Short Communication
Interphase FISH characterization of mosaicism using uncultured amniocytes
and cultured stimulated cord blood lymphocytes in prenatally detected
Pallister-Killian syndrome
Chih-Ping Chen a,b,c,d,e,f *, Cheng-Ran Peng a, Schu-Rern Chern b, Yu-Ling Kuo g, Peih-Shan Wu h, Dai-Dyi Town a, Chen-Wen Pan a, Chien-Wen Yang b and Wayseen Wang b,i
a Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan b Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan
c Department of Biotechnology, Asia University, Taichung, Taiwan
d School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan e Institute of Clinical and Community Health Nursing, National Yang-Ming University, Taipei, Taiwan f Department of Obstetrics and Gynecology, School of Medicine, National Yang-Ming University, Taipei,
Taiwan
g Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical
University, Kaohsiung, Taiwan
h Gene Biodesign Co. Ltd, Taipei, Taiwan
i 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: cpc_mmh@yahoo.com
Abstract
Objective: To present molecular cytogenetic characterization of Pallister-Killian syndrome (PKS). Materials and methods: A 37-year-old woman underwent amniocentesis at 18 weeks of gestation.
Amniocentesis revealed a karyotype of 47,XY,+i(12)(p10)[6]/48,XY,+i(12)(p10)2[1]/ 46,XY[6]. Repeated amniocentesis was performed at 20 weeks of gestation. aCGH was performed using uncultured amniocytes, cord blood and skin. QF-PCR was performed using uncultured amniocytes and parental bloods. Interphase FISH analysis was performed using uncultured amniocytes and cultured stimulated cord blood lymphocytes. Conventional cytogenetic analysis was performed using cultured cells from amniotic fluid, skin, placenta, umbilical cord and cord blood.
Results: Repeated amniocentesis revealed a mosaic tetrasomy 12p level of 25% (10/40), cultured
cord blood lymphocytes had no mosaicism, cultured skin fibroblasts had a mosaic tetrasomy 12p level of 52.5% (21/40), umbilical cord fibroblasts had a mosaic tetrasomy 12p level of 72.5% (29/40), and the placental cells had a mosaic tetrasomy 12p level of 2.5% (1/40). aCGH analysis revealed that the gene dosage increase in 12p for uncultured amniocytes, skin and cord blood was the log2 ratio of 0.9, 0.7 and 0.7, respectively. Interphase FISH on uncultured amniocytes revealed a mosaic level of 73.1% (49/67) (tetrasomy 12p: 33; hexasomy 12p: 16). Interphase FISH analysis of stimulated cultured cord blood lymphocytes revealed a mosaic level of 58.3% (60/103) (tetrasomy 12p: 51; hexasomy 12p: 9).
Conclusions: In the diagnosis of PKS by conventional culture cytogenetics, cord blood samplings
and placental samplings are prone to a negative result when compared with amniocentesis. Whenever cord blood sampling is applied for prenatal diagnosis of PKS, aCGH on uncultured cord blood or interphase FISH on cultured cord blood can be used for the diagnosis in addition to conventional cytogenetics.
Key words: blood lymphocyte, interphase FISH, mosaic tetrasomy 12p, Pallister-Killian
Introduction
Pallister-Killian syndrome (PKS; OMIM 601803), a dysmorphic syndrome characterized by tissue-limited mosaicism for tetrasomy 12p because of a supernumerary isochromosome for the short arm of chromosome 12 [i(12)(p10)], has the clinical features of dysmorphic facial appearance of prominent forehead, sparse anterior scalp hair, hypertelorism, short nose, flat nasal bridge, a short neck and flat occiput, mental retardation, seizures, and pigmentary skin lesions [1-5]. Hexasomy 12p caused by the presence of two copies of i(12)(p10) may present in individuals with PKS. The population of abnormal cells with i(12)(p10) in lymphocytes and fibroblasts does not correlate with the severity of clinical phenotype [1].
Here, we present our experience of molecular cytogenetic characterization of mosaicism using uncultured amniocytes and cultured phytohemagglutinin (PHA)-stimulated cord blood lymphocytes in prenatally detected PKS.
