Generation of a novel disease model mouse for mucopolysaccharidosis type VI via c. 252T>C human ARSB mutation knock-in

Abstract

Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a mutation in the ARSB gene, which encodes arylsulfatase B (ARSB), and is characterized by glycosaminoglycan accumulation. Some pathogenic mutations have been identified in or near the substrate-binding pocket of ARSB, whereas many missense mutations present far from the substrate-binding pocket. Each MPS VI patient shows different severity of clinical symptoms. To understand the relationship between mutation patterns and the severity of MPS VI clinical symptoms, mutations located far from the substrate-binding pocket must be investigated using mutation knock-in mice. Here, I generated a knock-in mouse model of human ARSB Y85H mutation identified in Japanese MPS VI patients using a CRISPR-Cas9-mediated approach. The generated mouse model exhibited phenotypes similar to those of MPS VI patients, including facial features, mucopolysaccharide accumulation, and smaller body size, suggesting that this mouse will be a valuable model for understanding MPS VI pathology.

Keywords: Arylsulfatase B; CRISPR-Cas9 system; Disease model; Mucopolysaccharidosis type VI.

Fig. 1

Y85 conservation and strategy of mutation knock-in. (A) Scheme of the human ARSB gene; 252T is located in exon 1 of the human ARSB gene. (B) Conserved Y85 in mouse. Human and mouse ARSB genomic sequences are aligned. The amino acid sequences are also shown on top of the genomic sequences. Y85 is conserved in mice asY86. Red nucleotides and amino acids indicate target bases and amino acids. The blue nucleotides and amino acids indicate non-conserved bases and amino acid. (C) The strategy of CRISPR-Cas9-mediated mutation knock-in. We used 73 ssODN bases as the donor template. The red nucleotides show the targeted bases. The dotted line indicates PAM, and the normal line indicates the target sequence. (D) The sequence results in F2 mice generation. The center panel shows the ARSB sequence of heterozygous mutant mice, and the right panel shows the sequence of homozygous mutant mice.

Fig. 2

MPS VI-like phenotypes in Y86H homozygous mutant mice. (A) Wild type and homozygous mutant 10-months-old male mice. The body size of mutant mice is smaller than wild type mice. Bar: 2 cm (B) 10-months-old male mutant mice show characteristic facial features. Bar: 2 cm (C) X-ray CT analysis results. Shortened bone size was observed in homozygous mice. Bar: 1 cm (D) The graph of male mice body weight at each time point. The graph shows growth defects in mutant mice. The values represent means ± SD (n＝4). **P < 0.01 (one-way ANOVA, Tukey-Kramer test).

Fig. 3

The biochemical and histological analysis of the generated mutant mice. (A) ARSB enzyme activity in liver extracts from each 10-months-old male mouse. Enzyme activity in the liver extract decreased significantly in mutant mice. Activity was not detected in antibody treated samples. The values represent means ± SD (n＝3). **P < 0.01 (one-way ANOVA, Tukey-Kramer test). (B) ARSB enzyme activity in kidney extracts derived from each 10-months-old male mice. Enzyme activity in the kidney extract decreased significantly in mutant mice. Activity was not detected in antibody treated samples. The values represent means ± SD (n＝3). **P < 0.01 (one-way ANOVA, Tukey-Kramer test). (C) Colloid iron staining using liver sections derived from each 10-months-old male mouse. Acidic mucopolysaccharide was detected in the liver tissue sections of homozygous mutant mice. The red arrowheads indicate accumulated mucopolysaccharides. Bar: 50 μm.

Supplementary Fig. 1

Analysis of ARSA, ARSC, ARSD and ARSE loci off-target mutations in the generated mutant mice. (A) Sequence results for the ARSA locus. (B) Sequence results of ARSC locus. (C) Sequence results for the ARSD locus. (D) Sequence results for the ARSE locus. Off-target mutations were not identified in these genes. Blue indicates non-conserved bases.

Supplementary Fig. 2

Analysis of ARSG, ARSH, ARSI and ARSJ loci off-target mutations in the generated mutant mice. (A) Sequence results for the ARSG locus. (B) Sequence results of ARSH locus. (C) Sequence results for the ARSI locus. (D) Sequence results for the ARSJ locus. Off-target mutations were not identified in these genes. Blue indicates non-conserved bases.

Supplementary Fig. 3

Analysis of GALNS, SGSH, GNS and IDS loci off-target mutations in the generated mutant mice. (A) Sequence results for the GALNS locus. (B) Sequence results of SGSH locus. (C) Sequence results for the GNS locus. (D) Sequence results for the IDS locus. Off-target mutations were not identified in these genes. Blue indicates non-conserved bases.

Supplementary Fig. 4

Analysis of SULF1, SULF2 and TSULF loci off-target mutations in the generated mutant mice. (A) Sequence results for the SULF1 locus. (B) Sequence results of SULF2 locus. (C) Sequence results for the TSULF locus. Off-target mutations were not identified in these genes. Blue indicates non-conserved bases.

Supplementary Fig. 5

Analysis of off-target mutations in candidate sequences identified via the COSMID search. (A) Sequence results for Chr 11:22695672–22695694 (B) Sequence results for Chr 14:32167864–32167886. Off-target mutations were not detected in the mutant mice. Blue indicates non-conserved bases.

Supplementary Fig. 6

Immunofluorescence microscopy of liver tissues derived from each 10 months-old male mouse using anti-ARSB antibodies. The Y86H mutant protein aggregated in liver tissues. The wild type ARSB protein showed a diffusible localization. Alexa 488 signals were not detected in negative control samples. Bar: 20 μm

Supplementary Fig. 7

Immunofluorescence microscopy of kidney tissues derived from each 10 months-old male mouse using anti-ARSB antibodies. The Y86H mutant protein aggregated in kidney tissues. The wild type ARSB protein showed a diffusible localization. Alexa 488 signals were not detected in negative control samples. Bar: 20 μm

Supplementary Fig. 8

Detection of Y86H mutant protein by western blotting analysis. (A) Y86H mutant protein in liver lysates was detected by western blotting. (B) Y86H mutant protein in kidney lysates was detected by western blotting. Lysates were prepared from each 10-months-old male mice. GAPDH was used for loading control.

Supplementary Fig. 9

The uncropped images of Supplementary Fig. 8. Three experiments were performed using each tissue lysate from different mice.