Prenat Diagn 2006; 26: 898–902.
Published online 17 August 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/pd.1520
CASE REPORT
Prenatal detection and characterization of a small
supernumerary marker chromosome (sSMC) derived from
chromosome 22 with apparently normal phenotype
†
Chyi-Chyang Lin1, Yao-Yuan Hsieh2,5, Chung-Hsing Wang3, Yueh-Chun Li4, Lie-Jiau Hsieh1,
Chien-Chung Lee2, Chang-Hai Tsai3,6 and Fuu-Jen Tsai3,6*
1Department of Medical Research, China Medical University Hospital, Taichung, Taiwan
2Department of Obstetrics and Gynecology, China Medical University Hospital, Taichung, Taiwan 3Department of Pediatrics and Medical Genetics, China Medical University Hospital, Taichung, Taiwan 4Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan
5Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan 6Department of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwan
Objective To present prenatal findings and molecular cytogenetic characterization of a small supernumerary marker chromosome (sSMC) derived from chromosome 22 with apparently normal phenotype.
Case and Methods An amniocentesis was performed at 15 weeks’ gestation and a small marker chromosome in the female fetus of a twin pregnancy was noted. A second amniocentesis was performed at 18 weeks; G-banding analysis on amniotic cells confirmed the small marker chromosome found in the female fetus. Both parents and the male twin fetus had normal karyotypes. Spectral karyotyping (SKY), Fluorescence in situ hybridization (FISH) analyses with chromosomal specific whole chromosome painting probe (WCP 22) and alphoid satellite DNA probe (D22Z4) were used to identify the origin of the sSMC. The make-up of the sSMC was characterized by further FISH studies with chromosome region specific probes. The twin babies were delivered normally at 35 weeks’ gestation. The female neonate with sSMC did not show any dysmorphic features, except for a type II atrial septum defect (ASD) at birth. She was found to be developing and growing normally at her 2-year follow-up.
Results Conventional G-banding study confirmed the presence of a sSMC with bi-satellites. SKY and FISH with D22Z4 probes showed that the marker originated from chromosome 22. FISH studies using 4 locus-specific DNA probes in the 22q11.2 region (N25 probe to detect the D22S75 locus within the velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS) critical region, a clone to detect the Bid locus just distal to the cat eye syndrome (CES) critical region and two clones 77H2 and 109L3 to detect the proximal end of the CES critical region, (CECR2 and CECR7), did not reveal any hybridization signal with the marker chromosome. The karyotype of the fetus was 47,XX,+mar. ish der(22) (SKY+,D22Z42+,CECR7−,CECR2−, BID−,D22S75−). Conclusion The supernumerary marker chromosome in this case was a de novo inv dup(22)(q11.2) and contained a duplicated proximal long arm region <400 kb from the centromere; it did not appear to affect the phenotype of the child. Copyright 2006 John Wiley & Sons, Ltd.
KEY WORDS: marker chromosome 22; small supernumerary marker chromosome (sSMC); SKY; CES critical
region; VCFS/DGS critical region
INTRODUCTION
Small supernumerary marker chromosomes (sSMCs) are structurally abnormal chromosomes equal in size or smaller than chromosome 20 and cannot be identified or characterized unambiguously by conventional cytoge-netic banding techniques (Liehr et al., 2004). sSMCs are
*Correspondence to: Fuu-Jen Tsai, Department of Pediatrics and Medical Genetics, China Medical University Hospital, 2 Yuh-Der Road, North District, Taichung, Taiwan.
