aCGH Analysis as a Standard of Care, First-Tier Test
Individuals with Developmental/Intellectual Disabilities or Congenital Anomalies
Manning M and Hudgins L, “Array-based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities” (2010) American College of Medical Genetics Practice Guidelines. http://www.acmg.net/StaticContent/PPG/CMA_2010.pdf
Miller DT et al., “Consensus Statement: Chromosomal Microarray Is a First-Tier Clinical Diagnostic Test for Individuals with Developmental Disabilities or Congenital Anomalies”. 2010. Am J M Hum Genet. 86(5):749-764. PMID: 20466091 (http://www.ncbi.nlm.nih.gov/pubmed/20466091)
ABSTRACT
Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%–20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype (~3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.
Individuals with Autism Spectrum Disorders
Manning M and Hudgins L, “Array-based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities” (2010) American College of Medical Genetics Practice Guidelines. http://www.acmg.net/StaticContent/PPG/CMA_2010.pdf
Shen Y et al., “Clinical Genetic Testing for Patients With Autism Spectrum Disorders”. 2010. Pediatrics. 125(4):e727-735. PMID: 20231187 (http://www.ncbi.nlm.nih.gov/pubmed/20231187)
ABSTRACT
BACKGROUND: Multiple lines of evidence indicate a strong genetic contribution to autism spectrum disorders (ASDs). Current guidelines for clinical genetic testing recommend a G-banded karyotype to detect chromosomal abnormalities and fragile X DNA testing, but guidelinesfor chromosomal microarray analysis have not been established.PATIENTS AND METHODS: A cohort of 933 patients received clinical genetic testing for a diagnosis of ASD between January 2006 and December 2008. Clinical genetic testing included G-banded karyotype, fragile X testing, and chromosomal microarray (CMA) to test for submicroscopic genomic deletions and duplications. Diagnostic yield of clinically significant genetic changes was compared.
RESULTS: Karyotype yielded abnormal results in 19 of 852 patients (2.23% [95% confidence interval (CI): 1.73%–2.73%]), fragile X testing was abnormal in 4 of 861 (0.46% [95% CI: 0.36%– 0.56%]), and CMA identified deletions or duplications in 154 of 848 patients (18.2% [95% CI: 14.76%–21.64%]). CMA results for 59 of 848 patients (7.0% [95% CI: 5.5%– 8.5%]) were considered abnormal, which includes variants associated with known genomic disorders or variants of possible significance. CMA results were normal in 10 of 852 patients (1.2%) with abnormal karyotype due to balanced rearrangements or unidentified marker chromosome. CMA with whole-genome coverage and CMA with targeted genomic regions detected clinically relevant copy-number changes in 7.3% (51 of 697) and 5.3% (8 of 151) of patients, respectively, both higher than karyotype. With the exception of recurrent deletion and duplication of chromosome 16p11.2 and 15q13.2q13.3, most copy number changes were unique or identified in only a small subset of patients.
CONCLUSIONS: CMA had the highest detection rate among clinically available genetic tests for patients with ASD. Interpretation of microarray data is complicated by the presence of both novel and recurrent copy-number variants of unknown significance. Despite these limitations, CMA should be considered as part of the initial diagnostic evaluation of patients with ASD.
Review Articles on aCGH
Bejjani BA and Natowicz MR, “Array-based Cytogenetic Testing: Poised for a Transformative Role in the Lab and Clinical Genetics”. May 2010. Vol 36(5).
http://www.aacc.org/publications/cln/2010/may/Pages/SeriesArticle.aspx
Fruhman G and Van den Veyver IB, “Applications of Array Comparative Genomic Hybridization in Obstetrics”. Obstet Gynecol Clin N Am. 37(1):71-85. PMID: 20494259(http://www.ncbi.nlm.nih.gov/pubmed/20494259)
ACOG Committee Opinion No. 446: array comparative genomic hybridization in prenatal diagnosis. Obstet Gynecol. 2009 Nov; 114(5):1161-3. PMID: 20168129(http://www.ncbi.nlm.nih.gov/pubmed/20168129)
ABSTRACT
The widespread use of array comparative genomic hybridization (CGH) for the diagnosis of genomic rearrangements in children with idiopathic mental retardation, developmental delay, and multiple congenital anomalies has spurred interest in applying array CGH technology to prenatal diagnosis. The use of array CGH technology in prenatal diagnosis is currently limited by several factors, including the inability to detect balanced chromosomal rearrangements, the detection of copy number variations of uncertain clinical significance, and significantly higher costs than conventional karyotype analysis. Although array CGH has distinct advantages over classic cytogenetics in certain applications, the technology is not currently a replacement for classic cytogenetics in prenatal
diagnosis.
