Examples

This page lists a number of examples of variants that can be analyzed using GTGT, sometimes with surprising results

Frameshift mutation in SDHD

The first example concerns a frameshift mutation in exon 2 of SDHD: ENST00000375549.8:c.149_150dup. Click here to view the results in GTGT.

This variant is located in exon 2, which is an in frame exon. As expected, skipping exon 2 is one of the therapies that is suggested by GTGT.

GTGT also lists skipping both exon 2 and 3 together, but as can be seen from the annotations this is not a viable therapy. (This therapy was included in the results due to the fact that it directly modifies the frameshift variant of the patient).

However, GTGT also suggests a third therapy which will restore the reading frame of the transcript, namely skipping exon 3. In the wildtype transcript, exon 3 is not in an frame exon, and thus should not be eligible to skip. However, since the patient as a frame shift mutation in exon 2, skipping exon 3 restores the reading frame again. Whether or not this will be beneficial for the patient will depend on the exact features of the protein which are preserved and the role they play in the disease. For example, we can see from the results that exon 2 contains most of the mitochondrion trans membrane peptide, which is lost when exon 2 is skipped. GTGT shows us that most of this domain can be preserved if we skip exon 3 instead.

Premature STOP in DMD

This example highlights how GTGT supports multiple variants on a single allele, and correctly predict the resulting protein function:

NC_000023.11(NM_004006.3):c.2504_2505delinsAG. Click here to view the results in GTGT.

This example shows two mutations in exon 20 of DMD, which together introduce a STOP codon. As can be seen from the transcript visualization in GTGT from ExonViz, exon 20 is not an in frame exon, so it cannot be skipped to exclude the introduced STOP codon.

However, we can also see from the results of GTGT that the combination of exon 19 and 20 and the combination of 20 and 21 are in frame, and lead to slightly different losses of the Spectrin repeat 4 and 5. Determining which of these therapies is most likely to benefit the patients in left to the user.

Multi-exon deletion in CLN3

CLN3 is a gene which is involved in the lysosome and loss of CLN3 is associated with severe condition called Batten disease. In patients with Batten involving CLN3, the most common mutation is a deletion of exon 8 and 9 (ENST00000636147.2:c.461_677del). Click here to analyze this mutation using GTGT.

As can be seen from Transcript visualization, deletion of exon 8 and 9 causes a frameshift mutation in the transcript. The frameshift leads to the loss of the Lysosomal targetting motif, which is essential for the localisation of CLN3 to the lysosome.

As can be seen from the figure, the reading frame can be restored by skipping either the exon before the deletion (exon 7) or the exon after the deletion (exon 10). Note that skipping exon 10 does not fully preserve the lysosomal targeting motif.

However, GTGT has found another therapy which restores the reading frame of the transcript, namely skipping exon 6. This solution is not intuitive at all, and also difficult to see from the transript visualization of CLN3. The best way to explain it is that skipping exon 6 causes a frameshift when translating from exon 5 to exon 7, which is then cancled by the frameshift caused by the exon 8+9 deletion.

Interestingly, exon 6 is a vulnerable exon that is already partially skipped in healthy individuals. Skipping exon 6 has been shown to restore CLN3 function and localisation to the lysosome in patient cell lines (Centa2020). Note that they use a different transcript of CLN3 which lacks the first noncoding exon, so the deletion is exon 7+8, and the exon they skip is exon 5.