Chromosomal Microarray Versus Karyotyping For Prenatal Diagnosis - 2012
Chromosomal Microarray Versus Karyotyping For Prenatal Diagnosis - 2012
Chromosomal Microarray Versus Karyotyping For Prenatal Diagnosis - 2012
journal of medicine
The
established in 1812
december 6, 2012
A bs t r ac t
Background
Chromosomal microarray analysis has emerged as a primary diagnostic tool for the
evaluation of developmental delay and structural malformations in children. We
aimed to evaluate the accuracy, efficacy, and incremental yield of chromosomal
microarray analysis as compared with karyotyping for routine prenatal diagnosis.
Methods
We enrolled a total of 4406 women. Indications for prenatal diagnosis were advanced maternal age (46.6%), abnormal result on Downs syndrome screening
(18.8%), structural anomalies on ultrasonography (25.2%), and other indications
(9.4%). In 4340 (98.8%) of the fetal samples, microarray analysis was successful;
87.9% of samples could be used without tissue culture. Microarray analysis of the
4282 nonmosaic samples identified all the aneuploidies and unbalanced rearrangements identified on karyotyping but did not identify balanced translocations and
fetal triploidy. In samples with a normal karyotype, microarray analysis revealed
clinically relevant deletions or duplications in 6.0% with a structural anomaly and
in 1.7% of those whose indications were advanced maternal age or positive screening results.
Conclusions
In the context of prenatal diagnostic testing, chromosomal microarray analysis identified additional, clinically significant cytogenetic information as compared with
karyotyping and was equally efficacious in identifying aneuploidies and unbalanced rearrangements but did not identify balanced translocations and triploidies.
(Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT01279733.)
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Me thods
Study Conduct
The study was approved by the institutional review boards of all participating sites. The authors
vouch for the accuracy of the data and the fidelity of the study to the protocol, which is available
with the full text of this article at NEJM.org. All
the authors were involved in the design and conduct of the study and made the decision to submit
the manuscript for publication, and all approved
the content of the article. The academic authors
performed the data analysis. Agilent Technologies and Affymetrix donated all microarray kits
and reagents and provided training without reimbursement but were not otherwise involved in
either the conduct of the study or the preparation
of the manuscript. Integrated Genetics received
study funding to cover staff costs for handling
study-specific sample and conventional cytogenetic data but was not involved in any aspect of
the microarray analysis or results or in manuscript preparation. Integrated Genetics approved
the content of the manuscript without changes.
Participant Recruitment and Sample
Collection
For purposes of the primary analysis, each microarray result was assessed as being true positive,
true negative, false positive, or false negative
relative to the karyotype finding. Karyotyping was
considered the standard against which the performance of chromosomal microarray in identifying common autosomal and sex-chromosome
aneuploidies was measured. Per protocol, participants for whom mosaicism was determined
by means of karyotyping were excluded from the
primary analysis. Secondary outcomes included
the overall occurrence and classification of copynumber variants identified with the use of chromosomal microarray in the presence of a normal
karyotype, the success (or failure) of microarray
analysis, and the ability of chromosomal microarray to identify uncommon cytogenetic abnormalities seen on karyotyping (e.g., marker chromosomes, rearrangements, or polyploidy).
Microarray Laboratory Procedures
Karyotype and array results from each array laboratory were submitted separately to an independent
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Table 1. Baseline Characteristics and Primary Indication for Prenatal Testing and Characteristics of the 4406 Study Participants with Adequate
Samples for Analysis.*
Characteristic
Maternal age yr
Anomaly on
Ultrasonography
(N=1109)
Maternal
Advanced Age
(N=2054)
Positive Result on
Downs Syndrome
Screening (N=827)
Other (N=416)
All (N=4406)
32.25.8
38.52.5
34.05.2
33.14.5
35.65.1
12.51.6
11.80.8
12.80.8
11.90.8
12.11.1
Amniocentesis
21.14.0
17.41.3
18.31.9
17.82.1
18.83.1
114 (10.3)
80 (3.9)
65 (7.9)
27 (6.5)
286 (6.5)
164 (14.8)
163 (7.9)
110 (13.3)
46 (11.1)
483 (11.0)
Other
831 (74.9)
1811 (88.2)
652 (78.8)
343 (82.5)
3637 (82.5)
* Plusminus values are means SD. Pregnancies in which the fetus had a nuchal translucency of 3.5 mm or greater or a septated cystic hygroma are included as anomalies on ultrasonography. Nuchal translucencies of less than 3.5 mm were considered a component of Downs
syndrome screening. Other indications for prenatal testing include family history, previous pregnancy with chromosome abnormality, and
elective decision. Of the 4406 study participants, 2275 had chorionic-villus sampling and 2131 had amniocentesis.