Materials and Methods
Clinical description
A 37-year-old, gravida 2, para 0, woman underwent amniocentesis at 18 weeks of gestation because of advanced maternal age. Her husband was 30 years old. She and her husband were non-consanguineous and healthy, and there was no family history of congenital malformations. Prenatal ultrasound findings were unremarkable. Amniocentesis revealed a karyotype of 47,XY, +i(12) (p10)[6]/48,XY,+i(12)(p10)2[1]/46,XY[6]. The parental karyotypes were normal. The woman requested repeated amniocentesis which was performed at 20 weeks of gestation. Array comparative genomic hybridization (aCGH) was performed on the DNA extracted from uncultured amniocytes to determine the genomic imbalance. Quantitative fluorescent polymerase chain reaction (QF-PCR) analysis was performed on the DNA extracted from uncultured amniocytes and parental bloods to determine the parental origin of the chromosomal aberration. Interphase fluorescence in situ hybridization (FISH) analysis was performed on uncultured amniocytes to determine the level of mosaicism. Conventional cytogenetic analysis was performed on cultured amniocytes. Repeated amniocentesis confirmed a diagnosis of PKS with a high level of mosaicism. The pregnancy was subsequently terminated at 22 weeks of gestation, and a 576-g malformed male fetus was delivered. Conventional cytogenetic analysis was performed on cultured skin fibroblasts, placental cells, umbilical cord cells and cord blood lymphocytes. Interphase FISH analysis was
performed on cultured PHA-stimulated cord blood lymphocytes. aCGH analysis was performed on the DNA extracted from uncultured cord blood and skin samples.
Methods for detection
1. Conventional cytogenetic analysis
Routine cytogenetic analysis of G-banding techniques was performed. About 20 mL of amniotic fluid was collected, and the sample was subjected to in situ amniocyte culture according to the standard cytogenetic protocol. Placental tissue, umbilical cord, skin and cord blood were collected at delivery, and the samples were subjected to culture according to the standard cytogenetic protocol.
2. QF-PCR
QF-PCR analysis was performed on the DNA extracted from uncultured amniocytes acquired through 5 mL of amniotic fluid. Informative polymorphic DNA markers of D12S823 (12p12.1) and D12S390 (12q13.3) were used to determine the parental origin of the duplication.
3. FISH
Interphase FISH analysis was performed on uncultured amniocytes and cultured PHA-stimulated cord blood lymphocytes using a 12p11.21-specific bacterial artificial chromosome (BAC) probe RP11-205G1 (dye FITC) (31,197,953-31,361,902) [hg19] and a 12q24.21-specific BAC probe RP11-119J23 (dye Texas red) (115,504,136-115,667,155) [hg19] according to the standard FISH protocol.
4. aCGH
Whole-genome aCGH on the DNA extracted from uncultured amniocytes derived from 10 mL of amniotic fluid, uncultured skin sample and uncultured cord blood was performed using NimbleGen ISCA Plus Cytogenetic Array (Roche NimbleGen, Madison, WI, USA). The NimbleGen ISCA Plus Cytogenetic Array has 630,000 probes and a median resolution of 15-20 kb across the entire genome according to the manufacturer's instruction. The DNA from the cells in the collected samples was extracted first. It was done by following the manufacturer's protocol of QIAamp DNA Mini kit (Qiagen, Inc., Valencia, CA, USA). Then, the 0.5μg of the extracted DNA was labeled in Cy5 dye compared with equivalent amount of normal female gDNA (G1521, Promega) labeled in Cy3 dye to perform the aCGH experiment. The experiment was performed according to the procedures recommended from Roche NimbleGen ISCA plus Cytogenetic Array's user guide. The
Results
Conventional cytogenetic analysis revealed a karyotype of 47,XY,+idic(12)(q12)[10]/46,XY[30] in cultured amniocytes at repeated amniocentesis, a karyotype of 46,XY (40 cells) in cultured cord blood lymphocytes, a karyotype of 47,XY,+idic(12)(q12)[21]/46,XY[19] in cultured skin fibroblasts, a karyotype of 47,XY,+idic(12)(q12)[29]/46,XY[11] in cultured umbilical cord fibroblasts, and a karyotype of 47,XY,+idic(12)(q12)[1]/46,XY[39] in cultured placental cells (Fig. 1). aCGH analysis revealed a positive gene dosage increase in 12p13.33-12q12, or arr 12p13.33q12 (1-40,154,456)3.73 in uncultured amniocytes, and a positive gene dosage increase in 12p13.33-12q12, or arr 12p13.33q12 (1-40,166,628)3.24 in uncultured cord blood and arr 12p13.33q12 (184,873-40,154,456)3.24 in uncultured skin. The log2 ratio for uncultured amniocytes, uncultured skin and uncultured cord blood was 0.9, 0.7 and 0.7, respectively (Fig. 2). Interphase FISH analysis of 67 uncultured amniocytes showed that 18 cells had disomy 12p, 33 cells had tetrasomy 12p, and 16 cells had hexasomy 12p (Fig. 3). Interphase FISH analysis of 103 PHA-stimulated cultured cord blood interphase lymphocytes showed that 43 cells had disomy 12p, 51 cells had tetrasomy 12p, and 9 cells had hexasomy 12p (Fig. 4). QF-PCR assays showed a 3.14-fold increase of the maternal allele in the informative marker of D12S823 (12p12.1) with a maternal allele (143bp): paternal allele (147bp) ratio of 4.14:1, indicating a maternal origin of the duplication (Fig. 5). The malformed fetus manifested a prominent forehead, low-set ears, hypertelorism, a short nose with flat nasal bridge, a short neck and flat occiput (Fig. 6).