E-mail: [email protected]
†Chyi-Chyang Lin and Yao-Yuan Hsieh contributed equally to this work
present in about 0.043% of newborn and in 0.076% of prenatal cases (Liehr et al., 2004). Until very recently, there was no way of predicting precisely the outcome of the pregnancy when a de novo sSMC is ascertained prenatally. About 70% of sSMCs derive from acrocen-tric chromosomes (Viersbach et al., 1998; Liehr et al., 2004). In general, the risk for an abnormal phenotype is about 7% when de novo sSMC deriving from chromo-somes 13, 14, 21 and 22 ascertained prenatally (Crolla, 1998; Warburton, 1991). Patients with small deriva-tives of chromosome 15 tend to have a normal phe-notype and sSMC derived from chromosomes 13, 21 and 14 also appear to have low risk of abnormalities
(Vierbach et al., 1998). The 22q11 region is partic-ularly susceptible to chromosomal rearrangement and sSMC formation because it contains various low copy repeat sequences (Edelmann et al., 1999; Shaikh et al., 2000; Crolla et al., 2005). Three different congenital malformation syndromes are known to occur in this region: cat eye syndrome (CES), derivative 22 syndrome [der(22)] and velocardiofacial syndrome/DiGeorge syn-drome (VCFS/DGS) (McDermid and Morrow, 2002). However, sSMC cases with inv dup(22) (q10→ q11.2) do not necessarily result in abnormal phenotypes (Mears et al., 1994; Gravholt and Friedrich, 1995; Vierbach et al., 1998; Hastings et al., 1999; Engelen et al., 2000; Lohmann et al., 2000; Bartels et al., 2003; Starke et al., 2003; von Eggeling et al., 2003). Bartsch et al. (2005) recently identified six diagnostically relevant intervals on chromosome 22q by fluorescence in situ hybridiza-tion (FISH) and proposed using DNA probes corre-sponding to these intervals to characterize sSMC. In this study, we used a combination of spectral karyotyping (SKY) and FISH with chromosome-specific and locus-specific DNA probes in the region 22q11 to delineate the breakpoint of a prenatally detected sSMC originated from chromosome 22.
CASE REPORT AND METHODS
A 34-year-old Taiwanese woman, gravida 1, para 0, abortion 0, who had become pregnant after having undergone controlled ovarian hyperstimulation, in vitro fertilization and embryo transfer was referred to us from a local clinic to evaluate an earlier amniocentesis find-ing of abnormal karyotypfind-ing (47,XX,+mar) in one of her twin fetuses (twin-A). Family history was noncon-tributory except for her long-term infertility. Sonogra-phy demonstrated a dichorion–diamnionic twin preg-nancy with normal fetuses. A second amniocentesis was performed at 18 weeks’ gestation to confirm the aberrant karyotype. Conventional G-banding revealed that both parents and one of the twin fetuses (twin-B,
male) had normal karyotypes. However, an extra small bi-satellited marker chromosome was observed in all the 20 cells analyzed (47, XX, +mar) from the other twin (twin-A, female). The SKY analysis identified that the marker chromosome was derived from chromosome 22 (Figure 1(a)). FISH studies with chromosome 22 specific alphoid satellite DNA probe D22Z4 (Rocchi et al., 1994) under stringent conditions showed that the marker chromosome was positive for D22Z4 (Figure 1(b)). These findings indicated that the marker originated on chromosome 22 and might retain the chromosome 22 centromeric/pericentromeric region 22p11.1- q11.1. Further FISH experiments were con-ducted using locus-specific DNA probes for fine map-ping of the marker chromosome breakpoint within the VCFS/DGS and CES critical regions. LSI DiGeorge N25 probe (Vysis) was used to detect the D22S75 locus and nearby CTP and CLTD genes (Carlson et al., 1997) located within the VCFS/DGS