Shaffer LG et al., “Comparison of microarray-based detection rates for cytogenetic abnormalities in prenatal and neonatal specimens” Prenat Diagn. 2008. 28: 789–795. PMID: 18646242 (http://www.ncbi.nlm.nih.gov/pubmed/18646242)
ABSTRACT
OBJECTIVE: To compare the detection rate by microarray analysis for chromosome abnormalities in a prenatal population to that of a neonatal population referred for diagnostic testing.METHODS: Array comparative genomic hybridization (aCGH) analysis was performed for 151 prenatal cases and compared with the results from 1375 postnatal cases less than 3 months of age.
RESULTS: Two of 151 prenatal cases (1.3%) showed a clinically significant cytogenetic abnormality. In contrast, of the 1375 postnatal cases studied, 11.4% showed a cytogenetic abnormality by aCGH. Many of these (40%) were referred for aCGH because of dysmorphic features, a clinical indication unlikely to be identified in the prenatal population.
CONCLUSIONS: The chance of detecting a chromosome abnormality in a prenatal population that has already been screened by routine cytogenetics is ∼1.3%. However, given that many of the abnormal array results in the neonatal population were among those with dysmorphic features as the primary indication for testing, which are not easily identifiable by ultrasound, offering prenatal testing by aCGH to a wider population would likely result in a higher detection rate.
Van den Veyver IB et al. “Clinical use of array comparative genomic hybridization (aCGH) for prenatal diagnosis in 300 cases” Prenat Diagn. 2009 Jan;29(1):29-39. PMID: 19012303 (http://www.ncbi.nlm.nih.gov/pubmed/19012303)
ABSTRACT
OBJECTIVE: To evaluate the use of array comparative genomic hybridization (aCGH) for prenatal diagnosis, including assessment of variants of uncertain significance, and the ability to detect abnormalities not detected by karyotype, and vice versa.METHODS: Women undergoing amniocentesis or chorionic villus sampling (CVS) for karyotype were offered aCGH analysis using a targeted microarray. Parental samples were obtained concurrently to exclude maternal cell contamination and determine if copy number variants (CNVs) were de novo, or inherited prior to issuing a report.
RESULTS: We analyzed 300 samples, most were amniotic fluid (82%) and CVS (17%). The most common indications were advanced maternal age (N=123) and abnormal ultrasound findings (N=84). We detected 58 CNVs (19.3%). Of these, 40 (13.3%) were interpreted as likely benign, 15 (5.0%) were of defined pathological significance, while 3 (1.0%) were of uncertain clinical significance. For seven (approximately 2.3% or 1/43), aCGH contributed important new information. For two of these (1% or approximately 1/150), the abnormality would not have been detected without aCGH analysis.
CONCLUSION: Although aCGH-detected benign inherited variants in 13.3% of cases, these did not present major counseling difficulties, and the procedure is an improved diagnostic tool for prenatal detection of chromosomal abnormalities.
Kleeman L et al. “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. PMID: 19862770 (http://www.ncbi.nlm.nih.gov/pubmed/19862770)
ABSTRACT
OBJECTIVE: To prospectively study the addition of array comparative genomic hybridization (CGH) to the prenatal evaluation of fetal structural anomalies.METHODS: Pregnant women carrying fetuses with a major structural abnormality were recruited at the time of invasive procedure for chromosome analysis. Only women whose fetuses had a normal karyotype (n = 50) were subsequently evaluated by array CGH using one of two arrays (1887 clones covering 622 loci or subsequently 4685 clones covering 1500 loci).
RESULTS: The mean gestational age of the fetuses was 24.5 weeks (range 11-38 weeks). The most prevalent anomalies were cardiac, central nervous system, skeletal, and urogenital. The median turnaround time for culturing and array CGH diagnosis was 18 days (range 2-72). Four of 50 fetuses had abnormal array results. One (2%) was clinically significant and three (6%) were inherited or benign variants.
CONCLUSIONS: Array CGH studies in fetuses with sonographic anomalies and normal metaphase karyotype detected clinically significant copy number alterations in 1 of 50 cases. This percentage (2%) is consistent with prior prenatal reports. Further studies are warranted to more precisely identify which fetal anomalies are associated with copy number alterations of clinical significance.
aCGH Analysis of Products of Conception
Scott SA et al., “Detection of low-level mosaicism and placental mosaicism by oligonucleotide array comparative genomic hybridization”. Genet Med. 2010 Feb;12(2):85-92. PMID: 20084009 (http://www.ncbi.nlm.nih.gov/pubmed/20084009)
ABSTRACT
PURPOSE: To determine the sensitivity of whole-genome oligonucleotide array comparative genomic hybridization for the detection of mosaic cytogenetic abnormalities.METHODS: Mosaicism sensitivity was evaluated by testing artificially derived whole chromosome and segmental aneuploidies ranging from 0% to 100% abnormal and additional naturally occurring mosaic specimens.
