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BACs-on-beads: a new robust and rapid detection method for prenatal diagnosis Expert Rev. Mol. Diagn. 14(3), 273–280 (2014)
Richard Kwong Wai Choy*1,2, Ying Chen3, Xiao-fang Sun4,5, Yvonne Ka Yin Kwok1 and Tak Yeung Leung1,2 1 Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China 2 Shenzhen Research Institute, School of Biomedical Sciences, The Chinese University of Hong Kong, Shenzhen, China 3 Center for Reproduction and Genetics, Nanjing Medical University Affiliated Suzhou Hospital, Suzhou, Jiangsu Province, China 4 Research Institute of Gynaecology and Obstetrics, Third Affiliated Hospital of Guangzhou Medical College, Guangzhou, China 5 Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institute, Guangzhou Medical University, Guangzhou, China *Author for correspondence: Tel.: +852 2632 3099 Fax: +852 2636 0008 [email protected]
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Karyotyping, the gold standard used for diagnosis of chromosomal abnormalities, is being progressively replaced by rapid aneuploidy testing (RAT) techniques such as quantitative fluorescence-PCR, FISH and multiplex ligation-dependent probe amplification for diagnosing the common aneuploidies or chromosomal microarray analysis for comprehensive genome-wide testing. However, due to technical limitations, current RATs are confined to the detection of common aneuploidies 13, 18, 21 and sex chromosomes. To overcome the limitations of RATs, a bacterial artificial chromosomes-on-beads (BoBsTM ) assay technology has been introduced for the detection of the common aneuploidies as well as specific microdeletion syndromes. The BoBs assay is a bead-based multiplex assay using polystyrene beads impregnated with two spectrally distinct infrared fluorochromes to create a liquid array of up to 100 unique spectral signatures that supports the analysis of that scale of simultaneous hybridization assays on a minute DNA sample. This review gives an overview on the collective experiences of BoBs applications in prenatal diagnosis. KEYWORDS: BACs-on-beads • POC • prenatal diagnosis • RAT
Invasive procedures in prenatal diagnosis are most frequently performed for the purpose of cytogenetic analysis, commonly in the form of conventional karyotyping. Karyotyping is highly reliable for the detection of all types of aneuploidies and significant chromosomal structural abnormalities, such as inversion, translocation, duplication and deletion (to a resolution of ~5 Mb) [1]. However, karyotyping is a labor-intensive process that requires days for cell culture to yield a sufficient number of active cells for karyotyping analysis. Hence, the pregnant woman has to undergo a long period of anxious waiting before reassurance or advice on intervention can be given. With the advent of molecular cytogenetic techniques, quick results are possible using rapid aneuploidy testing (RAT), such as quantitative fluorescence-PCR (QF-PCR), FISH and multiplex ligation-dependent probe amplification (MLPA). Consequently, different RAT methods have become well-established molecular genetics technique for routine use in prenatal diagnosis [1–3]. Nevertheless, these RATs are currently confined to the detection of the common aneuploidies of 13, 18, 21 and sex
10.1586/14737159.2014.899468
chromosomes in most routine cases, because the inclusion of other abnormalities requires the use of the additional reactions which would, in turn, increase the cost and time. To overcome the limitations of QF-PCR, MLPA and FISH [2–5], chromosomal microarray analysis (CMA) has been introduced for a comprehensive genome-wide identification of submicroscopic abnormalities in both the postnatal and prenatal settings [6–9]. While superior in detecting small chromosomal deletion/ duplication when compared with karyotyping analysis, CMA as a diagnostic tool also creates uncertainties when copy number variations of unknown pathogenic significance (VOUS) are detected and the test is relatively costly [6,8]. Recently, detection of aneuploidies, involving chromosomes 13, 18, 21, X and Y, and at least nine microdeletion regions using the technique of bacterial artificial chromosomes (BACs)-on-beads (BoBs) have been reported [10–15]. The BoBs assay is a bead-based multiplex assay, based on PerkinElmer’s BoBs and Luminex’s xMAP technologies [10]. The aim of the BoBs test is to extend from the currently available RATs to cover a more
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comprehensive set of microdeletion/microduplication syndromes while also avoiding the problem of uncertain results detected by genome-wide CMA analysis. In this review, we summarize the key findings from recent prenatal studies using BoBs analysis, describe the application of this technique in the clinical genetics evaluation of products of conception (POC) and discuss its implications for prenatal diagnosis and human health.
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 10/13/14 For personal use only.
