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Chih-Ping Chen1,2,3,4,5,6†*, Yung-Ting Kuo7†, Shuan-Pei Lin2,8, Yi-Ning Su9, Yann-Jang Chen10, Rui-Yuan Hsueh11, Yi-Hui Lin12, Pei-Chen Wu1, Chen-Chi Lee1,
Yu-Ting Chen2, Wayseen Wang2,13
Departments of 1Obstetrics and Gynecology, 2Medical Research, and 8Pediatrics, Mackay Memorial Hospital, 5Institute of Clinical and Community Health Nursing, 6Department of Obstetrics and Gynecology, School of Medicine,
and 10Faculty of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Departments of 7Pediatrics and 11Obstetrics and Gynecology, Taipei Medical University-Shuang Ho Hospital, 9Department of
Medical Genetics, National Taiwan University Hospital, 12Department of Obstetrics and Gynecology, Taipei Medical University-Wan Fang Hospital, and 13Department of Bioengineering,
Tatung University, Taipei; 3Department of Biotechnology, Asia University, and 4School of Chinese Medicine, College of Chinese Medicine,
China Medical University, Taichung, Taiwan.
SUMMARY
Objective:To present the perinatal findings and molecular cytogenetic analysis of a rare chromosomal abnor-mality involving structural and numerical abnormalities of chromosome 18.
Materials, Methods and Results:A 36-year-old woman, gravida 5, para 3, underwent amniocentesis because of her advanced maternal age. Amniocentesis revealed a karyotype of 46,XY,r(18) [27]/45,XY,-18[5]/46,XY[5]. The parents decided to continue the pregnancy. Level II ultrasound revealed ventriculomegaly. At 38 weeks of gestation, a 3,725 g male fetus was delivered. The fetus had microcephaly, hypertelorism, epicanthal folds, cleft palate, a broad flat nose, simian creases, broad hands, tapered fingers, clubfeet, micropenis, a sacral dimple, hypotonia, ventriculomegaly, and a ventricular septal defect. The peripheral blood lymphocytes revealed a kary-otype of 46,XY,r(18)[81]/45,XY,-18[3]/46,XY,idic r(18)[3]/46,XY[13]. Fluorescence in situ hybridization using chromosome 18 centromeric probe (cep18) and subtelomeric (18pter, 18qter) identified four types of cells, r(18), idic r(18), monosomy 18, and disomy 18. Array comparative genomic hybridization analysis of the blood demonstrated a 14.9-Mb deletion at chromosome 18p [arr cgh 18p11.32p11.21 (0–14,941,330)× 1] and a 29.6-Mb deletion at chromosome 18q [arr cgh 18q21.2q23 (46,533,430–76,117,153)× 1]. The proband’s karyotype was 46,XY,r(18)(p11.21q21.2)[81]/45,XY,-18[3]/46,XY,idic r(18)(p11.21q21.2;p11.21q21.2)[3]/46,XY[13].
*Correspondence to: Dr Chih-Ping Chen, Department of Obstetrics and Gynecology, Mackay Memorial Hospital,
92, Section 2, Chung-Shan North Road, Taipei, Taiwan. E-mail: cpc_mmh@yahoo.com
Accepted: June 8, 2010
Introduction
The 18q deletion syndrome (OMIM 601808) is caused by variable deletions ranging from 18q21.2, 18q21.3, or 18q22.2 to 18qter [1–3]. The diverse phenotypic fea-tures of the 18q deletion syndrome include low birth weight, short stature, microcephaly, midfacial hypopla-sia, prognathism, a carp-shaped mouth, a protuberant lower lip, dysplastic ears with prominent antihelix and antitragus, abnormal skull, vertebrae and ribs, atretic ear canals, clubfeet, vertical tali, tapered fingers, dimples over limb joints, hypoplasia of labia or scrotum, micro-penis, cryptorchidism, hypospadias, nystagmus, strabis-mus, glaucoma, tapetoretinal degeneration, bilateral optic atrophy, hypotonia, seizures, deafness, enlarged ventricles, hydrocephalus, porencephaly, holoprosen-cephaly (HPE), cerebellar hypoplasia, decreased white matter, impaired or delayed myelination, and congeni-tal heart defects [3–12].
