ISPD 2013 MEETING PRESENTATION

Controversies in prenatal diagnosis 3: should everyoneundergoing invasive testing have a microarray?John A. Crolla1, Ronald Wapner2 and Jan M. M. Van Lith3*

1Wessex Regional Genetics Laboratory, Salisbury, UK2Columbia University College of Physicians and Surgeons, New York, NY, USA3Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands*Correspondence to: Jan M. M. Van Lith. E-mail: j.m.m.van_lith@lumc.nl

Funding sources: NoneConflicts of interest: None declared

The possibility to obtain fetal cells during pregnancy hasopened the way to prenatal diagnosis of genetic disorders. Thisstarted in 1996/1967, when Steele and Breg,1 and Jacobson andBarter,2 reported the possibility to karyotype fetal cells andsecond trimester amniocentesis for chromosomal disorders.In 1983, Simoni et al.3 described karyotyping of unculturedchorionic villi, moving prenatal diagnosis forward to the firsttrimester. Until recently, these invasive procedures andkaryotyping remained the gold standard in prenatal diagnosisfor genetic disorders.

Down syndrome is related to maternal age, and this was themain indication for invasive procedures until the introductionof risk assessment in early pregnancy in 1988.4 This riskassessment refined to the combination screening test and/orthe quadruple test.

Another important development in diagnosing fetal anomalieswas the introduction of ultrasound. This developed from the1980s onwards. Structural anomalies detected by ultrasoundare in most cases followed by invasive prenatal diagnosis. Thisis the other main indication to offer invasive testing.

In the late 1990s, new genetic techniques, such asfluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification, and PCR were implemented,referred to as rapid aneuploidy testing. These techniques ledto earlier results when compared with karyotyping, were oftencheaper; however, only selected chromosome regions weretested. Some thought this to be advantageous, others thoughtit to be too limited.5

This change started discussions on what to test for. Thescreening tests mainly focused on Down syndrome, whereasthe invasive tests covered amuchbroader range of chromosomaldisorders. In case of ultrasound anomalies, a broader test rangeis needed. The genetic techniques further developed, andmicroarrays are routinely used in postnatal genetic diagnosiscovering a broader range of genetic disorders. There are goodreasons to introducemicroarrays in prenatal diagnosis; however,it does have disadvantages.6

The question for the debaters (John Crolla and RonaldWapner) at the 17th International Conference was: ‘Shouldeveryone undergoing invasive testing have a microarray?’.

IN FAVOR (RONALD WAPNER)For over 50 years, karyotyping has been the only technology usedfor prenatal cytogenetic diagnosis, but – more recently – newerandmore robust techniques have entered the field. One of theseis chromosomal microarray analysis (CMA). CMA has anumber of significant advantages over karyotyping, whichsuggest that it should be made available to all patientsundergoing invasive prenatal testing (and perhaps should beoffered to all pregnant women).

The most important technical advantage of CMA is theability to detect all of the unbalanced cytogenetic findingspresently identified by karyotyping, whereas – in addition –

having markedly superior resolution allowing identificationof much smaller genomic alterations. At best, a karyotypehas a resolution between 7 and 10 million base pairs(bp), whereas chromosomal microarray has the ability toidentify microdeletions and duplications in the 100 to300 kb range.

Identification of cytogenetic alterations too small to be seenby karyotype has major clinical importance. Most of the welldescribed microdeletion and microduplication syndromes aresecondary to copy number variants in the 1.5 to 3.5Mb range.In addition, there are now well described non-syndromaleffects of microdeletions and duplications resulting from copynumber variants smaller than 1 Mb.7 This improved resolutionhas led to CMA becoming the first tier test for postnatalevaluation of children with congenital anomalies, dysmorphicfeatures, and neurocognitive difficulties. In these children,microarray analysis has an incremental 10% to 15% yield ofclinically relevant findings over karyotype. This advantagealone should be sufficient to recommend microarray analysison all prenatal samples.7

Prenatal Diagnosis 2014, 34, 18–22 © 2013 John Wiley & Sons, Ltd.

