ABSTRACT
Purpose Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of a tractable animal model (Caenorhabditis elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a cilia gene associated with ciliopathies.
Methods CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene (mks-3). Quantitative phenotypic assays of sensory cilia structure and function measured if the variants affect mks-3 gene function. Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signaling function.
Results Assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile, Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg, Ser961Tyr).
Conclusion Efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model that allows rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.
INTRODUCTION
Exome and genome sequencing have revolutionised our ability to identify the genetic causes of disease. Missense variants (single codon altered to encode a different amino acid) are the most numerous class of protein-altering variants1 but only a subset are associated with disease2. Based on disease features and patterns of inheritance, identified variants are classified as benign, likely benign, uncertain significance, likely pathogenic or pathogenic (as defined by the Association for Clinical Genomic Science; ACGS)3. For novel or previously uncharacterized variants, the only evidence available to assess their pathogenicity is population allele frequency and analysis by in silico tools (e.g. SIFT/PolyPhen/CADD), which are not sufficient to meet the threshold for a ‘likely pathogenic’ classification according to best practices established by ACGS3. A VUS (variant of uncertain significance) classification is made when there is insufficient evidence to conclude on pathogenicity3,4. Currently, most missense variants are classified as VUS (116,215/198,080=58.7%, accessed from ClinVar5 September 2021). Since VUS classifications cannot be used for molecular diagnosis of disease, they cannot be acted upon clinically. Therefore, a VUS classification can delay or prohibit accurate disease management or genetic counselling, and prevents patients from accessing gene-specific therapies and clinical trials6,7. Given the wealth of genomic data available, there is a pressing clinical need to definitively reclassify VUS as benign or pathogenic. Effective experimental strategies for the functional interpretation of VUS are required to address this problem8. With the emergence of advanced genetics tools such as CRISPR-Cas9 gene editing, non-rodent model organisms such as zebrafish, Drosophila and C. elegans are emerging as robust in vivo experimental platforms for determining variant pathogenicity9–11.
Ciliopathies are a heterogenous group of at least 25 inherited disorders with clinically overlapping phenotypes, caused by pathogenic variants in almost 200 genes12. Ciliopathies are caused by defects in cilia, which are microtubule-based organelles, typically 2-20 microns long, that extend from the surfaces of most cell types. Motile cilia propel cells through a fluid or push fluid across a tissue surface. Primary cilia act as cellular “antennae”13, transducing a wide variety of extrinsic chemical and physical (eg. light, odorants) signals into the cell14.
Primary cilia are especially important for coordinating cell-cell communication signaling pathways (eg. Shh, Wnt, PDGF-a) essential for development and homeostasis15. Ciliopathies affect many organ systems, causing a broad range of clinical phenotypes of varied severity and penetrance that include cystic kidneys, retinal dystrophy, bone abnormalities, organ laterality defects, respiratory tract defects, infertility, obesity, neurodevelopmental defects and cognitive impairment16.
TMEM67 is a transmembrane protein that localizes specifically to the proximal 0.2-1.0 μm region of the ciliary axoneme called the transition zone17,18. Pathogenic variants in TMEM67 cause several ciliopathies19 including Meckel Syndrome (OMIM #607361), COACH Syndrome (OMIM #216360), and are a major contributor to Joubert Syndrome7,20 (OMIM #610688). Most reported missense variants in TMEM67 have uncertain or conflicting interpretations of their clinical significance (88/141=62.4%, accessed from ClinVar5 September 2021). The abundance of VUS alleles makes TMEM67 an excellent candidate to explore if modelling VUS in C. elegans can generate evidence about their pathogenicity. We used CRISPR-Cas9 technology to model eight TMEM67 missense VUS in C. elegans. Using quantifiable assays of cilium structure and function, we determined whether the variants are damaging or benign. We then validated the worm findings using a genetic complementation-based approach in TMEM67 null human cells. Our study indicates that C. elegans is a tractable model system that can provide evidence of variant pathogenicity.
