Chromosomal microarray analysis (CMA)

Array-based comparative genomic hybridisation marked a major advance in the genome-wide analysis of chromosomal imbalances, offering substantially higher resolution than conventional chromosome analysis. While traditional karyotyping typically detects changes at a resolution of approximately 5 Mb, array-CGH can achieve a resolution ranging from approximately 100 kb to less than 10 kb, depending on the number, distribution and density of DNA probes used.

In array-CGH, patient DNA and reference DNA are labelled with different fluorochromes (dyes) and applied in equal quantities to a microarray chip. The chip consists of a glass slide containing thousands of immobilised DNA probes arranged in a high-density grid. Patient and reference DNA compete to bind to these probes according to their relative quantities.

After hybridisation, fluorescence signals are detected using a laser scanner and processed computationally. Genomic regions showing reduced signal from patient DNA indicate a deletion, while regions showing increased signal indicate a duplication.

In addition to genome-wide analysis, array-CGH can be performed using specialised formats, including very high-resolution chromosome-specific arrays or custom-designed arrays targeting selected chromosomal regions.

CMA provides substantially higher resolution than conventional chromosome analysis. Depending on the array design, probe density and genomic region, the resolution may range from approximately 100 kb to less than 10 kb.

The method is suitable for detecting clinically relevant chromosomal copy number imbalances like deletions or duplications. It may also help refine known or suspected imbalances, including those involving marker chromosomes or rearrangements that appear balanced by conventional chromosome analysis.

The main analytical limitation is that CMA detects unbalanced copy number changes only. Balanced translocations, inversions and low-level mosaicism are not reliably detected. CMA also does not reliably detect single nucleotide variants, small insertions or deletions, repeat expansions, methylation abnormalities or imprinting defects. If these mechanisms are suspected, additional cytogenetic or molecular testing may be required.

CMA is used when a chromosomal imbalance is suspected and higher resolution than conventional chromosome analysis is required. CMA may be indicated for:

• Patients with developmental delay, neurological abnormalities and dysmorphic features despite an unremarkable chromosome analysis
• Patients with chromosomal changes that appear balanced by light microscopy
• Patients with a known imbalance requiring more precise characterisation
• Patients with a marker chromosome

CMA is an established genome-wide diagnostic method for detecting chromosomal copy number changes, particularly in patients with developmental delay, intellectual disability, autism spectrum disorder, multiple congenital anomalies or dysmorphic/syndromic features.

Detected CNVs are interpreted using recognised professional standards for constitutional CNV classification and reporting. Genetic counselling is recommended before and after testing to explain the indication, possible outcomes, limitations, inheritance, recurrence risk and whether parental or additional testing may be indicated.

The non-detection of clinically relevant CNVs does not exclude an underlying genetic condition. Further testing may be required if the clinical presentation suggests a balanced rearrangement, low-level mosaicism, sequence-level variant, methylation disorder, repeat expansion disorder or another mechanism not reliably detected by CMA.

• Heinrich U, Rost I, Brown A, Gordon T, Haan N, Massie J. Array comparative genomic hybridisation in clinical diagnostics: principles and applications / Array-CGH in der klinischen Diagnostik: Prinzipien und Anwendungen. LaboratoriumsMedizin. 2009;33(5): 255-266.
• Hochstenbach R, van Binsbergen E, Engelen J, Nieuwint A, Polstra A, Poddighe P, Ruivenkamp C, Sikkema-Raddatz B, Smeets D, Poot M. Array analysis and karyotyping: workflow consequences based on a retrospective study of 36,325 patients with idiopathic developmental delay in the Netherlands. Eur J Med Genet. 2009 Jul-Aug;52(4):161-9. doi: 10.1016/j.ejmg.2009.03.015. Epub 2009 Apr 9. PMID: .
• Kleeman L, Bianchi DW, Shaffer LG, Rorem E, Cowan J, Craigo SD, Tighiouart H, Wilkins-Haug LE. Use of array comparative genomic hybridization for prenatal diagnosis of fetuses with sonographic anomalies and normal metaphase karyotype. Prenat Diagn. 2009 Dec;29(13):1213-7. doi: 10.1002/pd.2367. PMID: ; PMCID: PMC4459708.
• Menten B, Maas N, Thienpont B, Buysse K, Vandesompele J, Melotte C, de Ravel T, Van Vooren S, Balikova I, Backx L, Janssens S, De Paepe A, De Moor B, Moreau Y, Marynen P, Fryns JP, Mortier G, Devriendt K, Speleman F, Vermeesch JR. Emerging patterns of cryptic chromosomal imbalance in patients with idiopathic mental retardation and multiple congenital anomalies: a new series of 140 patients and review of published reports. J Med Genet. 2006 Aug;43(8):625-33. doi: 10.1136/jmg.2005.039453. Epub 2006 Feb 20. PMID: ; PMCID: PMC2564583.
• Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR, Carter NP, Church DM, Crolla JA, Eichler EE, Epstein CJ, Faucett WA, Feuk L, Friedman JM, Hamosh A, Jackson L, Kaminsky EB, Kok K, Krantz ID, Kuhn RM, Lee C, Ostell JM, Rosenberg C, Scherer SW, Spinner NB, Stavropoulos DJ, Tepperberg JH, Thorland EC, Vermeesch JR, Waggoner DJ, Watson MS, Martin CL, Ledbetter DH. Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet. 2010 May 14;86(5):749-64. doi: 10.1016/j.ajhg.2010.04.006. PMID: ; PMCID: PMC2869000.
• Riggs ER, Andersen EF, Cherry AM, Kantarci S, Kearney H, Patel A, Raca G, Ritter DI, South ST, Thorland EC, Pineda-Alvarez D, Aradhya S, Martin CL. Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen). Genet Med. 2020 Feb;22(2):245-257. doi: 10.1038/s41436-019-0686-8. Epub 2019 Nov 6. Erratum in: Genet Med. 2021 Nov;23(11):2230. doi: 10.1038/s41436-021-01150-9. PMID: ; PMCID: PMC7313390.