Short Communication
Detection of no isochromosome 20q by interphase FISH on uncultured
amniocytes in a pregnancy with mosaic isochromosome 20q in cultured
amniocytes at amniocentesis
Chih-Ping Chen a,b,c,d,e,f*, Jun-Wei Su g, Schu-Rern Chern b, Yu-Ling Kuo h, Peih-Shan Wu i, Meng-Shan Lee a, Chien-Wen Yang b and Wayseen Wang b,j
a Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan b Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan
c Department of Biotechnology, Asia University, Taichung, Taiwan
d 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, Taiwan f Department of Obstetrics and Gynecology, School of Medicine, National Yang-Ming University, Taipei,
Taiwan
g Department of Obstetrics and Gynecology, China Medical University Hospital, Taichung, Taiwan
h Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical
University, Kaohsiung, Taiwan
i Gene Biodesign Co. Ltd, Taipei, Taiwan
j Department of Bioengineering, Tatung University, Taipei, Taiwan
* Correspondence to: Chih-Ping Chen, MD
Department of Obstetrics and Gynecology, Mackay Memorial Hospital 92, Section 2, Chung-Shan North Road, Taipei, Taiwan
Tel: +886-2-25433535; Fax: +886-2-25433642, +886-2-25232448 E-mail: cpc_mmh@yahoo.com
Abstract
Objective: To present prenatal diagnosis and molecular cytogenetic characterization of mosaic
isochromosome 20q at amniocentesis.
Materials and methods: A 36-year-old woman underwent amniocentesis at 17 weeks of gestation
because of advanced maternal age, and conventional cytogenetic analysis revealed a karyotype of 46,XY,i(20)(q10)[12]/46,XY[7]. Repeated amniocentesis was performed at 20 weeks of gestation. During repeated amniocentesis, array comparative genomic hybridization (aCGH), interphase fluorescence in situ hybridization (FISH) and quantitative fluorescent polymerase chain reaction (QF-PCR) were applied on uncultured amniocytes, and conventional cytogenetic analysis and interphase FISH were applied on cultured amniocytes.
Results: Conventional cytogenetic analysis of cultured amniocytes revealed a karyotype of
46,XY,i(20)(q10)[4]/46,XY[16]. Interphase FISH analysis on 217 uncultured amniocytes detected no isochromosome 20q, aCGH on the DNA extracted from uncultured amniocytes detected no genomic imbalance, and QF-PCR analysis on the DNA extracted from uncultured amniocytes excluded uniparental disomy (UPD) 20. Interphase FISH analysis on 115 cultured untouched amniocytes revealed 13% (15/115 cells) mosaicism for isochromosome 20q.
Conclusions: Mosaic isochromosome 20q detected at amniocentesis can be a cell culture artifact.
Detail ultrasound examination, application of interphase FISH and/or aCGH on uncultured amniocytes for confirmation of true mosaicism, and application of QF-PCR to exclude UPD 20 may be useful under such a circumstance.
Introduction
Mosaic isochromosome 20q identified at amniocentesis has been shown to be a benign condition in most reported cases [1-6]. Robinson et al [3] suggested that the isochromosome 20q arises through a postzygotic error, and its growth persists in vitro in a few specific cell types of amniocytes. We previously reported cytogenetic discrepancy between uncultured amniocytes and cultured amniocytes and suggested that mosaic isochromosome 20q detected at amniocentesis is a cell culture artifact [4-6]. Here, we present an additional case of detection of no isochromosome 20q by interphase fluorescence in situ hybridization (FISH) on uncultured amniocytes in a pregnancy with mosaic isochromosome 20q in cultured amniocytes at amniocentesis.
Materials and Methods
Clinical description
A 36-year-old, primigravid woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Conventional cytogenetic analysis using cultured amniocytes revealed a karyotype of 46,XY,i(20)(q10)[12]/46,XY[7]. Among 19 colonies of cultured amniocytes, 12 colonies had a karyotype of 46,XY,i(20)(q10), whereas the other 7 colonies had a karyotype of 46,XY. Prenatal ultrasound findings were unremarkable. The woman requested repeated amniocentesis at 20 weeks of gestation. During repeated amniocentesis, array comparative genomic hybridization (aCGH) was applied on the DNA extracted from uncultured amniocytes, interphase FISH was applied on uncultured amniocytes and cultured amniocytes, and quantitative fluorescent polymerase chain reaction (QF-PCR) analysis using informative polymorphic DNA markers was performed on the DNAs extracted from uncultured amniocytes and parental bloods. Conventional cytogenetic analysis was performed on cultured amniocytes and parental bloods.
