Abstract

Four siblings of consanguineous Bedouin kindred presented at infancy with an autosomal recessive syndrome of congenital microcephaly, facial dysmorphism, strabismus, developmental delay and ataxia with positive pyramidal signs. Toward the end of their first decade, they developed areflexia, multiple cranial neuropathies and severe polyneuropathy with progressive muscle weakness, affecting proximal and distal extremities. Physical assessment exhibited kyphoscoliosis, bilateral syndactyly and distal muscle wasting with drop-foot and pes cavus. Magnetic resonance imaging (MRI) showed profound cerebellar atrophy with highly unique findings at the pontine and mesencephalic levels, previously described as fork and bracket signs. Genome-wide linkage analysis identified a single similar to 1.5Mbp disease-associated locus on chromosome 22q13.33. Whole exome sequencing identified a single novel homozygous deleterious splice-site mutation within this locus in SET binding factor 1 (SBF1). SBF1 missense mutations were shown to underlie Charcot-Marie-Tooth (CMT) type 4B3 disease, a rare autosomal recessive subtype of CMT4. The novel SBF1 null mutation highlights distinct severe phenotypic manifestations, broadening the clinical spectrum of SBF1-related neuropathies: cerebellar and pyramidal signs evident in the first months of life with peripheral polyneuropathy emerging only toward the end of the first decade, together with unique MRI findings.


  • pre-publication peer review (FINAL ROUND)
    Decision Letter
    2018/07/13

    Dear Dr. Ohad Birk:

    I am very pleased to inform you that your manuscript entitled ""Novel SBF1 splice-site null mutation broadens the clinical spectrum of Charcot-Marie-Tooth type 4B3 disease"" (CGE-00590-2018), has been received and accepted for publication in Clinical Genetics.

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    Decision letter by
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    Reviewer report
    2018/07/11

    It seems that most comments were properly addressed.

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    Author Response
    2018/07/08

    We thank the editor and the reviewers for their helpful comments. All comments have been addressed

    (see detailed response below). Per editorial request, the manuscript is now shorter (2500 words).

    Thanks and best wishes, Ohad Birk

    Reviewer: 1 Comments to the Author: This is a well-written report. SBF1 recessive mutations are rare and this report helps to confirm the phenotype associated with recessive mutations in this gene. Most helpful is the confirmation that unlike the other MTMR related forms of CMT, this is predominantly a syndromic condition with an axonal as opposed to a demyelinating neuropathy. The evidence for pathogenicity is strong, i.e., mutation in known gene in a mapped region and confirmation that the intronic mutation affects splicing lead to a premature stop codon and likely NMD.

    I have a few minor comments: 1. Appropriate references to the 4 previously reported SBF1 families is required in the introduction. RESPONSE: Done as requested.

    1. Figure 1A: double line for consanguineous parents. RESPONSE: Done as requested.

    2. Fig 2C: please expand the abbreviations within the figure, e.g. what does Pfam SBF2 refer to? RESPONSE: Done – appears in the legend of figure 2.

    Reviewer: 2 Comments to the Author: The authors identified a novel homozygous splice-site mutation in SBF1 in four siblings of consanguineous Bedouin kindred, which may be the underlying cause of severe polyneuropathy with progressive muscle weakness as well as congenital microcephaly, developmental delay, and ataxia. They confirmed that the splice-site mutation was related to 4-bp insertion in cDNA and nonsense-mediated decay. It seems to be reasonable the suggestion that SBF1 mutations may cause a syndromic form of axonal neuropathy with multiple cranial involvements. However, several points are needed to be addressed:

    1. Genome-wide homozygosity mapping identified a ~1.5 Mbp homozygous segment on chromosome 22q13.33 with a LOD score of 2.3 at rs8142256. The LOD score was not high. Is the 22q13.33 the unique candidate region? Are all other regions excluded? It is recommended to provide more specific genome mapping results at the supplementary data. RESPONSE: As now stated clearly in the text, the 22q13.33 was indeed the only homozygous diseaseassociated locus found to segregate within the family as expected for autosomal recessive heredity. The LOD score at the shared locus was calculated using SUPERLINK ONLINE SNP 1.1 (http://cblhap.cs.technion.ac.il/superlink-snp/), assuming an autosomal recessive mode of inheritance with penetrance of 0.99 and disease mutant gene frequency of 0.01. We added this information, regarding LOD score calculations, in the Materials and methods section. We further provide a supplementary figure demonstrating loci with segregating haplotype (none of them is in a homozygous manner, except for the aforementioned 22q13.33 locus). As now clearly stated, in these loci, no compound heterozygous mutations were found to co-segregate within the pedigree (mentioned in an enclosed legend).

