ORIGINAL ARTICLES: GENETICS

Genetic mechanisms leading to

primary amenorrhea in balanced
X-autosome translocations
Mariana Moyse
s-Oliveira, M.Sc.,a Roberta dos Santos Guilherme, M.Sc.,a,b Anelisa Gollo Dantas, B.S.,a
Renata Ueta, B.S.,a Ana Beatriz Perez, Ph.D.,a Mauro Haidar, Ph.D.,c Rosane Canonaco, M.D.,d
Vera Ayres Meloni, M.D.,a Nadezda Kosyakova, Ph.D.,b Thomas Liehr, Ph.D.,b
Gianna Maria Carvalheira, Ph.D.,a and Maria Isabel Melaragno, Ph.D.a
a
Genetics Division, Department of Morphology and Genetics, and c Departament of Gynecology, Universidade Federal de
Sa~o Paulo; d Genetics Division, Hospital do Servidor Pu ~o Paulo, Sa

blico do Estado de Sa ~ o Paulo, Brazil; and b Institute of
Human Genetics, Jena University Hospital, Friedrich Schiller University, Jena, Germany

Objective: To map the X-chromosome and autosome breakpoints in women with balanced X-autosome translocations and primary
amenorrhea, searching candidate genomic loci for female infertility.
Design: Retrospective and case–control study.
Setting: University-based research laboratory.
Patient(s): Three women with balanced X-autosome translocation and primary amenorrhea.
Intervention(s): Conventional cytogenetic methods, genomic array, array painting, fluorescence in situ hybridization, and quantitative
reverse transcription–polymerase chain reaction.
Main Outcome Measure(s): Karyotype, copy number variation, breakpoint mapping, and gene expression levels.
Result(s): All patients presented with breakpoints in the Xq13q21 region. In two patients, the X-chromosome breakpoint disrupted
coding sequences (KIAA2022 and ZDHHC15 genes). Although both gene disruptions caused absence of transcription in peripheral
blood, there is no evidence that supports the involvement of these genes with ovarian function. The ZDHHC15 gene belongs to a
conserved syntenic region that encompasses the FGF16 gene, which plays a role in female germ line development. The break in the
FGF16 syntenic block may have disrupted the interaction between the FGF16 promoter and its cis-regulatory element. In the third pa-
tient, although both breakpoints are intergenic, a gene that plays a role in the DAX1 pathway (FHL2 gene) flanks distally the autosome
breakpoint. The FHL2 gene may be subject to position effect due to the attachment of an autosome segment in Xq21 region.
Conclusion(s): The etiology of primary amenorrhea in balanced X-autosome translocation patients may underlie more complex mech-
anisms than interruption of specific X-linked candidate genes, such as position effect. The fine mapping of the rearrangement break-
points may be a tool for identifying genetic pathogenic mechanisms for primary amenorrhea.
(Fertil SterilÒ 2015;103:1289–96. Ó2015 by American Society for Reproductive Medicine.) Use your smartphone
Key Words: X-chromosome, reciprocal translocation, female fertility, primary amenorrhea, to scan this QR code
position effect and connect to the
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this article now.*
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P
remature ovarian insufficiency degined as the cessation of ovarian UI/L) before the age of 40 years (1). Pre-
(POI, OMIM [Online Mendelian function associated with elevated go- mature ovarian insufficiency causing
Inheritance in Man] 311360) is nadotropins serum levels (FSH R40 hypergonadotropic hypogonadism oc-
curs in 1% of reproductive-age women,
Received November 25, 2014; revised January 15, 2015; accepted January 21, 2015; published online and the most severe cases exhibit
March 4, 2015. gonadal dysgenesis and primary amen-
M.M.-O. has nothing to disclose. R.d.S.G. has nothing to disclose. A.G.D. has nothing to disclose. R.U.
has nothing to disclose. A.B.P. has nothing to disclose. M.H. has nothing to disclose. R.C. has orrhea (2). The pathogenic mechanisms
nothing to disclose. V.A.M. has nothing to disclose. N.K. has nothing to disclose. T.L. has nothing leading to POI are complex and hetero-
to disclose. G.M.C. has nothing to disclose. M.I.M. has nothing to disclose.
This study was supported by Fundaça ~o de Amparo a  Pesquisa do Estado de Sa ~o Paulo (grant 2011/
geneous, including chromosomal,
51690-1). genetic, autoimmune, metabolic, infec-
Reprint requests: Maria Isabel Melaragno, Ph.D., Department of Morphology and Genetics, Universi- tious, and iatrogenic factors (3, 4).
dade Federal de Sa ~o Paulo, Rua Botucatu 740, CEP 04023-900, Sa ~o Paulo, SP, Brazil (E-mail:
melaragno.morf@epm.br). Despite diagnostic advances, 50% of
cases remain idiopathic (5). Genetic
Fertility and Sterility® Vol. 103, No. 5, May 2015 0015-0282/$36.00
Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc.
causes can be identified in
http://dx.doi.org/10.1016/j.fertnstert.2015.01.030 approximately 20%–25% of cases,

