Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 248e251
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Prenatal diagnosis and molecular cytogenetic characterization of chromosome 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 Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan 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 Gene Biodesign Co. Ltd, Taipei, Taiwan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 16 April 2014
Objective: To report prenatal diagnosis of 22q11.2 deletion syndrome in a pregnancy with congenital heart defects in the fetus. Case report: A 26-year-old, primigravid woman was referred for counseling at 24 weeks of gestation because of abnormal ultrasound ﬁndings of fetal congenital heart defects. The Level II ultrasound revealed a singleton fetus with heart defects including overriding aorta, small pulmonary artery, and ventricular septal defect. Cordocentesis was performed. The DNA extracted from the cord blood was analyzed by multiplex ligation-dependent ampliﬁcation (MLPA). The MLPA showed deletion in the DiGeorge syndrome (DGS) critical region of chromosome 22 low copy number repeat (LCR) 22-A~C. Conventional cytogenetic analysis revealed a normal male karyotype. Repeated amniocentesis and cordocentesis were performed. Whole-genome 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 abnormalities, 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.
Keywords: 22q11.2 deletion syndrome congenital heart defects prenatal diagnosis
* Corresponding author. Department of Obstetrics and Gynecology, Mackay Memorial Hospital, 92, Section 2, Chung-Shan North Road, Taipei, Taiwan. E-mail address: [email protected]
Chromosome 22q11.2 deletion syndrome occurs in approximately one in 4000 births, and is the most common human deletion syndrome. It encompasses a wide spectrum of abnormalities including DiGeorge syndrome [Online Mendelian Inheritance in 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
192430). Patients with 22q11.2 deletion syndrome can suffer from congenital heart diseases, palatal abnormalities, learning difﬁculties, immune deﬁciency, characteristic facial features, and hypocalcemia [1,2]. Individuals with this syndrome have an estimate high rate of 74% of congenital heart disease, including tetralogy of Fallot, ventricular septal defect, interrupted aortic arch, and truncus arteriosus [1,2]. With the advent of ultrasound and molecular genetic technology, many cases with 22q11.2 deletion have been diagnosed in the prenatal period by the use of ﬂuorescence in situ hybridization (FISH), multiplex ligation-dependent ampliﬁcation (MLPA) and array comparative genomic hybridization (aCGH) [3e9]. We present our experience of prenatal diagnosis of 22q11.2 microdeletion syndrome by MLPA and aCGH in a fetus with congenital heart defects. Case presentation A 26-year-old, primigravid woman was referred for counseling at 24 weeks of gestation because of abnormal ultrasound ﬁndings of fetal congenital heart defects. Her husband was 27 years old. She and her husband were healthy and nonconsanguineous. There was no family history of congenital malformations. She denied any recent infections or exposure to teratogens during this pregnancy. The pregnancy was uneventful until 24 weeks of gestation when Level II ultrasound revealed a singleton fetus with heart defects including overriding aorta, small pulmonary artery, and ventricular septal defect. The amniotic ﬂuid amount and fetal growth were normal. Other internal organs were unremarkable. Cordocentesis was performed for cytogenetic analysis and molecular diagnosis of DiGeorge syndrome. The DNA extracted from cord blood was analyzed by MLPA