Race or ethnic group was self-reported.
R e sult s
We screened 6537 women from October 2008
through July 2011. Of these, 4450 were eligible
and consented to participate in the study; we obtained adequate samples from 4406 (with 2275 undergoing chorionic-villus sampling and 2131 undergoing amniocentesis) (Fig. 1). Characteristics
of the study population, including indications for
prenatal testing, are provided in Table 1.
We obtained an adequate sample for microarray analysis for 4391 (99.7%) of these 4406 participants. Overall, microarray was successful in
98.8% of cases (4340 of 4391). The microarray
analysis was performed on uncultured samples
for 3860 (87.9%) of the 4391 participants. We successfully obtained study results in 3408 (88.3%)
of these 3860 uncultured samples: 1781 (93.2%)
of the 1910 chorionic-villus samples and 1627
(83.4%) of the 1950 amniotic-fluid samples. The
study result was derived from the cultured, rather
than uncultured, sample in the remaining 932
(21.5%) of the 4340 cases of successful microarray.
Fifty-eight samples showed mosaicism on
karyotyping and were excluded from this study.
The remaining 4282 samples were included in the
primary analysis (Table 2). Of these, 317 (7.4%)
common autosomal and 57 (1.3%) sex-chromosome aneuploidies were identified by means of
standard karyotyping. Microarray analysis identi-
fied all of these aneuploidies. Eight of these cases, all from uncultured chorionic-villus samples,
were mosaic on the microarray and could represent mosaicism not detected on karyotyping. None
of these cases had maternal-cell contamination.
All 22 unbalanced rearrangements also were identified by microarray (1 as a mosaic).
As expected, none of the apparently balanced
rearrangements identified on karyotyping were
identified with the use of microarray analysis,
suggesting that these rearrangements were truly
balanced. Of the three marker chromosomes detected on karyotyping, we detected two on microarray. Results obtained on FISH suggested that
the third marker chromosome contained no euchromatin and thus was unlikely to contain genes,
be detected by means of microarray, or have clinical significance. Seventeen triploid samples (0.4%)
were present in our series; none were identified on
microarray; in 15 (88.2%), the test for maternalcell contamination revealed aberrant findings suggestive of triploidy. One other was recorded as
mosaic 47,XXY on microarray. Three of the 17
triploid fetuses had normal ultrasonography images at the time of chorionic-villus sampling.
Microarray analysis revealed clinically significant, segmental aneuploidies not detected on
karyotyping (Table 3, and Table S1 in the Supplementary Appendix). On microarray, 1399 samples
were identified as having copy-number variants;
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Table 2. Results of Karyotype and Microarray Analysis in 4282 Samples with a Nonmosaic Karyotype, According to
Cytogenetic Abnormality.
Detected on
Karyotyping
Abnormality
Detected on Microarray*
Total
Full
Complement
Mosaic
Complement
no. (%)
no. (%)
no.
no.
374 (8.7)
374 (100)
366
317 (7.4)
317 (100)
312
Trisomy 21
188
188 (100)
185
Trisomy 18
93
93 (100)
91
Trisomy 13
36
36 (100)
36
4 (0.1)
4 (100)
57 (1.3)
57 (100)
54
45,X
39
39 (100)
36
18
18 (100)
18
21
Structural rearrangement
65 (1.5)
Balanced
40
Unbalanced
22
22 (100)
2 (66.7)
Marker
Triploidy
17 (0.4)
* All results are reported from uncultured samples where available, and otherwise from cultured samples.
No euchromatin was identified on fluorescence in situ hybridization in the marker with a normal result on chromosomal microarray.
A total of 15 of the 17 triploidy cases (88.2%) were identified in maternal-cell contamination studies. One other was recorded as mosaic 47,XXY on microarray.
confidence interval [CI], 2.1 to 3.1) had a microdeletion or duplication of clinical significance.
