A special issue of the journal Prenatal Diagnosis published this month provides an overview of new cytogenetic technologies, ranging from arrays to next-generation sequencing, and also provides two commentaries that address the ethical questions surrounding the use of such tools in prenatal testing.
Lisa Shaffer, chief scientific officer at PerkinElmer and co-founder of the company's Signature Genomic Laboratories subsidiary, told BioArray News this week that the journal's editorial board, of which she is a member, decided to devote an issue to new technologies for prenatal diagnosis of chromosome abnormalities because the field is "moving quite fast."
Indeed, the cytogenetics community is awaiting the publication of a five-year study on array-based prenatal testing, expected later this year (BAN 2/14/2012). Preliminary results of the National Institute of Child Health and Human Development-funded project demonstrated that arrays can detect more abnormalities than microscope-based karyotyping, the method of choice for the past 40 years.
The early results of the NICHD study prompted some to argue that professional societies, such as the American College of Medical Genetics and the American Congress of Obstetricians and Gynecologists, should revise their guidelines in favor of expanded use of arrays in the prenatal setting (BAN 2/21/2012).
Both organizations' current guidelines support the use of arrays only in cases of an abnormal ultrasound finding followed by a normal karyotype.
Shaffer said that she hoped that the special issue of Prenatal Diagnosis might serve as an additional driver for ACMG and ACOG to revisit their recommendations. "I hope that these papers demonstrate the swift need for guidelines from both organizations," said Shaffer. "The field is moving fast and it would be helpful."
Two papers in the new issue specifically address the ethical questions that arise in the prenatal setting when arrays pick up variants of unknown significance (see related article, this issue). As Shaffer and co-author Ignatia Van den Veyver acknowledge in the issue's editorial, counseling in such cases is "more difficult" than in more clear-cut cases and "requires integration of all available information, including data from the literature on penetrance for an abnormal phenotype and experience from the diagnostic laboratory." Van den Veyver is an associate professor of obstetrics and gynecology at Baylor College of Medicine.
Still, the new papers are not only geared toward professional societies, but also to the community, Shaffer said. By discussing not only chromosomal microarray analysis, but other methods, including fluorescence in situ hybridization-based microfluidics, multiplex ligation-dependent probe amplification, and sequencing, Shaffer said the issue will "likely be an important reference for clinicians deciding the best tests for their patients."
'State of the Art'
According to the editorial, the purpose of the issue was to "compile state-of-the-art information" on new cytogenetic technologies that are being adopted to identify alterations of the genome leading to human disease. Each of the methods described in the issue is aimed at uncovering chromosome imbalance — either whole-chromosome aneuploidies or locus-specific gains and losses of the genome, Shaffer and Van den Veyver wrote.
The majority of articles, though, focus on the use of arrays in prenatal testing. Signature contributed a paper on the ordering practices of physicians desiring microarray testing for their patients, comparing 1,400 arrays performed for indications on two different platforms for labs in the US, Canada, and Israel. One platform was a 55,000-probe, lower-resolution array, and the other a 135,000-probe, high-resolution array. The 55,000-probe array had fewer probes covering the genomic 'backbone' between targeted regions known to contain pathogenic variants.
Signature compared the detection rates of the platforms in identifying variants of unknown clinical significance. It also compared which arrays were being requested based on location. It found that the lower-resolution array reported 32 percent fewer results of unclear clinical significance as compared to the higher-resolution array, while retaining the ability to detect all but one significant abnormality identified by the higher-resolution array. Though it was more likely to identify variants of unclear significance, labs in the US and Canada tended to favor the higher-resolution array, while the lab in Israel opted for the lower-resolution chip.
Shaffer said that the Israeli lab might have preferred the lower-resolution array because the lab offers invasive prenatal testing to all pregnant women and therefore wanted to reduce the risk of finding results of unclear clinical significance. Most of the cases in all three countries were referred because of abnormal ultrasound findings relative to those referred because of advanced maternal age and maternal anxiety.
Based on these findings, Shaffer and fellow authors noted that a "thoughtful array design that includes relevant clinical content, necessary density of probes over targeted regions, and decreased density of probes in backbone regions will decrease detection of findings of uncertain clinical relevance without interfering with the detection of clinically significant alterations." This was not an endorsement, however, of using a lower-resolution array in all prenatal cases. Rather, the authors concluded that array design is another consideration for clinicians, many of whom, without specific guidance from professional obstetric and genetic organizations, have been left to "use their own judgment on how and when to use microarrays."
