Untersuchungen zu familiären und rassespezifischen ...

Material and methods
MITF as candidate for deafness in Dalmatian dogs 117
Blood samples were available from 88 not closely related Dalmatian dogs. Pedigree
records included five to eight generations. This sample consisted of 44 unilaterally
(n=32) or bilaterally (n=12) deaf Dalmatian dogs and 44 bilaterally hearing Dalmatian
dogs. The hearing status of these dogs was diagnosed by veterinarians via BAER
(brain stem auditory evoked response). In addition to the hearing status, we recorded
the eye pigmentation status of these dogs. Our sample was composed of 22 deaf
dogs with brown eyes and 22 deaf dogs with partially or totally blue eyes and 22
bilaterally hearing dogs with brown eyes and 22 bilaterally hearing dogs with partially
or totally blue eyes.
We genotyped eight MITF-associated markers of which six were intragenic and two
in the adjacent regions (Table 1). Five of the intragenic SNPs were adopted from the
study of Karlsson et al. (2007). An intronic MITF-SNP of those five SNPs was
previously reported to be associated with white spotting in dogs (Rothschild et al.
2006). Another intronic SNP was identified from the NCBI dbsnp list
(http://www.ncbi.nlm.nih.gov/projects/SNP/index.html). MITF-flanking markers
included the LP-MITF-M (Karlsson et al. 2007) and two microsatellites previously
identified in Dalmatian dogs (Distl & Mieskes, unpublished results). The microsatellite
RPCI81-119P24 was located within the ~100 kb region containing the S locus and
the gene MITF. REN100J13 was about 0.8 Mb downstream of this associated region.
SNP primers were designed using the Primer3 software (http://frodo.wi.mit.edu/cgi-
bin/primer3/primer3_www.cgi) after masking repetitive sequences by the
RepeatMasker (http://www.repeatmasker.org/). PCR-amplicons were sequenced
after purification using the automated sequencer MegaBACE 1000 (GE Healthcare,
Freiburg, Germany). Sequencher 4.7 (GeneCodes, Ann Arbor, MI, USA) was used to
analyze the sequence data. For genotyping of the microsatellites and the LP-MITF-
M, forward primers were labeled with IRD700 or IRD800. PCR-products were length-
fractionated on 6% polyacrylamid gels using the automated sequencer LI-COR
4200/S-2 (Lincoln, NE, USA). Size determination of alleles was performed by visual
examination in comparison to a DNA-ladder.