FYCO1 Frameshift Deletion in Wirehaired Pointing Griffon Dogs with Juvenile Cataract

Round 1

Reviewer 1 Report

Summary

The authors present a study aiming to ascertain the genetic cause of juvenile cataracts in the Wirehaired Pointing Griffon dog breed. Pedigree information collected by the authors suggested an autosomal recessive mode of inheritance. Using a whole genome sequencing approached they subsequently identified a convincing candidate variant in FYCO1:c.2024delG, and predicted it results in the truncation of half the protein.

This is a nice study and a well written paper. Some rewording will improve readability, and some further discussion points will also improve the manuscript.

General comments

There is no mention of structural variants. At the very least you should acknowledge that SVs were not considered at all and therefore cannot formally be excluded.

The relevance of the findings in European and US dogs should be discussed, i.e. do you have sufficient evidence to suggest that the variant is private to this family, or could it be segregating in other unrelated European dogs.

Your strategy excluded variants that were heterozygous in any of the control dogs. In light of your subsequent findings, this is probably a reasonable approach, but is presumably based on the assumption that the variant must be private to the breed. You can’t know that for certain, so you should at least acknowledge this limitation, and that it is possible to have missed something as a result.

Specific Comments

Line 50: Change “Another single base deletion” to “A single base deletion”

Line 76-77: Wording is confusing, consider changing to “An Illumina TruSeq PCR-free DNA library with an insert size of ~400 bp was prepared from the DNA of an affected Wirehaired Pointing Griffon dog.”

Line 77: You don’t need the “2 x” and “paired-end”. Delete “2 x “

Line 90: Change “variants at which” to “variants for which” or “loci at which”

Line 97: XM_038566669.1 and XP_038422597.1 have both been made obsolete on NCBI. Are there updated records to reference, and do they change anything present in your manuscript?

Line 124-126: Were all 3 puppies examined ophthalmalogically? And was it by a qualified veterinary ophthalmologist or a primary practice veterinarian? Were any of the unaffected littermates examined?

Line 125: I don’t think “demonstrated” is quite the right word to use. Perhaps “revealed”?

Line 129-130: Had any of the reportedly affected puppies in previous litter been officially diagnosed by a qualified professional?

Line 144: Please describe in more detail how you prioritised genes according to functional knowledge. The method/process should be in the materials and methods. Did you use specific databases (OMIM, Genecards etc)?

Line 150-152: Reference the table showing this data – currently table S3, but I think the data warrants a table in the manuscript instead of being supplemental.

Figure 2: The figure would be improved by zooming out and adding graphics that demonstrate the exon-intron boundaries and wildtype and altered amino acid sequence.

Supplementary table S3: Half of this information is in table 2. I suggest adding the other half to the main manuscript (either as a separate table, or combined with table 2), and removing this table entirely.

Line 161: Based on the data in table S3, the unrelated dogs were not genotyped for the CRYGB variant.

Lines 167-168: You should mention that the unrelated dogs were all homozygous wt, and comment on if the fact that they are US dogs is relevant.

Author Response

Summary

The authors present a study aiming to ascertain the genetic cause of juvenile cataracts in the Wirehaired Pointing Griffon dog breed. Pedigree information collected by the authors suggested an autosomal recessive mode of inheritance. Using a whole genome sequencing approached they subsequently identified a convincing candidate variant in FYCO1:c.2024delG, and predicted it results in the truncation of half the protein.

This is a nice study and a well written paper. Some rewording will improve readability, and some further discussion points will also improve the manuscript.

Response: Thank you for the favorable comments!

General comments

There is no mention of structural variants. At the very least you should acknowledge that SVs were not considered at all and therefore cannot formally be excluded.

Response: The reviewer is absolutely right. We thought that this limitation is obvious from our description of the methodology. To address the comment, we now added a paragraph to the discussion, in which we emphasize once more that structural variants were not investigated.

The relevance of the findings in European and US dogs should be discussed, i.e. do you have sufficient evidence to suggest that the variant is private to this family, or could it be segregating in other unrelated European dogs.

Response: Our cohort was too small to answer this question. We added two sentences at the beginning of the discussion that explicitly state that we do not know how far this genetic defect has already spread.

Your strategy excluded variants that were heterozygous in any of the control dogs. In light of your subsequent findings, this is probably a reasonable approach, but is presumably based on the assumption that the variant must be private to the breed. You can’t know that for certain, so you should at least acknowledge this limitation, and that it is possible to have missed something as a result.

Response: Agreed. We added this limitation to the paragraph discussing the structural variants.