Conventional cytogenetic analysis
Routine cytogenetic analysis by G-banding techniques at the 550 bands of resolution was performed. Amniotic fluid and parental bloods were collected, and the samples were subjected to cell culture according to the standard cytogenetic protocol.
aCGH
Whole-genome aCGH on the DNA extracted from uncultured amniocytes derived from 10 mL of amniotic fluid was performed using NimbleGen ISCA Plus Cytogenetic Array (Roche NimbleGen,
Madison, WI, USA). The NimbleGen ISCA Plus Cytogenetic Array has 630,000 probes and a median resolution of 15-20 kb across the entire genome according to the manufacturer's instruction. The DNA from uncultured amniocytes was extracted first. It was done by following the manufacturer's protocol of QIAamp DNA Mini kit (Qiagen, Inc., Valencia, CA, USA). Then, the 0.5μg of the extracted DNA was labeled in Cy5 dye compared with equivalent amount of normal female gDNA (G1521, Promega, Madison, WI, USA) labeled in Cy3 dye to perform the aCGH experiment. The experiment was performed according to the procedures recommended from Roche NimbleGen ISCA plus Cytogenetic Array's user guide. The data were finally represented by using Nexus 6.1 (BioDiscovery, Hawthorne, CA, USA).
FISH
Interphase FISH analysis was performed on 100 uncultured amniocytes using a 20q13.33-specific bacterial artificial chromosome (BAC) probe (RP11-266K16) (62,706,872-62,959,918) [hg 19] (FITC, green spectrum) according to the standard FISH protocol. Interphase FISH analysis was performed on another 117 uncultured amniocytes using a 20q11.21-specific BAC probe (RP11-702M8) (30,180,454-30,348,569) [hg 19] (FITC, green spectrum) according to the standard FISH protocol. For the RP11-702M8 probe, a control study was performed on 136 interphase amniocytes from normal database from untouched cultured amniocytes. Interphase FISH analysis was performed on 115 cultured untouched amniocytes of this case using a 20q13.3-specific BAC probe of RP11-266K16 (FITC, green spectrum) and a 20p12.2-specific BAC probe of RP11-2E8 (10,613,237-10,817,493) [hg 19] (red spectrum) according to the standard FISH protocol.
QF-PCR
QF-PCR analysis was performed by using genomic DNAs extracted from uncultured amniocytes and parental bloods. Primers specifically flanking polymorphic markers on chromosome 20 such as D20S605 (20p12.1) and D20S1083 (20q13.2) were used to exclude uniparental disomy (UPD) 20.
Results
G-banded chromosome analysis of cultured amniocytes at repeated amniocentesis revealed a karyotype of 46,XY,i(20)(q10)[4]/46,XY[16]. Among 20 colonies of cultured amniocytes, 4 colonies had a karyotype of 46,XY,i(20)(q10) (Fig. 1), whereas the other 16 colonies had a karyotype of 46,XY. Whole-genome aCGH analysis on the DNA extracted from uncultured
amniocytes showed no genomic imbalance. Interphase FISH analysis on 100 uncultured amniocytes using a 20q13.33-specific probe of RP11-266K16 (green spectrum) revealed two green signals in all 100 cells (Fig. 2). Interphase FISH analysis on another 117 uncultured amniocytes using a 20q11.21-specific probe of RP11-702M8 (green spectrum) revealed two green signals in all 117 cells (Fig. 2). In the 136 interphase cultured amniocytes of normal control, 130 cells had two green signals, 4 cells had one green signal, and two cells had three signals. Interphase FISH analysis on 115 cultured amniocytes of this case using a 20q13.3-specific probe of RP11-266K16 (green spectrum) and a 20p12.2-specific probe of RP11-2E8 (red spectrum) showed two green signals and two red signals in 100 cultured untouched amniocytes, and one red signal and three green signals in 15 cultured untouched amniocytes (Fig. 3). QF-PCR analysis of uncultured amniocytes excluded UPD 20 (Fig. 4). The woman was advised to continue the pregnancy.
