CNV and Array Tests

Whole-Genome Chromosomal Microarray (CMA-ISCA) via the aCGH and SNP Test #2000

New York State Approved Test

New York State Approved Test

BRIEF DESCRIPTION AND RATIONALE

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 only larger chromosomal abnormalities (>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 disorders^1-5. 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. AOH 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 disorders^6-8. 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.

THE TECHNOLOGY

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 AOH 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 intellectual disability regions.

METHODS

• 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.

INTERPRETATION

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 area of homozygosity will only be reported if it is larger than 5 Mb for telomeric AOH for any chromosome, or larger than 10 Mb in size for interstitial AOH on imprinted chromosomes, or larger than 15 Mb in size for interstitial AOH on nonimprinted chromosomes, or cumulatively >25 Mb if multiple areas exist on a single chromosome. An exception may be homozygosity for a genomic region with clinically relevant genes that overlap with the patient’s phenotype. Multiple regions of homozygosity on several chromosomes suggestive of parental consanguinity will be reported only if it cumulatively exceeds 50 Mb.

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.

INDICATIONS

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

ANALYTICAL LIMITATIONS

• 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 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 alone^9-10.

SPECIMEN REQUIREMENTS

Specimen Requirements

TURNAROUND TIME

3 weeks on average for standard orders (abnormal findings are typically issued in preliminary report). Cases requiring confirmatory tests will delay issue of final report.

References

1. 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. PubMed ID: 20962661

2. 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. PubMed ID: 20466091

3. 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. PubMed ID: 21844811

4. 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. PubMed ID: 21681106

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. PubMed ID: 24071793

6. 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. PubMed ID: 22118739

7. 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. PubMed ID: 24053112

8. 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. PubMed ID: 22858719

9. Tucker T et al., Uniparental disomy: can SNP array data be used for diagnosis? Genet Med. 2012 Apr 26. PubMed ID: 22538256

10. 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. PubMed ID: 21594998

Contacts

Genetic Counselors: GC Team - support@preventiongenetics.com

Geneticist: Megan Piazza, PhD, FACMG - megan.piazza@preventiongenetics.com

Alias

CGH, SNP array, CMA, Copy Number array, Cytogenomic array, ISCA, Uniparental Disomy, Deletion, Duplication, Mosaicism,

Rapid Chromosomal Microarray via aCGH and SNP Test #12684

BRIEF DESCRIPTION AND RATIONALE

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 disorders^1-5. 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.

The custom microarray used for Rapid Chromosomal Microarray 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 disorders^6-8. 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.

THE TECHNOLOGY

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. Allele Diagnostics’ custom Agilent 180K CGH+SNP microarray contains ~107,000 distinct CGH probes distributed across the entire genome with a resolution of 20 kb in targeted regions and 80 kb in the backbone, and ~60,000 SNP probes resulting in ~5-10 Mb resolution for LOH detection. Targeted regions include over 255 recognized genetic syndromes and over 980 gene regions of functional significance in human development. These targeted regions include telomere and unique centromere FISH clone regions, microdeletion/duplication regions, genes of known haploinsufficiency, and X-linked intellectual disability regions.

METHODS

This testing is performed by Allele Diagnostics (CLIA #50D2086351/CAP #9018482). Please refer to Allele Diagnostics’ website for more information about their specific methodology (https://www.allelediagnostics.com/).

INTERPRETATION

A written summary and interpretation of the microarray findings are provided using standard aCGH nomenclature and in line with the ACMG guidelines for microarray interpretation ^4-5 . 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.

We will report known genomic disorders (microdeletion and microduplication syndromes) in addition to whole or partial chromosome aneuploidies.

AOH can be indicative of uniparental disomy (UPD) if within a chromosome, or an increased risk of a recessive disorder if identified across multiple chromosomes. Note that for some regions of the genome, AOH of 8-10 Mb may be within normal limits and will not be reported. There are a small number of chromosome regions which are associated with imprinting or UPD disorders. AOH of 5 Mb or more will be reported for regions that have previously been associated with imprinting conditions. The SNP component of this assay also has the ability to determine an overall level of homozygosity throughout the genome of 5% or more. If identified, this will be reported as it may be reflective of identity by descent and an increased risk for recessive disorders. Clinical correlation is required to properly assess a potential for relatedness. In addition, this assay cannot determine whether the alteration is paternal, maternal or de novo in origin. Additional UPD or molecular testing may be required.

INDICATIONS

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 RAPID CHROMOSOMAL MICROARRAY

• Genome-wide detection of genomic copy number imbalances, including pericentromeric/subtelomeric regions
• Genome-wide coverage for 255 curated and well characterized microdeletion and microduplication syndromes
• Includes coverage for over 980 gene regions of functional significance in human development
• Superior resolution to "Gold Standard" G-banded karyotype analysis (20-80 kb 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

ANALYTICAL LIMITATIONS

• 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
  • Point mutations and indels
  • Minimal detectable CNV size may be ~20 kb in targeted regions and ~80 kb in the backbone.
  • Complete uniparental heterodisomy for the entire chromosome (it can only detect uniparental isodisomy, and segmental heterodisomy)
  • Imbalances in the mitochondrial genome
  • Imbalances when mosaicism for reciprocal CNVs exist. For example, when mosaicism for 46,XX, 45,X and 47,XXX 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.

PARENTAL STUDIES

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 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 CNVs in the proband, to rule out the possibility of a balanced rearrangement in a parent.

