Somatic Mutations in TSC1 and TSC2 Cause Focal Cortical Dysplasia
Jae Seok Lim1, Ramu Gopalappa2, Se Hoon Kim3
1Brain Korea 21 Plus Project, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science & Technology, Daejeon 34141, South Korea.
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.
Insights
Brain somatic mutations in TSC1 and TSC2 genes cause focal cortical dysplasia (FCDII) in individuals lacking MTOR mutations. These mutations hyperactivate the mTOR pathway, leading to epilepsy and developmental abnormalities.
Area of Science:
- Neurogenetics
- Molecular Biology
- Epilepsy Research
Background:
- Focal cortical dysplasia (FCD) is a primary cause of intractable focal epilepsies requiring surgery.
- Brain somatic mutations in MTOR explain 15-25% of FCD type II (FCDII) cases, characterized by cortical malformations.
- The genetic causes for FCDII in patients without MTOR mutations are largely unknown.
Purpose of the Study:
- To investigate the genetic etiologies of FCDII in individuals negative for MTOR mutations.
- To identify novel gene mutations within the mTOR pathway associated with FCDII.
- To establish a disease model for FCDII using CRISPR-Cas9 technology.
Main Methods:
- Deep sequencing of five key mTOR pathway genes (PIK3CA, PIK3R2, AKT3, TSC1, TSC2) in paired brain and saliva samples from 40 FCDII patients.
- Validation of identified mutations on two independent sequencing platforms.
- In utero CRISPR-Cas9 genome editing in mouse models to study the functional consequences of Tsc1/Tsc2 mutations.
Main Results:
- Somatic mutations in TSC1 and TSC2 were identified in 12.5% (5 of 40) of FCDII patients lacking MTOR mutations.
- All identified TSC1 and TSC2 mutations led to hyperactivation of the mTOR pathway by impairing the TSC1-TSC2 complex.
- CRISPR-Cas9-induced mutations in Tsc1 or Tsc2 in utero resulted in epilepsy, cytomegalic neurons, and cortical dyslamination.
Conclusions:
- Brain somatic mutations in TSC1 and TSC2 are a significant cause of FCDII.
- Disruption of the TSC1-TSC2 complex and subsequent mTOR hyperactivation are key mechanisms in FCDII pathogenesis.
- In utero CRISPR-Cas9 genome editing provides a valuable tool for modeling neurodevelopmental disorders caused by somatic mutations.
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