9. Example 4 Likely Pathogenic EGFR In-Frame Insertion

The final example is variant number 4 and is an in-frame insertion in EGFR. Select this variant card and look at the Population tab. This tab shows that the variant is novel by looking at the frequency tracks, so includes the PM2 criteria. Next, move to the Gene Impact tab.

PM1 scoring criteria hot spot

Figure 9-1: Analyzing the PM1 hot spot scoring criteria.

Expanding the Gene Region and Mutation Profile section and showing the details for PM1 explain that this 12 bp in-frame insertion is located in a mutational hotspot, with 2 other variants within 6 amino acids having been shown to be pathogenic and none shown to be benign. Along with scoring PM1 (Hot spot), we can score PM4 as this insertion changes the protein lengthby adding 4 additional residues while not being in a repeat region. Finally, we can see that in our Computation Evidence section that this position is conserved by GERP++ and PhyloP across 100 vertebrate species and we can score PP3.

While looking at this section, we can consider the representation of any insertion or deletion with the display of the alignment of the mutation against the transcript strand. The standard procedure is to represent an insertion or deletion in the right aligned format, or 3’ alignment against the transcript. However, in forward strand genes, this position is opposite of how a variant is represented in VCF file (left-aligned to the reference sequence).

VSClinical automatically re-aligns the variant to the canonical 3’ transcript position, but allows you to view the variant in either alignment. This particular variant moves 12bp and across a intron-exon boundary and would be completely mis-classified by any tool not performing right-shifting normalization.

variant alignment direction

Figure 9-2: Comparing the influence of alignment direction.

Next, take a look at populating a report with the ACMG results.