Two mouse models carrying truncating mutations in Magel2 show distinct phenotypes

Abstract

Schaaf-Yang syndrome (SYS) is a neurodevelopmental disorder caused by truncating variants in the paternal allele of MAGEL2, located in the Prader-Willi critical region, 15q11-q13. Although the phenotypes of SYS overlap those of Prader-Willi syndrome (PWS), including neonatal hypotonia, feeding problems, and developmental delay/intellectual disability, SYS patients show autism spectrum disorder and joint contractures, which are atypical phenotypes for PWS. Therefore, we hypothesized that the truncated Magel2 protein could potentially produce gain-of-function toxic effects. To test the hypothesis, we generated two engineered mouse models; one, an overexpression model that expressed the N-terminal region of Magel2 that was FLAG tagged with a strong ubiquitous promoter, and another, a genome-edited model that carried a truncating variant in Magel2 generated using the CRISPR/Cas9 system. In the overexpression model, all transgenic mice died in the fetal or neonatal period indicating embryonic or neonatal lethality of the transgene. Therefore, overexpression of the truncated Magel2 could show toxic effects. In the genome-edited model, we generated a mouse model carrying a frameshift variant (c.1690_1924del; p(Glu564Serfs*130)) in Magel2. Model mice carrying the frameshift variant in the paternal or maternal allele of Magel2 were termed Magel2P:fs and Magel2M:fs, respectively. The imprinted expression and spatial distribution of truncating Magel2 transcripts in the brain were maintained. Although neonatal Magel2P:fs mice were lighter than wildtype littermates, Magel2P:fs males and females weighed the same as their wildtype littermates by eight and four weeks of age, respectively. Collectively, the overexpression mouse model may recapitulate fetal or neonatal death, which are the severest phenotypes for SYS. In contrast, the genome-edited mouse model maintains genomic imprinting and distribution of truncated Magel2 transcripts in the brain, but only partially recapitulates SYS phenotypes. Therefore, our results imply that simple gain-of-function toxic effects may not explain the patho-mechanism of SYS, but rather suggest a range of effects due to Magel2 variants as in human SYS patients.

Fig 1. Schematic structure of the human MAGEL2 and mice Magel2.

(A) Human MAGEL2 contains a proline-rich region (residues 13–700), USP7 binding site (U7BS: residues 949–1004), and MAGE homolog domain (MHD: residues 1020–1219). Truncating variants reported previously are indicated by their positions (top; frameshift variants, bottom: nonsense variants). The mutation hotspot is located at nucleotides c.1990-1996. Over half of SYS patients carried c.1996dupC:p.(Q666Pfs*47) in MAGEL2 (in red text). (B) Mouse Magel2 contains proline-rich region (residues 13–646) and MHD (residues 1052–1251).

Fig 2. Strategies to generate transgenic mice.

(A) Strategy to generate the overexpression model which overexpresses the N-terminal region of Magel2 with FLAG-tag. (B) Strategy to generate genome-edited model which carries truncating variant in Magel2. Target sequence for CRISPR/Cas9 (5´-CCACAGGAGCTCCCGGTGCCACA-3´) is located on c.1702-1724, c.1720-1742, c.1810-1832, c.1882-1904 and c.1900-1922 (black triangles).

Fig 3. Generation of a mouse model carrying a frameshift variant in Magel2.

(A) We obtained genome-edited mice carrying different variants in Magel2. We selected #1.10 as the founder mouse. (B) Comparison of the base sequence and amino acid residues in Magel2. Founder mouse #1.10 carried a homozygous frameshift variant in Magel2. (C) The pedigree of our mouse model. Mice carrying a variant in the paternal allele of Magel2 were termed ‘Magel2^P:fs’. Mice carrying a variant in the maternal allele of Magel2 were termed ‘Magel2 ^M:fs’. Littermates not carrying a variant in Magel2 were termed ‘Magel2⁺’.

Fig 4. Expression and distribution of Magel2 in the mouse brain.

(A) Expression of Magel2 transcripts in the neonatal mouse brain. Only the paternal allele of Magel2 is expressed in the brain. (B) Distribution of Magel2 transcripts in young-adult brains was similar in WT and Magel2^P:fs mice. Magel2 mRNAs were expressed in the SCN of the hypothalamus in both groups. SCN: suprachiasmatic nucleus. Scale bar: 100 μm.

Fig 5. Comparison of body weight of affected and WT mice.

(A) Mean weight ± SEM at P10. The weight of Magel2^P:fs was reduced compared to Magel2⁺ (5.44 ± 0.12 g vs 6.11 ± 0.13 g, P = 0.0003). There was no difference between Magel2^M:fs and Magel2⁺ mice (6.00 ± 0.13 g vs 5.72 ± 0.11 g, P = 0.058). Both sexes were included. (B, C) Mean weight ± SEM at four, eight and 12 weeks of age in both sexes. At four weeks, Magel2^P:fs males were lighter than Magel2⁺ (13.71 ± 0.49 g vs 15.84 ± 0.50 g, P = 0.0032), but by eight weeks of age, there was no difference. In females, there was no difference between Magel2^P:fs mice and Magel2⁺ at four, eight and 12 weeks of age.