Controversies in prenatal diagnosis 3 should everyone


Published on

Published in: Health & Medicine
  • Be the first to comment

  • Be the first to like this

Controversies in prenatal diagnosis 3 should everyone

  1. 1. ISPD 2013 MEETING PRESENTATION Controversies in prenatal diagnosis 3: should everyone undergoing invasive testing have a microarray? John A. Crolla1 , Ronald Wapner2 and Jan M. M. Van Lith3* 1 Wessex Regional Genetics Laboratory, Salisbury, UK 2 Columbia University College of Physicians and Surgeons, New York, NY, USA 3 Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands *Correspondence to: Jan M. M. Van Lith. E-mail: Funding sources: None Conflicts of interest: None declared The possibility to obtain fetal cells during pregnancy has opened the way to prenatal diagnosis of genetic disorders. This started in 1996/1967, when Steele and Breg,1 and Jacobson and Barter,2 reported the possibility to karyotype fetal cells and second trimester amniocentesis for chromosomal disorders. In 1983, Simoni et al.3 described karyotyping of uncultured chorionic villi, moving prenatal diagnosis forward to the first trimester. Until recently, these invasive procedures and karyotyping remained the gold standard in prenatal diagnosis for genetic disorders. Down syndrome is related to maternal age, and this was the main indication for invasive procedures until the introduction of risk assessment in early pregnancy in 1988.4 This risk assessment refined to the combination screening test and/or the quadruple test. Another important development in diagnosing fetal anomalies was the introduction of ultrasound. This developed from the 1980s onwards. Structural anomalies detected by ultrasound are in most cases followed by invasive prenatal diagnosis. This is the other main indication to offer invasive testing. In the late 1990s, new genetic techniques, such as fluorescence in situ hybridization (FISH), multiplex ligation- dependent probe amplification, and PCR were implemented, referred to as rapid aneuploidy testing. These techniques led to earlier results when compared with karyotyping, were often cheaper; however, only selected chromosome regions were tested. Some thought this to be advantageous, others thought it to be too limited.5 This change started discussions on what to test for. The screening tests mainly focused on Down syndrome, whereas the invasive tests covered a much broader range of chromosomal disorders. In case of ultrasound anomalies, a broader test range is needed. The genetic techniques further developed, and microarrays are routinely used in postnatal genetic diagnosis covering a broader range of genetic disorders. There are good reasons to introduce microarrays in prenatal diagnosis; however, it does have disadvantages.6 The question for the debaters (John Crolla and Ronald Wapner) at the 17th International Conference was: ‘Should everyone undergoing invasive testing have a microarray?’. IN FAVOR (RONALD WAPNER) For over 50 years, karyotyping has been the only technology used for prenatal cytogenetic diagnosis, but – more recently – newer and more robust techniques have entered the field. One of these is chromosomal microarray analysis (CMA). CMA has a number of significant advantages over karyotyping, which suggest that it should be made available to all patients undergoing invasive prenatal testing (and perhaps should be offered to all pregnant women). The most important technical advantage of CMA is the ability to detect all of the unbalanced cytogenetic findings presently identified by karyotyping, whereas – in addition – having markedly superior resolution allowing identification of much smaller genomic alterations. At best, a karyotype has a resolution between 7 and 10 million base pairs (bp), whereas chromosomal microarray has the ability to identify microdeletions and duplications in the 100 to 300 kb range. Identification of cytogenetic alterations too small to be seen by karyotype has major clinical importance. Most of the well described microdeletion and microduplication syndromes are secondary to copy number variants in the 1.5 to 3.5 Mb range. In addition, there are now well described non-syndromal effects of microdeletions and duplications resulting from copy number variants smaller than 1 Mb.7 This improved resolution has led to CMA becoming the first tier test for postnatal evaluation of children with congenital anomalies, dysmorphic features, and neurocognitive difficulties. In these children, microarray analysis has an incremental 10% to 15% yield of clinically relevant findings over karyotype. This advantage alone should be sufficient to recommend microarray analysis on all prenatal samples.7 Prenatal Diagnosis 2014, 34, 18–22 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/pd.4287
