Appraisal of fluorescence in situ hybridization (FISH) techniques in prenatal diagnosis

Early Human Development, 33 (1993) 101-108 0 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved.

101

037%3782/93/$06.00

EHD 01396

Appraisal of fluorescence in situ hybridization (FISH) techniques in prenatal diagnosis P. Grao”, M. Guitartb, ‘Departament

(Received

M.D. Coil” and J. Egozcuea

de Biologia Cellular i Fisiologia. Universitat Autbnoma de Barcelona, Bellaterra and bDepartament de Gektica, Hospital de Sabadell, Barcelona (Spain) 23 October

1992; revision

received

15 January

1993; accepted

24 January

1993)

Summary

The reliability of FISH was appraised using probes for the X and Y chromosome and for chromosomes 12 and 18 in prenatal and adult interphase nuclei. Detection of a single hybridization spot proved to be quite reliable (80-92% positive nuclei). Detection of two hybridization spots was more difficult; percentages of nuclei showing two signals varied between 62-72%. The percentages of nuclei with the correct number of spots was higher in the metaphases occasionally found. Thus, FISH may complement but not replace cytogenetic analysis. For sex determination and for the detection of mosaicism, we suggest the use of two different probes in separate regions of the same preparation. Key words: in situ hybridization;

amniocytes; chorionic villi; probes

Introduction

Numerical and structural chromosome abnormalities can be prenatally diagnosed through the cytogenetic analysis of the metaphases obtained from amniocytes (AF) [5] or from chorionic villi (CVS) [8]. Although in general the results are good, in some cases relatively long periods of culture may be needed; furthermore, if direct CVS preparations are obtained, the number and/or quality of the metaphaes may not always be adequate for a correct diagnosis. The use of fluorescence in situ hybridization (FISH) using biotin-labelled probes specific for some chromosomes could help in establishing a faster and more reliable Correspondence to; P. Grao, Departament C), Universitat Autbnoma de Barcelona,

de Biologia Cellular i Fisiologia, Facultat 08193 Bellaterra, Barcelona, Spain.

de Ciencies (Editici

102

diagnosis of numerical chromosomes anomalies, especially because such probes can also be applied to interphase nuclei [ 1,191. The advantages of FISH are: (1) quick and high resolution detection of numerical anomalies, (2) detection of DNA sequences both in metaphase chromosomes and in interphase nuclei, thus increasing the number of cells that can be analysed, even in direct preparations and (3) commercial availability of labelled probes. However, since probes are chromosome-specific, they can only detect numerical anomalies affecting the chromosomes that they will label. Thus, other possible chromosome abnormalities will go undetected. As a result, FISH may complement and improve, but not replace the traditional cytogenetic analysis of prenatal samples. In this work, we have appraised the reliability of FISH techniques in interphase nuclei from amniocytes, chorionic villus cells and fetal blood as compared to other types of cells, using centromeric probes from chromosomes 12 and 18 and for the X chromosome, as well as a probe for the polymorphic (Yq) region of the Y chromosome. Materials and Methods We analysed direct CVS preparations, cultured CVS, cultured amniocytes and cultured fetal blood; for comparision, we used cultured adult lymphocytes, cultured adult skin tibroblasts and cultured gonadal biopsy from an XOEiY mosaic. When metaphases were found they were scored apart. The karyotype of all samples had been previously established, but was not known to the observer. The biotinilated chromosome-specific probes (ONCOR) used were: DXZl (pBam X7) which is a fragment of the repetitive o-satellite sequences of the centromere of the X-chromosome; DYZl, corresponding to repetitive satellite III DNA of the polymorphic region of the Y-chromosome; D12Z3 a-satellite sequences of the centromere of chromosome 12; and D18Zl a-satellite specific for the centromere region of chromosome 18. FISH was carried out following the protocol recommended by ONCOR, with some modifications: preparations were kept at -20°C and prior to hybridization they were kept for 20 min at 37°C; preparations were incubated in RNase (100 pg/ml in 2 x SSC) for 1 h at 37”C, washed four times with 2 x SSC pH 7, dehydrated in ethanol (70, 80 and 96%), denaturated in a 70% formamide solution in 2 x SSC for 2 min at 70°C and immediately immersed in cold 70% ethanol. The hybridization solution, consisting of a 65% formamide solution in 2 x SSC was always prepared with a DNA probe concentration of 15 @ml. Hybridization was carried out for about 14 h at 37°C. Then, preparations were washed in a 65% formamide solution in 2 x SSC at 43°C. Under these stringency conditions, all possible unspecific hybridizations (false positive-false negative) were avoided. Detection was carried out by means of three 20-min incubations at 37°C the first one in avidine-fluorescein, the second in biotinilated anti-avidin and the third one again in avidin-fluorescein. After each incubation, the preparations were washed with PBD buffer. Finally, preparations were mounted with a propidium iodide-antifade solution and analysed in a fluorescent Zeiss-Axioplan microscope with a UV 487709 combination (excitation at 450-490 nm).

