Somatic Mutations in TSC1 and TSC2 Cause Focal Cortical Dysplasia

Am J Hum Genet. 2017 Mar 2;100(3):454-472. doi: 10.1016/j.ajhg.2017.01.030. Epub 2017 Feb 16.

Abstract

Focal cortical dysplasia (FCD) is a major cause of the sporadic form of intractable focal epilepsies that require surgical treatment. It has recently been reported that brain somatic mutations in MTOR account for 15%-25% of FCD type II (FCDII), characterized by cortical dyslamination and dysmorphic neurons. However, the genetic etiologies of FCDII-affected individuals who lack the MTOR mutation remain unclear. Here, we performed deep hybrid capture and amplicon sequencing (read depth of 100×-20,012×) of five important mTOR pathway genes-PIK3CA, PIK3R2, AKT3, TSC1, and TSC2-by using paired brain and saliva samples from 40 FCDII individuals negative for MTOR mutations. We found that 5 of 40 individuals (12.5%) had brain somatic mutations in TSC1 (c.64C>T [p.Arg22Trp] and c.610C>T [p.Arg204Cys]) and TSC2 (c.4639G>A [p.Val1547Ile]), and these results were reproducible on two different sequencing platforms. All identified mutations induced hyperactivation of the mTOR pathway by disrupting the formation or function of the TSC1-TSC2 complex. Furthermore, in utero CRISPR-Cas9-mediated genome editing of Tsc1 or Tsc2 induced the development of spontaneous behavioral seizures, as well as cytomegalic neurons and cortical dyslamination. These results show that brain somatic mutations in TSC1 and TSC2 cause FCD and that in utero application of the CRISPR-Cas9 system is useful for generating neurodevelopmental disease models of somatic mutations in the brain.

Keywords: CRISPR-Cas9 genome editing; TSC1; TSC2; brain mosaicism; brain somatic mutation; focal cortical dysplasia; intractable epilepsy.

MeSH terms

  • Adolescent
  • Animals
  • Brain / metabolism
  • CRISPR-Cas Systems
  • Cell Line, Tumor
  • Child
  • Class I Phosphatidylinositol 3-Kinases
  • Cloning, Molecular
  • Disease Models, Animal
  • Epilepsy / genetics*
  • Female
  • HEK293 Cells
  • Humans
  • Male
  • Malformations of Cortical Development, Group I / genetics*
  • Mice
  • Mutation
  • Neurons
  • Phosphatidylinositol 3-Kinases / genetics
  • Proto-Oncogene Proteins c-akt / genetics
  • Saliva / chemistry
  • Sequence Analysis, DNA
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases / genetics
  • Tuberous Sclerosis Complex 1 Protein
  • Tuberous Sclerosis Complex 2 Protein
  • Tumor Suppressor Proteins / genetics*

Substances

  • TSC1 protein, human
  • TSC2 protein, human
  • Tsc1 protein, mouse
  • Tsc2 protein, mouse
  • Tuberous Sclerosis Complex 1 Protein
  • Tuberous Sclerosis Complex 2 Protein
  • Tumor Suppressor Proteins
  • Phosphatidylinositol 3-Kinases
  • phosphoinositol-3 kinase regulatory subunit 2, human
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • Class I Phosphatidylinositol 3-Kinases
  • PIK3CA protein, human
  • AKT3 protein, human
  • Proto-Oncogene Proteins c-akt
  • Sirolimus

Supplementary concepts

  • Focal cortical dysplasia of Taylor