National Medical Policy

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1 National Medical Policy Subject: Policy Number: Single Nucleotide Polymorphism (SNP) Chromosomal Microarray Analysis for Prenatal Testing and Intellectual Disability, Developmental Delay, and Multiple Congenital Anomalies NMP516 Effective Date*: December 2012 Updated: January 2017 This National Medical Policy is subject to the terms in the IMPORTANT NOTICE at the end of this document For Medicaid Plans: Please refer to the appropriate State s Medicaid manual(s), publication(s), citation(s), and documented guidance for coverage criteria and benefit guidelines prior to applying Health Net Medical Policies The Centers for Medicare & Medicaid Services (CMS) For Medicare Advantage members please refer to the following for coverage guidelines first: Use Source Reference/Website Link National Coverage Determination (NCD) National Coverage Manual Citation Local Coverage Determination (LCD)* Article (Local)* X Other Palmetto GBA. MolDX. Molecular Diagnostic Services Program: cscathome/moldx None Use Health Net Policy Instructions Medicare NCDs and National Coverage Manuals apply to ALL Medicare members in ALL regions. Medicare LCDs and Articles apply to members in specific regions. To access your specific region, select the link provided under Reference/Website and follow the search instructions. Enter the topic and your specific state to find the coverage determinations for your region. *Note: Health Net must follow local coverage determinations (LCDs) of Medicare Administration Contractors (MACs) located outside their service area when those MACs have exclusive coverage of an item or service. (CMS Manual Chapter 4 Section 90.2) Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 1

2 If more than one source is checked, you need to access all sources as, on occasion, an LCD or article contains additional coverage information than contained in the NCD or National Coverage Manual. If there is no NCD, National Coverage Manual or region specific LCD/Article, follow the Health Net Hierarchy of Medical Resources for guidance. Current Policy Statement (Please refer the Health Net NMP on Comparative Genomic Hybridization) Health Net Inc. considers chromosomal microarray analysis (CMA), which includes single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) testing, as medically necessary in any of the following situations*: 1. In patients with a fetus with one or more major structural abnormalities identified in ultrasonographic examination and who are undergoing invasive prenatal diagnosis. This test replaces the need for fetal karyotype. 2. In patients with a structurally normal fetus undergoing invasive prenatal diagnostic testing; (In this situation, either fetal karyotyping or a CMA can be performed). 3. In cases of intrauterine fetal demise or stillbirth when further cytogenetic analysis is desired, fetal tissue testing, (i.e., amniotic fluid, placenta, or products of conception) is recommended because of its increased likelihood of obtaining results and improved detection of causative abnormalities. 4. For the diagnostic evaluation of post-natal genetic abnormality in children with developmental delay (DD), intellectual delay (ID), or autism spectrum disorder (ASD) only if the examination is suggestive of an inherited disease or the results will have an impact on the member s subsequent treatment plan, pregnancy planning, or family member genetic counseling. Important Notes: 1. Most genetic mutations identified by chromosomal microarray analysis are not associated with increasing maternal age; therefore, the use of this test for prenatal diagnosis should not be restricted to women aged 35 years and older. 2. Comprehensive patient pretest and posttest genetic counseling from qualified personnel such as a genetic counselor or geneticist regarding the benefits, limitations, and test results, is essential. Not Medically Necessary Health Net, Inc. considers chromosomal microarray analysis (CMA) which includes SNP and CGH, not medically necessary to evaluate first-trimester and secondtrimester pregnancy losses, since limited data are available on the clinical utility of this testing. *Note: Chromosomal microarray analysis should not be ordered without informed consent, which should be documented in the medical record and include discussion of the potential to identify findings of uncertain significance, nonpaternity, consanguinity, and adult-onset disease. (The recommendations noted above are from ACOG, 2013) Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 2

3 Definitions DD Developmental delay ID Intellectual disability MCA Multiple congenital anomalies FISH Fluorescence in situ hybridization acgh Comparative genomic hybridization SNP Single nucleotide polymorphism UPD Uniparental disomy CNV Copy number variants GDD Global developmental delay CMA Cytogenetic microarray Codes Related To This Policy NOTE: The codes listed in this policy are for reference purposes only. Listing of a code in this policy does not imply that the service described by this code is a covered or noncovered health service. Coverage is determined by the benefit documents and medical necessity criteria. This list of codes may not be all inclusive. On October 1, 2015, the ICD-9 code sets used to report medical diagnoses and inpatient procedures have been replaced by ICD-10 code sets. ICD-9 Codes Autistic disorder Mental Retardation 632 Missed abortion Other pregnancy with abortive outcome Chromosomal abnormality in fetus Congenital anomalies Lack of expected normal physiologic development Nonspecific abnormal findings on chromosomal analysis V V27.9 Outcomes of delivery. Single multiple births with stillborn V28.0 V28.9 Encounter for antenatal screening of mother V79.2 Special screening for mental retardation V82.71 V82.79 Genetic screening V82.89 Special screening for other specified conditions ICD-10 Codes F70-F79 F80.0-F89 F84.0-F84.9 P95 Intellectual Disabilities Pervasive and specific developmental disorders Autistic disorder Stillbirth Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 3

