Additional Information

METHODS

Chromosomal microarray (CMA) of genomic DNA using the Illumina Infinium Global Diversity Array with Cytogenetics-8 v1.0 BeadChip. The array resolution is approximately 5kb for regions targeting 4885 dosage sensitive genes and the backbone resolution is 20kb. The copy number results are assessed in silico and analyzed using the BioDiscovery NxClinical v6.2 software and interpreted in accordance with the ACMG standards and guidelines (Riggs et al Genet Med. 2020 Feb;22(2):245-257; Gonzales et al Genet Med. 2022;24(2):255-261; Brandt et al Genet Med. 2020;22(2):336-344.) using Human Genome build 19 (GRCh37/hg19 Feb 2009) as a reference. Cytogenomic nomenclature is reported per ISCN 2020 guidelines (McGowan-Jordan et al Cytogenet Genome Res 2020;160:341-503).  

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 array. 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 (Riggs et al Genet Med. 2020 Feb;22(2):245-257). Variants of uncertain significance (VUS) will be included when the region contains protein-coding genes.  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 (Gonzales et al Genet Med. 2022;24(2):255-261; del Gaudio et al Genet Med. 2020;22:113-1141). Genome-wide ROH segments that total 2% or more of the autosomal complement will be reported. 

Short tandem repeat (STR) analysis is performed to compare maternal and neonatal specimens to assess the presence of maternal cell contamination in neonatal (cord blood) samples.  Chromosome analysis on 68-70 hour culture with mitogens, Fluorescence in situ hybridization (FISH), or ddPCR copy number analysis may be performed to further characterize anomalies detected by CMA. These 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
This microarray will not detect balanced alterations (reciprocal translocations, Robertsonian translocations, inversions, or balanced insertions), point mutations, or insertions/deletions (indels) of regions not covered by the platform. It may not detect low-level of mosaicism, marker chromosomes that only contain heterochromatin, or tetraploidy.  Failure to detect an alteration at any locus does not exclude all anomalies at that locus or diagnosis of any of the clinical disorders tested for with this array. 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.