POSTNATAL TESTING
Germline testing for pediatric and adult individuals. See information on specimen requirements and billing below.
Chromosomal microarray (CMA) is a molecular cytogenomic test for detecting both copy number changes (deletions and duplications) and copy-neutral absence of heterozygosity (AOH).
Indications
ACMG recommends CMA as a first-tier test for individuals with:
- Unexplained developmental delay
- Intellectual disability
- Autism spectrum disorder
- Congenital anomalies.
- Other neurological or developmental disorders, such as seizures
- Family history of a copy number variant
Test Uses
CMA can be used for detection of the following genomic changes:
- Copy Number Variants (CNV) – duplications or deletions of chromosomal material
- Pathogenic and Likely Pathogenic CNVs are always reported
- Variants of Uncertain Significance (VUS) are reported if they meet reportable criteria
- Benign and Likely Benign CNVs are not reported
- Absence of Heterozygosity (AOH) – areas of identical chromosomal material
- AOH is not diagnostic but may narrow down areas and/or autosomal recessive genes of interest to direct further testing
- Shared ancestry/identity by descent is more likely when 2% or more of the autosomal genome is identical. Close consanguinity is predicted when 10% or more of the autosomal genome is identical
- Uniparental Isodisomy is detectable by CMA while heterodisomy is not. Additional testing for imprinted disorders may be recommended
- Mosaicism of a second cell population at greater than ~20%
- CNV classification is determined according to Riggs, et al. (2020).
Follow-Up Testing
CGL often recommends follow-up studies to further elucidate positive CMA results. Recommendations, if any, appear in the proband’s result report. Follow-up testing may include:
- proband and/or parental karyotype to evaluate for a chromosome rearrangement
- parental and/or family member CMA testing for the CNV detected
- When a VUS is detected, two close biological family members can be tested by CMA without additional charge if relevant to patient care
- proband molecular, methylation, or biochemical testing of a gene or region of interest at an outside laboratory
Limitations
CMA will NOT detect balanced rearrangements (i.e. inversions, translocations, etc.), heterodisomy, or reliably identify low-level mosaicism (below ~20%). It will NOT detect sequence variants or small duplications/deletions. CNVs that are smaller than the resolution of the CMA may not be detected or reported.
CMA will NOT detect balanced rearrangements (i.e. inversions, translocations, etc.), heterodisomy, or reliably identify low-level mosaicism (below ~20%). For mosaic conditions, CMA cannot differentiate between specific number of copies present (e.g. trisomy of a whole chromosome arm vs. tetrasomy in 50% of cells tested). It also does not detect deletions and duplications with limited probe coverage, gene expansions, variants within mitochondrial DNA, or single nucleotide variants/polymorphisms. CNVs that are smaller than the resolution of the CMA may not be detected or reported. CNVs determined to be benign or likely benign are not reported. Typically, carrier status is not reported unless possibly relevant to the patient’s reported symptoms to guide further testing. Contact the laboratory if there is a specific region or gene(s) of interest.
Reporting Thresholds
| Finding | Reportable Size* |
|---|---|
| Loss/Deletion | 200 kb or larger |
| Gain/Duplication | 400 kb or larger |
| Absence of Heterozygosity | 10 Mb or larger always reported 3 Mb and larger are analyzed and may be reported if total AOH is 2% or greater |
*Smaller AOH or copy number variants may be reported if likely to have relevant clinical significance.
Methods
Extracted DNA is processed on the Illumina® Infinium Global Diversity bead array. The Infinium Global Diversity bead array targets approximately 4885 genes in accordance with the most recent ICCG recommendations. The Infinium Global Diversity bead array is scanned on Illumina’s iScan Platform. Using single nucleotide polymorphism (SNP) probes, the array can detect genomic copy number losses and gains, known as copy number variants (CNVs), with a resolution of approximately 5 kb across the genome. NxClinical software (Bionano Inc.) is used to analyze and interpret the data. Detected CNVs are reported when found to have evident or suspected clinical relevance. CNVs devoid of relevant gene content or that are common CNVs in the general population (listed in the Database of Genomic Variants) may not be reported. Due to probe placement on the chromosomal microarray (CMA), the smallest size CNV that is detectable is variable by region. When applicable, CGL may complete confirmatory testing of a CNV prior to reporting.