    2. The examined patients were from a consanguineous Bedouin family. For the Fig 1A, it is recommended to extend the pedigree to the upper ancestral generation showing the consanguinity. RESPONSE: Both the father and mother are relatives from the same tribe, with the same ancestral founder. However, due to lack of cooperation regarding this specific issue, we could not extend the ancestral pedigree and decipher their exact familial relations. To emphasize this pedigree's consanguinity, we duplicated the line connecting the parents in figure 1A.

    3. To perform the real-time quantitative PCR, what kind of tissue was used to extract total RNA? RESPONSE: Total RNA was isolated from patients' lymphocytes. We now emphasized this point in the Materials and methods section.

    4. Page 2 (line 25): ~1.5Mbs1.5~ > Mbp RESPONSE: Done.

    5. Page 4 (line 25): For QIAGEN’s Ingenuity® Variant Analysis™ software, provide the company information, web address or related reference. RESPONSE: Done.

    6. Page 4 (line 29): For the 1000 genomes project and the Allele Frequency Community, provide web addresses. the 1000 genomes project > the 1000 Genomes Project. RESPONSE: Done.

    7. Page 4 (line 46-53): For Geneaid Biotech, Thermo Scientific, and Roche, provide more information, such as City and Country names. RESPONSE: Done.

    8. Page 5 (line 12): Provide unabbreviated full words for OFC. 29cm > 29 cm RESPONSE: Done.

    9. Page 6 (line 18, 39, 46): 50-53m/s, ~1.5Mbp 50-53 > m/s, ~1.5 Mbp RESPONSE: Done.

    10. Page 6 (line 26): Provide unabbreviated full words for CPK. RESPONSE: Done.

    11. Page 7 (line 18, 22, 42): SBF1 > italic RESPONSE: Done.

    Reviewer: 3 Comments to the Author: The manuscript titled ‘Novel splice-site mutation in SBF1 broadens the clinical spectrum of CharcotMarie-Tooth type 4B3 disease’, introduces four patients in a single family with neurological diseases with a novel SBF1 mutation. The authors carefully examined the various symptoms of neurological diseases and analyzed the clinical characteristics of the patients very well. They also structured the procedures for identifying the genetic cause well, and found novel mutation in SBF1 that is appropriate for the clinical characteristics. This mutation within the splicing site was experimentally proved that could lead to fatal consequences such as amino acid sequence changes and SBF1 transcript reduction. By presenting previous reported cases caused by mutations in the same gene, authors confirmed that this case is similar to the reported cases. Some new features of these patients were also presented. However, I would like to give some recommendations.Minor revision:

    1. In the 'Genome-wide linkage and sequence analysis' section of Materials and Methods, the authors said 'Following the above filtering, of the remaining variants we selected only homozygous variants which were located on chromosome 22q13.33 between the physical positions of SNPs rs9616639 and rs756638'. But it seems that the reason why the authors chose this part was not clearly mentioned in the text. I think it is necessary to mention more clearly whether the SBF1 was originally filtered in the first place, or there were no possible variations among other loci except this gene . RESPONSE: Thanks. We rephrased the text in the 'Genome-wide linkage and sequence analysis' section. We further elaborated on the linkage analysis in the 'genetic analysis' section, and provided a supplementary figure (Fig. S1), demonstrating two other loci with shared haplotype (none of them in a homozygous manner, except for the aforementioned 22q13.33 locus). In these loci, no compound heterozygous mutations were found to segregate within the pedigree as expected for autosomal recessive heredity (mentioned in the text).

