the axoneme consists of nine outer doublet microtubules (DMTs) surrounding two central singlets (9 + 2 structure) known as the central pair complex (CPC). DMTs carry several components that drive and
regulate ciliary or flagellar motility including axonemal dynein motors subdivided into inner (IDA) and outer arms (ODA), radial spokes, nexin links and many others (Satir and Christensen, 2008). DNAH1 is located in the inner dynein arm which drives axoneme bending and ciliary or flagellar motility (Ben Khelifa et al., 2014); CFAP43 and CFAP44 seem to be related to the tether and tether head (T/TH) complex, which connects dynein motor domains to DMTs (Fu et al., 2018; Urbanska et al., 2018); WDR66 is a component of the spoke-associated complex (CSC) which mediates regulatory signals between the radial spokes and dynein arms (Urbanska et al., 2015); and CEP135 is located in the centrosomal complex (Ohta et al., 2002) (Supplementary Fig. S1).
In this work, we analysed genetic data obtained by whole-exome sequencing (WES) from a large cohort of 167 MMAF patients, and identified two patients carrying a homozygous deleterious mutation in the CFAP70 gene, which encodes an axonemal protein that is localised at the base of the outer dynein arm (ODA) and regulates ciliary motility (Shamoto et al., 2018). Therefore, this work shows for the first time the involvement of an ODA-linked protein to the MMAF phenotype and confirmed that the ODA complex, in addition to its motility function, plays an important role in the assembly and/or stability of the flagellum axoneme.

Case report
WES was performed on a large cohort of 167 MMAF patients as previously described (Coutton et al., 2019). All individuals presented with a typical MMAF phenotype characterised by severe astheno- zoospermia (total sperm motility below 10%) with at least three of the following flagellar abnormalities present in >5% of the spermatozoa: short, absent, coiled, bent or irregular flagella (for details, see Coutton et al., 2019). Sperm analysis was carried out in the clinical laborato- ries during routine biological examination of the patient according to

. World Health Organization (WHO) guidelines (Wang et al., 2014).
. The morphology of the sperm cells from the CFAP70-mutated patients
. was assessed with Papanicolaou staining (Fig. 1A). Detailed semen
. parameters of the two mutated patients are presented in Table I.
. All individuals have a normal somatic karyotype (46,XY) with normal
. bilateral testicular size, normal hormone levels and secondary sexual
. characteristics. Informed consent was obtained from all the subjects
. participating in the study according to local protocols and the principles
. of the Declaration of Helsinki. The study was approved by local ethics
. committees, and samples were then stored in the CRB Germethèque
. (certification under ISO-9001 and NF-S 96-900) following a standard-
. ised procedure or were part of the Fertithèque collection declared to
. the French Ministry of health (DC-2015-2580) and the French Data
. Protection Authority (DR-2016-392).
. Bioinformatics analysis was performed according to our previously
. described protocol (Coutton et al., 2019). Data analysis of the whole
. cohort of 167 MMAF individuals permitted the identification of 54
. individuals (32.3%) with harmful mutations in known MMAF-related
. genes (Coutton et al., 2019). In addition, we identified here two
. patients (CFAP70_1 and CFAP70_2) (accounting for 1.2% of our
. cohort) with homozygous deleterious variants of CFAP70, a gene not
. previously associated with any pathology. The CFAP70 mutations were
. subsequently validated by Sanger sequencing (Fig. 1B). PCR primers
. are listed in Supplementary Table S1, and a detailed protocol can
. be found in Coutton et al. (2019). The first variant identified in the
. patient CFAP70_1 is a splice variant c.1723-1G>T, altering a consensus
. splice acceptor site of CFAP70 exon 16 (Fig. 1C). The second variant
. identified in individual CFAP70_2 is a missense variation c.178T>A
. (p.Phe60Ile) located in exon 3 (Fig. 1C). Both variants were absent
. from gnomAD, the largest control sequence database regrouping
. sequence data from over 120 000 individuals. No mRNA analysis or
. immunostaining could be performed on sperm cells from CFAP70_2
. due to the lack of biological samples. However, using prediction
. software for non-synonymous SNPs, we found that this missense
. change is predicted to be deleterious by SIFT (score of 0) and probably
. damaging by PolyPhen (score of 0.996). Moreover, the concerned
. amino acid (Phe60) was found to be conserved in CFAP70 orthologues
. (Fig. 1D). For these two patients, no other candidate variants reported
. to be associated with cilia, flagella or male fertility were found.
. CFAP70 (NM_145170.3, formerly reported as TTC18) is located on
. chromosome 10 and contains 27 exons encoding a predicted 1121-
. amino acid protein (Q5T0N1). CFAP70 is predominantly expressed
. in the testis according to data from GTEx (GTEx Consortium, 2015)
. and is described to be associated with cilia and flagella (Shamoto
. et al., 2018). RT-qPCR experiments performed with a panel of 10
. human tissues including other ciliated tissues such as trachea con-
. firmed the results showing that CFAP70 transcripts are most highly
. expressed in the testis compared with all the other tested tissues
. (Supplementary Fig. S2). Primer sequences and RT-qPCR conditions
. are indicated in Supplementary Table SII and in Coutton et al. (2019).
. According to the UniProt server, CFAP70 contains 8 tetratricopeptide
. repeats (TPR repeats), which are known to form scaffolds which
. mediate protein–protein interactions (The UniProt Consortium, 2017)
. (Fig. 1C).
. We performed immunofluorescence (IF) assays on sperm cells from
. a fertile control, a patient CFAP70_1 carrying the splice variant c.1723-
. 1G>T and other MMAF patients mutated in other genes. For each

