Melissa Stosic, Jessica L. Giordano, Brynn Levy, and Ronald Wapner
Chromosomal microarray analysis (CMA) by which chromosomal anomalies beyond the resolution of traditional karyotyping can be identified has become a mainstay of prenatal diagnosis over the past several years. In 2012, a landmark study was published comparing CMA to the previous standard of care, karyotype analysis, which can identify aneuploidy and chromosomal structural changes bigger than 7–10 million base pairs (Mb) (1). It was shown that CMA was able to detect all of the unbalanced changes identified on karyotype as well as additional microdeletions and duplications (copy number changes [CNVs]) in 1.7% of pregnancies without ultrasound anomalies. In pregnancies with a structural anomaly and a normal karyotype, 6% of fetuses had a clinically significant CNV identified by CMA. This incremental yield of CMA over karyotype led the American College of Obstetricians and Gynecologists (ACOG) along with the Society for Maternal-Fetal Medicine (SMFM) to publish guidelines suggesting that microarray be the preferred test for anomalous pregnancies. They also commented that in average-risk pregnancies undergoing invasive testing for indications such as advanced maternal age or abnormal screening, either karyotype or CMA is appropriate (2).
Basics of chromosomal microarray analysis
Standard karyotype is able to detect CNVs that are 7–10 megabases (Mb), or million base pairs in size or greater, such as a monosomy, trisomy, or a large deletion or duplication. Fluorescence in situ hybridization (FISH) can identify smaller copy number changes in specific regions of the genome by utilizing probes specific to that region or chromosome. Comparatively, CMA has the ability to find microdeletions and duplications as small as 10 kilobases (kb) or thousand base pairs, across the entire genome. These small deletions and duplications are missed by standard karyotyping and can cause genetic syndromes with intellectual disabilities and/or structural abnormalities. Table 36.1 lists common microdeletion and microduplication syndromes and the major phenotypic features associated with each condition.
Table 36.1 Common microdeletion and microduplication syndromes
Condition; genomic location
Major phenotypic features
Normal to DD, ASD, ADHD, microcephaly, psychiatric conditions
ID/DD, ASD, ADHD, macrocephaly, psychiatric conditions
ID/DD, seizures, schizophrenia
Normal to motor skill and articulation difficulty, ID/DD, ASD, ADHD, scoliosis, abnormal gait, scoliosis, macrocephaly, short stature, psychiatric conditions, CHD
22q11.2 deletion syndrome (DiGeorge, VCFS)
CHD (most commonly conotruncal), palate abnormalities, characteristic facies, ID/DD, immune deficiency, hypocalcemia, psychiatric conditions in young adulthood, etc.
Normal to ID/DD, growth retardation, hypotonia
1p36 deletion syndrome
ID/DD, hypotonia, seizures, structural brain abnormalities, CHD, vision and hearing issues, skeletal anomalies, characteristic facies
Charcot–Marie–Tooth Type 1A; 17p12 duplication
Slowly progressive neuropathy causing distal muscle weakness and atrophy, sensory loss, and slow nerve conduction velocity first noticeable in the first or second decade
X-linked ichthyosis; Xp22.31 deletion
ID/DD, ichthyosis, Kallmann syndrome, short stature, ocular albinism
Williams syndrome; 7q11.23 deletion
ID/DD, cardiovascular disease, characteristic facies, connective tissue abnormalities, specific personality, growth anomalies, endocrine abnormalities
DD, normal to ID intellectually, speech problems, hypotonia, problems with movement and walking, behavioral abnormalities, seizures, aortic enlargement
Prader–Willi syndrome; 15q11.2 paternal deletion
ID/DD, hypotonia and feeding difficulties in infancy, excessive eating, obesity, behavioral difficulties, hypogonadism, short stature
Angelman syndrome; 15q11.2 maternal deletion
ID/DD, severe speech impairment, gait ataxia, inappropriate happy affect, microcephaly, seizures
Kidney/urinary abnormalities, diabetes, ID/DD, ASD, psychiatric conditions
Sotos syndrome; 5q35 deletion
ID/DD, overgrowth, characteristic facies
Smith–Magenis syndrome; 17p11.2 deletion
ID/DD, characteristic facies, sleep disturbances, behavioral issues including self-injury and self-hugging and aggression, characteristic facies, reduced sensitivity to pain and temperature,
Cri-du-chat; 5p15 deletion
High-pitched cry, microcephaly, hypotonia, characteristic facies, ID/DD, CHD
Koolen–de Vries; 17q21 deletion
ID/DD, sociable personality, hypotonia, seizures, distinct facial features, CHD, kidney anomalies, foot deformities
Potocki–Lupski syndrome; 17p11.2 duplication
ID/DD, ASD, hypotonia, CHD
Abbreviations: ADHD, attention deficit hyperactivity disorder; ASD, autism spectrum disorder; CHD, congenital heart defect; DD, developmental delay; ID, intellectual disability.