    2. In the first sentence of the discussion, the authors wrote 'with distinct CMT4B3 clinical manifestations'. They also mentioned 'unique CMT subtype' in the second sentence of the last paragraph. Of course, the patients presented in this manuscript have some of those CMT symptoms, but strictly speaking, such as congenital microcephaly or facial dysmorphism. It was already mentioned enough in the text by the authors, such as the fifth paragraph of the discussion. Therefore, it would be a good idea to switch the phrase to more appropriate words than 'subtype of CMT'. As there have been reports of similar phenotypes, it would not be too bad to establish a new disease type with the SBF1 mutation. RESPONSE: We rephrased the sentence on 'subtype of CMT' which appears in the last paragraph and added a comment regarding your recommendation – establishing a new disease type with the SBF1 mutation.

    3. All the symptoms of the patients presented by the authors are likely to be due to a single SBF1 mutation. Nevertheless, in the case of certain symptoms, it may not be easy to completely exclude the possibility of other causes. I think it would be good to write a comment about it. RESPONSE: Done as requested (in the fifth paragraph of the discussion).

    4. As the authors noted, the SBF1 mutations were previously reported in four families. If this manuscript is published, then this is the fifth report. There are commonalities with the previously reported cases, but there are other features as well. So I think it is needed to make a more systematic comparison with each of the other cases. Although there are not many cases that have been reported yet, at this point, it may be a good option to summarize the variations and symptoms in each case using a simple table so that the reader can get better understanding. RESPONSE: Done as requested (Table 1).



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  • pre-publication peer review (ROUND 1)
    Decision Letter
    2018/05/04

    Dear Dr. Birk:

    Thank you for submitting your manuscript entitled “Novel splice-site mutation in SBF1 broadens the clinical spectrum of Charcot-Marie-Tooth type 4B3 disease ”. It has now been carefully reviewed.

    The reviewers have suggested Major revisions. For details, please see the commentary below.

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    Prof. Reiner A. Veitia, Ph.D. Acad. Europaea, French National Acad. Med. Editor-In-Chief, Clinical Genetics

    Reviewer: 1

    Comments to the Author This is a well written report. SBF1 recessive mutations are rare and this report helps to confirm the phenotype associated with recessive mutations in this gene. Most helpful is the confirmation that unlike the other MTMR related forms of CMT, this is predominantly a syndromic condition with an axonal as opposed to a demyelinating neuropathy. The evidence for pathogenicity is strong, ie, mutation in known gene in a mapped region and confirmation that the intronic mutation affects splicing lead to a premature stop codon and likely NMD.

    I have a few minor comments:

    Appropriate references to the 4 previously reported SBF1 families is required in the introduction Figure 1A : double line for consanguineous parents Fig 2C please expand the abbreviations within the figure, e.g. what does Pfam SBF2 refer to?

    Reviewer: 2

    Comments to the Author The authors identified a novel homozygous splice-site mutation in SBF1 in four siblings of consanguineous Bedouin kindred, which may underlying cause of evere polyneuropathy with progressive muscle weakness as well as congenital microcephaly, developmental delay, and ataxia. They confirmed that the spling site mutation was related to 4-bp insertion in cDNA and nonsense-mediated decay. It seems to be reasonable the suggestion that SBF1 mutations may cause a syndromic form of axonal neuropathy with multiple cranial involvements. However, several points are needed to be addressed.

    Genome-wide homozygosity mapping identified a ~1.5 Mbp homozygous segment on chromosome 22q13.33 with a LOD score of 2.3 at rs8142256. The LOD score was not high. Is the 22q13.33 the unique canditate region? Are all other regions excluded? It is recommended to provide more specific genome mapping results at the supplementary data.

    The examined patients were from a consanguineous Bedouin family. For the Fig 1A, it is recommended to extend the pedigree to the upper ancestral generation showing the consanguinity.

    To perform the real-time quantitative PCR, what kind of tissue was used to extract total RNA?

    Page 2 (line 25): ~1.5Mbs > ~1.5 Mbp

    Page 4 (line 25): For QIAGEN’s Ingenuity® Variant Analysis™ software, provide the company information, web address or related reference.

    Page 4 (line 29): For the 1000 genomes project and the Allele Frequency Community, provide web addresses. the 1000 genomes project > the 1000 Genomes Project.