Figure 1 Morphology of normal and CFAP70 mutant spermatozoa, and the mutations identified in the CFAP70 gene. (A) Light microscopy analysis of spermatozoa from fertile control and individual CFAP70_1. All spermatozoa from the patient have shorter flagella than controls. Additional features of morphological abnormalities of the sperm flagella (MMAF) spermatozoa such as thick and coiled flagella are also observed. Scale bars 10 μm. (B) Electropherograms of Sanger sequencing showing the two CFAP70 variants identified in the MMAF cohort. The substituted nucleotides are highlighted in red. (C) Location of CFAP70 mutations in the intron–exon structure and in the protein representation. Blue square stands for tetratricopeptide repeats (TPR) as predicted by the InterPro server (https://www.ebi.ac.uk/interpro/). Mutations are annotated in accordance to the Human Genome Variation Society’s recommendations. (D) Amino acid sequence alignment of CFAP70 in different vertebrate species showing the conservation of the amino acid (Phe, F) in position 60 (highlighted in red) as well as the surrounding amino acids.
MMAF patient studied, 200 sperm cells were analysed by two different experienced operators and the IF staining intensity and pattern were compared with a fertile control. CFAP70 immunostaining in sperm cells from the control was present all along the flagellum, with a marked signal at the base of the flagellum. In the CFAP70_1 patient, CFAP70 staining was totally absent in all analysed spermatozoa (Fig. 2), con- firming the deleterious effect of this mutation. In order to determine if the loss of CFAP70 was specifically linked to the CFAP70 mutation and was not a hallmark of the MMAF phenotype, secondary to structural defects, we performed IF assays on sperm cells from five other MMAF patients including four patients previously identified with a mutation in ARMC2, FSIP2, CFAP43 or WDR66 and one with no genetic cause identified (Fig. 2). Genotype information of the four additional MMAF patients mutated in ARMC2, FSIP2, CFAP43 and WDR66 is indicated in Supplementary Table SIII. In all but the WDR66-mutated patient, CFAP70 immunostaining was comparable with that observed on fertile controls (Fig. 2), indicating that CFAP70 expression and localisation were not altered in the sperm from MMAF patients with mutations in these genes. Interestingly, CFAP70 staining was mostly absent in the WDR66-mutated patient irrespective of the sperm morphology (Supplementary Fig. S3), suggesting a possible link, direct or indirect, between, two axonemal components in the sperm flagellum (Fig. 2, Supplementary Fig. S1).

To further characterise the molecular defects induced by CFAP70
mutations in human sperm, we used an indirect approach studying, by IF, the presence and localisation of some proteins belonging to different substructures of the axoneme. The presence of the only two proteins was investigated: SPAG6 as a marker of the CPC and DNAI2 as markers of the ODA. We observed that in sperm from individual CFAP70_1, staining of SPAG6, an axoneme CPC protein (Sapiro et al., 2000), was totally absent from the flagellum, suggesting defects in the CPC structure (Fig. 3). This finding is in concordance with previous results showing that the absence of CPC is a frequent characteristic of MMAF spermatozoa (Coutton et al., 2015). Similarly, the staining of DNAI2 was totally absent or dramatically reduced in most of the analysed sperm irrespective of the sperm morphology (Fig. 4). This result is consistent with the function of CFAP70 as a regulatory component of the ODA in motile cilia and flagella (Shamoto et al., 2018). To formally conclude, all the potential ultrastructural defects observed using IF should be further confirmed by transmission electron microscopy (TEM). However, TEM could not be performed in this present work due to a very low number of sperm cells available. Moreover, due to limited sample availability, IF analyses could not be repeated on sperm from the other patient with the CFAP70_2 mutation.

