CHROMOSOMAL MICROARRAY (CGH+SNP)

Chromosome imbalances are a significant cause of developmental delay, mental retardation, autism spectrum disorders, dysmorphic features and/or birth defects. The imbalance of genetic material may be due to additional copies or loss of whole chromosomes, or deletions and/or duplications of chromosome segments, some of which may be visible under the microscope while others are not. Routine high resolution chromosome analysis can detect microscopically visible genomic imbalances but cannot detect smaller, submicroscopic differences in DNA copy number caused by microdeletions and microduplications. Both the American Society of Human Genetics (ASHG) and the American College of Medical Genetics (ACMG) currently recommend acgh as the first-tier testing for individuals with multiple congenital anomalies, nonsyndromic developmental delay or intellectual disability, and/or autism spectrum disorders. 1,2 The diagnosis of a chromosome abnormality could change your patient s medical management by leading to screening for occult birth defects and anticipatory monitoring/screening for specific health problems. It may also help reveal other at-risk family members or an unrecognized reproductive risk for the family (which could lead them to change the management of a future pregnancy). What is Chromosomal Microarray? Chromosomal Microarray evaluates DNA copy number (by comparative genomic hybridization, or CGH) across the genome at higher resolution than routine chromosome analysis. It can detect submicroscopic genomic imbalances not detectable by routine chromosome analysis, as well as large imbalances detectable by routine chromosome analysis. A special slide (chip) is used that contains thousands of spots neatly arranged in a checkerboard pattern known as microarray. Each spot contains DNA segments (DNA probes) that are specific to certain genomic locations. Any increase or decrease in signal intensity ratios at these spots indicates DNA copy number differences within the patient s genome. This allows simultaneous analysis of multiple genomic regions to detect deletions (losses) or duplications (gains) in the patient s entire genome. Abnormal findings are followed-up with standard targeted fluorescence in situ hybridization (FISH) analysis when FISH probes are available. Chromosomal microarrays that incorporate single nucleotide polymorphism (SNP) probes may detect regions of homozygosity (ROH). The possible clinical significance of ROH relates to two different classes of genetic disorders: disorders of imprinting (uniparental disomy; UPD) and recessive diseases. Evidence suggestive of a blood relationship between the parents (parental consanguinity) also may be revealed. Chromosomal Microarray (CGH+SNP) at ChildLab ChildLab uses a chromosomal microarray that has two components, one for copy number analysis (CGH) and one for SNP analysis. The copy-number probes on the array (~135,000) have an average spacing of one probe every 35 kb throughout the genome and one probe every 10 kb in regions of the genome associated with a syndrome or clinical phenotype known to have clinical significance. The SNP probes (~67,000) have an average spacing of ~49 kb across the genome. This allows us to detect regions of regions of homozygosity (ROH) with a resolution down to 8-10 Mb across the genome. The location of each probe in the human genome is known, allowing direct correlation of what genes are present within the DNA copy number variation or ROH detected in the patient. Note For additional information, a Technical Brief is available. Results Reporting CGH Analysis Abnormal CGH findings are reported as genomic gain (duplication) or genomic loss (deletion). ChildLab categorizes each microarray finding as: Likely Pathogenic, Copy Number Change of Unknown Significance, or Likely Benign Copy Number Variation (CNV). Likely Pathogenic refers to a DNA copy number change known to be associated with an abnormal phenotype or syndrome based on previously published peer-reviewed literature. Copy Change of Unknown Significance suggests that there is not enough scientific evidence to support that the DNA copy number change as definitively associated with an abnormal phenotype or syndrome. In this case, parental studies (continued on back) CHROMOSOMAL MICROARRAY (CGH+SNP) ChildLab. Trust in the results. 700 Children s Drive, Columbus, Ohio 43205 T 800-934-6575 F 877-722-5478 ChildLab.com CGH+SNP0613

