Rapid-Fluorescence-in-Situ-Hybridization (FISH) on

Uncultured Amniocytes for Avoiding Birth Defects due to

Common Chromosomal Aberrations

Theme Symposium

INTRODUCTION

Birth defects are structural or functional abnormalitiespresent at birth that can cause physical or mental disability innew borns. Birth defects can be due to genetic problemscaused by mutation in one or more genes, chromosomeaneuploidy or environmental factors in women exposedduring pregnancy. Aneuploidies of 5 chromosomes (13, 18,21, X, Y) account for 95% of the chromosomal aberrationsthat cause infants born with birth defects. It is evident thatmore than 50% of first trimester spontaneous abortions aredue to chromosomal abnormality [1,2]. It has been reportedthat the most common cause of spontaneous abortion isnumerical chromosome imbalances (aneuploidies),particularly of chromosomes 13, 14, 15, 16, 18, 21, 22 and X[1,3-5].

It’s important to analyze chromosome abnormalitiesthrough prenatal diagnosis by procedure like amniocentesis

and chorionic villus sampling (CVS) for avoiding birthdefects in high risk pregnancy. The conventional fullkaryotype of amniocyte and cytotrophoblast is laborintensive, time consuming and require highly skilled personfor analysis. In recent time’s development of newertechniques guarantees shorter time of obtaining results thatincludes FISH (fluorescence in situ hybridization), multi-PRINS (primed in situ labeling), QF-PCR (quantitativefluorescence polymerase chain reaction) and MLPA(multiplex ligation-dependent probe amplification) [6-10].FISH allows the analysis of chromosome aberrations usingchromosome specific probes. Currently still mostlaboratories perform rapid tests for aneuploidy like FISHtogether with full karyotype as a stand-alone test in prenataldiagnosis. The aneuploidy analysis through Rapid-FISH(rapid fluorescence in situ hybridization), on unculturedaminocytes has been described world wide for detectingnumerical aberrations of chromosomes 13, 18, 21, X and Y

163 Apollo Medicine, Vol. 7, No. 3, September 2010

RAPID FLUORESCENCE-IN-SITU-HYBRIDIZATION (FISH) ON UNCULTURED

AMNIOCYTES FOR AVOIDING BIRTH DEFECTS DUE TO COMMON

CHROMOSOMAL ABERRATIONS

U Kandpal*, M Mishra**, A Fauzdar***, M Chowdhry@, RN Makroo+ and A Kaul#

* Technologist, ** Junior Molecular Biologist, *** Molecular Biologist,@ Associate Consultant, +Director, Department of Transfusion Medicine, Immunology and Molecular Biology, #Senior Consultant, Department of Fetal Medicine,

Indraprastha Apollo Hospitals, Sarita Vihar, New Delhi 110 076, India.

Correspondence to: Dr RN Makroo, Director, Department of Transfusion Medicine, Immunology & Molecular Biology,Indraprastha Apollo Hospitals, Sarita Vihar, New Delhi 110 076, India.

E-mail: makroo@apollohospitals.com

Background: Birth defects are structural or functional abnormalities present at birth that can cause physical ormental disability in new borns. Birth defects can be due to genetic problems caused by mutation in one or moregenes, chromosome aneuploidy or environmental factors in women exposed during pregnancy. Objective:

The aim of the study was to evaluate the effectiveness of the Rapid-Fluorescence-in-Situ-Hybridization

(Rapid-FISH) technique in detecting numerical chromosome aberrations of 13, 21, 18, X and Y in amniocytenuclei from amniotic fluid for avoiding birth defects due to chromosome abnormalities performed on 138 highrisk pregnancies. Methods: The FISH was performed using AneuVysion kit (Vysis, Inc), according to astandard protocol. Results: A total of one hundred thirty eight samples were received for full karyotype andFISH. The average age of mothers and their gestational ages were 33 years and 17.5 weeks respectively.Triple test screening was positive in 52.9% of the women followed by advanced maternal age andultrasonographic abnormalities. Interphase FISH was performed on seventy eight specimens. All normal andabnormal results were confirmed by classical cytogenetic method (GTG banding and karyotyping). The overalldetection rate for aneuploidies through FISH was 100%. Aneuploidy was identified in 5 out of 78 specimens(four cases of trisomy 21 and one case of Turner syndrome) taken for FISH. It was concluded that Rapid-FISHis a reliable and fast method for detecting numerical chromosomal aberrations through prenatal diagnosis andhas been implemented as a routine diagnostic procedure in pregnancies with high risk of fetal aneuploidy foravoiding chromosomes 13, 18, 21, X, Y related birth defects.