critical region at 22q11.2. BAC clone (RPCI11-91O6) (CHORI BAC/PAC resources) was used to detect BID locus at the distal end of CES critical region (McDermid and Morrow, 2002). Two clones, 77H2 and 109L3 which cover the CECR2 and CECR7 loci in the CES criti-cal region were also used to map the breakpoints about 800 and 400 kb away from the centromere, respec-tively (Footz et al., 2001). No hybridization signals were observed in the marker chromosome by any of the four DNA probes used; however, positive hybridization sig-nals of the 4 locus-specific probes were detected in chromosome 22s (Figure 2(a–d)). Together, G-banding, SKY and FISH studies revealed that the karyotype of fetus-A was 47, XX,+mar.ish der(22) (SKY+, D22Z4+,CECR7−,CECR2−,BID−,D22S75−). Our molecular cytogenetic studies indicated that the marker chromosome did not contain the N25 locus in the VCFS/DGS critical region and did not contain the BID, CECR2 or CECR7 gene sequences within the CES crit-ical region. Therefore, the breakpoint of the marker should have occurred somewhere <400 kb from the centromere (Footz et al., 2001) (Figure 3). The preg-nancy was allowed to continue. At 35 weeks’ gestation,
Figure 1—Spectral karyotyping (SKY) and FISH analysis of the marker chromosome. (a) SKY shows that the marker chromosome (indicated) is derived from chromosome 22. (b) FISH with a chromosome-specific alphoid satellite probe (D22Z4) shows positive hybridization signals on chromosome 22s as well as on the marker (indicated by an arrow). Chromosome 14s is also indicated
Figure 2—FISH study with locus-specific probes located within the velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS) critical region and cat eye syndrome (CES) critical region. (a) A metaphase cell hybridized with LSI DiGeorge N25 region probe (Vysis). Both number 22 chromosomes (indicated) show presence of the spectrumorange N25 probe and the specteumgreen LSI ARSA control probe. The G-banding patterns of chromosomes are visible by the inverted DAPI counterstain and show the bi-satellite nature of the marker, which lack a hybridization signal (indicated by an arrow). (b) The same metaphase as in Figure 1(b) hybridized with a BAC clone probe, RPCI11-91O6(CHORI) containing the BID locus in the CES critical region. Both chromosome 22s (indicated) show presence of the probe with green fluorescent signals, whereas the marker chromosome (indicated by an arrow) shows lack of hybridization, indicating that the BID locus is missing. (c) A metaphase cell hybridized with 77H2 clone probe of the CECR2 locus. Both chromosome 22s have positive red hybridization signals (indicated). The marker chromosome has no hybridization signal (indicated by an arrow), which indicates the absence of CECR2 locus in the marker. (d) A metaphase cell and an interphase cells hybridized with 109L3 clone probe containing the CECR7 locus. Both number 22 chromosomes (indicated) show presence of the109L3 probe (green signals). The marker chromosome shows absence of hybridization signal (indicated by an arrow), indicating that the CECR7 locus is not present. Two positive green hybridization signals are seen in the interphase cell
premature rupture of the membrane occurred and two babies were delivered normally. The neonatal course of both babies was uneventful. A serial examination revealed that twin-A did not have any dysmorphic fea-tures except for type II atrial septum defect (ASD). She was developing normally [head girth: 50 cm (50th percentile), body length: 92 cm (95th percentile), body weight: 12 kg (50th percentile)] comparable to her nor-mal karyotype twin brother at her 2-year follow-up. Spontaneous closure of type II ASD was also noted.
