In this issue of the
Journal, three research teams report on the application of the microarray in prenatal and reproductive genetics.
1-3 These reports highlight the power and complexity, and some of the pitfalls, of using new genomic technology in clinical practice. Chromosomal microarrays can detect almost all the chromosomal imbalances detected in conventional cytogenetic analysis in addition to submicroscopic deletions and duplications that are at least 1 kb in size, termed copy-number variants. These variants are typically classified as benign, pathogenic, or of uncertain clinical significance.
4 Chromosomal microarrays are recommended as the first-tier diagnostic test for the postnatal evaluation of patients with developmental delay or intellectual disability, autism spectrum disorders, or multiple congenital anomalies; clinically significant findings have been reported in up to 15% of patients with normal conventional karyotypes.
5 In addition to providing higher resolution, the chromosomal microarray offers potential advantages over conventional karyotyping, such as automation (and thus faster turnaround times) and elimination of the need to culture amniocytes or chorionic villi. Because microarray analysis does not require dividing cells, it is useful in cases of fetal death, when it is often not possible to culture cells. In pediatric practice, identifying a diagnosis is important to parents for many reasons (e.g., ending the diagnostic odyssey, obtaining resources and benefits, and planning for the future management of care), so the odds of finding a clinically significant abnormality on array may offset the downside of finding a copy-number variant of uncertain clinical significance. During pregnancy, the detection of a copy-number variant of uncertain clinical significance may cause considerable stress and anxiety for the parents, who may be considering termination of the pregnancy. Are the three studies reported in this issue helpful in deciding how to implement microarray analysis in clinical practice? Wapner et al. compare chromosomal microarray with standard karyotyping in 4406 women undergoing prenatal diagnosis.
1 Microarray analysis identified all of the common autosomal and sex-chromosome abnormalities and the unbalanced rearrangements identified on standard karyotyping. Microdeletions or duplications of clinical significance were found in 96 of 3822 fetal samples with normal karyotypes (2.5%; 95% confidence interval [CI], 2.1 to 3.1), including 6.0% of cases in which fetal anomalies were detected on ultrasonography. There were 94 copy-number variants of uncertain clinical significance that required adjudication by the Clinical Advisory Committee, and in 61 of these instances (65%) the findings were deemed to be of sufficient clinical relevance to be reported to the participant. A review of the copy-number variants of uncertain significance with data from copy-number variant databases updated in 2012 resulted in the reclassification of 30 copy-number variants as pathogenic and 8 as benign. With this updated information, the pathogenicity of 1.5% of copy-number variants detected on microarray analysis remained uncertain. Reddy et al. report their experience with single-nucleotide polymorphism (SNP) oligonucleotide microarray analysis and conventional karyotyping in 532 stillbirths.
2 Microarray analysis yielded results more frequently than conventional cytogenetic analysis (87.4% vs. 70.5%, P<0 .001=".001" 19.4="19.4" 2.6="2.6" 465="465" 5.8="5.8" 67="67" a="a" abnormalities="abnormalities" addition="addition" among="among" analysis="analysis" aneuploidy="aneuploidy" anomalies="anomalies" as="as" clinical="clinical" compared="compared" congenital="congenital" copy-number="copy-number" detection="detection" genomic="genomic" identified="identified" improved="improved" in="in" karyotype="karyotype" microarray="microarray" more="more" of="of" on="on" p="p" pathogenic="pathogenic" plus="plus" reported="reported" samples.="samples." significance="significance" stillbirths="stillbirths" than="than" the="the" uncertain="uncertain" variant="variant" vs.="vs." was="was" were="were" with="with"> Talkowski et al. report on a prenatal case study in which a new, apparently balanced translocation between chromosomes 6 and 8 was identified after an isolated fetal heart defect was detected on ultrasonography at 18.8 weeks' gestation.
