RESEARCH LETTER
Usefulness of interphase FISH on uncultured amniocytes for rapid
confirmation of low-level trisomy 7 mosaicism in a pregnancy with fetal
intrauterine growth restriction and microcephaly
Chih-Ping Chen a,b,c,d,e,f,g *, Yi-Ning Su h, Yi-Yung Chen b, Schu-Rern Chern c, Jun-Wei Su b,i, Yu-Ting Chen c, Dai-Dyi Town b and Wayseen Wang c,j
a Department of Medicine, Mackay Medical College, New Taipei City, 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 Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan
i Department of Obstetrics and Gynecology, China Medical University Hospital, Taichung, Taiwan j Department of Bioengineering, Tatung University, Taipei, Taiwan
* Correspondence to: Chih-Ping Chen, MD
Department of Obstetrics and Gynecology, Mackay Memorial Hospital 92, Section 2, Chung-Shan North Road, Taipei, Taiwan
Tel: +886-2-25433535; Fax: +886-2-25433642, +886-2-25232448 E-mail: [email protected]
Genetic counseling for trisomy 7 mosaicism at amniocentesis remains difficult because of the phenotypic variability [1]. Trisomy 7 mosaicism may present variable features ranging from normal to facial dysmorphisms, sparse hair, enamel dysplasia, pigmentary abnormalities, hypomelanosis of Ito, radial defects, Potter syndrome, Blaschkolinear malformation syndrome and Goldenhar syndrome [2]. Trisomy 7 mosaicism may also be associated with maternal uniparental disomy for chromosome 7 (UPD 7) and Silver-Russell syndrome (SRS) [3-6]. Here, we report prenatal diagnosis of low-level trisomy 7 mosaicism by amniocentesis using cultured and uncultured amniocytes in the second trimester in a pregnancy with fetal intrauterine growth restriction (IUGR) and microcephaly. In this presentation, we demonstrate the application of molecular cytogenetic technologies in uncultured amniocytes for rapid confirmation of low-level trisomy 7 mosaicism and exclusion of UPD 7.
A 30-year-old primigravid woman underwent amniocentesis at 24 weeks of gestation because of microcephaly and intrauterine growth restriction (IUGR). Prenatal ultrasound revealed a singleton fetus with a biparietal diameter (BPD) of 4.3 cm (17 weeks), an abdominal circumference (AC) of 16.2 cm (20 weeks) and a femur length (FL) of 3.5 cm (21 weeks). Array comparative genomic hybridization (aCGH) investigation using CytoChip Oligo Array (BlueGnome, Cambridge, UK) on uncultured amniocytes revealed no gene dosage variation in all chromosomes (Fig. 1). In three out of 25 separated colonies of cultured amniocytes, an abnormal karyotype of 47,XX,+7 was noted, while the other 22 colonies had a karyotype of 46,XX (Fig. 2). The cytogenetic result of amniocentesis was 47,XX,+7[3]/46,XX[22]. The parental karyotypes were normal.
She underwent repeated amniocentesis at 26 weeks of gestation. Prenatal ultrasound at 26 weeks of gestation revealed a BPD of 5.35 cm (22 weeks), an AC of 17.71 cm (22 weeks) and an FL of 4.27 cm (24 weeks). Interphase fluorescence in situ hybridization (FISH), quantitative fluorescent polymerase chain reaction (QF-PCR) and a PEG1/MEST methylation-sensitive high-resolution melting PCR assay were applied to the uncultured amniocytes. Interphase FISH analysis on uncultured amniocytes using a 7q11.1-specific probe (RP11-432A1) showed three 7q-specific signals in 6/30 (20%) and two signals in 24/30 (80%) of uncultured amniocytes (Fig. 3).