DISCUSSION
Great achievement has been made in recent years, both in the characterization of sSMC and in exploration of its clinical impact. sSMC have been characterized by
molecular cytogenetic study for their chromosomal ori-gin in over 1500 patients (Liehr et al., 2004). A num-ber of clinical syndromes associated with sSMC have also been documented including the der(22) syndrome and CES (Fraccaro et al., 1980; Schinzel et al., 1981), Pallister–Killian syndrome (Peltomaki et al., 1987) and i(18p) syndrome (Callen et al., 1990). A large portion of sSMCs (30.3%) have been shown to have origi-nated from inverted duplication of chromosome 15, inv dup(15) (Liehr et al., 2004). It has been noticed that patients with small inv dup(15) have normal pheno-types but those with large inv dup(15) show phenotypic anomalities (Viersbach et al., 1998). It is now known that the clinical severity of sSMC with inv dup(15) is associated with the dosage of the Prader-Willi/Angelman syndrome critical region (Nietzel et al., 2003). How-ever, about 62% of sSMC are not correlated with a
Figure 3—A graphic presentation of the small supernumerary marker chromosome (sSMC) and a map of the CES and VCFS/DGS critical regions in 22q11.1-q11.2. Horizontal lines show relative order of the genes in those regions. Locus-specific probes used in the study are shown by bold horizontal lines. Distances between the CECR2 and the centromere, and CECR7 and the centromere are indicated. Breakpoint of the sSMC could have occurred somewhere in the 22q11.1, <400 kb from the centromere
specific clinical syndrome (Liehr et al., 2004). Further-more, based on the recent studies by Starke et al. (2003) and Liehr et al. (2004), it is believed that sSMCs with-out euchromatin and no uniparental disomy (UPD) are harmless, for example, it was reported that a patient with maternal UPD 22 did not have clinical abnor-malities (Bartels et al., 2003). Duplications in the jux-tacentromeric region of 2q, 3p, 4q, 5q, 7p, 8p, 17p, 18p, 19p, 19q and 22q have been shown to be clini-cally insignificant. At least 22 patients with sSMC(22) derived from chromosome region 22q10-22q11.2 have been documented to be clinically normal (see electronic-database: sSMC derived from chromosome 22 created by Liehr et al., 2004). Among those, crossover break-points occurred at 22q10-22q11.1 in 17 patients and at 22q11.1-22q11.2 in the remaining 5 patients. sSMCs in most of the patients were due to inverted duplication of a part of chromosome 22, inv dup(22)(q10-q11.2) result-ing from crossover errors in meiosis (Schreck et al., 1977).
In this study, we were able to map the crossover breakpoint of an sSMC(22) somewhere <400 kb away from the centromere in 22q11.1 by using region spe-cific DNA probes. The breakpoint location is somewhere between interval 1 (centromere heterochromatin) and interval 2 (the CECR) of 22q as defined by the FISH study reported by Bartsch et al. (2005). This sSMC has centromeric/pericentromeric heterochromatin but may contain very minimal amounts of euchromatin in the proximal long arm of chromosome 22. The affected neonate’s phenotype appeared to be normal with the exception of type II ASD, which spontaneously closed before the age of two; the infant is growing and devel-oping normally. Thus, the sSMC(22) observed in this study will add another case to the growing list of benign sSMC derived from chromosome 22. We were unable to determine the exact amounts of euchromatin present in the sSMC. Therefore, the possibility that the neonate
may develop some mild features of CES cannot be com-pletely ruled out. This risk was mentioned to the parents during prenatal counseling. Parental origin studies of the sSMC and microsatellite analysis to exclude a UPD of the sSMC’s sister-chromosome were not performed.
In conclusion, we have presented the prenatal diagno-sis of a de novo sSMC derived from chromosome 22 in a second-trimester fetus. This sSMC did not contain the region covering the CTP-ClTD genes in the VCFS/DGS critical region and also did not harbour the BID, CECR 2 and CECR 7 loci in the CES critical region. There-fore, the breakpoint must have occurred at the proximal region of 22q11.1 <400 kb from the centromere. The sSMC appeared to have little effect on the fetal phe-notype and the neonate continues to develop normally. Molecular cytogenetic analyses enable a more detailed and accurate characterization of the marker, which is helpful in prenatal counseling and in preventing unnec-essary termination of pregnancy.
ELECTRONIC-DATABASE INFORMATION sSMC derived from chromosome 22. http://mit-n.mti. uni-jena.de/∼huwww/MOL ZYTO/sSMAC/22.htm
ACKNOWLEDGEMENTS
We would like to thank Professor Heather E. McDermid, University of Alberta, Edmonton, Canada for providing us with the 77H2 and 109L3 clones and would also like to thank Professor Mariano Rocchi, University of Bari, Bari, Italy and Dr. Charles Lee, Harvard Medical School, Boston, USA for providing us with the D22Z4 probe. The technical assistance of Ms I-Chia Lien is also greatly appreciated. This study was supported by grants (BH92-GC03-1) from Bureau of Health Promotion, Department
of Health, Taiwan and (NHRI EX92-9207SI) from National Health Research Institute, Taiwan.
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