VOL. 103 NO. 5 / MAY 2015 1289

ORIGINAL ARTICLE: GENETICS
with X-chromosome abnormalities being the most frequent
genetic cause of POI, including aneuploidies, deletions,
isochromosomes, and X-autosome translocations (6, 7).
Women with balanced X-autosome translocations are a
rare and clinically heterogeneous group of patients whose
most common phenotype is POI (8, 9). The consequences of
balanced X-autosome translocations in female carriers
depend mostly on the location of the breakpoints and on
the X-chromosome inactivation pattern (10). In these
women the normal X-chromosome is preferentially
inactivated, and most X-linked genes remain functional
only in the derivative chromosomes. Thus, high-resolution
breakpoint mapping in women with balanced X-autosome
translocations has proven to be valuable in searching candi-
date genomic loci for diseases (11–13).
Studies on women with rearrangements involving
X-chromosomes indicate that breakpoints in patients
with POI are usually concentrated in the long arm of the X-
chromosome, in a specific region that spans from Xq13 to
Xq27 called the ‘‘Xq critical region,’’ which is assumed to be
responsible for the maintenance of ovarian function and
normal reproductive lifespan (9, 14). This Xq critical region
for ovarian function is often divided into two portions,
Xq13-Xq21 and Xq23-Xq27, the former being frequently dis-
rupted in X-autosome translocations (9, 15, 16). Despite the
fact that the importance of the Xq critical region in POI is
well known, the molecular pathogenic mechanism that
causes this association is still unclear. The majority of the
genes disrupted at the X-chromosome breakpoints were
discarded as candidates because they apparently are not
expressed in human or mouse ovaries, or their mutation
analysis failed to demonstrate a role in POI (16–18). This fact
points to the hypothesis that the interruption of genes in the
Xq critical region is not the cause of ovarian failure. Several
other hypotheses have been considered, most of them related
to an effect position mechanism altering gene expression in
the X-chromosome breakpoint flanking regions or in the
autosomes involved in the rearrangements (19–21).
We therefore used the array painting method to map the
rearrangement breakpoints in three women with balanced
X-autosome translocations and primary amenorrhea, deter-
mining whether the breakpoints disrupted gene sequences.
For disrupted genes, expression assays were performed to
address the impact of the gene interruption in transcription.
MATERIALS AND METHODS
Subjects
We studied three women with balanced X-autosome translo-
cation and primary amenorrhea, who originally presented to
genetic and endocrinologic public services in S~ao Paulo state,
Brazil. Eight female individuals, aged 7–51 years, were
selected from the healthy population as control individuals
for gene expression investigation.
Ethical Approval
This study was approved by the Institutional Ethics Commit-


tee ‘‘Comit^e de Etica em Pesquisa da Universidade Federal de
1290
S~ao Paulo/Hospital S~ao Paulo’’ (CEP 0394/10) and performed
after written informed consent was obtained from the pa-
tients, controls, or their parents.
Classic Banding Cytogenetic Analysis and Clinical
Report
Chromosome analyses with G and replication banding, as well
as nucleolar organiser regions (NOR) staining, were performed
on 72-hour lymphocyte cultures from the patients and, when
possible, from their parents according to standard procedures,
based on a total of 40 metaphase cells.
Patient 1 was a 26-year-old woman, second child of a
nonconsanguineous and healthy couple. She was born at
term with a birth weight of 2,500 g (3rd–10th centile) and
with an undocumented length and birth head circumference.
Her developmental milestones were delayed. She presented
with severe impairment in all cognitive and adaptive behavior
domains, including total dependence on self-care and
communicative skills, and autistic behavior. At 24 years old
she did not speak or communicate and was unable to read
or write. At the same age the patient had not reached
menarche, and pelvic magnetic resonance imaging showed
reduced uterine size and nonvisualized ovaries. At that age
her serum FSH concentration was 119.35 mUI/L, LH 293 UI/
L, E2 12.1 pg/mL, and P 0.5 ng/mL. Additionally, the clinical
evaluation showed short stature and minor facial dysmorphic
features. Standard blood karyotype revealed a de novo,
apparently balanced translocation between chromosomes X
and 3, as follows: 46,X,t(X;3)(q13;q11)dn (Fig. 1A).
Patient 2 was a 42-year-old woman with normal hall-
mark developmental milestones. At the age of 16 years she
had not reached menarche, and her serum FSH concentration
was 76.0 mUI/L and LH 16.5 UI/L. At 17 years of age, pelvic
ultrasound showed reduced uterine and ovarian size. Two
years later the patient started hormone replacement therapy.
Overall, she had a normal clinical morphologic evaluation.
Standard blood karyotype showed an apparently balanced
translocation between chromosomes X and 9:
46,X,t(X;9)(q13;p11.1) (Fig. 2A).
Patient 3 was a 35-year-old woman with normal hall-
mark developmental milestones. At the age of 33 years she
had not reached menarche, and transvaginal ultrasound
showed reduced uterine dimensions and nonvisualized
ovaries. Her serum FSH concentration was 161.5 mUI/L, LH
51.4 UI/L, E2 13 pg/mL, and P 0.4 ng/mL. At 33 years
of age, hormone replacement therapy was initiated. She pre-
sented with normal clinical morphologic evaluation. Stan-
dard blood karyotype showed an apparently balanced
translocation between chromosomes X and 2: 46,X,t(X;2)
(q21;q12) (Fig. 3A).
Genomic Array
Genomic DNA was extracted from peripheral blood leuko-
cytes using the Gentra Puregene kit (Qiagen-Sciences). The
Affymetrix Genome-Wide Human SNP Nsp/Sty 6.0 array
was used to detect copy number variation, and the data
were analyzed with Genotyping Console 3.0.2 and
VOL. 103 NO. 5 / MAY 2015