Discussion
In this present work, we identified two infertile patients with a typical MMAF phenotype carrying deleterious homozygous mutations in the CFAP70 gene. The first patient harbours a deleterious splicing mutation leading to the absence of the protein in the sperm flagellum (Fig. 2). The second patient has a missense mutation at position Phe60 predicted to be deleterious using mutation-prediction software. However, bioinfor- matics predictions are not sufficient to conclude to the pathogenicity of this mutation and additional supporting functional work (e.g. mRNA or protein studies, IF experiments or CRISPR/Cas9-targeted muta- genesis in different animal models) could be useful to reinforce this

 

assumption. Mutations in CFAP70 appeared as the best candidates to explain the MMAF phenotype observed in these two patients based on robust evidence in the literature supporting its critical function for cilia/flagella formation and function. First, transcriptomic studies showed that Cfap70 was strongly upregulated in differentiating mouse tracheal epithelial cells (Xu et al., 2015). Furthermore, inactivation of ttc18, the orthologue of the human CFAP70 in zebrafish, led to a significant reduced cilia length and number in Kupffer’s vesicle (van Dam et al., 2017). Moreover, extensive work on Chlamydomonas FAP70 demonstrated that the protein is a regulator of the ODA (Shamoto et al., 2018). Importantly, the authors demonstrated that FAP70 is not formally a component of the ODA complex but seems to be involved in the coordination of the ODA activity in response to changes in
. the surrounding mechanical and/or chemical conditions to produce
. proper ciliary motility (Shamoto et al., 2018). In this work, IF exper-
. iments indicated ODA defects in sperm cells from the patient with the
. CFAP70_1 mutation (Fig. 4). Moreover, previous studies demonstrated
. that the ODAs were not disorganised in the sperm flagellum from
. MMAF patients mutated in other axonemal genes suggesting that ODA
. defects may be specifically linked to CFAP70 mutations. In Chlamy-
. domonas, the lack of FAP70 did not affect the ODA ultrastructure
. (Shamoto et al., 2018), in contrast to what was observed in the sperm
. cells from our patient (Fig. 4). However, such a discrepancy between
. human and flagellate flagella was already found for two other genes
. involved in MMAF (CFAP43 and CFAP44) (Coutton et al., 2018). These
. IF results should nevertheless be confirmed by TEM analysis in sperm

Figure 3 The central pair complex (CPC) is affected by CFAP70 mutations. Sperm cells from a fertile control and the patient CFAP70_1 were stained with anti-SPAG6 (HPA038440, Sigma-Aldrich, rabbit, 1:500, green), a protein located in the CPC, and anti-acetylated tubulin (32-2500, Thermo Fisher, mouse, 1:1000, red) antibodies. DNA was counterstained with DAPI. SPAG6 staining is mostly absent in sperm from the patient mutated in WDR66 irrespective of the sperm morphology (short, long, coiled or disorganised). The histogram showing the percentage of analysed spermatozoa with or without SPAG6 staining according to their morphology is presented (number of sperm cells analysed = 200). Scale bars 10 μm.

 

cells from CFAP70-mutated patients to allow reliable and definitive conclusions. CFAP70 encodes a TPR protein composed of eight TPR repeats located at the C-terminal domain. TPR domains are known to mediate protein–protein interactions, and it has been proposed that FAP70 localises at the base of ODA through the N-terminal end of the protein and the C-terminal region may define the binding partner that regulates ODA function (Shamoto et al., 2018). The partners of CFAP70 are still unknown, but Shamoto et al. (2018) suggested that CFAP70 could interact with various ciliary regulatory proteins, such as protein phosphatases (Elam et al., 2011) and calcium-binding proteins (Mizuno et al., 2012). Interestingly, we showed in the present study that CFAP70 staining is absent in a patient with mutation in the

WDR66 gene. WDR66 is a MMAF-related gene previously reported
to encode an axonemal protein containing a calcium-regulating EF-
hand domain in its C-terminal end (Auguste et al., 2018; Kherraf et al.,
2018). Interestingly, the WDR66 patient included in this present work
harboured the recurrent terminal deletion previously described to
remove the C-terminal domain of the protein (Kherraf et al., 2018).
Therefore, this observation may suggest an additional role of the C-
ter domain of CFAP251 in the recruitment and assembly of the other
component of the flagellum, as CFAP70 may be. Moreover, WDR66
has been described to localise to the calmodulin- and spoke-associated
(CSC) complex at the base of radial spoke 3 in Tetrahymena and Chlamy-
domonas (Urbanska et al., 2015). Interestingly, the CSC is involved

