We have shown that microarray analysis is equivalent to standard karyotype analysis for the prenatal diagnosis of common aneuploidies. Microarray analysis provided additional clinically relevant information in 1.7% of pregnancies with standard indications for prenatal diagnosis (such as advanced maternal age and positive aneuploid screening result) and in 6.0% of cases with an anomaly on ultrasonography. These data indicate a benefit to chromosomal microarray analysis as a standard part of prenatal testing, bearing in mind that, as with karyotyping, the detection of variants of uncertain clinical significance present a challenge for counseling and cause anxiety.15
We used an array design that maximized the detection of well-characterized microdeletions and duplications but also included oligonucleotides representing regions distributed throughout the genome to identify additional chromosomal imbalances. Uncertain findings occurred in 3.4% (130 of 3822) of all karyotypically normal cases analyzed with the use of microarray. Of these 130 cases, 94 (72.3%) had findings that were not easily dismissed as likely to be benign and therefore required expert adjudication for clinical relevance. Since the start of our study 5 years ago, the literature and databases of array results and associated phenotypes have expanded, providing additional information with which to predict the phenotype.1,3,4
The laboratory directors therefore reinterpreted their initial categorization on the basis of the current literature. Were data available in 2012 used for the ascertainment, only 56 of the original 94 uncertain results requiring evaluation by the clinical advisory committee would remain in that category; 30 are now clearly pathogenic and 8 are now likely to be benign (Table S1 in the Supplementary Appendix
). With this additional information, the pathogenicity of only 1.5% of copy-number variants detected on microarray in karyotypically normal samples remains uncertain, and this number should continue to fall as additional experience is acquired. The interpretation of uncertain results will continue to require a close working relationship among laboratory directors, clinical geneticists, counselors, and practitioners.
We chose to obtain results preferentially from uncultured samples so as to avoid the additional time needed for, and the artifacts of, cell and tissue culture. However, experience with traditional cytogenetic analysis and confined placental mosaicism in chorionic-villus samples has occasionally revealed discrepant results between 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 typically smaller microdeletions or microduplications than those identified with the use of chromosome banding or FISH and have much greater variability in their associated phenotypes.5
Copy-number variants associated with substantial phenotypic variability are listed in Table S1 in the Supplementary Appendix
. When encountered in the prenatal setting, this increased range of phenotypic features can make genetic counseling challenging; many of these copy-number variants do not always result in severe impairments. Because of their smaller size and milder phenotypic effects and the possibility that these copy-number variants exert a phenotypic effect only in the presence of other genetic variants,21-23
these copy-number variants may be inherited from a parent with minimal or no recognizable features. Although data from symptomatic infants evaluated postnatally gives some guidance for prenatal counseling, this group almost certainly represents a biased, more severe, and incomplete characterization of the phenotype. To address this bias, we are following the children with copy-number variants ascertained in this prenatal study, as well as others discovered in utero, to understand the associated phenotypic variability more comprehensively and to gauge the relative contribution of copy-number variants to the 13 to 14% of children who receive a diagnosis of developmental delay.24
The comparatively high rate of discovery, with the use of microarray, of clinically relevant genomic disorders may result in more requests for invasive prenatal diagnostic testing. At present, on the basis of the relative balance between the genetic risk and the risk of procedure-induced miscarriage, a risk of aneuploidy of 1:270 or higher is the generally accepted threshold for offering invasive testing.25
However, the decision to have an amniocentesis or chorionic-villus sampling is based on many factors, including the risk that the fetus will have an abnormality, the risk of pregnancy loss from an invasive procedure, and the consequences of having an affected child. Potential parents weigh these potential outcomes in different ways.26-28
If the observed 1.7% (1:60) frequency of clinically relevant microdeletions and microduplications in pregnancies sampled for indications other than fetal structural anomalies is confirmed by others, offering invasive testing and microarray analysis to all pregnant women would seem to be appropriate. This is consistent with the recommendations of the American Congress of Obstetricians and Gynecologists, who suggest that all women, regardless of risk, should be offered the option of invasive testing.25
Counseling should include a discussion of the risk of invasive testing, the frequency and severity of clinically relevant microarray findings, and the more limited identification of common aneuploidies currently achievable with the use of noninvasive screening.29
We are still in the process of gauging the extent of incremental information that should be sought in the context of prenatal testing and how that information should be introduced into care. Lessons learned from microarray analysis will be helpful when whole-genome sequencing of the fetus, perhaps with the use of maternal blood samples, becomes clinically available30-32
and should help to ensure the sensible application of new technology.