FIGURE 1
Breakpoint definition for patient 1. (A) Partial G-banding karyotype and ideogram of the chromosomes involved in the rearrangement. (B, C) Array
painting results showing (B) X-chromosomal breakpoint and (C) chromosome 3 breakpoint in the color change region. (D, E) FISH in metaphase cells
using (D) RP11-34P8 and (E) RP11-1072D12 probes, showing three hybridization signals, including split signals in the derivative chromosomes.
Moys
es-Oliveira. Amenorrhea in X-autosome translocations. Fertil Steril 2015.
Chromosome Analysis Suite software (Affymetrix) based on
GRCh37/hg19. To determine the imbalance pathogenicity,
gene content and overlapping benign copy number variations
regions were considered, according to the Database of
Genomic Variants (http://projects.tcag.ca/variation/).
Microdissection, Amplification of the Derivative
Chromosomes, and Reverse Chromosome Painting
Glass needle–based chromosome microdissection was per-
formed as previously described by Liehr et al. (22). Briefly,
both derivative chromosomes involved in each patient's
translocation were collected from lymphocyte culture–
derived metaphase cells spread on coverslips. Collection was
done with extended glass micro-needles controlled by a
micro-manipulator under an inverted microscope. Approxi-
mately 10 copies of the entire derivative chromosome or the
chromosome arm affected by the translocations were
collected. The DNA collected was amplified by degenerated
oligonucleotide primed polymerase chain reaction (PCR), as
previously described (23). The chromosomal origin of the mi-
crodissected segment was assessed by reverse chromosome
painting (i.e., fluorescence in situ hybridization [FISH] on
normal chromosome spreads), as previously described by
Liehr et al. (22).
VOL. 103 NO. 5 / MAY 2015
Fertility and Sterility®
FISH for Breakpoint Definition in Heterochromatic
Regions
For patient 2, FISH was performed in lymphocyte metaphase
using chromosome 9 pericentromeric probes, according to
Weise et al. (24), to determine the autosome breakpoint local-
ization. Another FISH was performed using commercially
available alphoid probes for chromosomes X and 9 centro-
meric regions to further characterize the rearrangement.
Array-CGH of the Dissected and Amplified DNA
For each array-CGH reaction, the dissected and degener-
ated oligonucleotide primed amplified derivative chromo-
some's DNA from the same patient—der(X) and
der(A)—was labeled: one of them with Cy3 and the other
with Cy5, using the Universal Linkage System Labeling
Kit (Agilent Technologies). Both labeled dissected DNA
were hybridized to the same CGH array SurePrint G3 (Agi-
lent Technologies). The hybridization resulted in the chro-
mosomal material from one side of the translocation
breakpoint being labeled in a different color than the ma-
terial on the other side of the breakpoint. After scanning
with a DNA Microarray scanner (Agilent Technologies),
the differential labeling of the two derivative chromosomes
resulted in high hybridization ratios proximal to the
1291
ORIGINAL ARTICLE: GENETICS

FIGURE 2

Region affected by X-chromosome breakpoints in patients 1 and 2. (A) Partial G-banding karyotype and ideogram of the chromosomes involved in
patient 2's rearrangement. (B) Genome view of the Xq13.3 cytoband and the FGF16 syntenic block, including breakpoints in patients 1 and 2 (BP,
red bars), the microduplication (blue bar) described by Dudding et al. (35), and the genes conserved in the syntenic block in red (ZDHHC15 - FGF16 -
ATRX). The FGF16 gene is located approximately 2 Mb downstream of patient 2's breakpoint.
Moys
es-Oliveira. Amenorrhea in X-autosome translocations. Fertil Steril 2015.

breakpoint and low ratios distal to the breakpoint, or vice
versa (for an example, see Fig. 1B and C).
Two different slide designs were used: SurePrint G3
Custom CGH 8x60k (Agilent Technologies) for the X-chromo-
some breakpoint definition, and SurePrint G3 Unrestricted
1x1M (Agilent Technologies) for the autosome breakpoint
definition. The SurePrint G3 Custom CGH 8x60k array was
designed with 46,928 probes for chromosome X and 13,072
backbone probes, selected from the best-performing oligos
in Agilent's electronic library. The SurePrint G3 Unrestricted
1x1M array followed the standard manufacturer design
from the catalog (AMADID 021529) that covers the entire
genome, excluding repetitive sequences. For the 8x60k
design, each CGH-array reaction defined the X-chromosome
breakpoint from one patient. For the 1x1M design, pairs of
dissected derivative chromosomes from different patients
were mixed in a pool before the labeling step, thus one
CGH-array reaction defined the three autosome breakpoints.
FISH for Breakpoint Mapping Validation
Bacterial artificial chromosome probes spanning the break-
point regions degined by the array-CGH results were selected
through the University of California, Santa Cruz genome
browser. The following probes were applied: RP11-34P8
(chrX:73,914,389–74,105,538), RP11-1072D12 (chr3:95,897,
890–96,067,315), RP11-203K12 (chrX:74,592,669–74,748,
513), RP11-980D8 (chrX:94,336,369–94,495,561), and RP
11-1148D23 (chr2:105,763,767–105,913,091). They were
fluorescein isothiocyanate or tetramethyl rhodamine isothio-
cyanate labeled by nick-translation and hybridized on meta-
phase spread as previously described (25).
Expression Analysis of the Disrupted Genes
Whole-blood RNA was extracted from the patients and con-
trol individuals using the PAXgene Blood RNA MDx Kit (Qia-
gen-Sciences). Ribonucleic acid concentration and purity
were assessed using a NanoDrop ND-1000 Spectrophotometer
(Thermo Fisher Scientific), and integrity was assessed by
agarose gel electrophoresis. The complementary DNA
(cDNA) was synthesized using High Capacity cDNA Reverse
Transcription (Life Technologies) using 770 ng of total RNA.
For quantitative reverse transcription–PCR, Gene Expression
Assays (Life Technologies) with TaqMan probes were per-
formed. The assays Hs00911606_m1 (ZDHHC15) and
Hs02339405_m1 (KIAA2022) were used for genes of
interest, and the assays Hs99999905_m1 (GAPDH) and
Hs99999903_m1 (ACTB) were used as internal controls. All
samples of cDNA were assayed in technical triplicate, in 12-
mL reaction volumes with standard PCR conditions of 96-
Well Fast Thermal Cycling (Life Technologies). The cycle
threshold (CT) was determined during the geometric phase
of the PCR ampligication plots, as recommended by the