Keywords : Fluorescence in situ Hybridization (FISH), Chromosome abnormality, Prenatal diagnosis,Aneuploidy, Amniotic fluid, Chorionic villus sample (CVS).

Apollo Medicine, Vol. 7, No. 3, September 2010 164

Theme Symposium

results in 2-5 days [6,11-15].

The aim of the study was to evaluate the accuracy,sensitivity of the Rapid-FISH technique for the earlydetection numerical chromosome aberrations of 13, 21, 18,X and Y in interphase nuclei of uncultured amniocytes/cytotrophoblast cells in 138 high risk pregnancies incomparison to conventional cytogenetics.

METHODS

Prenatal Diagnosis was performed on 138 high riskpregnancies (Chorionic Villus Sampling n=21,Amniocentesis n=116 and fetal skin biopsy n=1) during theyear November 2006 to December 2009 at the Departmentof Fetal Medicine and Immunology and Molecular Biology,Indraprastha Apollo Hospitals, New Delhi. Indication’s forprenatal diagnosis to classify high risk pregnancies aredepicted in Table 1, Fig. 1. The Fluorescence in-situHybridization (FISH) analysis was performed on theuncultured amniocytes using DNA probes specific forchromosome 13, 18, 21, X and Y (Abbott Molecular, USA).Invasive prenatal sampling i.e. either chorionic villussampling (at 11-13 weeks) or amniocentesis (16-18 weeks)was performed by the Fetal Unit Consultant underultrasound guidance.

Sample preparation

10-20 mL of amniotic fluid or 05–10 mg of chorionicvillus was obtained from each patient depending on thegestation stage at the time of sampling. Amniotic fluid andchorionic villi samples were then subjected to either to FISHor full karyotype or both for chromosome analysis as perrequest of fetal unit consultant. 5-10 mL of amniotic fluidand centrifuged (1000 rpm, 5 min) and the pellet issuspended in 5 mL 1X Trypsin/EDTA and incubated for 30

min at 37ºC. After centrifugation, the pellet is resuspendedin 7 mL 0.075 M KCl and incubated at 37ºC for 15 min.After addition of few drops of Carnoy fixative [methanol:acetic acid (3:1)], centrifugation, resuspension, andwashing of pellet at least 3 times and finally the cells areincubated at –20ºC for 20 min in 5 mL Carnoy fixative andthe preparation is finalized with a last centrifugation. Thesupernatant is discarded, and the cells are diluted in 200 Lof the remaining supernatant and dropped on a slide andkept for overnight (~16 hrs) at 37ºC for ageing.

Probes

We exclusively applied FDA-approved FISH test forrapid aneuploidy screening in uncultivated amniocytes, theAneuVysion kit (commercially available at ABBOTT/Vysis; Downers Grove, IL) consisting of three satellite DNAprobes for chromosomes X, Y, and 18 (CEP X, CEPY, andCEP18) and two locus-specific probes for 13q14 (LSI 13)and 21q22.13~22.2 (LSI 21). The three centromeric probesand the two locus-specific probes are applied to the samplesin two different hybridizations.

FISH procedure

Rapid-FISH protocol was performed using DNA probesfor detecting numerical chromosome aberrations of 13, 21,18, X and Y in amniotic fluid and chorionic villus sample(CVS) sample. Rapid aneuploidy screening through FISHon uncultivated amniocytes was performed by standardizedrecommended AneuVysion kit protocol. After the post-hybridization washings, the slides are mounted with 6-8 LDAPI containing antifade with coverslip an incubated at -20ºC for at least 10 min for signal enumeration in thefluorescence microscope.