METHODS
Modelling of protein secondary structure
Human TMEM67 (NP_714915.3) and C. elegans MKS-3 (NP_495591.2) protein sequences were analysed by the RaptorX protein structure prediction server, using default settings for the deep dilated convolutional residual neural networks method21. Absolute model quality was assessed by ranking GDT (Global Distance Test) scores defined as 1*N(1)+0.75*N(2)+0.5*N(4)+0.25*N(8), where N(x) is the number of residues with estimated modelling error (in Å) smaller than x, divided by protein length and multiplied by 100. GDT scores >50 indicate a good quality model. However, the highest ranking models for TMEM67 (GDT = 28.968) and MKS-3 (GDT = 19.269) suggest that portions of these models are lower quality. Models in the .pdb format were visualized and annotated in EzMol (http://www.sbg.bio.ic.ac.uk/ezmol/).
C. elegans maintenance
All C. elegans strains in this study were maintained at 20°C or 15°C on nematode growth medium seeded with OP50 E. coli using standard techniques 22. All worm strains are listed in Table S2.
CRISPR/Cas9 to engineer mks-3 mutants in C. elegans
CRISPR protocols were performed as previously described23 in nphp-4(tm925) using an unc-58 co-CRISPR strategy. crRNA are listed in Table S3. The CRISPR efficiency (defined as the percent of F1 pools that were positive for the edit by PCR) varied from 1-35% with an average 15%. Accuracy of the engineered variants was confirmed with Sanger sequencing. unc-58 was also sequenced and unintended unc-58 mutations24 were outcrossed. PCR and sequencing primers are listed in Table S4.
C. elegans quantitative phenotyping assays
Dye filling, roaming, and chemotaxis assays were performed as previously described25 with young adult hermaphrodites at 20°C. Assays were performed blinded to genotype with at least three independent biological replicates.
Generating transgenic worms expressing extrachromosomal MKS-3::GFP
mks-3::gfp transgenes were generated with PCR-based fusion of mks-3 gDNA (including 485 bp of 5’ UTR sequence) with GFP (pPD95_77, gift from Andrew Fire, Addgene plasmid #1495). All primers are listed in Table S4. mks-3(tm2547) hermaphrodites were injected with 0.25ng/μl mks-3::gfp and 100ng/μl coel::dsRed (gift from Piali Sengupta, Addgene plasmid #8938) to generate extrachromosomal arrays (1-7 lines each). PCR confirmed the presence of mks-3::gfp in the stable extrachromosomal arrays.
C. elegans wide-field imaging and quantification of fluorescence
Young adult hermaphrodites were immobilised on 4% agarose pads in 40 mM tetramisole (Sigma). Images were acquired with a 100x (1.40 NA) oil objective on an upright Leica DM5000B epifluorescence microscope and captured with an Andor iXon+ camera. Image analysis was performed with FIJI/ImageJ (NIH). MKS-3::GFP fluorescence was quantified as previously described23.
Transmission electron microscopy
Young adult hermaphrodites were processed as previously described25. Briefly, worms were fixed in 2.5% glutaraldehyde (Merck) in Sørensen’s phosphate buffer (0.1 M, pH 7.4) for 48 hours at 4°C, post-fixed in 1% osmium tetroxide (EMS) for 1 hour, and dehydrated through an increasing ethanol gradient. Samples were treated with propylene oxide (Sigma) and embedded in EPON resin (Agar Scientific) for 24 hours at 60°C. Serial, ultra-thin (90 nm) sections of the worm nose tissue were cut using a Leica EM UC6 Ultramicrotome, collected on copper grids (EMS), stained with 2% uranyl acetate (Agar Scientific) for 20 min followed by 3% lead citrate (LabTech) for 5 min, and imaged on a Tecnai 12 (FEI software) with an acceleration voltage of 120 kV.
Cell culture
Human hTERT-immortalized retinal pigmentary epithelial (hTERT-RPE1, American Type Culture Collection; ATCC) wild-type and crispant cell-lines were grown in Dulbecco’s minimum essential medium (DMEM)/Ham’s F12 medium supplemented with GlutaMAX (Gibco #10565018) and 10% fetal bovine serum (FBS). For selected experiments involving cilia, cells following passage were serum-starved in DMEM/F-12 media containing 0.2% FBS. Cells were cultured in an incubator at 37°C with 5% CO2.
