A Novel Homozygous Founder Variant of RTN4IP1 in Two Consanguineous Saudi Families

Round 1

Reviewer 1 Report

The manuscript by Aldosary et al. describes two familial cases with the same homozygous variant in RTN4IP1.

The variant is novel (and likely causative) but it is not clear if the reported patients present any new phenotype compared with previous cases.

1-OMIM defines the RTN4IP1-related disease as “Optic atrophy 10 with or without ataxia, mental retardation, and seizures”.

Could the authors exclude optic atrophy in Family 2? OCT needs to be performed, at least in the index case.

Regarding F1-II-2, there are confusing data. Line 193: “pupil is poorly reactive to light and severE optic atrophy in both eyes” while line 378 “Magnetic resonance imaging was normal and failed to demonstrate any … definitive optic nerve abnormality”; it is better to just say that the patient has optic neuropathy although not visible by MRI.

2-Nystagmus is a common symptom in family 2, but also reported in F1-II-1 and diverse previous cases. Could the authors comment on this in the discussion?

3-Based on the reported consanguinity, it is important to exclude a second genetic defect that can explain some unexpected features in some subjects (for instance dysmorphisms or arteriovenous malformation in F1-II-2). Did WES analysis reveal any additional candidate pathogenic variant?

4-It would be interesting to assess the presence of a mitochondrial defects of the respiratory chain complexes (in particular complex I), since fibroblasts from F1-II-2 and muscle from F1-II-4 were available and have been analysed.

5-the Conclusions are missing and the final sentence (lines 405-406) has to be removed.

Table/Figures

-There are a lot of mistakes/inaccuracies in the main table.

Age at presentation is missing in most of the cases.

Cerebral atrophy is reported as “congenital anomalies” in patients 4 and 6 but then there is “No” in the specific row for cerebral atrophy in these two subjects.

Patient 4 has nystagmus beside optic atrophy as eye examination. Why for family 1 there is Yes for “cerebral encephalopathy” while for family 2 “mitochondrial encephalopathy” is indicated?

-Figure 1. I suggest to clearly show the “shared ROH block” and indicate the subjects corresponding to each line.

Typos, rephrasing

l.193 sever =>severe

l.396 the variant is functionally an important residue => the variant affects a functionally important residue

Final comment

I don't think that this manucript is appropriate for this special issue

"The purpose of this Special Issue is to overview the current status of the field and highlight the new findings about the role of impaired Ca²⁺ homeostasis in neurodegenerative diseases"

Author Response

Prof. Dr. Ghulam Ullah

Section Editor-in-Chief

Genes

Dear Prof. Ghunam Ullah,

We greatly appreciate the time and effort that you, the editorial team, and the reviewers have devoted to our paper and thank for the valuable comments, criticism, and feedback on our manuscript. We incorporated all the requested changes and suggestions into the revised version. We have highlighted the changes within the manuscript by using track changes option in Word.

Here is a point-by-point response (in bold) to the reviewers’ comments and concerns.

Yours Sincerely,

Namik Kaya

Reviewer 1

The manuscript by Aldosary et al. describes two familial cases with the same homozygous variant in RTN4IP1. The variant is novel (and likely causative) but it is not clear if the reported patients present any new phenotype compared with previous cases.

1-OMIM defines the RTN4IP1-related disease as “Optic atrophy 10 with or without ataxia, mental retardation, and seizures”. 

Could the authors exclude optic atrophy in Family 2? OCT needs to be performed, at least in the index case.

Response: We thank the reviewer for these insightful suggestions. We apologize for the oversights. We cannot exclude optic atrophy. We now present MRI images as a new additional figure. A neuroradiologist reviewed all the available images and indeed optic atrophy was observed in the MRIs. In addition, a small lactate peak was also seen in the patients.

Regarding F1-II-2, there are confusing data. Line 193: “pupil is poorly reactive to light and severE optic atrophy in both eyes” while line 378 “Magnetic resonance imaging was normal and failed to demonstrate any … definitive optic nerve abnormality”; it is better to just say that the patient has optic neuropathy although not visible by MRI.

Response: We apologize for the oversight. Indeed, the MRIs reviewed by a neuroradiologist indicate that there are optic atrophies in our patients. Fortunately, we were able to review all the MRS by the same neuroradiologist where we found small lactate peak in all the 5 patients examined.

