Uppsala, Sweden, September 12, 2014 To: Tim Sands, Executive

Uppsala, Sweden, September 12, 2014
To: Tim Sands, Executive Editor
BMC-series Journals
Thank you for your mail on September 4 regarding MS:1363857294134363 (“A novel
AP4M1 mutation in autosomal recessive cerebral palsy syndrome and clinical
expansion of AP-4 deficiency”) intended for BMC Medical Genetics. We have now
considered the reviewer’s constructive comments and suggestions in a modified
version of the MS with a point-by-point response below. The manuscript is modified
Reviewer 1
Title:A novel AP4M1 mutation in autosomal recessive cerebral palsy syndrome and
clinical expansion of AP-4 deficiency
Version:2 Date:8 August 2014
Reviewer:Kerstin Kutsche
Reviewer's report:
Is the question posed by the authors well defined?
Are the methods appropriate and well described?
Are the data sound?
4. Does the manuscript adhere to the relevant standards for reporting and data
5. Are the discussion and conclusions well balanced and adequately supported
by the data?
I recommend some changes.
6. Are limitations of the work clearly stated?
7. Do the authors clearly acknowledge any work upon which they are building,
both published and unpublished?
No (see review).
8. Do the title and abstract accurately convey what has been found?
Yes, although I recommend some changes (see review).
9. Is the writing acceptable?
The authors describe a novel homozygous mutation in the AP4M1 gene in two
siblings with cerebral palsy, intellectual disability, spastic paraparesis and
reduced head circumference. They suggest unspecific dysmorphic features and
aggressive behavior as novel clinical findings associated with AP-4 complex
deficiency. However, except aggressive behavior the clinical features are the2ame in
the two affected brothers described by Jameel et al. compared with
previously published cases.
Major Compulsory Revisions (which the author must respond to before a
decision on publication can be reached)
- Results, Whole exome sequencing, 1st paragraph: The homozygous mutation
identified in the AP4M1 gene is described as c.189_190delCC / p.(Y65Ffs*50). A
second homozygous sequence variant, c.194A>C, in AP4M1 has been noted by
the authors. Sequence electropherograms showing the two mutations are
presented in Figure 2A. I noticed that at position c.189 a cytosine is changed by
a thymine (c.189C>T) in the homozygous mutant sequence (bottom).
Nucleotides from c.190 to c.193 in the homozygous mutant sequence (bottom)
are identical with the wild-type sequence (top). The c.189C>T transition is a
synonymous variant that is present in dbSNP with reference number
rs367614875. I therefore conclude that the pathogenic mutation is not correctly
described by the authors. The true frameshift starts at position c.194 (adenine;
middle and bottom panels). I suggest the description c.194_195delAT /
p.(Y65Ffs*50) of the pathogenic mutation, while the synonymous variant
c.189C>T should be considered as SNP rs367614875 in the text. According to
this description, the above mentioned transversion c.194A>C does not exist.
Please double-check the description of the mutations and correct!
Reply: Thanks for these valuable comments and that you observed that position
c.189 is in fact polymorphic (rs367614875) – our mistake! Your correct assignment of
the mutation as c.194_195delAT /p.Y65Ffs*50 is now incorporated throughout the
- Results, mRNA analysis (text and legend of Figure 2B): The AP4M1 mRNA is
reduced by 3-fold and not 2-fold in the patients compared with controls (see
Figure 2B). Please also correct in the discussion.
Reply: Thanks, this is now corrected (Resuls, page 7, lines 206-208) and in legend
to Figure 2C).
- Abstract, discussion and conclusion: The authors describe unspecific
dysmorphic features in their patients with an AP4M1 mutation and suggest that
the absence of specific dysmorphic features adds to the clinical variability in AP-4
deficiency. Characteristic facial features have been described in all patients
reported by Tüysüz et al. 2014, and they are similar similar to those seen in the
two brothers in this manuscript. I suggest referring to Tüysüz et al. 2014 when
describing facial features in the two affected siblings. The combination of facial
dysmorphism together with reduced head circumference, para- or tetraplegia,
severe intellectual disability and some brain imaging features underlies the
common clinical features in patients with AP-4 deficiency and should prompt
testing of the four genes. The only novel clinical finding of Jameel and colleagues
is aggressive behavior. Please correct the text accordingly.
Reply: As suggested we are now more cautious in stating differences between our
two brothers and previous cases with AP4M1 mutations. We now more clearly state
that the aggressive behavior together with relatively late onset, normal body height
and retained upper limb function are the most obvious differences when compared to
previous reports (discussion, page 8, lines 244-247) and that the dysmorphic
features are similar to those described by Tüysüz et al. (discussion, page 8, lines
235-237). This is also stated in the abstract and the conclusion.
- Figure 2A: the sequence elctropherograms are of poor quality. The color of the
peaks cannot be distinguished on an out-print. Please improve significantly!
Reply: Thanks. We now submit a modified figure 2A at higher resolution.
- Figure 2B: Please describe the unit of the X-axis. What does 0.300% for
example mean?
Reply: We have now modified Figure 2B (now Figure 2C) and we did set the relative
and normalized AP4M1 expression level to one in controls. This is explainedin the
figure legend.
Minor Essential Revisions (such as missing labels on figures, or the wrong use of
a term, which the author can be trusted to correct)
Change “mental retardation” to “intellectual disability” throughout the text.3
Reply: Done.
Introduction, 1st paragraph: “….for vesicular assembly [9, 10].” This is not the right
term. Please correct to vesicular trafficking or vesicular transport.
Reply: Done.
In addition, reference number 9 is incorrect and reference number 10 corresponds
to a review published in 2004. Please try to find a more recent review on AP-4
proteins such as Hirst et al. 2013, Traffic.
Reply: Thanks, we have now included the refence to Hirst et al.
- Introduction, end of 1st paragraph: a reference is missing for description of the
clinical features associated with AP-4 complex deficiency.
Reply: Two references (14 and 15) are now added.
- Results, whole exome sequencing, 1st paragraph: Please insert “sequences
were” before “….aligned to the human reference genome (hg19).”
Reply: Done
- Results, whole exome sequencing, 1st paragraph: “The two variants
c.189_190delCC and c.194A>C in AP4M1 is located in the region encoding a
highly conserved longin-like domain (Figure 2A).” Figure 2A should be replaced
by figure 2C in the text (but see my comment below). If you refer to the
longin-like domain of the AP4M1 protein, please briefly explain its function.
Reply: We now briefly describe the function of the longin-domain with a reference
(page 7, lines 198-199 and on page 7, lines 223-226). We agree on modifying the
order of figures but we feel that the best logistic of the paper is to present the
chromatogram as Figure 2A and then to put this into context by showing the relative
positions of all known mutations as figure 2B (previous figure 2C). The previous
Figure 2B is now 2C.
- Discussion, 1st paragraph: “The deletion was associated with a two-fold
reduction in mRNA levels presumably because of incomplete non-sense
mediated mRNA decay [9].” Reference 9 does not seem to be appropriate to
refer to nonsense-mediated mRNA decay. Please find a better one.
Reply: Thanks, we agree. As we show an incomplete nonsense mediated mRNA
decay we have now replaced this reference for the two references 19 and 20.
- Figure 2C: All reported pathogenic mutations in the AP4M1 gene are indicated
in the domain structure of the protein. However, all mutations found in this gene
most likely lead to nonsense-mediated mRNA decay, and if at all, only very small
amounts of aberrant (C-terminal truncated) proteins will be produced in the
patients. I suggest to delete Figure 2C. If you decide to leave the figure, please
describe the mutations according to the current version of the “Nomenclature for
the description of sequence variants” (e.g. p.Arg318* and not p.Arg318Ter). The
amino acid alignment below the domain structure is not explained in the legend
of the figure.
