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Taiwanese Journal of Obstetrics & Gynecology 55 (2016) 705e711

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Taiwanese Journal of Obstetrics & Gynecology

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Short Communication

Molecular cytogenetic characterization of Xp22.32/pter deletion andXq26.3/qter duplication in a male fetus associated with 46,Y,rec(X)dup(Xq) inv(X)(p22.3q26.3), a hypoplastic left heart, short stature, andmaternal X chromosome pericentric inversion

Chih-Ping Chen a, b, c, d, e, f, *, Chen-Yu Chen a, g, h, Schu-Rern Chern b, Peih-Shan Wu i,Yen-Ni Chen a, Shin-Wen Chen a, Chen-Chi Lee a, Dai-Dyi Town a, Meng-Shan Lee a,Chien-Wen Yang b, Wayseen Wang b, j

a Department of Obstetrics and Gynecology, MacKay Memorial Hospital, Taipei, Taiwanb Department of Medical Research, MacKay Memorial Hospital, Taipei, Taiwanc Department of Biotechnology, Asia University, Taichung, Taiwand School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwane Institute of Clinical and Community Health Nursing, National Yang-Ming University, Taipei, Taiwanf Department of Obstetrics and Gynecology, School of Medicine, National Yang-Ming University, Taipei, Taiwang Department of Medicine, MacKay Medical College, New Taipei City, Taiwanh MacKay Junior College of Medicine, Nursing and Management, Taipei, Taiwani Gene Biodesign Co. Ltd, Taipei, Taiwanj Department of Bioengineering, Tatung University, Taipei, Taiwan

a r t i c l e i n f o

Article history:Accepted 19 May 2016

Keywords:hypotrophic heartshort statureXp22.32/pter deletionXq26.3/qter duplicationYp11.31/pter deletion

* Corresponding author. Department of ObstetricMemorial Hospital, 92, Section 2, Chung-Shan North

E-mail address: cpc_mmh@yahoo.com (C.-P. Chen

http://dx.doi.org/10.1016/j.tjog.2016.05.0091028-4559/Copyright © 2016, Taiwan Association of O(http://creativecommons.org/licenses/by-nc-nd/4.0/).

a b s t r a c t

Objective: We present molecular cytogenetic characterization of an Xp22.32/pter deletion and anXq26.3/qter duplication in a male fetus with congenital malformations and maternal X chromosomepericentric inversion.Materials and Methods: A 22-year-old woman underwent amniocentesis at 17 weeks of gestationbecause of an abnormal maternal serum screening result. Prenatal ultrasound revealed a hypoplastic leftheart and short limbs. Amniocentesis revealed a karyotype of 46,Y,der(X) t(X;?)(p22.31;?). The preg-nancy was subsequently terminated, and a malformed fetus was delivered with short stature and facialdysmorphism. Repeat amniocentesis was performed before termination of the pregnancy. Arraycomparative genomic hybridization was performed on uncultured amniocytes and maternal blood.Conventional cytogenetic analysis was performed on cultured amniocytes, cord blood, and blood fromboth parents. Fluorescence in situ hybridization was performed on cultured amniocytes.Results: The maternal karyotype was 46,X,inv(X)(p22.3q26.3). The fetal karyotype was 46,Y, rec(X)dup(Xq)inv(X)(p22.3q26.3) or 46,Y, rec(X)(qter/q26.3::p22.3/qter). Array comparative genomic hy-bridization on uncultured amniocytes revealed a 4.56-Mb deletion of Xp22.33ep22.32 encompassingSHOX, CSF2RA, and ARSE, and a 19.22-Mb duplication of Xq26.3eq28 encompassing SOX3, FMR1, MECP2,RAB39B, and CLIC2 in the fetus. The mother did not have X chromosome imbalance.Conclusion: Detection of X chromosome aberration in a male fetus should give suspicion of a recombi-nant X chromosome derived from maternal X chromosome pericentric inversion.Copyright © 2016, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. Thisis an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/

4.0/).

s and Gynecology, MacKayRoad, Taipei 10449, Taiwan.).

bstetrics & Gynecology. Published b

Introduction

Xp-Xq rearrangements with duplication/deletion eventsinvolving Xq terminal duplication and Xp terminal deletion have

y Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license

Figure 1. A karyotype of 46,X,inv(X)(p22.3q26.3) in the mother.

C.-P. Chen et al. / Taiwanese Journal of Obstetrics & Gynecology 55 (2016) 705e711706

rarely been reported. The derivative or recombinant X chromosomewith Xq duplication/Xp deletion can be the result of a meioticrecombination event in a parent carrying a pericentric X inversionor the result of direct X inheritance from a phenotypically normalmother carrying the same aberrant derivative X chromosome[1e9].

