A Generic Assay to Detect Aberrant ARSB Splicing and mRNA Degradation for the Molecular Diagnosis of MPS VI

Broeders, Mike; Smits, Kasper; Goynuk, Busra; Oussoren, Esmée; Hout, Hannerieke J.M.P. van den; Bergsma, Atze J.; Ploeg, Ans T. van der; Pijnappel, W.W.M. Pim

doi:10.1016/j.omtm.2020.09.004

Cited by 7 publications

(8 citation statements)

References 44 publications

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“…Patient #4 was homozygous for ARSB c.937C>T, and a sibling of patient #1. No disease-associated variant for patient #5 had been identified, but we recently confirmed the molecular diagnosis at the RNA level (Broeders et al, 2020). hiPSC lines were generated from primary fibroblasts using lentiviral expression of Oct4, Sox2, Klf-4, and C-Myc as described (van der Wal et al, 2018).…”

Section: Generation Of Patient-derived Hipscs and Gene Editing To Gen...mentioning

confidence: 74%

Modeling cartilage pathology in mucopolysaccharidosis VI using iPSCs reveals early dysregulation of chondrogenic and metabolic gene expression

Broeders

Rooij

Oussoren

et al. 2022

Front. Bioeng. Biotechnol.

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Mucopolysaccharidosis type VI (MPS VI) is a metabolic disorder caused by disease-associated variants in the Arylsulfatase B (ARSB) gene, resulting in ARSB enzyme deficiency, lysosomal glycosaminoglycan accumulation, and cartilage and bone pathology. The molecular response to MPS VI that results in cartilage pathology in human patients is largely unknown. Here, we generated a disease model to study the early stages of cartilage pathology in MPS VI. We generated iPSCs from four patients and isogenic controls by inserting the ARSB cDNA in the AAVS1 safe harbor locus using CRISPR/Cas9. Using an optimized chondrogenic differentiation protocol, we found Periodic acid–Schiff positive inclusions in hiPSC-derived chondrogenic cells with MPS VI. Genome-wide mRNA expression analysis showed that hiPSC-derived chondrogenic cells with MPS VI downregulated expression of genes involved in TGF-β/BMP signalling, and upregulated expression of inhibitors of the Wnt/β-catenin signalling pathway. Expression of genes involved in apoptosis and growth was upregulated, while expression of genes involved in glycosaminoglycan metabolism was dysregulated in hiPSC-derived chondrogenic cells with MPS VI. These results suggest that human ARSB deficiency in MPS VI causes changes in the transcriptional program underlying the early stages of chondrogenic differentiation and metabolism.

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Section: Generation Of Patient-derived Hipscs and Gene Editing To Gen...mentioning

confidence: 74%

Modeling cartilage pathology in mucopolysaccharidosis VI using iPSCs reveals early dysregulation of chondrogenic and metabolic gene expression

Broeders

Rooij

Oussoren

et al. 2022

Front. Bioeng. Biotechnol.

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“…4 ). Residual pseudoexon inclusion leading to a nonfunctional transcript in control individuals has been detected for several genes involved in disease [ 63 , 64 ]. It was initially considered an undesired unproductive splicing event.…”

Section: Discussionmentioning

confidence: 99%

Antisense Oligonucleotide Rescue of Deep-Intronic Variants Activating Pseudoexons in the 6-Pyruvoyl-Tetrahydropterin Synthase Gene

Martínez-Pizarro

Leal

Holm

et al. 2022

Nucleic Acid Therapeutics

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We report two new 6-pyruvoyl-tetrahydropterin synthase splicing variants identified through genomic sequencing and transcript analysis in a patient with tetrahydrobiopterin deficiency, presenting with hyperphenylalaninemia and monoamine neurotransmitter deficiency. Variant c.243 + 3A>G causes exon 4 skipping. The deep-intronic c.164–672C>T variant creates a potential 5′ splice site that leads to the inclusion of four overlapping pseudoexons, corresponding to exonizations of an antisense short interspersed nuclear element AluSq repeat sequence. Two of the identified pseudoexons have been reported previously, activated by different deep-intronic variants, and were also detected at residual levels in control cells. Interestingly, the predominant pseudoexon is nearly identical to a disease causing activated pseudoexon in the F8 gene, with the same 3′ and 5′ splice sites. Splice switching antisense oligonucleotides (SSOs) were designed to hybridize with splice sites and/or predicted binding sites for regulatory splice factors. Different SSOs corrected the aberrant pseudoexon inclusion, both in minigenes and in fibroblasts from patients carrying the new variant c.164–672C>T or the previously described c.164–716A>T. With SSO treatment PTPS protein was recovered, illustrating the therapeutic potential of the approach, for patients with different pseudoexon activating variants in the region. In addition, the natural presence of pseudoexons in the wild type context suggests the possibility of applying the antisense strategy in patients with hypomorphic PTS variants with the purpose of upregulating their expression to increase overall protein and activity.

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“…3.3.1 | This CE in Arylsulfatase B (ARSB-OMIM #300461) was discovered by Broeders et al (2020) as a sporadic inclusion in both patient and healthy control RNAs from primary human fibroblasts treated with cycloheximide, an NMD inhibitor. Broeders and colleagues noted that the donor site of this CE, which bears a non-canonical AT flanking dinucleotide in the reference sequence, was not predicted by any of the algorithms they tested.…”

Section: Potential Snptic Exonsmentioning

confidence: 99%

A spotter’s guide to SNPtic exons: The common splice variants underlying some SNP–phenotype correlations

Keegan

Fletcher

2021

Molec Gen & Gen Med

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A Generic Assay to Detect Aberrant ARSB Splicing and mRNA Degradation for the Molecular Diagnosis of MPS VI

Cited by 7 publications

References 44 publications

Modeling cartilage pathology in mucopolysaccharidosis VI using iPSCs reveals early dysregulation of chondrogenic and metabolic gene expression

Modeling cartilage pathology in mucopolysaccharidosis VI using iPSCs reveals early dysregulation of chondrogenic and metabolic gene expression

Antisense Oligonucleotide Rescue of Deep-Intronic Variants Activating Pseudoexons in the 6-Pyruvoyl-Tetrahydropterin Synthase Gene

A spotter’s guide to SNPtic exons: The common splice variants underlying some SNP–phenotype correlations

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