4 ICD-10 Codes O35.1XX0- Maternal care for (suspected) chromosomal abnormality in O35.9XX0 fetus, not applicable or unspecified QØØ.Ø-Q07.00 Congenital malformations of the nervous system Q10.0-Q18.9 Congenital malformations of eye, ear, face and neck R62.50-R62.51 Other and unspecified lack of expected normal physiological developments in childhood Z13.4 Encounter for screening for certain developmental disorders in childhood Z13.71-Z13.79 Encounter for screening for genetic and chromosomal anomalies Z36 Encounter for antenatal screening of mother Z37.1-Z37.9 Outcome of delivery. Single stillbirth multiple births. Some live, some stillbirths CPT Codes Cytogenetic constitutional (genome-wide) microarray analysis; Interrogation of genomic regions for copy number variants (eg, Bacterial Artificial Chromosome (BAC) or oligobased comparative genomic hybridization (CGH) microarray analysis Cytogenetic constitutional (genome-wide) microarray analysis; interrogation of genomic regions for copy number and single nucleotide polymorphism (SNP) variants for chromosomal abnormalities. HCPCS Codes N/A N/A Scientific Rationale Update January 2016 Per the American College of Obstetricians and Gynecologists (ACOG, Number 11, 2014), the Technology Assessment if Obstetrics and Gynecology, notes: In contrast to the conventional karyotype, which can detect genetic abnormalities that result from changes in the number or structure of chromosomes, microarray analysis can provide information at the submicroscopic level, demonstrating duplications and deletions of DNA throughout the human genome. DNA microarray technology is a molecular method used to study gene expression. Duplicated or deleted sections of DNA are known as copy number variants; the high resolution of chromosomal microarray analysis enables the detection of copy number variants that are 1/100 the size of those identified by current conventional G-banded karyotyping. It can be used to identify cytogenetic changes in tumors, to characterize subtle unbalanced translocations, to identify the origin of marker or supernumerary chromosomes, and to detect intrachromosomal duplications or deletions. With comparative genomic hybridization (CGH), the fetal DNA is labeled with one color of fluorescent dye, whereas the validated, control DNA is labeled with another color. Arrays of cdna clones are spotted onto a glass slide, known as the array, and hybridized with labeled cdna prepared from the tissue of interest. The more the gene is expressed in the tissue, the more intense the hybridization signal. The relative intensity of the different colors, and therefore relative DNA content of fetal versus control DNA, is compared. Scientific Rationale Update January 2015 A Blue Cross Blue Shield Technology Evaluation Center Special Report: Array Comparative Genomic Hybridization (acgh) for the Genetic Evaluation of Patients Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 4

5 with Developmental Delay/Mental Retardation and Autism Spectrum Disorder, updated October 2014, reported: Clinical utility is determined by the impact of a genetic diagnosis on outcomes that matter to the patient and family. Several outcomes are regarded as important in the setting of DD/MR or ASD, including estimation of recurrence rate for reproductive decision-making, avoidance of additional diagnostic tests and specialist consultations, and early and improved access to behavioral and educational services. However, neither standard cytogenetic analysis nor acgh have been systematically studied for impact on these kinds of outcomes. Rather, clinical utility of genetic testing is primarily inferred based on the value of knowledge to the family, estimation of recurrence risk, and on the importance of early detection and early intervention. In addition, the special report noted that current guidelines for early assessment of DD/MR and for ASD recommend genetic evaluation for those cases that cannot be readily diagnosed from clinical characteristics or other specific tests. Conventional cytogenetic analysis (e.g., G- banded karyotype, specific FISH assays, and subtelomeric screening) has been routinely used for many years, but has low resolution and low diagnostic yield. Array CGH specifically detects CNVs, which account for the majority of genomic abnormalities currently detectable by conventional cytogenetic testing. Array CGH does not detect balanced translocations or inversions, which account for a minority of clinically important genomic abnormalities. However, acgh has shown that a substantial proportion of apparently balanced translocations by conventional methods contain submicroscopic deletions (i.e., are actually unbalanced). CGH arrays have the advantage of greatly improved resolution, which allows detection of smaller, clinically significant genomic abnormalities not detectable by conventional assays (improving diagnostic yield), and more exact locus definition of conventionally detectable abnormalities (improving information for genotypephenotype correlation). While acgh technology is relatively new, the results are conceptually similar to those obtained by conventional methods, and should be evaluated as an extension of those methods. The results of neither conventional cytogenetic evaluation nor of acgh evaluation have been systematically studied for impact on patient outcomes other than diagnostic yield, which is an intermediate outcome. Impact of testing on the kinds of outcomes that matter to the patient and family has been directly addressed in very few studies. Thus, it is not possible to draw evidence-based conclusions regarding the clinical utility of acgh genetic evaluation. The same may also be said of conventional cytogenetic evaluation. Li et al (2014) investigated clinically relevant copy number variations (CNV) in patients with unexplained mental retardation/developmental delay (MR/DD) through chromosomal microarray analysis (CMA). Sixty-six patients with unexplained MR/DD were analyzed with CMA. All identified CNVs were verified with database of genomic variations (DGV), DECIPHER, ISCA and the literature. Nineteen clinically relevant CNVs were detected among the 16 individuals. The diagnostic yield for the MR/DD patients was 24.2%. The most common abnormality was Phelan-McDermid syndrome (3/16, 18.8%), which was followed by Prader-Willi syndrome/angelman syndrome (2/16, 12.5%) and Wolf-Hirschhorn syndrome (2/16, 12.5%). The authors concluded CNVs are one of the most common causes for unexplained MR/DD. CMA can improve the detection rate of CNVs and confer genetic testing with greater sensitivity in elucidating the diagnosis for unexplained MR/DD. Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 5

