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Whole-genome sequencing (WGS) as a first-tier diagnostic test could transform medical genetic assessments, but there are limited data regarding its clinical use. We previously showed that WGS could feasibly be deployed as a single molecular test capable of a higher diagnostic rate than current practices, in a prospectively recruited cohort of 100 children meeting criteria for chromosomal microarray analysis. In this study, we report on the added diagnostic yield with re-annotation and reanalysis of these WGS data ~2 years later. Explanatory variants have been discovered in seven (10.9%) of 64 previously undiagnosed cases, in emerging disease genes like HMGA2. No new genetic diagnoses were made by any other method in the interval period as part of ongoing clinical care. The results increase the cumulative diagnostic yield of WGS in the study cohort to 41%. This represents a greater than 5-fold increase over the chromosomal microarrays, and a greater than 3-fold increase over all the clinical genetic testing ordered in practice. These findings highlight periodic reanalysis as yet another advantage of genomic sequencing in heterogeneous disorders. We recommend reanalysis of an individual’s genome-wide sequencing data every 1–2 years until diagnosis, or sooner if their phenotype evolves.

Purpose RAC3 is an underexamined member of the Rho GTPase gene family that is expressed in the developing brain and linked to key cellular functions. De novo missense variants in the homolog RAC1 were recently associated with developmental disorders. In the RAC subfamily, transforming missense changes at certain shared residues have been observed in human cancers and previously characterized in experimental studies. The purpose of this study was to determine whether constitutional dysregulation of RAC3 is associated with human disease. Methods We discovered a RAC3 variant in the index case using genome sequencing, and searched for additional variants using international data-sharing initiatives. Functional effects of the variants were assessed using a multifaceted approach generalizable to most clinical laboratory settings. Results We rapidly identified five individuals with de novo monoallelic missense variants in RAC3 , including one recurrent change. Every participant had severe intellectual disability and brain malformations. In silico protein modeling, and prior in vivo and in situ experiments, supported a transforming effect for each of the three different RAC3 variants. All variants were observed in databases of somatic variation in cancer. Conclusions Missense variants in RAC3 cause a novel brain disorder, likely through a mechanism of constitutive protein activation.

Children with medical complexity (CMC) represent a growing population in the pediatric health care system, with high resource use and associated health care costs. A genetic diagnosis can inform prognosis, anticipatory care, management, and reproductive planning. Conventional genetic testing strategies for CMC are often costly, time consuming, and ultimately unsuccessful. To evaluate the analytical and clinical validity of genome sequencing as a comprehensive diagnostic genetic test for CMC. In this cohort study of the prospective use of genome sequencing and comparison with standard-of-care genetic testing, CMC were recruited from May 1, 2017, to November 30, 2018, from a structured complex care program based at a tertiary care pediatric hospital in Toronto, Canada. Recruited CMC had at least 1 chronic condition, technology dependence (child is dependent at least part of each day on mechanical ventilators, and/or child requires prolonged intravenous administration of nutritional substances or drugs, and/or child is expected to have prolonged dependence on other device-based support), multiple subspecialist involvement, and substantial health care use. Review of the care plans for 545 CMC identified 143 suspected of having an undiagnosed genetic condition. Fifty-four families met inclusion criteria and were interested in participating, and 49 completed the study. Probands, similarly affected siblings, and biological parents were eligible for genome sequencing. Genome sequencing was performed using blood-derived DNA from probands and family members using established methods and a bioinformatics pipeline for clinical genome annotation. The primary study outcome was the diagnostic yield of genome sequencing (proportion of CMC for whom the test result yielded a new diagnosis). Genome sequencing was performed for 138 individuals from 49 families of CMC (29 male and 20 female probands; mean [SD] age, 7.0 [4.5] years). Genome sequencing detected all genomic variation previously identified by conventional genetic testing. A total of 15 probands (30.6%; 95% CI 19.5%-44.6%) received a new primary molecular genetic diagnosis after genome sequencing. Three individuals had novel diseases and an additional 9 had either ultrarare genetic conditions or rare genetic conditions with atypical features. At least 11 families received diagnostic information that had clinical management implications beyond genetic and reproductive counseling. This study suggests that genome sequencing has high analytical and clinical validity and can result in new diagnoses in CMC even in the setting of extensive prior investigations. This clinical population may be enriched for ultrarare and novel genetic disorders. Genome sequencing is a potentially first-tier genetic test for CMC.

Abstract Genetic testing is often pursued in children with medical complexity (CMC), in an attempt to establish a unifying diagnosis, understand pathogenicity and disease progression, guide care and inform reproductive planning. CMC are defined by at least one chronic condition, technology dependence, multiple subspecialist involvement, and high healthcare utilization. Despite multiple efforts to confirm clinical suspicion of an underlying genetic condition, many remain undiagnosed. Whole genome sequencing (WGS) is becoming increasingly available as an informative diagnostic tool. The application of genomic technology to this population has the potential to increase the proportion of CMC for whom diagnoses are established, in an effort to reduce time and emotional burden of the diagnostic process, and reduce health care system costs. The main purpose of this study was to optimize the clinical implementation of state-of-the-art genome diagnostics for CMC, in terms of diagnostic yield. We conducted a prospective study using patients followed by the Complex Care program at a large urban tertiary care center. Research ethics board approval was obtained. Of 435 patients screened, 114 were eligible for inclusion as an underlying genetic condition was clinically suspected but not established to date by conventional genetic testing. To date, 21 participants were evaluated through a clinical genetic assessment, previous genetic testing review and peripheral blood-derived DNA sequence. A laboratory team identified candidate genetic variants associated with patients' clinical symptoms, as well as other paediatric medically actionable variants. When found, these variants were validated as clinically significant by comparing the child's DNA to his parents'. WGS diagnostic yield was then determined by calculating the proportion of cases for which a genetic diagnosis was established. Of the 21 patients recruited, nine WGS analysis were completed thus far. Among these, four participants were diagnosed with established diseases, two of which were considered as novel diseases. One case was identified with a possible diagnosis, however, the interpretation of this clinical phenotype remains of unknown significance. The other four patients of the study remained undiagnosed. Given these preliminary results, the diagnostic yield of WGS was predicted at 44% in CMC. This can be compared to a previous study performed at our center in which the diagnostic rate for chromosomal microarray alone was reported to be 8% and microarray plus targeted gene sequencing 13%. This study has shown WGS to be feasible and achieve a higher diagnostic yield in our complex care population. As detection rates improve and laboratory costs decrease overtime, WGS will undoubtedly become a more informative diagnostic tool, particularly in this population. Optimizing the application of this increasingly sophisticated genomic technology warrants further consideration.