RESULTS: Using combined dye-reversed replicates and an unfiltered analysis, oligonucleotide array comparative genomic hybridization detected as low as 10% and 20-30% mosaicism from whole chromosome and segmental aneuploidies, respectively. To investigate discrepancies between cultured and uncultured specimens, array comparative genomic hybridization was performed on DNA from additional direct product of conception specimens with abnormal karyotypes in culture. Interestingly, 5 of 10 product of conception specimens with double trisomies on cultured cell analysis had only a single trisomy by array comparative genomic hybridization and quantitative polymerase chain reaction on DNA from the uncultured direct specimen, and all harbored the more commonly observed trisomy. Thus, oligonucleotide array comparative genomic hybridization revealed previously unidentified placental mosaicism in half of the products of conception with double-aneuploid conventional karyotypes.
CONCLUSION: Oligonucleotide array comparative genomic hybridization can detect low-level mosaicism for whole chromosome ( approximately 10%) and segmental ( approximately 20-30%) aneuploidies when using specific detection criteria. In addition, careful interpretation is required when performing array comparative genomic hybridization on DNA isolated from direct specimens as the results may differ from the cultured chromosome analysis.
Menten B et al. “Array comparative genomic hybridization and flow cytometry analysis of spontaneous abortions and mors in utero samples”. BMC Med Genet. 2009 Sep 14;10:89. PMID: 19751515 (http://www.ncbi.nlm.nih.gov/pubmed/19751515)
ABSTRACT
BACKGROUND: It is estimated that 10-15% of all clinically recognised pregnancies result in a spontaneous abortion or miscarriage. Previous studies have indicated that in up to 50% of first trimester miscarriages, chromosomal abnormalities can be identified. For several decades chromosome analysis has been the golden standard to detect these genomic imbalances. A major drawback of this method is the requirement of short term cultures of fetal cells. In this study we evaluated the combined use of array CGH and flow cytometry (FCM), for detection of chromosomal abnormalities, as an alternative for karyotyping.
METHODS: In total 100 spontaneous abortions and mors in utero samples were investigated by karyotyping and array CGH in combination with FCM in order to compare the results for both methods.
RESULTS: Chromosome analysis revealed 17 abnormal karyotypes whereas array CGH in combination with FCM identified 26 aberrations due to the increased test success rate. Karyotyping was unsuccessful in 28% of cases as compared to only two out of hundred samples with inconclusive results for combined array CGH and FCM analysis.
CONCLUSION: This study convincingly shows that array CGH analysis for detection of numerical and segmental imbalances in combination with flow cytometry for detection of ploidy status has a significant higher detection rate for chromosomal abnormalities as compared to karyotyping of miscarriages samples.
Evans MI et al., "Automated microscopy of amniotic fluid cells: detection of FISH signals using the FastFISH imaging system”. Fetal Diagn Ther. 2006;21(6):523-7. PMID: 16969008 (http://www.ncbi.nlm.nih.gov/pubmed/16969008)
ABSTRACT
OBJECTIVE: FISH (fluorescence in situ hybridization) analysis is a valuable adjunct to cytogenetics that provides a rapid screen for common abnormalities. However, FISH is expensive, labor-intensive, and requires a high skill level and subjective signal interpretation. A fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs.METHODS: In this study we blindly compared automated FISH signal acquisition and display against standard FISH analysis. A total of 62 amniocentesis samples were prepared using the AneuVysion multicolor DNA probe kit and probed for chromosomes 13, 18, 21, X, and Y. Two sets of slides were produced from each sample. Fifty cells were scored in each slide. One set was evaluated using standard manual microscopy and the other using the automated image acquisition and display capabilities of the Ikoniscope fastFISH amnio Test System. This system uses epifluorescence optics, along with optimized slide management to process slides automatically.
RESULTS: A 100% concordance was observed between the results obtained using manual microscopy and the automated system. There was also 100% concordance between the FISH results and those obtained by conventional karyotyping.
CONCLUSION: Our data suggest that the automated system is capable of providing accurate and rapid identification and display of cells and FISH signals.