  2. 2. Chromosomal microarray analysis has additional technical advantages beyond improved resolution. It provides direct mapping of aberrations to their location in the genome sequence, which significantly improves the ability to define marker chromosomes, to confirm that de novo balanced translocations are actually balanced, and to identify the specific start and stop points of these changes. In contrast, in karyotyping the location of the abnormality is not nearly as precise. Another technological advantage of CMA is that it is amenable to automation and quite easily undergoes quality control, whereas the resolution and reliability of karyotyping is dependent on the experience and abilities of the cytogenetics laboratory. CMA is also capable of being performed on uncultured cells and does not require good metaphase spreads, which should result in higher throughput with more rapid reporting time.8 In the long run, because CMA requires less manpower, it should become more cost-effective than karyotyping. The incremental information provided by CMA improves our ability to counsel patients identified with a pregnancy having a fetal structural anomaly. Multiple studies9,10 have demonstrated that in these cases a clinically relevant copy number variant will be identified, which can alter prognostic counseling. Routine use of CMA reveals that this occurs in approximately 6% of pregnancies with a normal karyotype.8,11 For example, counseling a patient whose fetus has a cardiac defect secondary to a 22q11.2 deletion requires (in addition to details about surgical correction of the defect) discussion of the associated learning and developmental disabilities.12 Understanding the etiology therefore is an important adjunct to the parental counseling session. Although 22q11.2 deletions are routinely tested for when a cardiac structural abnormality is identified, it is not the only microdeletion or duplication, in which cardiac malformations are associated with intellectual disability (Table 1). In our experience with the National Institute of Child Health and Human Development (NICHD) prenatal microarray study,13 in which 297 fetuses with structural cardiac defects were evaluated, only one third of the causative copy number variants were deletions of 22q11.2. In other words, if only FISH for the 22q11.2 deletion were performed, two thirds of the families having a copy number variant that could alter the prognosis would not be aware of this. Presently, most practitioners recommend invasive prenatal diagnosis when the ultrasound findings are suggestive of a common autosomal aneuploidy. We are now aware that microdeletions and duplicatons may have more subtle in utero phenotypes so that identification of these requires expanding the ultrasound criteria used for invasive diagnosis. Table 2 demonstrates a number of single fetal structural abnormalities identified in the NICHD prenatal array study13 that are not routinely associated with aneuploidy but have a high frequency of having a microdeletion or duplication. Although it is clear that CMA has incremental value in evaluating structurally abnormal pregnancies, it is also important to explore its use in patients undergoing invasive testing for more routine indications, such as advanced maternal age (AMA) or positive aneuploid screening. Of the slightly less than 3000 patients without anomalies evaluated in the NICHD prenatal microarray study, just short of 2000 were sampled for AMA and 729 for positive screening. In each of these categories, approximately 1 in 60 pregnancies were identified as having a clinically relevant microdeletion or duplication.8 Although some of these microdeletions and duplications had mild phenotypes, approximately 1 in 125 without a structural abnormality is known to be associated with significant neurocognitive impairment.8 This information can be valuable for many parents in making reproductive decisions, and it will have significant value in the future management of the child. When testing a structurally normal pregnancy, the identification of a copy number variant early in gestation may suggest the potential of finding a subsequent fetal structural anomaly later in gestation. In essence, the genotype identified with CMA will give the practicing physician the knowledge to do a targeted fetal surveillance study later in gestation to better delineate the phenotype. One of the concerns about prenatal CMA is that there may be results that do not have severe and/or lethal consequences. Table 2 Genetic abnormalities seen in patients with a Single Fetal Structural Defect (N = 845) System N Abnormal karyotype (%) Abnormal CMA (%) Cardiac 92 16 13 IUGR 49 10 9 NT ≥ 3.5 mm 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 congenital heart disease Copy number Variant Syndrome Additional phenotype Del 1p36 ID Del 1q21.1 Mild ID Del p16.3 Wolf–Hirschhorn syndrome Microcephaly, severe ID, and seizure Del 5p15.2 Cri-du-chat Severe ID Del 5q35.2 ASD and conduction defect Del 7q11.23 Williams–Bueren syndrome Cognitive deficits and infantile hypocalcemia 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.
