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Secondary constriction region (qh) variations in individuals from Jilin Province with
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reproductive failure
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Ru-Lin Dai, M.D., Yun-Qing Yang, M.D., Rong-Rong Han, M.D., Xin Yun, M.D., Yuan Dong,
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M.D., and Rui-Zhi Liu, Ph.D.*
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Center for Reproductive Medicine, First Hospital, Jilin University, Changchun, China
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*Corresponding
address: Rui-Zhi Liu, Center for Reproductive Medicine, First Hospital, Jilin
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University, Changchun 130021, China. Tel: +86-13331643399; Fax：+86-431-85654528. Email:
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lrz420@126.com
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Running title : Secondary constriction region (qh) variations
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Abstract
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Objective: To investigate the relationship between secondary constriction region (qh) variations and
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abnormal reproduction in individuals from Jilin Province, China. The study also included a
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comparison of qh variations with populations in the United Kingdom, U.S.S.R. and India.
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Materials & Methods: Four hundred and twenty-eight individuals with reproductive failure and 200
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control individuals were enrolled. Karyotype analysis using Giemsa (G)-banding was performed.
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Results: There was no statistically significant difference in the frequency of 1qh+ (P = 0.21), 9qh+
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(P = 0.65) and 16qh+ (P = 0.44) between Chinese individuals with reproductive failure and controls
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enrolled in this study. There was also no statistically significant difference in the frequency of 1qh+,
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9qh+ and 16qh+ between normally reproductive Chinese people in this study and the data of people
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from other countries who were included in this study (P > 0.05).
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Conclusion: In contrast to the findings of other researchers for other populations, we conclude that
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there is no relationship between qh variations and reproductive failure.
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Keywords: 1qh+; 9qh+; 16qh+; abnormal reproduction
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Introduction
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Infertility is best defined as the inability to conceive or produce a child after one year of
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unprotected intercourse (1, 2). It is caused by many factors, including tubal damage, reduced sperm
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count, motility and morphology, hormone or chromosomal abnormalities, and others. Recently, there
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has been great interest in the putative relationship between chromosomal abnormalities and infertility
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(3,4). The role of variations in the secondary constriction (qh) regions of chromosomes on
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reproduction is not well studied.
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Variations in the heterochromatic qh regions are classified by comparison with the short (p)
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arm of chromosome 16. The qh may be a significantly enlarged heterochromatic region of the long (q)
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arm which is greater than, or equal to, twice the size of the p arm of chromosome 16 (qh+).
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Conversely, the variant may be a very short or deficient region in the q arm (qh-) (5). In the past,
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most clinicians considered the qh variations to be a chromosomal polymorphism indicative of no
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abnormality, as qh resides in heterochromatin which has no coding potential, and nucleolar
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organizing regions contain the genes coding for rRNA (6). However, recent reports indicate that
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chromosomal polymorphisms are related to infertility and recurrent abortions (6-9). In order to
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clarify whether the qh influences reproduction, we analyzed the incidence of qh variations in patients
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who had experienced an abnormal reproductive outcome.
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Materials and Methods
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Patients
From January 2007 to March 2010, 428 patients with any kind of reproductive failure including
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infertility and complete or missed abortion, and attending the First Affiliated Hospital and Cell
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Biology Department of Norman Bethune College of Jilin University, were enrolled in this study. A
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detailed medical history was taken from each patient. We also investigated patients’ medical
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histories, family backgrounds, reproductive problems, and possible consanguinities. A physical
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examination was conducted in all cases, in order to identify anatomical problems. Two hundred
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individuals with normal reproductive function were enrolled as a control group. The study was
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approved by the Chinese Association of Humanitarianism and Ethics.
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In order to analyze whether there is a statistical difference in qh variants between various
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normal populations, we compared chromosomal data from our control groups with data from the
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United Kingdom, U.S.S.R. and India.
