PRENATAL DIAGNOSIS, VOL. 14: 583-588 (1994)

CHARACTERIZATION OF MARKER CHROMOSOMES BY MICRODISSECTION AND

FLUORESCENCE IN SITU HYBRIDIZATION MAYA THANGAVELU*, EUGENE PERGAMENT*, RAFAEL ESPINOSA nrt AND STEFAN K. BOHLANDER~

*Department of Obstetrics and Gynecology, Northwestern University Medical School, Chicago, U. S. A.; ?Department of Medicine (Section of HematologylOncology), University of Chicago, Chicago, U. S. A.

Received June 1993 Revised December 1993

Accepted 21 December I993

SUMMARY We characterized by microdissection and fluorescence in situ hybridization (FISH) two marker chromosomes: (1)

a de novo, acrocentric marker chromosome detected in 88 per cent of the amniotic fluid cells of one of two physically and developmentally normal twins; and (2) a metacentric marker chromosome present in a phenotypically normal female. Analysis of FISH probes developed from the marker chromosomes indicated that the marker chromosomes in cases 1 and 2 were del(l4)(qll) and a derivative chromosome from a Robertsonian translocation, respectively. Microdissection in combination with FISH may prove to be a valuable technique in determining the chromosomal origin of de novo marker chromosomes and unbalanced structural rearrangements detected during prenatal diagnosis.

KEY woms-Marker chromosome, microdissection, in situ hybridization.

INTRODUCTION

Marker chromosomes are chromosomes whose genetic constitution cannot be determined by con- ventional cytogenetic techniques. The frequency of supernumerary marker chromosomes in humans is estimated to be 1.5/1000, with approximately 40 per cent being familial (Sachs et a l , 1987). Marker chromosomes of de novo origin are of particular concern when identified in fetal cells during prena- tal diagnosis. The risk of congenital abnormalities from de novo marker chromosomes detected during prenatal diagnosis may be as high as 13 per cent (confidence limits 6.9-19.1 per cent) (Warburton, 1991). Conventional cytogenetic techniques used in characterizing marker chromosomes include stain- ing of nucleolar organizer regions (NORs) with silver nitrate, C-banding, and distamycin-A/DAPI

Addressee for correspondence: M. Thangavelu, PhD, Department of Obstetrics and Gynecology, Northwestern University Medical School, Room 1564, Prentice Women’s Hospital and Maternity Center, 333 E. Superior St., Chicago, IL 60611, U.S.A.

0 1994 by John Wiley & Sons, Ltd. CCC 0 197-3 85 11941070583-06

staining. Positive NOR staining suggests that the marker chromosome consists of material from the short arms of acrocentric chromosomes. Positive staining with the distamycin-A/DAPI staining techniques suggests the presence of heterochroma- tin material from chromosomes 1, 9, 15, 16, and the Y chromosome. More recently, in situ hybrid- ization with probes derived from pericentric re- peats have been used in determining the origin of chromosomes (Callen et al., 1990, 1991, 1992; Stetten et al., 1992; Crolla et al., 1992; Plattner et al., 1993; Verschraegen-Spae et al., 1993). We have used microdissection and fluorescence in situ hybridization (FISH) in characterizing two supernumerary marker chromosomes.

CASE REPORTS AND METHODS Case 1

A 36-year-old primigravida underwent prenatal diagnosis because of advanced maternal age. Ultrasound revealed a twin gestation and amniocentesis was performed in the 16th week of

584 M. THANGAVELU ET AL

Fig. 1-Partial metaphase cells from cases 1 and 2 before and after microdissection of marker chromosomes (arrows)

pregnancy. A de novo acrocentric marker chro- mosome was detected in 88 per cent of the cultured cells from the amniotic fluid of one of two twins. Postnatally, the marker chromosome was observed in 87 per cent of lymphocytes analysed from the cord blood of this twin. At the age of 18 months, both infants were physically and developmentally normal. The centromere of this maker chromosome stained positive for C-band and did not stain with the distamycin-A/ DAPI staining technique. This chromosome could not be identified on preparations processed for NOR staining with silver nitrate.

