Special Report: Array Comparative Genomic Hybridization (aCGH) for the Genetic Evaluation of Patients with Developmental Delay/Mental Retardation and Autism Spectrum Disorder
Background: Children with signs of neurodevelopmental delays or disorders in the first few years of life may eventually be diagnosed with mental retardation or autism syndromes, serious and lifelong conditions that present significant challenges to families and to public health. A large proportion of cases of developmental delay/mental retardation and of autism are associated with genetic abnormalities. Current guidelines for these patients recommend cytogenetic evaluation to look for certain kinds of chromosomal abnormalities that may be causally related to their condition. Most commonly these abnormalities are deletions and duplications of large segments of genomic material, which are called “copy number variants,” or CNVs. For many well-described syndromes, the type and location of the chromosomal abnormality has been established with the study of a large number of cases and constitutes a genetic diagnosis; for others, only a small number of patients with similar abnormalities may exist to support a genotype-phenotype correlation. Finally, for some patients, cytogenetic analysis will discover entirely new chromosomal abnormalities that will require additional study to determine their significance.
Conventional methods of cytogenetic analysis, including karyotyping (e.g., G-banded) and fluorescence in situ hybridization (FISH), have relatively low resolution and a low diagnostic yield (i.e., proportion of tested patients with clinically relevant genomic abnormalities), leaving the majority of cases without identification of a chromosomal abnormality associated with the child’s condition. Array comparative genomic hybridization (aCGH) is a newer cytogenetic analysis method that increases the chromosomal resolution for detection of CNVs, and, as a result, increases the diagnostic yield and the genomic detail beyond that of conventional methods. Array CGH results are clinically informative in the same way as results derived from conventional methods and thus aCGH represents an extension of standard methods with increased resolution. Array CGH may be ordered when conventional results are negative, although some believe it will soon replace conventional technology.
There are two types of CGH arrays. The first kind, targeted CGH arrays, provide high-resolution coverage of the genome primarily in areas containing known, clinically significant CNVs. The second kind, whole-genome arrays, provide high resolution coverage of the entire genome. Thus, in addition to detecting known CNVs like targeted arrays, whole genome arrays also promote discovery of new CNVs. Such discoveries have resulted in the characterization of several new genetic syndromes by aCGH, with other potential candidates currently under study. However, the whole-genome arrays also have the disadvantage of potentially high numbers of apparent false-positive results, because benign CNVs are found in phenotypically normal populations; both benign and pathogenic CNVs are continuously cataloged and made available in public reference databases to aid in clinical interpretation. Additionally, some new CNVs are neither known to be benign nor causal; these CNVs may require detailed family history and family genetic testing to determine clinical significance, and/or may require confirmation by subsequent accumulation of similar cases and so, for a time, may be considered a CNV of undetermined significance (some may eventually be confirmed true positives or causal, others false positives or benign).
This Special Report will summarize the current status of aCGH, including what is known about how the technology improves diagnostic yield and outcomes in patients with clinically diagnosed developmental disability/mental retardation (DD/MR) and/or autism spectrum disorder (ASD), but normal results by conventional cytogenetic evaluation.
Search strategy: An initial MEDLINE® (via PubMed) search strategy of ("Developmental Disabilities"[MeSH®] OR "Mental Retardation"[MeSH®] OR "Autistic Disorder"[MeSH®]) AND ("Genetic Screening"[MeSH®] OR "Genetic Predisposition to Disease"[MeSH®] OR "Genetic Phenomena"[MeSH®] OR "Genetic Techniques"[MeSH®]), limited to human subjects, English language, and published within the last 3 years, yielded 2,172 citations. Specific searches for studies of the clinical outcomes of cytogenetic testing in DD/MR and ASD were also conducted. Relevant, earlier articles were selected from bibliographies of recent publications.
Selection criteria: Included studies were case reports, case series, or cohort studies published in peer-reviewed journals that enrolled patients with clinical diagnoses of developmental delay/mental retardation or of autism with known or suspicion of genetic abnormalities but negative results by conventional cytogenetic evaluation, and that conducted molecular karyotyping by aCGH on enrolled patients. Studies were also included if they examined management decisions and/or patient outcomes based on genetic evaluation results, either by conventional methods or by molecular karyotyping.
Question 1A. What is the clinical sensitivity of molecular karyotyping by aCGH for known chromosomal abnormalities? What is the false-positive rate in normal controls? (Clinical Validity) Several studies have conducted aCGH on samples with known chromosomal abnormalities by standard karyotyping. In general, currently available aCGH clinical services using higher-resolution bacterial artificial chromosome (BAC) arrays or high-resolution oligo CGH arrays achieve 100% sensitivity for known chromosomal abnormalities. False-positive rates (i.e., CNVs of undetermined clinical significance) were inconsistently reported and are not summarized here. However, it should be noted that as array resolution increases, the likelihood of indeterminate CNV results also increases. This is somewhat mitigated by the continual addition of new abnormalities, as well as new benign polymorphic CNVs to public databases available for consultation.
