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Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 248e251

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Taiwanese Journal of Obstetrics & Gynecology

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Case Report Prenatal diagnosis and molecular cytogenetic characterization of 22q11.2 deletion syndrome associated with congenital heart defects

Yu-Ling Kuo a, Chih-Ping Chen b, c, d, e, f, g, *, Liang-Kai Wang b, Tsang-Ming Ko h, Tung-Yao Chang i, Schu-Rern Chern c, Peih-Shan Wu j, Yu-Ting Chen c, Shu-Yuan Chang b

a Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan b Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan c Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan d Department of Biotechnology, Asia University, Taichung, Taiwan e School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan f Institute of Clinical and Community Health Nursing, National Yang-Ming University, Taipei, Taiwan g Department of Obstetrics and Gynecology, School of Medicine, National Yang-Ming University, Taipei, Taiwan h GenePhile Bioscience Laboratory, Ko's Obstetrics and Gynecology, Taipei, Taiwan i Taiji Fetal Medicine Center, Taipei, Taiwan j Biodesign Co. Ltd, Taipei, Taiwan

article info abstract

Article history: Objective: To report prenatal diagnosis of 22q11.2 deletion syndrome in a pregnancy with congenital Accepted 16 April 2014 heart defects in the fetus. Case report: A 26-year-old, primigravid woman was referred for counseling at 24 weeks of gestation Keywords: because of abnormal ultrasound findings of fetal congenital heart defects. The Level II ultrasound 22q11.2 deletion syndrome revealed a singleton fetus with heart defects including overriding aorta, small pulmonary artery, and congenital heart defects ventricular septal defect. Cordocentesis was performed. The DNA extracted from the cord blood was prenatal diagnosis analyzed by multiplex ligation-dependent amplification (MLPA). The MLPA showed deletion in the DiGeorge syndrome (DGS) critical region of low copy number repeat (LCR) 22-A~C. Conventional cytogenetic analysis revealed a normal male karyotype. Repeated amniocentesis and cor- docentesis were performed. Whole- array comparative genomic hybridization (aCGH) on cord blood was performed. aCGH detected a 3.07-Mb deletion at 22q11.21. Conventional cytogenetic analysis of cultured amniocytes revealed a karyotype 46,XY. Metaphase fluorescence in situ hybridization (FISH) analysis on cultured amniocytes confirmed an interstitial 22q11.2 deletion. Conclusion: Prenatal ultrasound findings of congenital heart defects indicate that the fetuses are at increased risk for chromosome abnormalities. Studies for 22q11.2 deletion syndrome should be considered adjunct to conventional karyotyping. Although FISH has become a standard procedure for diagnosis of 22q11.2 deletion syndrome, MLPA can potentially diagnose a broader spectrum of abnor- malities, and aCGH analysis has the advantage of refining the 22q11.2 deletion breakpoints and detecting uncharacterized chromosome rearrangements or genomic imbalances. Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.

Introduction

Chromosome 22q11.2 deletion syndrome occurs in approxi- mately one in 4000 births, and is the most common dele- tion syndrome. It encompasses a wide spectrum of abnormalities * Corresponding author. Department of Obstetrics and Gynecology, Mackay Memorial Hospital, 92, Section 2, Chung-Shan North Road, Taipei, Taiwan. including DiGeorge syndrome [Online Mendelian Inheritance in E-mail address: [email protected] (C.-P. Chen). Man (OMIM) 188400] and velocardiofacial syndrome (OMIM

http://dx.doi.org/10.1016/j.tjog.2014.04.021 1028-4559/Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved. Y.-L. Kuo et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 248e251 249