The slides are screened for 50 interphase nuclei per caseand probe combination counterstained with 4,6-diamidino-2-phenylindole (DAPI) and mounted with an antifadesolution. The signal analysis was done by Olympus BX 60fluorescent microscope with a 100-watt mercury bulb using

Table 1. Indications for prenatal diagnosis

Different type indication’s for No. of casesprenatal diagnosis

Triple test 60

Ultrasonography (USG) 30

First trimester screening 6

Advanced maternal age 10

More than one indication for prenatal diagnosis 26

Microbial infection in mother 1

â-thalassemia carrier parents 2

Chromosome abnormality in parents 2

Enzyme disorder in parents 1

Total no. of cases 138 Fig.1. Pie chart showing indications for prenatal diagnosis

Theme Symposium

165 Apollo Medicine, Vol. 7, No. 3, September 2010

100X plane appochromatic objective and single band passfilter for DAPI, FITC and TRITC and a dual band pass filterfor TRITC and FITC (Olympus Japan). Image acquisitionwas performed with an epifluorescence microscope(Olympus BX60) equipped with a cooled charge-coupleddevice (CCD) camera with karyotype software package(Cytovision, Applied Imaging, Sunderland, UK).

RESULTS

A total of 138 patient undergone prenatal diagnoses hadmedian maternal age of 36 years (range 24-45), the mediangestational age was 16 weeks (range 14-24). In our studymain indication for prenatal diagnosis was Triple test (44%)followed by ultrasonograpy (22%). FISH was offered as anadjunct test to the standard chromosome analysis (FullKaryotype) at the request of clinician & patient. FISH signalwere enumerated as disomic, when normal signal patternwere observed in ≥80% nuclei and aneuploidy if ≥70%nuclei have abnormal signal pattern. Out of 138 samplesobtained, FISH result were obtained for 100 cases (Table 2),92 cases were disomic for chromosome 13, 18 and 21 withnormal sex chromosome constitution and later confirmed

with cytogenetics whereas there were only 8 abnormal cases(5.8%) (Table 3, Fig. 2a, Fig. 2b). There were no false-positive and false- negative autosomal or sex chromosomalresults. Our date shows aneuploidy detection rate of 100%and signal hybrization of probe was 100% accurate in allattempted for FISH cases.

DISCUSSION

Aneuploidy of chromosome X, Y, 13, 18 & 21 compriseapproximately 95% cases of chromosomal abnormalitiesthat accompany by birth defects in newborns. Cytogeneticdisorders are present in almost 1% of live births, in about 2%of pregnancies in women older than 35 years, in 6% of stillbirths and in 50% of first trimester spontaneous abortions[16]. In India almost half million babies are born annuallywith malformations and the figure for Down syndrome(trisomy 21) is 21,000 or 1 per 1150 births [17]. This load ismore than the load of any other genetic disease. Moreover,in pregnancy with ultrasound detected malformations, itsincidence is much higher. It may vary between 17% [18] to27% [19]. This chromosomally abnormal birth ispreventable by prenatal cytogenetic diagnosis and selective

Fig.2(a). Abnormal karyotpe patient P1 with 47,**+21 and FISH picture showing trisomy 21 with three spectrum orange signalsfor chromosome 21 and two spectrum green signal for chromosome 13.

Fig.2(b). Abnormal karyotype of patient P5 with 45* with FISH picture with only one spectrum green signal for chromosome X.

Apollo Medicine, Vol. 7, No. 3, September 2010 166

Theme Symposium

Table 2. Summary of FISH and karyotpe results

Indication for prenatal Sample type FISH Karyotype Abnormaldiagnosis (n) Amniotic Fluie (AF) results results (%)

Chorionic VillusSample (CVS)Fetal Skin (FS)

Triple test risk (6) AF-58, CVS-2 42 43 1 (0.72%)

Ultrasonography (USG) (30) AF-25, CVS-4; FS-1 23 17 3 (2.1%)

More than one indication for AF-21; CVS-5 20 20 2 (0.66%)

prenatal diagnosis (26)

Maternal age (10) AF-8; CVS-2 7 8 0

First timester screening (6) AF-2; CVS-4 5 4 1 (0.72%)

Other’s (6) AF-3; CVS-4 3 3 1 (0.72%)