The BoBs technology & its laboratory workflow
The BoBs assay is a bead-based multiplex assay, based on PerkinElmer’s BoBs and Luminex’s xMAP technologies [10]. xMAP utilizes polystyrene beads impregnated with a specific ratio of two spectrally distinct infrared and red fluorochromes enabling bead identification during the assay readout. Utilizing different concentrations of each of these two fluorochromes, a liquid array is created consisting up to 100 different dye/bead combinations representing unique spectral signatures. In BoBs assay, PCR products generated from selected BAC clones are immobilized as hybridization probes on beads with specific fluorometric signatures. The assay is designed for the detection of gains and losses of DNA in chromosomal regions associated with at least nine microdeletion syndromes, as well as the copy number of chromosomes 13, 18, 21, X and Y in human DNA. For the BoBs laboratory workflow SUPPLEMENTARY FIGURE 1 (supplementary material can be found online at www.informahealthcare. com/suppl/10.1586/14737159.2014.899468), 50–250 ng of genomic DNA was required for enzymatic biotin-dNTP labeling. Normal male and female DNAs (Promega, Madison, WI, USA) were also labeled and hybridized as references. After a series of wash steps before and after fluorescent streptavidin–phycoerythrin reporter incubation, the beads were read out using the Luminex 200 analyzer. The fluorescence assay data were analyzed with the assay’s companion BoBsoftTM software v1.0 (PerkinElmer, Turku, Finland). Five independent probes are utilized for each of the chromosome 13, 18, 21, X and Y; and 4–8 probes for each of the tested microdeletion regions generated results with both numeric and graphic presentations of probe and signal intensity ratios. Ratios are generated against both female (red line in the plots) and male (blue line in the plots) reference controls. Sample acceptance requires a coefficient of variation of all of the autosomal ratios to be 6% or less. A sample is defined as normal disomic when the ratio of the fluorescence intensities is within the lower and upper threshold (mean ratio ± 2 standard deviation). A sample is defined as deleted/ duplicated when the ratio of the fluorescence intensities in a chromosome locus is lower or higher than that of the lower and upper threshold. Typical abnormalities produce ratios ranging below 0.8 (deleted) and greater than 1.3 (duplicated) (FIGURE 1). In this manner, the assay allows detection of the common aneuploidies and microdeletions, yielding results within 24 h [11–15]. 274
Prospective performance of BoBs in the routine clinical setting
A search in the PubMed database using the term ‘BACs on beads’ displays the presence of a total of 12 related articles (TABLES 1 & 2) [10–21], of which 4 were in 2011, 3 in 2012 and 5 in 2013, thus demonstrating that it is a very new technique, which has not yet been widely used. The majority of these studies were on prenatal cases (TABLE 1) [10–16,18]. Except those case reports, all these pioneering works by Gross et al. [10], Vialard et al. [11,13], Shaffer et al. [12] and Piotrowski et al. [14] have demonstrated the ability of the BoBs assay in the rapid diagnosis of abnormalities involving chromosomes 13, 18, 21, X and Y, and the selected microdeletion syndromes. The assay is robust and with a relatively low failure rate of less than 4%. Based on the 2823 prenatal cases reported, the BoBs assay is superior to current RAT as a first-tier rapid test for prenatal diagnosis. All of the data support the proposal that the BoBs assay provides an extra diagnostic yield of common microdeletion syndromes including 22q11.2 and Williams–Beuren syndrome [16] compared with QF-PCR excepting polyploidies. An estimated additional diagnostic yield of Prenatal BoBsTM assay as compared with conventional karyotyping was reported to be 1 in 145 (11 out of 1599, ~0.7%) [13] and 1 in 50 (1 out of 50, ~2%), respectively [14]. The assay showed a very low false-positive and false-negative rate (25
>30
>30
N/A
†
The percentage represent overall all the studies combined.
(44.4%) than QF-PCR (33.3%) because of its ability to detect copy number changes for the additional target regions representing nine common microdeletion syndromes. When comparing the sensitivity in detecting chromosomes involving common aneuploidies, both BoBs and QF-PCR can detect six out of nine cases. The capability of the Prenatal BoBs assay for detecting chromosome mosaicism and maternal cell contamination (MCC) was also explored. Shaffer et al. reported that a male with 20% MCC could not be distinguished from 69,XXY [12]. They recommend that in this situation, MCC studies are recommended in conjunction with molecular assay to confirm that the DNA tested is predominantly fetal. Limitations of the BoBs assay
As BoBs technology has been validated in studies using prospective and retrospective samples, a small fraction of samples have been reported to be not suitable for the analysis by BoBs due to low DNA concentration or quality. The overall technical failure rate has been reported as 3–4% (TABLES 1 & 2). The failures were due to poor quality of the DNA, which in turn, produced noisy assay results with suboptimal autosomal ratio coefficient of variation values. Many of these should be able to be recovered by use of an improved DNA extraction method. In addition, due to the principle of assay, detection of polyploidy is not possible with BoBs, similar to microarrays. Vialard et al. reported six discordant results out of 212 prenatal samples, which were due to triploidies [11]. Shaffer et al. reported three discordant results out of 430 cases: one, 69, XXX, incorrectly identified as a normal female, and two, 69, XXY, initially considered as uninterpretable by the BoBs assay [12]. False-positive cases have also been reported by Vialard et al. [13]. They reported three false-positive results out of 1653 cases (0.18%). Two of them showed a borderline trend for clones mapping in the Williams–Beuren syndrome and resembling a mosaic haploinsufficiency condition. The third case was regarded a single clone deviation in Xp22. These findings emphasize the need to confirm any positive result by a second method. informahealthcare.com