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FIGURE 3

Region affected by autosomal breakpoint in patient 3. (A) Partial G-banding karyotype and ideogram of the chromosomes involved in the
rearrangement. (B) Genome view of the region downstream to chromosome 2 breakpoint in patient 3, including segment affected by the
autosomal breakpoint of patient 3 (BP, red bar) and the FHL2 gene, located 121.6 kb downstream of the breakpoint.
Moys
es-Oliveira. Amenorrhea in X-autosome translocations. Fertil Steril 2015.

manufacturer. Acquired data were analyzed using ViiA 7
software v1.2.2 (Life Technologies). A qualitative analysis
was performed to assess presence or absence of expression
in patients and controls. The presence of expression in con-
trols was considered only when the TaqMan assay showed
amplification in all control individuals with a maximum CT
value of 38 and maximum CT deviation of 3.
autosomal breakpoints with a resolution range of 1.8–95.6 kb,
depending on the amount of repetitive sequences in the break-
point region.
The breakpoint definition of patient 1 is described in full
to illustrate the methods used (Fig. 1), whereas results for the
others are summarized in Table 1.

RESULTS
Banding cytogenetic analyses and genomic array indicated
that all patients presented with balanced X-autosome trans-
locations, without cryptic genomic imbalances. Addition-
ally, replication banding studies showed that the normal
X-chromosome was preferentially inactivated in all three
cases.
Array-CGH of the microdissected and amplified DNA from
the derivative chromosomes defined the X-chromosomal and
Patient 1
For patient 1, breakpoint definition by array painting indi-
cated that the X-chromosomal breakpoint in Xq13.3 disrup-
ted the KIAA2022 gene and that the autosomal breakpoint
in 3q11.2 did not affect gene coding sequences (Table 1).
The localization of both breakpoints was validated by FISH,
using RP11-34P8 (Xq13.3) and RP11-1072D12 (3q11.2)
probes. For each probe a split signal on each of the derivative
chromosomes was observed (Fig. 1D and E). The gene expres-
sion assay did not detect any KIAA2022 gene expression in

TABLE 1

X-chromosome and autosome breakpoints definition by array painting.

Resolution of the
Patient Karyotype Breakpoints genomic coordinates breakpoint definition (kb) Gene disruption at the breakpoint
1 46,X,t(X;3)(q13.3;q11.2)dn chrX:73,977,371–74,073,029 95.6 KIAA2022
chr3:95,953,332–96,033,144 79.8 Intergenic breakpoint
2 46,X,t(X;9)(Xq13.3; p11.1) chrX:74,628,683–74,633,728 5.0 ZDHHC15
chr9 - array painting not informative – –
3 46,X,t(X;2)(q21.33;q12.1) chrX:94,376,126–94,380,742 4.6 Intergenic breakpoint
chr2:105,853,844–105,855,644 1.8 Intergenic breakpoint
Moys
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VOL. 103 NO. 5 / MAY 2015 1293