Figure 4 Outer dynein arms (ODAs) are affected by CFAP70 mutations. Sperm cells from a fertile control and the patient CFAP70_1 were stained with anti-DNAI2 (H00064446-M01, Abnova, mouse, 1:400, red), a protein located in the ODA, and anti-acetylated tubulin (PA5-19489, Thermo Fisher, rabbit, 1:500, green) antibodies. DNA was counterstained with DAPI. The DNAI2 immunostaining is mostly absent or dramatically reduced in sperm from the patient CFAP70_1 compared with the fertile control and irrespective of the sperm morphology (short, long, coiled or disorganised). The histogram showing the percentage of analysed spermatozoa with or without the DNAI2 staining according to their morphology is presented (number of sperm cells analysed = 200). Scale bars 10 μm.

in the modulation of dynein activity and therefore in the regulation of flagellar motility (Heuser et al., 2012). These findings support the fact that WDR66 is a potential partner of CFAP70 and may work in synergy to modulate inner and outer dynein activity. However, further investigations (such as co-immunoprecipitation (co-IP) experiments) are necessary to clarify this hypothesis and will permit elucidation of the mechanisms by which CFAP70 regulates ODA function and flagellum and cilia length in mammals.

It has been demonstrated that Cfap70 inactivation in mouse ependy- mal cells led to shortened cilia in addition to motility defects (Shamoto et al., 2018). This observation echoes the sperm phenotype observed

in our two MMAF patients presenting sperm with short and immotile
flagella. Moreover, this supports our findings that CFAP70 may be
involved in axoneme biogenesis or stability beyond its function in ODA
regulation.
As observed for the other MMAF individuals of the cohort (Coutton
et al., 2019), analysis of sperm morphology from patients CFAP70_1–2
showed a high proportion of spermatozoa with abnormal flagella
(Fig. 1, Table I). Interestingly, we also found low sperm concentra-
tions in the ejaculates (oligozoospermia) as was observed for patients
mutated in CFAP69 (Dong et al., 2018). However, the two CFAP70
patients presented a different pattern of morphological defects of the

sperm flagellum with only short flagella for patient 1 while a mosaic of defects (short, absent or irregular caliber) was observed in patient 2. In addition, patient CFAP70_1 with the most severe variant presents spermatozoa with 0% motility in contrast with the patient with the missense variant (p.Phe60Ile), who presents a milder phenotype with 13% motility (Table I). This could suggest that the Phe60Ile-mutated protein may preserve residual activity, as reported for other missense mutations in other MMAF-related genes (Ben Khelifa et al., 2014; Amiri-Yekta et al., 2016). Moreover, this may reinforce the hypothesis of a possible phenotype continuum depending on the severity of muta- tions in MMAF genes, as shown for DNAH1 (Ben Khelifa et al., 2014). Concerning the prognosis of intracytoplasmic sperm injection (ICSI) using sperm cells from CFAP70-mutated subjects, only ICSI outcomes for patient CFAP70_1 were available, and one clinical pregnancy with one live birth after 2 cycles was reported. This positive outcome is consistent with previous studies demonstrating that MMAF-affected individuals have a good prognosis following ICSI (Coutton et al., 2015; Wambergue et al., 2016; Sha et al., 2017b).
Finally, we know that CFAP70 is expressed in different ciliated tissues
such as trachea or ependymal epithelium. However, the two patients with CFAP70 mutations presented only with primary infertility without any other clinical features, thus excluding a phenotype of primary cilia dyskinesia (PCD), as has been observed for all genes associated with a MMAF phenotype. These recurrent observations confirm once again that flagellum biogenesis requires some proteins and pathways that are at least partly different from those necessary for cilia biogenesis. Inter- estingly, we observed that many of these MMAF-related genes encode proteins which are linked directly or indirectly with the protein complex close to the radial spoke 3 (RS3) (DNAH1, WDR66, CFAP43, CFAP44 and now CFAP70), suggesting that the RS3 biogenesis and function may be one of the major differences between cilia and sperm flagella.

Supplementary data
Supplementary data are available at Human Reproduction online.

Acknowledgements
We thank all patients and control individuals for their participation.

Authors’ roles
J.B., C.C., G.M., P.F.R., C.A., S.B. and A.T. analysed the data and wrote the manuscript; Z-E.K., N.T-M., P.F.R. and C.C. performed and analysed the genetic data; J.B., C.C. and G.M. performed the IF experiments. G.P. and M.B. performed the RT-qPCR experiments; A.A-Y. and V.S. provided clinical samples and data; C.C., P.F.R., C.A. and G.M. designed the study, supervised all molecular laboratory work, had full access to all of the data in the study and took responsibility for the integrity of the data and its accuracy. All authors contributed to the report.

Funding
‘MAS-Flagella’ project financed by Agence Nationale de la Recherche and Direction Générale de l’Offre de Soins for the program PRTS
2014; ‘Whole genome sequencing of patients with Flagellar Growth
Defects (FGD)’ project financed by the Fondation Maladies Rares for
the program Séquençage à haut débit 2012.

Conflict of interest

The authors have declared that no conflict of interest exists.

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