  3. 3. This raises the question of whether these disorders should be diagnosed in utero. However, we must realize that as new prenatal tests deliver improved resolution, they offer increasing options for use of the genetic information. Discovery of many of these findings can assist the parents in seeking appropriate care early in the infant’s life. For example, fetuses identified to have a microdeletion or duplication associated with a high risk for autism could initiate treatment much earlier, which has been demonstrated to have a marked improvement in the long-term prognosis for the child. Similarly, children at risk for learning disorders could have early intervention before starting standard schooling. One other concern mentioned about CMA is the identification of variants of uncertain clinical significance. This is a relatively minor concern that will shrink over time. Our NICHD study8 demonstrated that between 2007 and 2012, the classification of copy number variants discovered in utero and classified as uncertain decreased from 2.5% to 1.5%. Interestingly, the majority of findings initially identified as uncertain have subsequently been confirmed as pathogenic.8 As additional information from the use of CMA and more cases of similar microdeletions and duplications are documented, the number of findings of uncertain clinical significance will continue to fall. One must remember also that findings of unknown clinical significance occur routinely with karyotyping. Han et al.14 in 2008 demonstrated with karyotyping that findings of uncertain clinical significance occurred in almost 1% of cases. Chang et al.15 in 2012 demonstrated a similar 1% incidence of findings of uncertain significance in a similar cohort. Many of the uncertain findings in karyotyping, such as de novo, apparently balanced translocations or marker chromosomes are actually better defined using CMA. Therefore, the primary use of CMA will minimize these numbers as well. In conclusion, it is relatively clear that the increased detection afforded by CMA makes it the ideal test to become the first tier tool for prenatal diagnosis. Not only should one recommend it as the first tier test for anyone having invasive testing, the over 1% frequency of clinically relevant findings in all pregnancies suggests that all patients should be made aware of the technology and have the option of having their pregnancy evaluated by CMA. AGAINST (JOHN A. CROLLA) For the past 40years, conventional cytogenetics has been the main laboratory technique for the prenatal diagnosis of chromosomal abnormalities. Ironically, in the context of this discussion, the principal purpose of cytogenetic prenatal diagnosis has been intrinsically linked to risk factors associated with Down syndrome, initially increased maternal age. Over the past two decades, the maternal age risk has been combined with maternal serum and ultrasound markers to provide more robust risk algorithms associated with a risk of a Down syndrome pregnancy.16 The irony alluded to above is that, from the outset, conventional cytogenetics was capable of detecting and reporting structural (e.g. balanced and unbalanced translocations) as well as the common autosomal and sex chromosome numerical abnormalities (including trisomy 21) and so has a long history of dealing with chromosome abnormalities incidental to the primary referral reason. Conventional cytogenetics remained the primary prenatal diagnostic test until recently when molecular techniques were developed to provide fast and accurate methods for the identification of the common autosomal and sex chromosome aneuploidies. In many countries, quantitative fluorescence polymerase chain reaction (QF-PCR) is now the primary invasive prenatal diagnostic test for trisomies 13, 18, 21, and for numerical sex chromosome abnormalities17 but because of concerns about missing structural chromosome abnormalities,18 QF-PCR is usually supplemented with a karyotype result. Although technical advances in prenatal cytogenetic diagnostics have remained relatively static, by comparison the past decade has seen a revolution in postnatal cytogenetics driven by the introduction of array comparative genome hybridization (aCGH) driven initially by proof of principal studies which showed that if aCGH was targeted to defined clinical populations diagnostic yields (of pathogenic sub- microscopic deletions and duplications) were~ 20% higher than achieved using karyotyping.19,20 Contemporaneously, aCGH studies also showed that genomic imbalances, called Copy Number Variants (CNV) were also present in clinically normal control