6 A review by Xu et al (2014) compared the accuracy of prenatal diagnosis for abnormal chromosome diseases by chromosome microarray technology and karyotyping. The studies obtained were filtered by using the QUADAS tool, and studies conforming to the quality standard were fully analyzed. There was one paper conforming to the QUADAS standards including 4406 gravidas with adaptability syndromes of prenatal diagnosis including elderly parturient women, abnormal structure by type-b ultrasound, and other abnormalities. Microarray technology yielded successful diagnoses in 4340 cases (98.8%), and there was no need for tissue culture in 87.9% of the samples. All aneuploids and non-parallel translocations in 4282 cases of non-chimera identified by karyotyping could be detected using microarray analysis technology, whereas parallel translocations and fetal triploids could not be detected by microarray analysis technology. In the samples with normal karyotyping results, type-b ultrasound showed that 6% of chromosomal deficiencies or chromosome duplications could be detected by microarray technology, and the same abnormal chromosomes were detected in 1.7% of elderly parturient women and samples with positive serology screening results. The reviewers concluded in the prenatal diagnosis test, compared with karyotyping, microarray technology could identify the extra cell genetic information with clinical significance, aneuploids, and non-parallel translocations; however, its disadvantage is that it could not identify parallel translocations and triploids. Tobler et al (2014) compared single nucleotide polymorphism (SNP) and comparative genomic hybridization (acgh) microarray platforms to evaluate embryos for parental translocation imbalances and aneuploidy. A retrospective review of preimplantation genetic diagnosis and screening (PGD/PGS) results of 498 embryos from 63 couples undergoing 75 in vitro fertilization cycles due to parental carriers of a reciprocal translocation was performed. There was no significant difference between SNP and acgh microarrays when comparing the prevalence of embryos that were euploid with no translocation imbalance, euploidy with a parental translocation imbalance or aneuploid with or without the parental chromosome imbalance. The clinical pregnancy rates were also equivalent for SNP (60 %) versus acgh (65 %) microarrays. Of 498 diagnosed embryos, 45 % (226/498) were chromosomally normal without translocation errors or aneuploidy, 22 % (112/498) were euploid but had a parentally derived unbalanced chromosomal segregant, 8 % (42/498) harbored both a translocation imbalance and aneuploidy and 24 % (118/498) of embryos were genetically balanced for the parental reciprocal translocation but were aneuploid for other chromosomes. The overall clinical pregnancy rate per IVF cycle following SNP or acgh microarray analysis was 61 % and was higher if the biopsy was done on blastocysts (65 %) versus cleavage stage embryos (59 %), although not statistically significant. The reviews concluded SNP or acgh microarray technologies demonstrate equivalent clinical findings that maximize the pregnancy potential in patients with known parental reciprocal chromosomal translocations. Levy et al (2014) sought to report the full cohort of identifiable anomalies, regardless of known clinical significance, in a large-scale cohort of postmiscarriage products-ofconception samples analyzed using a high-resolution single-nucleotide polymorphism (SNP)-based microarray platform. High-resolution chromosomal microarray analysis allows for the identification of visible and submicroscopic cytogenomic imbalances; the specific use of SNPs permits detection of maternal cell contamination, triploidy, and uniparental disomy. Miscarriage specimens were sent to a single laboratory for cytogenomic analysis. Chromosomal microarray analysis was performed using a SNP-based genotyping microarray platform. Results were evaluated at the cytogenetic and microscopic (greater than 10 Mb) and submicroscopic (less than 10 Mb) levels. Maternal cell contamination was assessed using information derived from fetal and maternal SNPs. Results were obtained on 2,389 of 2,392 specimens (99.9%) that were less than 20 weeks of gestation. Maternal cell contamination was Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 6

