Precise detection of a murine germline mutation of the Notch3 gene associated with kyphosis and developmental disorders

Abstract

Objective: Humpback (hpbk) mice harbor a pathogenic mutation in the Notch3 gene and can serve as a beneficial animal model for investigating human myopathy, kyphosis, and developmental disorders, including lateral meningocele syndrome. Detection of the point mutation in hpbk mice is important for maintaining strains and scrutinizing genetic rescues, especially considering that homozygous mice are infertile and indistinguishable from their littermates at a young age. This study aimed for the development of a novel, precise, and time-saving genotyping method to identify the mutation in hpbk mice.

Materials and methods: In order to study the hpbk mouse line, we describe how we applied several tools, including quantitative polymerase chain reaction (qPCR), multiplex tetra-primer amplification-refractory mutation system (ARMS-PCR) and Sanger sequencing, toward the recognition of heterozygous and homozygous mice.

Results: The Notch3 mutation was clearly identified using qPCR and ARMS assays, but the latter was a more precise and cost-effective approach. The lengths of the ARMS-PCR amplicons are 210 bp and 164 bp for the wild-type and hpbk alleles, respectively. Moreover, the genotyping results for each mouse were corroborated by Sanger DNA sequencing.

Conclusion: Our newly developed PCR-based ARMS system affords a swift and precise way to genotype the hpbk mice. ARMS-PCR does not rely on any advanced equipment and is useful as a genotyping method for other model organisms that harbor a pathogenic variant.

Keywords: ARMS; Lateral meningocele syndrome; Notch3 mutation; genotyping; skeletal disease.

Figure 1.. Location of PCR primers and partial sequences for the Notch3 receptor mutation and wild-type alleles (NCBI, Gene ID: 18131). The primers for ARMS-PCR system were designed using NCBI Primer-Blast tool. Underline indicates the point mutation (G→A) in the splice donor site at the exon 31-intron 31 boundary of the Notch3 receptor. Specificity is increased by introducing a deliberate second mismatch at −2 base pairs (bp) from the 3’ end of both inner forward and reverse primers, which are indicated in lowercase.

Figure 2.. Detection of the hpbk mutation by the tetra-primer ARMS-PCR analysis. (A) Schematic summary of ARMS-PCR primer design and amplicon gel patterns for the different genotypes. The two outer primers amplify a common large fragment of the Notch3 gene that contains a G→A point mutation (red and underlined). The remaining two inner primers amplify the two allelic states (i.e., in a G→A point mutation, one primer will amplify the A allele (homozygous mutant, Hom) and the other the G allele (wild-type, WT) of Notch3 gene. (B) Genotyping of hpbk mice through tetra-primer ARMS-PCR. M: 100 bp ladder.

Figure 3.. Optimization of the qPCR conditions with newly designed primers. (A) A list of primers (two pairs for the wild-type allele and two pairs for the hpbk allele), primer sequence, and size of amplicons. Red colors indicate the point mutation base pair (G–A). (B) A representative gel image for PCR products from tails of three mice (one per genotype) using two pairs of wild-type primers (WT P1 and WT P2) with PCR conditions (annealing temperature at 60°C and a 36-cycle reaction). (C) A representative gel image for PCR from three mice (one per genotype) using two pairs of primers (mutant Mut P1 and Mut P2) under the same PCR condition (annealing temperature at 60°C and a 36-cycle reaction). (D) Optimization of the PCR cycles (28 and 32) using the same annealing temperature (63°C) for the Mut P1 pair of primers.

Figure 4.. Detection and verification of hpbk mutation with qPCR and Sanger sequencing methods. (A) An example of a qPCR amplification plot showing homozygous (purple), heterozygous (blue), and wild-type (pink) mice with values of Ct. (cycle threshold) at 24.07, 25.39, and 32.76, respectively. (B) Representative Sanger sequencing chromatograms showing a mutation allele (point mutation “A”) in a homozygous hpbk/hpbk mouse (top panel) and a heterozygous mouse (bottom panel) with a black arrow pointing to “G/A” mutation site and wild-type allele. (C) Photo of 8-week-old Hom and their Het littermate. (D) X-ray image of 8-week-old Hom and their Het littermate.

Figure 5.. Sensitivity and stability test of our ARMS-PCR genotyping method. Thirty genomic DNA samples were tested using our established genotyping method. These genomic DNA samples were run after storing a few months to up to 2 years kept at −20°C after extraction. These results indicate that our genotyping method can work well even with old samples. M: 100 bp DNA ladder (the most left and right lanes). WT: 319 + 210 bp, wild-type mice; Het: 319 + 210 + 164 bp, heterozygous mice; Hom: 319 + 164 bp, mutant homozygous mice. Each lane represents one independent sample.