DOI: 10.1002/pd.4287

Chromosomal microarray analysis has additional technicaladvantages beyond improved resolution. It provides directmapping of aberrations to their location in the genomesequence, which significantly improves the ability to definemarker chromosomes, to confirm that de novo balancedtranslocations are actually balanced, and to identify the specificstart and stoppoints of these changes. In contrast, in karyotypingthe location of the abnormality is not nearly as precise.

Another technological advantage of CMA is that it is amenableto automation and quite easily undergoes quality control,whereas the resolution and reliability of karyotyping is dependenton the experience and abilities of the cytogenetics laboratory.CMA is also capable of being performed on uncultured cells anddoes not require good metaphase spreads, which should resultin higher throughputwithmore rapid reporting time.8 In the longrun, because CMA requires less manpower, it should becomemore cost-effective than karyotyping.

The incremental information provided by CMA improvesour ability to counsel patients identified with a pregnancyhaving a fetal structural anomaly. Multiple studies9,10 havedemonstrated that in these cases a clinically relevant copynumber variant will be identified, which can alter prognosticcounseling. Routine use of CMA reveals that this occurs inapproximately 6% of pregnancies with a normal karyotype.8,11

For example, counseling a patient whose fetus has a cardiacdefect secondary to a 22q11.2 deletion requires (in additionto details about surgical correction of the defect) discussionof the associated learning and developmental disabilities.12

Understanding the etiology therefore is an important adjunctto the parental counseling session.

Although 22q11.2 deletions are routinely tested for when acardiac structural abnormality is identified, it is not the onlymicrodeletion or duplication, in which cardiac malformationsare associated with intellectual disability (Table 1). In our

experience with the National Institute of Child Health andHuman Development (NICHD) prenatal microarray study,13

in which 297 fetuses with structural cardiac defects wereevaluated, only one third of the causative copy numbervariants were deletions of 22q11.2. In other words, if only FISHfor the 22q11.2 deletion were performed, two thirds of thefamilies having a copy number variant that could alter theprognosis would not be aware of this.

Presently, most practitioners recommend invasive prenataldiagnosis when the ultrasound findings are suggestive of acommon autosomal aneuploidy. We are now aware thatmicrodeletions and duplicatons may have more subtle in uterophenotypes so that identification of these requires expandingthe ultrasound criteria used for invasive diagnosis. Table 2demonstrates a number of single fetal structural abnormalitiesidentified in the NICHD prenatal array study13 that are notroutinely associated with aneuploidy but have a highfrequency of having a microdeletion or duplication.

Although it is clear that CMA has incremental value inevaluating structurally abnormal pregnancies, it is alsoimportant to explore its use in patients undergoing invasivetesting for more routine indications, such as advancedmaternal age (AMA) or positive aneuploid screening. Of theslightly less than 3000 patients without anomalies evaluatedin the NICHD prenatal microarray study, just short of 2000were sampled for AMA and 729 for positive screening. In eachof these categories, approximately 1 in 60 pregnancies wereidentified as having a clinically relevant microdeletion orduplication.8 Although some of these microdeletions andduplications had mild phenotypes, approximately 1 in 125without a structural abnormality is known to be associatedwith significant neurocognitive impairment.8 This informationcan be valuable for many parents in making reproductivedecisions, and it will have significant value in the futuremanagement of the child.

When testing a structurally normal pregnancy, theidentification of a copy number variant early in gestationmay suggest the potential of finding a subsequent fetalstructural anomaly later in gestation. In essence, the genotypeidentified with CMA will give the practicing physician theknowledge to do a targeted fetal surveillance study later ingestation to better delineate the phenotype.

One of the concerns about prenatal CMA is that there maybe results that do not have severe and/or lethal consequences.

Table 2 Genetic abnormalities seen in patients with a SingleFetal Structural Defect (N=845)

System NAbnormal

karyotype (%) Abnormal CMA (%)

Cardiac 92 16 13

IUGR 49 10 9

NT≥3.5mm 337 50 4

CNS 95 13 4

Skeletal 23 4 4

Others 234 12 5

CMA, chromosomal microarrays; IUGR, intrauterine growth restriction.

