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Taiwanese Journal of Obstetrics & Gynecology 52 (2013) 450e453www.tjog-online.com

Research Letter

Application of interphase fluorescence in situ hybridization to unculturedamniocytes for differential diagnosis of pseudomosaicism from truemosaicism in mosaic isochromosome 20q detected at amniocentesis

Chih-Ping Chen a,b,c,d,e,f,*, Schu-Rern Chern b, Peih-Shan Wu g, Jun-Wei Su a,h, Yu-Ting Chen b,Li-Feng Chen a, Chen-Wen Pan a, Wayseen Wang b,i

aDepartment of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, TaiwanbDepartment of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan

cDepartment of Biotechnology, Asia University, Taichung, Taiwand School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan

e Institute of Clinical and Community Health Nursing, National Yang-Ming University, Taipei, TaiwanfDepartment of Obstetrics and Gynecology, School of Medicine, National Yang-Ming University, Taipei, Taiwan

gGene Biodesign Co. Ltd, Taipei, TaiwanhDepartment of Obstetrics and Gynecology, China Medical University Hospital, Taichung, Taiwan

iDepartment of Bioengineering, Tatung University, Taipei, Taiwan

Accepted 13 June 2013

A 31-year-old, gravida 4, para 0, woman underwentamniocentesis at 17 weeks of gestation because of maternalanxiety. In four of 25 separated colonies of culturedamniocytes, an abnormal karyotype of 46,XX,i(20)(q10)was noted, whereas the other 21 colonies had a karyotype of46,XX. The cytogenetic result of cultured amniocytes was46,XX,i(20)(q10)[4]/46,XX[21]. The prenatal ultrasoundfindings were unremarkable. The parental karyotypes werenormal.

The woman requested repeated amniocentesis, which wasperformed at 23 weeks of gestation. The aspired amniotic fluidwas sent to another laboratory for interphase fluorescence insitu hybridization (FISH), array comparative genomic hy-bridization (aCGH), quantitative fluorescent polymerase chainreaction (QF-PCR), and conventional cytogenetic analyses. Aninterphase FISH study of 100 uncultured amniocytes using a20q13.33-specific bacterial artificial chromosome (BAC)probe (RP11-266K16) (red spectrum) and a 20p13-specificBAC probe (RP11-530N10) [green spectrum, fluorescein iso-thiocyanate (FITC)] revealed two red signals and two greensignals in all 100 uncultured amniocytes, indicating no evi-dence of isochromosome 20q in uncultured amniocytes(Fig. 1). Whole-genome aCGH analysis on the DNA extracted

* Corresponding author. Department of Obstetrics and Gynecology, Mackay

Memorial Hospital, 92, Section 2, Chung-Shan North Road, Taipei, Taiwan.

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

1028-4559/$ - see front matter Copyright � 2013, Taiwan Association of Obstetri

http://dx.doi.org/10.1016/j.tjog.2013.06.007

from uncultured amniocytes using NimbleGen ISCA PlusCytogenetic Array (Roche NimbleGen, Madison, WI, USA)showed no genomic imbalance (Fig. 2). Polymorphic DNAmarker analysis by QF-PCR on the DNA extracted from un-cultured amniocytes using informative markers D20S604(20p12.1) and D20S454 (20q13.12) revealed a diallelic bipa-rental pattern with equal fluorescent activity and thus excludeduniparental disomy 20 in the fetus (Fig. 3).

Cytogenetic analysis of cultured amniocytes revealed akaryotype of 46,XX,i(20)(q10)[1]/46,XX[26] (Fig. 4). Among27 colonies of the cultured amniocytes, one colony had akaryotype of 46,XX,i(20)(q10), whereas the remaining 26colonies had a karyotype of 46,XX. An interphase FISH studyof 73 cultured amniocytes using the probes RP11-266K16(20q13.33) (red spectrum) and RP11-530N10 (20p13) (greenspectrum, FITC) showed three red signals and two greensignals in 16 cells (21.9%), whereas the remaining 57 cellsshowed two red signals and two green signals, indicatingmosaicism for isochromosome 20q in cultured amniocytes(Fig. 5).

The parents decided to continue the pregnancy. At38 weeks of gestation, a 2800-g healthy female baby wasdelivered uneventfully. The neonate manifested normal growthand normal psychomotor development during routine follow-up at 10 months of age. Cytogenetic analysis of peripheralblood revealed a karyotype of 46,XX (40 cells). An interphaseFISH analysis of uncultured urinary cells using the probesRP11-266K16 (20q13.33) (red spectrum) and RP11-530N10

cs & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.

Fig. 2. Array comparative genomic hybridization on the DNA extracted from uncultured amniocytes shows no genomic imbalance. (A) Whole-genome view and

(B) chromosomal view.