Case 2 A 27-year-old woman, P2G1, was scheduled for

amniocentesis due to a triple screen (alpha- fetoprotein, unconjugated oestriol, and human chorionic gonadotropin) suggestive of increased risk of Down syndrome (1 in 47). Ultrasound prior to amniocentesis revealed intrauterine fetal demise at 16 weeks of gestation. The fetus had a large cystic hygroma. Chromosome analysis of the prod- ucts of conception revealed a karyotype of 46,X, +mar. The metacentric marker chromosome was maternal in origin. The maternal karyotype was 47,XX,+mar and was associated with a nor- mal female phenotype. The centromere of this marker chromosome stained positive with C-banding and did not stain with the distamycin- ADAPI staining technique. Both arms of this marker stained positive for NOR with silver nitrate staining.

Microdissection of marker chromosomes Metaphase cells obtained from PHA-stimulated

lymphocytes by conventional cytogenetic tech- niques were spread on coverslips and trypsin- Giemsa banded. Microdissection was performed on an inverted microscope (Zeiss) with fine glass needles that were moved by an electronically con- trolled micromanipulator (Eppendorf, Model 5170) (Fig. 1).

Amplijication and labelling of microdissected DNA Four to eight microdissected chromosomes

were transferred to Eppendorf tubes and amplified by a sequence-independent amplification (SIA) protocol essentially as described by Bohlander et al. (1991, 1992). The amplified pieces were over- laid with 5pl of buffer A [40mM Tris-HC1 (PH7.5), 10mM MgCl,, 50mM NaC1, 5 m M dithiothreitol, 50 pg/ml bovine serum albumin, 300pM of each dNTP, and 3 p M primer A (5'-TGGTAGCTCTTGATCANNN"-3')]. The DNA was denatured at 94°C for 2 min and cooled to 4°C to permit primer A to anneal to random sites. One unit of T7 DNA polymerase (USB: Sequenase version 2.0) was added in 2 . 5 ~ 1 of buffer A and the temperature was ramped to 37°C over an 8-min interval and kept at 37°C for 8 min. After denaturation and annealing, this synthesis step was repeated one more time by adding fresh T7 enzyme in 2 . 5 ~ 1 of buffer A. Polymerase chain reaction (PCR) was carried out by adding 90p1 of buffer B [6.6mM Tris-HC1 (pH9.0), 55mM KC1, 0.01 per cent (w/v) gelatin, 77pM

CHARACTERIZATION OF TWO MARKER CHROMOSOMES 585

Fig. 2-FISH analysis with the probes developed from the marker chromosome in case 1. There is clear hybridization to the centromeres of chromosomes 14 and 22 (arrows)

of each dNTP, 3.33pM primer B (5'-

Taq DNA polymerase]. The underlined part of primer B is identical to the 15 5' nucleotides of primer A. Five low stringency cycles with denatur- ation at 94°C for 50 s, annealing at 42°C for 5 min, a ramp to 72°C for 3 min, and synthesis at 72°C for 3 min were followed by 33 PCR cycles with dena- turation at 94°C for 50s, annealing at 56°C for 1 min, and synthesis at 72'C for 2 min.

A 1 pl aliquot of the SIA products which ranged in size from 200 to 600 bp were labelled in a second PCR reaction by incorporation of Bio-11-dUTP under the following conditions: 1.5 mM MgC12, 50 mM KC1, 10 mM Tris-HC1 (PH %3), 0.01 per cent (wlv) gelatin, 150pM of each dATP, dGTP and dCTP, 11OpM dTTP, 40pM Bio-11-dUTP, 1 5 p M primer B, and 1 U Taq DNA polymerase in a 3Opl reaction volume. Eighteen cycles of 50 s at 94"C, 1 min at 56"C, and 2min at 72°C were performed.

FISH was performed as described previously (Rowley et af., 1990). Metaphase chromosomes were identified by DAPI staining (4',6-diamidino- 2-phenylindole dihydrochloride). The hybridiz- ation signals of the biotin-labelled probes were detected by fluorescein-isothiocyanate (F1TC)- conjugated-avidin (Vector Laboratories).

Separate gray-scale images for the DAPI stain- ing and the FITC signal were captured using a

AGAGTTGGTAGCTCTTGATC-3'), and 2.5 U liquidcooled charge coupled device camera (KAF 1420, Photometrics). The gray-scale images were overlaid with the GeneJoin M d i x software (Tim Rand, Yale University).

RESULTS

FISH probes derived from the marker chromo- somes were hybridized to normal metaphase cells to determine the origin of the amplified DNA sequences.

