Pathogenic Yield of Genetic Testing in Autism Spectrum Disorder

We conducted a retrospective chart review of 500 toddlers (18–36 months) with an ASD diagnosis made in the Developmental Medicine Center at Boston Children’s Hospital (BCH) via an interdisciplinary team assessment from July 1, 2013, to May 1, 2016. Five children were excluded because they were seen by a single clinician, rather than a team, for the initial ASD evaluation. In the team assessments, a developmental-behavioral pediatrician obtained a medical and developmental history and conducted a physical examination and a clinical child psychologist obtained neuropsychological measures. Diagnoses were formulated and agreed on by the pediatrician and psychologist through a clinical team meeting that occurred immediately after the diagnostic assessment and through which consensus diagnoses were made. The Bayley Scales of Infant and Toddler Development, Third Edition (Bayley)¹⁸  was used to obtain cognitive, language, and motor standard scores. The Vineland Adaptive Behavior Scales, Second or Third Editions^19,20  were used to obtain scores related to adaptive functioning. The time frame for data abstraction corresponded with a clinic-wide shift to use of a checklist to ensure fidelity to the new DSM-5 criteria.²¹  We identified subjects via electronic medical record search based on a billing code signifying a team assessment (99245 AND 96118 or 96119) and an International Classification of Diseases, Ninth Revision or 10th Revision code for ASD (F84.0, F84.9, or F84.5 or 299.0, 299.8, or 299.9). Inclusion criteria were a documented DSM-5 diagnosis of ASD made via a team assessment. Patients are referred to the center by primary care clinicians, early childhood intervention providers, or parents on the basis of concern for delayed or atypical development. It is standard practice for clinicians in this practice to recommend genetic testing (CMA and fragile X) for all children diagnosed with ASD. The study was approved by the BCH Institutional Review Board with a waiver of informed consent.

We abstracted demographic, medical, and neuropsychological information for all subjects (Table 1). We recorded whether subjects completed genetic testing, along with the results of all CMA and fragile X testing. CMA analysis was performed via custom whole genome array with enhanced probe coverage on targeted genes and with single nucleotide polymorphism or loss of heterozygosity probes (Optimized Version 3.0 on Agilent 180k platform). The array uses human genome build 19, has a resolution of ∼50 kb for targeted regions or genes, and can detect copy number changes as small as ∼150 kb between targeted regions. CMA findings were reported as follows: (1) no clinically significant copy number variants or areas of copy number neutral homozygosity (≥10 Mb) were identified (which we refer to as “negative or normal”), (2) variants of unknown or uncertain significance, and (3) known or likely pathogenic finding. For fragile X testing, DNA was amplified across the region of CGG repeats in Fragile X mental retardation 1 by using Asuragen Amplidex Fragile X mental retardation 1 polymerase chain reaction reagents. Fragile X results were reported by CGG repeats, categorized as (1) normal (5–44 repeats), (2) “grey zone” (45–54 repeats), (3) premutation (55–200 repeats), and (4) full mutation (>200 repeats). All tests were performed at Claritas Genomics and followed the requirements of Clinical Laboratory Improvement Amendment 88 for clinical testing. Analyses were performed on DNA extracted by standard methodologies from blood samples. Results were reviewed, and the pathogenicity of those results determined by laboratory geneticists with Claritas Genomics. Pathogenicity determination was included in the report that was returned to the ordering clinician who, in turn, communicated the results to the parent. For pathogenic findings, clinicians often referred to a geneticist who then made additional, specific recommendations. For purposes of analyses, we considered known or likely pathogenic CMA findings and any abnormal fragile X findings to be “pathogenic.”

For patients with a pathogenic CMA finding, we performed further manual chart review to determine if a medical recommendation was subsequently made on the basis of the genetic result. The following documented medical recommendations would not have otherwise been made before the return of a pathogenic result: referral to other specialists (endocrinology, metabolism, and sleep medicine in our sample), audiology examination, ophthalmology examination, monitoring for seizures, echocardiogram, renal ultrasound, or other laboratory evaluation (urine amino acids, creatine kinase level). For pathogenic CMA results, referral to genetics, parental testing, or genetic counseling were not considered a recommendation. Those with a pathogenic finding on fragile X testing were referred to a fragile X clinic. This was considered a further medical recommendation because this referral informed family planning and resulted in ongoing monitoring. Although genetic testing is recommended at the time of diagnosis, factors such as family preference, logistics, and insurance authorization often delay testing. Data abstraction occurred at least 2 to 3 years after the diagnosis of ASD and thus reflects any genetic testing that occurred following the ASD diagnosis. Two trained reviewers (including author H.H.) recorded data in the Research Electronic Data Capture (REDCap)²²  database with >96% interrater reliability as determined by bimonthly review of 10% of the records that were double-coded. A third reviewer (E.H.) arbitrated any coding discrepancies.

