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Single germline or somatic activating mutations of mammalian target of rapamycin (mTOR) pathway genes are emerging as a major cause of type II focal cortical dysplasia (FCD), hemimegalencephaly (HME) and tuberous sclerosis complex (TSC). A double-hit mechanism, based on a primary germline mutation in one allele and a secondary somatic hit affecting the other allele of the same gene in a small number of cells, has been documented in some patients with TSC or FCD. In a patient with HME, severe intellectual disability, intractable seizures and hypochromic skin patches, we identified the ribosomal protein S6 (RPS6) p.R232H variant, present as somatic mosaicism at ~15.1% in dysplastic brain tissue and ~11% in blood, and the MTOR p.S2215F variant, detected as ~8.8% mosaicism in brain tissue, but not in blood. Overexpressing the two variants independently in animal models, we demonstrated that MTOR p.S2215F caused neuronal migration delay and cytomegaly, while RPS6 p.R232H prompted increased cell proliferation. Double mutants exhibited a more severe phenotype, with increased proliferation and migration defects at embryonic stage and, at postnatal stage, cytomegalic cells exhibiting eccentric nuclei and binucleation, which are typical features of balloon cells. These findings suggest a synergistic effect of the two variants. This study indicates that, in addition to single activating mutations and double-hit inactivating mutations in mTOR pathway genes, severe forms of cortical dysplasia can also result from activating mutations affecting different genes in this pathway. RPS6 is a potential novel disease-related gene.

The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the α/β-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of β-tubulin to fold or become assembled into the α/β-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects.