Responses to reviewers’ comments
Article title: The identification of a spontaneous 47,XX,+21/46,XY chimeric fetus
with male genitalia
MS ID: 1803744262712374
Authors: Kuei-Fang Lee, Chun-Shuo Hsu, Pao-Lin Kuo, Jin-Liang Chen,
Yuan-Hong Jiang and Ingrid Y Liu
Journal: BMC Medical Genetics
Responses to comments made by reviewer 1
Major compulsory revision:
1. Title modification: Pathenogenesis refers to the growth or division of an
unfertilized ovum. It is therefore confusing to refer to a “fertilized
parthenogenetic chimeric fetus”. The word “parthenogenetic” should be used
carefully because once fertilization has taken place the fetus is no longer
parthenogenic, even though the very first division of the ovum may have been a
pathogenic division. The use of the word should be considered throughout the
manuscript. It has been used in this context in other published reviews, but is not
strictly correct.
Response:
We appreciate reviewer’s comment and suggestion. Indeed, we adapted the term
“parthenogenesis” from some review papers. We agree that pathenogensis should
be strictly referred to the growth or division of and unfertilized ovum, therefore,
we have changed the title as “A spontaneous 47,XX,+21/46,XY chimeric fetus
with male genitalia”. In addition, in the revised version, we use “parthenogenic”
only in referring to the unfertilized ovum, not to this chimeric fetus.
2. The GenePhile-G-plex: This kit detect 16 STRs of which one distinguishes the
sex chromosomes and the remaining 15 are autosomal STRs, of which one is on
chromosome 21 (D21S1437). It does not cover the whole genome at 10cM
resolution. The additional three chromosome 21 STRs have been designed by the
researchers. AMEL amplifies X and Y chromosome sequences (resulting in
different fragment lengths). The kit is produced by a Taiwanese company called
“GenePhile Bioscience Co., Ltd.” And, according to the webpage, not by ABI,
USA. The entire section on “Short-tandem-repeat genotyping” should be revised
for accuracy.
Response:
We appreciate reviewer for pointing out this error and have revised the
“Short-tandem-repeat genotyping” section as following:
Short-tandem-repeat genotyping was performed according to the manual of
Human Sex and Autosomal STR Mapping Set (GenePhile G-Plex Kit, GenePhile
Bioscience Co., Ltd, Taiwan). A total of 16 STR markers were applied: AMEL
amplifies different length of fragments from X and Y chromosome and the
remaining 15 are autosomal markers, of which one is on chromosome 21
(D21S1437). Additional 3 STR markers specific to chromosome 21 were
obtained from AmpFlSTR ® Identifiler ™ PCR Amplification Kit (Applied
Biosystems, Foster City, CA, USA) (D21S11) and Bioscience Co., Ltd
(D21S1436 and D21S1270). GenePhile G-Plex PCR Amplification Kit
(GenePhile Bioscience Co., Ltd, Taiwan) was used for STR genotyping.
Genotypes were scored using Gene Scan® and Genotyper® softwares (Applied
Biosystems, USA) and were verified blindly by three technicians.
Minor essential revisions:
1. Karyotypes: it is stated that 20 metaphases were analyzed for each tissue type, yet
for the skin and kidney only 16 are reported.
Response:
Thank you for pointing out the error. We have revised the sentence as the
following:
Twenty cells from the cortex and placenta and sixteen cells from the skin and
kidney were analyzed. The ratio of (46,XY) (Figure 1C) to (47,XX, +21) (Figure
1D) was 11:9 in the cortex and placenta and 11:5 in the skin and kidney.
2. STR analysis: reference made to “hermaphroditic chimera”-this is the first time
the term hermaphroditic is used. Hermaphrodite is a term usually applied to a
phenotype of ambiguous genitalia.
Response:
We have revised “hermaphroditic chimera” to “chimera”, since in our reported
case the genitalia appeared to be male at its embryonic stage.
3. Discussion: This section starts with the phrase- “True sex-chromosome
chimerism”-a different description would be clearer- e.g. Chimera with two cell
lines discordant for sex, or sex chromosome discordant chimera.
Response:
We have replaced “true sex-chromosome chimerism” with “Sex-chromosome
discordant chimera (46,XX/46,XY)” in the first sentence and later sentences of
the discussion section.
4. Discussion 5th sentence-The case described in reference 6 was histologically
confirmed to be a true hermaphrodite- it is incorrect the say “appear to be”
Response:
We have replaced “appear to be” with “was” in the sentence mentioning the case
reported in reference 6.
5. Discussion 7th sentence- “fifth sex chromosome discordant chimerism case—“and
later in the same paragraph and section.
Response:
We have used “sex-chromosome discordant chimera (chimerism case)”
consistently throughout the whole manuscript.