Specific Comments

Line 50: Change “Another single base deletion” to “A single base deletion”

Response: Revised accordingly.

Line 76-77: Wording is confusing, consider changing to “An Illumina TruSeq PCR-free DNA library with an insert size of ~400 bp was prepared from the DNA of an affected Wirehaired Pointing Griffon dog.”

Response: Revised accordingly.

Line 77: You don’t need the “2 x” and “paired-end”. Delete “2 x “

Response: Revised accordingly.

Line 90: Change “variants at which” to “variants for which” or “loci at which”

Response: Revised accordingly.

Line 97: XM_038566669.1 and XP_038422597.1 have both been made obsolete on NCBI. Are there updated records to reference, and do they change anything present in your manuscript?

Response: NCBI annotation release 106 currently proposes XM_038427989.1 and XP_038283917.1 derived from the ROS_CFam_1.0 genome reference assembly (male Labrador Retriever) as default representations of the canine FYCO1 transcript and protein. These RefSeqs are only minimally different from XM_038566669.1 and XP_038422597.1, which were also predicted during the NCBI annotation release 106 on the alternative UU_Cfam_GSD_1.0 reference assembly (female German Shepherd). Our variant designations c.2024del and p.(Ser675Thrfs*5) are identical for both sets of RefSeqs. None of these RefSeqs is likely to remain stable for very long. We therefore think it is appropriate to give the RefSeq accessions, with which we actually worked during our analyses. To the best of our knowledge, RefSeqs may be declared “obsolete” by NCBI, but they will remain accessible. In this case, the term “obsolete” is somewhat misleading as both sets of RefSeqs were predicted at the same time during NCBI annotation release 106, they just refer to 2 different reference genome assemblies derived from 2 different dogs. The minimal differences between the two sets of RefSeqs only reflect true variation in genomic sequence between the two sequenced dogs. The predicted transcription start site, start codon, stop codon and length of coding sequence are identical.

Line 124-126: Were all 3 puppies examined ophthalmologically? And was it by a qualified veterinary ophthalmologist or a primary practice veterinarian? Were any of the unaffected littermates examined?

Response: All phenotypic information was provided by the breeder. She remembered that she had taken one of the affected puppies to an ophthalmologist, but she could not provide any written reports. It would indeed be very desirable to obtain a much more detailed characterization of the phenotype, but this is beyond the scope of our study.

Line 125: I don’t think “demonstrated” is quite the right word to use. Perhaps “revealed”?

Response: Revised accordingly.

Line 129-130: Had any of the reportedly affected puppies in previous litter been officially diagnosed by a qualified professional?

Response: This is not fully clear to us. We tried to obtain this information from the breeder who provided the samples, but she could not provide any written records on the previous examinations.

Line 144: Please describe in more detail how you prioritised genes according to functional knowledge. The method/process should be in the materials and methods. Did you use specific databases (OMIM, Genecards etc)?

Response: We added the missing information to section 2.4.

Line 150-152: Reference the table showing this data – currently table S3, but I think the data warrants a table in the manuscript instead of being supplemental.

Response: We integrated these data into Table 2.

Figure 2: The figure would be improved by zooming out and adding graphics that demonstrate the exon-intron boundaries and wildtype and altered amino acid sequence.

Response: We modified the figure according to these suggestions.

Supplementary table S3: Half of this information is in table 2. I suggest adding the other half to the main manuscript (either as a separate table, or combined with table 2), and removing this table entirely.

Response: We integrated the information from Table S3 into Table 2 and removed Table S3 as requested.

Line 161: Based on the data in table S3, the unrelated dogs were not genotyped for the CRYGB variant.

Response: We now determined the CRYGB:c.367C>T genotypes in all dogs of the study. The frequency of the alternate allele was 17% in the unrelated North American dogs. Such a high frequency would be very unusual for an allele causing a severe early onset disease. The new data are now also included in Table 2.

Lines 167-168: You should mention that the unrelated dogs were all homozygous wt, and comment on if the fact that they are US dogs is relevant.

Response: Revised accordingly.

Reviewer 2 Report

The authors present strong evidence supporting a frameshift deletion in the FYCO1 gene, which is very likely to cause juvenile cataracts in an inbred family of Wirehaired Pointing Griffon dogs. I believe that the impact of the frameshift deletion, together with its perfect segregation with the disease and the reports of similar gene-disease relations in humans and mice warrant the publication of this finding.

The authors could have gone on to genotype a few more missense variants outside of the two candidate genes to ensure that no additional sites match the disease segregation patterns. However, given how compelling the FYCO1 frameshift variant is, I think it is OK to leave it there. 

