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Review: How a Small Worm is Helping Uncover the Pathogenicity of Gene Variants


We have previously discussed how the advancement of genomics technology has led to an increase in variants of unknown significance (VUS), and how important it is to identify whether or not they are disease-causingIn this review we discuss a recent publication from Dr. Oliver Blacque and collaborators (Lange et al., 2022), where they leveraged the genetically tractable C. elegans system to model and classify clinically relevant variants associated with human ciliopathies. 

The genomic revolution and accompanying technological advancement have enabled researchers and clinicians to identify genomic variation, with the intent of gaining better insight into human health and disease. However, not all genetic variants are disease-causing. Indeed, genomic reports use a scale to classify variants as pathogenic, likely pathogenic, variants of uncertain significance (VUS), likely benign, and benign [Figure 1] (Clinical Sequencing Exploratory Research (CSER) Consortium, 2017).

Figure 1. Genomic variants are typically classified on a five-point scale to indicate the likelihood that the particular variant is associated with disease ((CSER) Consortium, 2017). 

Thus, while the increase in genetic testing has provided clinicians and researchers with more genetic data than ever before, we now find ourselves with the pressing need to classify these variants in order to provide actionable information for patients.  

One such gene of interest to researchers and clinicians is the transmembrane protein TMEM67 (also called MKS3 for Meckel syndrome type 3). This gene is known for encoding the meckelin protein, which is a key regulator of cilia function (Liu et al., 2021). Notably, pathogenic mutations in theTMEM67 gene have been found to play a central role in ciliopathies such as Meckel Syndrome, COACH Syndrome, and Joubert Syndrome. 

Ciliopathies are a collection of heterogeneous diseases categorized/grouped by their abnormal formation or function of cilia. Cilia are hair-like organelles that protrude from the surface of nearly every cell type and are essential for many signaling pathways and processes associated with development and disease. Consequently, when they are dysfunctional, these small structures cause severely debilitating to deadly disorders — for instance, Meckel Syndrome has a 100% mortality rate either in utero or shortly after birth (Liu et al., 2021). 

While TMEM67 is known to be associated with these catastrophic disorders, the majority of their variants currently have unknown clinical significance (52.1% according to ClinVar) (Lange et al., 2022). In light of this need, many researchers (such as Oliver Blacque and his lab) are turning to alternative animal models such as zebrafish, Drosophila, and C. elegans to evaluate these variants. These models are attractive to researchers as they offer the invaluable advantages of being relatively inexpensive and fast in vivo platforms for preclinical studies and therapeutics discovery [Figure 2].

Figure 2. C. elegans knock-in model as in vivo platform for classifying variants of unknown significance.

C. elegans may seem like a particularly surprising choice for modeling ciliopathies since they do not possess many of the organs that are affected in humans (such as bones or a liver) [Figure 3] (Kim, Underwood, Greenwald & Shaye, 2018). However, this being said, C. elegans are actually one of the most widely used models for cilia-related disorders as the vast majority of genes and associated pathways are conserved between humans and worms [Figure 4]. Thus, establishing C. elegans as a suitable model for interpreting ciliopathy-associated VUS has the potential to be extremely impactful.

For example, these 35 Joubert syndrome

genes that cause Joubert syndrome in humans,

23 of them, are conserved in C. elegans

– Dr. Oliver Blacque, an Associate Professor in cell biology and genetics at the University College Dublin, 17 Minutes of Science

Figure 3. Uncovering roles of Colopathy-associated genes. ALMS, Alström syndrome; BBS, Bardet-Biedl syndrome; CORS, cerebello-oculo-renal syndrome; EVC, Ellis-van Creveld syndrome; JATD, Jeune asphyxiating thoracic dystrophy; JBTS, Joubert syndrome; LCA, Leber congenital amaurosis; MKS, Meckel syndrome; NPHP, nephronophthisis; OFD1, oral-facial-digital syndrome type 1; PCD, primary ciliary dyskinesia; PKD, polycystic kidney disease (Lee & Gleeson, 2011).

Figure 4. C. elegans and human.

One research group that has embraced C. elegans as a ciliopathy model is Dr. Oliver Blacque, an Associate Professor in cell biology and genetics at the University College Dublin who has been working with C. elegans for almost two decades. In their recent publication, Dr. Blacque and his colleagues utilized transgenic C. elegans to determine the pathogenicity of TMEM67 variants.