Total (138) AF-117, CVS-20, FS-1 100 95 8 (5.8%)

Table 3. Details of patient with abnormal FISH and karyotype results

Patient Age Indication for prenatal Karyotype FISH resultsNo. diagnosis results

P1 24/F 1st trimester screening test risk 47**+21 nuc ish 13q14 (RB1×2), 18cen (D18Z1×2),21q22.13-q22.2 (D21S259×3, D21S341×3,D21S342×3)

P2 30/F USG: Increased Nuchal thickness 45* nuc ish13q14(RB1×2), 18cen(D18Z1×2),21q22.13-q22.2(D21S259×2, D21S341×2,D21S342×2), Xp11.1-q11.1(DXZ1×1)

P3 30/F Previous child with trisomy 21 – nuc ish 13q14(RB1×2), 18cen (D18Z1×2),21q22.13-q22.2 (D21S259×3, D21S341×3,D21S342×3)

P4 33/F Triple test 1; 123 – nuc ish 13q14 (RB1×2), 18cen(D18Z1×2),21q22.13-q22.2 (D21S259×3,D21S341×3, D21S342×3)

P5 33/F USG: Cystic hygroma 45* nuc ish 13q14, (RB1×2), 18cen(D18Z1×2),21q22.13-q22.2(D21S259×3, D21S341×3,D21S342×3)Xp11.1-q11.1(DXZ1×1)

P6 35/F Maternal age, triple test 47,**, +21 nuc ish 13q14(RB1×2), 18cen(D18Z1×2),risk, 1:50, hyperchogenic bowel 21q22.13-q22.2 (D21S259×3, D21S341×3,

D21S342×3)

abortion thus reducing burden on family, and society. Themost stress-laden cytogenetic studies involve prenataldiagnosis, which demand accuracy and speed because ofpotential abnormal outcome and time constraint limitingpossible intervention. This is also associated with ethical &legal issues, activities limited under Prenatal Diagnostic Act(1994, implemented in 1996) in India. Prenatalchromosomal analysis is done mainly by conventionalcytogenetics from amniotic fluid (80-85% cases) orchorionic villous (10-20% of cases) or fetal blood (very

rarely) cells. Conventional cytogenetics from amniotic fluidcells is the gold standard for prenatal fetal chromosomalanalysis. However, these require great technical expertiseand sometime culture fails to grow. Furthermore, long delayin conventional method is not acceptable to many parentsand obstetricians in second half of pregnancy because, inmany countries, legal limit of pregnancy termination is 20-22 weeks. After this period, termination of pregnancy mayalso be associated with increased maternal complications.This is a typical picture with ultrasound detected

Theme Symposium

167 Apollo Medicine, Vol. 7, No. 3, September 2010

malformations as most of them are recognized in secondhalf of pregnancy. Hence, there is a need for rapidalternative method. These factors have led investigators toseek alternative methods for identifying chromosomalabnormalities quickly. It seems at this moment thatappropriate answer is cytogenetics at molecular level(conventional FISH & QF PCR). FISH using probesspecific for chromosome 13, 18, 21, X and Y has thepotential to find answer quickly (can be made within 24-72hours of receiving the specimen) in these situations andcapable to reduce parental anxiety as well as to guide furtherobstetric management. FISH has been used and tested inprenatal diagnostic situation like advanced maternal age[20], abnormal biochemical serum screening [21],abnormal nuchal thickness [22], fetal dysmorphismsuggestive of aneuploidy [23], previous trisomy [24], etc. Alarger study of 4500 prenatal specimens resulted in anoverall accuracy of 99.8% in informative cases where FISHwas compared with conventional cytogenetics [25]. Severalresearchers [12,14,25,26] have confirmed the potentialapplicability of the technique. Rapid and accurate detectionof chromosomal aneuploidies has been demonstrated in ablind prospective study which compared aneuploidydetection by interphase FISH for chromosome 13, 18, 21, X& Y with conventional cytogenetics [6]. The sensitivity ofFISH (to pick up chromosomal abnormality) varies withdifferent clinical indications. For example in advancedmaternal age sensitivity is approximately 95% whereas inearly abnormal ultrasound suspicion the figure isapproximately 85% and in late abnormal ultrasound signcorresponding value is 73% [27]. American College ofMedical Genetics (ACMG) & American Society of HumanGenetics (ASHG) has published guidelines endorsing useof FISH tests for prenatal testing (ACMG 2000) [28,29]. Itsays that prenatal FISH tests provide highly accurate resultsfor selected chromosomal abnormalities. It also states thatFISH results may be reported to the physician before theconventional chromosome analysis results are available.Similar to other areas of dignostic testing, the Collegerecommends that irreversible decisions to act on positiveresults should be supported by two of the three possiblepieces of information, i.e., FISH, conventional cytogenetics& clinical information.

