Next-Generation Sequencing
Implementation of NGS for discovering MPS variants has offered a number of advantages in this study. Firstly, as a low-throughput technique, Sanger sequencing works on a gene-dependent exon-by-exon basis. Therefore, lack of a clinically designated MPS type would lead to rounds of labor-intensive gene-by-gene analyses. Due to the wide range of symptoms shared by MPS types, a misdiagnosis of the MPS type is also probable. Secondly, CNV analysis, which conventionally required separate genetic investigations, was integrated within our NGS workflow that resulted in an improvement of the diagnostic yield at a fraction of time and cost.
As recorded in the IMPRESsion study, NGS could overcome these limitations by sequencing all MPS genes in a single test per patient. This capability was also expanded to screen for other MPS differential diagnoses, which led to the identification of 4 cases with other lysosomal storage disorders including sialidosis I/II, galactosialidosis, mucolipidosis II/III alpha/beta and mucolipidosis III gamma and one case of L-2-Hydroxyglutaric Aciduria. Also, such comprehensive analysis diagnosed 2 cases with dual molecular diagnosis; the first with homozygous variants in GALNS and IDUA and the second with homozygous variants in GALNS and HOGA1genes and the dual diagnoses of MPS IV and primary hyperoxaluria type III. These variant combinations could support the recently proposed concept of complex genetic models where disease-causing variants in multiple genes cause or modify the development of one or several disease phenotypes, and challenge the classic concept of one gene leading to a particular phenotype that now appears to be an oversimplification (Papadimitriou et al. 2019).
Overall, the applied genetic workflow in group B cases could not confirm MPS in 16.8%, including 26 negative patients and 5 non-MPS IEMs. Lack of a clinically designated MPS type was evident in 34.6% (9/26) of negative cases, which was more than four times higher than that of positive cases (7.8%, 12/154). Such uncertainty could be as a result of Inconclusive biochemical assays and/or non-specific clinical symptoms. The most common symptoms in these negative patients were intellectual disability, developmental delay, and short stature which are not unique to MPS and overlap with other genetic or non-genetic disorders. In addition, the lack of finding variants in these patients might be due to an NGS shortcoming in sequencing GC rich regions. Although complementary sanger sequencing has been performed to compensate for these limitations, the possibility of uncovered pathogenic variants in these regions, as well as upstream, downstream, deep intronic regions and even in unknown MPS associated genes should still be considered.
The workflow also revealed an incompatibility between the genetic and clinical type of MPS in four patients of group B (2.8%). To further elaborate on the four discordant cases, MA-032 had inconclusive enzyme assay results, and MPS-154 was not tested by an enzyme assay, and so their diagnosis was based only on the clinical features. Since MPS VI shares a variety of symptoms with MPS I and MPS IV, making a differential diagnosis based solely on the clinical signs and symptoms could be error prone. Additionally, in MPS-213 and MPS-142, clinical diagnoses were based on the enzymatic assays conducted on dried blood samples (DBS), once for each patient. These limitations may have led to a discordant diagnosis as the standard diagnostic guidelines strongly recommend confirmation of DBS findings by a second independent assay on DBS, since the number of cells in DBS is lower than in other methods, and the sample is more susceptible to environmental factors (Wood et al. 2013). Moreover, diagnosis of MPS I with DBS is even more unreliable since α-iduronidase becomes less stable in DBS compared to the other enzymes (Cobos et al. 2015; Khan et al. 2017; Tomatsu et al. 2011). As two of these discordant cases were under enzyme replacement therapy based on their clinical diagnosis, this finding was of paramount importance in providing appropriate treatment for the patients.