Complementary Sanger Sequencing
IGV review of the gene/genes matched with the clinical MPS type was
carried out for each of the cases with no primary IEM-panel findings.
This approach led to the identification of zero or poor coverage in exon
1 of ARSB , IDUA , GALNS , and NAGLU genes.
Complementary Sanger sequencing of these poorly covered regions resulted
in discovering 26 additional patients, improving the diagnostic yield by
14%. This finding is in accordance with the study reported by
Brusius-Facchin, et.al (2019) in which they observed failure of NGS in
sequencing exon 1 of IDUA , ARSB , NAGLU ,HGSNAT , and GALNS genes likely as a result of high GC
content and repeat regions. As a result, complementary Sanger sequencing
for exon 1 of the aforementioned genes is hereby suggested to ensure
that a pathogenic variant is not overlooked because of this shortcoming.
Furthermore, based on the same approach, the IGV review led to the
identification of 4 variants not previously annotated by the Ion
Reporter®. MPS-23 was found to have a single-nucleotide deletion in exon
12 of GALNS (c.1279del) overlooked by Ion Reporter®, which was
confirmed by Sanger sequencing. Additionally, two homozygous splicing
variants in IDUA (g.997263G>A,
c.1650+5G>A), and HGSNAT(g.43033381A>G, c.1012+4A>G) were uncovered in
MPS-144 and MPS-155 respectively. It is likely that the Ion Reporter®
failed to annotate these variants because they were more than 3 base
pairs downstream of the exons. Review of the IGV data also revealed that
MPS-166 harbored a pathogenic variant adjacent to a polymorphism. The
pathogenic variant was not annotated because these two adjacent variants
were grouped together as one multi-nucleotide variant which was then
filtered out due to its high population frequency. Overall,
complementary Sanger Sequencing, carried out following the IGV review,
allowed us to rule out false positive and false negative results with a
significant reduction in time and resources.