    Page 4 (line 46-53): For Geneaid Biotech, Thermo Scientific, and Roche, provide more informations, shuch as City and Country names.

    Page 5 (line 12): Provide unabbreviated full words for OFC. 29cm > 29 cm

    Page 6 (line 18, 39, 46): 50-53m/s, ~1.5Mbp > 50-53 m/s, ~1.5 Mbp

    Page 6 (line 26): Provide unabbreviated full words for CPK.

    Page 7 (line 18, 22, 42): SBF1 > italic

    Reviewer: 3

    Comments to the Author The manuscript titled ‘Novel splice-site mutation in SBF1 broadens the clinical spectrum of Charcot-Marie-Tooth type 4B3 disease’, introduces four patients in a single family with neurological diseases with a novel SBF1 mutation. The authors carefully examined the various symptoms of neurological diseases and analyzed the clinical characteristics of the patients very well. They also structured the procedures for identifying the genetic cause well, and found novel mutation in SBF1 that is appropriate for the clinical characteristics. This mutation within the splicing site was experimentally proved that could lead to fatal consequences such as amino acid sequence changes and SBF1 transcript reduction. By presenting previous reported cases caused by mutations in the same gene, authors confirmed that this case is similar to the reported cases. Some new features of these patients were also presented.

    However, I would like to give some recommendations. Minor revision In the 'Genome-wide linkage and sequence analysis' section of Materials and Methods, the authors said 'Following the above filtering, of the remaining variants we selected only homozygous variants which were located on chromosome 22q13.33 between the physical positions of SNPs rs9616639 and rs756638'. But it seems that the reason why the authors chose this part was not clearly mentioned in the text. I think it is necessary to mention more clearly whether the SBF1 was originally filtered in the first place, or there were no possible variations among other loci except this gene. In the first sentence of the discussion, the authors wrote 'with distinct CMT4B3 clinical manifestations'. They also mentioned 'unique CMT subtype' in the second sentence of the last paragraph. Of course, the patients presented in this manuscript have some of those CMT symptoms, but strictly speaking, many other symptoms are different from those of typical CMT4 patients such as congenital microcephaly or facial dysmorphism. It was already mentioned enough in the text by the authors, such as the fifth paragraph of the discussion. Therefore, it would be a good idea to switch the phrase to more appropriate words than 'subtype of CMT'. As there have been reports of similar phenotypes, it would not be too bad to establish a new disease type with the SBF1 mutation. All the symptoms of the patients presented by the authors are likely to be due to a single SBF1 mutation. Nevertheless, in the case of certain symptoms, it may not be easy to completely exclude the possibility of other causes. I think it would be good to write a comment about it. As the authors noted, the SBF1 mutations were previously reported in four families. If this manuscript is published, then this is the fifth report. There are commonalities with the previously reported cases, but there are other features as well. So I think it is needed to make a more systematic comparison with each of the other cases. Although there are not many cases that have been reported yet, at this point, it may be a good option to summarize the variations and symptoms in each case using a simple table so that the reader can get better understanding.

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    Reviewer report
    2018/05/02

    The manuscript titled ‘Novel splice-site mutation in SBF1 broadens the clinical spectrum of Charcot-Marie-Tooth type 4B3 disease’, introduces four patients in a single family with neurological diseases with a novel SBF1 mutation. The authors carefully examined the various symptoms of neurological diseases and analyzed the clinical characteristics of the patients very well. They also structured the procedures for identifying the genetic cause well, and found novel mutation in SBF1 that is appropriate for the clinical characteristics. This mutation within the splicing site was experimentally proved that could lead to fatal consequences such as amino acid sequence changes and SBF1 transcript reduction. By presenting previous reported cases caused by mutations in the same gene, authors confirmed that this case is similar to the reported cases. Some new features of these patients were also presented.