2-Nystagmus is a common symptom in family 2, but also reported in F1-II-1 and diverse previous cases. Could the authors comment on this in the discussion?

Response: We made a change in the discussion related to the raised issue.

3-Based on the reported consanguinity, it is important to exclude a second genetic defect that can explain some unexpected features in some subjects (for instance dysmorphisms or arteriovenous malformation in F1-II-2). Did WES analysis reveal any additional candidate pathogenic variant?

Response: After an iterative filtering and using in-house data sets, we were indeed confident of the WES analysis that did not reveal any other plausible candidate. This was also confirmed with the diagnostic reports provided by independent diagnostic labs. The report from Centogene (a highly reliable private molecular laboratory) revealed another candidate gene, OMA1. However, the variant in OMA1 was not segregated in the family members (Family 1); therefore, excluded from the further analysis.

4-It would be interesting to assess the presence of a mitochondrial defects of the respiratory chain complexes (in particular complex I), since fibroblasts from F1-II-2 and muscle from F1-II-4 were available and have been analysed.

Response: Indeed, we concur with the respected reviewer. We performed very initial experiments such as WB on the cultured fibroblasts. Then we froze the cells for further experimental procedures. However, the cells had never been grown again in the culture (in our lab as well as in UK at Dr. Rob Taylor’s lab). Hence, unfortunately, we had no cells left to carry any further experiments. As for the muscle biopsy, we now provide histopathology results. However, since the biopsy was taken more than 10 years ago, we couldn’t get remaining muscle tissue to perform additional experiments. Our hospital does not maintain older samples in the archive. Our efforts to perform additional biopsies (skin and muscle) were also refused by the family.

5-the Conclusions are missing, and the final sentence (lines 405-406) has to be removed.

Response: We apologize for the oversights. We add a conclusion and remove the lines as requested.

Table/Figures

-There are a lot of mistakes/inaccuracies in the main table.

Response: We apologize for the oversights. These are corrected now in the revised table. Repetitions, unnecessary and irrelevant information are removed. 

Age at presentation is missing in most of the cases. 

Response: We updated the information as requested.

Cerebral atrophy is reported as “congenital anomalies” in patients 4 and 6 but then there is “No” in the specific row for cerebral atrophy in these two subjects. 

Response: After consulting with a neuroradiologist we removed the line related to this and revised the manuscript accordingly. Please see new section related to MRI findings in the main text.

Patient 4 has nystagmus beside optic atrophy as eye examination. Why for family 1 there is Yes for “cerebral encephalopathy” while for family 2 “mitochondrial encephalopathy” is indicated?

Response: Again, we apologize for the oversight. To avoid confusion, we have selected “optic atrophy” in the table under the ophthalmological findings.

-Figure 1. I suggest to clearly show the “shared ROH block” and indicate the subjects corresponding to each line.

Response: The figure 1 is revised, accordingly.

Typos, rephrasing

l.193 sever =>severe

l.396 the variant is functionally an important residue => the variant affects a functionally important residue

Response: Typos are corrected.

Final comment

I don't think that this manucript is appropriate for this special issue

"The purpose of this Special Issue is to overview the current status of the field and highlight the new findings about the role of impaired Ca²⁺ homeostasis in neurodegenerative diseases"

Author Response File: Author Response.pdf

Reviewer 2 Report

 In this manuscript the authors describe the identification, by WES, of a new a missense variant (RTN4IP1: NM_032730.5; c.475G>T, p.Val159Phe) in two unrelated consanguineous families from the northern region of Saudi Arabia. The affected patients presented with intellectual disability, encephalopathy, ataxia, optic atrophy and seizures.

In silico, structural modeling and immunoblot analyses classified this variant to be pathogenic. Genome-wide SNP genotyping and autozygosity mapping predicted that the variant is likely to be a founder and to have appeared approximately 1400 years ago.

This is an interesting work, which confirm the implication of RTN4IP1 variants in syndromic and non syndromic optic atrophy and present a newly identified founder RTN4IP1 variant. While I agree with the approach and study is of interest, I have few suggestions for authors to improve the paper.

