Indian Journal of Human Genetics Medknow Publications on behalf of Indian Society of Human Genetics ISSN: 0971-6866 EISSN: 1998-362x Vol. 8, Num. 1, 2002, pp. 11-14

Indian Journal of Human Genetics, Vol. 8, No. 1, Jan-Jun, 2002 pp. 11-14

Detection of Human Aneuploidies in Prenatal and Postnatal Diagnosis using Molecular Cytogenetics

Kiran Kucheria, Vaidehi Jobanputra, Rashmi Talwar, M. E. Ahmed, Rima Dada, T. A.Sivakumaran

Department of Anatomy, All India Institute of Medical Sciences, New Delhi-110029, India.
Address for correspondence: Dr. Kiran Kucheria, Professor and Head, Division of Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi-110029, India, Fax: 91-11-6862663, 6521041, E-mail: kkucheria@hotmail.com

Code Number: hg02003

Chromosomal aneuploidies especially trisomies 13, 18, 21, monosomy X and 47, XXY account for up to 95% of live born cytogenetic abnormalities. The diagnosis of aneuploidies usually done by conventional cytogenetic analysis (CCA) is associated with technical difficulties and requires about 1-3 weeks for providing a result, especially in prenatal diagnosis. In the present study, Fluorescence In Situ Hybridization (FISH) was used on interphase cells for rapid prenatal and postnatal detection of aneuploidies.

The frequent indications of high pregnancies included for prenatal diagnosis were previous child with chromosomal abnormalities, abnormal ultrasound scan and advanced maternal age (> 35 years). Interphase FISH was done using probes specific for chromosomes 13, 18, 21, X and Y on uncultured chorionic villi and amniotic fluid samples. All samples were analyzed subsequently using conventional cytogenetics. The analysis of aneuploidies for chromosomes 13, 15, 16, 18, 21, 22, X and Y using FISH was extended to abortuses from spontaneous abortion cases. In cases where cytogenetics was not informative, a diagnosis could be made using interphase FISH.

For postnatal diagnosis, interphase FISH was done to confirm low-level mosaicism in patients with primary amenorrhea, suspected cases of Klinefelter syndrome, and mental retardation using probes specific for various autosomes, X and Y chromosomes. FISH was also done using probe specific for the sex-determining region (SRY) on the Y chromosome in cases with ambiguous genitalia. The SRY region could be identified in cases that lacked the Y chromosome on conventional cytogenetic analysis thereby emphasizing on the high resolution of FISH technique in detecting sub-microscopic rearrangements.

To conclude, interphase FISH decreases the time interval between sampling and diagnosis. This is of tremendous value in prenatal diagnosis of urgent high-risk pregnancies, management of ambiguous genitalia and low-level mosaicism where result can be obtained within 24 hours.

Key words: FISH, prenatal, postnatal, ambiguous genitalia, interphase, mosaicism

Chromosomal imbalance has been identified as a major cause for spontaneous pregnancy loss and childhood disability thereby, contributing significantly to the genetic burden on society. The incidence of karyotypic abnormalities at birth is about 0.5%-1%, most of which are aneuploidies (Handyslide and Delhanty, 1997). The cytogenetic abnormalities that account for up to 95% of liveborn cytogenetic abnormalities include trisomies 13, 18, 21, monosomy X and 47, XXY (Whiteman and Klinger, 1991).

Classically, the diagnosis of aneuploidies was made by conventional cytogenetic analysis. This involves karyotyping of GTG banded chromosome preparations obtained from various prenatal and postnatal tissues. Although this technique is highly accurate and reliable, it can be applied only to mitotic (dividing) cells. But it is often difficult to obtain good quality metaphases from certain tissues like amniocytes and chorionic villi (Jobanputra et al., 1998). Further, the time required for reporting especially in prenatal diagnosis is about 1-3 weeks, as most of the prenatal tissues (amniotic fluid and chorionic villus sampling) require long-term culturing. Hence, there was a need for highly sensitive technique that could provide a rapid and accurate postnatal as well as prenatal diagnosis.

Fluorescence In Situ Hybridization (FISH) technique is a highly sensitive molecular cytogenetic technique that has the potential to detect genetic changes in the interphase nuclei (Jobanputra et al., 1998; Vosanova et al., 2000). The use of interphase FISH can therefore be exploited for obtaining faster results.

In the present study, FISH technique was used on interphase cells for rapid prenatal and postnatal diagnosis of aneuploidies. The diagnostic value of interphase FISH was evaluated by comparing FISH results with those of conventional cytogenetics.

