Somatic recombination underlies frequent revertant mosaicism in loricrin keratoderma

We demonstrate that revertant mosaicism frequently occurs in loricrin keratoderma and that somatic recombination is the major mechanism underlying this therapeutically important phenomenon.

As you will see, the reviewers appreciate your data and only have a few suggestions on how to further strengthen your work. We would be thus happy to publish your paper in Life Science Alliance pending final revisions to address the comments raised by the reviewers as well as the following editorial points: -please deposit the whole exome sequencing data in a repository (https://ega-archive.org or https://www.ncbi.nlm.nih.gov/clinvar/) -please add the description for supplementary figure S6E to the legend -please add the missing panel 'D' to supplementary figure S7 -please upload all supplementary figures as individual files To upload the final version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plex You will be guided to complete the submission of your revised manuscript and to fill in all necessary information.
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Sincerely, Andrea Leibfried, PhD Executive Editor Life Science Alliance Meyerhofstr. 1 69117 Heidelberg, Germany t +49 6221 8891 502 e a.leibfried@life-science-alliance.org www.life-science-alliance.org Suzuki et al. report on two unrelated families with loricrin keratoderma where two different pathogenic germline variants were identified in LOR. The probands were exhibiting dozens of clinically and histologically normalized skin areas in which the authors demonstrated that the causal variants were corrected. In the normalized skin spots,the authors also identified loss-ofheterozygosity on chromosome 1q from regions centrometic of LOR to the telomere. In contrast, the affected skin did not show any LOH. The authors therefore proposed that somatic recombination is the mechanism for the reversion of the LOR mutations. Based on in vitro studies the authors furthermore demonstrate that the reversion confers a growth and/or survival advantage to cells in vitro.
The authors demonstrate for the first time the occurrence of somatic revertant mosaicim in LOR. The data is solid and the manuscript is well written.

Minor comment
In the material and methods there is limited amount of information how the whole exome sequencing and analysis was performed. Was only the proband sequenced or were additional family members included in the analysis? Was in silico panels used? How was the filtering of variants performed?
Reviewer #2 (Comments to the Authors (Required)): This is a well-written and illustrated account of the phenomenon of revertant mosaicism occurring in the setting of a skin disease, loricrin keratoderma, an autosomal dominant disorder in which heterozygous frameshift mutations in LOR usually result in an arginine rich tail to the protein, nuclear retention of the mutant protein, and the disruption to the cornification process in skin. The clinical, skin microscopy and functional data collectively provide new insight into this observation. Minor comments only.
[1] In the introduction, bottom of page 4, it would be helpful to mention the specific genes involved since there are at least 2 genes showing RM in junctional EB (COL17A1 and LAMB3) and two in IWC (KRT1 and KRT10).
[2] Perhaps the extrapolation to therapy for patients should be downplayed a bit (abstract and discussion). Thus far, the most successful translation (with patient benefit) has been punch grafting for LAMB3 reported 5 years ago. Answer: In accordance with the editor's suggestion, we have (1) modified the legend for Figure   S6 to add the description for Figure S6E, (2)  We thank the reviewer for the positive and supportive comments. We performed whole exome sequencing in 3 affected and 2 unaffected individuals in the family 1 without using in silico panels. Because the diagnosis of loricrin keratoderma was clinically and histologically suspected, we could easily identify the pathogenic mutation in LOR in this family. The revised manuscript now mentions these points and the filtering strategy used in this study as follows:

Results, 1 st paragraph
"Whole-exome and Sanger sequencing in 3 affected and 2 unaffected individuals in the family revealed that the affected individuals were all heterozygous for a 1-bp insertion mutation in LOR, c.545_546insG (p.Gly183ArgfsTer153) (Fig 1G), which had previously been reported to cause LK (Song et al, 2008), further verifying the diagnosis of LK. By contrast, the unaffected individuals were both wild-type for the mutation."

Answer:
We thank the reviewer for these insightful comments. In accordance with the reviewer's suggestion, we have revised the manuscript as shown below. Perhaps the extrapolation to therapy for patients should be downplayed a bit (abstract and discussion). Thus far, the most successful translation (with patient benefit) has been punch grafting for LAMB3 reported 5 years ago.

Answer:
We fully understand the reviewer's concern. In accordance with the reviewer's suggestion, we have revised the manuscript as shown below.
Abstract "Nevertheless, the identification of revertant mosaicism in LK might pave the way for revertant therapy for this intractable disease." Discussion, 3 rd paragraph "Future research into the molecular basis of revertant mosaicism might hold potential to benefit patients with LK and/or other currently intractable genetic diseases."