would be recommended and may be necessary to evaluate the inheritance and may help determine the significance of the DNA copy number change observed in the patient. Likely Benign Copy Number Variation implies that information based on literature searches and electronic databases suggest that the DNA copy number change is likely a population polymorphism and is not associated with a given phenotypic finding. Please note: Copy number changes strongly suggestive of benign CNVs (as determined by literature, CNV databases, and frequent observation in the ChildLab Laboratory) are not included in the reports. SNP Analysis The final report will comment on the presence or absence of reportable ROH in each case. ROH greater than or equal to 10 Mb will be reported (but may report down to 8 Mb in regions that encompass imprinting centers or key imprinted genes). Evidence suggestive of a second-degree union or closer (~10-12% genomic homozygosity or greater) will be reported. It is the clinician s responsibility to follow-up on suspicions of parental relatedness. ChildLab reports contain a detailed interpretation of each DNA copy number change and ROH identified. Every effort is made to provide ordering clinicians with up-to-date knowledge of genes contained within a given finding as well as clinical information available in the literature or genomic databases. In addition, abnormal results categorized as Likely Pathogenic and Unknown Significance will be called to the physician by one of the laboratory directors or genetic counselors, who are available at any time to discuss results and recommendations. Turn-Around Time (TAT) Results of the microarray analysis are available within 3-4 weeks, with the TAT dependent on the number of follow-up FISH analyses needed. If specific chromosomal syndromes are suspected and urgent results are needed, verbal preliminary results can be called to ordering clinician upon request. Indications for Chromosomal Microarray Testing Patients with unexplained developmental delay, autism spectrum disorders, mental retardation, dysmorphic features, and/or congenital birth defects. Patients with an abnormal phenotype and/or family history suggestive of a chromosome imbalance, but with previously normal routine chromosome analysis or targeted FISH analysis. Patients with some features of a clinically recognized microdeletion or microduplication syndrome and unusual features. Patients whose phenotype is consistent with an imprinting disorder or other UPD-phenotype. To determine the size, precise breakpoints, and gene content of a chromosomal imbalance previously detected by routine chromosome analysis or FISH analysis. To rule out submicroscopic deletions or duplications in patients with an abnormal phenotype but with an apparently balanced chromosome rearrangement (e.g. translocation, inversion, etc) detected by routine chromosome analysis. This may also be useful for patients with known parental consanguinity, as homozygosity in the genome raises the possibility of recessive diseases. Identifying these regions of homozygosity may aid in determining specific genes of interest. Specimen Requirements Peripheral blood: 4mL EDTA (purple top) and 3mL Sodium Heparin (NaHep, green top) Signed requisition form with clinical indications for study *Microarray analysis with reflex to chromosome analysis is available* Limitations The following are limitations to CGH+SNP technology: Balanced translocations, insertions, and inversions cannot be detected, as these do not generally lead to a gain or loss of genetic information. Chromosome analysis is still required to detect most balanced chromosome rearrangements. Low level mosaicism (<30%) for chromosome imbalances cannot be detected by this analysis. Chromosome and FISH analyses are available if low level mosaicism for a specific chromosome imbalance is suspected. CGH does not provide any information for genomic regions not represented on the microarray (genomic regions where microarray has no DNA probe coverage). Small changes, such as point mutations, within a gene cannot be detected by chromosomal microarray. Some disorders are more commonly caused by mutations in specific genes rather than copy number changes.

Failure to detect evidence of UPD does not exclude the clinical diagnosis of an imprinting associated disorder. There are two reasons for this: 1) UPD may be due to heterodisomy type, which is not detected by the array. 2) Mechanisms other than UPD can cause a given disorder. Failure to detect ROH does not exclude the clinical diagnosis of a recessive disorder. The existence of human polymorphisms in DNA copy number is continuously being investigated. Parental blood samples may be needed for full interpretation of results. Although CGH results confirm the presence of DNA copy number changes, follow-up FISH analyses are performed when possible as another confirmatory testing method and to rule out complex chromosome rearrangements. However, deletions <200 kb and duplications <1 megabases (Mb) are often not visible by FISH analysis due to technical limitations. References 1. Miller DT, et al. Consensus statement: Chromosomal microarray is a first0tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet 86:749-764 (2010). 2. Manning M and Hudgins L. Professional Practice and Guidelines Committee: Array-based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities. Genet Med 12(11):742-745 (2010). For questions regarding test availability, genomic coverage, pre-test considerations, and sample requirements, please contact ChildLab Client Services at (800) 934-6575 and ask to be directed to the Cytogenetics and Molecular Genetics Laboratory.

