Interpreting the pathogenicity of Joubert syndrome missense variants in Caenorhabditis elegans
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Abstract
Ciliopathies are inherited disorders caused by defects in motile and non-motile (primary) cilia. Ciliopathy syndromes and associated gene variants are often highly pleiotropic and represent exemplars for interrogating genotype-phenotype correlations. Towards understanding disease mechanisms in the context of ciliopathy mutations, we have used a leading model organism for cilia and ciliopathy research, Caenorhabditis elegans, together with gene editing, to characterise two missense variants (P74S and G155S) in mksr-2/B9D2 associated with Joubert syndrome (JBTS). B9D2 functions within the Meckel syndrome (MKS) module at the ciliary base transition zone (TZ) compartment and regulates the molecular composition and sensory/signalling functions of the cilium. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm that both variant alleles are pathogenic in worms. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that compound heterozygous worms phenocopy worms homozygous for P74S. The P74S and G155S alleles also reveal evidence of a very close functional association between the B9D2-associated B9 complex and MKS-2/TMEM216. Together, these data establish C. elegans as a model for interpreting JBTS mutations and provide further insight into MKS module organisation.
This article has an associated First Person interview with the first author of the paper.
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###Reviewer #3:
Summary:
The authors evaluate functional implications of two B9D2 missense variants identified in an individual with Joubert syndrome, by engineering the variants into a C. elegans model system. Few studies have evaluated the functional consequences of patient variants in model systems (rather than null alleles). Overall, the experiments are elegant and rigorous. The functional defects evaluated include decreased and altered localization of the variant proteins at the TZ, altered TZ function, altered cilium function (dye filling and behavioral assays), and reduced TZ protein localization, especially for TMEM216. The functional effects of homozygous null, homozygous missense variants, and compound heterozygous missense variants are compared. While most of the conclusions are well-supported, the work does not connect the …
###Reviewer #3:
Summary:
The authors evaluate functional implications of two B9D2 missense variants identified in an individual with Joubert syndrome, by engineering the variants into a C. elegans model system. Few studies have evaluated the functional consequences of patient variants in model systems (rather than null alleles). Overall, the experiments are elegant and rigorous. The functional defects evaluated include decreased and altered localization of the variant proteins at the TZ, altered TZ function, altered cilium function (dye filling and behavioral assays), and reduced TZ protein localization, especially for TMEM216. The functional effects of homozygous null, homozygous missense variants, and compound heterozygous missense variants are compared. While most of the conclusions are well-supported, the work does not connect the functional consequences in C. elegans to phenotypic severity in humans, a critical validation of methods to test pathogenicity of human variants.
Major comment:
The authors introduce the concept of using C. elegans for genotype-phenotype correlations in the Abstract and Introduction, but do not interrogate an allelic series from humans with more and less severe phenotypes. The claims of genotype-phenotype correlation could be de-emphasized (eliminated? restricted to C. elegans), or the work could be strengthened by including more of an allelic series including variants predicted to be more deleterious (p.Ser101Arg identified in families segregating a Meckel syndrome phenotype. and p.His5Gln identified in a family segregating a possible Meckel syndrome phenotype), less deleterious (the p.Leu36Pro variant in a possibly less severely affected person with JBTS, also published in the Bachmann-Gagescu paper), and benign (i.e. common variants that are found homozygous in population databases like gnomAD and are unlikely to impair B9D2 function). It seems that this would be a lot of additional work; however, the Discussion highlights "it should be possible to generate hundreds of alleles in a relatively short time frame at relatively low cost and manpower compared to other multicellular systems. The workflow to generate and characterize ciliopathy associated variants described here can also be extended to other conserved cilia genes and ciliopathies."
Other comments:
- Important considerations for data presentation and statistical analysis:
-Use dot (or violin) plots rather than bar graphs to show data structure for length, intensity, and other measurements (see PMID 32346721). For the curves of linescan intensities, it would be helpful to include supplemental figures with all of individual curves to see their shapes and variability.
-t-tests on all data points together may over estimate statistical significance; consider whether it would be more appropriate to compare mean measurements for each animal (or median if the data are not normally distributed). At a minimum, list the number of cilia and the number of animals for each experiment.
- Could the lower levels of mutant protein in the TZ be due to lower levels of total mutant protein? Although there is no MKSR-2 antibody, this could be evaluated by Western blots of mNG::MKSR-2/mNG::MKSR-2(P74S) and mNG::MKSR-2/mNG::MKSR-2(G155S) animals.
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###Reviewer #2:
In this manuscript, the authors analyzed the function of two pathogenic missense variants (P74S, G155S) of Joubert Syndrome protein B9D2/ mksr-2 using a C. elegans model. The data shows that both P74S and G155S mutations change the distribution of MKSR-2 on TZ and disrupt the structure and function of cilia in C. elegans, indicating that both mutations are pathogenic.
Characterizing the function of pathogenic mutations associated with ciliopathies is important for us to understand the function of ciliopathy genes and the pathogenesis of ciliopathies, therefore, the topic of the manuscript is very important and interesting.
The manuscript is well organized, and the data is of high experimental quality. However, there is a lack of new insights about the function of MKSR-2 protein or the formation of TZ.