Table 1 Significant microdeletions associated with congenitalheart disease

Copy number

Variant Syndrome Additional phenotype

Del 1p36 ID

Del 1q21.1 Mild ID

Del p16.3 Wolf–Hirschhornsyndrome

Microcephaly, severe ID, andseizure

Del 5p15.2 Cri-du-chat Severe ID

Del 5q35.2 ASD and conductiondefect

Del 7q11.23 Williams–Buerensyndrome

Cognitive deficits and infantilehypocalcemia

Del 8p23.1 ID

Del 9 q34

Del 11 q23-qter Jacobsen syndrome ID

Del 16p13.3 Rubinstein Taybi +CHD ID

Del 20p12.2 Alagille syndrome Liver disease

Del 22q11.2 DiGeorge syndrome ID, schizophrenia

ID, intellectual disability.

Controversies in prenatal diagnosis 3 19

Prenatal Diagnosis 2014, 34, 18–22 © 2013 John Wiley & Sons, Ltd.

This raises the question of whether these disorders should bediagnosed in utero. However, we must realize that as newprenatal tests deliver improved resolution, they offerincreasing options for use of the genetic information.Discovery of many of these findings can assist the parents inseeking appropriate care early in the infant’s life. For example,fetuses identified to have a microdeletion or duplicationassociated with a high risk for autism could initiate treatmentmuch earlier, which has been demonstrated to have a markedimprovement in the long-term prognosis for the child.Similarly, children at risk for learning disorders could haveearly intervention before starting standard schooling.

One other concern mentioned about CMA is theidentification of variants of uncertain clinical significance. Thisis a relatively minor concern that will shrink over time. OurNICHD study8 demonstrated that between 2007 and 2012, theclassification of copy number variants discovered in uteroand classified as uncertain decreased from 2.5% to 1.5%.Interestingly, the majority of findings initially identified asuncertain have subsequently been confirmed as pathogenic.8

As additional information from the use of CMA and more casesof similar microdeletions and duplications are documented,the number of findings of uncertain clinical significance willcontinue to fall. One must remember also that findings ofunknown clinical significance occur routinely withkaryotyping. Han et al.14 in 2008 demonstrated withkaryotyping that findings of uncertain clinical significanceoccurred in almost 1% of cases. Chang et al.15 in 2012demonstrated a similar 1% incidence of findings of uncertainsignificance in a similar cohort. Many of the uncertain findingsin karyotyping, such as de novo, apparently balancedtranslocations or marker chromosomes are actually betterdefined using CMA. Therefore, the primary use of CMA willminimize these numbers as well.

In conclusion, it is relatively clear that the increaseddetection afforded by CMA makes it the ideal test to becomethe first tier tool for prenatal diagnosis. Not only should onerecommend it as the first tier test for anyone having invasivetesting, the over 1% frequency of clinically relevant findingsin all pregnancies suggests that all patients should be madeaware of the technology and have the option of having theirpregnancy evaluated by CMA.

AGAINST (JOHN A. CROLLA)For the past 40years, conventional cytogenetics has been themain laboratory technique for the prenatal diagnosis ofchromosomal abnormalities. Ironically, in the context of thisdiscussion, the principal purpose of cytogenetic prenataldiagnosis has been intrinsically linked to risk factors associatedwith Down syndrome, initially increased maternal age. Over thepast two decades, the maternal age risk has been combined withmaternal serum and ultrasound markers to provide more robustrisk algorithms associated with a risk of a Down syndromepregnancy.16 The irony alluded to above is that, from the outset,conventional cytogenetics was capable of detecting and reportingstructural (e.g. balanced and unbalanced translocations) as wellas the common autosomal and sex chromosome numericalabnormalities (including trisomy 21) and so has a long history of

dealing with chromosome abnormalities incidental to theprimary referral reason.

Conventional cytogenetics remained the primary prenataldiagnostic test until recently when molecular techniques weredeveloped to provide fast and accurate methods for theidentification of the common autosomal and sex chromosomeaneuploidies. In many countries, quantitative fluorescencepolymerase chain reaction (QF-PCR) is now the primary invasiveprenatal diagnostic test for trisomies 13, 18, 21, and for numericalsex chromosome abnormalities17 but because of concerns aboutmissing structural chromosome abnormalities,18 QF-PCR isusually supplemented with a karyotype result.

