Tuberous Sclerosis Complex: Epidemiology and manifestations

Neurofibromatosis type 1 (NF1) is a cancer predisposition syndrome that results from dominant loss-of-function mutations mainly in the NF1 gene. Large rearrangements are present in 5–10% of affected patients, generally encompass NF1 neighboring genes, and are correlated with a more severe NF1 phenotype. Evident genotype–phenotype correlations and the importance of the co-deleted genes are difficult to establish. In our study we employed an evolutionary approach to provide further insights into the understanding of the fundamental function of genes that are co-deleted in subjects with NF1 microdeletions. Our goal was to access the ortholog and paralog relationship of these genes in primates and verify if purifying or positive selection are acting on these genes. Fourteen genes were analyzed in twelve mammalian species. Of these, four and ten genes showed positive selection and purifying selection, respectively. The protein, RNF135, showed three sites under positive selection at the RING finger domain, which may have been selected to increase efficiency in ubiquitination routes in primates. The phylogenetic analysis suggests distinct evolutionary constraint between the analyzed genes. With these analyses, we hope to help clarify the correlation of the co-deletion of these genes and the more severe phenotype of NF1.

Neurofibromatosis type (NF1) is a syndrome characterized by varied symptoms, ranging from mild to more aggressive phenotypes. The variation is not explained only by genetic and epigenetic changes in the NF1 gene and the concept of phenotype-modifier genes in extensively discussed in an attempt to explain this variability. Many datasets and tools are already available to explore the relationship between genetic variation and disease, including systems biology and expression data. To suggest potential NF1 modifier genes, we selected proteins related to NF1 phenotype and NF1 gene ontologies. Protein–protein interaction (PPI) networks were assembled, and network statistics were obtained by using forward and reverse genetics strategies. We also evaluated the heterogeneous networks comprising the phenotype ontologies selected, gene expression data, and the PPI network. Finally, the hypothesized phenotype-modifier genes were verified by a random-walk mathematical model. The network statistics analyses combined with the forward and reverse genetics strategies, and the assembly of heterogeneous networks, resulted in ten potential phenotype-modifier genes: AKT1, BRAF, EGFR, LIMK1, PAK1, PTEN, RAF1, SDC2, SMARCA4, and VCP. Mathematical models using the random-walk approach suggested SDC2 and VCP as the main candidate genes for phenotype-modifiers.

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by the presence of pathogenic germline variants in TSC1 or TSC2 genes. However, most TSC1 and TSC2 genetic testing strategies are limited and about 20% of patients have inconclusive or negative results. In this study, we standardized a low-cost technique based on RT-PCR followed by Sanger sequencing, aimed to complement genetic testing strategies. Patients with clinical suspicion of TSC and TSC1 and TSC2 inconclusive (n=3) or negative (n=1) genetic testing results were recruited. Exon 5 skipping in TSC1 gene was identified in one patient with a previous negative genetic testing result. In addition, a 9-base-pair intronic inclusion was found in the TSC1 of an individual with a previously identified variant of uncertain significance, c.664-10A>C. No alterations were found in the other two patients. The present study was successful in identifying errors in mRNA processing in two TSC patients with previously negative or inconclusive genetic testing results. Furthermore, this strategy was useful to reclassify a VUS to pathogenic. Finally, the validated technique could be used to complement coding region genetic tests and to investigate causality of VUS in TSC and other monogenic diseases. Highlights: - A novel cDNA analysis method revealed the presence of exon 5 skipping in the - TSC1 gene - A nine-base-pair retention in TSC1 mRNA is caused by the intronic variant c.664-10A>C - The variant of uncertain significance c.664-10A>C in TSC1 was reclassified as pathogenic - A strategy using cDNA could be a good complement to conventional molecular diagnosis protocols