We calculated frequencies and percentages to represent the baseline characteristics of the sample. We used independent sample t tests to evaluate for significant differences between patients who completed versus did not complete genetic testing on continuous variables and χ² tests for categorical variables. Power levels were in excess of 99% for either test (Supplemental Information). Effect sizes involved Glass δ statistic, which is a variant of Cohen d, for which values of 0.2, 0.5, and 0.8 are indicative of small, medium, and large effects, respectively. In hypotheses involving percentages, χ² statistics were also presented with this effect size metric by using SDs. Thus, effects are comparable across all tests.

Among subjects in the study (N = 500, 79.2% male), 299 (59.8%) completed genetic testing (mean age: 25.8 months at diagnosis). There were no differences in demographic characteristics between those children that completed genetic testing versus those that did not (Table 2). Subjects who completed testing were more likely to have dysmorphic features, be nonverbal, and have lower Bayley language scores and lower scores on parent report of overall adaptive functioning, communication, motor, and socialization skills (Table 2).

Of the 299 subjects who completed testing, 186 (62.2%) had negative or normal findings, 77 (25.8%) had variants of unknown significance on CMA, and 36 (12%) had a pathogenic finding (Fig 1). Of those with pathogenic findings, 27 subjects had pathogenic variants on CMA (9.0%) and 9 subjects had pathogenic fragile X findings (3.0%). The most common copy number variants in this sample were deletions or duplications on 15q (n = 10) and 22q (n = 2). Among subjects with fragile X findings, there were 3 full mutations, 3 premutations, 2 intermediate or “grey zone” mutations, and 1 patient with mosaicism (Table 3). The role of fragile X premutations, “grey zone” mutations, and mosaicism in ASD is an ongoing area of investigation. Because current literature suggests potential relevance, we considered these mutations pathogenic.^23–25 

There were no significant differences in cognitive, language, or motor standard scores or in patient age, insurance, or median household income across genetic result categories (Table 4). Among subjects who completed genetic testing, there was a higher rate of variants of unknown significance for girls versus boys (25 of the 65 girls, 38.5%; compared with 52 of the 234 boys, 22.2%; P = .03). There was a significant difference in mean motor standard score on the Vineland Adaptive Behavior Scales across genetic finding categories (86.54 for negative or normal finding, 85.60 for variants of unknown significance, and 80.10 for pathogenic findings; P = .01).

Of those children with a pathogenic finding (n = 36), manual chart review was conducted to determine if a medical recommendation was made on the basis of the finding. All children with positive fragile X results (n = 9) received a further recommendation via referral to a fragile X clinic for genetic counseling and comprehensive care. Of subjects with pathogenic CMA findings (n = 27), 17 children (63%) had a medical recommendation made on the basis of their finding(s). The most common medical recommendations included education regarding risk and management of potential seizures and seizure monitoring (n = 5) and referrals to subspecialists (endocrinology, metabolism, or sleep medicine; n = 4). Other common recommendations included echocardiogram, renal ultrasound, audiology examination, and ophthalmology examination (n = 3 for each). In summary, including both pathogenic CMA findings and positive fragile X findings, 26 of 36 subjects (72.2%) had subsequent medical recommendations made on the basis of their results.

In this study, 59.8% of toddlers with a DSM-5 diagnosis of ASD completed genetic testing. Many factors may influence completion rate, including clinician practices or family considerations such as the likelihood of a positive result, the potential for results with unclear significance, or family planning implications. Additional barriers include discomfort of phlebotomy, cost, and third-party payer policies. Although the rate of completed testing in our sample is higher than previously reported (parent surveys report rates of 28%–41%), it is low in the context of guidelines that recommend genetic testing in all patients with a new ASD diagnosis.^26,27  In this sample, parents’ likelihood to pursue genetic testing for their child did not vary on the basis of subject age, insurance status, sex, or median household income. Patients who had dysmorphic features, were nonverbal, and had lower language scores were more likely to complete testing. Additionally, for those children completing testing, parent report across domains of functioning (adaptive, communication, motor, social) was lower compared with children who did not complete testing.

Among subjects who did complete CMA and fragile X testing, the pathogenic yield of 12.0% is consistent with previous reports.^9,14,28  We did not find significant differences in age at diagnosis, insurance status, or median household income between those with and without pathogenic findings. There was a higher percentage of female subjects in the group with variants of unknown significance. This finding may be consistent with the hypothesis that girls require a higher burden of mutations to reach a threshold required to manifest ASD, known as the female protective effect.²⁹  Ho et al¹⁵  also demonstrated a higher rate of pathogenic mutations in female patients with complex ASD.