TMEM67 cloning, plasmid constructs and transfections
Full-length H. sapiens TMEM67 isoform 1 (RefSeq JF432845, plasmid ID HsCD00505975, DNASU Plasmid Repository) was cloned into pENTR223. The ORF was Gateway cloned into a C-terminal GFP-tagged Gateway pcDNA-DEST47 vector (ThermoFisher Scientific), sequence verified, and sub-cloned into pcDNA3.1 myc/HisA vector with HiFi cloning (New England Biolabs). We also introduced an endogenous Kozak sequence prior to the start site, the first 30 nucleotides of the main transcript that were missing from the DNASU sequence, and a GS linker between the ORF and myc tag. To generate TMEM67 variants, we used the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent) according to the manufacturer’s protocol. Primer sequences are listed in Table S5. The final constructs were verified by sequencing. Cells at 80% confluency were transfected with plasmids using Lipofectamine 2000 (Life Technologies Ltd.) as described previously26.
CRISPR/Cas9 genome editing in cell culture
GFP-expressing pSpCas9(BB)-2A-GFP (PX458) (gift from Feng Zhang, Addgene plasmid #48138). Three crRNAs targeting human TMEM67 (RefSeq NM_153704.5) were designed using Benchling (https://benchling.com), selected for the highest ranking on- and off-target effects. crRNAs were ordered as HPLC-purified oligos from Integrated DNA Technologies in addition to Alt-R CRISPR-Cas9 tracrRNA-ATTO550 conjugates. crRNA sequences are listed in Table S6. Lyophilised pellets were resuspended in Tris-EDTA (TE) buffer (Qiagen) to give 100mM stocks. crRNA and tracrRNA were mixed (1:1), and incubated in nuclease-free duplex buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate) to make 300nM guide RNA master mixes. Before transfecting into cells the crRNA:tracrRNA duplexes were incubated with Lipofectamine 2000 at an RNA:Lipofetamine ratio of 2:1 in 200μl/well Opti-MEM for 20 minutes. 1 ml of media from 6-well plate wells was removed, the transfection reagents applied, and the cells incubated overnight. Media was changed to fresh DMEM/F-12 with 10% FBS after 16 hours, and cells incubated for 48 hours. Following transfection, fluorescence-activated cell sorting (FACS) was performed to enrich cells expressing GFP and to produce clonal populations. 96 well plates (Corning) were treated with 200 μl of 4% bovine serum albumin (BSA) per well for one hour. Wells were then filled with 100 μl of filter-sterilised collection buffer (20% FCS, 1% penicillin-streptomycin, 50% conditioned media, 29% fresh DMEM/F-12 media). Transfected cells were prepared for FACS by removing media, washing in PBS, and treating with trypsin for 5 min before resuspending in filter-sterilised sorting buffer (1x Ca2+/Mg2+-free PBS, 5 mM EDTA, 25 mM HEPES pH 7.0). A 70 μm filter was used to disperse cells into 4% BSA-treated polystyrene FACS tubes. A BD Influx 6-way cell sorter (BD Biosciences) was used to index sort GFP-positive cells, calibrated against un-transfected control cells. When an abundance of GFP positive cells were present, the top 5% were targeted for index sorting. After sorting, cells were incubated for 3 weeks at 37°C with 5% CO2, with weekly checks for growing colonies.
PCR and sequence validation of crispant cell-lines
To extract DNA from colonies within the 96-well plates, cells were washed with 1x Ca2+/Mg2+-free PBS and resuspended in 50 μl of DirectPCR Lysis Reagent (Viagen Biotech) containing 0.4 mg/ml Proteinase K (Sigma, # P4850). Suspensions were incubated at 55°C for 5 hours, followed by 85°C for 45 minutes. 1 μl of DNA extracts were used in PCR reactions. Primers are listed in Table S7. variants were identified by Sanger sequencing (GeneWiz Inc.) followed by analysis using the Synthego ICE v227. The following clones were chosen for further study: clone 40, heterozygous for c.369delC (p.Glu124Lysfs*12); and clone 16 carrying biallelic variants [c.519delT]+[ c.519dupT] ([p.Cys173Trpfs*20]+[ p.Glu174*]). All variants were predicted to result in nonsense mediated decay by the Ensembl Variant Effect Predictor28. Clone 21 was a negative control cell-line that was mock-transfected, underwent FACS, but was verified to carry wild-type TMEM67.