using the SALSA MLPA P250 DiGeorge Probemix (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer's protocol. The MLPA showed a deletion in the DiGeorge syndrome (DGS) critical region of chromosome 22 low copy number repeat (LCR) 22A~C or mlpa 22q LCR22-A~C(P250) 1. Conventional cytogenetic analysis revealed a normal male karyotype. The parents requested repeated amniocentesis. Repeated amniocentesis and cordocentesis were performed. Whole-genome aCGH on cord blood was performed using a NimbleGen ISCA Plus Cytogenetic Array (Roche NimbleGen, Madison, WI, USA). aCGH detected a 3.07-Mb deletion at 22q11.21, or arr 22q11.21 (18,657,470e21,724,242) 1 (Fig. 1). The deleted region encompasses 126 genes including 43 OMIM genes: USP18, DGCR6, PRODH, DGCR2, DGCR14, TSSK2, GSC2, SLC25A1, CLTCL1, DVL1P1, HIRA, MRPL40, UFD1L, CDC45, CLDN5, SEPT5, GP1BB, TBX1, GNB1L, TXNRD2, COMT, ARVCF, DGCR8, TRMT2A, RANBP1, ZDHHC8, RTN4R, DGCR6L, GGTLC3, RIMBP3, ZNF74, SCARF2, MED15, PI4KA, SERPIND1, SNAP29, CRKL, LZTR1, THAP7, P2RX6, SLC7A4, BCRP2, and GGT2. Whole-genome aCCH analysis on parental bloods revealed no genomic imbalance. Conventional cytogenetic analysis of cultured amniocytes revealed a karyotype of 46,XY. FISH analysis was used for conﬁrmation. Metaphase FISH analysis on cultured amniocytes using Vysis DiGeorge region probe [Vysis, LSI TUPLE 1 (Histone cell cycle regulation defective, s. cerevisiae, homolog of, A (HIRA)) spectrum orange/LSI Arylsulfatase A (ARSA) spectrum green; Abbott Laboratories, Chicago, IL, USA] showed the presence of only one orange signal and two green signals, indicating a deletion of DiGeorge syndrome Tup-like enhancer of split 1 (TUPLE 1) locus at 22q11.2 in the fetus (Fig. 2). The karyotype after FISH analysis was 46,XY.ish del(22)(q11.21)(TUPLE 1 ). The fetus had facial dysmorphism of hypertelorism, prominent nasal root, bulbous nasal tip, micrognathia, and low-set ears 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) 1]. (A) Chromosomal view; (B) zoom-in view.
Y.-L. Kuo et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 248e251
Fig. 2. Fluorescence in situ hybridization analysis using Vysis LSI TUPLE 1 (HIRA) spectrum orange/LSI ARSA spectrum green probe set (Abbott Laboratories) shows a normal chromosome 22 (one orange signal and one green signal) and a del(22) chromosome (only one green signal) in a metaphase amniocyte.
Discussion The present case was associated with a 3.07-Mb deletion at 22q11.2 encompassing the DGCR6, DGCR2, DGCR14, UFD1L, TBX1, GNB1L, COMT, DGCR8, DGCR6L, and MED15 genes. Most patients with 22q11.2 deletion syndrome have been found to have a large interstitial deletion of 3 Mb containing A-D LCRs. Others have an atypical shorter deletion within this typically deleted region of A-D LCRs . In addition to a deletion in the 22q11.2 region, a small number of patients with features of DiGeorge anomaly have other chromosome aberrations . Haploinsufﬁciency of the TBX1 gene (OMIM 602054) is responsible for most of the physical malformations associated with 22q11.2 deletion syndrome . Other possible responsible genes include DGCR6 (OMIM 601279), DGCR2 (OMIM 600594), DGCR14 (OMIM 601755), UFD1L (OMIM 601754), GNB1L (OMIM 610778), COMT (OMIM 116790), DGCR8 (OMIM 609030), DGCR6L (OMIM 609459) and MED15 (OMIM 607372) [2,7]. The present case perinatally manifested conotruncal heart malformations and craniofacial dysmorphism. Among fetuses with congenital heart defects, the incidence of chromosomal abnormalities ranges from 16% to 56% [3,11e15]. Therefore, molecular and cytogenetic analysis for chromosomal abnormalities should be considered in prenatally diagnosed congenital heart defects. Testing for chromosome 22q11.2 deletion is warranted, especially for conotruncal heart defects [3,4,9,12]. In a prospective study of 1510 cases with structural heart defects identiﬁed on prenatal ultrasound, Moore et al  found that 41.3% (624 cases) had abnormal karyotypes. In those fetuses with normal karyotypes that underwent FISH testing for the