There were no differences in medical characteristics of interest, nor was there a statistically significant difference in Bayley cognitive scores across subjects in the 3 categories of genetic test results (normal or negative findings, variants of unknown significance, and pathogenic findings). This finding is in contrast to previous work suggesting a higher pathogenic yield of testing in complex ASD, which often includes intellectual disability or cognitive delay, but is consistent with a recent study by McGrew et al,¹⁷  who compared characteristics between children with and without pathogenic findings and found no differences.^10,12,14  Although measuring cognitive functioning in young children is challenging, our results suggest that clinicians should continue to recommend genetic testing regardless of cognitive level.

In our sample, children with known pathogenic findings had lower parent-reported motor functioning than children with negative or normal results or variants of unknown significance. This raises the question of the role of motor delays in suggesting a potential underlying genetic etiology, an area of recent investigation.³⁰  However, although this difference was statistically significant in our sample, scores in both groups remained in the “low adequate” to “moderately low” range on the Vineland Scales; such differences may not be appreciable clinically.

In children with pathogenic CMA or fragile X findings, 72.2% had further medical recommendations made on the basis of their genetic results. We combined fragile X and CMA when reporting on percentage receiving medical recommendations given our focus on implications of genetic testing in ASD overall. All subjects with abnormal fragile X results received referral to a genetic specialist. For CMA alone, 17 of 27 (63%) received medical recommendations. Additionally, all patients with a pathogenic finding received genetic counseling around family planning. When considering the entire group of subjects (N = 299) who completed genetic testing, 8.7% had a medical recommendation made on the basis of their results. This finding is consistent with one previous report of 6.2%.¹⁴  Our results suggest that genetic test findings do not merely provide information for families but also result in further medical recommendations and monitoring, arguing for broader, more uniform third-party payer coverage. Furthermore, a pathogenic genetic finding may provide an answer for families seeking to understand why their child has ASD.

A potential limitation to this study is our retrospective methodology; thus, data are limited to information documented in the medical record. For example, some demographic information (race, ethnicity, maternal education) was not routinely collected. All genetic testing available for review was performed at the BCH laboratory. It is possible that some children may have had genetic testing conducted at other approved laboratories and that these results were not documented in their medical records. Although all clinicians within the practice are instructed to recommend testing and documented such recommendations in the note, the strength with which the recommendation was communicated verbally cannot be confirmed. Only 60% of subjects completed genetic testing, and on the basis of our comparisons, it is possible that the perception of greater impairment on the part of parent and/or clinician increased the likelihood that a patient completed testing, which could have influenced our findings; however, differences between those who did versus did not complete testing were unlikely to be clinically important, as indicated with effect sizes being below an accepted medium-level threshold.³¹  Of the sample that did ultimately complete testing, there were no significant differences in medical characteristics or objectively measured cognitive, language, or motor functioning between subjects across result categories. We used billing and diagnostic codes to identify cases, and thus there is the potential for false-negatives (children who have ASD were not billed in this manner), although this would be highly unlikely because toddlers are usually presenting for the first time, evaluated through the team assessment, and billed as such. The study sample comes from a single tertiary care site. However, the majority of children in our region with a concern for ASD are referred to a tertiary center for developmental evaluation because this is the means to access services. Thus, we believe our sample is representative of young children receiving a diagnosis of ASD in our region. Although our sample size is large, the number of children with pathogenic genetic findings is small (n = 36) and must be considered when evaluating the frequency with which medical recommendations are made within this group. A strength of the study is the large sample size of toddlers with a DSM-5 diagnosis. Additionally, the standardized format of the comprehensive interdisciplinary team visit allowed us to evaluate numerous medical and neuropsychological characteristics for the sample.

In a clinical sample of toddlers with DSM-5 ASD, almost 60% completed genetic testing (CMA and/or fragile X). Of those who completed genetic testing, 12% had a pathogenic finding. There was no association between medical characteristics or cognitive, language, and motor functioning and the likelihood of a pathogenic finding. Our results suggest that genetic testing (CMA and/or fragile X) should continue to be recommended for patients receiving a diagnosis of ASD, regardless of phenotypic characteristics at diagnosis. Further medical recommendations were made in 72.2% of patients with pathogenic findings, indicating that genetic testing in this population has significant clinical implications. With these findings, we highlight the importance of genetic testing for toddlers with a diagnosis of ASD.

We thank Collin Lee for assistance with data abstraction and Hyejin “Jasmine” Jeon for assistance with article preparation.

Study data were collected and managed by using REDCap electronic data capture tools hosted at BCH. REDCap is a secure, web-based application designed to support data capture for research studies, providing (1) an intuitive interface for validated data entry, (2) audit trails for tracking data manipulation and export procedures, (3) automated export procedures for seamless data downloads to common statistical packages, and (4) procedures for importing data from external sources.

ASD

autism spectrum disorder

Bayley

Bayley Scales of Infant and Toddler Development, Third Edition

BCH

Boston Children’s Hospital

CMA

chromosomal microarray

DSM-5

Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition

REDCap

Research Electronic Data Capture