Clinical features, functional consequences, and rescue pharmacology of missense GRID1 and GRID2 human variants.

GRID1 and GRID2 encode the enigmatic GluD1 and GluD2 proteins, which form tetrameric receptors that play important roles in synapse organization and development of the central nervous system. Variation in these genes has been implicated in neurodevelopmental phenotypes. We evaluated GRID1 and GRID2 human variants from the literature, ClinVar, and clinical laboratories and found that many of these variants reside in intolerant domains, including the amino terminal domain of both GRID1 and GRID2. Other conserved regions, such as the M3 transmembrane domain, show different intolerance between GRID1 and GRID2. We introduced these variants into GluD1 and GluD2 cDNA and performed electrophysiological and biochemical assays to investigate the mechanisms of dysfunction of GRID1/2 variants. One variant in the GRID1 distal amino terminal domain resides at a position predicted to interact with Cbln2/Cbln4, and the variant disrupts complex formation between GluD1 and Cbln2, which could perturb its role in synapse organization. We also discovered that, like the lurcher mutation (GluD2-A654T), other rare variants in the GRID2 M3 domain create constitutively active receptors that share similar pathogenic phenotypes. We also found that the SCHEMA schizophrenia M3 variant GluD1-A650T produced constitutively active receptors. We tested a variety of compounds for their ability to inhibit constitutive currents of GluD receptor variants and found that pentamidine potently inhibited GluD2-T649A constitutive channels (IC50 50 nM). These results identify regions of intolerance to variation in the GRID genes, illustrate the functional consequences of GRID1 and GRID2 variants, and suggest how these receptors function normally and in disease.

ATP6V0C variants impair V-ATPase function causing a neurodevelopmental disorder often associated with epilepsy.

The vacuolar H+-ATPase is an enzymatic complex that functions in an ATP-dependent manner to pump protons across membranes and acidify organelles, thereby creating the proton/pH gradient required for membrane trafficking by several different types of transporters. We describe heterozygous point variants in ATP6V0C, encoding the c-subunit in the membrane bound integral domain of the vacuolar H+-ATPase, in 27 patients with neurodevelopmental abnormalities with or without epilepsy. Corpus callosum hypoplasia and cardiac abnormalities were also present in some patients. In silico modelling suggested that the patient variants interfere with the interactions between the ATP6V0C and ATP6V0A subunits during ATP hydrolysis. Consistent with decreased vacuolar H+-ATPase activity, functional analyses conducted in Saccharomyces cerevisiae revealed reduced LysoSensor fluorescence and reduced growth in media containing varying concentrations of CaCl2. Knockdown of ATP6V0C in Drosophila resulted in increased duration of seizure-like behaviour, and the expression of selected patient variants in Caenorhabditis elegans led to reduced growth, motor dysfunction and reduced lifespan. In summary, this study establishes ATP6V0C as an important disease gene, describes the clinical features of the associated neurodevelopmental disorder and provides insight into disease mechanisms.

Inherited germline ATRX mutation in two brothers with ATR-X syndrome and osteosarcoma.

We report a family in which two brothers had an undiagnosed genetic disorder comprised of dysmorphic features, microcephaly, severe intellectual disability (non-verbal), mild anemia, and cryptorchidism. Both developed osteosarcoma. Trio exome sequencing (using blood samples from the younger brother and both parents) was performed and a nonsense NM_000489.4:c.7156C>T (p.Arg2386*) mutation in the ATRX gene was identified in the proband (hemizygous) and in the mother's peripheral blood DNA (heterozygous). The mother is healthy, does not exhibit any clinical manifestations of ATR-X syndrome and there was no family history of cancer. The same hemizygous pathogenic variant was confirmed in the affected older brother's skin tissue by subsequent Sanger sequencing. Chromosomal microarray studies of both brothers' osteosarcomas revealed complex copy number alterations consistent with the clinical diagnosis of osteosarcoma. Recently, somatic mutations in the ATRX gene have been observed as recurrent alterations in both osteosarcoma and brain tumors. However, it is unclear if there is any association between osteosarcoma and germline ATRX mutations, specifically in patients with constitutional ATR-X syndrome. This is the first report of osteosarcoma diagnosed in two males with ATR-X syndrome, suggesting a potential increased risk for cancer in patients with this disorder.

Diagnostic Yield of Epilepsy-Genes Sequencing and Chromosomal Microarray in Pediatric Epilepsy.