Major concerns:
What…
###Reviewer #2:
In this manuscript, the authors analyzed the function of two pathogenic missense variants (P74S, G155S) of Joubert Syndrome protein B9D2/ mksr-2 using a C. elegans model. The data shows that both P74S and G155S mutations change the distribution of MKSR-2 on TZ and disrupt the structure and function of cilia in C. elegans, indicating that both mutations are pathogenic.
Characterizing the function of pathogenic mutations associated with ciliopathies is important for us to understand the function of ciliopathy genes and the pathogenesis of ciliopathies, therefore, the topic of the manuscript is very important and interesting.
The manuscript is well organized, and the data is of high experimental quality. However, there is a lack of new insights about the function of MKSR-2 protein or the formation of TZ.
Major concerns:
What are the possible mechanisms by which P74S and G155S mutations affect the function of MKSR-2? Do these mutations affect the interaction between MKSR-2 with other TZ proteins? I do think some (even a little) new insights into the function of MKSR-2 are needed.
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###Reviewer #1:
The experiments are elegant, take advantage of the strengths of the model and the conclusions are mostly supported by the results, even if the discussion should address potential limitations a little more. Overall, this is thorough work of potential high impact.
Major comments:
The authors test the localization of the mutant B9D2 protein at the base of the cilium, show decreased fluorescent signal and conclude that the patient mutations affect the TZ localization of the protein. It seems important to me to also demonstrate that the overall protein stability is not affected by measuring the protein levels by western blot if possible. The competition assay between wildtype and mutant alleles with and without transgene somewhat supports the presence of product from the mutant alleles, but an objective measure of the amount …
###Reviewer #1:
The experiments are elegant, take advantage of the strengths of the model and the conclusions are mostly supported by the results, even if the discussion should address potential limitations a little more. Overall, this is thorough work of potential high impact.
Major comments:
The authors test the localization of the mutant B9D2 protein at the base of the cilium, show decreased fluorescent signal and conclude that the patient mutations affect the TZ localization of the protein. It seems important to me to also demonstrate that the overall protein stability is not affected by measuring the protein levels by western blot if possible. The competition assay between wildtype and mutant alleles with and without transgene somewhat supports the presence of product from the mutant alleles, but an objective measure of the amount would further strengthen their point. (One could imagine that the G155S mutation leads to decreased protein stability with increased degradation and this may explain why it is more similar to the knock-out than the P74S allele?).
One major claim made by the authors is that the experiments allow to classify the severity of the effect caused by the individual mutations, showing that the G155S is more severe than P74S. What I find puzzling, is that the ultrastructural consequences on the TZ appear to be similar in both mutants, whereas the TZ gating function is affected only in the G155S mutant. How do the authors explain this discrepancy? If the morphology of the gate is affected similarly, why is the function not affected similarly? Maybe some quantification of the ultrastructural defects would show that the ZT is more disrupted in the G155S mutant?
A more detailed discussion of the differences between C.elegans and mammalian cilia appears necessary to me, since these difference may prove to limit the applicability of the proposed assays (for example differences in the basal body of C.elegans may limit this approach for basal body resident proteins? Even for the TZ, in humans mutations in only on TZ component cause phenotypes, while in worms, double mutants are necessary for most genes, suggesting differences in the function of the individual proteins or the structure of the TZ). Beyond the species differences, evidence is appearing for cell-type specific roles of ciliary proteins, so that results from one type of cilium (including those shown here in worms), do not necessarily guarantee that this holds true in all cilia types, which would limit the interpretation for patients with many different cilia types. This being said, I still support the relevance of the current work, but just think that these potential limitations should be mentioned in the discussion in a more detailed manner.
Figure 2D: the curve for the P74S mutant overlays with the WT curve with respect to height (fluorescence intensity) and length (x-axis). Does this not contradict panels A-C where the signal is weaker and shorter?
Figure 6C: my impression from the graphs is that nphp4 and cep290 are just as much affected as mks14 and mks6 in both P74S and G155S mutants? The text does not mention that mksr2 mutants have any effect on nphp4, cep290 or mks5 whereas the graphs do show a mild effect? Wouldn't this contradict the model of how the TZ is built? Figure 6D again seems to show a different result than Figure 6C (mainly for mks6)?
Statistics: correction for multiple testing should be performed everywhere (no pair-wise t-tests).
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
The manuscript by Lange et al describes how C. elegans can be used to generate functional assays to interpret the significance of missense variants in known human ciliopathy genes. This work thus aims at being a proof-of-principle for a way to address the major problem of VUSs (variants of unknown significance) faced by human geneticists today and is therefore of high relevance to the field (even if no major novel biological insights with respect to ciliary biology are described). The reviewers agreed that …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
The manuscript by Lange et al describes how C. elegans can be used to generate functional assays to interpret the significance of missense variants in known human ciliopathy genes. This work thus aims at being a proof-of-principle for a way to address the major problem of VUSs (variants of unknown significance) faced by human geneticists today and is therefore of high relevance to the field (even if no major novel biological insights with respect to ciliary biology are described). The reviewers agreed that characterizing the function of pathogenic mutations associated with ciliopathies is important for us to understand the function of ciliopathy genes and the pathogenesis of ciliopathies. The manuscript is well organized and the data are of high experimental quality.
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