Whole cell extract preparation and western immunoblotting
Whole cell extracts containing total soluble proteins were prepared from hTERT-RPE1 cells that were transiently transfected with 1.0 μg plasmid constructs in 90 mm tissue culture dishes, or scaled down as appropriate. 10 μg total soluble protein was analysed by SDS-PAGE (4-12% polyacrylamide gradient) and western blotting according to standard protocols. Primary antibodies used: mouse anti-β actin (1:10000, clone AC-15, Abcam Ltd., Cambridge, UK); rabbit polyclonal anti-TMEM67 (1:500, 13975-1-AP; ProteinTech Inc., Rosemont, IL, USA); goat anti-ROR2 (1:1000, AF2064; R&D Systems Inc., Minneapolis, MN, USA). Appropriate HRP-conjugated secondary antibodies (Dako UK Ltd.) were used (final dilutions of 1:10000-25000) for detection by the enhanced chemiluminescence “Femto West” western blotting detection system (Thermo Fisher Scientific Inc., Rockford, IL, USA) and visualized using a ChemiDoc MP imaging system (BioRad Inc., Hercules, CA, USA). Ratios of active phosphorylated ROR2 : unphosphorylated ROR2 isoforms were calculated by quantitating band intensity using ImageLab 5.2.1 software (BioRad Inc.) for three biological replicates, as described previously26.
Statistical analyses
All C. elegans statistical analyses were performed in Microsoft Excel with the Real Statistics Resource Pack Version 7.2 (www.real-statistics.com). A Shapiro-Wilk test determined if data were normally distributed. Statistical significance was determined with an ANOVA followed by Tukey’s post hoc (chemotaxis and GFP quantification) or Kruskal-Wallis followed by Dunn’s (roaming) or Schaich-Hammerle (dye filling) post hoc tests. For cell culture results, a normal distribution of data was confirmed using the Kolmogorov-Smirnov test (GraphPad Prism). Pairwise comparisons were analysed with Student’s two-tailed t-test using InStat (GraphPad Software Inc.). *, **, and *** refer to p-values of <0.05, <0.01, and <0.001, respectively.
RESULTS
Selection of TMEM67 variants for analysis
TMEM67 variants were selected using two criteria: (i) conservation of the mutated amino acid in the worm orthologue (MKS-3) (Figure S1), and (ii) presence of an adjacent Cas9 PAM site in the C. elegans genome to facilitate CRISPR gene editing. Using these criteria, we modelled eight missense TMEM67 VUS in C. elegans mks-3 (Figure 1A, Table S1). We also included two known benign and two pathogenic variants as controls.
Benign1(Asp359Glu) and VUS4(His782Arg) were identified on the Ensembl variation database29. Benign2(Thr360Ala), Pathogenic1(Glu361Ter), Pathogenic2(Gln376Pro), VUS2(Cys173Arg), and VUS5(Gly786Glu) were identified on ClinVar5. VUS1(Cys170Tyr), VUS3(Thr176Ile), VUS6(His790Arg), VUS7(Gly979Arg), and VUS8(Ser961Tyr) were identified from clinical exome sequencing of Meckel syndrome foetuses. For simplicity, we refer to the variants using a shorthand notation (e.g. Benign1, Pathogenic1, VUS1). A de novo protein structure prediction program, Raptor X, revealed that the human and worm TMEM67 proteins show remarkable similarity in their overall predicted domain organization and secondary structure (Figure 1B). The targeted pathogenic, benign and VUS residues are present in comparable regions of secondary structure (Figure 1B). For example, the known benign variant residues are in exposed loops and the known pathogenic residues are buried in β-sheets (Figure 1B, Figure S2).