Expert commentary
Regarding prenatal diagnosis, RAT is recommended as a standalone approach as part of Down syndrome screening programs in the UK and some parts of Europe [2,21]. A cost-effectiveness analysis of RAT by Gekas et al. demonstrated that only a very small number of clinically significant chromosomal abnormalities were missed when RAT was used as a standalone approach [24]. Further analysis demonstrated that healthcare savings can be substantial depending on the screening strategy used. Emerging studies also demonstrated that BoBs assay is a high-quality tool that provides rapid, accurate results and yields important additional information beyond that obtained by FISH and QF-PCR (TABLE 3) and overcomes any uncertainties of VOUS detected by CMA. Therefore, replacing current RAT with the BoBs assay would allow greater sensitivity for detecting additional chromosomal abnormalities and microdeletions missed by QF-PCR. This approach may be of great relevance to the global public health community. In many developing countries, there are insufficient cytogeneticists to analyze large numbers of amniocentesis samples or chorionic villus sampling, thus necessitating a molecular approach such as BoBs technology that would also be cost-effective. To date, there is no cost-effectiveness study comparing BoBs as standalone analysis with other standard methods of analysis in prenatal setting including QF-PCR and targeted interphase FISH analysis. In our opinion, the total hands-on and reporting time for BoBs assay was estimated to be much faster than QF-PCR and karyotyping, while the total hands-on time for QF-PCR was estimated to be 3 h for 12 samples (TABLE 3). The total reagent and consumable cost per sample for BoBs assay and QF-PCR was estimated to be similar (value for DNA extraction and equipment maintenance was not included). However, the hands-on time per sample for BoBs assay is much faster than QF-PCR (3 h for 12 samples vs 6 h for 92 samples), and therefore, the cost-effectiveness of BoBs is superior than QF-PCR. The BoBs assay is a newly developed test that is not yet widely used in many countries, probably due to the fact that most of the laboratories have already developed and validated their own RAT assays, for example, QF-PCR and FISH. In 277
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Table 3. Comparing the cost–effectiveness of various molecular methods versus karyotyping based on reagent cost in Hong Kong. Karyotyping
QF-PCR
Array CGH/CMA
BoBs
Hands-on work (h)
8–10
3
6–10
6
Cell culture
Yes
No
No
No
No cases/run
1
12
4–12
92
Reporting time (day)
14–20
1–2
2–3
2
Coverage
23 pair of chromosome
Chr. 13, 18, 21, X and Y
23 pair of chromosome down to 100 kb resolution
Chr. 13, 18, 21, X and Y and 9 microdeletion syndromes
Estimated cost per test (HKD)
1000
450
3500
520
BoBs: Bacterial artificial chromosomes-on-beads; CGH: Comparative genomic hybridization; CMA: Chromosomal microarray analysis; QF-PCR: Quantitative fluorescence PCR.
addition, the financial investment for bead array equipment purchase also is a contributor to the slow adoption of this technology. As compared with CMA, for laboratories that aim to develop RAT for prenatal diagnosis, BoBs assay may be the better option because this assay can run in high throughput, in a cheaper cost and can detect small chromosomal deletion/ duplication without creating uncertainties when copy number VOUS are detected. Therefore, it could be introduced to resource-limited countries where a rapid test is the only feasible method in managing patients with advanced maternal age or increased risk identified after maternal serum screening. Furthermore, the bead-based BoBs assay is highly flexible, and the target conditions to be detected could be added or removed according to the prevalence and characteristics of the local population [10]. Five-year view
Given that CMA is becoming more popular and widely recommended in prenatal diagnosis, it is hard to predict the value of BoBs for high-risk pregnancy with ultrasound structural abnormalities. However, considering cost-effectiveness and to avoid the findings of VOUS by CMA, BoBs assay will still be one of the best choices for women who only want or need to rule out the presence of common aneuploidies and microdeletion syndromes. The cost-effectiveness in terms of low cost and high throughput is a point particularly worth considering for higher volume prenatal laboratories that handle thousands of samples annually. On the other hand, since KaryoLite BoBs can screen for common chromosomal abnormalities among the 23 pairs of chromosomes, it may also have a great potential for preimplantation genetic screening [25]. It is well-known that numerical chromosomal abnormalities are present between 20 and 50% of IVF embryos. Emerging data also demonstrate that comprehensive embryo screening at numerical chromosome resolution at day 3 blastomeres and day 5 blastocysts can significantly improve the pregnancy rate from 41 to over 69% [26]. Recently, non-invasive prenatal screening (NIPS) has become poised to change the standard of care for prenatal 278