In case of one of the parents with a chromosome inversion, thereis about 5% recurrence risk for the recombinant chromosome in theoffspring [10,11]. Affected male patients with a recombinant Xchromosome with terminal Xq duplication/Xp deletion may pre-sent: terminal Xq duplication syndrome, such as growth retarda-tion, developmental delay, hypotonia, and genital abnormalities;and terminal Xp deletion syndrome, such as short stature, mentalretardation, ichthyosis, Kallmann syndrome, skeletal dysplasia,chondrodysplasia punctata, and hypogonadism [6].

Here, we present molecular cytogenetic characterization of arecombinant X chromosome with terminal Xq duplication/Xpdeletion in a fetus with congenital malformations and maternal Xchromosome pericentric inversion.

Materials and methods

Clinical description

During the first pregnancy, a 22-year-old, primigravid womanunderwent amniocentesis at 17 weeks of gestation because of anabnormal maternal serum screening result showing a trisomy 18risk of 1/262. Her husband was aged 26 years, and there was nofamily history of congenital malformations. The woman had a bodyheight of 147 cm, and her husband had a body height of 170 cm.Prenatal ultrasound at 20 weeks of gestation revealed a hypoplastic

left heart and short limbs. The biparietal diameter was 5.19 cm(21.36 weeks), abdominal circumference 15.34 cm (20.54 weeks),femur length 2.69 cm (18.2 weeks), tibia length 2.3 cm (18 weeks),humerus length 1.7 cm (15 weeks), and ulna length 2.4 cm(17 weeks). Amniocentesis revealed a karyotype of 46,Y,der(X)t(X;?)(p22.31;?). The pregnancy was terminated at 24 weeks ofgestation, and a malformed male fetus was delivered with a bodyweight of 412 g, a body length of 23.5 cm, short stature, short limbs,and facial dysmorphism of hypertelorism and low-set ears. Repeatamniocentesis was performed before termination of the pregnancy.Array comparative genomic hybridization (aCGH) was performedon the DNA extracted from uncultured amniocytes and maternalblood. Conventional cytogenetic analysis was performed oncultured amniocytes, cord blood, and parental bloods. Metaphasefluorescence in situ hybridization (FISH) was performed on culturedamniocytes. During the second pregnancy, the same mother un-derwent amniocentesis at 23 weeks of gestation, which revealed akaryotype of 46,Y,inv(X)(p22.3q26.3). aCGH was performed on theDNA extracted from cultured amniocytes, and metaphase FISH wasperformed on cultured amniocytes. Preterm birth occurred at29weeks of gestation, and a babywas deliveredwith a bodyweightof 1489 g, a body length of 45 cm, and a head circumference of28.5 cm. The baby was normal when followed up at age 4 monthswith a body weight of 4.6 kg and a body length of 45 cm with nophenotypic abnormalities.

aCGH

Whole-genome aCGH on the DNA extracted from unculturedamniocytes in the first pregnancy was performed using ISCA PlusCytogenetic Array (Roche NimbleGen, Madison, WI, USA). This

Figure 2. A karyotype of 46,Y,rec(X)dup(Xq)inv(X)(p22.3q26.3) or 46,Y,rec(X)(qter/q26.3::p22.3/qter) in the fetus of the first pregnancy.

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array has 630,000 probes and a median resolution of 15e20 kbacross the entire genome according to the manufacturer's in-struction. Whole-genome aCGH on the DNAs extracted frommaternal blood and cultured amniocytes in the second pregnancywas performed using CytoChip ISCA Array (Illumina, San Diego, CA,USA), which has 60,000 probes and a median resolution of 51 kbacross the entire genome according to the manufacturer'sinstruction.

Conventional cytogenetic analysis

Routine cytogenetic analysis by G-banding techniques at the550 bands of resolution was performed. Amniotic fluid, cord bloodand parental blood were collected, and the samples were subjectedto cell culture according to the standard blood cytogenetic protocol.

FISH

Metaphase FISH analysis on cultured amniocytes was performedusing the bacterial artificial chromosome probes of RP11-513H18(Xq27.3, 142,900,963e143,081,146; dye: FITC, spectrum green),RP11-155F12 (Xp22.3, 2,456,240e2,612,008; Yp11.32-p11.31, 2,406,240e2,562,008; dye: Texas red, spectrum red), and RP11-22P2(Xq24, 118,252,205e118,409,958; dye: Cy5, spectrum yellow) ac-cording to the standard FISH protocol.