The probes developed from the marker in case 1 hybridized to the centromeres of chromosomes 14 and 22 (Fig. 2).

In case 2, the probes developed from the entire marker hybridized to the centromeres and short arms of all acrocentric chromosomes as well as to the heterochromatic region of chromosomes 1 and 9 (Fig. 3). The probes developed from the arms of the marker in case 2 hybridized to the centromeres and short arms of all acrocentric chromosomes but did not hybridize to the heterochromatic regions of chromosomes 1 and 9 (Fig. 4).

DISCUSSION

We have used FISH probes developed by micro- dissection to determine the chromosomal origin of

586 M. THANGAVELU ET A L

Fig. >FISH analysis with the probes developed from the whole marker in case 2. All acrocentric centromeres and short arms are labelled as well as the hetero- chromatic region of chromosomes 1 and 9

Fig. &FISH analysis with the probes developed from the arms of the marker in case 2. The same pattern of hybridization is seen as that in Fig. 3, except that the heterochromatic regions of 1 and 9 are not labelled

two marker chromosomes. The probes developed from the marker chromosome in case 1 hybridized to the centromeres of chromosomes 14 and 22, suggesting that the origin of this marker chromo- some was from either one or both of these chromosomes. Chromosome 22-specific probes (Integrated Genetics) did not hybridize to this marker chromosome, indicating that this chromo-

some was originally derived from chromosome 14. Such a marker chromosome, initially identified in amniocytes with a normal clinical outcome, has been reported previously (Stetten et al., 1992). The cross-hybridization of probes derived from this marker chromosome to the centromeres of chromosomes 14 and 22 was attributed to the presence of the alpha-repeat subfamily shared by

CHARACTERIZATION OF TWO MARKER CHROMOSOMES 587

the centromeres of both of these chromosomes (Jorgensen e t al. , 1988).

Hybridization of the probes developed from the arms of the marker chromosome in case 2 to the short arm and centromeres of acrocentric chromosomes suggested that this marker repre- sented a derivative from a Robertsonian trans- location involving acrocentric chromosomes. This was substantiated by the presence of NORs on both arms of the marker chromosome. Alpha- repeat subfamilies from the centromeric region of this marker chromosome account for the hybrid- ization of the FISH probes developed from this marker to the heterochromatic regions of lq and 9q.

Microdissection offers several advantages over previously described molecular cytogenetic tech- niques for the identification of chromosomes. Unlike other molecular cytogenetic techniques requiring probes from multiple chromosomes, the number of hybridizations required by our approach is significantly reduced. FISH probes could be developed by flow sorting of the marker chromosome (Carter et al., 1992; Blennow et al., 1993), but as shown in case 2, probes developed from the entire marker chromosome were not as specific as probes developed from the arms of the marker. Microdissection offers a greater likelihood of identifying the origin of marker chromosomes, since FISH probes may be generated from these regions of the marker chromosome that are more likely to contain unique sequences. Until recently, the use of microdissection was limited to a few laboratories, primarily because of technical d 8 i - culties in cloning or amplifying the microdissected material (Ludecke et al., 1989). As demonstrated in this study, DNA from as few as four marker chromosomes was successfully amplified by a simple sequence-independent DNA amplification protocol (Bohlander et al., 1991). This technical development should contribute to the widespread application of microdissection in the characteriz- ation of chromosomes.

In both cases 1 and 2, carriers of the marker chromosomes had a normal phenotype. The cystic hygroma observed in the proband (fetus) in case 2 is consistent with the phenotype of Turner syn- drome resulting from monosomy for the sex chro- mosome. Since this marker was observed in the phenotypically normal maternal parent, it may be concluded that the presence of the marker in the fetal cells had no effect in the manifestation of the abnormal phenotype.

At the present time, the clinical effects of extra copies of unique coding DNA sequences in a cell are unknown. It would be useful to study a series of marker chromosomes associated with a normal phenotype to determine whether or not they con- tain unique coding DNA sequences. Similar molecular studies on marker chromosomes from individuals with congenital abnormalities could identify unique coding DNA sequences associated with clinically abnormal development. Micro- dissection, in conjunction with other techniques of molecular genetics, may prove to be a valuable approach in evaluating the clinical significance of de novo marker chromosomes, particularly those identified prenatally.

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