populations.21,22 By 2010, aCGH had largely replaced karyotyping for patients with neurodevelopmental and/or congenital abnormalities in most major cytogenetic centers in the USA, UK, Europe, and Australasia, and its implementation was fostered in part by large-scale collaborations such as the International Standard for Cytogenomic Array Consortium, which in turn resulted in a key consensus statement .7 Compared with postnatal cytogenetics, the uptake and use of aCGH in the prenatal area has been slower with a more cautious approach to it’s implementation.23 Early proof of principles studies utilized previously characterized chromosomally abnormal prenatal samples and a targeted array to determine the sensitivity of detection rates between conventional cytogenetics and aCGH.24 However, over the past 2 years, large prenatal aCGH scale studies have been published, which a priori appear to show that aCGH can improve the detection of ‘clinically relevant CNVs’ in the presence of a normal karyotype by ~ 3%.8,9,25,26 This diagnostic pick up rate is significantly higher if aCGH is targeted to fetuses with ultrasound abnormalities and this ranges from 6% to 13% depending on the study design.27–29 Amongst women undergoing prenatal, aCGH for maternal age or other indications other than fetal anomalies, the detection rate of clinically significant CNVs was reported to be ~ 1%.8 The articles quoted earlier have recently been collated by Callaway et al.30 and on the face of it, the increased detection rate achieved by prenatal aCGH appears compelling. However, it is important to take into account that at least some of the studies quoted have been carried out within a defined research protocol8 and so aspects of the clinical use of prenatal aCGH requires further consideration before the technology can be used to routinely replace karyotyping either partly or completely. The principal reasons for caution at this stage are summarized below. At the heart of a debate about prenatal microarrays lies the interpretation 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.
  4. 4. (fetal medicine consultants, obstetricians, and geneticists) and the laboratory scientist will have relatively limited clinical and phenotypic information to make a judgment call on the possible clinical consequences of a significant number of aCGH detected imbalances. Clearly, at one end of the spectrum, there are clearly defined micro deletion and micro duplication syndromes, which show high penetrance, are associated with well characterized clinical phenotypes and, if found prenatally, can be associated with relatively robust and accurate genetic counseling. However, such clear cut correlations in the data quoted earlier represent the minority of cases reported, and at the other end of the same spectrum are rare or unique CNVs with no know associated clinical phenotypes [classified as Variants of Uncertain Clinical Significance (VOUS)]. Between these two ends of the spectrum lie a number of CNVs with highly variable clinical expressivity associated with incomplete penetrance, which are problematic enough in the postnatal area but in the context of a prenatal diagnosis cause significant diagnostic, counseling, and ethical dilemmas.31 Into this already complex mix, there will also be the admittedly rare but nevertheless difficult cases where the CNV(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 before widespread prenatal aCGH can be implemented. The proponents for an immediate implementation of prenatal aCGH suggest that the speed with which data derived from the clinical use of postnatal aCGH is accumulating means that the proportion of cases currently defined as VOUS will decline as more genotype/phenotype data emerge, but this does not take into consideration how prenatally detected CNVs with variable penetrance or VOUS should be handled now. The study by Wapner et al.8 in 2012 and an ongoing research study in the UK called EACH (Evaluation of Array Comparative Genomic Hybridization in prenatal diagnosis of fetal abnormalities) both have made use of an expert review panel to determine whether or not specific VOUS should be reported to the patient or not. In this context, to date approximately 3.5% of aCGH results had been referred to the EACH review committee, which consists of five consultant clinical geneticists and five consultant clinical cytogeneticists. Approximately half of the cases referred for EACH review were recommended for reporting and half for not reporting. Although tertiary review of this type already exists within some postnatal aCGH centers, it is vital that before