The clinical phenotype of the 18p deletion syndrome (OMIM 146390) usually includes growth and mental retardation, hypotonia, epicanthic folds, ptosis, a low nasal bridge, a rounded face, micrognathia, a short neck, abnormal ears, small hands and feet, clinodactyly of the fifth finger, cardiac defects, abnormal genitalia, and cerebral malformations [13–15].
A ring chromosome 18, or r(18) exhibits breakage and reunion at the breakpoints on the long and short arms of chromosome 18, with deletions of the chromo-somal segments distal to the breakpoints. The r(18) phenotype is associated with the anomalies of 18p deletion and 18q deletion, and can be associated with the features of both 18p deletion and 18q deletion syn-dromes [16]. Here, we present the perinatal findings and array comparative genomic hybridization (aCGH) char-acterization of a rare chromosomal abnormality asso-ciated with different cell lines involving structural and numerical abnormalities of chromosome 18.
Materials, Methods and Results
A 36-year-old woman, gravida 5, para 3, underwent amniocentesis at a community obstetric clinic because
of her advanced maternal age. Her husband was 40 years old. She and her husband were both healthy and unre-lated, and there was no family history of congenital malformations. The parents had two healthy daughters, one was 12 years old and the other was 9 years old. Amniocentesis at 16 weeks of gestation revealed a male fetus with mosaic ring chromosome 18 [r(18)], monosomy 18 and disomy 18, or 46,XY,r(18)[27]/ 45,XY,-18[5]/46,XY[5]. The parents decided to continue the pregnancy. Level II ultrasound was unremarkable, except for ventriculomegaly. A 3,725 g male fetus was delivered uneventfully at 38 weeks of gestation. The baby had a head circumference of 33 cm (15thcentile) and a
body length of 51 cm (60thcentile). On examination, the baby manifested microcephaly, hypertelorism, epicanthal folds, cleft palate, a broad flat nose, simian creases, broad hands, tapered fingers, clubfeet, micropenis, a sacral dimple, and hypotonia. Brain ultrasound revealed ventriculomegaly, and echocardiography revealed a ven-tricular septal defect (VSD). Conventional cytogenetic analysis of peripheral blood lymphocytes revealed a karyotype of 46,XY,r(18)[81]/45,XY,-18[3]/46,XY,idic r(18)[3]/46,XY[13] (Figures 1–4). The deletion of dis-tal 18p and disdis-tal 18q on r(18) and idic r(18), and the duplication of the centromere of chromosome 18 were demonstrated by fluorescence in situ hybridization (FISH) using an 18p terminal probe (18pter, RP11-324G2) Conclusion:Array comparative genomic hybridization is useful to determine the breakpoints of a ring chromo-some, particularly in cases where the ring chromosome comprises the majority of the mosaicism. [Taiwan J
Obstet Gynecol 2010;49(3):327–332]
Key Words:array comparative genomic hybridization, fluorescence in situ hybridization, mosaic ring chromosome, prenatal diagnosis, ring chromosome 18 duplication/deletion
1 6 13 19 20 21 22 Y X 14 15 16 17 r(18) 18 7 8 9 10 11 12 2 3 4 5
(spectrum green) at 18p11.32, an 18q terminal probe (18qter, RP11-89N1) (spectrum yellow) at 18q23, and an 18 centromeric probe (cep18) (spectrum red) (TelVysion, Downers Grove, IL, USA) (Figure 5). A normal
chromosome 18 contained one cep18 signal (red), one 18pter signal (green), and one 18qter (yellow). The r(18) contained only one cep18 signal (red) and lacked the 18pter signal (green) and the 18qter signal (yellow). The idic r(18) contained two cep18 signals (red) and lacked the 18pter signal (green) and the 18qter signal (yellow). A monosomy 18 cell contained only one normal chromosome 18. A disomy 18 cell contained only two normal chromosomes 18. Oligonucleotide-based aCGH of the blood using Oligo HD Scan (CMDX, Irvine, CA, USA) revealed a 14.9-Mb deletion at chro-mosome 18p [arr cgh 18p11.32p11.21 (0–14,941, 330)× 1] and a 29.6-Mb deletion at chromosome 18q [arr cgh 18q21.2q23 (46,533,430–76,117,153)× 1] (Figure 5). Therefore, the proband’s karyotype was 46, XY,r(18)(p11.21q21.2)[81]/45,XY,-18[3]/46,XY,idic r(18)(p11.21q21.2;p11.21q21.2)[3]/46,XY[13].