Although technical advances in prenatal cytogeneticdiagnostics have remained relatively static, by comparison thepast decade has seen a revolution in postnatal cytogeneticsdriven by the introduction of array comparative genomehybridization (aCGH) driven initially by proof of principalstudies which showed that if aCGH was targeted to definedclinical populations diagnostic yields (of pathogenic sub-microscopic deletions and duplications) were~20% higher thanachieved using karyotyping.19,20 Contemporaneously, aCGHstudies also showed that genomic imbalances, called CopyNumber Variants (CNV) were also present in clinically normalcontrol populations.21,22 By 2010, aCGH had largely replacedkaryotyping for patients with neurodevelopmental and/orcongenital abnormalities in most major cytogenetic centers inthe USA, UK, Europe, and Australasia, and its implementationwas fostered in part by large-scale collaborations such as theInternational Standard for Cytogenomic Array Consortium,which in turn resulted in a key consensus statement .7

Compared with postnatal cytogenetics, the uptake and use ofaCGH in the prenatal area has been slower with a more cautiousapproach to it’s implementation.23 Early proof of principlesstudies utilized previously characterized chromosomallyabnormal prenatal samples and a targeted array to determinethe sensitivity of detection rates between conventionalcytogenetics and aCGH.24 However, over the past 2 years, largeprenatal aCGH scale studies have been published, which a prioriappear to show that aCGH can improve the detection of‘clinically relevant CNVs’ in the presence of a normal karyotypeby~3%.8,9,25,26 This diagnostic pick up rate is significantly higherif aCGH is targeted to fetuses with ultrasound abnormalities andthis ranges from 6% to 13% depending on the study design.27–29

Amongst women undergoing prenatal, aCGH for maternal ageor other indications other than fetal anomalies, the detectionrate of clinically significant CNVs was reported to be~1%.8

The articles quoted earlier have recently been collated byCallaway et al.30 and on the face of it, the increased detectionrate achieved by prenatal aCGH appears compelling. However,it is important to take into account that at least some of thestudies quoted have been carried out within a defined researchprotocol8 and so aspects of the clinical use of prenatal aCGHrequires further consideration before the technology can beused to routinely replace karyotyping either partly orcompletely. The principal reasons for caution at this stage aresummarized below.

At the heart of a debate about prenatal microarrays lies theinterpretation of a CNV (or CNVs) against which the clinician

J. A. Crolla et al.20

Prenatal Diagnosis 2014, 34, 18–22 © 2013 John Wiley & Sons, Ltd.

(fetal medicine consultants, obstetricians, and geneticists) andthe laboratory scientist will have relatively limited clinical andphenotypic information to make a judgment call on thepossible clinical consequences of a significant number ofaCGH detected imbalances. Clearly, at one end of thespectrum, there are clearly defined micro deletion and microduplication syndromes, which show high penetrance, areassociated with well characterized clinical phenotypes and, iffound prenatally, can be associated with relatively robust andaccurate genetic counseling. However, such clear cutcorrelations in the data quoted earlier represent the minorityof cases reported, and at the other end of the same spectrumare rare or unique CNVs with no know associated clinicalphenotypes [classified as Variants of Uncertain ClinicalSignificance (VOUS)]. Between these two ends of the spectrumlie a number of CNVs with highly variable clinical expressivityassociated with incomplete penetrance, which are problematicenough in the postnatal area but in the context of a prenataldiagnosis cause significant diagnostic, counseling, and ethicaldilemmas.31 Into this already complex mix, there will also bethe admittedly rare but nevertheless difficult cases where theCNV(s) detected may be incidental to the referral reason (e.g.dominant cancer gene loci and other late onset disorders)but for which a well defined protocol must be in place beforewidespread prenatal aCGH can be implemented.