Types of CMA and coverage
CMA is performed by mixing a control normal sample of fragmented DNA with DNA from the sample to be tested. Each sample is attached to a florescent dye of a different color (usually red and green). The mixture is then heated to denature (separate) the double-stranded DNA fragments from the patient and the control into single strands. The mixture is then hybridized to a slide embedded with short probes of known DNA sequences. If an equal number of strands from the control and the patient are present at a location on the chip, this will be represented by an equal amount of red and green fragments. The slide is then read by a laser that evaluates all the locations on the slide and interprets their color. Deletions and duplications will appear as too much or too little of one color since the matching strand is either missing or overrepresented. Oligonucleotide arrays are “comparative” in that they compare the sample to a reference genome and are hence described as comparative genomic hybridization arrays (aCGH). This is illustrated in Figure 36.1.
Figure 36.1 Array CGH process—The aCGH process leads to the generation of a ratio of the fluorescence signal intensity (after normalization) of the reference to test DNAs at each oligonucleotide position on the array. This ratio is usually plotted on the Log2 scale and shows when there is an excess (increased Log2 ratio) or deficiency (decreased Log2 ratio) of test DNA and is used to identify imbalances such as whole chromosome aneuploidy or microdeletions or duplications.
A second type of array is the SNP microarray that uses high-density oligonucleotide-based arrays in which target probes are chosen from DNA locations known to vary between individuals by a single base pair, i.e., single nucleotide polymorphisms (SNPs). Today, most CMAs utilized for prenatal diagnosis are hybrid arrays that contain both SNP probes and copy number probes. By including SNPs on the array, additional clinically useful information such as uniparental disomy (UPD), zygosity, maternal cell contamination, parent of origin, and consanguinity, may be extracted from the genotype plots generated from the SNPs. Consanguinity and UPD can be responsible for recessive genetic conditions in the SNP regions with long contiguous stretches of homozygosity (LCSH), and UPD can cause disease when it occurs on an imprinted chromosome. Examples of imprinting disorders include Russell–Silver syndrome, which is caused by UPD 7; Prader–Willi syndrome, which results from maternal UPD 15; and Beckwith-Wiedemann syndrome, which occurs from partial UPD 11. In addition, triploidy, which cannot be detected by aCGH, can easily be identified by SNP oligonucleotide microarray analysis (SOMA) by assessing the SNP allele patterns on the array. (The allele plots should show four distinct tracks for every autosome, see Figure 36.2.)
Figure 36.2 CMA findings and corresponding ultrasound anomalies. For the CMA images, a loss is indicated in the software call panel by the presence of a red bar and a gain by the presence of a blue bar. Losses are identified by a decrease in the Log2 ratio from zero as seen in the Log2 Ratio panel while gains are observed as an increase in the Log2 ratio. The gene content within the regions of imbalance can be ascertained using the genome coordinates using Human Genome Build Hg19. The allele difference panel indicates the genotype for each SNP probe. For normal copy number of 2, there are only three 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. When there is a duplication (copy number of 3), the genotype options are AAA, AAB, ABB, and BBB giving rise to four visible tracks 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, and a blue bar indicates a duplication. (A-i) A 4.693 Mb terminal deletion of the short arm of chromosome 5 which is associated with a clinical diagnosis of cri du chat microdeletion syndrome. The precise coordinates of the deletion correspond to chr5:113,576–4,806,382. (A-ii) 23-week ultrasound revealing estimated fetal weight less than third percentile and a thickened placenta (arrow). (B-i) A 4.573 Mb interstitial deletion of the long arm of chromosome 14. The precise coordinates of the deletion correspond to chr14:76,837,875–81,411,347. (B-ii) Routine ultrasound scan identifying a small subarachnoid cyst of the brain (arrows). (C-i) A 2.816-Mb deletion of the proximal long arm region of chromosome 22 which is associated with a clinical diagnosis of DiGeorge/velocardiofacial microdeletion syndrome. The precise coordinates of the deletion correspond to chr22:18,649,166–21,465,659. (C-ii) Fetal echocardiogram demonstrating a fetal truncus arteriosis. (D-i) Loss of the entire short arm and gain of the entire long arm of chromosome 18 resulting from the formation of an isochromosome of 18q. Monosomy 18p and trisomy 18q are together associated with severe anomalies such as holoprosencephaly and cloacal dysgenesis. (D-ii) 18-week scan with multiple fetal anomalies. Shown is a single midline monoventricle and fused thalamus consistent with alobar holoprosencephaly.