Materials and Methods

Prenatal diagnosis was carried out in high-risk pregnancies (singleton and twin). The pregnant women were classified as high-risk pregnancies on the basis of following indications: previous child with chromosomal abnormalities, advanced maternal age (age>35 years), fetal abnormality detected through ultrasound examination, abnormal levels of maternal serum alpha-fetoprotein (MS-AFP) and b-human chorionic gonadotropin (bhCG) or occurrence of chromosomal abnormality in either of the parents. The twin pregnancy had a hydatiform mole. The cytogenetic and FISH analysis was carried out in amniotic fluid or chorionic villi samples depending on the gestational age at which these women presented to the antenatal clinic. The analysis of aneuploidies was also conducted in abortuses from spontaneous abortion cases.

Postnatal diagnosis for various autosomal and sex chromosomal aneuploidies was carried out in peripheral blood samples from patients with primary amenorrhea, suspected cases of Klinefelter syndrome, ambiguous genitalia and mental retardation to analyze minor cell lines with aneuploidies.

Conventional cytogenetic analysis

Chromosome preparations were obtained from chorionic villus and amniotic fluid samples by direct preparations and long-term cultures (1-2 weeks). The remaining tissues (spontaneous abortion and peripheral blood samples) were cultured for 72 hours (Roulston and Beau, 1997). Karyotyping was done on GTG banded metaphases according to the ISCN nomenclature (ISCN, 1995).

Fluorescence In Situ Hybridization (FISH) analysis

FISH analysis was performed on interphase nuclei using commercially available centromeric and locus specific probes. For prenatal diagnosis, interphase FISH was done on uncultured chorionic villus and amniotic fluid samples using centromeric probes for chromosomes 18, X and Y and locus-specific probes for chromosomes 13 and 21. The probes used for detection of aneuploidies in abortuses from spontaneous abortion cases were specific for chromosomes 13, 15, 16, 18, 21, 22, X and Y. Postnatal detection of aneuploidies was done using centromeric probes for chromosomes X and Y in sex chromosomal aneuploidies and locus-specific probe for chromosome 21 in mentally retarded cases. The cases with ambiguous genitalia were analyzed using locus specific probe for sex-determining region (SRY) on the Y chromosome. All the probes were standardized previously in peripheral blood samples from normal, healthy individuals.

The slides prepared from fixed cell suspension obtained during cytogenetic analysis were denatured in 70% formamide/2X SSC at 73°C for 2-5 minutes. The slides were then dehydrated in ethanol (70%, 85% and 100%) series. After the slide was dried, 10ml of denatured probe was applied to the marked area of the slide with highest cell density. Hybridisation was carried out overnight at 37°C in a humidified chamber. Post-hybridisation washes were given as per manufacturers' instructions. The slide was then counterstained with DAPI and viewed under a Zeiss Axiophot fluorescence microscope (Lichter et al., 1998). Images were captured using a CCD camera attached to the microscope and analysed using CytoVision software (Applied Imaging). A minimum of 200 interphase nuclei was scored for each probe. Clumped cells and cells with low fluorescence intensity signals were excluded. Aneuploidy was identified when >15 per cent cells showed abnormal signals (Jobanputra et al., 1998; Vosanova et al., 2000).

Results

FISH analysis in prenatal diagnosis was successful in all the cases. FISH results revealed 5 aneuploid samples. Of the 5 abnormal samples, 3 revealed trisomy 21 in all cells while 2 were mosaics for trisomy 21. The hybridization efficiency of the 5 probes used for detection of aneuploidies in prenatal tissues was 100%. Some samples showed mosaicism using interphase FISH but no correlation was found between the percentage of nuclei with abnormal FISH results and conventional cytogenetic analysis. Further, FISH showed normal chromosome compliment on uncultured chorionic villus sampling in the case where conventional cytogenetics was not informative.

The detection of aneuploidies was extended to analyze abortuses from spontaneous abortion cases to determine the accuracy and efficiency of using multiplex FISH as a preliminary screening tool in these cases. In 3 cases cytogenetics was not informative due to culture contamination. Interphase FISH could identify mosaic trisomy 21 in one of these samples. Twenty-eight abortuses samples revealed chromosomal aneuploidies: 4 were triploid, 3 had monosomy X, 1 was mosaic twins and 1 had monosomy 21. Among the abnormal samples, trisomy for chromosome 16 was predominant within the single trisomies.

The cytogenic results in some cases of primary amenorrhea, suspected cases of Klinefelter syndrome and mental retardation revealed minor cell lines (1-5%) with abnormal chromosome constitution. FISH analysis was done on interphase cells and metaphase chromosomes to confirm the presence of these abnormal cell lines in these samples. Twelve cases with primary amenorrhea revealed monosomy X in 20-30% cells using FISH while 1 case revealed 3 cell lines: 46, XX (70%), 45, X (20%) and 47, XXX (10%). Similarly, 6 males with suspected Klinefelter syndrome revealed 47, XXY in 10-30% cells using FISH. Trisomy 21 was observed in all cases with mental retardation using FISH. The difference in the percentage of aneuploid cells using FISH and conventional cytogenetics was probably due to the larger number of cells analyzed by FISH (200-300) as compared to conventional cytogenetics (20-30).