The Results

Figure 4. Validation of C. elegans predictions of TMEM67 VUS pathogenicity in cell culture. Schematic summarizes the genetic complementation assay in hTERT-RPE1 TMEM67 knock-out cells. Left: in the presence of TMEM67, phosphorylation of the co-receptor ROR2 is stimulated by exogenous treatment with the non-canonical ligand Wnt5a in comparison to control treatment. Right: if TMEM67 is lost or disrupted, ROR2 phosphorylation is not stimulated by this treatment.

Blacque and his collaborators found that, of eight missense VUS evaluated, three were benign and five were pathogenic [see Table 1]. Furthermore, these results were validated in a complementary in vitro assay in a human cell line.













This study demonstrates how invaluable a C. elegans animal model can be for better understanding ciliopathies, and more broadly for classifying VUS, as the study’s authors subsequently tested the accuracy of five in silico tools to classify the same missense variants and found that the in vivo models were significantly more accurate prediction tools of pathogenicity. Thus, while in silico models are promising tools for future interpretation of genetic variation, in vivo models such as C. elegans are currently the most robust models for accurately capturing and functionally validating the effects of patient mutations to begin to address the growing VUS burden in clinical genetics.

To hear Oliver Blacque discuss his use of C. elegans in rare disease research, listen to our episode of 17 Minutes of Science.

To read more of Oliver’s publications: https://people.ucd.ie/oliver.blacque/publications


  1. Lee, J.E. & Gleeson, J. (2011). Emerging genetics of the ciliopathies, Semantic Scholar, https://www.semanticscholar.org/paper/Emerging-genetics-of-the-ciliopathies-Lee-Gleeson/cb452d7c81dff7f774460592783ca8e3b0fdb62d
  2. Lee, J.E., Gleeson, J.G. A systems-biology approach to understanding the ciliopathy disorders. Genome Med 3, 59 (2011). https://doi.org/10.1186/gm275
  3. The Kaplan Lab (2021). http://kaplanlab.com/
  4. Gerdes, Jantje M., Erica E. Davis, and Nicholas Katsanis. “The vertebrate primary cilium in development, homeostasis, and disease.” Cell 137.1 (2009): 32-45. https://doi.org/10.1016/j.cell.2009.03.023
  5. Focșa, Ina Ofelia, Magdalena Budișteanu, and Mihaela Bălgrădean. “Clinical and genetic heterogeneity of primary ciliopathies.” International journal of molecular medicine 48.3 (2021): 1-15. https://doi.org/10.3892/ijmm.2021.5009
  6. Best, S., Lord, J., Roche, M., Watson, C.M., Poulter, J.A., Bevers, R.P.J., Stuckey, A., Szymanska, K., Ellingford, J.M., Carmichael, J. et al. (2021) Molecular diagnoses in the congenital malformations caused by ciliopathies cohort of the 100,000 Genomes Project. J. Med. Genet, Published Online First: 29 October 2021. doi: 10.1136/jmedgenet-2021-108065.
  7. Clinical Sequencing Exploratory Research (CSER) Consortium (2017). Guide to Interpreting
    Genomic Reports: A Genomics Toolkit, cser. https://www.genome.gov/sites/default/files/media/files/2020-04/Guide_to_Interpreting_Genomic_Reports_Toolkit.pdf
  8. Liu, D., Qian, D., Shen, H., & Gong, D. (2021). Structure of the human Meckel-Gruber protein Meckelin. Science advances, 7(45), eabj9748. https://doi.org/10.1126/sciadv.abj9748

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About The Author

Ben Jussila

Ben is an R&D scientist at InVivo Biosystems, specializing in genome editing with CRISPR/Cas9 technology in zebrafish. He received his Bachelor of Science from the University of Minnesota in Genetics, Cell Biology & Development in 2014, and worked as a research technician in the laboratory of Dr. Kristen Kwan at the University of Utah prior to joining the InVivo Biosystems team in 2018. Ben is passionate about nature and conservation, and in his spare time enjoys science outreach, field herpetology, his cats, and raising and breeding reptiles and amphibians.

About The Author

Alexandra Narin

Alexandra is the Marketing Content Manager and Grant Writer for InVivo Biosystems. She graduated from the University of St Andrews in 2020 where she earned a Joint MA Honours Degree in English & Psychology/Neuroscience with BPS [British Psychology Society] Accreditation. She has worked as a research assistant, examining the LEC's (lateral entorhinal cortex) involvement in spatial memory and integrating long term multimodal item-context associations, and completed her dissertation on how the number and kinds of sensory cues affect memory persistence across timescales. Her hobbies include running, boxing, and reading.

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