Del/Dup and Array Tests
Deletion/Duplication Analysis via Gene-Centric aCGH Test (Test Code #600)
BRIEF DESCRIPTION AND RATIONALE Specimen Requirements
Array Comparative Genomic Hybridization (aCGH) enables the detection of deletions and duplications of single and multiple exons within a given gene (Tayeh et al. Genet Med 11:232-240, 2009). This test involves analysis only of the specific gene(s) of interest for each patient.
PreventionGenetics' high density gene-centric (HDGC) aCGH is designed to have comprehensive coverage for coding regions (18 bp median probe spacing) and non-coding regions (87 bp median probe spacing) for each targeted gene and includes coverage of all transcripts. We also include probe coverage 5kb upstream and downstream. In addition, the flanking 300-bp intronic sequence on either side of targeted exons has enriched probe coverage. Therefore, PreventionGenetics' aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene.
Deletion and duplication frequency varies among genes, yet it represents a significant fraction of the total pathogenic mutations in essentially every gene (Tayeh et al. 2009). This fraction ranges from values as low as 5% (ACADM gene; Andresen et al. Am J Hum Genet. 68:1408-1418, 2001) up to 80% (NPHP1 gene; Konrad et al. Hum Mol Genet 5:367-371, 1996). In cases where the majority of the reported mutations in a gene can be detected by DNA sequencing, PreventionGenetics' aCGH is an excellent complementary test when DNA sequencing fails to identify the causative mutation(s). In cases where the majority of the reported mutations in a gene are deletions and duplications, then PreventionGenetics' aCGH should be considered as a primary test even before DNA sequencing. Examples of genes where clinical sensitivity is higher by starting with del/dup testing are listed below:
SHOX: 70-75% deletions
DMD: 65-75% deletions/duplications
RAI1: 90-95% deletions
EHMT1: 75% deletions
The availability of both DNA sequencing and PreventionGenetics' HDGC custom aCGH significantly improves the sensitivity of molecular clinical testing at PreventionGenetics.
Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.
This test is not a Chromosome Microarray (CMA) test, and we will usually interpret and report copy number variants only in requested genes. However, due to the nature of the methodology, incidental findings cannot be entirely avoided. In the event that a medically actionable copy number variant is identified in a gene not requested, we will reach out and discuss the findings with the ordering healthcare provider.
Candidates for this test are:
- Patients with genetic disorders mainly caused by deletion and duplications.
- Patients with autosomal dominant disorders where no mutation has been identified by DNA sequencing.
- Patients with autosomal recessive disorders with one or no mutations have been identified by DNA sequencing.
- Patients with autosomal recessive disorders where one or more amplicons within the gene fails to PCR amplify.
- Male patients with X-linked disorders where no mutation has been identified by DNA sequencing, or PCR fails.
- Female patients with X-linked disorders with one or no mutations have been identified by DNA sequencing.
- Patients with gross genomic imbalances in a region harboring one or multiple genes targeted on PreventionGenetics' HDGC aCGH, to confirm involvement of such gene(s).
PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.
Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.
This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.
aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.
Breakpoints, if occurring outside the targeted gene, may be hard to define.
The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.
The great majority of tests are completed within 28 days
For STAT testing requests, we will attempt but cannot guarantee to complete the test within 10 days. The 25% surcharge will be added to the test price only if the test is completed in 10 days.
At this time, PreventionGenetics does not offer aCGH for prenatal specimens, however, if a copy number variation (CNV) was able to be confirmed by an alternate method (PCR) in a proband tested at PreventionGenetics, we may be able to offer targeted deletion or duplication testing prenatally. For any CNV where the mother is a carrier, we are unable to offer targeted prenatal for that CNV at this time.
|Number of Genes Ordered||Total Price|
|Over 100||Call for quote|
Targeted testing for relatives of probands tested at PreventionGenetics (Test Code 1400/$340) is available if we are able to test the del/dup by PCR or MLPA.
Please click here to search for the applicable CPT code(s).