Discussion
We previously demonstrated the usefulness of aCGH for prenatal detection of microdeletions and unbal-anced translocations [17,18]. In this report, we also demonstrate the use of aCGH to determine the break-points of a ring chromosome in a case in which the ring chromosome comprises the majority of the mosaicism. The present case had high-level mosaicism for r(18) and low-level mosaicism for disomy 18, r(18) duplication and r(18) deletion. The aCGH findings were consistent with a deletion of 18p11.21pter and a deletion of 18q21.2qter in the r(18). aCGH can detect DNA dosage imbalances, including deletions and duplica-tions, but shows limitations for the detection of low-level mosaicism, balanced translocations, inversions and polyploidy. Recent studies have suggested that aCGH can detect as little as 20% mosaicism in peripheral blood cells [19,20]. In this study, aCGH was unable to detect low-level mosaicism.
The present case had a 29.6-Mb 18q deletion, a 14.9-Mb 18p deletion, and phenotypic abnormalities included microcephaly, ventriculomegaly, clubfeet, ab-normal external genitalia, ventricular septal defect, cleft palate, hypotonia, and facial dysmorphism. The 18q 21.2qter deletion in this case encompassed the criti-cal regions for orofacial cleft, microcephaly, and the typ-ical 18q deletion phenotype. Dostal et al [21] suggested that the potential critical region for orofacial cleft is at 18q22.3, between markers D18S879 and D18S1141, and contains the orofacial cleft candidate genes, SALL3 and TSHZ1. Feenstra et al [12] suggested the critical region for the typical phenotype of 18q deletion syn-drome is 4.3-Mb region located within 18q22.3-q23, 1 6 13 19 20 21 22 Y X 14 15 16 17 18 idic r(18) 7 8 9 10 11 12 2 3 4 5
Figure 2. A karyotype of 46,XY,idic r(18)(p11.21q21.2; p11.21q21.2). 1 6 13 19 20 21 22 Y X 14 15 16 17 18 7 8 9 10 11 12 2 3 4 5
Figure 4.A karyotype of 46,XY.
1 6 13 19 20 21 22 Y X 14 15 16 17 18 7 8 9 10 11 12 2 3 4 5
and the critical region for microcephaly is at 18q21.33. The 18p11.21pter deletion in this case encompassed HPE4. However, the present case lacked the HPE phe-notype. An HPE critical region on 18p11.3 has been defined as HPE4 (OMIM 142946) [22] and the respon-sible gene is TGIF (OMIM 602630) [23]. Only ∼10% of cases with 18p deletion have HPE [13]. The low incidence of HPE in patients with 18p deletion and TGIF haploinsufficiency is because of the autosomal-dominant inheritance pattern of TGIF with low pene-trance [23], and the requirement for other genetic or environmental factors [24]. Nanni et al [25] suggested that either maternal retinoic acid levels or altered activity in another protein can modify the effects of TGIF, and thus lead to phenotypic variability among patients with a TGIF deletion.
Prenatal diagnosis of mosaic r(18) with r(18) dele-tion/duplication is rare [26–29]. Eiben et al [26] re-ported prenatal diagnosis of 45,XX,-18/46,XX,r(18) (p11q12) by amniocentesis in a fetus with cebocephaly and HPE. The skin fibroblasts had 26.7% (8/30) of
monosomy 18 and 73.3% (22/30) of r(18). Fischer et al [27] reported prenatal diagnosis of 46,XY,r(18)(p11 q23)[103]/45,XY,-18[13] by amniocentesis in a fetus with amniotic band syndrome. The cord blood had a karyotype of 46,XY,r(18). Carreira et al [28] reported prenatal diagnosis of mosaic r(18) and r(18) deletion/ duplication in two cases, and used multicolor banding to determine the breakpoints. In the first case, a fetus with HPE, 46,XY,r(18)(p11.1q22)[36]/45,XY,-18[7]/ 47,XY,-18,+r(18)(p11.1q22) × 2[1]/46,XY,dupr(18) (p11.1q22)[1] was diagnosed by amniocentesis. Post-natal cytogenetic analysis of fetal tissue revealed a karyo-type of 46,XY,r(18)[89]/45,XY,-18[22]/46,XY,dupr(18) [2]/46,XY,mar(18)[4]. In the second case, a fetus with congenital heart defects and obstructive uropathy, 46, XY,r(18)(p11.22q21.2)[12]/46,XY,r(18;18)(p11.22 q21.2;p11.22q21.2)[4]/47,XY,-18,+r(18)(p11.22 q21.2)× 2[1]/del(18)(:p11.21q11.2:)[1]/ace(18) (:q11.2q12.2:)[1] was diagnosed by amniocentesis. Mello et al [29] reported prenatal diagnosis of 46,XY, t(3;6)(p26.2;q15),r(18)[22]/45,XY,t(3;6)(p26.2; A 18 18 18 r(18) idic r(18) 18 18 B C D
Figure 5. Fluorescence in situ hybridization studies of four types of cells with an 18p terminal probe RP11-324G2 (spectrum green), an 18q terminal probe RP11-89N1 (spectrum yellow) and an 18 centromeric probe (spectrum red). (A) A metaphase cell with monosomy 18 and one chromosome 18 with three signals (green, red and yellow). (B) A metaphase cell with disomy 18 and two chromosomes 18. (C) A metaphase cell with r(18) and one chromosome 18 and one r(18), which has only one red signal. (D) A metaphase cell with idic r(18) and one chromosome 18 and one idic r(18), which has two red signals. r(18)= ring chromosome 18; idic r(18) = isodicentric ring chromosome 18; 18 = chromosome 18.
