Pathogenic Intronic Splice-Affecting Variants in MYBPC3 in Three Patients with Hypertrophic Cardiomyopathy

Round 1

Reviewer 1 Report

The paper by Wood and colleagues depicts a short but compelling study on the role of cryptic splicing alterations on disease pathogenesis and progression. For that, the effects of MYBPC3 intronic variants on splicing were evaluated with minigene assays in vitro, which is an appropriate approach.
Regarding the research presented, I have a few remarks:
1) In the introduction section, the authors present the general panorama regarding HCM genetics and the weight of splicing alterations on the pathogenesis of the disease. The major research on splice-affecting variants in MYBPC3 was mentioned however, I would like to know how the authors would have compared their results with the ones described in the paper by Lopes LR et al. (Circ Genom Precis Med. 2020, doi: 10.1161/CIRCGEN.120.002905).  
2) At the beginning of the results section the authors state they"... underwent in-depth genetic and molecular screening by next-generation sequencing (NGS)..." (lines 131, 132), leading to the impression that a whole-genome analysis was performed which is not correct.
3) It is not clear, for all the variants described if they were described elsewhere (out of the UK) and their overall prevalence/impact.
4) Did the authors considered assessing the effect of the splicing variation on protein production and quality?
5) Finally, could any of the variants described, mirror other abnormal spicing patterns described in other genes?
 

Author Response

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Reviewer 2 Report

In this manuscript, the authors studied the new intronic variants in MYBPC3 (that encode for Myosin-binding protein C, cardiac-type protein in the human) in patients with HCM. They have used a simple assay called “Minigene Splicing Assay” and in silico analysis and also investigated the patients' family history. The data is convincing by and large, and approaches appear sound. The lacking point in this manuscript is experimental details.  So the authors should add more experimental details, for example, how many times they have performed Minigene Splicing Assay, which is not indicated anywhere in the manuscript.

Some minor points:

The methods need to be improved. The authors may separate all methods by subsection, like one subsection for bioinformatics analysis and another for RET-PCR assay etc. 

In the abstract, in vitro word is not in italic, Which needs to be corrected by authors. For example, on page1, line no. 17.    

Author Response

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Reviewer 3 Report

1.  

When writing the results section, authors should avoid discussing or interpreting the results (the last few sentences of all sections, take examples, lines140-149, lines168-172 etc.), ignoring negative results, and repeating the same information more than once (line 163, 182).

 

2.  

3.1 Clinical characteristics associated with three intronic MYBPC3 variants,

Another variant (MYL2_Ala13Thr) harboring in the proband in family 3 (figure 2c) also should be listed in the genetic testing part in the results.

One of his sisters is also a light HCM (line 180). We might find out more about the variant if her genetic test were done.

3.  

3.2 In vitro minigene assays to assess the impact of intronic MYBPC3 splice-affecting variants,

You should show the sequence electropherogram figure (an additional 19bp) of the minigene assay of the c.927-8G>A variant.

 

4.  

Regarding section of the following discussion,

3rd paragraph (lines 234-246)

“We postulate the adjacent variants may have the same effect on MYBPC3 splicing as our c.927-8G>A variant (lines 240-241) ... ”

You should explain why you think the adjacent variants may have the same effect.

5.  

Also continued,

the novel c.927-8G>A variant has not been confirmed to be pathogenicity in patient RNA samples, at least in this article.

So, a sentence, which was stated "These data indicate that the c.927-8G>A variants of uncertain significance and ... should reclassified as likely pathogenic." (lines21-23), is unsuitable. The same can be said for the c.3815-10T>G variant. More investigations need to assign pathogenicity.

6.  

Last paragraph (lines 280-296)

Based on the above, we should reconsider the results of in silico analysis (splice AI score) again.

Author Response

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Round 2

Reviewer 3 Report

Also continued,

the novel c.927-8G>A variant has not been confirmed to be pathogenicity in patient RNA samples, at least in this article.

So, a sentence, which was stated "These data indicate that the c.927-8G>A variants of uncertain significance and ... should reclassified as likely pathogenic." (lines21-23), is unsuitable. The same can be said for the c.3815-10T>G variant. More investigations need to assign pathogenicity.

For both of these variants, we can now apply PS3 from the ACMG guidelines for assigning pathogenicity – we have shown, using a well-established in vitro functional study, which support a damaging effect of the variants by causing abnormal splicing. We have included this justification in the discussion (lines 250-254).   For assigning pathogenicity (likely pathogenicity) according to the scoring rules on the ACMG guidelines, in addition to the current PS3 evidence, more than moderate or/and supporting evidence also needs. Also the CADD score of this variant  is not so high. I recommend  that you should give one(s).   Researchers of ref27 succeeded to detect mutant mRNA from blood. We consider that the variant may not hardly influence to mRNA in the blood (living subject) rather than nonsense mediated mRNA decay. 

Author Response

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