Genetic Counselors: GC Team - email@example.com
Brief Description and Rationale Specimen Requirements
Chromosome analysis through the study of G-banded karyotype has been standard practice for the clinical diagnosis of idiopathic intellectual disability (ID), developmental delay (DD), autism spectrum disorder (ASD), and multiple congenital anomalies (MCA) since its introduction in the early 1970s. This and other conventional cytogenetic techniques including fluorescence in situ hybridization (FISH) are limited by their ability to detect chromosomal abnormalities beyond their detection limit (>5 MB for G-band and >100 kb for FISH).
Over the last several years chromosomal microarray (CMA) has replaced chromosomal G-banded karyotype analysis as a first-tier test for the detection of copy number variants (CNVs) associated with the diagnosis of genomic disorders1-5. CMA not only provides increased resolution to detect genomic CNVs, but also provides 10-15% higher diagnostic yield of clinically significant and pathogenic CNVs. CMA platforms cover all types of array-based analyses, including oligonucleotide-based array-comparative genomic hybridization (aCGH) that detects only copy number alterations of a set of sequences as compared with a control sample, and single nucleotide polymorphism (SNP) arrays that may be able to discriminate and/or quantify allelic copy number.
PreventionGenetics' CMA-ISCA array is a high density oligonucleotide aCGH array that combines both copy-number analyses and SNP-based allelic discrimination. The presence of SNP probes allows for the detection of relative changes in allelic distribution and also for the detection of copy neutral regions of homozygosity. Homozygous regions of the genome have been variously described using multiple terms such as absence/loss of heterozygosity (AOH/LOH), regions/runs of homozygosity (ROH), or long contiguous stretches of homozygosity (LCSH), with each conveying slightly different meaning. LCSH on any individual chromosome may result from uniparental disomy (UPD), or when present in multiple chromosomes may represent regions of the genome identical by descent (IBD) (e.g., consanguinity not suspected on the basis of clinical history), both of which increases the risk for autosomal recessive disorders6-8. In addition, depending on the density of the SNP probes, relative changes in the allelic distribution can provide confirmation of heterozygous CNV calls over the same interval. Thus the inclusion of SNP probes along with CNV probes improves the diagnostic yield of a CMA test.
CMA via aCGH compares a patient's genomic DNA with a gender-matched reference genomic DNA to detect small copy number gains (duplications) and losses (deletions) on all 46 chromosomes in a single test. PreventionGenetics' CMA-ISCA v1.0 (Whole-genome chromosomal microarray) contains ~110,000 distinct CGH probes distributed across the entire genome with a median probe spacing of ~25 kb, and ~59,000 SNP probes resulting in ~5-10 Mb resolution for LOH detection. The CGH probes consist of the entire ISCA (International Standards for Cytogenomic Arrays: https://www.iscaconsortium.org/) Consortium 8x60K version probe set and an additional 60,000 backbone probes. This includes high-density coverage of ~500 targeted regions with the spacing of 5 kb per probe or at least 20 probes per gene region. These targeted regions include telomere and unique centromere FISH clone regions, microdeletion/duplication regions, genes of known haploinsufficiency, and X-linked mental retardation regions.
- CMA starts with the extraction of genomic DNA from a patient specimen (e.g., whole blood) using a QIAamp DNA Blood Midi kit (Qiagen).
- Equal amounts of genomic DNA (~1.0 microgram) from a patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively.
- Labeled patient product is then purified, quantified, and combined with the same amount of reference product.
- The combined sample is loaded onto a microarray slide and hybridized for at least 22-24 hours at 65°C.
- Arrays are then washed and scanned immediately at 2.5 µM resolution.
CMA copy number data is analyzed following feature extraction of the scanned microarray, using Agilent CytoGenomics Software and in-house tools. The resolution for CNV detection depends on the region of the genome, its uniqueness and complexity. On average, across the targeted regions, this array can detect CNVs of minimal size between 500 bp to 30 kb.