Fig. 1. Interphase fluorescence in situ hybridization analysis of uncultured

amniocytes using the probes RP11-266K16 (20q13.33) (red spectrum) and

RP11-530N10 (20p13) (green spectrum) shows a normal cell (disomy 20) with

two red signals and two green signals.

Fig. 3. Representative electrophoretograms of quantitative fluorescent poly-

merase chain reaction assays. The markers D20S604 (20p12.1) and D20S454

(20q13.12) show two peaks (144 bp:148 bp; paternal:maternal) and

(170 bp:178 bp; paternal:maternal), respectively, of equal fluorescent activity

from two different parental alleles in uncultured amniocytes.

451C.-P. Chen et al. / Taiwanese Journal of Obstetrics & Gynecology 52 (2013) 450e453

Fig. 4. A karyotype of 46,XX,i(20)(q10).

452 C.-P. Chen et al. / Taiwanese Journal of Obstetrics & Gynecology 52 (2013) 450e453

(20p13) (green spectrum, FITC) showed two red signals andtwo green signals in all 40 of 40 urinary cells examined,indicating no evidence of isochromosome 20q in unculturedurinary epithelial cells.

The present case provides evidence for the usefulness of theapplication of interphase FISH to uncultured amniocytes for arapid differential diagnosis of pseudomosaicism from truemosaicism in mosaic isochromosome 20q detected byamniocentesis. The cytogenetic discrepancy between uncul-tured and cultured amniocytes in mosaic isochromosome 20qas presented in this case is in accordance with our previousobservations that mosaic isochromosome 20q detected in

Fig. 5. Interphase fluorescence in situ hybridization analysis of cultured amniocy

(20p13) (green spectrum) shows (A) a normal cell (disomy 20) with two red sig

with three red signals and one green signal.

cultured amniocytes can be a tissue-limited cell culture artifact[1,2]. However, true mosaicism for isochromosome 20q hasbeen associated with fetal abnormalities [3e6]. Therefore,genetic counseling for mosaic isochromosome 20q detected atamniocentesis should be provided with caution. We suggestthat molecular cytogenetic analyses by interphase FISH andaCGH on uncultured amniocytes are very useful for differen-tial diagnosis of pseudomosaicism from true mosaicism inisochromosome 20q detected by amniocentesis. Our casedemonstrates that mosaic isochromosome 20q detected byconventional cytogenetic analysis at amniocentesis in associ-ation with no fetal abnormalities on ultrasound and no

tes using probes RP11-266K16 (20q13.33) (red spectrum) and RP11-530N10

nals and two green signals, and (B) an abnormal cell (isochromosome 20q)

453C.-P. Chen et al. / Taiwanese Journal of Obstetrics & Gynecology 52 (2013) 450e453

isochromosome 20q in cultured amniocytes is likely to have afavorable outcome.

Acknowledgments

This work was supported by research grants from the Na-tional Science Council (Grant Nos. NSC-99-2628-B-195-001-MY3 and NSC-101-2314-B-195-011-MY3) and Mackay Me-morial Hospital, Taipei, Taiwan (Grant No. MMH-E-102-04).

References

[1] Chen CP, Liou JD, Chiang CH, Su YN, Chern SR, Tsai FJ, et al. Cyto-

genetic discrepancy between uncultured amniocytes and cultured amnio-

cytes in mosaic isochromosome 20q detected at amniocentesis. Taiwan J

Obstet Gynecol 2011;50:245e8.

[2] Chen CP, Chang SD, Chen YT, Su JW, Town DD, Wang W. Mosaic

isochromosome 20q detected at amniocentesis: a likely cell culture arti-

fact. Taiwan J Obstet Gynecol 2012;51:663e5.

[3] Chernos JE, McLeod DR, Cox DM. Prenatal diagnosis of mosaic

isochromosome 20q associated with abnormal phenotype. Am J Hum

Genet 1992;51:A288.

[4] Pfeiffer RA, Ulmer R, Rauch A, Trautmann U, Beinder E,

Rupprecht T, et al. True fetal mosaicism of an isochromosome of the

long arm of a chromosome 20: the dilemma persists. Prenat Diagn

1997;17:1171e5.

[5] Chen CP. Detection of mosaic isochromosome 20q in amniotic fluid in a

pregnancy with fetal arthrogryposis multiplex congenita and normal kar-

yotype in fetal blood and postnatal samples of placenta, skin, and liver.

Prenat Diagn 2003;23:85e7.

[6] Goumy C, Beaufrere AM, Francannet C, Tchirkov A, Laurichesse

Delmas H, Geissler F, et al. Prenatal detection of mosaic isochromosome

20q: a fourth report with abnormal phenotype. Prenat Diagn 2005;25:

653e5.