The proponents for an immediate implementation ofprenatal aCGH suggest that the speed with which data derivedfrom the clinical use of postnatal aCGH is accumulating meansthat the proportion of cases currently defined as VOUS willdecline as more genotype/phenotype data emerge, but thisdoes not take into consideration how prenatally detected CNVswith variable penetrance or VOUS should be handled now. Thestudy by Wapner et al.8 in 2012 and an ongoing research studyin the UK called EACH (Evaluation of Array ComparativeGenomic Hybridization in prenatal diagnosis of fetalabnormalities) both have made use of an expert review panelto determine whether or not specific VOUS should be reportedto the patient or not. In this context, to date approximately3.5% of aCGH results had been referred to the EACH reviewcommittee, which consists of five consultant clinical geneticistsand five consultant clinical cytogeneticists. Approximately halfof the cases referred for EACH review were recommended forreporting and half for not reporting. Although tertiary review ofthis type already exists within some postnatal aCGH centers, itis vital that before prenatal aCGH is widely adopted theresourcing, logictics, and resource implications of such tertiaryreview groups must be fully considered.

Another important aspect of prenatal aCGH implementationinvolves the design of the array and whether the analysesshould be targeted to known pathogenic regions together witha low density backbone coverage, or the backbone coverageshould be higher density together with higher probe coverageof known pathogenic CNVs.32 The final decision of whichplatform to adopt may also be complicated not only byscientific evidence but also by the health economics drivingthe method of delivery adopted by different countries.33 Thereis also the unresolved discussion on whether prenatal aCGHshould be limited to dosage analysis using oligos or single

nucleotide polymorphisms (SNPs) or should also routinelyincorporate the use of SNPs to detect possibly pathogenicsegmental or whole chromosome uniparental disomy.34

The lag between proof of principle studies showing, in aresearch context, the clinical utility of a novel technique andthe widespread application of the novel technique in clinicaland laboratory practice can be significant, and for thepostnatal application of aCGH, this took several years in manycountries including the UK. Technological advances are sorapid that important laboratory external quality assurancesschemes for novel technologies often lag behind theirimplementation, and for prenatal aCGH no such schemes haveyet been developed or incorporated into laboratory practice.Furthermore, to date there are no clear guidelines publishedfor the use of prenatal aCGH from either the USA or Europeanstakeholders although the International Society for PrenatalDiagnosis (ISPD) is currently drafting such guidelines.

Finally, in the ISPD debate in Lisbon, the question put was‘should everyone undergoing invasive testing have amicroarray’. The answer to this question is ‘not yet’ for twoprincipal reasons. First, there are simpler, cheaper, and morecost-effective ways of testing for Down syndrome and the othercommon aneuploidies following invasive prenatal diagnosis(e.g. QF-PCR). Furthermore, the availability and gradualadoption of non-invasive next generation sequencingapproaches to the diagnosis of Down syndrome is changing theprenatal screening and subsequent testing protocols for prenatalhealth care providers worldwide. Prenatal aCGH shouldtherefore only be used once the common aneuploidies havebeen excluded by other methods. Second, although it is clearthat postnatal aCGH has contributed greatly to ourunderstanding of the underlying pathology of many CNVs, thefact remains that many of the reported prenatal CNVs are eitherunique or VOUS and therefore require detailed interpretationutilizing multidisciplinary teams of laboratory scientists, geneticcounselors, and fetal medicine practitioners. The cost andresource implications of this needs to be considered so that,once implemented, prenatal aCGH can make a positivecontribution to improving diagnosis and prognosis.

CONCLUSIONAround 50 years ago, prenatal diagnosis became possible forchromosomal disorders. Ultrasound further opened up thepossibility to diagnose fetal abnormalities early in pregnancy.Nowadays, a great part of fetal structural and geneticanomalies can be diagnosed early in pregnancy, therebyproviding reproductive choices to future parents and earlytreatment options for newborns improving outcome andprognosis.

New genetic techniques have evolved rapidly in recent years.CMA has replaced karyotyping in postnatal diagnosis. Itsimplementation in prenatal diagnosis has been slower andmore cautious. The debate clearly shows that CMA increasesthe diagnostic scope and brings with it new challenges.

Both debaters agree that CMA will have a place in prenataldiagnosis. Its exact application needs further evaluation, andthe implementation of quality system is a prerequisite.