I

807

Dl8Z1

S.D., sex determitation;

DYZI

n, number

16.4

0.5 3.8 1.9 0.5

15.1

I9 23 7.3 3.1 7.6

I?.?

13.6

1.9 10.1 19.9

0

cells.

23.6

2.1 6.8 5 1.9

84.3

81 17 29.3 15.8 92.4

19.5

14.3

92. I 89.9 80.1

I

96.1 89.4 93. I 94.2

63.1 80.5

62.8

12.1

2

of nuclei with number

of analyzed

1492

518 500 580 1013

DXZI

Dl2Zl

360

DYZI

DYZI

DXZI

814 400 768 414 350

1304

DXZI

DYZI

659 602 1311

”

Percentage

DYZI DYZI DYZI

DNA Probe

0.88

0.1

0.1

3

of spots

2.2

0.2

4

with zero to four spots from FISH with a panel of specific probes.

Culture 46, XX Culture 45, X0/46, XY

villus sample;

biopsy

Gonadal

CVS, corion

fibroblasts

Normal

Normal blood lymphocytes

Direct (SD) Direct (SD) Culture 46, XY Culture 46, XX Culture 46, XY 2 Cultures 46 XY 2 Cultures 46, XX Culture 46, XY Culture 46, XY 3 Cultures 46, XX

Method

of nuclei and metaphases

fluid

Fetal blood

Amniotic

cvs

Cell type

Percentage

TABLE

37

6

7

13

28

14 16 80 4

290

n

2.1 1.4

8

1.1

21.5

2.5 2.5 2.3

92

13.5

92.3

I

of metaphases

78.5

0

Percentage of spots

91.5 94.8 96.3 100

86.5

100

100

2

with number

104

Results

As shown in Table I, the reliability of FISH varied depending on the material and the probes used. In interphase 46,XY nuclei from prenatal and adult material, probe DYZl was detected as a single spot in 80.1-92.4% of interphase nuclei (Fig. 1). Similar results were obtained in the metaphases found in one CVS culture and in adult lymphocyte culture. The centromeric probe for the X-chromosome (DXZl) was present as two spots in 63.1-80.5% of 46, XX interphase nuclei from CVS (Fig. 2) or amniocytes (Fig. 3); in adult lymphocytes, two spots were observed in 89.4-96.7% of the nuclei. Probe D18Zl dgtected two spots in only 62.8% of CVS diploid nuclei, while probe D12Zl was present as two spots in 94.2% of normal skin fibroblast. In gonadal tissue from a 45XO/46XY mosaic, 23.6% of the nuclei were DYZl positive, and 76.4% were DYZl negative, a percentage similar to that detected in cytogenetic studies of lymphocytes from this patient (30-70%). Discussion

Using the stringency conditions described, the reliability of FISH in prenatal and adult material can be appraised by means of the detection of a number of fluorescent spots corresponding to the karyotype of the cells. In all cases, the percentages of positive cells found by us are comparable to those described in the literature for similar material [1,4,10,12,17].

Fig. 1. FISH in a cultured

CVS preparation

using probe

DYZI.

Most nuclei show a single spot.

Fig. 2. FISH in a cultured be observed.

CVS preparation

using probe

Fig. 3. FISH in an AF preparation using probe DXZI. nuclei with two spots and nuclei with a single spot.

DXZI.

