Large-scale whole-exome sequencing of children with signs of neurodevelopmental disorders — including microcephaly, epilepsy, sensory and motor defects, and intellectual disability — identified Turkish families in which affected family members were homozygous for the same single-nucleotide substitution in the CLP1 gene. In both studies, the exact features that constitute the clinically distinguishable phenotype only became evident after the mutation had been discovered, which enabled the researchers to define the disease. Additionally, in the cohort sequenced by James R. Lupski (Baylor College of Medicine) and co-workers, distinction of shared clinical features in patients with the CLP1 mutation helped to identify additional affected families. “The interesting thing about this approach is that it reverses the usual paradigm of 'gene hunting',” says Joseph G. Gleeson (University of California, San Diego), who led the second study. “Starting with a group of patients, their DNA defines the clinical features, rather than the doctors.”
The CLP1 mutation leads to an Arg140His amino acid substitution that both teams predicted would change the protein structure. To elucidate the cellular consequences of this mutation, Gleeson and colleagues used patient-derived skin fibroblasts and induced their transdifferentiation into neurons by knockdown of polypyrimidine tract-binding protein 1 (PTBP1). In both cell types, in vitro kinase activity and nuclear localization of the CLP1 protein were severely disrupted. Meanwhile, Lupski had joined forces with teams led by Josef M. Penninger and Javier Martinez (Austrian Academy of Sciences, Vienna), and showed using in vitro assays that the kinase activity of mutant CLP1, albeit reduced, was still sufficient to phosphorylate RNA. However, they identified an impaired interaction of the mutant with TSEN proteins that correlated with decreased pre-tRNA splicing. The same effect on tRNA biogenesis was anticipated by Gleeson and colleagues. “The CLP1 mutation, we predicted, should cause a backlog of pre-tRNAs and depletion of mature tRNAs,” remembers Gleeson. “Indeed, we found that patient-derived induced neurons, but not fibroblasts, showed this defect, which points to a potential use of these cells for other nervous system-specific diseases.”
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