Genomic linear positions are given relative to the Genome Reference Consortium Human Genome build 37 (hg19) released by the National Center for Biotechnology Information (NCBI). CNVs are analyzed using publicly available databases and literature. See the following references regarding CMA analysis, interpretation, and reporting. PMID: 31690835, 34131312, 34906464.
Turn Around Time
7-14 days
Family members of probands (primary patient) identified with a copy number variant (CNV) of known, potential, or uncertain clinical significance may wish to be tested for the CNV. When performed on a parent, the results may help classify an uncertain finding and/or inform the recurrence risk for other offspring. When performed on a sibling, the results may be diagnostic without requiring a full CMA analysis.
A targeted CMA will be analyzed at only the locus/loci of interest, though any clinically significant incidental findings on the same chromosome may be reported. Other findings will not be reported. When a VUS is detected, two close biological family members can be tested by CGL without additional charge if relevant to patient care.
Methods
Extracted DNA is processed on the Infinium Global Diversity chromosomal microarray (CMA) (Illumina®). The CMA targets approximately 4885 genes in accordance with the most recent ICCG recommendations. The CMA is scanned on iScan Platform (Illumina®). Using single nucleotide polymorphism (SNP) probes, CMA can detect genomic copy number losses and gains, known as copy number variants (CNVs), with a resolution of approximately 5 kb across the genome. NxClinical software (Bionano Inc.) is used to analyze and interpret CMA data. Reporting for a “targeted” CMA is restricted to interpretation of a specific copy number change previously detected in a family member and does not represent a full CMA analysis. Absence of heterozygosity (AOH) is not typically reported in a targeted array. Result interpretation assumes the accuracy of any demographic, clinical, and familial relationship information provided. Genomic coordinates are given relative to the Genome Reference Consortium Human Genome build 37 (hg19) released by the National Center for Biotechnology Information (NCBI). CNVs are analyzed using publicly available databases and literature. See the following references regarding CMA analysis, interpretation, and reporting: PMID: 31690835, 34131312.
Turn Around Time
7-14 days
Chromosome analysis is performed on G-banded metaphase chromosomes for the purpose of detecting numerical and/or structural abnormalities that may be present. These include aneuploidy (such as trisomy or monosomy), mosaicism, unbalanced rearrangements such as interstitial or terminal deletions/duplications and/or balanced rearrangements such as translocations and inversions.
Indications
Standard Chromosome Analysis
- Suspicion of aneuploidy
- Congenital anomalies
- Growth delay
- Intellectual disabilities
- Infertility
- Personal or family history of recurrent miscarriage
High-Resolution Chromosome Analysis
- Known family history of a translocation or other rearrangement
- Testing (such as CMA) suggestive of a rearrangement
5-cell Chromosome Analysis
- As a supplement to Chromosomal Microarray (CMA),
five5 cells are evaluated to identify or rule out balanced chromosome rearrangements for the indications above. This is NOT a stand-alone test.
Methods
Peripheral blood cultures are mitogen-stimulated for lymphocyte proliferation. Metaphase chromosomes are prepared using methanol/acetic acid fixation and G-banding by trypsin-Leishman (GTL). The number of metaphases analyzed, counted, and karyotyped are enumerated in the patient’s report. Nomenclature is according to the current ISCN (2024).
When sex chromosome abnormalities are suspected in postnatal specimens, twenty cells are examined. If a single cell with a sex chromosome abnormality is detected, ten additional cells are examined (PMID: 20081420 and Correction). For other mosaic aneuploidies, additional cells may be counted as needed.
Limitations
The detection of abnormalities is limited by banding resolution and the presence of viable, dividing cells. Conventional chromosome analysis cannot reliably detect submicroscopic copy-number changes (below 5-10Mb), cryptic rearrangements, or low-level mosaicism. If performed, five-cell chromosome analysis may not detect mosaicism. Chromosome analysis cannot detect Uniparental Disomy (UPD), absence of heterozygosity (AOH), or sequence variants.