7 identified in 528 (22.0%) specimens. The remaining 1,861 specimens were considered to be of true fetal origin. Of these, 1,106 (59.4%) showed classical cytogenetic abnormalities: aneuploidy accounted for 945 (85.4%), triploidy for 114 (10.3%), and structural anomalies or tetraploidy for the remaining 47 (4.2%). Of the 755 (40.6%) cases considered normal at the cytogenetic level, SNP chromosomal microarray analysis revealed a clinically significant copy number change or wholegenome uniparental disomy in 12 (1.6%) and three (0.4%) cases, respectively. The authors concluded chromosomal microarray analysis of products-of-conception specimens yields a high diagnostic return. Using SNPs extends the scope of detectable genomic abnormalities and facilitates reporting "true" fetal results. This supports the use of SNP chromosomal microarray analysis for cytogenomic evaluation of miscarriage specimens when clinically indicated. Romero et al (2014) sought to characterize the proportion and type of genetic abnormalities in early pregnancy losses at different developmental stages; and hypothesized that these rates will differ. Women with pregnancy loss<20weeks gestation (n=86) were enrolled at the time of diagnosis. Maternal tissue without a fetal component was found in 13 samples, and one sample of monochorionic twins was treated as a single sample. An additional 9 samples were excluded from analysis due to 100% maternal cell contamination. Chromosomal microarray analysis (CMA) was performed on 64 samples. These included 15 pre-embryonic (no visible embryo on ultrasound), 31 embryonic (embryo measuring 60/7-96/7 weeks), and 27 fetal (100/7-196/7 weeks) losses. The overall rate of genetic abnormalities differed across developmental stages (9% pre-embryonic; 69.2% embryonic; 33.3% fetal, p<0.01). This difference persisted when comparing pre-embryonic to embryonic (p<0.01) and embryonic to fetal (p=0.02) but not pre-embryonic to fetal (p=0.12). The rate of aneuploidy also differed significantly across developmental stages (0% pre-embryonic vs 69.2% embryonic vs 25.9% fetal, p<0.001). Abnormalities were most common in embryonic cases, followed by fetal and then pre-embryonic. Maternal cell contamination (MCC) was noted in 47% of 46, XX cases assessed. The authors concluded genetic abnormalities detected by CMA are more likely to occur in the embryonic period compared to pre-embryonic or fetal stages. MCC is common in early pregnancy loss and should be excluded when results demonstrate 46,XX. Scientific Rationale Update January 2014 Chromosomal microarray analysis (CMA) which includes both comparative genetic hybridization and single nucleotide polymorphism arrays, have been shown in studies to have a higher diagnostic efficiency compared to routine chromosomal analysis for the identification of chromosomal aberrations. CMA has replaced florescence in situ hybridization based methods since it is efficient in identifying smaller rearrangements at FISH level of resolution. Therefore, CMA has been recommended by expert panels as the first tier testing for individuals with developmental delay, intellectual disability and/or multiple congenital anomalies in cases where there is no evidence of recognizable genetic syndromes. The American College of Medical Genetics (ACMG 2013 revisions) notes the following: Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 7

8 CMA has emerged as a powerful tool for clinical genetic testing. Currently, two methods of CMA are used in the clinical setting. Array-comparative genomic hybridization and single-nucleotide polymorphism arrays use different techniques to scan the genome for copy-number variants (CNVs). With the increased number of CMA studies, new information has emerged regarding the contribution of genome CNVs in autism spectrum disorders (ASDs). Estimates of CNV frequencies in unselected populations of individuals with an ASD are from 8 to 21%. Given this diagnostic yield, we have moved CMA to a first-tier test in place of a karyotype, as suggested in the 2008 guidelines. This is in keeping with recent consensus opinion that CMA is a first-tier test for individuals with developmental disabilities or congenital anomalies. Scientific Rationale Update December 2013 Genomic disorders are diseases that result from the loss or gain of chromosomal or DNA material. The most common genomic disorders are divided in two main categories, those resulting from copy number losses (i.e., deletion syndromes) and copy number gains (i.e., duplication syndromes). Chromosomal microarray analysis (CMA) has emerged as a powerful new tool to identify genomic abnormalities associated with a wide range of developmental disabilities including congenital malformations, cognitive impairment, and behavioral abnormalities. At this time, two main types of microarray-based chromosome tests or CMA are available for use in clinical diagnostics, array-based comparative genomic hybridization (acgh), also known as chromosome microarray or microarray-based comparative genomic hybridization, and single nucleotide polymorphism (SNP) microarray analysis or testing. These two tests significantly increase the detection rate in both prenatal and postnatal cases. Array comparative genomic hybridization (array CGH), also known as chromosome microarray or microarray-based comparative genomic hybridization, is the gold standard laboratory test for the detection of copy number variants (CNVs) that cause genomic disorders. The frequency of disease-causing CNVs is highest (20%-25%) in children with moderate to severe intellectual disability accompanied by malformations or dysmorphic features. Disease-causing CNVs are found in 5%-10% of cases of autism, being more frequent in severe phenotypes. CMA has replaced Giemsabanded karyotype as the first-tier test for genetic evaluation of children with developmental and behavioral disabilities. CMA is also known as molecular karyotyping, microarray-based genomic copy-number analysis or array-based comparative genomic hybridization (acgh). The use of CMA leads to a genetic diagnosis in 15 to 20% of patients with unexplained ID, which is substantially higher than chromosomal karyotyping. The higher yield of CMA is primarily because of its higher sensitivity for submicroscopic deletions and duplications. CMA provides much higher resolution than chromosomal karyotyping, identifying more genetic conditions, including single nucleotide polymorphisms (SNP). Chromosome abnormalities are a common cause of developmental delay (DD), intellectual disability (ID), multiple congenital anomalies (MCA), and other neurodevelopmental disorders. However, traditional cytogenetic techniques, such as karyotype analysis, will identify a chromosome abnormality in fewer than 10% of individuals with clinical features suggestive of a genetic syndrome. Standard Karyotype analysis identifies a chromosome abnormality in only 5% -10% of fetuses undergoing prenatal testing because of an increased risk of a chromosome disorder. Intellectual disability (ID) is a term synonymous with and is now preferred over the older term, mental retardation. It is a static encephalopathy with multiple etiologies Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 8