All patients with ambiguous genitalia were analyzed using probe specific for the centromere of chromosome X and locus-specific probe for sex-determining region (SRY) on the Y chromosome. Conventional cytogenetics revealed 46, XX karyotypes in most cases while the remaining had 46, XY karyotypes. FISH could detect the presence of SRY region on the X chromosome in 2 cases that were 46, XX by conventional cytogenetics.

Discussion

Conventional cytogenetics plays an important role in the identification of chromosomal aneuploidies associated with various human disorders in prenatal and postnatal diagnosis. Although traditional chromosomal banding techniques are critical in the assessment of karyotypic changes, these techniques have certain inherent limitations that complicate accurate characterization of genomes. These limitations which apply particularly to prenatal diagnosis are: (a) difficulty in culturing of fetal tissues (amniotic fluid, chorionic villus and fetal blood), which typically produce chromosomes of poor quality; (b) maternal cell contamination (in case of fetal tissues) that makes subsequent analyses problematic; (c) time-consuming (2-3 weeks) ; (d) labor intensive and (e) the presence of complex karyotypes, which often precludes reliable, comprehensive identification and characterization of chromosomal abnormalities (Cremer et al., 1996; Jobanputra et al., 1998).

The most stress-laden cytogenetic analysis is in case of prenatal diagnosis that demands accuracy and speed as the potential abnormal outcome and time constrains may limit possible intervention (Cremer et al., 1996). The rapid identification of aneuploidies can have a major impact on patient care and further reduce anxiety in pregnant women. The advent of Fluorescence In Situ Hybridization (FISH) that can be used in non-dividing (interphase) cells eliminates the need for dividing cells and reduces the time interval between sampling and diagnosis (Schwartz, 1993; Divane et al., 1994). In the present study, Interphase FISH has been used as a tool for providing rapid prenatal diagnosis. The results of prenatal diagnosis using FISH indicate that Interphase FISH is of immense importance in urgent high-risk pregnancies as it not only eliminates the need for long-term culturing procedures but can also provide accurate diagnosis within 24 hours. Interphase FISH could also provide a result in spontaneous abortion cases with culture failure and maternal contamination.

In the present study, multiplex FISH has been used on abortuses from spontaneous abortion cases for simultaneous detection of chromosomes 13, 15, 16, 18, 21, 22, X and Y in a single experiment (Divane et al., 1994; Feldman et al., 2000; Vorsonova et al., 2000). This advanced technique provides an unprecedented opportunity for screening of commonly occurring aneuploidies in a single experiment thereby enabling rapid diagnosis and patient management. However, the use of specific probes in FISH does not exclude other chromosomal abnormalities that may or may not be associated with a particular disorder.

The results of postnatal diagnosis using interphase FISH indicate that this technique is highly efficient, accurate and reliable tool for screening low-level mosaicism in autosomal and sex chromosomal aneuploidies.

The high resolution of FISH technique in detecting sub-microscopic rearrangements was highlighted in two patients with ambiguous genitalia who revealed the presence of sex determining region (normally present on the Y chromosome) using SRY-specific probe even in the absence of Y chromosome. The SRY gene has been known to be responsible for the initiation of a cascade reaction leading to male differentiation of the primitive gonad (Okada et al., 2001; Domenice et al., 2001). According to few reports, patients with ambiguous genitalia having Y chromosome or segments of Y chromosome have greater susceptibility for developing malignancies especially prostrate cancer (Berkovitz et al., 1992; Aviv et al., 2001). Hence it is essential to screen such patients for genes on Y chromosome (like SRY gene) using highly sensitive and powerful techniques like FISH.

To conclude, the present study stresses that interphase FISH is a highly sensitive, efficient, accurate and reproducible technique that can be used independently for screening of aneuploidies in prenatal and postnatal diagnosis and in abortuses from spontaneous abortion cases. Further, Interphase FISH can provide a result within 24 hours thereby aiding the patients as well as health-providers in rapid decision-making, management and counseling.

Acknowledgement

Authors acknowledge the financial support from Department of Biotechnology, New Delhi (BT/PRO 824/Med/15/031/97) and Indian Council of Medical Research, New Delhi (54/1/98). Two of the authors (VJ and RT) are grateful to Council of Scientific and Industrial Research, New Delhi and one of the authors (TAS) is grateful to Indian Council of Medical Research, New Delhi for their Senior Research Fellowship.

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