Nuclei with two, one or no spots can

Note metaphase

with two hybridization

spots,

106

When a single spot has to be detected, as is the case of probe DYZl, the method is highly reliable, with a mean of 85.4% of the nuclei being positive, and similar figures for prenatal and adult material. However, if two spots are to be detected, as with DXZl, D12Z3 and D18Z1, the percentage of cells with a single spot in prenatal material is quite high. This may be due to somatic pairing of the target regions, to factors that prevent the probes from reaching their target, or to technical defects. When a single spot is observed, the fluorescent signal may be less discrete than the normal fluorescent spot [4]. Thus, in these cases FISH is insufficient to establish a diagnosis of monosmy [3,16,11]. It is notable that the percentage of nuclei with two DXZl spots was higher in adult lymphocyte nuclei (89.4-96,7%) than in prenatal material (63.1-80.5%). When using autosomal probes, the percentage of CVS nuclei with two spots for probe D18Zl was only 62.8%, while in adult skin fibroblasts 94.2% had two spots for probe D12Z3. Although the autosomal probes used were different in prenatal and adult material due to the fact that each probe was used in the type of material where identification problems can usually be found, i.e. sex determination and common trisomies in prenatal material, mosaicisms in gonadal tissues and the possible presence in fibroblast of trisomies not found in peripheral blood lymphocytes, the results also indicated a higher reliability of the method in adult than prenatal material. This could be due to a different structural organization of some chromatin regions in fetal cells, as has been shown for some heterochromatic regions by Miguez et al. and Perez et al. [15,18]. In the 45,XO/46,XY mosaic studied, FISH gave a percentage of mosaicism similar to that detected in cytogenetic studies. In these cases, the use of probe DYZl can be considered as reliable, due to the high percentage of hybridization of this probe. In fact, Tantravahi et al. [20], using FISH, were able to detect the presence of a 69,XXY cell line in low percentages (l-3%) in a patient with some abnormal phenotypical characteristics cytogenetically diagnosed as 46,Xx. Hook and Warburton [8] proposed that all 45,X0 patients should be analysed for the presence of a mosaicism, because most if not all 45,X0 fetuses that survive are probably mosaics, with a second cell line in some organ or tissue containing the double dose of the genes in the short arm of the X chromosome needed for survival [7]. In summary, when using FISH in prenatal diagnosis one must take into account that if two spots are to be detected, between 14-29% of the cells may show only one signal, and between 3-13% may be negative. Thus, FISH may complement and improve, but not replace cytogenetic studies. For sex determination and to appraise cases of mosaicism, as well as to determine the efficiency of hybridization, we propose to use two different probes (e.g. DYZl and DXZl) on two different and separate regions of the same preparation. Such a procedure would allow a fast reliable determination of the fetal sex in carriers of sex-linked genetic diseases, with the only exceptions of cases with a non-fluorescent Y [14], with a Yq-deletion [2] or with a small heterochromatic region in the Y chromosome [6]. However, these problems could be solved using a centromere Y-chromosome probe such as DYZ3. Acknowledgments

We thank Dr M.M. Perez for providing CVS and AF samples, and Dr N. Clusellas

107

for skin fibroblast cultures. This work was supported by research grants from ‘Fond0 Investigaciones Sanitarias’ (project number 91-0635), Spain. References I