We examined the results from microarray
analysis in subgroups of women with normal
karyotypes (Table 3). In samples from fetuses
with suspected growth or structural anomalies,
45 of the 755 (6.0%; 95% CI, 4.5 to 7.9) had
clinically relevant findings on microarray that were
not found on karyotyping. A total of 34 of the
1966 women without ultrasonography-identified
anomalies who were tested because of advanced
maternal age had a normal karyotype and a
clinically relevant finding on microarray (1.7%;
95% CI, 1.2 to 2.4), as did 12 of the 729 women
who tested positive on Downs syndrome screening (1.6%; 95% CI, 0.9 to 2.9). Recurrent copynumber variants associated with autism and
neurocognitive alterations were relatively prevalent in our study series (Table 1 in the Supplementary Appendix)5; we detected these copynumber variants in 1.3% (51 of 3822) of
Table 3. Frequency and Clinical Interpretation of Microdeletions and Duplications on Chromosomal Microarray in the 3822 Samples
with a Normal Karyotype, According to Indication for Prenatal Testing.
Normal
Karyotype
Common
Benign
Uncertain Clinical
Significance (N=130)
Pathogenic
Likely to Be
Benign
no.
Potential
for Clinical
Significance
no. (%)
Any
3822
1234 (32.3)
35 (0.9)
69 (1.8)
61 (1.6)
96 (2.5) [2.13.1]
1966
628 (31.9)
9 (0.5)
37 (1.9)
25 (1.3)
34 (1.7) [1.22.4]
729
247 (33.9)
3 (0.4)
13 (1.8)
9 (1.2)
12 (1.6) [0.92.9]
Anomaly on ultrasonography
755
247 (32.7)
21 (2.8)
16 (2.1)
24 (3.2)
45 (6.0) [4.57.9]
Other
372
112 (30.1)
2 (0.5)
3 (0.8)
3 (0.8)
5 (1.3) [0.63.1]
* Total includes those predetermined as known to be pathogenic and those classified by the clinical advisory committee as clinically relevant.
CI denotes confidence interval.
Includes 36 samples determined likely to be benign by the study geneticist and 33 determined by the independent clinical advisory committee on the basis of size, gene content, inheritance, the literature, and ultrasonography findings.
Other indications include family history, previous pregnancy with chromosomal abnormalities, and elective decision.
karyotypically normal pregnancies: 3.6% (27 of fore required expert adjudication for clinical rel755) with and 0.8% (24 of 3067) without struc- evance. Since the start of our study 5 years ago,
the literature and databases of array results and
tural anomalies.
associated phenotypes have expanded, providing
additional information with which to predict the
Discussion
phenotype.1,3,4 The laboratory directors therefore
We have shown that microarray analysis is equiv- reinterpreted their initial categorization on the
alent to standard karyotype analysis for the pre- basis of the current literature. Were data available
natal diagnosis of common aneuploidies. Micro- in 2012 used for the ascertainment, only 56 of
array analysis provided additional clinically the original 94 uncertain results requiring evalurelevant information in 1.7% of pregnancies with ation by the clinical advisory committee would
standard indications for prenatal diagnosis (such remain in that category; 30 are now clearly pathoas advanced maternal age and positive aneuploid genic and 8 are now likely to be benign (Table
screening result) and in 6.0% of cases with an S1 in the Supplementary Appendix). With this
anomaly on ultrasonography. These data indicate additional information, the pathogenicity of only
a benefit to chromosomal microarray analysis as 1.5% of copy-number variants detected on microa standard part of prenatal testing, bearing in array in karyotypically normal samples remains
mind that, as with karyotyping, the detection of uncertain, and this number should continue to
variants of uncertain clinical significance present fall as additional experience is acquired. The ina challenge for counseling and cause anxiety.15
terpretation of uncertain results will continue to
We used an array design that maximized the require a close working relationship among labodetection of well-characterized microdeletions and ratory directors, clinical geneticists, counselors,
duplications but also included oligonucleotides and practitioners.
representing regions distributed throughout the
We chose to obtain results preferentially from
genome to identify additional chromosomal im- uncultured samples so as to avoid the additional
balances. Uncertain findings occurred in 3.4% time needed for, and the artifacts of, cell and tis(130 of 3822) of all karyotypically normal cases sue culture. However, experience with traditional
analyzed with the use of microarray. Of these cytogenetic analysis and confined placental
130 cases, 94 (72.3%) had findings that were not mosaicism in chorionic-villus samples has oceasily dismissed as likely to be benign and there- casionally revealed discrepant results between
n engl j med 367;23 nejm.org december 6, 2012
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direct (uncultured) analysis that evaluates predominantly the cytotrophoblast and cultured
samples that typically derive from the mesenchymal core of the villi.16 Microarray analysis of uncultured samples captures the genomic content
of both cell lineages. Although our initial comparison of microarray results from paired cultured and uncultured samples was reassuring,
the limited sample size makes further evaluation
necessary.