diagnosis. The NIPS is expected to be a preferred screening test for common aneuploidies, despite not being diagnostic. Because evidence on the performance of the testing in the general population is limited, so far only a few large-scale prospective studies in high-risk pregnancies have been performed [27,28]. Furthermore, the important metric for ‘positive predicative value’ has not yet been reported in any of the published literature. Expert opinion has suggested [29] that the clinical utility of NIPS in the general population has not yet been well established. Therefore, we assume that the clinical utility of NIPS for all pregnancies remains uncertain and there are requests that a large population randomized controlled study be done. Given that it is a screening test and a yet unproven utility, in general, for pregnant women, leading professional organizations recommend that NIPS be performed on ‘high-risk pregnancies’ only. In addition, we also recommend that positive results should be confirmed by invasive testing due to placental confined mosaicism and maternal factor [30]. In summary, due to the current limitation of NIPS on clinical utility in general population, the invasive test still remains an important role in prenatal diagnosis. Therefore, especially for a newly developed prenatal diagnostic laboratory, the BoBs assay may be a preferred cost-effective option over QF-PCR or other RATs. Acknowledgements
This work was partially supported by the Direct Grant for research, the Chinese University of Hong Kong and the Health and Medical Research fund from the Hong Kong Government. Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript. Expert Rev. Mol. Diagn. 14(3), (2014)
Rapid diagnostic testing by BACs-on-beads
Special Report
Key issues • Traditional karyotyping is the gold standard, but it is a labor-intensive method that requires days for cell culture to yield active cells for analysis. It will be progressively replaced by rapid aneuploidy testing as well as chromosomal microarray analysis. • Clinical needs drive the investigation of the high-throughput, multiplexed, quantitative and cost-effective new methods for prenatal diagnosis. • Bacterial artificial chromosomes-on-beads (BoBs) platform creates a liquid array of up to 100 different dye/bead combinations with unique spectral signatures and supports that scale of simultaneous hybridization assays on a minute DNA sample. • BoBs assay provides rapid, accurate results and yields important additional information beyond that obtained by the other rapid Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 10/13/14 For personal use only.
aneuploidy testing, such as FISH and quantitative fluorescence-PCR. • A negative result by BoBs does not fully exclude any of these syndromes, as there are other etiological bases for these disorders that are not covered in this platform. • False-positive and false-negative rates of BoBs are estimated to be approximately 0.2 and 1–3%, respectively.
disabilities or congenital anomalies. Am J Hum Genet 2010;86:749-64
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Choy KW, Setlur SR, Lee C, Lau TK. The impact of human copy number variation on a new era of genetic testing. BJOG 2010;117:391-8
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Gross SJ, Bajaj K, Garry D, et al. Rapid and novel prenatal molecular assay for detecting aneuploidies and microdeletion syndromes. Prenat Diagn 2011;31:259-66 First study that demonstrated the application of bacterial artificial chromosomes-on-beads (BoBs) technology in prenatal diagnosis of common aneuploidies and microdeletion syndrome. Vialard F, Simoni G, Aboura A, et al. Prenatal BACs-on-BeadsTM : a new technology for rapid detection of aneuploidies and microdeletions in prenatal diagnosis. Prenat Diagn 2011;31:500-8 Shaffer LG, Coppinger J, Morton SA, et al. The development of a rapid assay for prenatal testing of common aneuploidies
and microdeletion syndromes. Prenat Diagn 2011;31:778-87 •
The first study showing we can custom design the disease of interest by BoBs and also expand to 15 syndromes.
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Vialard F, Simoni G, Gomes DM, et al. Prenatal BACs-on-BeadsTM : the prospective experience of five prenatal diagnosis laboratories. Prenat Diagn 2012;32:329-35
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Piotrowski K, Henkelman M, Zajaczek S. Will the new molecular karyotyping BACs-on-Beads technique replace the traditional cytogenetic prenatal diagnostics? Preliminary reports. Ginekol Pol 2012;83: 284-90
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Cheng YK, Wong C, Wong HK, et al. The detection of mosaicism by prenatal BoBs. Prenat Diagn 2013;33:42-9
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The first and only study so far that evaluated the level of mosaicism detectable by BoBs.
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Popowski T, Vialard F, Leroy B, et al. Williams-Beuren syndrome: the prenatal phenotype. Am J Obstet Gynecol 2011; 205(6):e6-8
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Grati FR, Gomes DM, Ganesamoorthy D, et al. Application of a new molecular technique for the genetic evaluation of products of conception. Prenat Diagn 2013; 33(1):32-41
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The first study that demonstrated the advantage and application of BoBs for diagnosing numerical chromosomal abnormalities among product of conception.