192430). Patients with 22q11.2 deletion syndrome can suffer from using the SALSA MLPA P250 DiGeorge Probemix (MRC-Holland, congenital heart diseases, palatal abnormalities, learning diffi- Amsterdam, The Netherlands) according to the manufacturer'spro- culties, immune deficiency, characteristic facial features, and hy- tocol. The MLPA showed a deletion in the DiGeorge syndrome (DGS) pocalcemia [1,2]. Individuals with this syndrome have an estimate critical region of chromosome 22 low copy number repeat (LCR) 22- high rate of 74% of congenital heart disease, including tetralogy of A~C or mlpa 22q LCR22-A~C(P250) Â 1. Conventional cytogenetic Fallot, ventricular septal defect, interrupted aortic arch, and truncus analysis revealed a normal male karyotype. The parents requested arteriosus [1,2]. repeated amniocentesis. Repeated amniocentesis and cordocentesis With the advent of ultrasound and molecular genetic technol- were performed. Whole-genome aCGH on cord blood was performed ogy, many cases with 22q11.2 deletion have been diagnosed in the using a NimbleGen ISCA Plus Cytogenetic Array (Roche NimbleGen, prenatal period by the use of fluorescence in situ hybridization Madison, WI, USA). aCGH detected a 3.07-Mb deletion at 22q11.21, or (FISH), multiplex ligation-dependent amplification (MLPA) and arr 22q11.21 (18,657,470e21,724,242) Â 1(Fig. 1). The deleted region array comparative genomic hybridization (aCGH) [3e9].Wepre- encompasses 126 including 43 OMIM genes: USP18, DGCR6, sent our experience of prenatal diagnosis of 22q11.2 microdeletion PRODH, DGCR2, DGCR14, TSSK2, GSC2, SLC25A1, CLTCL1, DVL1P1, HIRA, syndrome by MLPA and aCGH in a fetus with congenital heart MRPL40, UFD1L, CDC45, CLDN5, SEPT5, GP1BB, TBX1, GNB1L, TXNRD2, defects. COMT, ARVCF, DGCR8, TRMT2A, RANBP1, ZDHHC8, RTN4R, DGCR6L, GGTLC3, RIMBP3, ZNF74, SCARF2, MED15, PI4KA, SERPIND1, SNAP29, Case presentation CRKL, LZTR1, THAP7, P2RX6, SLC7A4, BCRP2,andGGT2.Whole-genome aCCH analysis on parental bloods revealed no genomic imbalance. A 26-year-old, primigravid woman was referred for counseling at Conventional cytogenetic analysis of cultured amniocytes revealed a 24 weeks of gestation because of abnormal ultrasound findings of karyotype of 46,XY. FISH analysis was used for confirmation. Meta- fetal congenital heart defects. Her husband was 27 years old. She and phase FISH analysis on cultured amniocytes using Vysis DiGeorge her husband were healthy and nonconsanguineous. There was no region probe [Vysis, LSI TUPLE 1 (Histone cell cycle regulation family history of congenital malformations. She denied any recent defective, s. cerevisiae, homolog of, A (HIRA)) spectrum orange/LSI infections or exposure to teratogens during this pregnancy. The Arylsulfatase A (ARSA) spectrum green; Abbott Laboratories, Chicago, pregnancy was uneventful until 24 weeks of gestation when Level II IL, USA] showed the presence of only one orange signal and two green ultrasound revealed a singleton fetus with heart defects including signals, indicating a deletion of DiGeorge syndrome Tup-like overriding aorta, small pulmonary artery, and ventricular septal enhancer of split 1 (TUPLE 1) at 22q11.2 in the fetus (Fig. 2). The defect. The amniotic fluid amount and fetal growth were normal. karyotype after FISH analysis was 46,XY.ish del(22)(q11.21)(TUPLE Other internal organs were unremarkable. Cordocentesis was per- 1À). The fetus had facial dysmorphism of hypertelorism, prominent formed for cytogenetic analysis and molecular diagnosis of DiGeorge nasal root, bulbous nasal tip, micrognathia, and low-set ears syndrome. The DNA extracted from cord blood was analyzed by MLPA perinatally.

Fig. 1. Whole-genome array comparative genomic hybridization (aCGH) analysis on uncultured amniocytes shows a 3.07-Mb deletion at 22q11.21 or arr [hg 19] 22q11.21 (18,657,470e21,724,242) Â 1]. (A) Chromosomal view; (B) zoom-in view. 250 Y.-L. Kuo et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 248e251

