Restless legs syndrome (RLS) is a neurological disorder characterized by an urge to move and uncomfortable sensations. Genetic studies have identified polymorphisms in up to 19 risk loci, including MEIS1 and BTBD9. Rodents deficient in either homolog show RLS-like phenotypes. However, whether MEIS1 and BTBD9 interact in vivo is unclear. Here, with C. elegans, we observed that the hyperactive egg-laying behavior caused by loss of BTBD9 homolog was counteracted by knockdown of MEIS1 homolog. This was further investigated in mutant mice with Btbd9, Meis1, or both knocked out. The double knockout mice showed an earlier onset of the motor deficit in a wheel running test but did not have increased sensitivity to heat stimuli as observed in single knock outs. Meis1 protein level was not influenced by Btbd9 deficiency, and Btbd9 transcription was not affected by Meis1 haploinsufficiency. Our results demonstrate that MEIS1 and BTBD9 do not regulate each other.
Comments to the Author: The manuscript details mouse and worm experiments on BTBD9 and MEIS1, two major candidate genes of RLS. As RLS is a polygenic disease, the double knock-out experiment is of great value in determining the relationship as well as a possible additive effect of these genes to the susceptibility to RLS. However, there are concerns relating to the analysis of the data which should be addressed.
In Figures 2 and 3, individual measurements resulting from repeated measurements in the same animal should not be displayed as individual data points, but should be first averaged within animal.
In statistical tests relating to Figs 2-3, was repeated measures ANOVA used? The test used should be stated in the legend. If so, the appropriate way to analyse the data would be to average first within animal and then use for example one-way ANOVA.
Division into light and dark period is used for wheel running activity, as is often done for this kind of data. Rather, the period right before and after the lights-on should be looked at. This would correspond better to human RLS and allow comparison with previous data in MEIS1 knock-out mice.
In the introduction, the sentence describing the human genetics of RLS is somewhat confusing and should be reformulated.
What does “4 repeats for each genotype” mean? Does this mean the last 4 days that were used in the analysis according to Lyu et al. 2019 or did the mice spend four times seven days in the running wheel?
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Comments to the Author: In this paper Lyu et al. investigated the impact of two RLS candidate genes in the sensory-motor characteristics of C. elegans and mice. They also probed the potential interactions of these two genes based on the sensory-motor effects of their double KOs in the animals. This is an interesting paper that can provide useful information on the roles of MEIS1 and BTBD9 as two significant genes in RLS genetics.
The paper is well written, and the experiments are well conducted. The Western blot analysis results are not included in the supplemental data, and only graphs with quantitative measurement of the protein levels are provided.
1. Lines 39-41 "The double knockout mice showed an earlier onset of the motor deficit in the wheel running test but did not have increased sensitivity to the heat stimuli as observed in single KOs." Based on publications by Salminen et al. 2017, Meneely et al. 2018 and Spieler et al. 2014, the sensitivity to heat stimuli was not observed in mice with Meis1 deficiency. The authors could provide references supporting the observation of increased sensitivity to heat stimuli in BOTH KOs.
2. Could the authors elaborate on why Meis1 was only measured at protein level (WB), but Btbd9 at mRNA level (q-RT-PCR)? Would have been ideal if both genes were measured at both levels. The reason and limitations should be discussed in the paper.
3. Lines 84-85 "With or without unc-62 RNAi, hpo-9(tm3719) retained fewer eggs than N2." According to Figure 1, “N2” should be “N2(unc-62)”.
4. To make it easier for the readers, in the results section, it should be more clearly stated that which paragraph refers to which animal.
5. Line 87 "fo" is a typo?
6. Lines 90-91. The two sentences should be separated more clearly. The second sentence seems to continue the previous sentence about dark phase activity.
7. Line 122. "the two RLS risk genes work independently and have functional interactions in both worms and mice." The authors are suggesting that there are functional interactions between the two proteins. A protein-protein interaction assay would be ideal to confirm this interaction. If not feasible, the limitations of this conclusion should be discussed in the manuscript.
8. Please provide all the original Western blot figures in the supplemental data.
Overall Score 4.6 out of5
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