Polymorphic DNA marker analysis of uncultured amniocytes using QF-PCR and microsatellite markers specific for chromosome 7 revealed a biparental diallelic pattern with equal biparental inheritance of chromosome 7 and equal dosage in the biparental alleles (Fig. 4). The methylation-specific PCR assay identified the differential methylation of the imprinted
PEG1/MEST locus on 7q32 and revealed biparental inheritance of chromosome 7 in uncultured
amniocytes (Fig. 5). The cytogenetic result of the repeated amniocentesis was 46,XX (39/39 colonies). The aCGH investigation using CytoChip Oligo Array (BlueGnome, Cambridge, UK) on uncultured amniocytes did not detect genomic imbalance in chromosome 7.
Prenatal ultrasound at 30 weeks of gestation revealed a BPD of 5.67 cm (24 weeks), an AC of 19.56 cm (24 weeks), an FL of 4.8 cm (26 weeks), ventriculomegaly, microcephaly, micrognathia, a narrow chest and oligohydramnios. The fetus had intrauterine fetal death at 31 weeks. A 936-g malformed fetus was delivered with microcephaly, hypertelorism, micrognathia, a depressed nasal bridge and low-set ears (Fig. 6). Cytogenetic analysis of the amniotic membrane, placenta, umbilical cord and skin revealed a karyotype of 46,XX in 40 cells in all samples. The cultures of the blood and the tissues from lungs and liver were not successful. The present case provides evidence for the usefulness of application of interphase FISH on uncultured amniocytes for rapid confirmation of low-level trisomy 7 mosaicism at amniocentesis. Trisomy 7 mosaicism at amniocentesis has been associated with pseudomosaicism [7], and discrepancy between the cytogenetic results of cultured amniocytes and the molecular results of uncultured amniocytes [1]. In the present case, the first amniocentesis revealed 12% (3/25) mosaicism for trisomy 7 in cultured amniocytes, and the second amniocentesis revealed a normal karyotype in all 39 colonies of cultured amniocytes. In the second amniocentesis, the interphase FISH on uncultured amniocytes revealed 20% (6/30) mosaicism for trisomy 7, but QF-PCR and aCGH failed to detect trisomy 7 mosaicism on uncultured amniocytes. This is because QF-PCR and aCGH have the limitation in detecting low-level mosaicism of trisomies [8-9]. We previously reported the successful detection of trisomy 7 mosaicism by interphase FISH, QF-PCR and aCGH on uncultured amniocytes in a case with a higher level of 26% (13/50) mosaicism for trisomy 7 [2]. In the present case, however, only interphase FISH could detect a
lower level of 20% mosaicism for trisomy 7. Therefore, we suggest that interphase FISH should be the molecular cytogenetic method of choice for rapid confirmation of low-level mosaicism at amniocentesis. Our case also provides evidence for cytogenetic discrepancy on cultured amniocytes in low-level trisomy 7 mosaicism between the first and repeated amniocenteses. We think that the abnormal cell line might have disappeared after cell culture in the repeated amniocentesis.
In this study, cytogenetic analyses of the fetal skin and extraembryonic tissues such as membrane, placenta and cord failed to detect trisomy 7 mosaicism. We think that incomplete tissue samplings and fetal death might explain for this result, although the possibility of an origin of the abnormal cell line in the amnion could not be completely excluded [10-11].
Prenatal diagnosis of trisomy 7 mosaicism should raise a suspicion of UPD 7 and SRS [3-6]. The present case had severe IUGR, facial dysmorphism and microcephaly but without UPD 7. Trisomy 7 mosaicism has variable and nonspecific clinical features. Our case represents a very unusual case with low-level trisomy 7 mosaicism at amniocentesis and an abnormal phenotype of IUGR, microcephaly and facial dysmorphism but without UPD 7.
Acknowledgements
This work was supported by research grant NSC-99-2628-B-195-001-MY3 from the National Science Council and MMH-E-100-04 from Mackay Memorial Hospital, Taipei, Taiwan.