ORIGINAL ARTICLE: GENETICS
patient 1's peripheral blood, whereas normal KIAA2022
expression levels were found in all controls (CT mean 35.1;
CT SD 1.0).
Patient 2
For patient 2, array painting indicated that the X-chromo-
somal breakpoint in Xq13.3 disrupted the ZDHHC15 gene
(Table 1); data were validated by FISH, which showed split
signal in both derivative chromosomes with RP11-203K12
(Xq13.3) probe. No ZDHHC15 gene expression was detected
in patient 2's peripheral blood, whereas all control individuals
presented normal expression levels (CT mean 35.4; CT SD 1.1).
The array painting method was not informative for the auto-
somal breakpoint because that was located in the chromo-
some 9 heterochromatic region. Reverse chromosome
painting after chromosome microdissection showed cross-
hybridization in 9p12 and 9q13–21.1 due to sequences known
to be homologous to each other (26). Thus, the autosomal
breakpoint was further analyzed by FISH, to better charac-
terize the rearrangement. Fluorescence in situ hybridization
with centromeric and pericentromeric probes for chromosome
9 confirmed that the autosomal breakpoint affected the chro-
mosome 9 centromeric region. However, the probe for the
chromosome 9 centromere showed three signals: one in
normal chromosome 9 and one in each derivative chromo-
some, indicating that the autosomal breakpoint affected the
chromosome 9 centromeric region (Supplemental Fig. 1,
available online). Another FISH reaction was performed
with commercial centromeric probes for chromosomes X
and 9; it indicated that an autosomal break appeared in region
9p11.1 to 9q11.1. Thus, the derivative X chromosome con-
tains centromeric alphoid sequences from chromosomes X
and 9 (Supplemental Fig. 1). Overall, patient 2's karyotype
was defined as 46,X,t(X;9)(q13.3;cen).
Patient 3
For patient 3, the array painting indicated that the break-
points in Xq21.33 and 2q12.2 did not disrupt any gene
(Table 1). The localization of both breakpoints was validated
by FISH, using RP11-980D8 (Xq21.33) and RP11-1148D23
(2q12.1) probes. For each probe, a split signal on each of the
derivative chromosomes was observed.
DISCUSSION
To properly interpret the clinical impact of a balanced chro-
mosomal rearrangement, the precise breakpoint definition is
fundamental for determining the occurrence of gene disrup-
tions. For this reason, mapping balanced X-autosome trans-
location breakpoints with a high resolution is essential to
assess the genetic mechanisms leading to primary amenor-
rhea. Despite the recognition of this approach as a powerful
tool in searching pathogenic genomic regions, the investiga-
tion of balanced chromosomal rearrangements involving the
X chromosome is generally performed only by low-resolution
techniques, such as karyotyping. This occurs because the
breakpoint definition of balanced chromosomal aberrations
using cytogenomic and molecular methods (e.g., FISH and
1294
long-range PCR followed by Sanger sequencing) are generally
time-consuming or technically challenging. Furthermore,
because such rearrangements do not result in large gains or
losses of genetic material, they are undetected by chromo-
somal microarray-based genome-wide surveys (27). There-
fore, other methodologies, such as array painting, are more
appropriate for achieving breakpoint mapping with a high-
resolution definition (28).
In this study, three balanced X-autosome translocations
identified in women with primary amenorrhea were charac-
terized using the array painting technique, to better under-
stand the genetic contribution in primary amenorrhea.
Although it is not possible to exclude the involvement of
autosomal genes and other X-linked genomic regions in the
onset of ovarian insufficiency, the fact that all three patients
presented with breakpoints in the Xq13q21 region highlights
the importance of this chromosomal region in primary amen-
orrhea pathogenesis. The high-resolution breakpoint map-
ping revealed that two of our patients had neighboring
X-chromosome breakpoints, approximately 0.6 Mb apart, in
the Xq13.3 chromosome region. Considering the X-chromo-
some breakpoint distribution in patients with balanced
X-autosome translocation, independently of the phenotype,
there is not a higher frequency of breaks in Xq13q21 (29),
which excludes the hypothesis that this region is prone to
breakage.
In patient 1, the rearrangement disrupted the KIAA2022
gene, which is highly expressed in the brain and encodes a G
protein–coupled purinergic receptor related to neurite exten-
sion (30). The literature shows that the KIAA2022 loss of
function, caused by chromosomal rearrangements or point
mutations, results in cognitive impairment and autistic
behavior (11, 31), features compatible to our patient. Thus,
the severe intellectual disability in patient 1 could be
attributed to KIAA2022 gene disruption. However, the
literature does not provide links between KIAA2022 gene
and ovarian function or development. Additionally, the
functions of the breakpoint flanking genes located up to
1 Mb upstream and downstream of X-chromosome and
autosome breaks were assessed (Supplemental Table 1). In
patient 1, for the X-chromosome and autosome flanking
region, the function of the identified genes with respect to
ovarian function has not been reported.
In patient 2, the disrupted gene, ZDHHC15, encodes a
palmitoyl acyltransferase, an enzyme that promotes a post-
translational modigication of proteins by the addition of the
lipid palmitate (32). The literature reported a woman with a
balanced X-autosome translocation and ZDHHC15 disrup-
tion that also caused absence of gene expression in peripheral
blood (33). However, this patient presented with severe intel-
lectual disability, a feature not observed in our patient 2, and
a description of her ovarian function or endocrinologic eval-
uation was not given. Mansouri et al. (33) suggested that the
ZDHHC15 gene is a strong candidate for nonsyndromic se-
vere X-linked intellectual deficiency (#300577, MRX91);
however, recent studies indicated that this hypothesis needs
to be validated (34).
The palmitoylation is critical for protein localization and
function in several cell signaling pathways; however, this
VOL. 103 NO. 5 / MAY 2015