After approximately 12 weeks gestation, most
triploid pregnancies show abnormalities on ultrasonography, which could alert the physician
to request further evaluation by means of karyotyping. Ultrasonographic images obtained earlier than 12 weeks may miss these abnormalities.
Arrays including SNP probes can identify triploidy with the use of genotype data,17 but this
information was not included in our study design.
Our array analysis did not use the genotype data
derived from the SNP probes on the Affymetrix
array because we initiated the study before their
development for clinical use. However, a post hoc
review determined that had the SNP data been
analyzed, the triploid cases would have been detected. We therefore suggest that arrays used for
prenatal testing should contain SNP probes that
can reliably identify triploidy.
Balanced chromosomal translocations and inversions occur in approximately 0.08 to 0.09% of
prenatal diagnostic samples18 and are not detectable with the use of an array because there is no
gain or loss of genetic material. An inherited balanced rearrangement will have no consequences
for the current pregnancy but is relevant to future
reproductive counseling. A de novo, apparently
balanced rearrangement identified by means of
standard karyotyping is associated with a 6.7%
risk of congenital abnormalities,19 many of which
may be caused by a genomic gain or loss at the
breakpoints and may be discoverable with the
use of an array.20 Further investigation is necessary to quantify the residual risk of a balanced
rearrangement when a microarray analysis is normal and to determine when and whether additional genomic analysis is necessary.
One in 60 pregnancies that underwent genetic
testing because of advanced maternal age or
positive aneuploidy screening had a clinically
relevant copy-number variant in our study. However, many of these copy-number variants are
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Genetics and Celula and lecture fees and travel support from
Affymetrix and Agilent Technologies. Dr. Scholl reports being
an employee of LabCorp (Integrated Genetics). Dr. Simpson reports receiving consulting fees from Bio Dx, Bayer, and Novartis. No other potential conflict of interest relevant to this article
was reported.
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org
We thank Deborah A. Driscoll, M.D., Sherman Elias, M.D.,
Deborah L. Eunpu, M.S., Kurt Hirschhorn, M.D., Charles Lee,
Ph.D., Eugene Pergament, M.D., and Dorothy Warburton, Ph.D.,
for their work as the Clinical Advisory Committee; Maternal
Fetal Specialists, Atlanta (Daniel Eller, M.D.), Baylor College of
Medicine (Anthony Johnson, D.O.), NJ Perinatal Associates/St.
Barnabas Medical Center (Richard Miller, M.D.), Main Line
Health and Womens Health Care Group of PA (Alan Donnenfeld, M.D.), Phoenix Perinatal Associates (Rodney Edwards,
M.D.), Maternal Fetal Medicine of Central PA (Terry Tressler,
D.O.), Thomas Jefferson University (Stuart Weiner, M.D.), Fetal
Diagnostic Center (Greggory DeVore, M.D.), Virtua Health System (Ronald Librizzi, D.O.), Valley Health System (Andrei Rebarber, M.D.), Christiana Care Health System (Anthony Sciscione,
D.O.), San Francisco Perinatal Associates (James Goldberg,
M.D.), University of Medicine and Dentistry, New JerseyRobert
Wood Johnson (Todd Rosen, M.D.), University of California, Irvine (Deborah Wing, M.D.), Vanderbilt Center for Womens
Health (William Walsh, M.D.), Childrens Hospital of Philadelphia (Mark Johnson, M.D.), Ohio State University Maternal Fetal
Medicine (Richard OShaughnessy, M.D.), St. Vincent Womens
Hospital (Maurice Eggleston, M.D., and James Sumners, M.D.),
Englewood Hospital (Ying Chan, M.D.), University of Utah (Robert Silver, M.D.), and Magella Medical Group (Melissa Bush,
M.D.) for their role as participating recruitment sites; Nicolasa
H. Chavez, Jessica Feinberg, and Andrea M. Murad for their coordinating and recruitment efforts; Lindsay Doherty and Mark
McNellis from the George Washington University Data Coordinating Center; the laboratory technologists including Emily Carron from Columbia University, Vanessa Jump from Emory University, Caron Glotzbach from Signature Genomic Laboratories,
PerkinElmer, and Patricia Hixson at Baylor College of Medicine;
Eliza Sanchez for her work at Integrated Genetics; and the participants and the North American Fetal Therapy Network for
their support of the study.
References
1. Sagoo GS, Butterworth AS, Sanderson
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