Results

During the first pregnancy, cytogenetic analysis revealed a kar-yotype of 46,X,inv(X)(p22.3q26.3) (Figure 1) in the maternal blood,

a karyotype of 46,XY in the paternal blood and a karyotype of46,Y,rec(X)dup(Xq)inv(X)(p22.3q26.3) or 46,Y,rec(X)(qter/q26.3::p22.3/qter) (Figure 2) in the cultured amniocytes and the cordblood lymphocytes. aCGH on uncultured amniocytes revealed a4.56-Mb deletion of Xp22.33ep22.32 [arr Xp22.33 (1e2,697,663)�0.5, Xp22.33p22.32 (2,697,663e4,557,932)�0.2] encompassingSHOX, CSF2RA and ARSE, and a 19.22-Mb duplication of Xq26.3eq28[arr Xq26.3q28 (136,052,757e155,270,560)�2.0] encompassingSOX3, FMR1, MECP2, RAB39B, and CLIC2 in the fetus (Figure 3).Metaphase FISH analysis on the cultured amniocytes revealed twogreen signals in the terminal ends of the X chromosome andabsence of the red signal in the X chromosome, indicating an Xp/Xqrearrangement with terminal Xp deletion/terminal Xq duplication(Figure 4). During the second pregnancy, cytogenetic analysis of thecultured amniocytes revealed a karyotype of 46,Y,inv(X)(p22.3q26.3) (Figure 5). aCGH analysis on cultured amniocytes andmaternal blood revealed no genomic imbalance in X chromosome.Metaphase FISH analysis on the cultured amniocytes revealed theorder of greenecentromereeyellowered in the X chromosome,indicating a pericentric inversion of X chromosome in the fetus(Figure 6).

Discussion

The present case had a 19.22-Mb duplication of Xq26.3eq28encompassing SOX3, FMR1, MECP2, RAB39B, and CLIC2. MECP2(OMIM 300005) encodes methyl-CpG-binding protein thatcan both activate and repress transcription. MECP2 is a criticaldosage-sensitive gene. Deletion or loss-of-function mutation ofMECP2 is responsible for a progressive neurological disorder of Rett

Figure 3. Array comparative genomic hybridization on uncultured amniocytes of the first pregnancy shows 4.56-Mb deletion of Xp22.33ep22.32 encompassing SHOX, CSF2RA, andARSE, and a 19.22-Mb duplication of Xq26.3eq28 encompassing SOX3, FMR1, MECP2, RAB39B, and CLIC2. (A, B, C) Zoom-in view of X chromosome.

C.-P. Chen et al. / Taiwanese Journal of Obstetrics & Gynecology 55 (2016) 705e711708

Figure 4. Metaphase fluorescence in situ hybridization on the cultured amniocytes ofthe first pregnancy using the bacterial artificial chromosome probes of RP11-513H18(Xq27.3, green) and RP11-155F12 (Xp22.3, red) shows two green signals in the ter-minal ends of the X chromosome and absence of the red signal in the X chromosome,indicating a recombinant X chromosome in the fetus with an Xp/Xq rearrangementresulting in terminal Xp deletion/terminal Xq duplication.

Figure 5. A karyotype of 46,Y,inv(X)(p22.3q26

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syndrome (OMIM 312750). Duplication or gain-of-function muta-tion of MECP2 will cause MECP2 duplication syndrome, which ischaracterized by recurrent respiratory infections, severe mentalretardation, seizures, autism, developmental regression, absent ordelayed speech, infantile hypotonia, and progressive spasticity, andis 100% penetrant in affected male patients [12e14]. Fu et al [15]suggested that ventriculomegaly, hydrocephalus, agenesis of thecorpus callosum, choroid plexus cysts, intrauterine growth re-striction, and hydronephrosis might be the common ultrasoundfeatures in the fetuses with MECP2 duplication syndrome. MECP2duplication syndrome have been reported in rec(X)dup(Xq)inv(X)offspring born to a pericentric inv(X) carrier mother [4,7e9]. Bre-man et al [8] identified two boys withMECP2 duplication syndromewith terminal Xp deletion, terminal Xq duplication and a recom-binant X chromosome containing the duplicated material fromXq28 and Xp, resulting from a maternal pericentric X inversion assimilarly presented in this case.

