Prenatal diagnosis by chromosomal microarray analysis

Abstract

Chromosomal microarray analysis (CMA) is performed either by array comparative genomic hybridization or by using a single nucleotide polymorphism array. In the prenatal setting, CMA is on par with traditional karyotyping for detection of major chromosomal imbalances such as aneuploidy and unbalanced rearrangements. CMA offers additional diagnostic benefits by revealing sub-microscopic imbalances or copy number variations that are too small to be seen on a standard G-banded chromosome preparation. These submicroscopic imbalances are also referred to as microdeletions and microduplications, particularly when they include specific genomic regions that are associated with clinical sequelae. Not all microdeletions/duplications are associated with adverse clinical phenotypes and in many cases, their presence is benign. In other cases, they are associated with a spectrum of clinical phenotypes that may range from benign to severe, while in some situations, the clinical significance may simply be unknown. These scenarios present a challenge for prenatal diagnosis, and genetic counseling prior to prenatal CMA greatly facilitates delivery of complex results. In prenatal diagnostic samples with a normal karyotype, chromosomal microarray will diagnose a clinically significant subchromosomal deletion or duplication in approximately 1% of structurally normal pregnancies and 6% with a structural anomaly. Pre-test counseling is also necessary to distinguish the primary differences between the benefits, limitations and diagnostic scope of CMA versus the powerful but limited screening nature of non-invasive prenatal diagnosis using cell-free fetal DNA.

Keywords: Chromosomal microarray; VOUS; microdeletion; microduplication; prenatal diagnosis.

Figure 1

Screenshot from the Affymetrix Chromosome Analysis Suite Software (Version 3.1) showing an 1.837 Mb interstitial deletion of the proximal long arm region (17q12) of chromosome 17 which is associated with a clinical diagnosis of Renal Cysts and Diabetes Syndrome (OMIM#137920) and is caused by a loss of the HNF1B gene. The precise coordinates of the deletion correspond to chr17: 34,446,914–36,283,612 using Human Genome Build Hg19. The gene content within the deleted region can be ascertained using the genome coordinates. A deletion is indicated in the software call panel by the presence of a red bar. The deletion is identified by a decrease in the Log2 ratio from zero as seen in the Log2 Ratio panel. The smooth signal copy number panel indicates the exact copy number of each probe. This panel is helpful in identifying mosaicism which is evident when the smooth signal for multiple consecutive probes lies between an integer, e.g. between 2 and 3 indicates trisomy mosaicism. The allele difference panel indicates the genotype for each SNP probe. For normal copy number of 2, there are only 3 possible SNP combinations, AA, AB and BB which are plotted on the allele difference graph. When there is a deletion (copy number of 1), the genotype options are either A or B and thus only two distinct tracks are visible on the allele difference graph. The chromosome ideogram in the chromosome panel highlights the position (breakpoints) on the chromosome where copy number imbalances are present. The red bar in the chromosome panel represents a deletion. Gains would typically be shown as blue bars.

Figure 2

16 week ultrasound image of fetus diagnosed by CVS with deletion of 17q12. Noted in the scan is bilateral enlargement and hyperechogenicity of kidneys. Kidneys appeared normal at the time of CVS at 12 weeks gestation

Figure 3

Screenshot from the Affymetrix Chromosome Analysis Suite Software (Version 3.1) showing mosaic trisomy 21. The Log2 Ratio of chromosome 21 is clearly shifted upwards compared to chromosome 15 (shown for comparison). The smooth signal copy number panel indicates the exact copy number of each probe. This panel is helpful in identifying mosaicism which is evident when the smooth signal for multiple consecutive probes lies between an integer, e.g. An integer between 2 and 3 indicates trisomy mosaicism. In this example, the median copy number state across chromosome 21 is 2.62, indicating the level of trisomy 21 mosaicism to be around 62%. The allele difference panel indicates the genotype for each SNP probe. For normal copy number of 2, there are only 3 possible SNP combinations, AA, AB and BB (see Figure1) which are plotted on the allele difference graph. When there is mosaicism, additional genotypes will be visible representing those present in both normal and abnormal cell lines.

Figure 4

Whole Genome view showing the Log2 Ratio and Allele Difference for every chromosome. [A] Since the intensities of the probes are normalized, the Log2 ratios for a normal diploid sample and a triploid sample are indistinguishable, with both indicating a DNA copy number of 2. Genotypes, extracted from the SNP data must be utilized to identify triploidy. [B] The allele difference plots show the various SNP genotypes for each SNP locus. In the presence of 2 chromosomes, there are only 3 possible SNP combinations: AA, AB and BB, see Figures 1 and 2. In the presence of 3 chromosomes, there are 4 possible SNP combinations: AAA, AAB, ABB and BBB which results in four distinct tracks on the allele difference graph. A normal diploid female would show the characteristic three tracks for all the chromosomes while the triploid, 69,XXX fetus in this example displays the four characteristic tracks for every autosome as well as the X chromosome. Triploid fetuses with a 69,XXY constitution would show the four characteristic tracks for every autosome, three tracks representing the two X chromosomes and two tracks representing the single Y chromosome.