Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 248e251
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Taiwanese Journal of Obstetrics & Gynecology journal homepage: www.tjog-online.com
Case Report
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
Introduction
* Corresponding author. Department of Obstetrics and Gynecology, Mackay Memorial Hospital, 92, Section 2, Chung-Shan North Road, Taipei, Taiwan. E-mail address: [email protected] (C.-P. Chen).
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
249
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.
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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 [1]. In addition to a deletion in the 22q11.2 region, a small number of patients with features of DiGeorge anomaly have other chromosome aberrations [10]. Haploinsufficiency of the TBX1 gene (OMIM 602054) is responsible for most of the physical malformations associated with 22q11.2 deletion syndrome [2]. 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 [3] 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 [12] 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 [12]. In a retrospective study of 169 pregnancies that underwent FISH testing for 22q11.2 deletion mostly because of conotruncal heart defects, Bretelle et al [4] 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 [9] 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 [16]. 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 [10]. 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 [12] and Yan et al [32] reported the clinical value of aCGH in prenatally diagnosed congenital heart defects. Mademont-Soler et al [12] 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 [32] 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 [32]. 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 [1]. 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
Conflicts of interest All authors declare no conflicts of interest. Acknowledgments This work was supported by research grants NSC-99-2628-B195-001-MY3 and NSC-101-2314-B-195-011-MY3 from the National Science Council and MMH-E-102-04 from Mackay Memorial Hospital, Taipei, Taiwan. References [1] McDonald-McGinn DM, Emanuel BS, Zackai EH. 22q11.2 deletion syndrome. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong CT, Stephens K, editors. GeneReviews. Seattle: University of Washington; 1993. [2] McDonald-McGinn DM, Sullivan KE. Chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome). Medicine (Baltimore) 2011;90:1e18. [3] Moore JW, Binder GA, Berry R. Prenatal diagnosis of aneuploidy and deletion 22q11.2 in fetuses with ultrasound detection of cardiac defects. Am J Obstet Gynecol 2004;191:2068e73. [4] Bretelle F, Beyer L, Pellissier MC, Missirian C, Sigaudy S, Gamerre M, et al. Prenatal and postnatal diagnosis of 22q11.2 deletion syndrome. Eur J Med Genet 2010;53:367e70. [5] Levy-Mozziconacci A, Piquet C, Heurtevin PC, Philip N. Prenatal diagnosis of 22q11 microdeletion. Prenat Diagn 1997;17:1033e7. [6] Tokuyasu TA, Cotter PD, Segraves R, Harris J, Elder ME, Gonzales M, et al. Detection of single clone deletions using array CGH: identification of submicroscopic deletions in the 22q11.2 deletion syndrome as a model system. Am J Med Genet A 2007;143A:925e32. [7] Chen CP, Huang JP, Chen YY, Chern SR, Wu PS, Su JW, et al. Chromosome 22q11.2 deletion syndrome: prenatal diagnosis, array comparative genomic hybridization characterization using uncultured amniocytes and literature review. Gene 2013;527:405e9. [8] Mademont-Soler I, Morales C, Soler A, Clusellas N, Margarit E, MartinezBarrios E, et al. MLPA: a prenatal diagnostic tool for the study of congenital heart defects? Gene 2012;500:151e4. [9] Kjaergaard S, Sundberg K, Jorgensen FS, Rohde MD, Lind AM, Gerdes T, et al. Diagnostic yield by supplementing prenatal metaphase karyotyping with MLPA for microdeletion syndromes and subtelomere imbalances. Prenat Diagn 2010;30:995e9. [10] Chen CP, Ko TM, Su YN, Su JW, Chen YT, Lee CC, et al. Prenatal diagnosis and molecular cytogenetic characterization of a proximal deletion of 22q (22q11.2/q11.21). Taiwan J Obstet Gynecol 2013;52:147e51. [11] Bellucco FT, Belangero SI, Farah LM, Machado MV, Cruz AP, Lopes LM, et al. Investigating 22q11.2 deletion and other chromosomal aberrations in fetuses with heart defects detected by prenatal echocardiography. Pediatr Cardiol 2010;31:1146e50. [12] Mademont-Soler I, Morales C, Soler A, Martinez-Crespo JM, Shen Y, Margarit E, et al. Prenatal diagnosis of chromosomal abnormalities in fetuses with abnormal cardiac ultrasound findings: evaluation of chromosomal microarray-based analysis. Ultrasound Obstet Gynecol 2013;41:375e82. [13] Comas Gabriel C, Galindo A, Martinez JM, Carrera JM, Gutierrez-Larraya F, de la Fuente P, et al. Early prenatal diagnosis of major cardiac anomalies in a high-risk population. Prenat Diagn 2002;22:586e93. [14] Gembruch U, Baschat AA, Knopfle G, Hansmann M. Results of chromosomal analysis in fetuses with cardiac anomalies as diagnosed by first- and early second-trimester echocardiography. Ultrasound Obstet Gynecol 1997;10: 391e6.