a deletion in the DGS critical region of LCR22-A~C; and afterward aCGH detected a 3.07-Mb deletion at 22q11.21 and FISH detected a deletion of DiGeorge syndrome TUPLE 1 locus at 22q11.2. MLPA is a novel, PCR-based technique, which was first described by Schouten et al [16]. This molecular method concur- rently permits the relative quantification of up to 45 different target DNA sequences. It has been used for rapid aneuploidy diagnosis for common aneuploidy and for diagnosis of several microdeletion syndromes [9,17,18]. MLPA has proven to be a highly sensitive, accurate, rapid, and relatively inexpensive tool for detecting copy number changes in the 22q11.2 region [8,19e21]. The MLPA kit for 22q11.2 deletion syndrome is commercially available. The probes of the SALSA MLPA P250 DiGeorge Probemix (MRC-Holland) are arranged according to specific chromosomal locations at 22q11 including (1) the Cat eye sundrome (CES) region covering the genes IL17RA, SLC25A18, BID, MICAL3, and USP18; (2) Fig. 2. Fluorescence in situ hybridization analysis using Vysis LSI TUPLE 1 (HIRA) LCR22-A~G as follows: LCR22-A covering CLTCL1, HIRA, CDC45L, spectrum orange/LSI ARSA spectrum green probe set (Abbott Laboratories) shows a CLDN5, GP1BB, TBX1, TXNRD2, and DGCR8; LCR22-B covering normal chromosome 22 (one orange signal and one green signal) and a del(22) ZNF74, KLHL22, and MED15; LCR22-C covering SNAP29 and LZTR1; chromosome (only one green signal) in a metaphase amniocyte. LCR22-D covering HIC2, PPIL2, and TOP3B; LCR22-E covering RTDR1, GNAZ, RTDR1, and RAB36; LCR22-F covering SMARCB1; Discussion LCR22-G covering SNRPD3; and (3) DiGeorge anomaly-related re- gions of 4q35, 8p23, 9q34, 10p14 DGS2, 17p13, and 22q13. Features The present case was associated with a 3.07-Mb deletion at of DiGeorge anomaly have been observed in patients with chro- 22q11.2 encompassing the DGCR6, DGCR2, DGCR14, UFD1L, TBX1, mosome deletions outside chromosome 22 [10]. MLPA can identify GNB1L, COMT, DGCR8, DGCR6L, and MED15 genes. Most patients these atypical deletions. with 22q11.2 deletion syndrome have been found to have a large aCGH enables a high-resolution whole-genome screening of interstitial deletion of 3 Mb containing A-D LCRs. Others have an submicroscopic chromosomal imbalance. Several studies have re- atypical shorter deletion within this typically deleted region of A-D ported the usefulness of aCGH in prenatal diagnosis and the LCRs [1]. In addition to a deletion in the 22q11.2 region, a small increased detection rate of chromosomal imbalances, compared number of patients with features of DiGeorge anomaly have other with conventional karyotyping [22e31]. The establishment of the chromosome aberrations [10]. Haploinsufficiency of the TBX1 gene etiology of congenital heart defects diagnosed prenatally is still (OMIM 602054) is responsible for most of the physical malforma- challenging. Mademont-Soler et al [12] and Yan et al [32] reported tions associated with 22q11.2 deletion syndrome [2]. Other possible the clinical value of aCGH in prenatally diagnosed congenital heart responsible genes include DGCR6 (OMIM 601279), DGCR2 (OMIM defects. Mademont-Soler et al [12] found that the diagnostic yield 600594), DGCR14 (OMIM 601755), UFD1L (OMIM 601754), GNB1L may be increased by 2% if chromosomal microarray-based analysis (OMIM 610778), COMT (OMIM 116790), DGCR8 (OMIM 609030), is used as a complementary tool to conventional cytogenetics in DGCR6L (OMIM 609459) and MED15 (OMIM 607372) [2,7]. pregnancies with abnormal cardiac ultrasound findings. Among 76 The present case perinatally manifested conotruncal heart fetuses with abnormal cardiac ultrasound findings and normal malformations and craniofacial dysmorphism. Among fetuses with karyotype, negative or no FISH results for the 22q11.2 deletion congenital heart defects, the incidence of chromosomal abnor- syndrome, Yan et al [32] found that the incidence of pathogenic malities ranges from 16% to 56% [3,11e15]. Therefore, molecular and copy number variants was 6.6% (5/76). They suggested that aCGH cytogenetic analysis for chromosomal abnormalities should be can provide additional genetic information in fetuses with considered in prenatally diagnosed congenital heart defects. abnormal heart findings [32]. Testing for chromosome 22q11.2 deletion is warranted, especially FISH is the most widely used genetic diagnostic procedure for for conotruncal heart defects [3,4,9,12]. In a prospective study of the detection of 22q11.2 deletions [3e5,8,15,20]. The FISH study is 1510 cases with structural heart defects identified on prenatal ul- usually performed with two probes. One is a chromosome 22- trasound, Moore et al [3] found that 41.3% (624 cases) had abnormal specific probe that identifies the chromosome. A second probe karyotypes. In those fetuses with normal karyotypes that under- hybridizes to the commonly deleted region. If the second probe is went FISH testing for the deletion of 22q11.2, 3.1% (17 cases) had the absent on the specific chromosome, the diagnosis is established. 22q11.2 deletion. In a study of 276 pregnancies with abnormal The commercially available FISH probes for 22q11.2 analysis are cardiac ultrasound findings, Mademont-Solar et al [12] found that TUPLE and N25, which are located within LCRs A-B [1]. Despite the 15.9% (44 cases) had chromosomal abnormalities. Of the fetuses high reliability of the FISH analysis, it is very labor intensive and the with normal karyotypes, FISH testing revealed 6.4% (5/78) had technology requires substantial training. Another issue is that 22q11.2 deletion syndrome. Among fetuses with normal karyo- atypical deletions that do not include LCRs A-B cannot be detected types and negative or no 22q11.2 deletion syndrome study, one by commercially available FISH probes [1,2]. case had pathogenic copy number variants (2%; 1/51) diagnosed by Prenatal ultrasound findings of congenital heart defects indicate aCGH [12]. In a retrospective study of 169 pregnancies that un- that the fetuses are at increased risk for chromosome abnormal- derwent FISH testing for 22q11.2 deletion mostly because of con- ities. Studies for 22q11.2 deletion syndrome should be considered otruncal heart defects, Bretelle et al [4] found that 4.7% had the adjunct to conventional karyotyping. Although FISH has become a 22q11.2 deletion (8 cases). In a cohort of 61 fetuses with a heart standard procedure for diagnosis of 22q11.2 deletion syndrome, malformation and normal karyotype, Kjaergaard et al [9] found that MLPA can potentially diagnose a broader spectrum of abnormal- 10% (6 cases) had the 22q11 deletion diagnosed by MLPA. ities, and aCGH analysis has the advantage of refining the 22q11.2 In this case, 22q11.2 deletion syndrome was primarily diagnosed deletion breakpoints and detecting uncharacterized chromosome by MLPA and was later confirmed by aCGH and FISH. MLPA detected rearrangements or genomic imbalances. Y.-L. Kuo et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 248e251 251

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