Reply: Thanks. As suggested, we have now modified the description of mutations in
(previous) Figure 2C by using the correct nomenclature for the sequences. We prefer
to keep the figure as it gives the reader a didactic overview and put this mutation in
context. The figure is now included Figure 2B (see the query and reply above).
"Minor issues not for publication" (spelling, typographical errors, grammatical
errors, stylistic suggestions etc)
Reply: Grammatical and spelling mistakes are corrected throughout
- Please check for grammatical errors: lines 49-51, 76-80, 230-231, 257-259
Reply: Corrected
- Please check for spelling errors:
line 137: “bactin”
Reply: Done
line 190: “The two variants…….is located….”
Reply: Done
line 197: “..we quantified the AP4M1 mRNA in cultured fibroblasts from….”
Reply: Done
Table 1: stereotypic laughter and (cm) are correct.
Reply: Done
Level of interest:An article whose findings are important to those with closely
related research interests
Quality of written English:Acceptable
Statistical review:No, the manuscript does not need to be seen by a statistician.
Reviewer 2
Reviewer's report
Title:A novel AP4M1 mutation in autosomal recessive cerebral palsy syndrome
and clinical expansion of AP-4 deficiency
Version:2Date:2 August 2014
Reviewer:Annick Raas-Rothschild
Reviewer's report:
Jameel et al reported on a new family with CP and cognitive deficit due to a
homozygous new mutation in AP4M1;
Major compulsory revision:
Page 6- Line 179: Please write the protein change caused by c.194A>C
Reply: Done, page 6, line 187.
Page 6- ligne 184: Please explain why the second healthy brother was not
included in the segregation analysis ?
Reply: Done, page 6, line 191 (the brother was not available for sampling)
Page 6-line 192:
The authors decided to exclude the transversion; the frameshift mutation is still
very close to the cc deletion and the effect of the c.194A>C change could add to
the severity of the patients symptoms ; how frequent is this change in the
population studied? What is the computerized expected effect ; Please comment;
page 7 ; line 207
Reply: Please see the modified and corrected assignment of the mutation
(c.194_195delAT). In addition, the C/T variants at position c.189 of AP4M1 is a
known polymorphism (rs367614875).
How did the authors ruled out the option of X linked inheritance?
Reply: See below the list of all coding X-chromosome variants identified in both
brothers. All variants result in missense variants without predicted pathogenic effect.
Furthermore, mutations in these genes are not previously associated with the
phenotype observed in our family. This is now described in the results section page
6, lines 180-183 and mentioned in first part of discussion.
Chr Gene
Protein taster
chrX FANCB NM_001018113 c.706G>A p.V236M 0.02
chrX CCNB3 NM_033670
c.819G>A p.M273I 0
chrX RGAG1 NM_020769
c.2503C>T p.P835S 0.53
chrX GPR50 NM_004224
c.377G>A p.R126H 0.59
chrX HAUS7 NM_017518
c.397G>A p.A133T 0
How many homozygous regions were common to the individuals who had exome
or did the authors used SNP array in the non affected and more extended family
to focus on this specific region?
Reply: SNP array was not performed on DNA from this family. Filtered exome seq
data revealed homozygous variants in four autosomal genes shared by the two
brothers (see page 6, lines 183-187 and mentioned in first part of discussion). In
addition to the AP4M1 variant we detected the following three variants:
Protein taster
chr1 TNFRSF14 NM_003820
c.745G>A p.E249K na
chr15 RGMA
NM_001166283 c.415C>A p.P139T na
chr20 NINL
c.58G>A p.D20N 0.74
The clinical discussion should be shortened with more focus on the reason of the
late onset and aggressive behavior; all the patients described until now with AP4
mutations had mental retardation in addition to the other symptoms; it is confusing to
write CP “alone” without mental retardation in the text; please modify this;
Reply: We have now re-written the discussion and we also omitted part of the clinical
discussion according to the suggestion.
Y.o.a ? Please write the words
Reply: Done
Level of interest:An article of limited interest
Quality of written English:Acceptable
Statistical review:No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
'I declare that I have no competing interests'
Niklas Dahl
Corresponding author
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