Application of RNA Analysis and Whole Genome Sequencing for Confirmation of Diagnosis in Mucopolysaccharidosis Type VI Patients

Background/Objectives:

Mucopolysaccharidosis type VI (MPS VI) is a lysosomal storage disorder associated with biallelic pathogenic variants in the ARSB gene. Herein, we present three patients with biochemical and clinical pictures of MPS VI for whom the routine molecular genetic analysis using Sanger sequencing of ARSB identified one or both causative variants.

Methods:

Sanger sequencing of eight ARSB exons failed to identify one or both causative variants, so RNA analysis of patients’ white blood cells was performed, with one case requiring subsequent whole-genome sequencing.

Results:

RNA analysis of patients’ white blood cells revealed alterations in the expression of ARSB mRNA isoforms in all three cases. Patient 1 was heterozygous for one pathogenic variant with mRNA analysis demonstrating the allelic imbalance characteristic of premature transcription termination, that is, an almost complete absence of full-length mRNA isoforms from the chromosome with the unidentified variant coupled with reduced expression of isoforms ending after exon 4. Whole-genome sequencing identified a complex structural variant, a 52 kb insertion of the LHFPL2 gene fragment in the ARSB intron 4 derived from unequal non-allelic homologous recombination. RNA sequencing and RACE were performed to identify the cause of premature transcription termination, the 3’ end of the chimeric mRNA isoform. Patients 2 and 3 had a deep intronic c.1142+581AG variant in a homozygous state, leading to cryptic exon activation and intragenic deletion c.500-1756_562del in a homozygous state altering the integrity and splicing of ARSB exon 2, respectively.

Conclusion:

These results expand the mutational spectrum of the ARSB gene towards complex structural variants and novel molecular-genetic mechanisms of MPS VI pathogenesis and emphasise the utility of RNA analysis and whole-genome sequencing in establishing the MPS VI diagnosis.