ClinVar Genomic variation as it relates to human health

This sequence change falls in intron 26 of the TSC2 gene. It does not directly change the encoded amino acid sequence of the TSC2 protein. It affects a nucleotide within the consensus splice site. This variant is not present in population databases (gnomAD no frequency). This variant has not been reported in the literature in individuals affected with TSC2-related conditions. ClinVar contains an entry for this variant (Variation ID: 639030). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance.

The c.2967-1G>T intronic variant results from a G to T substitution one nucleotide upstream from coding exon 26 of the TSC2 gene. Alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. RNA studies have demonstrated that this alteration results in a transcript predicted to lead to a protein with an in-frame deletion of one amino acid; however, the exact functional impact of the deleted amino acid is unknown at this time (Ambry internal data). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site. Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear.

Canonical splice site variant predicted to result in an in-frame loss of the adjacent exon in a gene for which loss of function is a known mechanism of disease; Observed as a paternally inherited variant in an individual with epilepsy and developmental delaying undergoing whole exome sequencing; neither had any typical features of TSC (PMID: 35692821); Not observed at significant frequency in large population cohorts (gnomAD); This variant is associated with the following publications: (PMID: 30476936, 35692821)

c.2967-1G>T identified in a child with epilepsy and developmental delay, who does not have typical features of tuberous sclerosis. This variant is a novel canonical -1 splice site variant at the invariant acceptor site of exon 27 of the TSC2 gene, and is not present in population databases (1000 Genomes, Exome Aggregation Consortium, and Genome Aggregation Database). However, this variant was inherited from the patient's father, who had no history of seizures or other neurological problems. Although the patient and her father were not clinically considered for tuberous sclerosis, due to the genetic results, a pediatric neurologist conducted a clinical evaluation of the patient and his father. The patient and her father did not present with typical symptoms of TSC.  In addition, we found that this mutation is located in a NAGNAG receptor, which has been recorded in the TAndem Splice Site DataBase (TassDB, http://www.tassdb.info).The NAGNAG receptor includes two 3′ tandem acceptors and mainly results in the insertion/deletion of one amino acid. Through blood RNA analysis and minigene assay, we found this variant disrupts the upstream site leads to the activation of the downstream one and the subsequent mRNA is expected to encode only the isoform without one amino acid. We evaluated the loss of this amino acid in the context of two different protein sequences (NP_000539.2 and NP_066399.2) using the PROVEAN protein tool[24]. The difference between the two protein sequences is whether or not they contain the amino acid encoded by exon 26. The PROVEAN scores for the absence of this amino acid were 0.516(NP_000539.2) and -1.337(NP_066399.2), respectively. The prediction (cutoff = -2.5) were neutral. Our results suggest that the c.2967-1G>T variant disrupts the balance of an alternative splicing event (formerly called alternative splice to NAGNAG acceptors) which consists of the use of two alternative acceptors only 3 bp apart. Alternative splicing at NAGNAG acceptors is widespread and contributes to proteome plasticityand such NAGNAG acceptor sites can ameliorate or bypass the phenotype of a mutation. This may be the reason that this variant did not related to the typical symptoms of TSC.