Researchers at Baylor discussed another large study in the new issue, presenting their experience with more than 1,000 prospective prenatal array tests performed to date. Baylor's medical genetics lab was the first to introduce array-based prenatal testing in 2005 (BAN 4/11/2006).
The researchers found that 4.2 percent of cases had an abnormality detected by microarray after abnormal karyotypes were excluded. This is a "significant finding because many of these abnormalities would be undetectable with routine banded chromosome analysis," they noted.
At the same time, 1.6 percent of the cases had findings of unclear clinical significance, which the authors considered to be a "low frequency" of such results.
"With careful consideration of probe coverage, increasing the resolution of prenatal arrays offers the potential to detect more pathogenic CNVs without a corresponding increase in findings of uncertain clinical significance," the authors wrote. The Baylor team also argued that efforts should continue to focus on reducing the likelihood of results of uncertain clinical significance by adding parental studies when indicated and by continuing to contribute to CNV databases.
Other array-related papers included in the issue included a review of chromosomal microarrays, as well as studies focused on specific applications, such as the use of array data in fetuses with congenital heart defects; the use of arrays after cultured prenatal specimens or extraction of cell-free fetal DNA; application of arrays after fetal death; and the use of high-resolution SNP arrays to study samples from pregnancies with abnormal ultrasound findings.
Arrays weren't the only technology highlighted in the new issue, as a number of articles discussed the application of other genomic technologies in cytogenetics.
In one paper, researchers at the National University of Singapore described the use of FISH-based microfluidics to detect aneuploidy. The goal of the study was to integrate a FISH assay, which can typically take 24 to 48 hours, into a single microfluidic device capable of delivering an answer the same day. According to the paper's abstract, the assay includes fixing amniotic fluids with a fixative agent, and loading the samples into the microchannels of the developed nanostructured device coated with titanium dioxide to facilitate cell adhesion. Following hybridization, users are able to detect fetal aneuploidies. The entire assay can be completed within three hours of sample receipt, the authors claimed.
Another paper discussed the application of quantitative fluorescent PCR. According to the authors, researchers at Guy's & St. Thomas' National Health Foundation Trust in London, QF-PCR has been in diagnostic use in the UK for more than 10 years and is an "accurate" and "rapid" prenatal test for common aneuploidies. Specific advantages cited in the paper include detection of triploidy, mosaicism, and maternal cell contamination.
Baylor researchers contributed another paper to the issue detailing the use of multiplex ligation-dependent probe amplification, or MLPA, assays in prenatal testing. This "recent" technique can be applied to relatively quantify as many as 40 to 45 nucleic acid targets, according to the paper. The Baylor researchers said that the MLPA platform is now "extensively applied" for postnatal diagnosis of genetic disorders and has recently been used for prenatal diagnosis too. Uses of MLPA for prenatal diagnosis include detection of aneuploidies, common microdeletion syndromes, and subtelomeric copy-number changes; identification of marker chromosomes; and detection of familial copy-number changes in single genes, the authors wrote.
Another approach discussed as having a "quick turnaround time" is BACs-on-Beads. Sold by PerkinElmer, the assay relies on bacterial artificial chromosomes immobilized onto Luminex encoded beads that are, in turn, used to survey chromosomal gains and losses in samples in 96-well plates. In a paper in the issue, authors from several European cytogenetic labs described their use of BACs-on-Beads in more than 1,600 prospective prenatal samples. The featured assay detects chromosome 13, 18, 21, and X and Y aneuploidies, and the nine most frequent microdeletion syndromes, they noted.
The issue was rounded out by a review of the use of next-generation sequencing to noninvasively detect trisomy 21. The authors, from the Chinese University of Hong Kong, argue that sequencing of maternal plasma DNA is the "most effective approach" for assessing fetal chromosome dosage.
The authors invented a sequencing-based method for noninvasive prenatal trisomy testing that serves as the basis for Sequenom's commercially available MaterniT21 test.
In the paper, they noted that sequencing studies covering a total of 305 trisomy 21 pregnancies and 2,061 euploid pregnancies have been published to date and that the overall diagnostic sensitivity and specificity in both instances was 99 percent.
In addition to trisomy 21, massively parallel maternal plasma DNA sequencing has also been applied to the noninvasive detection of trisomy 18, trisomy 13, and fetal genetic sequences across the genome, they added.
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