9 that encompasses a broad spectrum of functioning, disability, and strengths. The term global developmental delay is usually used to describe children younger than age five with significant cognitive deficits, because intelligence quotient (IQ) testing is less reliable in this age group; the cognitive deficits may be accompanied by other developmental deficits as well. In 2007 the American Association on Mental Retardation (AAMR) changed its name to the American Association on Intellectual and Developmental Disabilities (AAIDD). The AAIDD encourages the use of the term "intellectual disability" in place of "mental retardation" but stresses that the terms are synonymous. Some laboratories incorporate specific algorithms in their processing to better detect triploidy, either through the use of single nucleotide polymorphisms (SNP) arrays or through an initial assessment of a limited number of cells by fluorescence in situ hybridization (FISH). The American Academy of Neurology (AAN, 2013), completed a policy on Chromosomal Microarray Analysis for Intellectual Disabilities. It notes that the chromosomal microarray is able to detect copy number variants with much finer resolution and is not reliant on staining and visual resolution limits. The modern chromosomal microarray instead uses oligonucleotides of about 80 bases in length to target specific, matching regions of the genome. Absent or duplicated targets, or regions of the subjects DNA are thereby easily detected. These more refined tests have made it possible to routinely detect smaller deletions and duplications (CNVs) of less than 50 thousand bases. Other than a few very specific circumstances, microarrays are far superior to the traditional Kayrotype in sensitivity and diagnostic yield. Chromosomal microarray testing is technically referred to as the array comparative genomic hybridization (acgh). Patients with intellectual disabilities (ID) and dysmorphic features are the potential beneficiaries from the results of CMA testing. Per Pivalizza, (UpToDate, 2013) The optimal order or timing of tests to identify the etiology of intellectual disability (ID) is not certain. The staged approach and assessments recommended by the practice parameter of the American Academy of Neurology and the Child Neurology Society for the evaluation of the child with global developmental delay is noted below: Complete history and physical examination Perform comprehensive developmental assessment, including ntellectual and adaptive testing Complete vision and auditory screening Review newborn screening results Blood lead screening Perform a chromosomal microarray (CMA, also known as genomic microarray analysis) in all children with ID, unless a specific syndrome is suspected based on phenotypic characteristics and diagnosed by specific testing. (If CMA is not available, then a G-banded karyotype is an appropriate substitute). Blue Cross, Blue Shield Association, Technology Evaluation Center (TEC, 2009) completed an executive summary on a Special Report: Array Comparative Genomic Hybridization (acgh) for the Genetic Evaluation of Patients with Developmental Delay/Mental Retardation and Autism Spectrum Disorder. It notes the following: Authors comments and conclusions: Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 9

10 Current guidelines for early assessment of DD/MR and for ASD recommend genetic evaluation for those cases that cannot be readily diagnosed from clinical characteristics or other specific tests. Array CGH specifically detects CNVs, which account for the majority of genomic abnormalities currently detectable by conventional cytogenetic testing. Array CGH does not detect balanced translocations or inversions, which account for a minority of clinically important genomic abnormalities. CGH arrays have the advantage of greatly improved resolution, which allows detection of smaller, clinically significant genomic abnormalities not detectable by conventional assays (improving diagnostic yield), and more exact locus definition of conventionally detectable abnormalities (improving information for genotype-phenotype correlation). While acgh technology is relatively new, the results are conceptually similar to those obtained by conventional methods, and should be evaluated as an extension of those methods. The results of neither conventional cytogenetic evaluation nor of acgh evaluation have been systematically studied for impact on patient outcomes other than diagnostic yield, which is an intermediate outcome. Impact of testing on the kinds of outcomes that matter to the patient and family has been directly addressed in very few studies. Thus, it is not possible to draw evidence-based conclusions regarding the clinical utility of acgh genetic evaluation. The same may also be said of conventional cytogenetic evaluation. Expert consensus and clinical guidelines state that genetic information is of value because it establishes a causal explanation that is helpful to families. It is suggested that such genetic information avoids additional consultations and various types of diagnostic tests, assists with early and improved access to community services that may ameliorate or improve behavioral and cognitive outcomes, provides estimates of recurrence rates to better guide reproductive decision-making, and enables an understanding of prognosis and future needs. However, little evidence supports these outcomes. Array CGH technology is rapidly evolving and different kinds of arrays with different capabilities of detecting genomic abnormalities are clinically available from different laboratories; it is up to the ordering physician to know the limits of the particular technology employed by the laboratory. Some have called for broader efforts to standardize protocols, define quality criteria for successful analysis, and develop reporting guidelines; in addition, a national multicenter trial to address accuracy, indications, and efficacy has been suggested. Currently, a consortium of scientists from academic cytogenetics laboratories have agreed to develop a uniform, evidence-based Molecular Karyotype and shared national database to accumulate data on pathogenic versus benign deletions and duplications in the human genome. Such cooperative efforts should lead to more comparable results across platforms, more complete databases to aid in individual results interpretation, more uniform reporting, and more rapid accumulation of genotype-phenotype correlation information for future reference. Hochstenbach et al. (2009) Anomalies of chromosome number and structure are considered to be the most frequent cause of unexplained, non-syndromic developmental delay and mental retardation (DD/MR). High-resolution, genomewide, array-based segmental aneusomy profiling has emerged as a highly sensitive technique for detecting pathogenic genomic imbalances. A retrospective review of 29 Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 10