    However, I would like to give some recommendations. Minor revision In the 'Genome-wide linkage and sequence analysis' section of Materials and Methods, the authors said 'Following the above filtering, of the remaining variants we selected only homozygous variants which were located on chromosome 22q13.33 between the physical positions of SNPs rs9616639 and rs756638'. But it seems that the reason why the authors chose this part was not clearly mentioned in the text. I think it is necessary to mention more clearly whether the SBF1 was originally filtered in the first place, or there were no possible variations among other loci except this gene. In the first sentence of the discussion, the authors wrote 'with distinct CMT4B3 clinical manifestations'. They also mentioned 'unique CMT subtype' in the second sentence of the last paragraph. Of course, the patients presented in this manuscript have some of those CMT symptoms, but strictly speaking, many other symptoms are different from those of typical CMT4 patients such as congenital microcephaly or facial dysmorphism. It was already mentioned enough in the text by the authors, such as the fifth paragraph of the discussion. Therefore, it would be a good idea to switch the phrase to more appropriate words than 'subtype of CMT'. As there have been reports of similar phenotypes, it would not be too bad to establish a new disease type with the SBF1 mutation. All the symptoms of the patients presented by the authors are likely to be due to a single SBF1 mutation. Nevertheless, in the case of certain symptoms, it may not be easy to completely exclude the possibility of other causes. I think it would be good to write a comment about it. As the authors noted, the SBF1 mutations were previously reported in four families. If this manuscript is published, then this is the fifth report. There are commonalities with the previously reported cases, but there are other features as well. So I think it is needed to make a more systematic comparison with each of the other cases. Although there are not many cases that have been reported yet, at this point, it may be a good option to summarize the variations and symptoms in each case using a simple table so that the reader can get better understanding.

    Reviewed by
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    Reviewer report
    2018/04/28

    The authors identified a novel homozygous splice-site mutation in SBF1 in four siblings of consanguineous Bedouin kindred, which may underlying cause of evere polyneuropathy with progressive muscle weakness as well as congenital microcephaly, developmental delay, and ataxia. They confirmed that the spling site mutation was related to 4-bp insertion in cDNA and nonsense-mediated decay. It seems to be reasonable the suggestion that SBF1 mutations may cause a syndromic form of axonal neuropathy with multiple cranial involvements. However, several points are needed to be addressed.

    Genome-wide homozygosity mapping identified a ~1.5 Mbp homozygous segment on chromosome 22q13.33 with a LOD score of 2.3 at rs8142256. The LOD score was not high. Is the 22q13.33 the unique canditate region? Are all other regions excluded? It is recommended to provide more specific genome mapping results at the supplementary data.

    The examined patients were from a consanguineous Bedouin family. For the Fig 1A, it is recommended to extend the pedigree to the upper ancestral generation showing the consanguinity.

    To perform the real-time quantitative PCR, what kind of tissue was used to extract total RNA?

    Page 2 (line 25): ~1.5Mbs > ~1.5 Mbp

    Page 4 (line 25): For QIAGEN’s Ingenuity® Variant Analysis™ software, provide the company information, web address or related reference.

    Page 4 (line 29): For the 1000 genomes project and the Allele Frequency Community, provide web addresses. the 1000 genomes project > the 1000 Genomes Project.

    Page 4 (line 46-53): For Geneaid Biotech, Thermo Scientific, and Roche, provide more informations, shuch as City and Country names.

    Page 5 (line 12): Provide unabbreviated full words for OFC. 29cm > 29 cm

    Page 6 (line 18, 39, 46): 50-53m/s, ~1.5Mbp > 50-53 m/s, ~1.5 Mbp

    Page 6 (line 26): Provide unabbreviated full words for CPK.

    Page 7 (line 18, 22, 42): SBF1 > italic

    Reviewed by
    Cite this review
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    Reviewer report
    2018/04/27

    This is a well written report. SBF1 recessive mutations are rare and this report helps to confirm the phenotype associated with recessive mutations in this gene. Most helpful is the confirmation that unlike the other MTMR related forms of CMT, this is predominantly a syndromic condition with an axonal as opposed to a demyelinating neuropathy. The evidence for pathogenicity is strong, ie, mutation in known gene in a mapped region and confirmation that the intronic mutation affects splicing lead to a premature stop codon and likely NMD.

    I have a few minor comments:

    Appropriate references to the 4 previously reported SBF1 families is required in the introduction Figure 1A : double line for consanguineous parents Fig 2C please expand the abbreviations within the figure, e.g. what does Pfam SBF2 refer to?

    Reviewed by
    Cite this review
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