TECHNICAL BRIEF: CHROMOSOMAL MICROARRAY (CGH+SNP) AT CHILDLAB ChildLab uses a custom-designed Agilent 180k CGH+SNP chromosomal microarray. Chromosomal microarray analysis is designed for the identification of DNA copy number gains and losses associated with chromosomal imbalances, and will detect aneuploidy, deletions, and duplications of the loci represented on the array. This analysis will also detect regions of uniparental isodisomy as well as regions of homozygosity (ROH). The possible clinical significance of ROH relates to two different classes of genetic disorders: disorders of imprinting and recessive diseases. CGH+SNP Platform Our chromosomal microarray offerings have two components: one for copy number analysis (CGH) and one for SNP analysis. The copy-number probes on the array (~135,000) have an average spacing of one probe every 35 kb throughout the genome and one probe every 10 kb in regions known to have clinical significance. The SNP probes (~67,000) have an average spacing of ~49 kb across the genome. This spacing allows us to detect regions of homozygosity (ROH) with a resolution down to 5-10 Mb across the genome. Data Derived From CGH+SNP Microarray Testing DNA copy number differences are evaluated by CGH probes. Any increase or decrease in signal intensity ratios at these spots indicates DNA copy number differences within the patient s genome. This allows simultaneous analysis of multiple genomic regions to detect deletions (losses) or duplications (gains) in the patient s entire genome. This analysis accurately detects copy-number changes below the resolution of florescence in situ hybridization (FISH) technology. Confirmatory FISH testing is performed at the laboratory s discretion and may be used to confirm results or rule-out a more complex chromosomal rearrangement. Data for the ~67,000 SNP probes are displayed to demonstrate either homozygosity (AA or BB) or heterozygosity (AB) at every SNP locus. Long contiguous stretches showing ROH indicate findings of potential clinical significance. ROH is indicative of hemizygosity if the corresponding copy number analysis shows a deletion of the region. If the copy number analysis is normal, the possible clinical significance of these stretches of homozygosity relates to two classes of disorders: 1) those involving imprinted genomic regions and 2) recessive diseases. Evidence suggestive of relatedness of the parents (parental consanguinity) may be revealed. Uniparental Disomy Uniparental disomy (UPD) is the presence of two copies of a chromosome or chromosomal region from a single parent. It is clinically relevant when it involves loci that undergo genomic imprinting. There are two types of UPD: isodisomy, in which the two parental copies are identical; and heterodisomy, in which the parental copies are derived from both parental homologs (and are therefore not identical). CGH+SNP microarrays will detect isodisomy but not heterodisomy since only isodisomy results in homozygosity. UPD is the most likely explanation for a long stretch of homozygosity that is restricted to a single chromosome, especially if the region is very large. Several chromosome regions are imprinted and lead to an abnormal phenotype in the presence of UPD derived from a particular parent (chromosomes 6, 7, 11, 14, 15). UPD in other regions of the genome, even if covering a large region, is generally not considered to be a pathogenic finding in and of itself (unless a recessive disease is unmasked, see discussion below). (continued on back) ChildLab. Trust in the results. 700 Children s Drive, Columbus, Ohio 43205 T 800-934-6575 F 877-722-5478 ChildLab.com TECHCGH+SNP0513

Consanguinity and Identity by Descent (IBD) The observation of multiple long stretches of homozygosity present on multiple chromosomes is generally assumed to reflect inheritance of these regions by descent from a common ancestor. This type of homozygosity is referred to as identity by descent. A single or a few small isolated stretches can be the result of a founder effect in an isolated population. The presence of especially long stretches of homozygosity on multiple chromosomes suggests the possibility of parental consanguinity. A consanguineous relationship refers to the sharing of a common ancestor, and the term consanguinity is generally used when individuals are second cousins or closer. It may also be observed for a distantly related couple who have multiple common ancestors. The latter circumstance may occur, for example, in individuals from an isolated population that arose recently from a small founding group. For the above reasons the SNP data themselves are not diagnostic of a specific degree of relatedness of the parents. It is up to the ordering clinician to decide, based on additional family and social history information, whether it is appropriate to pursue the question of parental consanguinity for individual families. Autosomal Recessive Disease Risk Regardless of whether ROH results from uniparental disomy (UPD) or identity by descent, homozygosity anywhere in the genome raises the possibility of recessive diseases. Our reports alert the clinician to the increased possibility of these conditions for regions of homozygosity greater than 10 Mb. The