Quantitative phenotypic analysis of mks-3 VUS alleles in C. elegans
We employed a CRISPR/Cas9 genome editing strategy to engineer homozygous mks-3 mutant worms (Figure 1A). Since mks-3 functions redundantly with NPHP module genes (nphp-1/4), we generated the mks-3 knock-in variants in an nphp-4(tm925) mutant background to facilitate phenotypic analysis18. nphp-4(tm925) is a 1109 bp deletion, subsequently referred to as nphp-4(Δ). In phenotypic assays, double mutant mks-3(variant); nphp-4(Δ) phenotypes were compared to mks-3(+); nphp-4(Δ) (wild-type, positive control) and mks-3(Δ); nphp-4(Δ) (949 bp deletion of mks-3, negative control)18. We hypothesized that pathogenic mks-3 patient alleles would be phenotypically similar to the mks-3(Δ) allele.
To assess cilia structure and function we performed three quantitative assays: dye filling, roaming/foraging, and chemotaxis. The dye filling assay indirectly assesses the structural integrity of cilia25. We assessed lipophilic dye (DiI/DiO) staining in the four ciliated phasmid sensory neurons in the tail. Wild type and mks-3(+); nphp-4(Δ) positive controls display robust dye filling, whereas the mks-3(Δ); nphp-4(Δ) negative control is dye filling defective (Figure 2A). Benign1 and Benign2-containing strains show robust dye uptake whilst strains with Pathogenic1 and Pathogenic2 are defective (Figure 2A). Five of the VUS alleles cause a severe dye filling defect (VUS1/4/5/6/8), whereas three (VUS2/3/7) do not (Figure 2A). C. elegans foraging behaviour is dependent on sensory cilia25. A single young adult worm is placed on a lawn of bacteria for 20 hours and the extent of its roaming across the plate is quantified (Figure 2B). mks-3(+); nphp-4(Δ) positive control worms show a slight decrease in roaming compared to wild-type worms, whereas mks-3(Δ); nphp-4(Δ) negative controls exhibit a severe roaming defect (Figure 2B). As expected, Benign1 and Benign2-containing strains exhibit normal roaming behaviour, while Pathogenic1 and Pathogenic2-containing worm strains are roaming defective (Figure 2B). VUS4/5/6/8 exhibit a roaming defect, whereas worms containing VUS1/2/3/7 are roaming normal (Figure 2B). C. elegans chemotaxis towards benzaldehyde is also dependent on sensory cilia25. A population of 50-300 worms is placed in the center of a plate, equidistant from spots of control (ethanol) and benzaldehyde (1:200 in ethanol) solutions. mks-3(+); nphp-4(Δ) positive control worms show a slight reduction in chemotaxis compared to wild type, whereas mks-3(Δ); nphp-4(Δ) negative controls exhibit a chemotaxis defective phenotype (Figure 2C). As expected, Benign1 and Benign2-containing strains are chemotaxis normal while Pathogenic1 and Pathogenic2-containing strains are defective (Figure 2C). VUS1/4/5/6-containing strains are severely chemotaxis defective, VUS3/8-containing worms exhibit an intermediate phenotype, and VUS2/7-containing worms are chemotaxis normal (Figure 2C).
To derive a predictive ‘interpretation’ score for the VUS alleles, we integrated the results from the three phenotypic assays into a single value (equal weighting; averages normalized to the mks-3(+); nphp-4(Δ) positive control; maximum score of 1.0 per assay) (Figure 2D). The Benign1 and Benign2 variants score similarly to the mks-3(+); nphp-4(Δ) positive control, whereas Pathogenic1 and Pathogenic2 variants score similarly to the mks-3(Δ); nphp-4(Δ) negative control (Figure 2D). Strains with VUS2, VUS3, or VUS7 received high scores comparable to the benign variants, whereas those with VUS1, VUS4, VUS5, VUS6 or VUS8 received scores comparable to the pathogenic variants. Therefore, in C. elegans, we conclude that VUS2/3/7 are benign variants and VUS1/4/5/6/8 are pathogenic variants.