A written summary and interpretation of the microarray findings are provided using standard aCGH nomenclature and in line with the ACMG guidelines for microarray interpretation4-5. Common CNVs and rare CNVs with unknown clinical significance may be detected with this technology. CNVs in regions of the genome with no clinically relevant genes or in regions reported as common polymorphism in the general population will not be reported. In addition, CNVs confined to deep intronic sequences with no documented evidence of clinical significance will not be reported.
For CNVs with unknown clinical significance a number of considerations are taken into account, including the size, the gene content, whether the CNV is a deletion or a duplication, and the inheritance pattern. On some occasions, interpretation of whether a CNV is pathogenic depends on whether the CNV is inherited from one of the parents or de novo, and hence parental analysis is sometimes needed for optimal interpretation.
A single region of homozygosity will only be reported if it is larger than 10 Mb in size or cumulatively >25 Mb if multiple regions exists on a single chromosome. An exception may be homozygosity for a genomic region with OMIM genes that overlap with the clinical phenotype. Multiple regions of homozygosity on several chromosomes suggestive of parental consanguinity will be reported only if it cumulatively exceeds >2% of the genome.
A confirmatory test for all reported pathogenic CNVs will be attempted. Depending on the size and density of the probes, the choice of the method may be FISH, PCR, qPCR or MLPA. For microarray results indicative of a structural chromosomal abnormality, reflexive chromosome studies may be warranted for the patients and/or parents to guide recurrence risk estimation.
CMA is indicated for the following reasons:
- Individuals with an unexplained abnormal phenotype, such as:
- Global developmental delay/intellectual disability, with or without dysmorphic features
- Autism/autism spectrum disorder (ASD)/pervasive developmental disorder (PDD)
- Dysmophic features, multiple congenital anomalies, or birth defects
- Heart defects
- Epilepsy and seizures
- Individuals with normal G-banded chromosome analysis, but with an abnormal phenotype
- Evaluation of stillbirth, neonatal deaths and spontaneous pregnancy loss
- Screening for microdeletions and microduplications associated with known syndromes/clinical phenotypes
- Screening for unique microdeletions and microduplications not associated with known syndromes
- Suspected UPD syndrome
- Increased risk of a genetic heterogeneity and recessive disorder due to suspected common ancestry
- Further characterization of a previously identified chromosomal abnormality such as marker chromosomes, ring chromosomes, apparent terminal deletions, unbalanced translocations
- Analysis of an apparently balanced de novo chromosomal rearrangement seen in patients with abnormal phenotypes
Advantages of CMA-ISCA
- Genome-wide detection of genomic copy number imbalances, including pericentromeric/subtelomeric regions
- Genome-wide coverage for ~50 curated and well characterized microdeletion and microduplication syndromes
- Includes coverage for ~500 ISCA targeted regions
- Superior resolution to "Gold Standard" G-banded karyotype analysis (100-200kb vs 5-10 Mb)
- Superior diagnostic yield to "Gold Standard" G-banded karyotype analysis (15-20% vs 3%) for individuals with idiopathic intellectual disability, developmental delay, autism spectrum disorder, and multiple congenital abnormalities
- Concurrent analysis of CGH and SNP data allow detection of UPD, AOH/LOH, parental consanguinity, and ploidy changes
- CMA can only detect only gross genomic copy number imbalances, and LCSH in the nuclear genome. It cannot detect balanced chromosomal rearrangements such as inversions, balanced insertions, and reciprocal translocations.
- CMA cannot detect:
- Genomic copy number changes in the regions of the genome not represented on the microarray (including regions with repeat sequences such as segmental duplications, repeat sequences in the short arms of acrocentric chromosomes, and heterochromatic regions)
- Low levels of mosaicism for regions <10-15 Mb in size (although, it can reliable detect mosaicism as low as 25% for whole chromosome or for whole chromosomal arm imbalances)
- Point mutations and indels
- Minimal detectable CNV size may be 500bp in targeted regions.