Controversies in prenatal diagnosis 3 21

Prenatal Diagnosis 2014, 34, 18–22 © 2013 John Wiley & Sons, Ltd.

WHAT’S ALREADY KNOWN ABOUT THIS TOPIC?

• Chromosomal microarrays (CMA) are routinely used in postnatalgenetic diagnosis.

• CMA is technically applicable in prenatal diagnosis.• Pros and cons of routine use are discussed as follows: technical

aspects and design of array, yield, interpretation of CNV andvariances of unknown significance (VOUS), quality control regimens.

WHAT DOES THIS STUDY ADD?

• Pros and cons of routine use are discussed as follows:technical aspects, and design of array, yield, interpretationof CNV and variances of unknown significance (VOUS), qualitycontrol regimens.

REFERENCES1. Steele M, Breg WJ. Chromosome analysis of human amniotic-fluid cells.

Lancet 1966;1:383–5.2. Jacobson CB, Barter RH. Intrauterine diagnosis and management of

genetic defects. Am J Obstet Gynecol 1967;99:796–807.3. Simoni G, Brambati B, Danesino C, et al. Efficient direct chromosome

analysis and enzyme determinations from chorionic villus samples inthe first trimester of pregnancy. Hum Genet 1983;63:349–57.

4. Wald NJ, Cuckle HS, Densem JW, et al. Maternal serum screening forDown’s syndrome in early pregnancy. BMJ 1988;883–7.

5. De Jong A, Dondorp WJ, Timmermans DRM, et al. Rapid aneuploidydetection or karyotyping? Ethical reflection. Eur J Hum Genet2011;19:1020–5.

6. De Jong A, Dondorp WJ, Macvill MVE, et al. Microarrays as a diagnostictool in prenatal screening strategies. Ethical reflection. Hum Genet 2013[Epub ahead of print].

7. Miller DT, Adam MP, Aradhya S, et al. Consensus statement:chromosomal microarray is a first-tier clinical diagnostic test forindividuals with developmental disabilities or congenital anomalies. AmJ Hum Genet 2010;86:749–64.

8. Wapner RJ, Martin CL, Levy B, et al. Chromosomal microarray versuskaryotyping for prenatal diagnosis. N Engl J Med 2012;367:2175–84.

9. Fiorentino F, Caiazzo F, Napolitano S, et al. Introducing arraycomparative genomic hybridization into routine prenatal diagnosispractice: a prospective study on over 1000 consecutive clinical cases.Prenatal diagnosis 2011;31:1270–82.

10. Schwartz S. Clinical utility of single nucleotide polymorphism arrays.Clinics in Laboratory Medicine 2011;31:581–94, viii.

11. Shaffer LG, Rosenfeld JA. Microarray-based prenatal diagnosis for theidentification of fetal chromosome abnormalities. Expert Rev Mol Diagn2013;13:601–1.

12. Tang SX, Yi JJ, Calkins ME, et al. Psychiatric disorders in 22q11.2deletion syndrome are prevalent but undertreated. PsychologicalMedicine 2013: available on CJO2013. DOI: 10.1017/S0033291713001669.

13. Donnelly J, Platt L, Rebarber A, Wapner R. Association of Copy NumberVariants With Single and Multiple Structural Anomalies of Specific FetalOrgan Systems Presented at 17th International Conference on PrenatalDiagnosis and Therapy, 2013; Lisbon Portugal.

14. Han SH, An JW, Jeong GY, et al. Clinical and cytogenetic findings on31,615 mid-trimester amniocenteses. The Korean Journal of LaboratoryMedicine 2008;28:378–85.

15. Chang YW, Chang CM, Sung PL, et al. An overview of a 30-yearexperience with amniocentesis in a single tertiary medical center inTaiwan. Taiwanese Journal of Obstetrics & Gynecology 2012;51:206–11.

16. Wald NJ, Rodeck C, Hackshaw AK, et al. First and second trimesterantenatal screening for Down’s syndrome: the results of the Serum,Urine and Ultrasound Screening Study (SURUSS). J Med Screen2003;10:56–104.