SOX3 (OMIM 313430) encodes the SRY-BOX3 protein which is atranscription factor that is important in the regulation of nervoussystem development and embryogenesis such as gastrulation,neural induction, specification, and differentiation of many types ofcells [16,17]. The dysfunction of the SOX3 protein disturbs pituitarydevelopment, and over- and underdosage of SOX3 is associatedwith infundibular hypoplasia and hypopituitarism [18]. Stankie-wicz et al [19] reported a duplication of Xq26.2eq27.1 includingSOX3 in a mother and daughter with short stature and dyslalia, andsuggested a dosage effect of SOX3 in speech disorder in addition toshort stature secondary to hypopituitarism. Stagi et al [20] reporteda duplication of SOX3 in a boy with growth hormone deficiency,ocular dyspraxia, and intellectual disability, and suggested that

.3) in the fetus of the second pregnancy.

Figure 6. Metaphase fluorescence in situ hybridization on the cultured amniocytes ofthe second pregnancy using the bacterial artificial chromosome probes of RP11-22P2(Xq24, yellow), RP11-513H18 (Xq27.3, green), and RP11-155F12 (Xp22.3, red) showsthe order of greenecentromereeyellowered in the X chromosome, indicating a peri-centric inversion of X chromosome in the fetus.

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SOX3 duplication should be considered in a male patient with shortstature due to growth hormone deficiency associated with intel-lectual disability. Uguen et al [21] reported a duplication of SOX3 inthree male fetuses with spina bifida and suggested that SOX3duplication is a risk factor of neural tube defect.

FMR1 (OMIM 309550) encodes fragile X mental retardationprotein. A loss of expression of FMR1 results in fragile X mentalretardation syndrome and fragile X tremor/ataxia syndrome.However, a duplication of FMR1 has been reported to be associatedwith a newX-linkedmental retardation syndrome characterized byshort stature, hypogonadism and facial dysmorphism [22,23]. Rioet al [22] reported familial interstitial Xq27.3eq28 duplicationencompassing FMR1 but not MECP2 associated with an X-linkedmental retardation syndrome in affected males. Hickey et al [23]reported a duplication of Xq27.3eq28 including FMR1 in associa-tion with an X-linked hypogonadism, gynecomastia, intellectualdisability, short stature, and obesity syndrome in affected malepatients.

CLIC2 (OMIM 300138) encodes chloride intracellular channel 2protein, which plays a role in the regulation of ryanodine receptor(RyR) intracellular Ca2þ release channels. Gain-of-function muta-tion of CLIC2 enhancing RyR channel activity has been associatedwith an X-linked intellectual disability, atrial fibrillation, car-diomegaly, congenital heart failure, and seizures [24,25]. RAB39B(OMIM 300774) encodes RAS-associated protein RAB39B whichis a small GTPase involved in the regulation of vascular traffickingbetween membrane compartments. Vanmarsenille et al [26] foundcopy number gains of RAB39B in Xq28 in 4 male patientswith mild intellectual disability and behavioral problems andsuggested that increased dosage of RAB39B causes a disturbedneuronal development leading to cognitive impairment. Recently,

an int22h1/int22h2-mediated Xq28 duplication syndrome has beenidentified in males with X-linked intellectual disability, cognitiveimpairment, behavioral and psychiatric problems, recurrent in-fections and atopic diseases, obesity, distinctive facial features, andan Xq28 duplication overlapping the int22h1/int22h2 region andincluding RAB39B and CLIC2 [27e29].

The present case had a 4.56-Mb deletion of Xp22.33ep22.32encompassing SHOX, CSF2RA, and ARSE. SHOX (OMIM 312865) en-codes short stature homeobox protein. Deletions or mutations inSHOX cause LerieWeill dyschondrosteosis (OMIM 127300), Langermesomelic dysplasia (OMIM 249700), and X-linked idiopathic fa-milial short stature (OMIM 300582). CSF2RA (OMIM 306250) en-codes colony-stimulating factor 2 receptor-a (CSF2RA). Mutationsin CSF2RA cause pulmonary alveolar proteinosis due to CSF2RAdeficiency. ARSE (OMIM 300180) encodes arylsulfatase E. Deletionsor mutations in ARSE cause X-linked recessive chondrodysplasiapunctata (OMIM 302950).

In summary, we have presented molecular cytogenetic charac-terization of Xp deletion/Xq duplication in a malformed fetus witha recombinant X chromosome due to maternal X chromosomepericentric inversion, and we discuss the genotypeephenotypecorrelation. We suggest that detection of X chromosome aberrationin a male fetus should give suspicion of a recombinant X chromo-some derived from maternal X chromosome pericentric inversion.

Conflict of interest

The authors have no conflicts of interest relevant to this article.

Acknowledgments

This work was supported by research grants MOST-103-2314-B-195-010 and MOST-104-2314-B-195-009 from the Ministry of Sci-ence and Technology and MMH-E-105-04 from MacKay MemorialHospital, Taipei, Taiwan.

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