BACKGROUND: Around 40% of individuals with epilepsy have an underlying identifiable genetic etiology. Common methods for epilepsy genetic testing are chromosomal microarray (CMA) and epilepsy-genes sequencing (EGS). Historically, CMA was the first-line test for patients with epilepsy, but recent studies have shown that EGS has a superior diagnostic yield. To further optimize testing algorithms for epilepsy, we compared these tests' diagnostic yields and explored how they are influenced by age of onset and phenotype complexity. METHODS: Genetic test results from a cohort of patients with epilepsy were used to determine the diagnostic yield of CMA (n = 366) versus EGS (n = 370) for genetic epilepsy etiologies. Further analysis examined the probability of diagnostic results based on age of seizure onset and patients' phenotype complexity. RESULTS: For patients who underwent CMA, causative variants were found in 28 of 366 cases (7.7%), and 60 of 366 patients (16.4%) had at least one variant of uncertain significance (VUS). For EGS, 65 of 370 (17.6%) cases had causative variants, whereas 155 of 370 (41.9%) had at least one VUS. EGS had a significantly higher diagnostic yield than CMA (odds ratio [OR] = 2.63, P < 0.001). This difference in diagnostic yield was further pronounced among patients with infantile seizure onset (OR = 4.69, P < 0.001) and patients with additional neurological findings (OR = 2.99, P < 0.001). CONCLUSION: To minimize the time and resources required to reach a diagnosis, clinicians and insurers alike should consider using EGS as an initial diagnostic tool.

Custom Pediatric Oncology Next-Generation Sequencing Panel Identifies Somatic Mosaicism in Archival Tissue and Enhances Targeted Clinical Care.

BACKGROUND: Disorders in the PIK3CA-related overgrowth spectrum because of somatic mosaicism are associated with segmental overgrowth of the body in conjunction with vascular, skeletal, and brain malformations such as hemimegalencephaly. A pathogenic variant may only be detectable in affected tissue and not in peripheral blood or saliva samples; therefore archival tissue may be the only relevant available specimen for testing. Although this is a common approach for cancer testing, it is not typically used for constitutional genetic disorders. METHODS: PIK3CA mosaicism was assessed with a custom pediatric oncology next-generation sequencing panel (OncoKids) designed to capture somatic mutations in pediatric malignancies. The panel covers a wide range of targets including PIK3CA and AKT1 hotspots. We used OncoKids on archival formalin-fixed, paraffin-embedded or frozen samples from seven patients with facial hemihypertrophy and lipomas, hemimegalencephaly, or hemihypertrophy with a lymphovascular malformation. The age of the archival tissue examined by next-generation sequencing ranged from two to 13 years (median 5 years). Every patient had clinical manifestations within the PIK3CA-related overgrowth spectrum and had a sample of an affected tissue available for testing from a prior surgical intervention. RESULTS: PIK3CA mosaicism was detected in all seven patients and the mutant allele fraction was lower in the lymphovascular malformation tissues (8% to 11%) than in brain (20% to 32%) and lipomatous (16% to 23%) tissues. CONCLUSIONS: Our study highlights the clinical utility of using a robust, oncology-focused next-generation sequencing assay to identify PIK3CA mosaicism in noncancer cases. It is feasible to use archival samples that are more than a decade old to obtain a molecular diagnosis, which can then be used to improve health care management.

A semiautomated whole-exome sequencing workflow leads to increased diagnostic yield and identification of novel candidate variants.

Advancing the clinical utility of whole-exome sequencing (WES) for patients with suspected genetic disorders is largely driven by bioinformatics approaches that streamline data processing and analysis. Herein, we describe our experience with implementing a semiautomated and phenotype-driven WES diagnostic workflow, incorporating both the DRAGEN pipeline and the Exomiser variant prioritization tool, at an academic children's hospital with an ethnically diverse pediatric patient population. We achieved a 41% molecular diagnostic rate for 66 duo-, quad-, or trio-WES cases, and 28% for 40 singleton-WES cases. Preliminary results were returned to ordering physicians within 1 wk for 12 of 38 (32%) probands with positive findings, which were instrumental in guiding the appropriate clinical management for a variety of patients, especially in critical care settings. The semiautomated and streamlined WES workflow also enabled us to identify novel variants in candidate disease genes in patients with developmental delay and autism and immune disorders and cancer, including ANK2, BPTF, BCL11A, FOXN1, PLAA, ATRX, DNAJC21, and RAD50 Together, we demonstrated the implementation of a streamlined WES workflow that was successfully applied for both clinical and research purposes.