17. Mann K, Hills A, Donaghue C, et al. Quantitative fluorescence PCRanalysis of >40,000 prenatal samples for the rapid diagnosis of trisomies13, 18 and 21 and monosomy X. Prenat Diagn 2012;32:1197–204

18. Caine A, Maltby AE, Parkin CA, et al. UK Association of ClinicalCytogeneticists (ACC). Prenatal detection of Down’s syndrome by rapid

aneuploidy testing for chromosomes 13, 18, and 21 by FISH or PCRwithout a full karyotype: a cytogenetic risk assessment. Lancet2005;366:123–8.

19. Vissers LE, de Vries BB, Osoegawa K, et al. Array-based comparativegenomic hybridization for the genomewide detection ofsubmicroscopic chromosomal abnormalities. Am J Hum Genet2003;73:1261–70.

20. Shaw-Smith C, Redon R, Rickman L, et al. Microarray basedcomparative genomic hybridisation (array-CGH) detectssubmicroscopic chromosomal deletions and duplications in patientswith learning disability/mental retardation and dysmorphic features. JMed Genet 2004;41:241–8.

21. Sebat J, Lakshmi B, Troge J, et al. Large-scale copy numberpolymorphism in the human genome. Science 2004;305:525–8.

22. Lafrate AJ, Feuk L, Rivera MN, et al. Detection of large-scale variation inthe human genome. Nat Genet 2004;36:949–51.

23. Rickman L, Fiegler H, Carter NP, Bobrow M. Prenatal diagnosis byarray-CGH. Eur J Med Genet 2005;48:232–40.

24. Rickman L, Fiegler H, Shaw-Smith C, et al. Prenatal detection ofunbalanced chromosomal rearrangements by array CGH. J Med Genet2006;43:353–61.

25. Shaffer LG, Dabell MP, Fisher AJ, et al. Experience with microarray-basedcomparative genomic hybridization for prenatal diagnosis in over 5000pregnancies. Prenat Diag 2012;32:1–10.

26. Lee C-N, Lin S-Y, Lin C-H, et al. Clinical utility of array comparativegenomic hybridisation for prenatal diagnosis: a cohort study of 3171pregnancies. BJOG 2012;119:614–25.

27. Tyreman M, Abbott KM, Willatt LR, et al. High resolution array analysis:diagnosing pregnancies with abnormal ultrasound findings. J MedGenet 2009;46:531–41.

28. Srebniak MI, Boter M, Oudesluijs GO, et al. Genomic SNP array as a goldstandard for prenatal diagnosis of foetal ulltrasound abnormalities. MolCytogenet 2012;5:14–7.

29. Rooryck C, Toutain J, Cailley D, et al. Prenatal diagnosis using array-CGH:a French experience. Eur J Med Genet 2013. DOI: 10.1016/j.ejmg.2013.02.003.

30. Callaway JL, Shaffer LG, Chitty L, et al. The clinical utility of microarraytechnologies applied to prenatal cytogenetics in the presence of anormal conventional karyotype: a review of the literature. Prenat Diagn.2013 Aug 23. DOI: 10.1002/pd.4209. [Epub ahead of print].

31. Mikhaelian M, Veach PM, MacFarlane I, et al. Prenatal chromosomalmicroarray analysis: a survey of prenatal genetic counselors’experiences and attitudes. Prenat Diagn 2013;33:371–7.

32. Baldwin EL, Lee JY, Blake DM, et al. Enhanced detection of clinicallyrelevant genomic imbalances using a targeted plus whole genomeoligonucleotide microarray. Genet Med 2008;10:415–29.

33. Vetro A, Bouman K, Hastings R, et al. The introduction of arrays inprenatal diagnosis: a special challenge. Hum Mutat 2012;33:923–9.

34. Ganesamoorthy D, Bruno DL, McGillivray G, et al. Meeting thechallenge of interpreting high-resolution single nucleotidepolymorphism array data in prenatal diagnosis: does increaseddiagnostic power outweigh the dilemma of rare variants? BJOG2013;120:594–606.

J. A. Crolla et al.22

Prenatal Diagnosis 2014, 34, 18–22 © 2013 John Wiley & Sons, Ltd.