Discussion
PKS has been well known to be a phenotypic disorder for tissue-limited mosaicism which is characterized by a normal karyotype in cultured blood lymphocytes, and the cytogenetic diagnosis principally relies on the analysis of cultured fibroblasts [6]. Prenatal diagnosis of PKS may result in a false-negative result because of tissue-limited mosaicism. In a meta-analysis of 28 cases with cytogenetic discrepancy or variability among various tissue samplings in prenatally or postnatally detected PKS, Chen et al [2] observed that a false-negative diagnostic result can occur in 55% (12/22) of the cases from blood lymphocyte cultures, 43% (3/7) of the cases from short-term cultures/direct preparations of chorionic villus sampling (CVS), 50% (1/2) of the case from long-term cultures of CVS, and a 12% (3/25) of the cases from amniocyte cultures. It has been suggested that the abnormal cells with tetrasomy 12p in blood lymphocyte and amniocyte cultures are less stable than those in the fibroblast cultures derived from skin and other tissues, and the
young cultures at early passage are likely to have more abnormal cells with tetrasomy 12p than the old cultures [7]. During amniocyte subcultures, a rapid decrease of abnormal clones with tetrasomy 12p has been observed in PKS [8], and the cell death of abnormal clones with tetrasomy 12p has been implicated as the cause of tissue-limited mosaicism in PKS [9].
We previously applied interphase FISH on uncultured amniocytes for rapid confirmation of mosaicism at amniocentesis [10,11]. The present case provides evidence for the usefulness of interphase FISH on uncultured amniocytes and cultured cord blood lymphocytes for the confirmatory diagnosis of the true mosaic level as well as the different cell lines of abnormal cells in cases of PKS detected prenatally by conventional amniocyte cultures. In the present case, first amniocentesis revealed a mosaic level of 53.8% (7/13 colonies) (tetrasomy 12p: hexasomy 12p: disomy 12p = 6:1:6), repeated amniocentesis revealed a mosaic level of 25% (10/40 colonies) (tetrasomy 12p: disomy 12p = 10:30), cultured cord blood lymphocytes had no mosaicism, cultured skin fibroblasts had a mosaic level of 52.5% (21/40 cells) (tetrasomy 12p: disomy 12p = 21:19), umbilical cord fibroblasts had a mosaic level of 72.5% (29/40 cells) (tetrasomy 12p: disomy 12p = 29:11), and the placental cells had a mosaic level of 2.5% (1/40 cells) (tetrasomy 12p: disomy 12p = 1:39). Array CGH analysis revealed that the positive gene dosage increase in 12p for uncultured amniocytes, uncultured skin and uncultured cord blood was the log2 ratio of 0.9, 0.7 and 0.7, respectively. Interphase FISH analysis of uncultured amniocytes revealed a mosaic level of 73.1% (49/67 cells) (tetrasomy 12p: hexasomy 12p: disomy 12p = 33:16:18). Interphase FISH analysis of PHA-stimulated cultured cord blood interphase lymphocytes revealed a mosaic level of 58.3% (60/103 cells) (tetrasomy 12p: hexasomy 12p: disomy 12p = 51:9:43). The present case shows that in the diagnosis of PKS by conventional culture cytogenetics, cord blood samplings and placental samplings may be prone to a negative result when compared with amniocentesis. However, the present case confirms that in cases of cord blood samplings, by use of aCGH, cord blood DNA is useful for confirmation of PKS, and, by use of interphase FISH, interphase cord blood lymphocytes are useful for confirmation of PKS. Therefore, whenever cord blood sampling is applied for prenatal diagnosis of PKS, aCGH on uncultured cord blood or interphase FISH on cultured cord blood can be used for the diagnosis in addition to conventional cytogenetic analysis.