The article is well written, clear and concise. It is also similar in scope and length to other papers in the canine special issue #2, so there's no need for major revisions or further experimental work. I do however have a couple of quick analysis suggestions that could be done computationally. I am citing them in my personal order of relevance:

1. Discard structural variation (SV) involvement: If the authors preserve the affected sample bam file, they could very quickly run a read depth SV software like CNVnator (and LUMPY+SVtyper if they want to be more exhaustive) to rule out any SV oversights. Such an analysis shouldn't take more than a day and would ensure that all kinds of variation are screened at least in one of the affected samples. 
2. Annotate conservation scores for non-exonic variants: The authors find potentially up to 1965 private homozygous variants in introns and intergenic regions that could potentially affect regulatory regions. I'm not sure if PHYLOP/FastCons scores are available for the German Shepherd reference, but they are definitely available for Canfam3.1, and liftOver between the two references is possible. Therefore conservation scores could be annotated in Supplementary Table 2. The PROVEAN, MutPred2 and PredictSNP1 scores for all exonic variants could also be annotated in Supplementary Table 2 for completeness.
3. Deep dive into the protein structure effects of the FYCO1 and CRYGB variants:  Now that in silico protein structure simulations are posible using Alphafold 2 (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb), the authors could simulate the effects of the missense and frameshift variants in the proteins of interest and include them in Figure 2. This could give the article a more original touch and, additionally, it is quite fun to do. I was able to simulate both wt and CRYGB:p.(His123Tyr) in my laptop in about an hour each (results attached). That said, I'm not sure about the validity and caveats of this method, and the remote server may run out of memory when simulating big proteins like FYCO1.
4. Inclusion of the Sanger sequencing chromatograms: Maybe as a supplementary figure (for a wt/wt wt/del and del/del) or maybe integrated in with Figure 2.  
5. Add pictures of the dogs: if available, those always make canine genetics papers more attractive to general readeres!

None of these points are dealbreakers for me, but rather extra ideas to add to the paper for extra completeness. Additionally, here are a few minor corrections:

Line 22- change "were shown" for "have been shown"

Line 45- change "were described" for "have been described"

Line 98- change "Numbering within canine" for "Numbering within the canine"

Line 104 There's a space after the 10th letter in the 2nd primer that should be removed

Figure 1- Mark the sequenced individual in figure

Line 138- Change "With respect to UU_Cfam_GSD_1.0 reference" for "With respect to the UU_Cfam_GSD_1.0 reference"

Line 150- Give context to protein impact scores for CRYGB:p.(His123Tyr). This could be done simply by adding the impact scores from FYCO1:c.2024delG so the readers can tell the difference or, if they cannot be calculated for frameshift deletions, provide some reference values.

Line 203- Change "discovered" for "discover" 

Overall, the authors have written a really streamlined and well-presented manuscript which provides convincing evidence that they have found the causal variant for juvenile cataracts in the assessed WPG dog family.

Comments for author File: Comments.zip

Author Response

The authors present strong evidence supporting a frameshift deletion in the FYCO1 gene, which is very likely to cause juvenile cataracts in an inbred family of Wirehaired Pointing Griffon dogs. I believe that the impact of the frameshift deletion, together with its perfect segregation with the disease and the reports of similar gene-disease relations in humans and mice warrant the publication of this finding.

The authors could have gone on to genotype a few more missense variants outside of the two candidate genes to ensure that no additional sites match the disease segregation patterns. However, given how compelling the FYCO1 frameshift variant is, I think it is OK to leave it there.

The article is well written, clear and concise. It is also similar in scope and length to other papers in the canine special issue #2, so there's no need for major revisions or further experimental work. I do however have a couple of quick analysis suggestions that could be done computationally. I am citing them in my personal order of relevance:

Response: Thank you for the favorable comments!

(1)

Discard structural variation (SV) involvement: If the authors preserve the affected sample bam file, they could very quickly run a read depth SV software like CNVnator (and LUMPY+SVtyper if they want to be more exhaustive) to rule out any SV oversights. Such an analysis shouldn't take more than a day and would ensure that all kinds of variation are screened at least in one of the affected samples.

Response: In our experience, the available software tools for the detection of structural variants have poor sensitivity and specificity when working with less than perfect reference genome assemblies. The most common type of structural variants in dogs are SINE insertions. According to our experience, they are notoriously difficult to detect bioinformatically. We think that adding such an imperfect structural variant analysis may imply an unwarranted level of reliability. We added the limitation of the missing structural variant analysis to the discussion.