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[15] Manji S, Roberson JR, Wiktor A, Vats S, Rush P, Diment S, et al. Prenatal diagnosis of 22q11.2 deletion when ultrasound examination reveals a heart defect. Genet Med 2001;3:65e6. [16] Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplification. Nucleic Acids Res 2002;30:e57. [17] Chen CP, Su YN, Chang CL, Chen YY, Su JW, Chern SR, et al. Rapid aneuploidy diagnosis by multiplex ligation-dependent probe amplification using uncultured amniocytes in pregnancy with major fetal structural abnormalities. Taiwan J Obstet Gynecol 2012;51:123e8. [18] Chen CP, Su YN, Lin SY, Chang CL, Wang YL, Huang JP, et al. Rapid aneuploidy diagnosis by multiplex ligation-dependent probe amplification and array comparative genomic hybridization in pregnancy with major congenital malformations. Taiwan J Obstet Gynecol 2011;50:85e94. [19] Vorstman JA, Jalali GR, Rappaport EF, Hacker AM, Scott C, Emanuel BS. MLPA: a rapid, reliable, and sensitive method for detection and analysis of abnormalities of 22q. Hum Mutat 2006;27:814e21. [20] Fernandez L, Lapunzina P, Arjona D, Lopez Pajares I, Garcia-Guereta L, Elorza D, et al. Comparative study of three diagnostic approaches (FISH, STRs and MLPA) in 30 patients with 22q11.2 deletion syndrome. Clin Genet 2005;68:373e8. [21] Stachon AC, Baskin B, Smith AC, Shugar A, Cytrynbaum C, Fishman L, et al. Molecular diagnosis of 22q11.2 deletion and duplication by multiplex ligation dependent probe amplification. Am J Med Genet A 2007;143A:2924e30. [22] Hillman SC, Pretlove S, Coomarasamy A, McMullan DJ, Davison EV, Maher ER, et al. Additional information from array comparative genomic hybridization technology over conventional karyotyping in prenatal diagnosis: a systematic review and meta-analysis. Ultrasound Obstet Gynecol 2011;37:6e14. [23] D'Amours G, Kibar Z, Mathonnet G, Fetni R, Tihy F, Desilets V, et al. Wholegenome array CGH identifies pathogenic copy number variations in fetuses with major malformations and a normal karyotype. Clin Genet 2012;81: 128e41. [24] Armengol L, Nevado J, Serra-Juhe C, Plaja A, Mediano C, Garcia-Santiago FA, et al. Clinical utility of chromosomal microarray analysis in invasive prenatal diagnosis. Hum Genet 2012;131:513e23. [25] Lee CN, Lin SY, Lin CH, Shih JC, Lin TH, Su YN. Clinical utility of array comparative genomic hybridisation for prenatal diagnosis: a cohort study of 3171 pregnancies. BJOG 2012;119:614e25. [26] Van den Veyver IB, Patel A, Shaw CA, Pursley AN, Kang SH, Simovich MJ, et al. Clinical use of array comparative genomic hybridization (aCGH) for prenatal diagnosis in 300 cases. Prenat Diagn 2009;29:29e39. [27] Tyreman M, Abbott KM, Willatt LR, Nash R, Lees C, Whittaker J, et al. High resolution array analysis: diagnosing pregnancies with abnormal ultrasound findings. J Med Genet 2009;46:531e41. [28] Kleeman L, Bianchi DW, Shaffer LG, Rorem E, Cowan J, Craigo SD, et al. Use of array comparative genomic hybridization for prenatal diagnosis of fetuses with sonographic anomalies and normal metaphase karyotype. Prenat Diagn 2009;29:1213e7. [29] Coppinger J, Alliman S, Lamb AN, Torchia BS, Bejjani BA, Shaffer LG. Wholegenome microarray analysis in prenatal specimens identifies clinically significant chromosome alterations without increase in results of unclear significance compared to targeted microarray. Prenat Diagn 2009;29:1156e66. [30] Le Caignec C, Boceno M, Saugier-Veber P, Jacquemont S, Joubert M, David A, et al. Detection of genomic imbalances by array based comparative genomic hybridisation in fetuses with multiple malformations. J Med Genet 2005;42: 121e8. [31] Vialard F, Molina Gomes D, Leroy B, Quarello E, Escalona A, Le Sciellour C, et al. Array comparative genomic hybridization in prenatal diagnosis: another experience. Fetal Diagn Ther 2009;25:277e84. [32] Yan Y, Wu Q, Zhang L, Wang X, Dan S, Deng D, et al. Detection of submicroscopic chromosomal aberrations by array-based comparative genomic hybridization in fetuses with congenital heart disease. Ultrasound Obstet Gynecol 2014;43:404e12.
Contents lists available at ScienceDirect
Taiwanese Journal of Obstetrics & Gynecology journal homepage: www.tjog-online.com
Case Report
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
Introduction
* Corresponding author. Department of Obstetrics and Gynecology, Mackay Memorial Hospital, 92, Section 2, Chung-Shan North Road, Taipei, Taiwan. E-mail address: [email protected] (C.-P. Chen).
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
249
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.
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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 [1]. In addition to a deletion in the 22q11.2 region, a small number of patients with features of DiGeorge anomaly have other chromosome aberrations [10]. Haploinsufficiency of the TBX1 gene (OMIM 602054) is responsible for most of the physical malformations associated with 22q11.2 deletion syndrome [2]. 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 [3] 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 [12] 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 [12]. In a retrospective study of 169 pregnancies that underwent FISH testing for 22q11.2 deletion mostly because of conotruncal heart defects, Bretelle et al [4] 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 [9] 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 [16]. 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 [10]. 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 [12] and Yan et al [32] reported the clinical value of aCGH in prenatally diagnosed congenital heart defects. Mademont-Soler et al [12] 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 [32] 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 [32]. 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 [1]. 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.
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