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.