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Popowski T, Molina-Gomes D, Loeuillet L, et al. Prenatal diagnosis of the duplication 17p11.2 associated with Potocki-Lupski syndrome in a foetus presenting with mildly dysmorphic features. Eur J Med Genet 2012;55:723-6
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Sheath KL, Duffy L, Asquith P, et al. Bacterial artificial chromosomes (BACs)-onBeadsTM as a diagnostic platform for the rapid aneuploidy screening of products of conception. Mol Med Rep 2013;8:650-4 Baxter L, Adayapalam N. A comparative study of standard cytogenetic evaluation and molecular karyotyping for products of conception. Diagn Mol Pathol 2013. [Epub ahead of print] Paxton CN, Brothman AR, Geiersbach KB. Rapid aneusomy detection in products of conception using the KaryoLiteTM BACs-on-BeadsTM assay. Prenat Diagn 2013; 33(1):25-31 Peakman DC, Moreton MF, Corn BJ, Robinson A. Chromosomal mosaicism in amniotic fluid cell cultures. Am J Hum Genet 1979;31:149-55 Wang BB, Rubin CH, Williams J 3rd. Mosaicism in chorionic villus sampling: an analysis of incidence and chromosomes involved in 2612 consecutive cases. Prenat Diagn 1993;13:179-90
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Gekas J, van den Berg DG, Durand A, et al. Rapid testing versus karyotyping in Down’s syndrome screening: cost-effectiveness and detection of clinically significant chromosome abnormalities. Eur J Hum Genet 2011;19:3-9 Gutie´rrez-Mateo C, Colls P, Sa´nchez-Garcı´a J, et al. Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos. Fertil Steril 2011;95:953-8 Yang Z, Liu J, Collins GS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet 2012;5:24 Lau TK, Cheung SW, Lo PS, et al. Non-invasive prenatal testing for fetal chromosomal abnormalities by low coverage whole genome sequencing of maternal plasma DNA: review of 1,982 consecutive
cases in a single center. Ultrasound Obstet Gynecol 2013. [Epub ahead of print] 28.
Futch T, Spinosa J, Bhatt S, et al. Initial clinical laboratory experience in noninvasive prenatal testing for fetal aneuploidy from maternal plasma DNA samples. Prenat Diagn 2013;33:569-74
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Morain S, Greene MF, Mello MM. A new era in noninvasive prenatal testing. N Engl J Med 2013;369:499-501
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Lau TK, Jiang FM, Stevenson RJ, et al. Secondary findings from non-invasive prenatal testing for common fetal aneuploidies by whole genome sequencing as a clinical service. Prenat Diagn 2013; 33(6):602-8
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Expert Rev. Mol. Diagn. 14(3), (2014)
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BACs-on-beads: a new robust and rapid detection method for prenatal diagnosis Expert Rev. Mol. Diagn. 14(3), 273–280 (2014)
Richard Kwong Wai Choy*1,2, Ying Chen3, Xiao-fang Sun4,5, Yvonne Ka Yin Kwok1 and Tak Yeung Leung1,2 1 Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China 2 Shenzhen Research Institute, School of Biomedical Sciences, The Chinese University of Hong Kong, Shenzhen, China 3 Center for Reproduction and Genetics, Nanjing Medical University Affiliated Suzhou Hospital, Suzhou, Jiangsu Province, China 4 Research Institute of Gynaecology and Obstetrics, Third Affiliated Hospital of Guangzhou Medical College, Guangzhou, China 5 Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institute, Guangzhou Medical University, Guangzhou, China *Author for correspondence: Tel.: +852 2632 3099 Fax: +852 2636 0008 [email protected]
informahealthcare.com
Karyotyping, the gold standard used for diagnosis of chromosomal abnormalities, is being progressively replaced by rapid aneuploidy testing (RAT) techniques such as quantitative fluorescence-PCR, FISH and multiplex ligation-dependent probe amplification for diagnosing the common aneuploidies or chromosomal microarray analysis for comprehensive genome-wide testing. However, due to technical limitations, current RATs are confined to the detection of common aneuploidies 13, 18, 21 and sex chromosomes. To overcome the limitations of RATs, a bacterial artificial chromosomes-on-beads (BoBsTM ) assay technology has been introduced for the detection of the common aneuploidies as well as specific microdeletion syndromes. The BoBs assay is a bead-based multiplex assay using polystyrene beads impregnated with two spectrally distinct infrared fluorochromes to create a liquid array of up to 100 unique spectral signatures that supports the analysis of that scale of simultaneous hybridization assays on a minute DNA sample. This review gives an overview on the collective experiences of BoBs applications in prenatal diagnosis. KEYWORDS: BACs-on-beads • POC • prenatal diagnosis • RAT
Invasive procedures in prenatal diagnosis are most frequently performed for the purpose of cytogenetic analysis, commonly in the form of conventional karyotyping. Karyotyping is highly reliable for the detection of all types of aneuploidies and significant chromosomal structural abnormalities, such as inversion, translocation, duplication and deletion (to a resolution of ~5 Mb) [1]. However, karyotyping is a labor-intensive process that requires days for cell culture to yield a sufficient number of active cells for karyotyping analysis. Hence, the pregnant woman has to undergo a long period of anxious waiting before reassurance or advice on intervention can be given. With the advent of molecular cytogenetic techniques, quick results are possible using rapid aneuploidy testing (RAT), such as quantitative fluorescence-PCR (QF-PCR), FISH and multiplex ligation-dependent probe amplification (MLPA). Consequently, different RAT methods have become well-established molecular genetics technique for routine use in prenatal diagnosis [1–3]. Nevertheless, these RATs are currently confined to the detection of the common aneuploidies of 13, 18, 21 and sex