Follow-up Testing
Some chromosome abnormalities may warrant additional testing by CMA including unbalanced rearrangements, apparently balanced rearrangements in an individual with symptoms, and copy number variants to determine the extent of chromosome material involved and identify affected genes. Testing for family members may also be recommended when a chromosome rearrangement is identified.
Turn Around Time
10-14 daysFluorescence in situ hybridization (FISH) detects chromosome aneuploidy and submicroscopic genomic copy number changes such as deletions and duplications in specific regions.
Indications
- Suspicion of aneuploidy (X, Y, 13, 18, 21)
- Sex chromosome evaluation
- Xcen/Ycen, Xcen/STS, Xcen/SRY
- Suspicion for specific disorders:
- Williams Syndrome/ELN (7q11.2)
- Prader-Willi/Angelman Syndrome (15q11.2)
- 22q11.2 Microdeletion Syndrome
- X-linked Ichthyosis Xp22.21
- Familial testing after diagnosed proband
Limitations
FISH is not considered a diagnostic test but may guide the next steps of testing. FISH probes bind to a specific loci in the genome and can provide a rapid, preliminary copy number assessment of an area of interest. However, several factors may lead to inaccurate FISH results. Co-localized signals, interrupted binding sites, very small copy number variants, and other events contribute to occasional false positives and false negatives.
As FISH cannot determine the extent of the genetic material duplicated or deleted, abnormal FISH results require follow-up with CMA and/or chromosome analysis to further clarify the scope of the genomic imbalance and its medical significance. Critical and irreversible medical decisions based on FISH results alone are NOT recommended.
Occasionally, FISH may be recommended to clarify the results of chromosome analysis and/or CMA. Such additional recommendations will be discussed with the ordering provider prior to being performed.
Turn Around Time
1-3 days
Collection Instructions
All specimens must be labeled with the patient’s name and a second identifier (e.g. DOB, MRN).
- Blood should be collected in a 4mL sodium heparin (green top) tube and stored at room temperature. If a CMA is ordered, an additional EDTA tube is requested.
- Buccal swab kits are available by request.
- Skin biopsies should be collected using aseptic technique and placed in a sterile screw-top container with sterile transport media or sterile saline solution. Transport media is available upon request. Label with patient identifiers and tissue type. Store at room temperature or overnight at 4O C.
| Test | Volume | CPT Codes |
| Chromosome Analysis | Peripheral Blood: 4 mL* in NaHep Skin Biopsy: 3-4 mm3 | Standard 88230, 88262, 88280, 88291
5-Cell 88230, 88261, 88280, 88291
High-Resolution 88230, 88262, 88280, 88291, 88289 |
| Chromosome Microarray (CMA) | Peripheral Blood: 2 mL in EDTA Buccal Swab Skin Biopsy: 3-4 mm3 | 81229 |
FISH reflex to CMA or Chromosome Analysis A positive aneuploidy FISH will be followed by chromosome analysis. A negative aneuploidy FISH will be followed by CMA with optional 5-cell chromosome analysis. A positive microdup/del FISH will be confirmed as appropriate. | Peripheral Blood: 4 mL in NaHep and 2 mL in EDTA* Skin Biopsy: 3-4 mm3 | Aneuploidy FISH 88271 x 5, 88274 x4, and Chromosome Analysis or CMA codes above Microdeletion/Duplication FISH 88271 x 2, 88274, 88237, and Chromosome Analysis or CMA codes above Add 88291 if Chromosome Analysis is not performed. |
*Please contact CGL if insufficient blood volume is collected to discuss testing options.
REFERENCES
Gonzales PR, et al. Interpretation and reporting of large regions of homozygosity and suspected consanguinity/uniparental disomy, 2021 revision: A technical standard of the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2022 Feb;24(2):255-261. Epub 2021 Dec 3. PMID: 34906464.
Miller DT, et al. 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):749-64PMID: 20466091; PMCID: PMC2869000.
Riggs ER, et al. 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. Epub 2019 Nov 6. Erratum in: Genet Med. 2021 Nov;23(11):2230. PMID: 31690835; PMCID: PMC7313390.
Shao L, et al. Chromosomal microarray analysis, including constitutional and neoplastic disease applications, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2021 Oct;23(10):1818-1829. Epub 2021 Jun 15. PMID: 34131312.