-          In the title, “the consanguineous population” word is global and two families do not represent the entire Saudi population. So, we suggest to replace “the consanguineous population” by “two consanguineous Saudi Arabia families” 

-          The two families sharing similar ROH interval, so we can’t conclude that these families are unrelated and have no known close familial relationship.

-          Fibroblasts patient are available, why the authors don’t study the respiratory mitochondrial chain activity?

-          Why the authors don’t present the ACMG classification of the identified variant in the result section.

-          Affiliation 5 is absent in the list of authors

-          Line 36, the reference 3 concern this idea also “however, the oxygen consumption levels in these patient cells were normal”?

-          Line 81 “Also” there is a supplementary space

-          Replace “Mutation” by “variant” or “pathogenic variant”

Author Response

Prof. Dr. Ghulam Ullah

Section Editor-in-Chief

Genes

Dear Prof. Ghunam Ullah,

We appreciate the time and effort that you, the editorial team, and the reviewers have devoted to our paper and thank for the valuable comments, criticism, and feedback on our manuscript. We incorporated all the requested changes and suggestionsinto the revised version. We have highlighted the changes within the manuscript by using track changes option in Word.

Here is a point-by-point response (in bold) to the reviewers’ comments and concerns.

Yours Sincerely,

Namik Kaya

Comments and Suggestions for Authors

 In this manuscript the authors describe the identification, by WES, of a new a missense variant (RTN4IP1: NM_032730.5; c.475G>T, p.Val159Phe) in two unrelated consanguineous families from the northern region of Saudi Arabia. The affected patients presented with intellectual disability, encephalopathy, ataxia, optic atrophy and seizures.

In silico, structural modeling and immunoblot analyses classified this variant to be pathogenic. Genome-wide SNP genotyping and autozygosity mapping predicted that the variant is likely to be a founder and to have appeared approximately 1400 years ago. 

This is an interesting work, which confirm the implication of RTN4IP1 variants in syndromic and non syndromic optic atrophy and present a newly identified founder RTN4IP1 variant. While I agree with the approach and study is of interest, I have few suggestions for authors to improve the paper.

We thank the reviewer for these encouraging remarks. Certainly, our paper is improved significantly by the comments. We apologize for the oversights and made changes as requested.

-          In the title, “the consanguineous population” word is global and two families do not represent the entire Saudi population. So, we suggest to replace “the consanguineous population” by “two consanguineous Saudi Arabia families”  

Response: The title was changed as suggested.

-          The two families sharing similar ROH interval, so we can’t conclude that these families are unrelated and have no known close familial relationship. 

Response: Saudi Arabia has many tribes some of which consist of millions of members who live across the gulf region and beyond, such as northern Africa. Based on our interview with both families we couldn’t detect any “close familial relationship” but indeed as pointed, they belong to a single tribe. So, they have tribal relationship.

-          Fibroblasts patient are available, why the authors don’t study the respiratory mitochondrial chain activity?

Response: Indeed, the fibroblasts were available as the respected reviewer mentioned it. We performed very initial experiments such as WB on the fibroblasts. Then we froze the cells for further experiments as well as transferring abroad for examination of respiratory chain complexes. However, the cells had never been grown again in the culture. We and Prof. Rob’s group tried to regrow the cells and was not successful at all. Therefore, we had no additional cells to carry any further experiments. Our efforts to perform additional biopsies (skin and muscle) were refused by the family.

-          Why the authors don’t present the ACMG classification of the identified variant in the result section.

Response: We apologize for the oversights. Now, we provide it as requested.

-          Affiliation 5 is absent in the list of authors

Response: We apologize for the oversights. The affiliation is added and revised accordingly.

-          Line 36, the reference 3 concern this idea also “however, the oxygen consumption levels in these patient cells were normal”?

Response: We apologize for the typo. The reference is inserted into the end of the sentence.

-          Line 81 “Also” there is a supplementary space

Response: We apologize for the typo. It is corrected now.

-          Replace “Mutation” by “variant” or “pathogenic variant”

Response: Done as requested.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

In the revised version, the authors replied to most of my concerns.

Functional studies are still missing, reducing the value of the manuscript.

Despite being an interesting report, I think this manuscript does not fit the selected Collection: Ca2+ Signaling and Mitochondrial Function in Neurodegenerative Diseases