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].