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Chromosomal analysis
Chromosomal analysis of peripheral blood lymphocytes was performed according to the criteria
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established by the International System for Human Cytogenetic Nomenclature (10). In 428 infertile
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patients, chromosomal analysis was performed on peripheral blood lymphocytes by chromosome
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Giemsa (G)-banding. Briefly, peripheral blood lymphocytes with RPMI-1640 media (Gibco,
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Invitrogen, USA), phytohaemagglutinin (Shanghai Yihua Medical Technology, China), and fetal
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bovine serum (Beijing Dingguo Biotechnology, China) were cultured, followed by treatment with
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colcemid after a 72-h incubation period. G-banding of metaphase chromosomes was performed using
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the standard protocols for hypotonic fixation, trypsinization, and Giemsa staining. At least 50
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metaphase spreads were analyzed from each subject.
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Statistical analysis
Statistical analysis was carried out using Statistical Package for the Social Sciences (SPSS)
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11.5 (SPSS, Chicago, IL, USA) software. Student’s t-test was used as appropriate. Statistical
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significance was assessed at a probability (P)-value of P < 0.05.
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Results
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In the group of individuals with poor reproductive outcome, there was one patient with 1qh+
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(0.23%), 5 with 9qh+ (1.17%) and 2 with 16qh+ (0.47%). In the control group there were 2 patients
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with 1qh+ (1.00%), 4 patients with 9qh+ (2.00%) and one patient with 16qh+ (0.50%). There was no
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statistically significant difference in the frequency of 1qh+ (0.23% vs. 1.00%; P = 0.21), 9qh+
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(1.17% vs. 2.00%; P = 0.65) or 16qh+ (0.46 % vs. 0.50%; P = 0.44) between individuals with
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reproductive failure and controls (Figure 1).
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There are similar published data from other countries in people with normal reproductive
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outcome (11-13). While in the Chinese the frequencies of 1qh+, 9qh+, and 16qh+ were 1.00%,
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2.00% and 0.50%, respectively, the frequencies in the United Kingdom (11) were 0.5%, 1.20% and
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0.88% (P = 0.65, 0.51, 0.87), with no statistically significant difference between the two countries.
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For the U.S.S.R. (12), the frequencies of 1qh+ and 9qh+ were 0.9% and 5.7%, respectively, a result
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that is not statistically different from the Chinese (P = 1.00 and 0.15). In India (13) the frequency of
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16qh+ was 4%, which is not statistically different from the Chinese (P = 0.08).
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The different types of qh variants that were studied are shown in Figure 3.
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Clinical analysis
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Out of the 428 patients in the reproductive failure group, 8 had qh variants. They had no history
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of smoking or drinking, had no significant medical problems and were diagnosed by peripheral blood
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lymphocyte analysis to be phenotypically normal. The reproductive outcome of the 8 patients with
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qh variants are described below:
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Case 1: Male, with primary infertility.
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Case 2: Male, with primary infertility whose sperm analysis showed oligoasthenoteratozoospermia
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(sperm count <20 × 106 mL, motility <40%, normal forms <40%).
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Case 3: Male, whose wife had had 3 miscarriages, all at about 50 d gestation.
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Case.4: Female, with one spontaneous abortion in 2006, and another at about 50 d gestation in 2008.
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Case 5: Male. The first fetus had facial dysmorphosis, and the second fetus died at about 30 d
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gestation.
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Case 6: Male. Spontaneous abortion 3 times, with no other abnormality except the abnormal karyotype
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46,XX,9qh+.
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Case 7: Female; her uterus developed abnormally.
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Case 8: Male; infertility and his karyotype is 46, XY, 16qh+.
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Table 1 shows the profiles of all the qh detected in these patients. Among the 428 patients with
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abnormal reproduction, the total number of qh variants was 8, including two aberrant karyotypes:
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47,XXY,9qh+ and 46,XX,inv(9)(p11q12),16qh+.
