The Aey2 mutant was detected by slit lamp screening of
the offspring of ENU-treated male mice. The mutation has a complete
penetrance. The litter size in the matings of heterozygotes and of
homozygotes indicated normal fertility and viability of the mutants.
The
Aey2 mutant displays a progressive cataract. The
cataractous changes were observed as early as at eye opening (i.e., 12
days after birth) as a diffuse opacity in the cortex and abnormally
branched anterior suture
(Fig. 1a) . This type of opacity remained stationary until 8 to 11 weeks of age,
after which a total opacity developed
(Fig. 1b) .
The histologic sections of mutant eyes at 4 and 11 weeks of age
demonstrated striking irregularities in the anterior suture and a dark
subcortical zone (
Fig. 2a A higher power view showed swollen and liquefied fibers in the anterior
suture with dark particles
(Fig. 2b) . Abnormal dark bodies and dustlike
particles were also present in the subcortical zone
(Fig. 2c) .
The first 46 cataractous mice from the backcross (G3) were taken for
genome-wide mapping. The results indicate linkage with markers on mouse
chromosome 5. The following gene order was observed:
D5Mit15—(12.8 ± 5.4 cM)—
Aey2,
D5Mit10,
D5Mit25—(8.7 ± 4.2
cM)—
D5Mit138. These results are in good agreement with the
current report of the Chromosome Committee for mouse chromosome 5
(available at http://www.informatics.jax.org/bin/ccr/). A detailed
haplotype analysis is given in
Figure 3a and a partial map of mouse chromosome 5 in
Figure 3b .
Based on this mapping information, the cluster encoding the four Cryb genes (Cryba4, Crybb1, Crybb2, and Crybb3; 59–60 cM from the
centromere) was considered to contain good candidate genes for
progressive cataract formation. The corresponding cDNAs were amplified
both in the wild-type mice and in the homozygous mutants, by using cDNA
templates derived by reverse transcription of RNA from lenses of
4-week-old mice.
The only sequence alteration cosegregating with the mutant phenotype
was a T→A exchange at position 553 (exon 6) of the
Crybb2 gene
(Fig. 4) . The mutation lead also to the appearance of a new
DdeI
restriction site in the mutant mice that was not present in the
wild-type sequence of C3H and several other mouse strains. The presence
of the mutation was validated by the presence of the
DdeI
restriction site in five homozygous mutants
(Fig. 5) . Therefore, the new allele should be referred to as
Crybb2 Aey2.
The mutation is predicted to lead to a Val→Glu exchange at codon 187.
At this position Val occurred in four of the six mouse β-crystallins.
Only the acidic βA1/A3- and βA2-crystallins had an Ile at
this position. It is expected that this exchange prohibits the
formation of the fourth Greek key motif in the mutants. However, final
interpretations should include physicochemical data from corresponding
recombinant proteins, together with a more sophisticated computer
analysis.
Because
Crybb2 expression also has been reported recently in
the brain and testis,
3 we tested this expression also in
our wild-type C3H and cataractous mice. We observed conflicting results
with different sets of primers. The primer pair
Crybb2-L1
and -R1 amplified specifically the full-length cDNA of
Crybb2 in the lens only. No transcripts were detected,
either in brain or testis
(Fig. 6a) . The detection limit was determined for
10
− 18 moles
(Fig. 6b) .
However, using another 5′ primer,
Crybb2-L2, located inside
the
Crybb2 transcript, a transcript was observed in brain
and testis of the same size as in the lens
(Fig. 6c) . The sequence
analysis confirmed this short amplification product as part of
Crybb2. The absence of the entire
Crybb2 transcript but presence of a shorter 3′ end indicates the presence of a
novel splice product of
Crybb2 in brain and testis that is
missing the N-terminal part. This is in line with the observation by
Mugabo et al.
3 who presented just the C-terminal part ofβ
B2-crystallin, but not the entire protein. The presence of the
mutation in brain and testis was confirmed by the presence of the
DdeI restriction site as in the lens cDNA
(Fig. 6c) .