2 3

4

5

6

7

8

9

10

11

12

17

18

Cremer, T., Tesin, D., Hopman, A.H.N. and Manuelidis, L. (1988): Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodennal tumor cells by in situ hybridization with chemically modified DNA probes. Exp. Cell Res., 176, 199-220. Davis, R.M. (1981): Localization of male determining factors in man: a thorough review of structural anomalies of the Y chromosome. .I. Med. Genet., 11, 571-577. van Dekken, H., Pizzolo, J.G., Reuter, V.E. and Melamed, M.R. (1990): Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome-specific DNA probes. Cytogenet. Cell Genet., 54, 103-107. Eastmond, D.A. and Pinkel, D. (1990): Detection of aneuploidy and aneuploidy-indu -ing agents in human lymphocytes using fluorescence in situ hybridization with chromosome-specific DNA probes. Mutat. Res., 234, 303-318. Ferguson-Smith, M. and Yates, J. (1984): Maternal age specific rates for chromosome aberrations and factors influencing them: report of a collaborative European study on 52,965 amniocenteses. Prenatal Diagn., 4, 5-44. Gosden, J.R., Gosden, C.M., Cooke, H.J., Morsman, J.M. and Rodeck, C.H. (1984) The use of cloned Y chromosome-specific DNA probes for fetal sex determination in first trimester prenatal diagnosis. Hum. Genet., 66, 347-351. Held, K.R., Kerber, S., Kaminsky, E., Singh, S., Goetz, P., Seemanova, E. and Goedde, H.W. (1992): Mosaicism in 45, X Turner syndrome: does survival in early pregnancy depend on the presence of two sex chromosomes? Hum. Genet., 88, 288-294. Hook, E.B. and Warburton, D. (1983): The distribution of chromosomal genotypes associated with Turners syndrome: livebirth prevalence and evidence for diminished fetal mortality and severity in genotypes associated with structural X abnormalities or mosaicism. Hum. Genet., 64, 24-27. Hook, E.B., Cross, P.K., Jackson, L., Pergament, E. and Brambati, B. (1988): Maternal age-specific rates of 47, +21 and other cytogenetic abnormalities diagnosed the first trimester of pregnancy in chorionic villus biopsy specimens: comparison with rates expected from observations at amniocentesis. Am. J. Hum. Genet., 42, 797-807. Jauch, A., Daumer, C., Lichter, P., Murken, J., Schroeder-Kurth. T. and Cremer, T. (1990): Chromosomal in situ suppression of human gonosomes and autosomes and its use in clinical cytogenetics. Hum. Genet., 85, 145-150. Kiechle-Schwarz, M., Decker, H.J., Berger, C.S., Fiebig, H.H. and Sandberg, A.A. (1991): Detection of monosomy in interphase nuclei and identification of marker chromosomes using biotinylated Alpha-satellite DNA probes. Cancer Genet. Cytogenet., 51, 23-33. Kuo, W.-L., Tenjin, H., Segraves, R., Pinkel, D., Golbus, M.S. and Gray, J. (1991): Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes. Am. J. Hum. Genet., 49, 112-l 19. Lau, Y.-F., Huang, J.C., Dozy, A.M. and Kan, Y.W. (1984): A rapid screening test for antenatal sex determination. Lancet, i, 14-16. Madan, K., Gooren, L., and Schoemaker, J. (1979): Three cases of sex chromosome mosaicism with a nonfluorescent Y. Hum. Genet., 46, 295-304. Miguez, L., Fuster, C., Perez, M.M., Mire, R. and Egozcue, J. (1991): Spontaneous chromosome fragility in chorionic villus cells. Early Hum. Dev., 26, 93-99. Nederlof, P.M., van der Flier, S., Raap, A.K., Tanke, H.J., van der Ploeg, M., Kornips, F. and Geraedts, J.P.M. (1989): Detection of chromosome aberration in interphase tumor nuclei by nonradioactive in situ hybridization. Cancer Genet. Cytogenet., 42, 87-89. Perez, M.M., Miguez, L., Fuster, C., Mire, R., Genes&, A. and Egozcue, J. (1991): Heterochromatin decondensation in chromosomes from chorionic villus samples. Prenatal Diagn., 1 I, 696-704. Perez, A., Wessman, M., Tiainen, M., Hopman, A.H.N., Willard, H.F., Sole, F., Caballin, M.R.,

108

19 20

Woessner, S. and Knuutila, S. (1991): Trisomy 12 in chronic lymphocytic leukemia: an interphase cytogenetic study. Blood, 78, 775-779. Pinkel, D., Straume, T. and Gray, J.W. (1986): Cytogenetic analysis using quantitative, highsensivity, fluorescence hybridization. Proc. Nat]. Acad. Sci. U.S.A., 83, 2934-2938. Tantravahi, U., Bianchi, D.W., Haley, C., Destrempes, M.M., Ricker, A.T., Korf, B.R. and Latt, S.A. (1986): Use of Y chromosome mosaicism. Clin. Genet., 29, 445-448.