The Chor_muang phenotype was identified among G3 mice of the pedigree R6492, some of which showed reduced B to T cell ratios (Figure 1) as well as reduced frequencies of B cells (Figure 2) and NK cells (Figure 3) with concomitant increased frequencies of CD44+ CD8 T cells (Figure 4), central memory CD4 T cells in CD4 T cells (Figure 5), and effector memory CD4 T cells in CD4 T cells (Figure 6). Some mice showed reduced B220 expression on peripheral blood B cells (Figure 7). Some mice also showed reduced systolic blood pressures (Figure 8).

Whole exome HiSeq sequencing of the G1 grandsire identified 60 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Ptprc: a T to A transversion at base pair 138,113,562 (v38) on chromosome 1, or base pair 62,195 in the GenBank genomic region NC_000067 within the splice donor site of intron 9 (2-base pairs from exon 9). The strongest association was found with an additive model of inheritance to the B220 MFI, wherein two variant homozygotes and 22 heterozygous mice departed phenotypically from 15 homozygous reference mice with a P value of 1.013 x 10^-20 (Figure 9).  

The effect of the mutation at the cDNA and protein levels has not been examined, but the mutation is predicted to result in the use of a cryptic site in intron 9. The resulting transcript would have a 53-base pair insertion of intron 9 that would cause an in-frame protein product beginning after amino acid 203 of the protein [which is normally 1,293 amino acids in length (isoform 1)], and termination after the inclusion of 13 aberrant amino acids.

C57BL/6J:

            <--exon 8       <--exon 9 intron 9-->          exon 10-->

60126 ……AAGCAAACATGTG ……TATGTACCACCAG gtgaatgtcaatttctct…… GGACTGACAAG……

200   ……-K--Q--T--C-- ……-Y--V--P--P--                      G--T--D--K-……

Genomic numbering corresponds to NC_000067. The donor splice site of intron 9, which is destroyed by the Chor_muang mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red.

Ptprc encodes CD45, a receptor-like protein tyrosine phosphatase (PTP) expressed by cells of the immune system. It is known by several names, including T200 (1), B220 for the B cell form (2), the mouse allotypic marker Ly-5 (3), and CD45. CD45 is a type I transmembrane glycoprotein containing a large N-terminal extracellular domain of ~400-500 residues (depending on the expression of several alternative exons, see below), a single transmembrane domain (22 amino acids), and a C-terminal cytoplasmic domain of 707 residues containing tandem PTP domains only one of which is enzymatically active (Figure 10). Following the PTP domains is a 79 residue C-terminal tail. 

Exons 4, 5, and 6 of Ptprc are alternatively spliced to generate three protein isoforms with variations in the most N-terminal domain, furthest from the cell membrane. The three peptides are designated as A, B, and C, respectively. The protein isoforms are commonly named based on the exons included, with the largest isoform (RABC) including all three exons, RAB including exons 4 and 5, etc., and the smallest isoform lacking all three exons designated RO. The D2 domain is a protein tyrosine phosphatase (PTP) domain that is enzymatically inactive, but optimal phosphatase activity in cells requires both the D1 and the D2 domains (4). Within the D2 domain is a unique acidic region of 19 residues that contains multiple sites for serine phosphorylation by casein kinase II (5;6). This modification is important for optimal CD45 phosphatase activity toward a model substrate in vitro and for cellular signaling leading to Ca²⁺ flux in Jurkat T cells, although the mechanistic basis for these effects is unknown. The D2 domain may also modulate substrate access and localization, as suggested by the interaction of D2 with the CD45 substrate Lck (7).

The Chor_muang mutation is predicted to cause an in-frame protein product beginning after amino acid 203 of the protein [which is normally 1,293 amino acids in length (isoform 1)] and termination after the inclusion of 13 aberrant amino acids. The affected region is within an undefined area between the ABC domain and the first FNIII repeat.

Please see the record for belittle for information about CD45.

The physiological function of CD45 has been examined most extensively in T cells. Studies with CD45-deficient cell lines identified CD45 as an obligate positive regulator of antigen receptor signaling, since T cells lacking CD45 failed to proliferate or produce cytokines in response to TCR stimulation (8;9). CD45 can regulate both the activating and inhibitory tyrosines of Src family kinases.

Ptprc^-/- mice have profound defects in thymic development due to dysfunctional signaling through the preTCR and TCR, leading to a block in thymocyte development at β selection and at the DP stage (10-12). As a result, the absolute number of DP thymocytes is reduced twofold, and the number of single positive (SP) thymocytes is reduced five-fold. Peripheral B cell numbers are actually increased in CD45-deficient mice.  CD45 deficiency has less severe consequences for B cells than for T cells. Peripheral B cell numbers are actually increased in CD45-deficient mice (10-12). Marginal zone B cells are increased, while B1 cell production is decreased, and B cell development is blocked at the transitional 2 (T2) to mature follicular B cell transition (13). Humans deficient in CD45 develop severe combined immunodeficiency (SCID) with defects in T and B cell development and function (OMIM #608971) (14;15). 

The phenotype of the Chor_muang mice is similar to that of the Ptprc^-/- mice suggesting that the Gyoza mice do not express functional CD45.

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold

The following sequence of 405 nucleotides is amplified (chromosome 1, - strand):

1   ttccagctgc catgtttggg aacattactg tgaattacac ctatgaatct agtaatcaga
61  cttttaaggc agacctcaaa gatgtccaaa atgctaagtg tggaaatgag gattgtgaaa
121 acgtgttaaa taatctagaa gaatgctcac agataaaaaa catcagtgtg tctaatgact
181 catgtgctcc agctacaact atagatttat atgtaccacc aggtgaatgt caatttctct
241 ctatctgggt atttagagca acagaaggat ttcatgtatg tgttcctgtg cttcttcaag
301 ctaatgtggt cagaaaaaag aaccgaggga aagcaagtga gctttgataa gagtgtgttt
361 cctgctgaga accccggaga gcagagagaa atacacactt attcc

Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.