11 array-based studies of 36,325 patients with idiopathic developmental delay showed that a yield of at least approximately 19% pathogenic aberrations is attainable in unselected, consecutive DD/MR referrals if array platforms with kb median probe spacing are used as an initial genetic testing method. This corresponds to roughly twice the rate of classical cytogenetics. This raises the question whether chromosome banding studies, combined with targeted approaches, such as fluorescence in situ hybridisation for the detection of microdeletions, still hold substantial relevance for the clinical investigation of these patients. To address this question, the authors reviewed the outcome of cytogenetic studies in all the DD/MR referrals in the Netherlands during the period , a period before the advent of array-based genome investigation. The authors estimate that in a minimum of 0.78% of all referrals a balanced chromosomal rearrangement would have remained undetected by array-based investigation. These include familial rearrangements (0.48% of all referrals), de novo reciprocal translocations and inversions (0.23% of all referrals), de novo Robertsonian translocations (0.04% of all referrals), and 69,XXX triploidy (0.03% of all referrals). We conclude that karyotyping, following an initial array-based investigation, would give only a limited increase in the number of pathogenic abnormalities, i.e. 0.23% of all referrals with a de novo, apparently balanced, reciprocal translocation or inversion (assuming that all of these are pathogenic), and 0.03% of all referrals with 69,XXX triploidy. The authors propose that, because of its high diagnostic yield, high-resolution array-based genome investigation should be the first investigation performed in cases of DD/MR, detecting>99% of all pathogenic abnormalities. However, laboratories that supplant karyotyping by array-based investigation should be aware that, as shown here, a chromosomal abnormality, with possible pathogenic consequences for the patient or the family, will escape detection in about 0.78% of all DD/MR referrals. Miller et al. (2010) 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. The authors 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 ( approximately 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and lowlevel 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. Chromosomal Microarray Analysis in Prenatal Diagnosis Per the American College of Obstetricians and Gynecologists (ACOG), Committee Opinion. The Use of Chromosomal Microarray Analysis in Prenatal Diagnosis. Number 581. December The College and the Society for Maternal-Fetal Medicine offer the following recommendations for the use of chromosomal microarray analysis in prenatal diagnosis: Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 11

12 In patients with a fetus with one or more major structural abnormalities identified on ultrasonographic examination and who are undergoing invasive prenatal diagnosis, chromosomal microarray analysis is recommended. This test replaces the need for fetal karyotype. In patients with a structurally normal fetus undergoing invasive prenatal diagnostic testing, either fetal karyotyping or a chromosomal microarray analysis can be performed. Most genetic mutations identified by chromosomal microarray analysis are not associated with increasing maternal age; therefore, the use of this test for prenatal diagnosis should not be restricted to women aged 35 years and older. In cases of intrauterine fetal demise or stillbirth when further cytogenetic analysis is desired, chromosomal microarray analysis on fetal tissue (ie, amniotic fluid, placenta, or products of conception) is recommended because of its increased likelihood of obtaining results and improved detection of causative abnormalities. Limited data are available on the clinical utility of chromosomal microarray analysis to evaluate first-trimester and second-trimester pregnancy losses; therefore, this is not recommended at this time. Comprehensive patient pretest and posttest genetic counseling from qualified personnel such as a genetic counselor or geneticist regarding the benefits, limitations, and results of chromosomal microarray analysis is essential. Chromosomal microarray analysis should not be ordered without informed consent, which should be documented in the medical record and include discussion of the potential to identify findings of uncertain significance, nonpaternity, consanguinity, and adult-onset disease. Wapner et al. (2012) Chromosomal microarray analysis has emerged as a primary diagnostic tool for the evaluation of developmental delay and structural malformations in children. The authors aimed to evaluate the accuracy, efficacy, and incremental yield of chromosomal microarray analysis as compared with karyotyping for routine prenatal diagnosis. Samples from women undergoing prenatal diagnosis at 29 centers were sent to a central karyotyping laboratory. Each sample was split in two; standard karyotyping was performed on one portion and the other was sent to one of four laboratories for chromosomal microarray. The authors enrolled a total of 4406 women. Indications for prenatal diagnosis were advanced maternal age (46.6%), abnormal result on Down's syndrome screening (18.8%), structural anomalies on ultrasonography (25.2%), and other indications (9.4%). In 4340 (98.8%) of the fetal samples, microarray analysis was successful; 87.9% of samples could be used without tissue culture. Microarray analysis of the 4282 nonmosaic samples identified all the aneuploidies and unbalanced rearrangements identified on karyotyping but did not identify balanced translocations and fetal triploidy. In samples with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 6.0% with a structural anomaly and in 1.7% of those whose indications were advanced maternal age or positive screening results. In the context of prenatal diagnostic testing, chromosomal microarray analysis identified additional, clinically significant cytogenetic information as compared with karyotyping and was equally efficacious in identifying aneuploidies and unbalanced rearrangements but did not identify balanced translocations and triploidies. (Funded Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 12

