TUBEROUS SCLEROSIS COMPLEX: IDENTIFICATION OF THE GENETIC CAUSE IN PATIENTS WITH NO MUTATION DETECTED, AND ANALYSIS OF MOSAICISM

Tuberous sclerosis complex (TSC) is an autosomal dominant multisystem disorder characterized by development of hamartomas, intellectual disability, seizures and autism. TSC is caused by inactivating mutations in either the TSC1 or the TSC2 genes. A pathogenic variant is not identified in up to 10% of the patients with a clinical diagnosis of TSC despite full molecular assessment. These individuals are referred to as NMI (No Mutation Identified), and it is not clear if they should be monitored and/or treated in similar fashion to those with known etiology of TSC. To identify the genetic cause of TSC in these patients, we selected ten individuals with a definite clinical diagnosis of TSC and NMI and performed a pilot study. Three different technologies were used and their results were compared: chromosomal microarray, trio whole genome sequencing, and targeted deep-coverage next generation sequencing of TSC1 and TSC2. We identified mosaic variants in TSC1/TSC2 in six patients. No variants in other genes were detected in the remaining individuals. Based on the results of the pilot study and on recent literature, we then performed targeted deep-coverage TSC1/TSC2 sequencing on 200 affected individuals using peripheral blood DNA and saliva or other tissue samples where available. We identified 24 patients with mosaic pathogenic variants in TSC1 (n=2) or TSC2 (n=22), defining a rate of mosaicism of 12%. Mosaic variant allele frequency (VAF) ranged from 2% to 32% in blood and from 2% to 35% in saliva. Most affected individuals had low-level mosaicism (VAF ≤10%). We performed an extensive analysis of the phenotype, and show that individuals with a mosaic variant in TSC1/TSC2 often display normal cognitive functioning, although other TSC-associated neuropsychiatric disorders (TAND) are seen in 62% of the patients. Cortical tubers are invariably present and seizures are diagnosed in 54% of the cohort, but infantile spasms are rare. The number of cutaneous manifestations in these patients is often insufficient to meet diagnostic criteria, except for facial angiofibromas. We observed a high frequency of pulmonary and renal manifestations in our mosaic cohort, which are as severe as those seen in the general TSC population. None of the patients who have reproduced transmitted the variant to their offspring. In conclusion, our study shows that genome sequencing fails to identify rare variants in new genes related to TSC, and a third gene is therefore unlikely to exist. We demonstrated that at least one out of 10 patients with a clinical diagnosis of TSC carries a mosaic pathogenic variant in TSC1 or TSC2. We also showed that individuals with mosaic variants have a distinctive phenotypic severity, with important implications for surveillance.