Figure 2.

The frizz mutation was discovered while analyzing N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice using flow cytometry analysis of blood.  Frizz animals have reduced frequencies of peripheral CD4+ and CD8+ T cells and low expression of B220 (the B cell form of CD45; see the record for belittle), suggesting a block in B cell maturation (Figure 1).  In addition, these animals lack a T-dependent immunoglobin (IgG) response to model antigens encoded by a recombinant nonreplicating vector based on the Semliki Forest Virus (rSFV), but make normal T-independent IgM responses to haptenated ficoll (Figure 2).  The frizz phenotype is similar to that of frazz mutants with a mutation in the Dock2 gene.  Complementation testing between the two strains confirmed that frizz is also an allele of Dock2.

Whole genome sequencing of a homozygous frizz mouse using the SOLiD technique covered the coding/splicing region at least 1x or 3x with 75.9% and 46.5% coverage, respectively.  Validation sequencing using the Sanger method was attempted on all nucleotides for which discrepancies were seen at 3x or greater coverage, with 69 of 84 discrepancies successfully processed and mutations in three genes were identified. Whole genome sequencing did not identify the causative mutation in Dock2.  Standard Sanger sequencing of the Dock2 gene identified a T to A transversion at position 34158184 in the GenBank genomic region NC_000077 for the Dock2 gene on chromosome 11 (TGATCCCATAG  -> AGATCCCATAG) (Figure). In GenBank, the mutation is located near the acceptor splice site of intron 16 (intron 38 in Ensembl record ENSMUST00000093193) from the ATG exon, eleven nucleotides to the next exon.  Dock2 contains 30 exons according to Genbank record NM_033374 and 52 exons according to Ensembl record ENSMUST00000093193.  Multiple Dock2 transcripts are displayed on Ensembl and Vega. The effect of the mutation at the cDNA and protein level is currently being determined.  One possibility, shown below, is that aberrant splicing may result in skipping of the 90 bp exon 39, utilization of the acceptor splice site from intron 39 and in-frame splicing to exon 40 (depicted below as seen on Ensembl). This would result in deletion of 30 amino acids.

 

    <--exon 38 <--intron 38 exon 39-->  exon 40--> <--exon 52

     CACCAACAG……TGATCCCATAG ATGTGGGAA………ACCCAGCAG………AACATGTGA

1289 -K--G--K-              -M--W--E-   -T--Q--Q-………-N--M--*  1828

      correct                deleted          correct

       
The acceptor splice site of intron 38, which is destroyed by the mutation, is indicated in blue; the mutated nucleotide is indicated in red.

The frizz mutation likely results in abnormal splicing of Dock2 and may cause an internal deletion of amino acids 1292-1321 in the DHR-2 domain.

For more information on the Dock2 gene, see the record for frazz.