Additional Information

Method:

Chromosomal microarray (CMA) of genomic DNA uses the Illumina Infinium Global Diversity Array with Cytogenetics-8 v1.0 BeadChip. This BeadChip includes 1.8 million probes regularly spaced throughout the genome with higher density across exons of clinically relevant genes. The copy number results are analyzed using the BioDiscovery NxClinical v6.2 software with Human Genome build 19 (GRCh37/hg19 Feb 2009) as a reference. Lesions are interpreted in accordance with the ACMG standards and guidelines. Cytogenomic nomenclature is reported per ISCN 2020 guidelines.

Detection is limited to copy number losses of at least 6 kb in size containing a minimum of 15 consecutive probes and copy number gains greater than 50 kb containing a minimum of 40 consecutive probes. Mosaicism greater than 20% is generally detected by this CMA. Within this limit of detection, all pathogenic or likely pathogenic copy number variants will be reported, including incidental findings not associated with the indication for testing, following the ACMG recommendations. Variants of uncertain significance (VUS) may be included when the region contains protein-coding genes with a known or suspected disease association. Copy neutral Regions of Homozygosity (ROH) are reported when the pattern and size is suggestive of complete or partial uniparental disomy on a chromosome known to have UPD of clinical significance. Genome-wide ROH segments that total 2% or more of the autosomal complement will be reported. Additionally, a single ROH may be reported when it raises the possibility of a recessive disorder with a causative gene located in the region.

Additional studies may be performed to further characterize anomalies detected by CMA including chromosome analysis on 68-70 hour culture with mitogens, fluorescence in situ hybridization (FISH), or ddPCR copy number analysis. When cord blood and an accompanying maternal specimen are provided, short tandem repeat (STR) analysis is performed to assess for the presence of maternal cell contamination. These supplemental studies will be manually scored.

Chromosome Analysis

Whole blood is cultured with an additive to stimulate cell division. The cells are incubated for 48 hours at 37° Celsius. Cell cultures are synchronized with excess thymidine, arrested in metaphase with Colcemid, harvested, and fixed with methanol and acetic acid. Giemsa stain and trypsin are used to generate a characteristic banding pattern (G-banding) which permits unambiguous identification of individual chromosomes. (Arsham et al., The AGT Cytogenetics Laboratory Manual, Fourth Edition).

Twenty metaphases with an average band-level resolution of 400-700 are routinely examined. An additional 30 metaphases are analyzed in cases of suspected mosaicism. Two or more digital karyograms are prepared and stored using a computer-based imaging system. An abbreviated study of 5 metaphases may be performed for testing ordered to confirm a known familial chromosome abnormality or as an add-on to a normal CMA result. Chromosome abnormalities are confirmed in at least 2 different cultures. A minimum of 3 metaphases with the same chromosome loss, and a minimum of 2 metaphases with the same chromosome gain or structural abnormality are required to authenticate findings (ACMG, Technical Standards for Clinical Genetics Laboratories, 2021 Revision).

Limitations:
CMA
On its own, CMA does not provide information about the structural nature of a finding. CMA will not detect balanced alterations (reciprocal translocations, Robertsonian translocations, inversions, or balanced insertions). CMA will not detect methylation, point mutations, or small insertions/deletions (indels) below the resolution of this assay as described above. Additionally, it may not detect low-level mosaicism, tetraploidy, or marker chromosomes that only contain heterochromatin.  Given these limitations, if a genetic cause is still suspected, additional testing may be warranted. The results are not intended to be used as the sole means for clinical diagnosis or patient management decisions.

Chromosome Analysis
Chromosomes at the 400-700 band-level of resolution generally have a detection limit of 5-10 megabases in regions where the banding pattern is distinctive. The detection limit may be adversely affected in regions lacking contrasting bands. Smaller genomic abnormalities may not be detected via G-banded chromosome studies (cryptic abnormalities). Some chromosomal anomalies hinder cell growth and may be selected against during routine cell culture processes resulting in underrepresentation or exclusion during final analysis. This test is not appropriate for detecting acquired chromosome abnormalities.

References:

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