3 Microarray analysis did not reveal any clinically significant loss or gain of genetic material in the breakpoint regions. After delivery, a diagnosis of the CHARGE syndrome (coloboma of the eye, heart anomaly, atresia of the choanae, retardation, and genital and ear anomalies) was made on the basis of clinical features. Talkowski et al. used a 13-day protocol involving customized whole-genome “jumping libraries” to identify the precise translocation breakpoints, one of which directly disrupted
CHD7, a pathologic locus in the CHARGE syndrome. The three reports in this issue of the
Journal illustrate the diagnostic potential and advantages of microarray and sequencing technology as well as the limitations that present challenges for clinical diagnosis, counseling, and management of care. The major disadvantage of chromosomal microarrays and sequencing is our current inability to interpret the clinical significance of a novel, previously unreported copy-number variant or to accurately predict the phenotype of pathogenic copy-number variants associated with variable expression and penetrance. In order to promote more accurate assessment and interpretation, there has been a major international effort to catalogue genomic and clinical information, which will facilitate the classification of copy-number variants of uncertain clinical significance into benign or pathologic variants.
6 It is critical to develop large databases of copy-number variants identified prenatally to avoid the ascertainment bias associated with discovery in postnatal cases studied only because of existing abnormalities.
7 Longitudinal postnatal follow-up is also needed. It is important to consider that the chromosomal microarray can neither identify balanced chromosomal arrangements, such as translocations or inversions, nor differentiate free trisomies from unbalanced Robertsonian translocations. Marker chromosomes and low levels of mosaicism may be missed. Triploidy may not be recognized by some forms of microarray. Although microarrays are useful in detecting copy-number variants in apparent new balanced translocations or other structural rearrangements, microarray analysis may fail to detect mutations in some cases. The results of the study by Wapner et al. support the use of microarray analysis instead of conventional karyotyping when fetal structural anomalies have been identified on ultrasonography. Although the copy-number variant will be of uncertain clinical significance in a small percentage of these cases (which presents a real problem in counseling the couple), this disadvantage is outweighed by the higher percentage of well-characterized copy-number variants that are found. In contrast, the incremental value of microarray analysis as compared with conventional karyotyping alone is not clearly established when amniocentesis is performed for diagnostic purposes in cases of advanced maternal age or a positive screen for Down's syndrome. In such cases, the proportion of well-characterized copy-number variants as opposed to uncharacterized copy-number variants is much closer, and it is possible that the counseling conundrum may outweigh the value of the incremental information provided by the microarray analysis. In addition, the increased cost associated with microarray — including the cost of parental studies in some cases — as compared with the cost of conventional karyotyping may be significantly out of proportion to the information gained in these lower-yield cases. The good news is that additional information on the classification of copy-number variants is rapidly accumulating, as indicated by the improved yield of diagnostically certain results on the reinterpretation carried out by Wapner et al. (in which they used updated information on copy-number variants). However, a recent report in the
Journal showed that the effect of some copy-number variants is context-dependent in that some deletions that might not be considered clinically relevant may affect the phenotype in known deletion syndromes.
8 Both pretest and post-test counseling by trained genetics counselors and geneticists are critical. Pretest counseling must include a discussion of the genetic principles of uncertainty and variable expressivity, the lack of precise correlation between genotype and phenotype, and the possibility that genetic variants that cause adult-onset disorders may be identified in a fetus or a parent.
7 Post-test counseling should include interpretation of the results and an explanation of indicated follow-up studies and the possible implications for the family. The findings of Reddy et al. provide a good rationale for performing microarray analysis in cases of stillbirth, particularly when congenital anomalies are present. As in pediatric cases, the identification of an abnormal result may provide comfort, end the search for a cause, and help with the assessment of risk and the development of a plan of care for future reproduction. Talkowski's report illustrates the limitations of chromosomal microarray to detect mutations and illustrates the advantage of the sequencing approach: it can localize breakpoints of cytogenetically balanced chromosomal rearrangements to individual genes. Given the rapid advances and application of whole-exome sequencing in pediatric practice, it will not be long before this technology is ushered into the field of prenatal diagnosis. The future challenges of whole-exome sequencing include those of determining how to interpret large data sets and how to appropriately apply this information in clinical practice.
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