Question 1B. What is the diagnostic yield of molecular karyotyping by aCGH for patients suspected of having a chromosomal abnormality, but who are negative by conventional cytogenetic evaluation? (Clinical Validity) Several studies reported the diagnostic yield (i.e., proportion of patients with clinically relevant genomic abnormalities) of aCGH in patients with normal standard karyotype. Overall, diagnostic yield ranged from 5–16.7% in DD/MR patients and from 3.4–11.6% in patients with ASD. Although seemingly small, these yields represent significant improvements in identifying a genetic etiology for these patients. Based on the diagnostic yields reported, the number needed to test (NNT) by aCGH to detect 1 clinically relevant abnormality ranges from 6 to 25. The estimate depends on the resolution of the array used and on the stringency of the patient selection criteria.
Increasing array resolution is accompanied by increasing potential for CNVs of uncertain clinical relevance. To determine clinical relevance in individual cases, the following actions are taken:
- CNVs are confirmed by another laboratory method.
- CNVs detected are checked against public databases of benign and pathogenic CNVs.
- The laboratory may establish a size cutoff (e.g., 500 kb to 2 Mb); potentially pathogenic CNVs are likely to be larger than benign polymorphic CNVs.
- Parental studies are indicated when CNVs of appropriate size are detected and not found in available databases; CNVs inherited from a clinically normal parent are assumed to be benign, whereas those appearing de novo are likely pathogenic.
The final genetic assessment may not be definitive in all cases, but instead may report a CNV of undetermined significance. An attempt was made to abstract the rate of aCGH results requiring parental testing and of CNVs of undetermined significance for the studies included for this question; however, reporting was often unclear and dissimilar across studies and comparable rates could not be determined.
Question 2. How are management decisions changed and outcomes improved by the results of molecular karyotyping by aCGH? (Clinical Utility) 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.
A few published studies address aspects of clinical utility. The authors of one study interviewed 14 physicians (2 neurologists, 12 medical geneticists) regarding management changes as a result of positive aCGH testing results from the University of Utah Cytogenetics Laboratory for 48 patients with developmental delay or mental retardation and normal karyotypes. Only 29 percent of patients had no management changes reported. For significant proportions of patients, the diagnostic odyssey was ended, recurrence risk evaluation was possible in about one-third of families and patients had improved access to services. However, this study was only a survey and did not attempt to quantitate the diagnostic tests avoided, determine the impact of improved access to services on patient outcomes, or the effect of recurrence risk on reproductive planning. Therefore the results are only suggestive of, but do not confirm, clinical utility.
The authors of another published report studied the reproductive decisions of women from 38 families with X-linked mental retardation. Most of the women in these families spent many years knowing that they were at some risk of being carriers and of having a boy with MR. Prior to the availability of pathogenic mutation analysis, the birth rate for these families was well below average for the district (United Kingdom-New South Wales). After pathogenic mutation status was determined, both carriers and noncarriers (previously thought to be at risk) of the mutation had children at same rate with 74% of carriers choosing prenatal genetic evaluation. While the results of this study are suggestive, they do not show that knowledge of recurrence risk directly affected reproductive decisions.
A recent review of studies of early intervention for autism notes that while the evidence suggests that early intervention programs are beneficial, the evidence is generally not of the highest quality and whether early improvements in behavior and reductions in symptom severity lead to greater independence and social functioning in adulthood is unknown. Few randomized trials have been conducted and described interventions differ considerably, indicating that the field is still early in researching the elements of effective early intervention. Nevertheless, guidelines such as those published by the American Academy of Pediatrics and the American Academy of Neurology emphasize the importance of early diagnosis and intervention.
For DD/MR or ASD syndromes diagnosed by aCGH, it may be possible to predict the patient prognosis and the elements of care that will be most important to improving outcomes. However, the outcomes of prognosis and treatment planning have not been systematically studied.
Question 3. What is the evidence that the technical performance of clinically available aCGH assays accurately detects clinically significant CNVs and any other chromosomal abnormalities included in the array, and perform in a robust manner over time? (Analytic Validity) Molecular karyotyping is commercially available from several Clinical Laboratory Improvements Amendments (CLIA) -licensed laboratories as a laboratory-developed test (LDT). U.S. Food and Drug Administration (FDA) clearance for marketing is not required for LDTs; none of the commercially offered tests has been voluntarily submitted to the FDA for review. Clinically available molecular karyotyping tests currently differ considerably in technology, resolution, and likelihood of results with unknown significance. No laboratories were found that post a validation summary on their website. Some laboratories have published a summary of their assay validation procedure; examples highlight the complexity of designing and validating CGH arrays, as well as their analysis and interpretation.
Authors' comments and conclusions: 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 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.
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