10.1586/14737159.2014.899468
chromosomes in most routine cases, because the inclusion of other abnormalities requires the use of the additional reactions which would, in turn, increase the cost and time. To overcome the limitations of QF-PCR, MLPA and FISH [2–5], chromosomal microarray analysis (CMA) has been introduced for a comprehensive genome-wide identification of submicroscopic abnormalities in both the postnatal and prenatal settings [6–9]. While superior in detecting small chromosomal deletion/ duplication when compared with karyotyping analysis, CMA as a diagnostic tool also creates uncertainties when copy number variations of unknown pathogenic significance (VOUS) are detected and the test is relatively costly [6,8]. Recently, detection of aneuploidies, involving chromosomes 13, 18, 21, X and Y, and at least nine microdeletion regions using the technique of bacterial artificial chromosomes (BACs)-on-beads (BoBs) have been reported [10–15]. The BoBs assay is a bead-based multiplex assay, based on PerkinElmer’s BoBs and Luminex’s xMAP technologies [10]. The aim of the BoBs test is to extend from the currently available RATs to cover a more
2014 Informa UK Ltd
ISSN 1473-7159
273
Special Report
Choy, Chen, Sun, Kwok & Leung
comprehensive set of microdeletion/microduplication syndromes while also avoiding the problem of uncertain results detected by genome-wide CMA analysis. In this review, we summarize the key findings from recent prenatal studies using BoBs analysis, describe the application of this technique in the clinical genetics evaluation of products of conception (POC) and discuss its implications for prenatal diagnosis and human health.
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The BoBs technology & its laboratory workflow
The BoBs assay is a bead-based multiplex assay, based on PerkinElmer’s BoBs and Luminex’s xMAP technologies [10]. xMAP utilizes polystyrene beads impregnated with a specific ratio of two spectrally distinct infrared and red fluorochromes enabling bead identification during the assay readout. Utilizing different concentrations of each of these two fluorochromes, a liquid array is created consisting up to 100 different dye/bead combinations representing unique spectral signatures. In BoBs assay, PCR products generated from selected BAC clones are immobilized as hybridization probes on beads with specific fluorometric signatures. The assay is designed for the detection of gains and losses of DNA in chromosomal regions associated with at least nine microdeletion syndromes, as well as the copy number of chromosomes 13, 18, 21, X and Y in human DNA. For the BoBs laboratory workflow SUPPLEMENTARY FIGURE 1 (supplementary material can be found online at www.informahealthcare. com/suppl/10.1586/14737159.2014.899468), 50–250 ng of genomic DNA was required for enzymatic biotin-dNTP labeling. Normal male and female DNAs (Promega, Madison, WI, USA) were also labeled and hybridized as references. After a series of wash steps before and after fluorescent streptavidin–phycoerythrin reporter incubation, the beads were read out using the Luminex 200 analyzer. The fluorescence assay data were analyzed with the assay’s companion BoBsoftTM software v1.0 (PerkinElmer, Turku, Finland). Five independent probes are utilized for each of the chromosome 13, 18, 21, X and Y; and 4–8 probes for each of the tested microdeletion regions generated results with both numeric and graphic presentations of probe and signal intensity ratios. Ratios are generated against both female (red line in the plots) and male (blue line in the plots) reference controls. Sample acceptance requires a coefficient of variation of all of the autosomal ratios to be 6% or less. A sample is defined as normal disomic when the ratio of the fluorescence intensities is within the lower and upper threshold (mean ratio ± 2 standard deviation). A sample is defined as deleted/ duplicated when the ratio of the fluorescence intensities in a chromosome locus is lower or higher than that of the lower and upper threshold. Typical abnormalities produce ratios ranging below 0.8 (deleted) and greater than 1.3 (duplicated) (FIGURE 1). In this manner, the assay allows detection of the common aneuploidies and microdeletions, yielding results within 24 h [11–15]. 274
Prospective performance of BoBs in the routine clinical setting
A search in the PubMed database using the term ‘BACs on beads’ displays the presence of a total of 12 related articles (TABLES 1 & 2) [10–21], of which 4 were in 2011, 3 in 2012 and 5 in 2013, thus demonstrating that it is a very new technique, which has not yet been widely used. The majority of these studies were on prenatal cases (TABLE 1) [10–16,18]. Except those case reports, all these pioneering works by Gross et al. [10], Vialard et al. [11,13], Shaffer et al. [12] and Piotrowski et al. [14] have demonstrated the ability of the BoBs assay in the rapid diagnosis of abnormalities involving chromosomes 13, 18, 21, X and Y, and the selected microdeletion syndromes. The assay is robust and with a relatively low failure rate of less than 4%. Based on the 2823 prenatal cases reported, the BoBs assay is superior to current RAT as a first-tier rapid test for prenatal diagnosis. All of the data support the proposal that the BoBs assay provides an extra diagnostic yield of common microdeletion syndromes including 22q11.2 and Williams–Beuren syndrome [16] compared with QF-PCR excepting polyploidies. An estimated additional diagnostic yield of Prenatal BoBsTM assay as compared with conventional karyotyping was reported to be 1 in 145 (11 out of 1599, ~0.7%) [13] and 1 in 50 (1 out of 50, ~2%), respectively [14]. The assay showed a very low false-positive and false-negative rate (25
>30
>30
N/A
†
The percentage represent overall all the studies combined.