Our study was designed for prenatal diagnosis ofchromosomal abnormalities in high risk pregnancies usingRapid-FISH method in adjunct to conventional karyotypehelping patient and clinician for better management ofpregnancy. In this study we found FISH results werecomparable with conventional cytogenetic analysis wherewe find 100% accuracy with karyotype results in minimumturn around time. Rapid-FISH is an optimal strategy forrapid confirmation of potential numerical commonchromosome aneuploidies for avoiding Birth defect due to

trisomies 13, 18 & 21 and monosomy X in unculturedamniotic fluid cells/chorionic villus samples in less timeabout 24-72 hrs as compared to conventional karotype incomparison to earlier studies. Early receipt of normaldisomic results through Rapid-FISH has positive effect onmother and reduces anxiety. Rapid-FISH is a reliable andfast method for detecting numerical chromosomalaberrations through prenatal diagnosis and has beenimplemented as a routine diagnostic procedure inpregnancies with high risk of fetal aneuploidy for avoidingchromosomes 13, 18, 21, X, Y related birth defects.

REFERENCES

1. Hassold TJ. A cytogenetic study of repeated spontaneousabortions. Am J Hum Genet. 1980;32(5): 723-730.

2. Jacobs PA. The chromosome complement of humangametes. Oxf Rev Reprod Biol. 1992; 14: 47-72.

3. Chandley AC. Infertility and chromosome abnormality. OxfRev Reprod Biol. 1984; 6:1-46.

4. Zenzes MT, Casper RF. Cytogenetics of human oocytes,zygotes, and embryos after in vitro fertilization. HumGenet. 1992;88(4): 367-375.

5. Jacobs PA, Hassold TJ. The origin of numericalchromosome abnormalities. Adv Genet. 1995; 33:101-133.

6. Klinger K, et al. Rapid detection of chromosomeaneuploidies in uncultured amniocytes by usingfluorescence in situ Hybridization (FISH); Am J Hum Genet1992; 51: 55-65.

7. Pertl B, Yau S C, Sherlock J, Davies A F, Mathew C G,Adinolfi M. Rapid molecular method for prenatal detectionof Down’s syndrome; Lancet 1994; 343: 1197-1198.

8. Hochstenbach R, Meijer J, van de Brug J, et al. Rapiddetection of chromosomal aneuploidies in unculturedamniocytes by multiplex ligation-dependent probeamplification (MLPA). Prenat Diagn. 2005; 25(11):1032-1039.

9. Gadji M, Krabchi K, Drouin R. Simultaneous identificationof chromosomes 18, X and Y in uncultured amniocytes byusing multi-primed in situ labelling technique. Clin Genet.2005; 68(1):15-22.

10. Van Opstal D, Boter M, de Jong D, et al. Rapid aneuploidydetection with multiplex ligation-dependent probeamplification: a prospective study of 4000 amniotic fluidsamples. Eur J Hum Genet. 2009; 17(1):112-121.

11.Bryndorf T, Christensen B, Vad M, et al. Prenatal detection ofchromosome aneuploidies in uncultured chorionic villussamples by FISH. Am J Hum Genet. 1996; 59(4): 918-926.

12. Jobanputra V, Kucheria K. Prenatal detection ofaneuploidies using fluorescence in situ hybridization apreliminary experience in an Indian set up; J Biosci. 2002;27: 155-163.

