Functional characterization of a GFAP variant of uncertain significance in an Alexander disease case within the setting of an individualized medicine clinic.

Next‐generation sequencing has ushered in an era of individualized medicine enabling a patient's genome to be interrogated in search of variants implicated in disease. This has brought about diagnostic resolution for many patients suffering from previously undiagnosed genetic disorders. However, it has also created significant challenges in clinical interpretation, as many identified variants lack sufficient contextual information to allow clear association with disease and are classified as variants of uncertain significance (VUS). Here, we describe the sequential use of variant annotation, in silico protein modeling techniques, and in vitro functional studies to characterize a clinically reported VUS and reclassify it as a pathogenic variant. In this study, genetic panel testing in a 19‐month‐old boy with developmental delay, hypotonia, and abnormal brain MRI revealed a VUS, c.1126C>T, leading to p.R376W, in the glial fibrillary acidic protein (GFAP; OMIM:203450; NM_002055.4) which has previously been associated with Alexander disease (OMIM: 203450) 1. Alexander disease, a rare genetic disorder with more than 550 cases reported worldwide, is characterized as a progressive disorder of cerebral white matter 2. Alexander disease can present during infancy, childhood, or adulthood, with infantile onset being the most common presentation. The infantile form often presents with progressive psychomotor retardation, loss of developmental milestones, frontal bossing with megalencephaly, seizures, hyperreflexia, ataxia, and hydrocephalus, and frequently leads to death within the first decade 2. Juvenile and adult forms of Alexander disease often progress more slowly with ataxia, bulbar signs, and spasticity 3. The predominant genetic cause of Alexander disease was discovered after transgenic GFAP mice presented with astrocyte inclusions that pathologically mimicked the Rosenthal fibers in Alexander disease 4. The mouse model led to targeted GFAP sequencing in 11 cases of Alexander disease, 10 of which harbored a genetic variant. For cases with available parental samples, the variants were found to have arisen as de novo in the proband, and were absent in their larger cohort of control DNA samples 1. Since these initial studies, over 110 distinct pathogenic variants in GFAP have been identified 2. The vast majority of these variants are de novo missense heterozygous variants; however, there is some evidence supporting familial inheritance 5. Following the initial discovery of GFAP variants associated with Alexander disease, the functional characterization of newly identified variants was completed in mouse models 6. However, the vast majority of subsequently identified missense variants in GFAP were considered pathogenic without functional validation. Cellular‐based assays have been developed and used to examine the pathogenicity of variants in individuals with abnormal presentation or familial onset 3, 5. Results of these studies demonstrated that some variants elicit a wild‐type phenotype, suggesting they were benign in nature 3. These cases highlight the need for functional validation of GFAP variants associated with atypical Alexander disease. As our patient presented with developmental delay, hypotonia, and abnormal brain MRI, with no signs of regression, it could not confidently be concluded that the identified GFAP variant was associated with his disease phenotype. Because his phenotype may represent a subtle and atypical form of Alexander disease, we combined variant annotation using information on variant frequency in Alexander disease cases and publically available exome databases, protein studies via paralog modeling, and functional characterization utilizing a cellular model to determine the pathogenicity of p.R376W.