(44.4%) than QF-PCR (33.3%) because of its ability to detect copy number changes for the additional target regions representing nine common microdeletion syndromes. When comparing the sensitivity in detecting chromosomes involving common aneuploidies, both BoBs and QF-PCR can detect six out of nine cases. The capability of the Prenatal BoBs assay for detecting chromosome mosaicism and maternal cell contamination (MCC) was also explored. Shaffer et al. reported that a male with 20% MCC could not be distinguished from 69,XXY [12]. They recommend that in this situation, MCC studies are recommended in conjunction with molecular assay to confirm that the DNA tested is predominantly fetal. Limitations of the BoBs assay
As BoBs technology has been validated in studies using prospective and retrospective samples, a small fraction of samples have been reported to be not suitable for the analysis by BoBs due to low DNA concentration or quality. The overall technical failure rate has been reported as 3–4% (TABLES 1 & 2). The failures were due to poor quality of the DNA, which in turn, produced noisy assay results with suboptimal autosomal ratio coefficient of variation values. Many of these should be able to be recovered by use of an improved DNA extraction method. In addition, due to the principle of assay, detection of polyploidy is not possible with BoBs, similar to microarrays. Vialard et al. reported six discordant results out of 212 prenatal samples, which were due to triploidies [11]. Shaffer et al. reported three discordant results out of 430 cases: one, 69, XXX, incorrectly identified as a normal female, and two, 69, XXY, initially considered as uninterpretable by the BoBs assay [12]. False-positive cases have also been reported by Vialard et al. [13]. They reported three false-positive results out of 1653 cases (0.18%). Two of them showed a borderline trend for clones mapping in the Williams–Beuren syndrome and resembling a mosaic haploinsufficiency condition. The third case was regarded a single clone deviation in Xp22. These findings emphasize the need to confirm any positive result by a second method. informahealthcare.com
Expert commentary
Regarding prenatal diagnosis, RAT is recommended as a standalone approach as part of Down syndrome screening programs in the UK and some parts of Europe [2,21]. A cost-effectiveness analysis of RAT by Gekas et al. demonstrated that only a very small number of clinically significant chromosomal abnormalities were missed when RAT was used as a standalone approach [24]. Further analysis demonstrated that healthcare savings can be substantial depending on the screening strategy used. Emerging studies also demonstrated that BoBs assay is a high-quality tool that provides rapid, accurate results and yields important additional information beyond that obtained by FISH and QF-PCR (TABLE 3) and overcomes any uncertainties of VOUS detected by CMA. Therefore, replacing current RAT with the BoBs assay would allow greater sensitivity for detecting additional chromosomal abnormalities and microdeletions missed by QF-PCR. This approach may be of great relevance to the global public health community. In many developing countries, there are insufficient cytogeneticists to analyze large numbers of amniocentesis samples or chorionic villus sampling, thus necessitating a molecular approach such as BoBs technology that would also be cost-effective. To date, there is no cost-effectiveness study comparing BoBs as standalone analysis with other standard methods of analysis in prenatal setting including QF-PCR and targeted interphase FISH analysis. In our opinion, the total hands-on and reporting time for BoBs assay was estimated to be much faster than QF-PCR and karyotyping, while the total hands-on time for QF-PCR was estimated to be 3 h for 12 samples (TABLE 3). The total reagent and consumable cost per sample for BoBs assay and QF-PCR was estimated to be similar (value for DNA extraction and equipment maintenance was not included). However, the hands-on time per sample for BoBs assay is much faster than QF-PCR (3 h for 12 samples vs 6 h for 92 samples), and therefore, the cost-effectiveness of BoBs is superior than QF-PCR. The BoBs assay is a newly developed test that is not yet widely used in many countries, probably due to the fact that most of the laboratories have already developed and validated their own RAT assays, for example, QF-PCR and FISH. In 277
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Choy, Chen, Sun, Kwok & Leung
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Table 3. Comparing the cost–effectiveness of various molecular methods versus karyotyping based on reagent cost in Hong Kong. Karyotyping
QF-PCR
Array CGH/CMA
BoBs
Hands-on work (h)
8–10
3
6–10
6
Cell culture
Yes
No
No
No
No cases/run
1
12
4–12
92
Reporting time (day)
14–20
1–2
2–3
2
Coverage
23 pair of chromosome
Chr. 13, 18, 21, X and Y
23 pair of chromosome down to 100 kb resolution
Chr. 13, 18, 21, X and Y and 9 microdeletion syndromes
Estimated cost per test (HKD)
1000
450
3500
520
BoBs: Bacterial artificial chromosomes-on-beads; CGH: Comparative genomic hybridization; CMA: Chromosomal microarray analysis; QF-PCR: Quantitative fluorescence PCR.
