Birth defects occur in 3 percent of newborns and are the leading cause of neonatal disease and death.^1,2 Unbalanced chromosomal abnormalities, in which there is net loss or gain of genetic material, are believed to contribute to a substantial portion of birth defects; structural and numerical abnormalities are found in 25 percent of deaths of newborns with congenital anomalies.²

Additionally, more than half of first-trimester miscarriages result from fetal chromosome abnormalities.³ Detection of chromosome abnormalities during the prenatal and neonatal periods is critical for clinical management of pregnancies, anticipating treatment strategies following the birth of a potentially ill child and providing information to families regarding recurrence risk.

Visual Analysis
Since its development in the 1970s, visual analysis of the chromosomes under the light microscope after they have been stained to reveal the alternating light and dark bands has been the primary means by which cytogeneticists--those who study genetics at the chromosome, rather than DNA, level--have screened the chromosomes for abnormalities. In prenatal diagnosis, cytogenetic analysis has become an important tool for the identification of chromosome abnormalities in fetuses in women of advanced age, those ascertained through a screening test, and those with abnormal ultrasound findings.

Common numerical anomalies of chromosomes 13, 18, 21, X and Y have traditionally been detected during prenatal and neonatal diagnosis by conventional banding. Under ideal conditions, structural rearrangements as small as 5 million basepairs (Mb) of DNA can be identified in this manner, but small structural changes may escape detection.

Additionally, the need for cells to be cultured prior to chromosome analysis is of particular concern for prenatal and neonatal testing when, for example, a first-trimester ultrasound reveals anomalies that may be associated with a chromosome abnormality and timely diagnosis is essential for pregnancy management.

High-Resolution Assays
The high proportion of chromosome abnormalities that go undetected by chromosome analysis in both neonatal and prenatal samples demonstrates the need for high-resolution testing assays. Fortunately, in the last 15 years molecular methods of abnormality detection have increased the resolution and decreased the turnaround time (TAT) of genetic diagnosis of chromosome abnormalities.

Quantitative fluorescence polymerase chain reaction (QF-PCR) is one of several rapid aneuploidy diagnosis/detection (RAD) techniques that, because they do not require cell culture, have been used for detection of the common fetal aneuploidies with a fast 1- to 2-day TAT for results. In QF-PCR, chromosome-specific, repetitive DNA sequences known as short tandem repeats (STRs) are amplified by PCR using fluorescent primers. Automated DNA sequencers and specialized software can quantify the PCR products and compare the "peak heights" from the DNA of a patient to that of a control.⁴

One advantage to QF-PCR is its ability to detect maternal cell contamination of fetal cells. In combination with the analysis of parental DNAs, the technique can also determine whether an aneuploidy is maternally or paternally derived and whether it arose during meiosis I or meiosis II.

In multiple ligation-dependent probe amplification (MLPA), probes hybridizing to sites adjacent to the target region, rather than the target sequence itself, are amplified by PCR and quantified.⁵ MLPA provides a low-cost, simple means of quantifying a large number of DNA targets in a single reaction, with results in only 2-3 days. Although MLPA is sensitive to contamination and will not detect maternal cell contamination or triploidies, it has been successfully integrated in the clinical setting for prenatal detection of the common aneuploidies.^6-8

Fluorescence in situ hybridization (FISH), a technique using fluorescently-labeled DNA probes to detect DNA regions within cells in the interphase nucleus or in metaphase, the stage of active cell division when chromosomes are condensed enough to be microscopically visible, has been widely used in the cytogenetics laboratory since the early 1990s. FISH is usually a directed test in that laboratories use probes for individual disease loci based on clinical indications for a specific disorder, although probe sets for specific regions of the genome such as the subtelomeres or centromeres are commercially available.

Probe sets for the centromeres of the chromosomes most often involved in numerical anomalies are frequently used for prenatal diagnosis. However, clinical suspicion of a specific disorder may be difficult to ascertain during the neonatal period if the newborn presents with atypical clinical features or features shared by multiple syndromes, or if suggestive features manifesting at a later age are absent. Furthermore, if the alteration resulting in a genetic disorder is atypically sized, FISH may appear normal if the alteration lies outside the area assessed by the FISH probe; for example, the FISH probe to the TUPLE1 locus, which is commonly used for detection of DiGeorge syndrome will not detect the ~10 percent of deletions that do not encompass that locus.⁹

Comparative genomic hybridization (CGH) applies the principles of in situ hybridization to genome-wide detection of abnormalities. Two genomes, a patient and control, are fluorescently labeled and competitively hybridized to metaphase chromosomes. The relative fluorescent signal intensities of the labeled patient and control DNA can be visualized across each chromosome to show relative copy number changes.¹⁰ Although CGH can detect imbalances across the entire genome, its resolution is limited to that of metaphase chromosomes.^11-13 Therefore, CGH is no more powerful than conventional chromosome analysis, although it does not require dividing cells for analysis.¹⁴

Microarray-based comparative genomic hybridization (array CGH) combines the principles of CGH with our increased understanding of the human genome sequence and advances in robotically arraying genetic material on a solid surface.^15,16

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