Analytical and Clinical Validity Study of FirstStepDx PLUS: A Chromosomal Microarray Optimized for Patients with Neurodevelopmental Conditions

Introduction Chromosomal microarray analysis (CMA) is recognized as the first-tier test in the genetic evaluation of children with developmental delays, intellectual disabilities, congenital anomalies and autism spectrum disorders of unknown etiology. Array Design To optimize detection of clinically relevant copy number variants associated with these conditions, we designed a whole-genome microarray, FirstStepDx PLUS (FSDX). A set of 88,435 custom probes was added to the Affymetrix CytoScanHD platform targeting genomic regions strongly associated with these conditions. This combination of 2,784,985 total probes results in the highest probe coverage and clinical yield for these disorders. Results and Discussion Clinical testing of this patient population is validated on DNA from either non-invasive buccal swabs or traditional blood samples. In this report we provide data demonstrating the analytic and clinical validity of FSDX and provide an overview of results from the first 7,570 consecutive patients tested clinically. We further demonstrate that buccal sampling is an effective method of obtaining DNA samples, which may provide improved results compared to traditional blood sampling for patients with neurodevelopmental disorders who exhibit somatic mosaicism. Clinical scenario Neurodevelopmental disabilities, including developmental delays (DD), intellectual disabilities (ID), and autism spectrum disorders (ASD), affect up to 15% of children (1). In the majority of cases, a child’s clinical presentation does not allow for a definitive etiological diagnosis. In such cases, CMA is recommended as the first-tier test that should be used to evaluate for a potential genetic etiology (2–7). A definitive genetic diagnosis allows patients to more often receive appropriate medical care tailored to their condition, as reflected by medical management changes and improved access to necessary support and educational services (8–13). Test description FirstStepDx PLUS (FSDX) is an optimized clinical microarray test provided in the context of a comprehensive clinical service. Testing starts with either a non-invasive buccal swab sample or traditional blood sample from which DNA extraction using a Gentra Puregene® kit specific to the sample type (Qiagen, Inc., Valencia, CA) is performed in one of several contracted CLIA/CAP credentialed laboratories according to manufacturers’ protocols. High quality genomic DNA is fragmented, labeled and hybridized to FSDX arrays using reagents, equipment and methodology as specified by by the manufacturer (Affymetrix, Inc., Santa Clara, CA)(14). Washed arrays are scanned and raw data files are processed to CYCHP files using a reference file comprising at least 100 samples with normal array findings. Data analysis is performed using Chromosome Analysis Suite software version 2.0. 1 (Affymetrix). Hybridization of patient DNA to oligonucleotide and SNP probes is independently compared against a previously analyzed cohort of normal samples to call CNVs and allele genotypes. The percentage mosaicism of whole-chromosome aneuploidies is determined using the average log2 ratio of the entire chromosome (14). Microarray design FSDX was optimized by the addition of 88,435 custom probes targeting genomic regions strongly associated with ID/DD/ASD (15–24). This was effected, under GMP by Affymetrix, to the CytoScanHD platform using their microarray design process specifications which have been previously described (14). This is consistent with the ACMG recommendation of “enrichment of probes targeting dosage-sensitive genes known to result in phenotypes consistent with common indications for a genomic screen” (25). Critical regions that did not meet a desired probe density ≥1 probe/1000 bp on the CytoScanHD were supplemented with additional probe content to allow for improved detection of smaller deletions and duplications in these critical regions. Finally, additional probes were added to improve detection of CNVs encompassing genes involved in other well-characterized neurodevelopmental disorders, for example GAMT (26) and GATM (27). All incremental probes were added in substitution for probes deemed sub-optimal by Affymetrix and previously masked, bringing FSDX to a grand total of 2,784,985 probes. Custom SNP probes (n =416) on FSDX are targeted by 12 oligonucleotides, three for each strand of each allele, which is approximately double the typical probe coverage for SNPs. Test interpretation CYCHP files are evaluated by ABMG certified cytogeneticists. Determination of CNVs is consistent with established cytogenetic standards. A minimum of 25-consecutive impacted probes is the baseline determinant for deletions and 50 probes for duplications independent of variant size. Rare CNVs are determined to be "pathogenic" if there is sufficient evidence published (at least two independent publications) to indicate that haploinsufficiency or triplosensitivity of the region or gene(s) involved is causative of clinical features or of sufficient overall size (28). If however, there is insufficient but at least preliminary evidence for a causative role for the region or gene(s) therein they are classified as variants of unknown significance (VOUS) independent of CNV size. Areas of absence of heterozygosity (AOH) are also classed as VOUS if of sufficient size and location to increase the risk for conditions with autosomal recessive inheritance or conditions with parent-of-origin/imprinting effects. Other CNVs are typically not reported after determination that they most likely represent normal common population variants and are contained in databases documenting presumptively benign CNVs, e.g., the Database of Genomic Variants (DGV) (29). These parameters were standard independent of the microarray used for analysis in comparative studies. Public health importance A definitive genetic diagnosis facilitates patient access to appropriate and necessary medical and support services. Defining the underlying genetic cause of DD/ID/ASD and/or multiple congenital anomalies (MCA) in each unique patient is vital to understanding etiology, prognosis, and course. It informs physicians of potential comorbid conditions for which a patient should be evaluated and treated proactively and optimally. Improved understandings of the appropriate therapeutic and behavioral approaches to that patient are also enabled. Genetic testing is best provided in the context of an integrated service (30), so FSDX provides comprehensive, clear, readable, and personalized reports for the healthcare provider and a family-friendly section to facilitate understanding of the often-complex results. The report is complemented by availability of pre- and post-test genetic counseling and technical support to providers. Moreover, FSDX includes personalized insurance pre-authorization and appeals assistance to overcome barriers encountered by both providers and families that, in many circumstances, prevent access to crucial genetic testing services (10–11). Published reviews, recommendations and guidelines The American College of Medical Genetics (ACMG) (2,3), the American Academy of Child and Adolescent Psychiatry (4,7), the American Academy of Pediatrics (5), and the American Academy of Neurology/Child Neurology Society (6) recommend CMA as the first-tier test in the genetic evaluation of children with unexplained DD, ID, or ASD. Considerable data supporting these guidelines are documented in numerous reviews and publications (31–36). The ACMG has also published guidelines on both array design (25) and the validation of arrays, including validation of a new version of a platform in use by the laboratory from the same manufacturer, and of additional sample types (28).