SPECIMEN REQUIREMENTS

Specimen Requirements

TURNAROUND TIME

 2 weeks on average. Rarely, the need for confirmation and/or technical difficulties may increase this time.

References

1. 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. PubMed ID: 20962661

2. 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. PubMed ID: 20466091

3. 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. PubMed ID: 21844811

4. 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. PubMed ID: 21681106

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. PubMed ID: 24071793

6. 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. PubMed ID: 22118739

7. 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. PubMed ID: 24053112

8. 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. PubMed ID: 22858719

9. Tucker T et al., Uniparental disomy: can SNP array data be used for diagnosis? Genet Med. 2012 Apr 26. PubMed ID: 22538256

10. 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. PubMed ID: 21594998

Contacts

Genetic Counselors: GC Team - support@preventiongenetics.com

Geneticist: Megan Piazza, PhD, FACMG - megan.piazza@preventiongenetics.com

Alias

CGH, SNP array, CMA, Copy Number array, Cytogenomic array, ISCA, Uniparental Disomy, Deletion, Duplication, Mosaicism,

Rapid Prenatal Chromosomal Microarray via aCGH and SNP - Prenatal Test #3780

Prenatal Test Requisition Form

BRIEF DESCRIPTION AND RATIONALE

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 disorders^1-5. 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.

The custom microarray used for Rapid Prenatal Chromosomal Microarray 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 disorders^6-8. 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.

THE TECHNOLOGY

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. Allele Diagnostics’ custom Agilent 180K CGH+SNP microarray contains ~107,000 distinct CGH probes distributed across the entire genome with a resolution of 20 kb in targeted regions and 80 kb in the backbone, and ~60,000 SNP probes resulting in ~5-10 Mb resolution for LOH detection. Targeted regions include over 255 recognized genetic syndromes and over 980 gene regions of functional significance in human development. These targeted regions include telomere and unique centromere FISH clone regions, microdeletion/duplication regions, genes of known haploinsufficiency, and X-linked intellectual disability regions.

METHODS

This testing is performed by Allele Diagnostics (CLIA #50D2086351/CAP #9018482). Please refer to Allele Diagnostics’ website for more information about their specific methodology (https://www.allelediagnostics.com/). A backup cell culture for direct samples is always set up via Allele Diagnostics and may be used for testing if the direct sample fails the initial testing. In the event that both the direct and cultured samples fail testing, a failed analysis report will be issued. A complete sample failure will result in a charge of $350 and billed as test code #995 (cell culture). Maternal cell contamination studies are performed at PreventionGenetics with a maternal sample and the fetal sample used for the CMA by Allele Diagnostics. 

INTERPRETATION

A written summary and interpretation of the microarray findings are provided using standard aCGH nomenclature and in line with the ACMG guidelines for microarray interpretation 4-5 . 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.

We will report known genomic disorders (microdeletion and microduplication syndromes) in addition to whole or partial chromosome aneuploidies.

AOH can be indicative of uniparental disomy (UPD) if within a chromosome, or an increased risk of a recessive disorder if identified across multiple chromosomes. Note that for some regions of the genome, AOH of 8-10 Mb may be within normal limits and will not be reported. There are a small number of chromosome regions which are associated with imprinting or UPD disorders. AOH of 5 Mb or more will be reported for regions that have previously been associated with imprinting conditions. The SNP component of this assay also has the ability to determine an overall level of homozygosity throughout the genome of 5% or more. If identified, this will be reported as it may be reflective of identity by descent and an increased risk for recessive disorders. Clinical correlation is required to properly assess a potential for relatedness. In addition, this assay cannot determine whether the alteration is paternal, maternal or de novo in origin. Additional UPD or molecular testing may be required.

INDICATIONS

CMA is indicated for the following reasons:

• Abnormal fetal ultrasound findings
• Abnormal non-invasive prenatal testing (NIPT) screening
• Abnormal maternal serum screening
• Advanced maternal age
• Any patient undergoing invasive prenatal testing
• Family history of balanced rearrangement for the detection of an unbalanced rearrangement

ADVANTAGES OF RAPID PRENATAL CHROMOSOMAL MICROARRAY

• Genome-wide detection of genomic copy number imbalances, including pericentromeric/subtelomeric regions
• Genome-wide coverage for 255 curated and well characterized microdeletion and microduplication syndromes
• Includes coverage for over 980 gene regions of functional significance in human development
• Superior resolution to "Gold Standard" G-banded karyotype analysis (20-80 kb 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

ANALYTICAL LIMITATIONS

• 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
  • Point mutations and indels
  • Minimal detectable CNV size may be ~20 kb in targeted regions and ~80 kb in the backbone.
  • Complete uniparental heterodisomy for the entire chromosome (it can only detect uniparental isodisomy, and segmental heterodisomy)
  • Imbalances in the mitochondrial genome
  • Imbalances when mosaicism for reciprocal CNVs exist. For example, when mosaicism for 46,XX, 45,X and 47,XXX 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.

SPECIMEN REQUIREMENTS

Specimen Requirements

TURNAROUND TIME

1 week on average. Rarely, the need for confirmation and/or technical difficulties may increase this time.

References

1. 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. PubMed ID: 20962661

2. 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. PubMed ID: 20466091

3. 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. PubMed ID: 21844811

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Contacts

Genetic Counselors: GC Team - support@preventiongenetics.com

Geneticist: Megan Piazza, PhD, FACMG - megan.piazza@preventiongenetics.com

Alias

CGH, SNP array, CMA, Copy Number array, Cytogenomic array, ISCA, Uniparental Disomy, Deletion, Duplication, Mosaicism,