q15),-18[6] by amniocentesis in a fetus with a single umbilical artery, an increased nuchal fold thickness and tricuspid regurgitation. aCGH revealed a 2.8-Mb of 18p11.31p11.32 deletion and a 22.5-Mb 18q21.3 q23 deletion. The fetus was carried to term and had
microcephaly, micropenis, a short stature, congenital heart defects, a high-arched palate, abnormal hands, and facial dysmorphism.
To our knowledge, this is the first report of mosaic r(18), r(18) deletion/duplication and disomy 18 in which
10 Mb 20 Mb 30 Mb 40 Mb 50 Mb 60 Mb 70 Mb Genes Segmental Dups Decipher syndromes BacHDscanV2 Agilent 105KV6 CNVs EXons 180Kv1_CMDX 2.5 2.0 1.5 1.0 0.5 0.0 –0.5 –1.0 –1.5 –2.0 –2.5 0 p11.31 p11.21 q11.2 q12.1 q12.2 q12.3 q21.1 q21.2 q22.1 q22.3 q23 Chromosome 18 Chromosome 18 5.0 Mb 7.5 Mb 10.0 Mb 12.5 Mb 15.0 Mb 17.5 Mb 20.0 Mb Genes Segmental Dups Decipher syndromes BacHDscanV2 Agilent 105KV6 CNVs EXons 180Kv1_CMDX 2.5 2.0 1.5 1.0 0.5 0.0 –0.5 –1.0 –1.5 –2.0 –2.5 0 2.5 Mb p11.32 p11.31 p11.23 p11.22 p11.21 q11.2 Zoom-in Chromosome 18 Genes Segmental Dups Decipher syndromes BacHDscanV2 Agilent 105KV6 CNVs EXons 180Kv1_CMDX 2.5 2.0 1.5 1.0 0.5 0.0 –0.5 –1.0 –1.5 –2.0 –2.5 0 45 Mb 50 Mb 55 Mb 60 Mb 65 Mb 70 Mb 75 Mb q21.1 q21.2 q21.31 q21.32 q21.33 q22.1 q22.2 q22.3 q23 RP11-959J9 .2 Zoom-in Chromosome 18 2.50 2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 –0.25 –0.50 –0.75 –1.00 –1.25 –1.50 –1.75 –2.00 –2.25 –2.50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y 0.00
Whole genome view
Figure 6.Oligonucleotide-based array comparative genomic hybridization shows a 14.9-Mb deletion (0–14,941,330 bp) of the short arm of chromosome 18 and a 29.6-Mb deletion (46,533,430–76,117,153 bp) of the long arm of chromosome 18.
the baby was carried to term. Perinatally, this case rep-resents a difficulty for genetic counseling because of the complexity of the karyotype and the molecular evi-dence of large deletions of 18p and 18q. The parents elected to continue the pregnancy based on a desire for a son. Although the proband has many features of both 18p and 18q deletion syndromes, he is making great progress and surviving the neonatal period.
Acknowledgments
This work was supported by research grants NSC-96-2314-B-195-008-MY3 and NSC-97-2314-B-195-006-MY3 from the National Science Council, and MMH-E-99004 from Mackay Memorial Hospital, Taipei, Taiwan.
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