prenatal aCGH is widely adopted the resourcing, logictics, and resource implications of such tertiary review groups must be fully considered. Another important aspect of prenatal aCGH implementation involves the design of the array and whether the analyses should be targeted to known pathogenic regions together with a low density backbone coverage, or the backbone coverage should be higher density together with higher probe coverage of known pathogenic CNVs.32 The final decision of which platform to adopt may also be complicated not only by scientific evidence but also by the health economics driving the method of delivery adopted by different countries.33 There is also the unresolved discussion on whether prenatal aCGH should be limited to dosage analysis using oligos or single nucleotide polymorphisms (SNPs) or should also routinely incorporate the use of SNPs to detect possibly pathogenic segmental or whole chromosome uniparental disomy.34 The lag between proof of principle studies showing, in a research context, the clinical utility of a novel technique and the widespread application of the novel technique in clinical and laboratory practice can be significant, and for the postnatal application of aCGH, this took several years in many countries including the UK. Technological advances are so rapid that important laboratory external quality assurances schemes for novel technologies often lag behind their implementation, and for prenatal aCGH no such schemes have yet been developed or incorporated into laboratory practice. Furthermore, to date there are no clear guidelines published for the use of prenatal aCGH from either the USA or European stakeholders although the International Society for Prenatal Diagnosis (ISPD) is currently drafting such guidelines. Finally, in the ISPD debate in Lisbon, the question put was ‘should everyone undergoing invasive testing have a microarray’. The answer to this question is ‘not yet’ for two principal reasons. First, there are simpler, cheaper, and more cost-effective ways of testing for Down syndrome and the other common aneuploidies following invasive prenatal diagnosis (e.g. QF-PCR). Furthermore, the availability and gradual adoption of non-invasive next generation sequencing approaches to the diagnosis of Down syndrome is changing the prenatal screening and subsequent testing protocols for prenatal health care providers worldwide. Prenatal aCGH should therefore only be used once the common aneuploidies have been excluded by other methods. Second, although it is clear that postnatal aCGH has contributed greatly to our understanding of the underlying pathology of many CNVs, the fact remains that many of the reported prenatal CNVs are either unique or VOUS and therefore require detailed interpretation utilizing multidisciplinary teams of laboratory scientists, genetic counselors, and fetal medicine practitioners. The cost and resource implications of this needs to be considered so that, once implemented, prenatal aCGH can make a positive contribution to improving diagnosis and prognosis. CONCLUSION Around 50 years ago, prenatal diagnosis became possible for chromosomal disorders. Ultrasound further opened up the possibility to diagnose fetal abnormalities early in pregnancy. Nowadays, a great part of fetal structural and genetic anomalies can be diagnosed early in pregnancy, thereby providing reproductive choices to future parents and early treatment options for newborns improving outcome and prognosis. New genetic techniques have evolved rapidly in recent years. CMA has replaced karyotyping in postnatal diagnosis. Its implementation in prenatal diagnosis has been slower and more cautious. The debate clearly shows that CMA increases the diagnostic scope and brings with it new challenges. Both debaters agree that CMA will have a place in prenatal diagnosis. Its exact application needs further evaluation, and the implementation of quality system is a prerequisite. Controversies in prenatal diagnosis 3 21 Prenatal Diagnosis 2014, 34, 18–22 © 2013 John Wiley & Sons, Ltd.