modification does not have a clear involvement in gonadal
development, and the ZDHHC15 gene does not show partic-
ular expression in the ovary. In patient 2, the function of the
X-chromosome flanking genes with respect to ovarian func-
tion has not been reported.
Dudding et al. (35) screened for X-chromosome patho-
genic copy number variations in 50 women with normal kar-
yotype and POI, and microduplications were detected in two
cases, one of these encompassing the ABCB7, UPRT, and
ZDHHC15 genes. Despite these authors' reasoning that these
genes may be dose sensitive, they highlight that ZDHHC15
gene expression is restricted to a few tissues, not including
ovary.
The mechanism by which chromosomal rearrangements
involving the genomic region that encompasses ZDHHC15
causes ovarian dysfunction may be more complex than
dose sensitivity of disrupted or duplicated genes. The
ZDHHC15 gene belongs to a syntenic block highly conserved
through evolution (Fig. 2B). In this conserved syntenic region,
ZDHHC15, FGF16 and ATRX genes are maintained in this
exact gene order and in linkage on chromosomes of different
species, from human to fish genomes (36, 37). This
evolutionary constraint against chromosomal breakage is
thought to be necessary for distant cis-regulatory elements
to remain in the vicinity of the genes they act on (38, 39).
Therefore, conserved blocks of synteny can be used to
search for the likely target genes of position effect causing
disease (38, 39).
Recent studies have suggested that the FGF16 gene may
act as a key regulator in ovary development, especially in
oocyte growth (36, 37). Thus, in patient 2, the break in the
FGF16 syntenic block repositioning part of this genomic
segment to chromosome 9 may have disrupted the
interaction between the FGF16 promoter and its cis-
regulatory element, altering FGF16 gene expression and
causing primary amenorrhea (Fig. 2B). Depending on the
genomic architecture resulting from the microduplication
described by Dudding et al. (35) (e.g., position and
orientation of the extra copy), a position effect affecting
FGF16 gene expression could also explain the POI observed
in their patient (Fig. 2B).
In patient 3, the rearrangement affected the Xq21 chro-
mosome region, and neither her X chromosome nor her auto-
some breakpoints caused gene disruption. The association of
this region with primary amenorrhea is well known because it
is estimated that 80% of the X-chromosome breakpoints in
balanced translocations associated with primary amenorrhea
interrupted the Xq21 band (15). This band is a gene-poor re-
gion of the X chromosome, where large interstitial deletions
were identified in women who did not report reproductive
impairment (15). Thus, the explanation for the association be-
tween the Xq21 band and primary amenorrhea may be more
complex than impairment of X-linked genes involved in
ovarian function. It has been proposed that Xq21 may have
a unique mechanism of expression regulation that occurs spe-
cifically in the developing ovary (20). This occurs because,
during oocyte and ovarian follicle maturation, the Xq21 re-
gion undergoes a global transcription down-regulation
compared with other regions along the X-chromosome and
VOL. 103 NO. 5 / MAY 2015
Fertility and Sterility®
also to autosomal genes (21). Thus, an autosome segment
attached to Xq21 because of a translocation could be sub-
jected to this down-regulation, configuring a position effect
on autosomal genes, specifically in the developing ovary.
The region affected by the chromosome 2 breakpoint in
patient 3 presents a high density of regulatory elements,
including enhancers and insulators. The FHL2 gene is mapped
to 2q12.2, 121.6 kb from patient 3's autosomal breakpoint
(Fig. 3B), and is expressed in ovary granulosa cells (40). This
gene encodes a protein that coactivates the transcription of
factors that are involved in both testicular and ovarian differ-
entiation. In ovarian differentiation, the presence of FHL2
protein and its direct interaction with WT1 are responsible
for the transcriptional up-regulation of DAX1, which is
required for ovarian development (41). In accordance with
the hypothesis proposed by Rizzolio et al. (20), the FHL2
gene could be down-regulated in oocytes during ovary devel-
opment, disturbing gonadal formation and causing primary
amenorrhea. This hypothesis needs to be confirmed by further
analysis.
In conclusion, our study emphasizes the importance of
the precise breakpoints definition in balanced X-autosome
rearrangements, to better understand the genetic contribu-
tion to primary amenorrhea. Most studies of the genetic
mechanisms leading to ovarian insufficiency in balanced
X-autosome translocations tend to focus only on the X-chro-
mosome breakpoint localization, neglecting the impact of the
autosome regions on this disease. In patient 3 the autosome
breakpoint definition was extremely valuable, because a
gene flanking the chromosome 2 breakpoint may be subject
to a position effect, causing an alteration in ovarian develop-
ment. Although disruptions in coding sequences were
observed in two patients, there is no known evidence linking
these genes with ovarian function. Furthermore, the effect
position seems to be the most possible pathogenic mechanism
leading to ovarian dysfunction in patients 2 and 3. Thus, our
results support the hypothesis that the etiology of primary
amenorrhea in balanced X-autosome translocations may un-
derlie more complex mechanisms than interruption of spe-
cific X-linked candidate genes, such as position effect.
REFERENCES
1. Conway GS. Premature ovarian failure. Br Med Bull 2000;56:643–9.
2. Coulam CB, Adamson SC, Annegers JF. Incidence of premature ovarian fail-
ure. Obstet Gynecol 1986;67:604–6.
3. Goswami D, Conway GS. Premature ovarian failure. Hum Reprod Update
2005;11:391–410.
4. Cordts EB, Christofolini DM, Dos Santos AA, Bianco B, Barbosa CP. Genetic
aspects of premature ovarian failure: a literature review. Arch Gynecol Ob-
stet 2011;283:635–43.
5. Persani L, Rossetti R, Cacciatore C. Genes involved in human premature
ovarian failure. J Mol Endocrinol 2010;45:257–79.
6. Sala C, Arrigo G, Torri G, Martinazzi F, Riva P, Larizza L, et al. Eleven X chro-
mosome breakpoints associated with premature ovarian failure (POF) map
to a 15-Mb YAC contig spanning Xq21. Genomics 1997;40:123–31.
7. Sherman SL. Premature ovarian failure in the fragile X syndrome. Am J Med
Genet 2000;97:189–94.
8. Schmidt M, Du Sart D. Functional disomies of the X chromosome influence
the cell selection and hence the X inactivation pattern in females with
balanced X-autosome translocations: a review of 122 cases. Am J Med
Genet 1992;42:161–9.
1295
ORIGINAL ARTICLE: GENETICS
9. Therman E, Laxova R, Susman B. The critical region on the human Xq. Hum
Genet 1990;85:455–61.
