The manuscript titled “Exome sequencing utility in defining the genetic landscape of hearing loss and novel-gene discovery in Iran” by Mohseni and Babanejad et al. describes the continued follow-up of patients who had previously tested negative on a gene panel. An exome-sequencing approach was employed in 73 families. Twelve families were further diagnosed with variants in known genes and an additional nine were identified with candidate variants. This study is interesting in that it provides a unique look at potential candidate genes and other rare or newly identified genes that are poorly described that is likely to be of value to the community.

I recommend that the authors carefully edit for English errors. I have included my comments below:

Abstract:
Second paragraph, first sentence: this is a long and complicated sentence. I suggest to break it into two sentences. The most interesting part of this study is the nine candidate genes that should be included in the abstract text.

“To date, this study is the largest of its kind in hearing loss…” – this is incorrect. Even some of the co-authors who are included in this manuscript have been involved in larger studies and in the literature, there are examples of patient cohorts over 73 families.

Materials and Methods:
Were patients sequenced on both OtoSCOPE (V5 and V6) panels or only one of the other?

Was copy number variation assessed in these patients? A statement about this should be included.

Have the variants in known genes been deposited in genetic databases like DVD, LOVD or ClinVar?

Results:
One of the major weaknesses of the study is the classification of variants. The authors claim they used the ACMG guidelines but there was no indication anywhere how they did this, or which rules were applied for each variant. Adding this information would be good to see.

The identification of families with NARS2 variants is very interesting, as this gene is involved in both syndromic and non-syndromic hearing impairment. It is not clear to me if these two families fall into the non-syndromic or syndromic end of the phenotypic spectrum of this gene. There is a “-“ in the “Additional features” column of the table on page 33 of the proof. This needs to be very carefully checked, as to my knowledge, only one publication exists asserting a link between NARS2 and non-syndromic hearing loss. Was it because additional features were not present or were these families not available for detailed phenotyping? In those in whom additional clinical assessment was unable to be performed, this should be noted in the additional features column for all genes in which a syndrome is known for transparency. If these families are confirmed with non-syndromic hearing loss, a statement in the paper should be included that these are replicating families to provide evidence for non-syndromic hearing loss due to biallelic NARS2 variants. Furthermore, are the two NARS2 families with the same variant distantly related or from the same village?

In the second paragraph of the Results section, check that only six families had syndromic forms of deafness (ATP6V1B1 is missing here).

As this study involves a reasonable number of genes/families, genetic testing results should contain the genotype data (at least transcript, c. and p. position) of all variants without having to refer the reader elsewhere.

Figure 3H: LRRC16A is not mentioned at all in the results—I checked this and it is not the correct HGNC gene name. Please check and correct all gene names.

Results: it mentions that only one individual was subjected to exome sequencing with the exception of three families. In the materials and methods section, it mentions “…with mostly two or more affected members by exome sequencing…” – unify these statements so this point is clear to the reader.

Discussion:
Replace the word “mutation” with “variant.”

Add the variant for each of the different genes described.

What is meant by “clinical trials” and what was exactly assessed in the TOP3A section?

Have the investigators reached out to other labs studying families from the Greater Middle East to screen their cohorts? Finding additional families would greatly strengthen the confidence of each candidate gene. It could be that other labs are working on functional studies involving these genes and these families could be merged instead to these other studies.

Check the last sentence of the discussion for correctness in the body of literature covering genetic hearing loss using exome sequencing approaches.

Figures:
Figure 1: This flow-chart negates the two individuals who advanced straight to exome sequencing. It would be helpful to the reader to include this aspect in a revised figure.

Adding detailed audiometric data to all individuals shown in Figure 2 will enhance the quality of the manuscript.

Assemble Figures 1, 2 and 3 as you would expect them to appear in a paper (on one or two pages and not across so many pages).

Tables: check that some of the text is not cut off (like Table 1 header “Number of affected in…”

The revised manuscript (CGE-00961-2020-R1) has improved and I still find this study valuable and interesting. However, I still found many English errors that were distracting to the quality of the manuscript. I encourage the authors to edit this carefully. Some, but not all, are listed below.
Line 67: study cohort
Line 69: …with high rates of consanguineous marriages represent a…
The opening two sentences of the introduction are confusing. The authors state that thousands of genes are estimated to be involved, then estimate that 1% of genes are involved (this would correspond to 220-250 genes).
Rephrase the paragraph starting at line 129 (perhaps break this into two sentences).
Line 138: …except three families…
Line 151: …causal variants…
Line 159: …recessive consanguineous inheritance…
Line 170: Remove the word “Also” and replace “could be” with “were”.
Line 173: Remove the word “Totally” to start this sentence.
In the paragraph with the results starting line 176, if variants were previously published, be sure to include the reference.
Line 201: The causes of HL were found in eight… -- as a side note, we cannot conclusively say that these variants are causal due to the lack of replicating families or other functional studies. This aspect should be carefully checked throughout the manuscript.
Sentences start “By 3D” in several places like lines 339 and 377 – this should be written out to include “modelling” or something to that nature.
Line 378: …but in the variant form…
Line 380-381: The CARMIL1 multi-domain protein has critical roles in cell…
Ensure abbreviations are first defined, such as HHL, GATK, BWA, SNV (and other terms on line 161), ADHL, and NE.
Figure 1: GJB2 should be written in italics. Check for an extra space between the comma and “clinical examination”
As a side note, I was surprised to read in the response letter to reviewer 3 that there are functional studies underway for some of these genes but the authors have not had a chance to reach out to other labs in order to identify other families, which would be important to avoid being scooped. Exchange with other labs would have opened the possibility of finding other families. It is likely that, if these genes are indeed new hearing loss-associated genes, finding other families will be challenging in any case, as these likely represent rare forms of deafness. This outreach could have also benefited the community by finding substantial evidence (at least replication in other families) to accelerate integrating these genes into diagnostic settings with much stronger (clinical/functional) evidence. As someone in this community, it would be nice to avoid another “MYO1A” type of gene being published (a gene that was later retracted as a hearing loss gene), as this caused tremendous confusion in the field and negatively impacted diagnoses for many years until this was corrected.

The revised manuscript has improved following revision. I endorse the manuscript for publication.