Effect of VUSs on transition zone localization of MKS-3 and ultrastructure
To provide further insight into the damaging or benign nature of the TMEM67 VUS alleles, we examined the effect of the variants on the subcellular localisation of MKS-3. In C. elegans sensory neurons, transmembrane MKS-3 localizes to the ciliary transition zone (TZ), which corresponds to the most proximal 1 μm of the ciliary axoneme18. Fusion PCR was used to generate linear mks-3::gfp fragments (Figure S3A) containing variants of interest. Constructs were then expressed as extrachromosomal arrays in C. elegans, and a fluorescent lipophilic red dye (DiI) employed to co-stain the ciliary membrane. Pathogenic1 was excluded from this analysis because it is a nonsense allele with a premature stop codon. As expected, MKS-3(+)::GFP, Benign1::GFP, and Benign2::GFP exhibit TZ localization (Figure 3A, S3B). Pathogenic2::GFP showed no detectable fluorescence at the TZ, consistent with the finding that the human Pathogenic2 variant (Q376P) disrupts TMEM67 plasma membrane localization in cell culture30. Our conclusion that VUS2, VUS3, and VUS7 are benign (Figure 2D) predicts that the proteins should localize normally. Consistent with this hypothesis,VUS2::GFP, VUS3::GFP, and VUS7::GFP display TZ localizations in transgenic worms (Figure 3A, S3B). In contrast, transgenically expressed VUS1::GFP, VUS5::GFP, VUS6::GFP, and VUS8::GFP show no significant TZ localisation, consistent with these variants being pathogenic. Interestingly, despite a predicted pathogenic classification (Figure 2D), VUS4::GFP was TZ-localized in most transgenic worms, although signal levels were reduced (Figure 3A, S3B).
We also assessed the effect of the TMEM67 VUS variants on the ultrastructure of cilia and TZs using transmission electron microscopy (TEM). Specifically, we analyzed two alleles: VUS1 (predicted pathogenic) and VUS2 (predicted benign). Pathogenic2 was also examined as a control. Wild-type amphidal pores contain 10 rod-shaped ciliary axonemes emanating from 8 sensory neurons (2 neurons possess a pair of rods), each consisting of middle (doublet microtubules) and distal (singlet microtubules) segments, and a proximal TZ compartment that emerges from a swelling at the distal dendrite tip called the periciliary membrane compartment (PCMC) (Figure 3B). At the middle pore region, mks-3(+); nphp-4(Δ) positive controls display an almost full complement of 10 cilia, whereas at least two axonemes are missing in the mks-3(Δ); nphp-4(Δ) negative control and in Pathogenic2-containing worms (Figure 3C). We found that VUS1-containing worms are also missing some axonemes, whereas the VUS2-containing strain is not (Figure 3C). In cross-sections of the TZ and PCMC regions, approximately 50% of the TZs of mks-3(+); nphp-4(Δ) positive controls show a wild type phenotype (TZ membrane and microtubules in close apposition, connected by electron dense Y-linkers) (Figure 3B). As expected, the mks-3(Δ); nphp-4(Δ) negative control and the Pathogenic2-containing strain show a much more severe phenotype than nphp-4(Δ) alone (Figure 3D). The VUS2-containing strain displays a similar TZ phenotype to the positive control, whereas the VUS1-containing strain shows enhanced TZ membrane-MT detachment that is similar to the mks-3(Δ); nphp-4(Δ) negative control and Pathogenic2 (Figure 3D). Altogether, the ultrastructure data confirms the pathogenic and benign nature of VUS1 and VUS2, respectively.
In vitro genetic complementation assay of TMEM67 VUS function in human cell culture
To further validate our findings from C. elegans, we utilized an in vitro human cell culture-based assay of TMEM67 function. Previously, we demonstrated that TMEM67 is required for phosphorylation of the ROR2 co-receptor and subsequent activation of non-canonical Wnt signalling26 (Figure 4A). Here, we developed a genetic complementation approach using a validated hTERT-RPE1 crispant cell-line that has biallelic null mutations in TMEM67 (Figure S4). In the absence of TMEM67, phosphorylation of ROR2 was not stimulated by exogenous treatment with the non-canonical ligand Wnt5a. Transient transfection with full-length wild-type TMEM67 fully rescued ROR2 phosphorylation following Wnt5a treatment. These responses allowed us to develop a series of assays to determine the relative effects of VUS on biological function. In this assay, transfection of Benign1 allowed 204.7% induction of phospho-ROR2 levels by Wnt5a relative to control (Figure 4B). In contrast, Pathogenic2 did not rescue biological function (90.2% induction) (Figure 4B). Comparison of all VUS, normalized to wild-type TMEM67 responses across three independent biological replicates, enabled us to interpret VUS1, VUS4, VUS5, VUS6 and VUS8 as pathogenic, and VUS3 and VUS7 as benign (Figure 4C). VUS2 was not tested in this assay.