13 by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT ). Scientific Rationale Initial Chromosome abnormalities are a well-established cause of congenital anomalies, dysmorphic features, developmental delay (DD), intellectual disability (ID), and other neurodevelopmental disorders. However, less than 10% of individuals with DD, ID, and/or multiple congenital anomalies (MCA) will have abnormalities detectable by conventional karyotype analysis or fluorescence in situ hybridization (FISH). Laboratory evaluation of patients with DD/ID, congenital anomalies, and dysmorphic features has changed significantly in the last several years with the introduction of microarray technologies [array based comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) array analysis] for use in clinical diagnostics. With these techniques, a patient s genome is examined for detection of gains or losses of genetic material that typically are too small to be detectable by standard G- banded chromosome studies. acgh is able to detect much smaller chromosomal imbalances than standard karyotype and FISH analyses. acgh involves comparing the genomes of two individuals (the patient and a normal control). Chromosomal imbalances are identified through hybridization to a microarray containing thousands of DNA segments (i.e., bacterial artificial chromosome [BAC] or oligonucleotide probes) representing various regions throughout the genome. The microarray may be targeted in nature, assaying certain regions of the genome known to be associated with a specific syndrome or phenotype, or may be genome-wide. The microarrays vary by the number, size, and distribution of the probes, or targets, used. The use of a large number of small probes (oligonucleotides) that are more closely spaced leads to increased resolution and the ability to detect smaller abnormalities. Because of this increased resolution, acgh will identify chromosomal imbalances in approximately 10% of patients with a suspected chromosome disorder who have a normal karyotype by conventional cytogenetics. SNP microarrays are DNA sequence variations that occur when a single nucleotide in the genome sequence is altered. SNPs are sequence variants in which a single base pair differs from a specified reference sequence. For each SNP, a person generally has two alleles, one inherited from each parent. The absence of one allele in multiple contiguous SNPs indicates the presence of a chromosomal deletion, while an increase in SNP copy number indicates the presence of a chromosomal duplication. In addition to identifying genomic imbalances, SNP microarrays can detect copy number neutral abnormalities. Specifically, SNP microarrays may reveal long stretches of homozygosity that can indicate uniparental disomy (UPD) ( i.e., when both copies of a chromosome or chromosomal segment are inherited from a single parent) or possible consanguinity (i.e.parents related by blood). These copy number neutral abnormalities are associated with an increased chance of autosomal recessive conditions. The resolution of SNP microarrays is determined by the number of probes, their distribution or spacing, and by the statistical algorithms used to identify gains and losses (which differ among the various statistical software packages). SNP microarray testing involves hybridizing patient DNA to the microarray and comparing results with those obtained from a given set of control samples. A variety of software programs are used in the processing of microarray data. The resolution of SNP microarrays is determined by the length and spacing between probes and by the statistical algorithms used to identify gains and losses (which differ between the various statistical software packages available for analysis). Like acgh, SNP Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 13

14 microarrays offer a cytogenetic evaluation at a significantly higher resolution than a standard karyotype analysis, as well as the ability to look for genomic imbalances throughout the genome in a single assay. In addition, both types of microarray analysis may be used to evaluate apparently balanced translocations, as up to 40% of these anomalies are found to have submicroscopic imbalances at rearrangement breakpoints. The main issue with microarray testing, including SNP microarray analysis, is the identification of copy number variants (CNVs) of unknown clinical significance. CNVs are segments of DNA longer than 1 kilobase that differ in copy number from a reference genome. In addition, the microarrays will not detect balanced chromosome rearrangements (i.e., balanced translocations or inversions) or imbalances not covered by the probes on the microarray. SNP microarray testing has been investigated in patients with DD, ID, MCA, dysmorphic features, and/or related phenotypes. In addition, prenatal SNP microarray testing may be offered in cases of a suspected fetal chromosome abnormality. Testing may also be performed in the family members of individuals with previously identified chromosomal imbalances, and may be used for preimplantation genetic diagnosis. It has been suggested that establishing a clinical diagnosis by microarray analysis may allow for improved prognostication, a better assessment of treatment options, enhanced anticipatory guidance, a more accurate risk assessment for the patient and his or her family, and the avoidance of unnecessary tests and evaluations. However, no studies designed to evaluate the clinical utility of SNP microarray testing have been identified. SNP microarray testing is limited as the results of SNP microarray testing is dependant on the methods of data analysis, and currently there is no standard of practice when it comes to testing patients using this type of microarray. In addition, the identification of CNVs of unknown clinical significance poses a significant problem for SNP microarray testing. While CNV databases now exist and are gathering more data to facilitate the interpretation of such variants, it is evident that, at the present time, not all CNVs can be classified as pathogenic or benign. Recommendations from the ACMG include the following (Nov 2010): 1. CMA testing for CNV is recommended as a first-line test in the initial postnatal evaluation of individuals with the following: Multiple anomalies not specific to a well-delineated genetic syndrome. Apparently nonsyndromic developmental delay/intellectual disability (DD/ID). Autism spectrum disorders. 2. Further determination of the use of CMA testing for the evaluation of the child with growth retardation, speech delay, and other less well-studied indications is recommended, particularly by prospective studies and aftermarket analysis. 3. Appropriate follow-up is recommended in cases of chromosome imbalance identified by CMA, to include cytogenetic/fish studies of the patient, parental evaluation, and clinical genetic evaluation and counseling. In October 2011, the American Academy of Pediatrics endorsed the following publication, Evidence report: genetic and metabolic testing on children with global developmental delay: report of the Quality Standards Subcommittee of the American Academy of Neurology and the Practice Committee of the Child Neurology Society (Michelson et al 2011.) The report was a review of the evidence concerning the diagnostic yield of genetic and metabolic evaluation of children with global developmental delay or intellectual disability (GDD/ID). Per the report, In patients with GDD/ID, microarray testing is diagnostic on average in 7.8% (Class III), G- banded karyotyping is abnormal in at least 4% (Class II and III), and subtelomeric Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 14