13. Stephenson MD, Awartani KA, Robinson WP.Cytogenetic

Apollo Medicine, Vol. 7, No. 3, September 2010 168

Theme Symposium

analysis of miscarriages from couples with recurrentmiscarriage: a case-control study. Hum Reprod.2002;17(2): 446-451.

14. Luquet I, Mugneret F, Benzacken B. French multi-centricstudy of 2000 amniotic fluid interphase FISH analysesfrom high-risk pregnancies and review of the literature.Ann Genet. 2002; 45(2): 77-88.

15. Caine A, Maltby AE, Parkin CA, Waters JJ, Crolla JA. UKAssociation of Clinical Cytogeneticists (ACC). Prenataldetection of Down’s syndrome by rapid aneuploidy testingfor chromosomes 13, 18, and 21 by FISH or PCR without afull karyotype: a cytogenetic risk assessment. Lancet.2005; 366(9480):123-128.

16. Jackson L. Cytogenetics, Molecular Cytogenetics; ClinicalObstetrics & Gynecology 2002; 45: 622-639.

17. Verma I C, Anand NK, Modi U J, Bharucha B A. Study ofMalformations and Down syndrome in India - A MulticentricStudy; Department of Atomic Energy, Bhabha AtomicResearch Center, Mumbai. Burden of genetic disorders inIndia; Ind. J Pediatr 2000; 67: 893-898.

18. Wilson R D, Chitaya D, McGillivray B C. Fetal ultrasoundabnormalities: correlation with fetal karyotype, autopsyfindings & postnatal outcome-fiveyear prospective study;Am J Med Genet 1992; 44: 586-590.

19. Nicolaides K H, Rodeck C H, Gosden C M. Rapidkaryotyping in non-lethal fetal malformations; Lancet1986; 1: 283-286.

20. Hook E B. Rates of chromosomal abnormalities at differentmaternal ages; Obstet. Gynecol 1981; 58: 282-285.

21. Wald N J, Cuckle H S, Densem J W. Maternal serumscreening for Down syndrome in early pregnancy; BMJ1988; 297: 883.

22. Benacerraf B R, Barss V A, Laboda L A. A sonographic signfor the detection in the second trimester of the fetus withDown syndrome; Am. J Obstet. Gynecol 1985; 151: 1078 -1079.

23. Eydox P, Choiset P, Le Porrier N. Chromosomal prenataldiagnosis: study of 936 cases of intrauterine abnormalitiesafter ultrasound assessment; Prenat Diagn 1989; 9: 255.

24. Gardener R J M, Sutherland G R. In ChromosomeAbnormalities & Genetic Counseling, Second edition,Oxford University press, New York, 1996.

25. Ward B E, Gersen S L, Carelli M P, et al. Rapid prenataldiagnosis of chromosomal aneuploidies by fluorescencein situ hybridization: clinical experience with 4,500specimens; Am. J Hum. Genet. 1993; 52: 854-865.

26. Jobanputra V, Roy K K, Kriplani A, Kucheria K. Prenataldiagnosis of chromosomal abnormalities in women withhigh-risk pregnancies; Indian J Med. Res.2001; 114: 148-155.

27. Lewin P, Kleinfinger P, Bazin A, Mossafa H, Szipro- Tapia S.Defining the efficiency of uncultured amniocytes on aretrospective cohort of 27,407 prenatal diagnosis;Prenatal. Diagn. 2000; 20: 1-6.

28. American College of Medical Genetics. Prenatalinterphase fluorescence in situ Hybridization (FISH) policystatement; Am J Hum Genet 1993; 53: 526-527.

29. American College of Medical Genetics. Technical andclinical assessment of fluorescence in situ hybridization:An American College of Medical Genetics (ACMG) &American Society of Human Genetics (ASHG) positionstatement I 2000 Technical considerations; Genet Med2000; 2: 356-361.

Apollo hospitals: http://www.apollohospitals.com/Twitter: https://twitter.com/HospitalsApolloYoutube: http://www.youtube.com/apollohospitalsindiaFacebook: http://www.facebook.com/TheApolloHospitalsSlideshare: http://www.slideshare.net/Apollo_HospitalsLinkedin: http://www.linkedin.com/company/apollo-hospitalsBlog:Blog: http://www.letstalkhealth.in/