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Discussion
In this study, karyotype determination was solely based on G-banding. The existence of qh
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variants was easily detected, based on the location of heterochromatin. However, further
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specification of the breakpoints by band formation analysis was not possible. This limitation of
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secondary constriction region analysis based on G-banding may be the same as that found by most
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investigators in China.
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The lengths of the qh regions of chromosomes 1, 9, and 16 vary with the degree of contraction
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of the chromosomes, but these constrictions in the heterochromatin contract less than the achromatic
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portions (14). No relationship could be established between qh+ heteromorphisms and chromosomal
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anomalies or reproductive failures (15-17). These results are inconsistent (18) mainly due to the lack
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of objective C-band evaluation and well paired-controls (19).
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The variability of qh in chromosome 1 was associated with recurrent miscarriage in one study
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(8), but was not confirmed by another (20). In our study, there was only one case with 46,XY,1qh+,
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and its clinical symptom was primary infertility. The frequency of 1qh+ is lower than that of the
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control group. The total number of individuals with qh variants in our study is too small and the
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chance of bias therefore is greater. In our study, 1qh+ is not associated with reproductive failure.
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Some reports observed a high frequency of 9qh+ in parents of chromosomally abnormal
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abortuses (6,7,9) while in one study a significant difference in the 9qh+ regions between couples
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who had miscarriages and controls was found (20). There was one case of Klinefelter’s (47,XXY)
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syndrome in our study, with the karyotype 47,XXY,9qh+. This syndrome is the most frequent
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genetic cause of human infertility, occurring in 3% of infertile men (21). We cannot conclude
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whether the reproductive problem of the individual with this karyotype was caused by 9qh+ or by
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47,XXY.
In our study, the frequency of 9 qh+ was 2% in the control group and there was no significant
difference compared with individuals of abnormal reproductive outcome (1.17%).
Nielsen et al. (22) did not observe any association of 16qh+ with developmental or reproductive
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effects. In our study, there was another aberrant karyotype, and it is 46,XX,inv(9)(p11q12),16qh+.
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Some reports questioned the possibility of an association of inv(9)(p11q12) with fertility (23).
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However, we cannot distinguish whether the abnormal reproductive problem of this patient was due
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to either 16qh+, or inv(9)(p11q12). In our study, the frequency of 16qh+ is 0.47%, and there was no
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significant difference when compared with the control group. Therefore our study supports the
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opinion that 16 qh+ has no relationship with abnormal reproductive outcome.
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Conclusion
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In summary, we think that qh variations do not play a role in reproductive problems.
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However, our report is limited by only using cytogenetic detection methods, without confirmation by
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genetic testing. In some reports from other countries, the researchers concluded that abnormal
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reproduction is related to qh variations (6-9). Thus, there is no agreement regarding the putative
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association. Further research on qh variations is wanted, since pharmacological treatments for
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patients with such reproductive problems are not considered a productive line of research.
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Conflict of interest: None declared.
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References
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Figure legends：
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Figure 1. Comparison of the percentage of polymorphic variants in the study and control groups. ns:
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Not significant.
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Figure 2. Comparison of chromosomal qh variant rates between a Chinese population and other
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countries. ns: Not significant.
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Figure 3. Different types of qh variants
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Table 1. Eight variations in the secondary constriction (qh) region among 428 patients
Case Age
Gender
#
(y)
1
27
M
2
42
M
3
30
M
4
26
F
5
29
M
6
26
M
7
29
F
8
32
M
*F: female; M: male
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Reason for chromosomal
analysis
primary infertility
primary infertility
miscarriage
miscarriages
intrauterine fetal demise
spontaneous abortions
intrauterine fetal demise
primary infertility
Karyotype
46,XY,1qh+
47,XXY,9qh+
46,XY,9qh+
46,XX,9qh+
46,XY,9qh+
46,XX,9qh+
46,XX,inv(9)(p11q12),16qh+
46,XY,16qh+
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Figure 1
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Figure 2
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16qh+
9qh+
1qh+
46,XX,inv(9)(p11q12)(16qh+)
47,XYY(9qh+)
Figure 3
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