(2)

Annotate conservation scores for non-exonic variants: The authors find potentially up to 1965 private homozygous variants in introns and intergenic regions that could potentially affect regulatory regions. I'm not sure if PHYLOP/FastCons scores are available for the German Shepherd reference, but they are definitely available for Canfam3.1, and liftOver between the two references is possible. Therefore conservation scores could be annotated in Supplementary Table 2. The PROVEAN, MutPred2 and PredictSNP1 scores for all exonic variants could also be annotated in Supplementary Table 2 for completeness.

Response: We appreciate the comment of the reviewer. Adding the FastCons scores on UU_Cfam_GSD_1.0 variants would, however, indeed require a liftover of CanFam 3.1 data and that poses risk for additional errors.

In the specific context of this investigation, when we have a frameshift variant in an excellent functional candidate gene, we don’t think that even ultra-high conservation of a non-coding nucleotide or any in silico predicted consequence of missense variants would change the fact that the frameshift variant would be classified as the top candidate variant.

We added the in silico predictions for all missense variants and added some verbose comments for all protein-changing variants.

(3)

Deep dive into the protein structure effects of the FYCO1 and CRYGB variants:  Now that in silico protein structure simulations are possible using Alphafold 2 (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb), the authors could simulate the effects of the missense and frameshift variants in the proteins of interest and include them in Figure 2. This could give the article a more original touch and, additionally, it is quite fun to do. I was able to simulate both wt and CRYGB:p.(His123Tyr) in my laptop in about an hour each (results attached). That said, I'm not sure about the validity and caveats of this method, and the remote server may run out of memory when simulating big proteins like FYCO1.

Response: We also appreciate this comment by the reviewer. Unfortunately, my computational skills were insufficient to get this structure simulation to run successfully. We agree that protein structure simulations can provide additional supporting evidence that the CRYGB:p.His123Tyr variant is unlikely to have a major function impact on the protein. We are grateful to the reviewer for providing already the results of the analysis comparing the ¹²³His allele to the ¹²³Tyr allele, which postulates identical secondary and tertiary structures for the two alleles.

However, while the in silico analysis of potential CRYGB protein structures is relevant to the manuscript, comparable analyses on the FYCO1:c.2024delG variant are not possible. In our opinion, it is not possible to reliably predict, which mRNA transcript(s) will be generated from the mutant allele. It seems highly probably that any mutant transcript will be subject to nonsense mediated decay and then no protein would be expressed.

We revised Figure 2 with respect to the comment and to emphasize that the FYCO1:c.2024delG variant is indeed expected to completely inactivate the FYCO1 gene and to cause a true null allele.

(4)

Inclusion of the Sanger sequencing chromatograms: Maybe as a supplementary figure (for a wt/wt wt/del and del/del) or maybe integrated in with Figure 2. 

Response: We added Sanger electropherograms of wt/wt and del/del animals to figure 2. Unfortunately, our forward sequences had moderate dye blobs exactly at the position of the deletion. We therefore had to use the reverse sequences for the figure. Therefore, we could not display the Sanger sequence of a carrier animal (as this would have shown the overlapping “double peaks” to the left of the deletion).

(5)

Add pictures of the dogs: if available, those always make canine genetics papers more attractive to general readers!

Response: We fully agree with the reviewer and asked the breeder for photos of the dogs. Unfortunately, none were available.

(6)

None of these points are dealbreakers for me, but rather extra ideas to add to the paper for extra completeness. Additionally, here are a few minor corrections:

Line 22- change "were shown" for "have been shown"

Response: Revised accordingly.

(7)

Line 45- change "were described" for "have been described"

Response: Revised accordingly.

(8)

Line 98- change "Numbering within canine" for "Numbering within the canine"

Response: Revised accordingly.

(9)

Line 104 There's a space after the 10th letter in the 2nd primer that should be removed

Response: Revised accordingly.

(10)

Figure 1- Mark the sequenced individual in figure

Response: Revised accordingly.

(11)

Line 138- Change "With respect to UU_Cfam_GSD_1.0 reference" for "With respect to the UU_Cfam_GSD_1.0 reference"

Response: Revised accordingly.

(12)

Line 150- Give context to protein impact scores for CRYGB:p.(His123Tyr). This could be done simply by adding the impact scores from FYCO1:c.2024delG so the readers can tell the difference or, if they cannot be calculated for frameshift deletions, provide some reference values.

Response: We added the thresholds and meaning of the output scores to the methods section 2.7. Most predictors cannot handle frameshift variants. Therefore, it was not possible to apply the same set of predictors to the FYCO1:c.2024delG variant.

(13)

Line 203- Change "discovered" for "discover"

Response: We changed “discovered” into “reveal”.