  5. 5. WHAT’S ALREADY KNOWN ABOUT THIS TOPIC? • Chromosomal microarrays (CMA) are routinely used in postnatal genetic 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 and variances 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, interpretation of CNV and variances of unknown significance (VOUS), quality control regimens. REFERENCES 1. 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 in the first trimester of pregnancy. Hum Genet 1983;63:349–57. 4. Wald NJ, Cuckle HS, Densem JW, et al. Maternal serum screening for Down’s syndrome in early pregnancy. BMJ 1988;883–7. 5. De Jong A, Dondorp WJ, Timmermans DRM, et al. Rapid aneuploidy detection or karyotyping? Ethical reflection. Eur J Hum Genet 2011;19:1020–5. 6. De Jong A, Dondorp WJ, Macvill MVE, et al. Microarrays as a diagnostic tool 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 for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet 2010;86:749–64. 8. Wapner RJ, Martin CL, Levy B, et al. Chromosomal microarray versus karyotyping for prenatal diagnosis. N Engl J Med 2012;367:2175–84. 9. Fiorentino F, Caiazzo F, Napolitano S, et al. Introducing array comparative genomic hybridization into routine prenatal diagnosis practice: 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 the identification of fetal chromosome abnormalities. Expert Rev Mol Diagn 2013;13:601–1. 12. Tang SX, Yi JJ, Calkins ME, et al. Psychiatric disorders in 22q11.2 deletion syndrome are prevalent but undertreated. Psychological Medicine 2013: available on CJO2013. DOI: 10.1017/ S0033291713001669. 13. Donnelly J, Platt L, Rebarber A, Wapner R. Association of Copy Number Variants With Single and Multiple Structural Anomalies of Specific Fetal Organ Systems Presented at 17th International Conference on Prenatal Diagnosis and Therapy, 2013; Lisbon Portugal. 14. Han SH, An JW, Jeong GY, et al. Clinical and cytogenetic findings on 31,615 mid-trimester amniocenteses. The Korean Journal of Laboratory Medicine 2008;28:378–85. 15. Chang YW, Chang CM, Sung PL, et al. An overview of a 30-year experience with amniocentesis in a single tertiary medical center in Taiwan. Taiwanese Journal of Obstetrics & Gynecology 2012;51:206–11. 16. Wald NJ, Rodeck C, Hackshaw AK, et al. First and second trimester antenatal screening for Down’s syndrome: the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). J Med Screen 2003;10:56–104. 17. Mann K, Hills A, Donaghue C, et al. Quantitative fluorescence PCR analysis of >40,000 prenatal samples for the rapid diagnosis of trisomies 13, 18 and 21 and monosomy X. Prenat Diagn 2012;32:1197–204 18. Caine A, Maltby AE, Parkin CA, et al. UK Association of Clinical Cytogeneticists (ACC). Prenatal detection of Down’s syndrome by rapid aneuploidy testing for chromosomes 13, 18, and 21 by FISH or PCR without a full karyotype: a cytogenetic risk assessment. Lancet 2005;366:123–8. 19. Vissers LE, de Vries BB, Osoegawa K, et al. Array-based comparative genomic hybridization for the genomewide detection of submicroscopic chromosomal abnormalities. Am J Hum Genet 2003;73:1261–70. 20. Shaw-Smith C, Redon R, Rickman L, et al. Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features. J Med Genet 2004;41:241–8. 21. Sebat J, Lakshmi B, Troge J, et al. Large-scale copy number polymorphism in the human genome. Science 2004;305:525–8. 22. Lafrate AJ, Feuk L, Rivera MN, et al. Detection of large-scale variation in the human genome. Nat Genet 2004;36:949–51. 23. Rickman L, Fiegler H, Carter NP, Bobrow M. Prenatal diagnosis by array-CGH. Eur J Med Genet 2005;48:232–40. 24. Rickman L, Fiegler H, Shaw-Smith C, et al. Prenatal detection of unbalanced chromosomal rearrangements by array CGH. J Med Genet 2006;43:353–61. 25. Shaffer LG, Dabell MP, Fisher AJ, et al. Experience with microarray-based comparative genomic hybridization for prenatal diagnosis in over 5000 pregnancies. Prenat Diag 2012;32:1–10. 26. Lee C-N, Lin S-Y, Lin C-H, et al. Clinical utility of array comparative genomic hybridisation for prenatal diagnosis: a cohort study of 3171 pregnancies. 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 Med Genet 2009;46:531–41. 28. Srebniak MI, Boter M, Oudesluijs GO, et al. Genomic SNP array as a gold standard for prenatal diagnosis of foetal ulltrasound abnormalities. Mol Cytogenet 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 microarray technologies applied to prenatal cytogenetics in the presence of a normal 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 chromosomal microarray 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 clinically relevant genomic imbalances using a targeted plus whole genome oligonucleotide microarray. Genet Med 2008;10:415–29. 33. Vetro A, Bouman K, Hastings R, et al. The introduction of arrays in prenatal diagnosis: a special challenge. Hum Mutat 2012;33:923–9. 34. Ganesamoorthy D, Bruno DL, McGillivray G, et al. Meeting the challenge of interpreting high-resolution single nucleotide polymorphism array data in prenatal diagnosis: does increased diagnostic power outweigh the dilemma of rare variants? BJOG 2013;120:594–606. J. A. Crolla et al.22 Prenatal Diagnosis 2014, 34, 18–22 © 2013 John Wiley & Sons, Ltd.