Homozygous mice have a phenotype similar to human Chediak-Higashi syndrome patients, exhibiting lysosomal dysfunction with resultant protein storage; diluted coat color; abnormal melanogenesis; immune cell dysfunction resulting in increased susceptibility to bacterial, viral, and parasitic infections and decreased cytotoxic activity against tumor cells.
Figure 1. Hypopigmentation phenotype of the 50-cal mice (bottom). C57BL/6J mouse (top) is shown for reference.
Figure 2.50-calmice exhibited increased viral titers in the spleen after mouse cytomegalovirus (MCMV) infection. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 3.50-calmice exhibited increased viral titers in the liver after mouse cytomegalovirus (MCMV) infection. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
The 50-cal phenotype was identified among ENU-induced G3 mice of the pedigree R1475, some of which had a dark grey coat color (Figure 1). Some mice also showed increased mouse cytomegalovirus (MCMV) titers in the spleen (Figure 2) and liver (Figure 3) after MCMV infection.
Nature of Mutation
Figure 4.Linkage mapping of the increased MCMV titer in the spleen after MCMV infection using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 54 mutations (X-axis) identified in the G1 male of pedigree R1475. Normalized phenotype data are shown for single locus linkage analysis with consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.
Whole exome HiSeq sequencing of the G1 grandsire identified 54 mutations. The increased susceptibility to MCMV infection phenotype was linked by continuous variable mapping to a mutation in Lyst. The Lyst mutation is a T to A transversion at base pair 13,708,212 (v38) on chromosome 13, or base pair 117,881 in the GenBank genomic region NC_000079 within the donor splice site of intron 34. The strongest association was found with a recessive model of linkage to the normalized MCMV titer in the spleen, wherein 1 variant homozygote departed phenotypically from 10 homozygous reference mice and 9 heterozygous mice with a P value of 5.221 x 10-50 (Figure 4). The effect of the mutation at the cDNA and protein level have not examined, but the mutation is predicted to result in skipping of the 189-nucleotide exon 34 (out of 53 total exons), resulting in coding of one aberrant amino acid (S2857R); the rest of the protein product is unchanged.
The donor splice site of intron 34, which is destroyed by the 50-cal mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red. The highlighted amino acid (S2857) is aberrant as the result of the mutation.
Alternatively, the use of a cryptic site in exon 34 may be used resulting in deletion of 89 nucleotides in exon 34. The deletion would result in a frameshift, coding of four aberrant amino acids after amino acid 2890, and coding of a premature stop codon after amino acid 2894.
The donor splice site of intron 34, which is destroyed by the 50-cal mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red. The putative cryptic splice donor site is highlighted.
Figure 5. Domain structure of the Lyst protein. The Lyst protein is a 3788-amino acid protein whose biochemical functions remain unknown. The N-terminal portion of the protein contains approximately twenty repeats with homology to ARM and HEAT repeat motifs and a perilipin domain (PD). The C-terminal portion of Lyst contains a BEACH domain and seven WD40 motifs. The 50-cal mutation occurs in intron 34. This image is interactive. Click on the image to view other mutations found in Lyst (red). Click on the mutations for more specific information. There are two additional alleles in the Lyst gene: sooty and grey wolf. The locations of their mutations is unknown.
The Lyst gene encodes the protein Lyst (also CHS/Beige), a 3788-amino acid protein whose biochemical functions remain unknown (Figure 3). A large N-terminal portion of the protein (amino acids 1-3132) contains approximately twenty repeats with homology to ARM (Armadillo) and HEAT (huntingtin, elongation factor 3, A subunit of protein phosphatase A, target of rapamycin) repeat motifs (1;2). ARM and HEAT motifs are α-helical domains of about 50 amino acids that pack together to form elongated “solenoids” (3); evidence suggests they mediate protein associations at the membrane (4), and vesicle transport (5), respectively. The C-terminus of Lyst contains two distinct domains, a BEACH (beige and chediak) domain (amino acids 3132-3472) and seven WD40 motifs (1). The BEACH domain is a 345-amino acid region of unknown function (1), and WD40 motifs are protein interaction motifs that typically form β sheets arranged in a 7-bladed β propeller fold (6).The 50-cal mutation is predicted to affect the ARM/HEAT domain.
Please see the record for souris for information about Lyst.
In humans, mutations in the LYST gene cause Chediak-Higashi Syndrome (CHS, OMIM #214500), a rare autosomal recessive disorder characterized by oculocutaneous albinism, severe immune deficiency, bleeding tendency, recurrent pyogenic infection, progressive neurologic defects and a lymphoproliferative syndrome [(7;8); reviewed in (2)]. These defects are caused by the aberrant formation of giant granules within a variety of cell types, and disrupted intracellular protein trafficking (2;9;10). The enlarged granules consist of organelles such as lysosomes, melanosomes, cytolytic granules and platelet dense bodies, and it is thought that the increased size of these organelles inhibits their migration and fusion at the cell surface and/or organelle-organelle fusion. There is no clear understanding of the molecular mechanisms of Lyst protein function, or how its loss leads to the formation of enlarged lysosomes and lysosome-related organelles. In mice, mutations in Lyst cause the beige phenotype (7;8). As in humans, beige mice exhibit hypopigmentation, bleeding tendency, and defective immune cell function resulting from the formation of giant granules in melanosomes, lymphocytes, neutrophils, and other cell types (10-12). Beige mice have defective NK cell (13) and cytotoxic T lymphocyte function (14), and increased susceptibility to infections including MCMV (15;16). However, beige mice do not develop lymphoproliferative disorder, even after challenge with infection (15). The 50-cal hypopigmentation and MCMV susceptibility phenotype mimics other known alleles of Lyst (see MGI for a list of Lyst alleles as well as the Beutler alleles souris and charlotte_gray) indicating that there is loss of function in the Lyst50-cal protein.
50-cal(F):5'- CAGAGGCTCGATTTCAAATCCAAGGATA -3'
50-cal(R):5'- ctccctagccccCTCATAATGTACC -3'
50-cal_seq(F):5'- TCTCCCAAGGCATTGAAAGG -3'
50-cal_seq(R):5'- tccgtaatgagacctgacacc -3'