Functional characterization of the first missense variant in CEP78 , a founder allele associated with cone‐rod dystrophy, hearing loss, and reduced male fertility

Abstract

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. AbstractInactivating variants in the centrosomal CEP78 gene have been found in cone-rod dystrophy with hearing loss (CRDHL), a particular phenotype distinct from Usher syndrome. Here, we identified and functionally characterized the first CEP78 missense variant c.449T>C, p.(Leu150Ser) in three CRDHL families. The variant was found in a biallelic state in two Belgian families and in a compound heterozygous state-in trans with c.1462-1G>T-in a third German family. Haplotype reconstruction showed a founder effect. Homology modeling revealed a detrimental effect of p.(Leu150Ser) on protein stability, which was corroborated in patients' fibroblasts. Elongated primary cilia without clear ultrastructural abnormalities in sperm or nasal brushes suggest impaired cilia assembly. Two affected males from different families displayed sperm abnormalities causing infertility. One of these is a heterozygous carrier of a complex allele in SPAG17, a ciliary gene previously associated with autosomal recessive male infertility. Taken together, our data indicate that a missense founder allele in CEP78 underlies the same sensorineural CRDHL phenotype previously associated with inactivating variants. Interestingly, the CEP78 phenotype has been possibly expanded with male infertility. Finally, CEP78 loss-of-function variants may have an underestimated role in misdiagnosed Usher syndrome, with or without sperm abnormalities. K E Y W O R D S CEP78, cilia, cone-rod dystrophy with hearing loss (CRDHL), founder, male infertility, missense 1000 |

CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels

“…The latter result is in line with previous reports showing that the LRR region in the N-terminus of CEP78, which encompasses residue L150, is important for its localization to centrioles [27,35]. Both FLAG fusions were expressed at similar levels in the cells (Figure 2-figure supplement 1A, B); this was somewhat surprising given that endogenous CEP78 L150S is largely undetectable by western blot analysis of patient-derived HSFs suggesting its stability is compromised [30]. Therefore, we tested if the L150S mutation might affect binding of the CEP78 antibody used in [30] to CEP78.…”

“…Several studies have shown that loss-of-function mutations in CEP78 are causative of ciliopathy characterized by CRDHL [29][30][31][32], and that cells lacking CEP78 display reduced ciliation frequency [34] or abnormally long cilia [29,30]. However, the molecular mechanisms by which CEP78 regulates cilium biogenesis and length were so far unclear.…”

“…Previous studies showed that patient-derived fibroblasts lacking CEP78 display significantly elongated primary cilia compared to control cells [29,30] whereas depletion of CEP78 in Planarians and RPE1 cells was shown to significantly reduce cilia numbers [34]. To reconcile these seemingly contradictory findings we analyzed cilia numbers and length in serum-deprived wild type (WT) and CEP78 KO RPE1 cells [30] by immunofluorescence microscopy (IFM) with antibodies against ciliary (ARL13B) and centrosomal (CEP350) markers. We found that the frequency of ciliated cells is significantly reduced in CEP78 KO cells compared to WT cells ( Figure 1A, B) whereas the length of the remaining cilia is significantly increased in the CEP78 KO cells ( Figure 1A, C).…”

Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss

“…Apart from CRDHL, sperm abnormalities causing infertility have been reported in two unrelated affected males (Ascari et al, 2020). Functional studies pointed to a lossof-function effect with decreased amounts of protein, normal subcellular localization, and elongated primary cilia in patients' cells (Namburi et al, 2016;Nikopoulos et al, 2016;Ascari et al, 2020). Interestingly, a complex SV implicating CEP78 has been reported in one individual with CRDHL, being a homozygous deletion-inversion-deletion overlapping CEP78 (Sanchis-Juan et al, 2018).…”

“…One of the more recently identified disease genes that contribute to the genetic heterogeneity of IRD is the centrosomal gene CEP78 (MIM# 617110), in which several types of variants, inactivating sequence variants as well as a unique missense variant have been found in autosomal recessive conerod dystrophy with hearing loss (CRDHL; MIM# 617236), a recognizable phenotype distinct from Usher syndrome (Fu et al, 2016;Namburi et al, 2016;Nikopoulos et al, 2016;Ascari et al, 2020). Apart from CRDHL, sperm abnormalities causing infertility have been reported in two unrelated affected males (Ascari et al, 2020). Functional studies pointed to a lossof-function effect with decreased amounts of protein, normal subcellular localization, and elongated primary cilia in patients' cells (Namburi et al, 2016;Nikopoulos et al, 2016;Ascari et al, 2020).…”