15 fluorescence in situ hybridization is positive in 3.5% (Class I, II, and III). Testing for X-linked ID genes has a yield of up to 42% in males with an appropriate family history (Class III). FMR1 testing shows full expansion in at least 2%of patients with mild to moderate GDD/ID (Class II and III), and MeCP2 testing is diagnostic in 1.5% of females with moderate to severe GDD/ID (Class III). Tests for metabolic disorders have a yield of up to 5%, and tests for congenital disorders of glycosylation and cerebral creatine disorders have yields of up to 2.8% (Class III). Several genetic and metabolic screening tests have been shown to have a better than 1% diagnostic yield in selected populations of children with GDD/ID. These values should be among the many factors considered in planning the laboratory evaluation of such children. The report notes further, An etiologic diagnosis for GDD or ID only occasionally leads to a specific therapy that improves the child s outcome; however, it often leads to other benefits for the child and the child s family. These benefits include relieving caregivers of anxiety and uncertainty, empowering caregivers to become involved in support and research networks, limiting further diagnostic testing that may be costly or invasive, improving understanding of treatment and prognosis, anticipating and managing associated medical and behavioral comorbidities, allowing for counseling regarding recurrence risk, and preventing recurrence through screening for carriers and prenatal testing. Per the review, Microarray is the genetic test with the highest diagnostic yield in children with unexplained GDD/ID. The resolution of the current generation of commercially available, genome-wide, oligonucleotide-based microarray testing is 700 base pairs, 30 to 40 times higher than the oligo-based tests previously used in studies of GDD/ID and 1,000 times higher than older BAC-based microarrays. Laboratories now offer SNP microarray that detects and describes consanguinity or uniparental disomy. Studies on the yield of these more advanced microarray tests are anticipated in the near future. Currently, microarray testing can identify only unbalanced copy number changes and is insufficiently sensitive for detecting genetic disorders caused by inversions, balanced insertions, reciprocal translocations, polyploidy, low-level mosaicism (20% 25%), rearrangements in repeat sequences, point mutations, or duplications/deletions that are undetectable at the test s resolution level. The results of microarray testing are often complex and require confirmation and careful interpretation, often with the assistance of a medical geneticist. Faas et al (2012) evaluated both clinical and laboratory aspects of our new strategy offering quantitative fluorescence (QF)-PCR followed by non-targeted whole genome 250K single-nucleotide polymorphism array analysis instead of routine karyotyping for prenatal diagnosis of fetuses with structural anomalies. Upon the detection of structural fetal anomalies, parents were offered a choice between QF-PCR and 250K single-nucleotide polymorphism array analysis (QF/array) or QF-PCR and routine karyotyping (QF/karyo). Two hundred twenty fetal samples were included. In 153/220 cases (70%), QF/array analysis was requested. In 35/153 (23%), an abnormal QF-PCR result was found. The remaining samples were analyzed by array, which revealed clinically relevant aberrations, including two known microdeletions, in 5/118 cases. Inherited copy number variants were detected in 11/118 fetuses, copy number variants with uncertain clinical relevance in 3/118 and homozygous stretches in 2/118. In 67/220 (30%) fetuses, QF/karyo was requested: 23/67 (34%) were abnormal with QF-PCR, and in 3/67, an abnormal karyotype was found. Investigators concluded even though QF/array does not reveal a high percentage of submicroscopic aberrations in fetuses with unselected structural anomalies, it is preferred over QF/karyo, as it provides a whole genome scan at high resolution, without additional tests needed and with a low chance on findings not related to the ultrasound anomalies. Single Nucleotide Polymorphism Chromosomal Microarray Analysis Jan 17 15