Phenotype-oriented NGS panels for mucopolysaccharidoses: Validation and potential use in the diagnostic flowchart

Abstract

Mucopolysaccharidosis (MPS) are a group of rare genetic disorders caused by deficiency in the activity of specific lysosomal enzymes required for the degradation of glycosaminoglycans (GAGs). A defect in the activity of these enzymes will result in the abnormal accumulation of GAGs inside the lysosomes of most cells, inducing progressive cellular damage and multiple organ failure. DNA samples from 70 patients with biochemical diagnosis of different MPSs genotypes confirmed by Sanger sequencing were used to evaluate a Next Generation Sequencing (NGS) protocol. Eleven genes related to MPSs were divided into three different panels according to the clinical phenotype. This strategy led to the identification of several pathogenic mutations distributed across all exons of MPSs-related genes. We were able to identify 96% of all gene variants previously identified by Sanger sequencing, showing high sensitivity in detecting different types of mutations. Furthermore, new variants were not identified, representing 100% specificity of the NGS protocol. The use of this NGS approach for genotype identification in MPSs is an attractive option for diagnosis of patients. In addition, the MPS diagnosis workflow could be divided in a two-tier approach: NGS as a first-tier followed by biochemical confirmation as a second-tier.

Figure 1. Deletion visualized in the decrease coverage of log₂₅ values. Relative copy number derived from coverage data per target. The red squares mark the probable deletion of the exons IV to VIII of IDS gene.

Figure 2. IDUA gene amplicon coverage. (a) Intra-run relative depth of coverage (RDoC) at 14 exons corresponding to 8 samples sequenced in 6 runs; (b) IDUA gene amplicon coverage of second design. Intra-run RDoC at 14 exons corresponding to 8 samples sequenced in 1 run.

Figure 3. Flow Chart for MPS investigation.