10. Kalz-Fuller B, Sleegers E, Schwanitz G, Schubert R. Characterisation, pheno-
typic manifestations and X-inactivation pattern in 14 patients with X-auto-
some translocations. Clin Genet 1999;55:362–6.
11. Cantagrel V, Lossi AM, Boulanger S, Depetris D, Mattei MG, Gecz J, et al.
Disruption of a new X linked gene highly expressed in brain in a family
with two mentally retarded males. J Med Genet 2004;41:736–42.
12. Kalscheuer VM, Tao J, Donnelly A, Hollway G, Schwinger E, Kubart S, et al.
Disruption of the serine/threonine kinase 9 gene causes severe X-linked in-
fantile spasms and mental retardation. Am J Hum Genet 2003;72:1401–11.
13. Najm J, Horn D, Wimplinger I, Golden JA, Chizhikov VV, Sudi J, et al. Muta-
tions of CASK cause an X-linked brain malformation phenotype with micro-
cephaly and hypoplasia of the brainstem and cerebellum. Nat Genet 2008;
40:1065–7.
14. Sarto GE, Therman E, Patau K. X inactivation in man: a woman with t(Xq–;
12qþ). Am J Hum Genet 1973;25:262–70.
15. Rizzolio F, Bione S, Sala C, Goegan M, Gentile M, Gregato G, et al. Chromo-
somal rearrangements in Xq and premature ovarian failure: mapping of 25
new cases and review of the literature. Hum Reprod 2006;21:1477–83.
16. Schlessinger D, Herrera L, Crisponi L, Mumm S, Percesepe A, Pellegrini M,
et al. Genes and translocations involved in POF. Am J Med Genet 2002;
111:328–33.
17. Mumm S, Herrera L, Waeltz PW, Scardovi A, Nagaraja R, Esposito T, et al.
X/autosomal translocations in the Xq critical region associated with prema-
ture ovarian failure fall within and outside genes. Genomics 2001;76:30–6.
18. Portnoi MF, Aboura A, Tachdjian G, Bouchard P, Dewailly D, Bourcigaux N,
et al. Molecular cytogenetic studies of Xq critical regions in premature
ovarian failure patients. Hum Reprod 2006;21:2329–34.
19. Baronchelli S, Villa N, Redaelli S, Lissoni S, Saccheri F, Panzeri E, et al. Inves-
tigating the role of X chromosome breakpoints in premature ovarian failure.
Mol Cytogenet 2012;5:32.
20. Rizzolio F, Pramparo T, Sala C, Zuffardi O, De Santis L, Rabellotti E, et al.
Epigenetic analysis of the critical region I for premature ovarian failure:
demonstration of a highly heterochromatic domain on the long arm of
the mammalian X chromosome. J Med Genet 2009;46:585–92.
21. Rizzolio F, Sala C, Alboresi S, Bione S, Gilli S, Goegan M, et al. Epigenetic
control of the critical region for premature ovarian failure on autosomal
genes translocated to the X chromosome: a hypothesis. Hum Genet 2007;
121:441–50.
22. Liehr T, Heller A, Starke H, Rubtsov N, Trifonov V, Mrasek K, et al. Microdis-
section based high resolution multicolor banding for all 24 human chromo-
somes. Int J Mol Med 2002;9:335–9.
23. Backx L, Van Esch H, Melotte C, Kosyakova N, Starke H, Frijns JP, et al. Array
painting using microdissected chromosomes to map chromosomal break-
points. Cytogenet Genome Res 2007;116:158–66.
24. Weise A, Mrasek K, Fickelscher I, Claussen U, Cheung SW, Cai WW, et al.
Molecular definition of high-resolution multicolor banding probes: first
within the human DNA sequence anchored FISH banding probe set. J Histo-
chem Cytochem 2008;56:487–93.
25. de Carvalho AF, da Silva Bellucco FT, Kulikowski LD, Toralles MB,
Melaragno MI. Partial 5p monosomy or trisomy in 11 patients from a family
with a t(5;15)(p13.3;p12) translocation. Hum Genet 2008;124:387–92.
1296
26. Kosyakova N, Grigorian A, Liehr T, Manvelyan M, Simonyan I, Mkrtchyan H,
et al. Heteromorphic variants of chromosome 9. Mol Cytogenet 2013;6:14.
27. Talkowski ME, Ernst C, Heilbut A, Chiang C, Hanscom C, Lindgren A, et al.
Next-generation sequencing strategies enable routine detection of balanced
chromosome rearrangements for clinical diagnostics and genetic research.
Am J Hum Genet 2011;88:469–81.
28. Gribble SM, Kalaitzopoulos D, Burford DC, Prigmore E, Selzer RR, Ng BL,
et al. Ultra-high resolution array painting facilitates breakpoint sequencing.
J Med Genet 2007;44:51–8.
29. Waters JJ, Campbell PL, Crocker AJ, Campbell CM. Phenotypic effects
of balanced X-autosome translocations in females: a retrospective sur-
vey of 104 cases reported from UK laboratories. Hum Genet 2001;108:
318–27.
30. Magome T, Hattori T, Taniguchi M, Ishikawa T, Miyata S, Yamada K, et al.
XLMR protein related to neurite extension (Xpn/KIAA2022) regulates
cell-cell and cell-matrix adhesion and migration. Neurochem Int 2013;
63:561–9.
31. Van Maldergem L, Hou Q, Kalscheuer VM, Rio M, Doco-Fenzy M, Medeira A,
et al. Loss of function of KIAA2022 causes mild to severe intellectual
disability with an autism spectrum disorder and impairs neurite outgrowth.
Hum Mol Genet 2013;22:3306–14.
32. Young FB, Butland SL, Sanders SS, Sutton LM, Hayden MR. Putting proteins
in their place: palmitoylation in Huntington disease and other neuropsychi-
atric diseases. Prog Neurobiol 2012;97:220–38.
33. Mansouri MR, Marklund L, Gustavsson P, Davey E, Carlsson B, Larsson C,
et al. Loss of ZDHHC15 expression in a woman with a balanced translocation
t(X;15)(q13.3;cen) and severe mental retardation. Eur J Hum Genet 2005;
13:970–7.
34. Piton A, Redin C, Mandel JL. XLID-causing mutations and associated genes
challenged in light of data from large-scale human exome sequencing. Am J
Hum Genet 2013;93:368–83.
35. Dudding TE, Lawrence O, Winship I, Froyen G, Vandewalle J, Scott R, et al.
Array comparative genomic hybridization for the detection of submicro-
scopic copy number variations of the X chromosome in women with prema-
ture ovarian failure. Hum Reprod 2010;25:3159–60. author reply 60–1.
36. Forconi M, Canapa A, Barucca M, Biscotti MA, Capriglione T, Buonocore F,
et al. Characterization of sex determination and sex differentiation genes in
Latimeria. PLoS One 2013;8:e56006.
37. Sun YL, Zeng S, Ye K, Yang C, Li MH, Huang BF, et al. Involvement of FGF9/
16/20 subfamily in female germ cell development of the Nile tilapia, Oreo-
chromis niloticus. Fish Physiol Biochem 2012;38:1427–39.
38. Ahituv N, Prabhakar S, Poulin F, Rubin EM, Couronne O. Mapping cis-
regulatory domains in the human genome using multi-species conservation
of synteny. Hum Mol Genet 2005;14:3057–63.
39. Kikuta H, Laplante M, Navratilova P, Komisarczuk AZ, Engstrom PG, Fredman D,
et al. Genomic regulatory blocks encompass multiple neighboring genes and
maintain conserved synteny in vertebrates. Genome Res 2007;17:545–55.
40. Matulis CK, Mayo KE. The LIM domain protein FHL2 interacts with the NR5A
family of nuclear receptors and CREB to activate the inhibin-alpha subunit
gene in ovarian granulosa cells. Mol Endocrinol 2012;26:1278–90.
41. Du X, Hublitz P, Gunther T, Wilhelm D, Englert C, Schule R. The LIM-only co-
activator FHL2 modulates WT1 transcriptional activity during gonadal differ-
entiation. Biochim Biophys Acta 2002;1577:93–101.
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SUPPLEMENTAL FIGURE 1

