Array Comparative Genomic Hybridization in Prenatal Diagnosis

Completion of the Human Genome Project stimulated development of ancillary technologies that continue to revolutionize medical sciences and diagnostic techniques. Current conventional cytogenetic analysis (G-banded karyotype) can detect unbalanced structural rearrangements and numeric abnormalities, as well as apparently balanced rearrangements within the limits of resolution of the technique. The resolution of the current conventional cytogenetic analyses lies in the range of 3–10 Mb (1 Mb = 1 million base pairs) and requires dividing cells. Therefore, chromosomal microdeletions or microduplications (those smaller than 3 Mb) will go undetected with conventional cytogenetic analyses. These submicroscopic rearrangements may account for a sizable portion of the human genetic disease burden, with some estimates as high as 15% (1). Fluorescence in situ hybridization technology can be used to detect chromosomal abnormalities smaller than 3 Mb (DiGeorge syndrome for example), but because of technical limitations, it can only screen for a limited number of chromosomal abnormalities at one time.

Genomic microarray-based technologies can theoretically detect human genomic DNA variation at virtually any site in the human genome. Genomic microarrays can detect both duplications and deletions, also referred to in the literature as genomic copy number variants. Genomic copy number variants are defined as deletions and duplications of DNA segments larger than 1,000 bases and up to several megabases in size. Genomic microarrays can be used to perform karyotyping, which often is referred to as array comparative genomic hybridization or array CGH. Array CGH improves resolution over conventional G-banded karyotype in detecting chromosomal abnormalities smaller than 3 Mb. Array CGH has been reported to be useful in detecting causative genomic imbalances in as many as 10% of patients with unexplained mental retardation and previously normal conventional karyotype (2). In addition, array CGH has been a useful tool in discovering underlying genetic mutations in known, but genetically undefined, human genetic syndromes (3). The potential advantages of array CGH over conventional karyotyping in prenatal diagnosis include higher resolution, avoidance of culturing amniocytes or chorionic villi, automation, and faster turnaround times. In addition, array CGH does not require dividing cells, which is useful in the case of fetal demise where the ability to successfully culture cells may be compromised (4). The disadvantages of array CGH include the inability to detect balanced inversions or translocations as well as certain forms of triploidy, and array CGH costs significantly more than conventional karyotype analysis. The technique detects a large number of either benign copy number variants or copy number variants of uncertain clinical significance and is unlikely to detect mosaicism below 20% (5). The two types of arrays currently available are targeted and genome-wide arrays. Targeted arrays are currently preferred in clinical genetic practice because they can detect chromosomal abnormalities for known genetic syndromes. This allows genetic counseling with more certainty regarding phenotype and long-term prognosis. Targeted arrays, however, can miss novel pathologic copy number variants, which may explain a constellation of congenital anomalies that do not fit any particular known syndrome. In approximately 12–15% of samples, copy number variants of uncertain clinical significance will be detected among currently used targeted arrays (6). In such cases, DNA from the biological mother and father must also be analyzed by microarray to distinguish copy number variants that were inherited from the parents (inherited copy number variants) versus copy number variants that are absent in the parents and arose spontaneously in the child (de novo copy number variants). Inherited copy number variants are relatively common and represent benign polymorphisms in most cases. However, a recent report has indicated that inherited copy number variants from seemingly "normal" parents can cause major pathology in the offspring (7). De novo copy number variants are more likely to be the cause of congenital and developmental abnormalities compared with inherited copy number variants, but interpretation of copy number variant results can be clinically complex. A genetics professional should be involved in the interpretation of both inherited and de novo copy number variants. Genome-wide arrays, however, are designed to cover a greater portion of the human genome than targeted arrays. Genome-wide arrays have been particularly useful in research efforts to discover new submicroscopic syndromes (8, 9). However, at this point in time, genome-wide arrays will detect many more copy number variants of unknown clinical significance. Growing clinical experience with genome-wide arrays and the development of copy number variant databases of both healthy and affected individuals will reduce the number of copy number variants of unknown clinical significance and will make genome-wide arrays more useful in clinical practice. Several reports have now shown the potential utility of array CGH in prenatal diagnosis (10, 11). In a relatively large series of fetuses with ultrasound abnormalities and normal conventional karyotype, array CGH detected chromosomal abnormalities in 5% of fetuses and up to 10% in those with three or more anatomic abnormalities. In addition, array CGH was found to detect two genetic syndromes among 300 amniocentesis or chorionic villus samples that otherwise would not have been detected by conventional karyotype or aneuploidy fluorescence in situ hybridization (11). Data from this and other studies have generated increasing interest in the utilization of array CGH as an additional assay for fetal abnormalities, beyond conventional cytogenetic analysis. However, many of the known genomic disorders that can be detected on current targeted arrays do not show readily detectable fetal abnormalities on prenatal ultrasound examinations. Therefore, ordering array CGH only in the presence of ultrasound abnormalities may limit the diagnostic potential of this assay (11). The usefulness of array CGH as a first-line tool in detecting chromosomal abnormalities in all amniocentesis or chorionic villus samples is still unknown. The additional detection rate of chromosomal abnormalities using array CGH, as compared with conventional karyotype, for routine fetal chromosomal analysis, awaits a larger population-based study, which is currently underway in the United States. The answers to these and other questions are required before the routine clinical use of array CGH in prenatal diagnosis can be recommended.

Recommendations

• Conventional karyotyping remains the principal cytogenetic tool in prenatal diagnosis.
• Targeted array CGH, in concert with genetic counseling, can be offered as an adjunct tool in prenatal cases with abnormal anatomic findings and a normal conventional karyotype, as well as in cases of fetal demise with congenital anomalies and the inability to obtain a conventional karyotype.
• Couples choosing targeted array CGH should receive both pretest and posttest genetic counseling. Follow-up genetic counseling is required for interpretation of array CGH results. Couples should understand that array CGH will not detect all genetic pathologies and that array CGH results may be difficult to interpret.
• Targeted array CGH may be useful as a screening tool; however, further studies are necessary to fully determine its utility and its limitations.

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