clinician can use this information in conjunction with clinical features and family history to determine whether mutation testing of individual genes is warranted. Test Limitations Balanced translocations, insertions, and inversions cannot be detected by microarray analysis as these do not generally lead to a gain or loss of genetic information. Chromosome analysis is still required to detect balanced chromosome rearrangements. The failure to detect evidence of UPD does not exclude the clinical diagnosis of an imprinting associated disorder. There are two reasons for this: 1) UPD may be of the heterodisomy type, which is not detected by the array; and 2) for some of the disorders, mechanisms other than UPD can cause the disorder. Failure to detect ROH does not exclude the clinical diagnosis of a recessive disorder. Failure to detect a genomic imbalance in a region associated with a given genetic disorder does not rule out a diagnosis of that disorder. Microarray testing does not provide any information for genomic regions not represented on the microarray (genomic regions where the microarray has no DNA probe coverage). Small changes, such as point mutations, within a gene cannot be detected by microarray analysis. Some disorders are more commonly caused by mutations in specific genes rather than copy number changes. Low level mosaicism (<30%) for chromosome imbalances may not always be detected by this analysis. Chromosome and FISH analyses are available if low level mosaicism for a specific chromosome imbalance is suspected. Results Reporting CGH Analysis Abnormal array CGH findings are reported as genomic gain (duplication) or genomic loss (deletion). ChildLab categorizes each microarray finding as: Likely Pathogenic, Copy Number Change of Unknown Significance, or Likely Benign Copy Number Variation (CNV). Likely Pathogenic refers to a DNA copy number change known to be associated with an abnormal phenotype or syndrome based on previously published peer-reviewed literature. Copy Change of Unknown Significance suggests that there is not enough scientific evidence to support that the DNA copy number change as definitively associated with an abnormal phenotype or syndrome. In this case, parental studies would be recommended and may be necessary to evaluate the inheritance and may help determine the significance of the DNA copy number change observed in the patient. (continued)

Likely Benign Copy Number Variation implies that information based on literature searches and electronic databases suggest that the DNA copy number change is likely a population polymorphism and is not associated with a given phenotypic finding. Please note: Common population variants are found in all individuals. Copy number changes strongly suggestive of benign copy number variants (CNVs), as determined by literature analysis, CNV databases, and frequent observation in our laboratory, are not included in reports. SNP Analysis ROH greater than or equal to 10Mb will be reported (but may report down to 8Mb in regions that encompass imprinting centers or key imprinted genes). The final report will comment on the presence or absence of reportable ROH in each case. Evidence suggestive of a second-degree union or closer (~10-12% genomic homozygosity or greater) will be reported. It is the clinician s responsibility to follow-up on suspicions of parental relatedness. Every effort is made to provide ordering clinicians with a detailed interpretation of each abnormality identified. Parental Samples Parental samples may be requested to help determine the clinical significance of a copy number change (this testing is not free and is performed with charges). Parental samples may be requested to confirm uniparental disomy suggested by the array and to assign the parent of origin. This analysis is not performed on the microarray and may require additional send-out testing. This testing is also not free and is performed with charges. References Kearney HM, Kearney JB, Conlin LK (2011). Diagnostic implications of excessive homozygosity detected by SNPbased microarrays: Consanguinity, uniparental disomy and recessive single-gene mutations. Clin Lab Med 31(4):595-613. Kearney HM et al. (2011). American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants. Genet Med 13:680-685. Kearney HM et al. (2011). American College of Medical Genetics recommendations for the design and performance expectations for clinical genomic copy number micorarrays intended for the use in the postnatal setting for detection of constitutional abnormalities. Genet Med 13:676-679. Manning M and Hudgin L (2007). ACMG Practice Guidelines: Use of array-based technology in the practice of medical genetics. Genet Med 9:650-653. Manning M and Hudgins L (2010). ACMG Practice Guidelines: Array-based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities. Genet Med 12: 742-745. Miller D et al. (2010). Consensus statement: Chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Med Gen 86: 749-764. Shaffer LG, et al. (2007). Working Group of the American College of Medical Genetics Laboratory Quality Assurance Committee of the, ACMG Standards and Guidelines: Microarray analysis for constitutional cytogenetic abnormalities, Genet Med 9:654-662.