The present case had a maternal origin of the supernumerary isochromosome 12p. Dutly et al [12] found that the origin of the supernumerary isochromosome 12p is predominantly maternal because of maternal meiosis II non-disjunction followed by rearrangements leading to a duplication
of the short arm and a loss of the long arm. However, in rare occasions, the supernumerary isochromosome 12p can be of paternal origin and may occur post-zygotically [13].
Ballif et al [14] first reported the detection of a previously unsuspected case associated with PKS by aCGH using peripheral blood DNA and suggested that stimulated peripheral blood cultures may distort the percentage of abnormal cells and thus make the detection of PKS unlikely by conventional cytogenetic analysis using cultured lymphocytes. Cheung et al [15], Powis et al [16] and Theisen et al [17] later demonstrated the usefulness of aCGH using peripheral blood DNA to detect chromosomal mosaicism including PKS not revealed by conventional blood cytogenetics. However, aCGH on unstimulated blood has been shown to fail to detect all cases of PKS because of the limitation of aCGH to detect the cases with the tetrasomy 12p at less than 10% of the mosaic level [18]. Either a skin biopsy [18] or a buccal smear analysis [19] has been suggested as a diagnostic gold standard for PKS in postnatal cases.
Previous reports have shown that mosaic tetrasomy 12p is usually detected in cultured skin fibroblasts or amniocytes and rarely in cultured lymphocytes. In the present case, the interphase stimulated cultured T-lymphocytes showed a 58.3% mosaic level compared to a 52.5% mosaic level of cultured metaphase skin fibroblasts and no abnormal mosaicism in cultured metaphase stimulated cultured T-lymphocytes. The stimulation of T-lymphocytes has been suggested to distort the percentage of blood cells in PKS [15,17,20]. However, the present case provides evidence for cytogenetic discrepancy between interphase stimulated cultured lymphocytes and metaphase stimulated cultured lymphocytes in cases with PKS and suggests that abnormal cells still can be detected by interphase FISH in the blood slides prepared by conventional cytogenetic procedures in cases with PKS.
In summary, we present our novel observation of the detection of tetrasomy 12p and hexasomy 12p by interphase FISH in the cultured interphase cord blood cells following PHA stimulation in a PKS fetus with a karyotype of 46,XY in the cultured metaphase lymphocytes and a 12p dosage increase in the uncultured cord blood DNA.
Acknowledgements
This work was supported by research grants NSC-101-2314-B-195-011-MY3 and MOST 103-2314-B-195-010 from the Ministry of Science and Technology and MMH-E-103-04 from Mackay Memorial Hospital, Taipei, Taiwan.
References
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Figure Legends
Fig. 1. A karyotype of tetrasomy 12p. The arrow indicates a supernumerary isochromosome 12p.
Fig. 2. Array comparative genomic hybridization analysis shows a positive gene dosage increase in 12p in (A) uncultured amniocytes, (B) uncultured skin and (C) uncultured cord blood with the log2 ratios of 0.9, 0.7 and 0.7 for uncultured amniocytes, uncultured skin and uncultured cord blood, respectively. Fig. 3. Interphase fluorescence in situ hybridization (FISH) analysis of uncultured amniocytes shows (A)
presence of two red signals (RP11-119J23 (12q24.21) and two green signals (RP11-205G1), (12p11.21) in a cell with disomy 12p, (B) presence of two red signals and four green signals (arrows) in a cell with tetrasomy 12p, and (C) presence of two red signals and six green signals (arrows) in a cell with hexasomy 12p.
Fig. 4. Interphase FISH analysis of phytohemagglutinin-stimulated cultured cord blood interphase lymphocytes shows (A) presence of two red signals and four green signals (arrows) in a cell with tetrasomy 12p, and (B) presence of two red signals and six green signals (arrows) in a cell with hexasomy 12p.
Fig. 5. Representative electrophoretogram of quantitative fluorescent polymerase chain reaction assay using informative polymorphic markers of D12S823 (12p12.1) and D12S390 (12q13.3). The marker D12S390 (12q13.3) is outside the duplicated region and shows two peaks of equal fluorescent activity from two different parental alleles in the fetus. The marker D12S823 (12p12.1) is within the duplicated region and shows a 3.14-fold increase of the maternal allele with a maternal allele (143bp): paternal allele (147bp) ratio of 4.14:1.