6. Discussion last sentence- reword for clarity. a. the egg divided parthenogenically
and two cells were fertilized by two individual sperms, or b. the ovum and second
polar body were each fertilized by a different sperm. These two options could be
distinguished with much higher density genotyping across chromosome 21, but
not with only 4 chromosome 21 markers.
Response:
We agree with this comment and have revised our discussion as the following:
According to our STR analysis, this is the putative mechanism that led to the
genotype of the present case. This fetus may have been formed from a
parthenogenically-activated oocyte fertilized with two sperms with opposite sex
chromosomes, one of which harbored an extra chromosome 21 (Figure 3). The
possibility of double fertilization with the second polar body is considered very
rare because there is a lack of evidence of crossing over events, which requires
further analysis with more STR markers.
7. Discussion second paragraph- First sentence- the meaning of “mixed amniotic
cells” is unclear. 6th sentence option 2- clarify as per comments above and also
later in the paragraph and section.
Response:
1) We have replaced “mixed amniotic cells” with “heterogeneous amniotic cells”
to describe that composition of amniotic fluid is mixed cells originated from
various fetal tissues.
2) We have revised the discussion regarding the formation mechanism of the
reported chimera case as described in the response to question 6.
Discretionary revisions:
It would have been interesting to examine the gonads histologically for evidence of
early ovarian and testicular tissue to look for evidence of hermaphroditism, as has
been shown for other XX/XY chimeras.
Small corrections:
1. Legend Figure 1: Refer to (C) not [11]
Response:
We have replaced [11] with (C).
2. Reference 10- Is it a book chapter and can page numbers be given?
Response:
We have added page numbers to reference 10: p179-248
3. Reference 8 – Please check format
Response:
We have corrected the format of reference 8.
Responses to comments given by reviewer 2
Major compulsory revisions:
1.
The paper is poorly written, sometimes lacks stringency and needs rewriting.
Linguistic revision is needed.
Abstract: The abstract id difficult to follow. Especially the parts describing
phenotype, ratios of XX,+21/XY and the conclusion need rewriting. The
secondi line starting with Among them---- could be omitted
Response:
We have revised the whole manuscript sent for English editing. A certificate
for English editing is provided to the online submission system.
2.
Case Report: Please add information about why the 21-year-old mother asked for
amniocentesis? Please clarify acquired mutism in the husband. Was the couple
non-consanguineous?
Response:
We have revised this paragraph to provide more information of this couple:
A phenotypically normal 21-year-old female with an obstetric history of gravida
1 and para 0 (G1P0) requested an amniocentesis at the 18th week of her gestation
because of anxiety. Although prenatal ultrasonic examinations appeared normal,
amniocentesis revealed a sex chromosome discordant chimeric karyotype with
trisomy 21 in the XX lineage (47,XX,+21/46,XY). After receiving genetic
counseling, she and her non-consanguineous husband, a 29-year-old with
acquired mutism caused by suspected viral infection at 7 months old, decided to
terminate the pregnancy via induced vaginal delivery at the 21st week.
3.
Methods:
3.1. Autopsy: Replace the sentence “The lobes of ---systems“with “Autospy was
performed according to standard procedures. Replace “parts of the tissues” with
“tissues from heart, kidney and skin were collected for cell culture and DNA
was isolated directly from brain –for further genetic analysis.”
Response:
We have revised this paragraph as following:
Autopsy was performed according to standard procedures. Tissues from brain,
kidneys, skin, the placenta, and cord blood were collected for karyotyping and
DNA was isolated directly from the brain for further genetic analysis.
3.2. Preparation of genomic DNA: The descriptions are lengthy and should be
replaced by a sentences like; “DNA extraction was performed using standard
procedures.”
Response:
We have revised this paragraph as following:
Karyotying was performed as standard protocol. Genomic DNA was purified
according to the manual of DNA isolation kit produced by Gentra system,USA.
3.3. Short-tandem-repeat-genotyping, please shorten this section.
Response:
We have revised this section to more concisely and precisely state this method:
Short-tandem-repeat genotyping was performed according to the manual of
Human Sex and Autosomal STR Mapping Set (GenePhile G-Plex Kit,
GenePhile Bioscience Co., Ltd, Taiwan). A total of 16 STR markers were
applied: AMEL amplifies different length of fragments from X and Y
chromosome and the remaining 15 are autosomal markers, of which one is on
chromosome 21 (D21S1437). Additional 3 STR markers specific to
chromosome 21 were obtained from AmpFlSTR ® Identifiler ™ PCR
Amplification Kit (Applied Biosystems, Foster City, CA, USA) (D21S11) and
Bioscience Co., Ltd (D21S1436 and D21S1270). GenePhile G-Plex PCR
Amplification Kit (GenePhile Bioscience Co., Ltd, Taiwan) was used for STR
genotyping. Genotypes were scored using Gene Scan® and Genotyper®
softwares (Applied Biosystems, USA) and were verified blindly by three
technicians.