Discussion
We previously reported prenatal diagnosis of 50% (14/28 colonies) mosaic isochromosome 20q at amniocentesis using cultured amniocytes, and postnatal diagnosis of a karyotype of 46,XX in the blood, placenta, skin and liver in a fetus with arthrogryposis multiplex congenital, amyoplasia and a single umbilical artery [1]. We additionally reported a case with 26.9% (7/26 colonies) mosaicism for isochromosome 20q at amniocentesis using cultured amniocytes, and a karyotype of 46,XY in the cord blood, amniotic membrane, placenta, umbilical cord, liver, lung and skin in a fetus with no phenotypic abnormalities [2]. We later reported cytogenetic discrepancy between uncultured amniocytes and cultured amniocytes in mosaic isochromosome 20q detected at amniocentesis [4]. In that case, there were 23.5% (4/17 colonies) mosaicism for isochromosome 20q at the first amniocentesis and 33.3% (8/24 colonies) mosaicism for isochromosome 20q at repeated amniocentesis using cultured amniocytes. Interphase FISH analysis on 50 uncultured amniocytes detected no isochromosome 20q, and aCGH on the DNA extracted from uncultured amniocytes detected no genomic imbalance, however, interphase FISH analysis on cultured amniocytes detected 28% (7/25 cells) mosaic isochromosome 20q and aCGH on the DNA extracted from cultured amniocytes detected genomic imbalance of chromosome 20. The fetus was normal at birth, and the blood karyotype was normal. We subsequently reported an additional case and suggested that mosaic isochromosome 20q detected at amniocentesis is likely a cell culture artifact [5]. In that case, there were 19.2% (5/26 colonies) mosaicism for isochromosome
20q at the first amniocentesis and a normal karyotype of 46,XY (23/23 colonies) at repeated amniocentesis using cultured amniocytes. Interphase FISH analysis on 50 uncultured amniocytes detected no isochromosome 20q. Postnatal cytogenetic analysis of 50 cord blood lymphocytes and interphase FISH analysis of 100 uncultured urinary cells revealed no isochromosome 20q. The infant was normal in the neonatal period. We recently reported application of interphase FISH analysis on uncultured amniocytes for differential diagnosis of pseudomosaicism from true mosaicism in mosaic isochromosome 20q detected at amniocentesis [6]. The case had 16% (4/25 colonies) mosaicism for isochromosome 20q at the first amniocentesis and 3.7% (1/27 colonies) mosaicism for isochromosome 20q at repeated amniocentesis using cultured amniocytes. Interphase FISH analysis on 100 uncultured amniocytes detected no isochromosome 20q, and aCGH on the DNA extracted from uncultured amniocytes detected no genomic imbalance, but interphase FISH analysis on 73 cultured amniocytes detected 21.9% (16/73 cells) mosaicism for isochromosome 20q. Postnatal cytogenetic analysis of 40 peripheral blood lymphocytes and interphase FISH analysis of 40 uncultured urinary cells revealed no isochromosome 20q. The fetus was normal after birth.
The present case provides additional evidence that mosaic isochromosome 20q detected at amniocentesis can be a cell culture artifact and shows the value of using uncultured amniocytes for differential diagnosis of pseudomosaicism from true mosaicism in case of mosaic isochromosome 20q detected at prenatal diagnosis. Our observations can explain the benign status of mosaic isochromosome 20q detected at amniocentesis in most cases.
Although prenatal diagnosis of isochromosome 20q at amniocentesis has been regarded as a benign condition in most cases, genetic counseling still should be done with caution because at least four reports with mosaic isochromosome 20q detected at amniocentesis have been reported to be associated with structural abnormalities [1,7-9]. Therefore, detail ultrasound examination, application of interphase FISH and/or aCGH on uncultured amniocytes for confirmation of true mosaicism and application of QF-PCR to exclude UPD 20 may be useful under such a circumstance.
Acknowledgements
This work was supported by research grants NSC-101-2314-B-195-011-MY3 and MOST 103-2314-B-195-010 from the Ministry of Science and Technology and MMH-E-103-04 from Mackay Memorial Hospital, Taipei, Taiwan.
References
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Figure Legends
Fig. 1. A karyotype of 46,XY,i(20)(q10).
Fig. 2. (A) Interphase fluorescence in situ hybridization (FISH) analysis on 100 uncultured amniocytes using a 20q13.33-specific probe of RP11-266K16 (FITC, green spectrum) shows two green signals in all cells, indicating no isochromosome 20q in uncultured amniocytes.
. (B) Interphase FISH analysis on another 117 uncultured amniocytes using a 20q11.21-specific probe of RP11-702M8 (FITC, green spectrum) shows two green signals in all cells, indicating no isochromosome 20q in uncultured amniocytes.
Fig. 3. Interphase FISH analysis on 115 cultured untouched amniocytes using a 20q13.3-specific probe of RP11-266K16 (FITC, green spectrum) and a 20p12.2-specific probe of RP11-2E8 (red spectrum) shows (A) two red signals and two green signals in 100 cells, and (B) one red signal and three green signals in 15 cells, indicating presence of mosaic isochromosome 20q in cultured amniocytes. Fig. 4. Representative electrophoretograms of quantitative fluorescent polymerase chain reaction assays of
uncultured amniocytes shows two peaks of equal fluorescent activity from two different parental alleles in uncultured amniocytes, indicating no uniparetnal disomy 20 in uncultured amniocytes.