DISCUSSION
Ciliopathies are multisystem disorders that affect many organs including kidneys, liver, and retina. While the organ systems affected by cilia dysfunction are not present in C. elegans, the basic biology of primary cilia is conserved. Despite undoubted context-specific distinctions, cilia proteins are functionally conserved31,32, therefore allowing us to model missense variants of these proteins in worms.
In this study, we exploited efficient genome editing and quantitative phenotypic analysis in C. elegans to determine the pathogenicity of TMEM67 variants. This approach accurately classified known pathogenic and known benign variants. We also generated a pathogenicity prediction for all eight missense VUS alleles analyzed. Three VUS were phenotypically benign (VUS2(Cys173Arg), VUS3(Thr176Ile),VUS7(Gly979Arg)) and five were phenotypically pathogenic (VUS1(Cys170Tyr), VUS4(His782Arg), VUS5(Gly786Glu), VUS6(His790Arg), VUS8(Ser961Tyr)). These pathogenic predictions were validated in a human cell culture assay of TMEM67 function. We propose that experiments in C. elegans can interpret the pathogenicity of VUS and provide evidence towards their reclassification as benign or pathogenic.
Several in silico algorithms have been developed to predict the pathogenicity of missense variants. However, their accuracy is inconsistent33–35. Indeed, we tested five commonly used in silico tools (MISTIC, SIFT, Poly-Phen, CADD, and REVEL) and found that they return deleterious/damaging predictions for all 8 of the VUS examined in this project (Figure S5). The only exceptions are VUS1/3/8, where 1 or 2 of the tools returned non-deleterious or intermediate scores (Figure S5). Therefore, the algorithm predictions do not align with our observations in C. elegans and cell culture experiments. We conclude that in vivo modelling of missense variants in C. elegans more accurately predicts results in human cells than currently available prediction algorithms.
The quantitative assays employed in this study are suitable for high throughput analysis. Live animal fluorescence-activated cell sorting can be used for high throughput dye filling assays36 and automated worm tracking can be used for high throughput roaming and chemotaxis assays37. Additionally, machine learning can streamline the analysis of complex datasets to predict VUS pathogenicity38. One limitation to modelling patient variants in endogenous C. elegans genes is the conservation of amino acid residues. Although many cilia genes have clear orthologs in C. elegans, the amino acid conservation varies from 30-60%. C. elegans genes can be “humanized” to remove this limitation38–40. In summary, this study highlights that C. elegans is a practical model for variant interpretation of ciliary genes. Analysis of ciliopathy-associated VUS in C. elegans is accurate, quick, affordable, and easily interpretable. While this study focused on TMEM67, we anticipate that VUS alleles of any conserved ciliary genes can be modelled and characterized in C. elegans.
The authors declare no competing interests.
Acknowledgements
This work was funded by Science Foundation Ireland (SFI) in partnership with the Biotechnology and Biological Sciences Research Council (BBSRC) under grant number 16/BBSRC/3394 (to OEB) and BB/P007791/1 (to CAJ). SB acknowledges support from a Wellcome Trust clinical training fellowship (203914/Z/16/Z). We thank Joseph McNicholl for assistance with construction of the mks-3::gfp transgenes.
Footnotes
Figure 1B was updated to improve labels. A phenotypic analysis of VUS8 in C. elegans was added to the manuscript. This included quantitative analysis in three phenotypic assays (dye filling, roaming, and chemotaxis) and quantification of VUS8::GFP fluorescence at the transition zone. The worm strains containing VUS6 and VUS7 were outcrossed to remove unintended unc-58 mutations that were present in the original strains. These outcrossed worm strains were then re-characterized in the phenotypic assays. This analysis did not change the conclusions about VUS6 but VUS7 is now classified as benign. The transmission electron microscopy of VUS7 was removed because this analysis was performed on the original unc-58 mutation containing strain. Two supplemental figures that appeared in version 1 were removed (complementation test with mks-3 null of benign VUS alleles in dye filling assay and negative osmotic avoidance data).