4.
Results:
4.1 Morphological examination of the fetus: Please shorten this section.
Response:
We have revised this paragraph as following:
The morphologies of the fetal head, face, trunk, and extremities appeared to be
grossly normal, as well as internal organs including the heart, lungs,
gastrointestinal, hepatobiliary and genitourinary systems. The external genitalia
and gonads appeared to be normal male, though bilateral cryptorchidism was
observed at this embryonic stage (Figure 1A). Characteristics typical of Down
Syndrome were not obvious except enlarged gap between the large and the
second toes (Figure 1B).
4.2 Karyotypes: It is enough to write that the parents had normal karyotypes.
Thus XX---- for the mother and 46,XY for the father (karyotypes not shown)
could be omitted.
Response:
We have omitted the redundant sentence.
4.3 Please rewrite the part describing different ratios in different tissues since it is
hard to follow. Put placenta and brain together since they have the same ration.
Response:
We have revised this paragraph as following:
Chromosomal analyses of the parents revealed normal karyotypes (karyotypes
not shown). Karyotypes obtained from various cultured fetal tissues indicated the
presence of sex-chromosome discordant trisomy 21 chimerism but with different
ratios of (47,XX,+21) to (46,XY). Twenty cells from the cortex and placenta and
sixteen cells from the skin and kidney were analyzed. The ratio of (46,XY)
(Figure 1C) to (47,XX, +21) (Figure 1D) was 11:9 in the cortex and placenta and
11:5 in the skin and kidney.
4.4 STR analysis: Add information about the fact that only 5/19 markers were
informative (including AMEL) and that they were localized on chromosomes Y, 2,
8 and two markers on chromosome 21. It would have been informative if you had
included a figure with skewed ratios for some markers, with higher maternal
peaks compared to the paternal, indicating double, identical, haploid maternal
contributions,
Response:
We have add the sentence as following:
Only 5 out of the 19 markers were informative (including AMEL), of which 3
were localized on chromosomes Y, 2 (D2S1338), 8 (D8S1179), and 2 on
chromosome 21 (DS21S1270 and D21S1437).
We also mark the ratio of markers on Table 1.
5.
Discussion
5.1 The results strongly suggest the causative mechanism in your patient to be that of
dispermic fertilization of a parthenogenetically activated oocyte, however, polar
body fertilization is unlikely since there are no proofs of crossingover events
distinguishing the two maternal haploid genomes. This should be mentioned in
the discussion. More markers are need if the authors want to prove this.
Otherwise this part B could be removed from the figure.
Response:
We have revised the discussion to exclude the possibility of dispermic
fertilization of the second polar body and removed part B of Figure 3.
5.2 Please discuss the possibility of a post zygotic diploidization and non-disjunction
of chromosome 21 of a triploid as one very possible mechanism.
Response:
We have include this mechanism in the Discussion section.
5.3 Please clarify why the death of a twin could not result in the presence of XX and
XY cells in the amniotic fluid since reference 10 Hsu LY is difficult to find.
Response:
We have clarified this concept by modifying the sentence as following:
The death of a twin could not result in coexistence of XX and XY cells in the
amniotic fluid because cells in dead tissues stop dividing and would not be
detected by karyotyping [10].
5.4 Please explain androgenetic chimera in a better way (isodisomic paternal cell
line).
Response:
We have revised this sentence to clearly explain androgenetic chimera as
following:
An androgenetic chimera (isodisomic paternal cell lines) occurs when one normal
zygote fuses with another zygote formed via one sperm fertilizing with an egg
that is empty of genetic material.
Minor essential revisions
1. The word “abortus” should be replaced by the aborted fetus?
Response:
We have replaced “abortus” with “aborted fetus”.
2. Methods: Ethics statement, replace squares from IRB
Response:
We have modify the sentence in “Ethics statement” as following:
This research was performed abiding by the regulation of the institutional
review board (IRB) case 098-82 of the Tzu-Chi general hospital.
3. References: Perhaps add Wiley et al., Am J Med Genet. 2002 Jan 1;
107(1):64-6.”Dispermic chimerism with two abnormal cell lines, 47, XY+21
and 47, XX, +12” who reported a 47, XY,+21 and 47,XX,+12 karyotype in a
still born male fetus with multiple congenital anomalies
Response:
We appreciate this information and have included this case in Discussion
section.
4. Perhaps add Winberg J et al., Am J Med Genet A. 2010 Sep;
152A(9):2277-86.”Chimerism resulting from pathenogenetic activation and
dispermic fertilization” who reported a similar case but with trisomy 14.
Response:
We appreciate this information and have included this reference in Discussion
section.