Chromosome 9 breakpoint definition by FISH for patient 2 using centromeric and pericentromeric probes for chromosome 9. (A) Scheme of the
regions assessed by the centromeric and pericentromeric probes that were used, and the breakpoint localization indicated by the black line. (B)
FISH signals in the normal chromosome 9, derivative chromosome 9, and derivative chromosome X; regions investigated by the probes are
indicated on top, and intensities of the signals are represented on right. The probe for chromosome 9 centromere (labeled in green) showed
three signals: one in normal chromosome 9 and one in each derivative chromosome, indicating that the autosomal breakpoint affected the
chromosome 9 centromeric region. (C) FISH in metaphase cells using centromeric probes for chromosomes X and 9, labeled in green and red,
respectively, showing that the autosomal breakpoint appeared in the region covered by the chromosome 9 centromeric probe.
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ORIGINAL ARTICLE: GENETICS

SUPPLEMENTAL TABLE 1

Breakpoints flanking genes.

Patient 1 Patient 2 Patient 3
Genes Genomic coordinates (Hg19) Genes Genomic coordinates (Hg19) Genes Genomic coordinates (Hg19) Genes Genomic coordinates (Hg19)
TSIX chrX:73,012,039–73,049,066 MTHFD2P1 chr3:95,373,266–95,402,037 SLC16A2 chrX:73,641,327–73,753,764 LOC100287010 chr2:104,995,307–105,024,790
XIST chrX:73,040,485–73,072,588 MIR8060 chr3:96,078,807–96,078,883 RLIM chrX:73,802,810–73,834,461 LINC01102 chr2:105,050,804–105,129,215
JPX chrX:73,164,158–73,290,217 EPHA6 chr3:96,533,424–96,728,971 KIAA2022 chrX:73,952,690–74,145,287 LINC01103 chr2:105,104,913–105,126,967
FTX chrX:73,247,970–73,513,409 ABCB7 chrX:74,273,006–74,376,175 LINC01114 chr2:105,363,094–105,374,177
MIR421 chrX:73,438,211–73,438,296 UPRT chrX:74,493,893–74,524,732 LINC01158 chr2:105,421,882–105,467,934
MIR374B chrX:73,438,381–73,438,453 ZDHHC15 chrX:74,588,261–74,743,337 POU3F3 chr2:105,471,968–105,473,471
MIR374C chrX:73,438,383–73,438,453 TTC3P1 chrX:74,960,372–74,962,914 LINC01159 chr2:105,485,683–105,489,053
MIR545 chrX:73,506,938–73,507,044 MAGEE2 chrX:75,002,822–75,005,079 LOC102724691 chr2:105,552,698–105,654,954
MIR374A chrX:73,507,120–73,507,192 PBDC1 chrX:75,392,763–75,398,145 MRPS9 chr2:105,654,482–105,716,418
ZCCHC13 chrX:73,524,024–73,524,869 LOC101927492 chr2: 105,713,509–105,719,402
SLC16A2 chrX:73,641,327–73,753,764 GPR45 chr2: 105,858,199–105,859,924
RLIM chrX:73,802,810–73,834,461 TGFBRAP1 chr2:105,880,846–105,946,171
ABCB7 chrX:74,273,006–74,376,175 C2orf49 chr2:105,953,815–105,965,271
UPRT chrX:74,493,893–74,524,732 FHL2 chr2:105,977,282–106,015,575
ZDHHC15 chrX:74,588,261–74,743,337 LOC285000 chr2:106,209,553–106,227,016
TTC3P1 chrX:74,960,372–74,962,914 NCK2 chr2:106,361,519–106,510,730
MAGEE2 chrX:75,002,822–75,005,079 C2orf40 chr2:106,682,112–106,694,609
UXS1 chr2:106,709,758–106,810,795
Note: Refseq genes (Annotation Release 105) located up to 1 Mb upstream and downstream of X-chromosome and autosome breaks were considered as breakpoint flanking genes. In patient 2, the autosome breakpoint affects chromosome 9 centromeric region. In
patient 3, there are no genes flanking the X-chromosome breakpoint.
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