Two ENU-induced alleles of Atp2b2 cause deafness in mice

Abstract

Over 120 loci are known to cause inherited hearing loss in humans. The deafness gene has been identified for only half of these loci. With the aim of identifying some of the remaining deafness genes, we performed an ethylnitrosourea mutagenesis screen for deaf mice. We isolated two mutants with semi-dominant hearing loss, Deaf11 and Deaf13. Both contained causative mutations in Atp2b2, which encodes the plasma membrane calcium ATPase 2. The Atp2b2 (Deaf11) mutation leads to a p. I1023S substitution in the tenth transmembrane domain. The Atp2b2 (Deaf13) mutation leads to a p. R561S substitution in the catalytic core. Mice homozygous for these mutations display profound hearing loss. Heterozygotes display mild to moderate, progressive hearing loss.

Figure 1. Deaf11 and Deaf13 mice harbor point mutations in Atp2b2.

DNA sequence electropherograms of exon 17 in A) +/+ and B) Deaf11/Deaf11 mice. Results are representative of four +/+, 14 +/Deaf11 and 10 Deaf11/Deaf11 mice. DNA sequence electropherograms of exon 9 in C) +/+ and D) Deaf13/Deaf13 mice. Results are representative of two +/+, four +/Deaf13 and four Deaf13/Deaf13 mice. Mutation sites are marked by asterisks. Predicted effect on amino acid sequence is shown below. E) Amino acid sequence alignments for regions surrounding predicted Deaf11 and F) Deaf 13 amino acid substitutions (yellow). Alignments were performed with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) using protein sequences Q9R0K7.2, P11506.2, Q01814.2, NP_080758.1, NP_796210.2, AAI09173.1, NP_001033803.3, NP_001087020.1 and CAA09303.1.

Figure 2. Deaf11 and Deaf13 mice display hearing loss.

Mean ABR thresholds at A) 4 and B) 8 weeks of age; Atp2b2 ^+/+ n=3 female (F), 6 male (M) at 4 wks, n = 4F, 5M at 8 wks, Atp2b2 ^+/Deaf11 n = 6F, 2M at 4 wks, n = 7F, 4M at 8 wks, Atp2b2 ^+/Deaf13 n = 7F, 10M at 4 wks, n = 5F, 9M at 8 wks, Atp2b2 ^{Deaf11/Deaf11} n = 3F, 6M at 4 wks, n = 2F and 5M at 8 wks, Atp2b2 ^{Deaf13/Deaf13} n = 2F, 4M, Atp2b2 ^{Deaf11/Deaf13} n = 4F, 3M; * p<0.01 versus Atp2b2 ^+/+; Error bars show standard deviation.

Figure 3. The Deaf11 mutation is linked to chromosome 6.

A Deaf11/Deaf11 BALB/c mouse was crossed to a +/+ C57BL/6 mouse and the offspring intercrossed to generate 87 N ₁F₁ mice. Mice were divided into those with a click ABR threshold ≤55 dB SPL on left (+/+ or +/Deaf11) and those with a threshold ≥75 dB SPL on right (Deaf11/Deaf11). All mice were genotyped for SNPs on chromosome 6. SNP names and locations are listed on the left and right respectively (assembly GpCm38, database SNP, http://www.ncbi.nlm.nih.gov/projects/SNP/). The numbers of mice displaying each haplotype are listed below. Informative recombinations indicate that the Deaf11 causative mutation lies between rs13478976 at 112 Mb and rs3152403 at 121 Mb. Atp2b2 is located at 113 Mb.

Figure 4. Deaf11 and Deaf13 cochlear hair cells are morphologically abnormal.

Middle cochlear turn sections at 8 weeks of age; Upper panels 100x magnification, scale bar 200 µm; Lower panels 400x magnification, scale bar 50 µm; A, D) Atp2b2 ^+/+ B, E) Atp2b2 ^{Deaf11/Deaf11} C, F) Atp2b2 ^{Deaf13/Deaf13}; White arrowheads point to inner hair cells; Black arrowheads point to outer hair cells; Each section is representative of 4 cochleae.