addition, the financial investment for bead array equipment purchase also is a contributor to the slow adoption of this technology. As compared with CMA, for laboratories that aim to develop RAT for prenatal diagnosis, BoBs assay may be the better option because this assay can run in high throughput, in a cheaper cost and can detect small chromosomal deletion/ duplication without creating uncertainties when copy number VOUS are detected. Therefore, it could be introduced to resource-limited countries where a rapid test is the only feasible method in managing patients with advanced maternal age or increased risk identified after maternal serum screening. Furthermore, the bead-based BoBs assay is highly flexible, and the target conditions to be detected could be added or removed according to the prevalence and characteristics of the local population [10]. Five-year view
Given that CMA is becoming more popular and widely recommended in prenatal diagnosis, it is hard to predict the value of BoBs for high-risk pregnancy with ultrasound structural abnormalities. However, considering cost-effectiveness and to avoid the findings of VOUS by CMA, BoBs assay will still be one of the best choices for women who only want or need to rule out the presence of common aneuploidies and microdeletion syndromes. The cost-effectiveness in terms of low cost and high throughput is a point particularly worth considering for higher volume prenatal laboratories that handle thousands of samples annually. On the other hand, since KaryoLite BoBs can screen for common chromosomal abnormalities among the 23 pairs of chromosomes, it may also have a great potential for preimplantation genetic screening [25]. It is well-known that numerical chromosomal abnormalities are present between 20 and 50% of IVF embryos. Emerging data also demonstrate that comprehensive embryo screening at numerical chromosome resolution at day 3 blastomeres and day 5 blastocysts can significantly improve the pregnancy rate from 41 to over 69% [26]. Recently, non-invasive prenatal screening (NIPS) has become poised to change the standard of care for prenatal 278
diagnosis. The NIPS is expected to be a preferred screening test for common aneuploidies, despite not being diagnostic. Because evidence on the performance of the testing in the general population is limited, so far only a few large-scale prospective studies in high-risk pregnancies have been performed [27,28]. Furthermore, the important metric for ‘positive predicative value’ has not yet been reported in any of the published literature. Expert opinion has suggested [29] that the clinical utility of NIPS in the general population has not yet been well established. Therefore, we assume that the clinical utility of NIPS for all pregnancies remains uncertain and there are requests that a large population randomized controlled study be done. Given that it is a screening test and a yet unproven utility, in general, for pregnant women, leading professional organizations recommend that NIPS be performed on ‘high-risk pregnancies’ only. In addition, we also recommend that positive results should be confirmed by invasive testing due to placental confined mosaicism and maternal factor [30]. In summary, due to the current limitation of NIPS on clinical utility in general population, the invasive test still remains an important role in prenatal diagnosis. Therefore, especially for a newly developed prenatal diagnostic laboratory, the BoBs assay may be a preferred cost-effective option over QF-PCR or other RATs. Acknowledgements
This work was partially supported by the Direct Grant for research, the Chinese University of Hong Kong and the Health and Medical Research fund from the Hong Kong Government. Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript. Expert Rev. Mol. Diagn. 14(3), (2014)
Rapid diagnostic testing by BACs-on-beads
Special Report
Key issues • Traditional karyotyping is the gold standard, but it is a labor-intensive method that requires days for cell culture to yield active cells for analysis. It will be progressively replaced by rapid aneuploidy testing as well as chromosomal microarray analysis. • Clinical needs drive the investigation of the high-throughput, multiplexed, quantitative and cost-effective new methods for prenatal diagnosis. • Bacterial artificial chromosomes-on-beads (BoBs) platform creates a liquid array of up to 100 different dye/bead combinations with unique spectral signatures and supports that scale of simultaneous hybridization assays on a minute DNA sample. • BoBs assay provides rapid, accurate results and yields important additional information beyond that obtained by the other rapid Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 10/13/14 For personal use only.
aneuploidy testing, such as FISH and quantitative fluorescence-PCR. • A negative result by BoBs does not fully exclude any of these syndromes, as there are other etiological bases for these disorders that are not covered in this platform. • False-positive and false-negative rates of BoBs are estimated to be approximately 0.2 and 1–3%, respectively.
disabilities or congenital anomalies. Am J Hum Genet 2010;86:749-64
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Expert Rev. Mol. Diagn. 14(3), (2014)