- Complete uniparental heterodisomy for the entire chromosome (it can only detect uniparental isodisomy, and segmental heterodisomy)
- Imbalances in the mitochondrial genome
- CMA cannot detect imbalances when mosaicism for reciprocal CNVs exist. For example, when mosaicism for 46,XX, 45,X and 47XXX exist in approximate equal proportions, CMA will fail to detect the presence of the clinically significant 45,X line.
- Failure to detect an alteration at a specific locus does not rule out the diagnosis of a genetic disorder associated with that locus. Other abnormalities may be present that are undetectable by the microarray design.
- Failure to detect evidence of UPD does not exclude the clinical diagnosis of an imprinted associated disorder. For example complete heterodisomy that is seen in ~21-29% of UPD15 will show no evidence of recombination, leading to 8% of the individuals with Prader-Willi syndrome being missed by CMA alone9-10.
PreventionGenetics' policy for CMA is to provide free complimentary parental studies for copy number results of unknown significance, and for which the parental studies may clarify the clinical significance. Parental studies may be performed by FISH analysis for regions that are large enough (>0.5 Mb for deletions, and >1 Mb for duplications), and for which commercial FISH probes are available. For regions with no commercial FISH probes available, microarray may be performed on the parents for a charge. In addition, PCR and qPCR may be performed for parental analysis.
Free parental studies will not be performed for genomic imbalances for the following situations:
- Parental testing is needed to determine if the pathogenic abnormality is inherited or de novo. For example DiGeorge syndrome region CNVs may be inherited from a parent with no clinical phenotype.
- Presence of a large CNV in the proband, and there is general consensus regarding its pathogenicity and penetrance.
- Presence of known clinical microdeletion and microduplication syndromes in the proband.
- Terminal deletions/duplications in the proband, to rule out the possibility of a balanced rearrangement in a parent.
Currently, we do not accept prenatal specimens (chorionic villi/placental tissue and amniotic fluid samples) for this test, as these specimen types have not been validated.
The great majority of tests are completed within 20 days (abnormal findings are typically issued in preliminary report). Cases requiring confirmatory tests will delay issue of final report.
Test Code: #2000
CPT Coding: 81229
- Manning M and Hudgins L., Array based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities. Genet Med. 2010 Nov;12(11):742-5.
- 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):749-64.
- Kaminsky EB, et al., An evidence-based approach to establish the functional and clinical significance of copy number variants in intellectual and developmental disabilities. Genet Med. 2011 Sep;13(9):777-84.
- Kearney HM., American College of Medical Genetics standards and guidelines for the interpretation and reporting of postnatal constitutional copy number variants. Genet Med. 2011 Jul;13(7):680-5.
- South ST, et al, ACMG standards and Guidelines for constitutional cytogenomic microarray analysis, including postnatal and prenatal applications: revision 2013. Genet Med. 2013 Nov;15(11):901-9.
- Kearney HM, et al., Diagnostic implications of excessive homozygosity detected by SNP-based microarrays: consanguinity, uniparental disomy, and recessive single gene mutations. Clin Lab Med. 2011 Dec;31(4):595-613.
- Fan YS, et al., Frequent detection of parental consanguinity in children with developmental disorders by a combined CGH and SNP microarray. Mol Cytogenet. 2013 Sep 20;6(1):38.
- Sund KL, et al., Regions of homozygosity identified by SNP microarray analysis aid in the diagnosis of autosomal recessive disease and incidentally detect parental blood relationships. Genet Med. 2013 Jan;15(1):70-8.
- Tucker T et al., Uniparental disomy: can SNP array data be used for diagnosis? Genet Med. 2012 Apr 26.
- Papenhausen P, et al., UPD detection using homozygosity profiling with a SNP genotyping microarray. Am J Med Genet A. 2011 Apr;155A(4):757-68.
Genetic Counselors: GC Team - firstname.lastname@example.org
Geneticist: Srirangan Sampath, PhD, FACMG - email@example.com
CGH, SNP array, CMA, Copy Number array, Cytogenomic array, ISCA, Uniparental Disomy, Deletion, Duplication, Mosaicism,