deletion of 22q11.2, 3.1% (17 cases) had the 22q11.2 deletion. In a study of 276 pregnancies with abnormal cardiac ultrasound ﬁndings, Mademont-Solar et al  found that 15.9% (44 cases) had chromosomal abnormalities. Of the fetuses with normal karyotypes, FISH testing revealed 6.4% (5/78) had 22q11.2 deletion syndrome. Among fetuses with normal karyotypes and negative or no 22q11.2 deletion syndrome study, one case had pathogenic copy number variants (2%; 1/51) diagnosed by aCGH . In a retrospective study of 169 pregnancies that underwent FISH testing for 22q11.2 deletion mostly because of conotruncal heart defects, Bretelle et al  found that 4.7% had the 22q11.2 deletion (8 cases). In a cohort of 61 fetuses with a heart malformation and normal karyotype, Kjaergaard et al  found that 10% (6 cases) had the 22q11 deletion diagnosed by MLPA. In this case, 22q11.2 deletion syndrome was primarily diagnosed by MLPA and was later conﬁrmed by aCGH and FISH. MLPA detected
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 . This molecular method concurrently 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) LCR22-A~G as follows: LCR22-A covering CLTCL1, HIRA, CDC45L, CLDN5, GP1BB, TBX1, TXNRD2, and DGCR8; LCR22-B covering ZNF74, KLHL22, and MED15; LCR22-C covering SNAP29 and LZTR1; LCR22-D covering HIC2, PPIL2, and TOP3B; LCR22-E covering RTDR1, GNAZ, RTDR1, and RAB36; LCR22-F covering SMARCB1; LCR22-G covering SNRPD3; and (3) DiGeorge anomaly-related regions of 4q35, 8p23, 9q34, 10p14 DGS2, 17p13, and 22q13. Features of DiGeorge anomaly have been observed in patients with chromosome deletions outside chromosome 22 . MLPA can identify these atypical deletions. aCGH enables a high-resolution whole-genome screening of submicroscopic chromosomal imbalance. Several studies have reported the usefulness of aCGH in prenatal diagnosis and the increased detection rate of chromosomal imbalances, compared with conventional karyotyping [22e31]. The establishment of the etiology of congenital heart defects diagnosed prenatally is still challenging. Mademont-Soler et al  and Yan et al  reported the clinical value of aCGH in prenatally diagnosed congenital heart defects. Mademont-Soler et al  found that the diagnostic yield may be increased by 2% if chromosomal microarray-based analysis is used as a complementary tool to conventional cytogenetics in pregnancies with abnormal cardiac ultrasound ﬁndings. Among 76 fetuses with abnormal cardiac ultrasound ﬁndings and normal karyotype, negative or no FISH results for the 22q11.2 deletion syndrome, Yan et al  found that the incidence of pathogenic copy number variants was 6.6% (5/76). They suggested that aCGH can provide additional genetic information in fetuses with abnormal heart ﬁndings . FISH is the most widely used genetic diagnostic procedure for the detection of 22q11.2 deletions [3e5,8,15,20]. The FISH study is usually performed with two probes. One is a chromosome 22speciﬁc probe that identiﬁes the chromosome. A second probe hybridizes to the commonly deleted region. If the second probe is absent on the speciﬁc chromosome, the diagnosis is established. The commercially available FISH probes for 22q11.2 analysis are TUPLE and N25, which are located within LCRs A-B . Despite the high reliability of the FISH analysis, it is very labor intensive and the technology requires substantial training. Another issue is that atypical deletions that do not include LCRs A-B cannot be detected by commercially available FISH probes [1,2]. 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 abnormalities, and aCGH analysis has the advantage of reﬁning the 22q11.2 deletion breakpoints and detecting uncharacterized chromosome rearrangements or genomic imbalances.
Y.-L. Kuo et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 248e251
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