References
1. Chen C-P, Su Y-N, Chern S-R, Hwu Y-M, Lin S-P, Hsu C-H, et al. Mosaic trisomy 7 at amniocentesis: Prenatal diagnosis and molecular genetic analyses. Taiwan J Obstet Gynecol 2010; 49: 333-40.
2. Chen C-P, Huang H-K, Su Y-N, Chern S-R, Su J-W, Lee C-C, et al. Trisomy 7 mosaicism at amniocentesis: interphase FISH, QF-PCR and aCGH analyses on uncultured amniocytes for rapid distinguishing true mosaicism from pseudomosaicism. Taiwan J Obstet Gynecol 2012, in press. 3. Bilimoria KY, Rothenberg JM. Prenatal diagnosis of a trisomy 7/maternal uniparental heterodisomy 7
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5. Flori E, Girodon E, Samama B, Becmeur F, Viville B, Girard-Lemaire F, et al. Trisomy 7 mosaicism, maternal uniparental heterodisomy 7 and Hirschsprung's disease in a child with Silver-Russell syndrome. Eur J Hum Genet 2005; 13: 1013-8.
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7. Chen C-P, Chern S-R, Chen L-F, Chen W-L, Wang W. Prenatal diagnosis of low-level mosaic trisomy 7 by amniocentesis. Prenat Diagn 2005; 24: 1067-9.
8. Chen C-P, Lin M-H, Su Y-N, Chern S-R, Tsai F-J, Wu P-C, et al. Mosaic trisomy 9 at amniocentesis: Prenatal diagnosis and molecular genetic analyses. Taiwan J Obstet Gynecol 2010; 49: 341-50.
9. Chen C-P, Chen M, Pan Y-J, Su Y-N, Chern S-R, Tsai F-J, et al. Prenatal diagnosis of mosaic trisomy 8: Clinical report and literature review. Taiwan J Obstet Gynecol 2011; 50: 331-8.
10.Robinson WP, McFadden DE, Barrett IJ, Kuchinka B, Peñaherrera MS, Bruyère H, et al. Origin of amnion and implications for evaluation of the fetal genotype in cases of mosaicism. Prenat Diagn 2002; 22: 1076-85.
11.Chen C-P, Su Y-N, Lin S-Y, Chern S-R, Chen Y-T, Lee M-S, et al. Prenatal diagnosis of mosaic trisomy 2: discrepancy between molecular cytogenetic analyses of uncultured amniocytes and karyotyping of cultured amniocytes in a pregnancy with severe fetal intrauterine growth restriction.
Figure Legends
Fig. 1. Oligonucleotide-based array comparative genomic hybridization analysis using CytoChip Oligo array on uncultured amniocytes shows no genomic imbalance in chromosome 7.
Fig. 2. A karyotype of 47,XX,+7
Fig. 3. Interphase fluorescence in situ hybridization analysis on uncultured amniocytes using a bacterial artificial chromosome probe RP11-432A1 (7q11.1; spectrum green) shows (A) three green signals in an abnormal cell with trisomy 7 and (B) two green signals in a normal cell with disomy 7. Fig. 4. Representative electrophoretogram of quantitative fluorescent polymerase chain reaction
(QF-PCR) assay at a polymorphic DNA marker specific for chromosome 7 in the amniotic fluid sample with uncultured amniocytes shows a biparental diallelic pattern with equal biparental inheritance of chromosome 7 and equal dosage in the biparental alleles.
Fig. 5. PEG1/MEST methylation-sensitive high-resolution melting PCR assays show (A) a normal wild type (WT) with an unmethylated allele and a methylated allele, (B) a positive control of maternal uniparental disomy for chromosome 7 with only the methylated maternal allele, and (C) the uncultured amniocytes with an unmethylated allele and a methylated allele.
Fig. 6. (A) and (B) The fetus at birth with microcephaly, hypertelorism, micrognathia, a depressed nasal bridge and low-set ears.
