The aim of this work was to detect microsatellite markers associated with the epileptic phenotype in the Belgian shepherd. Using multiple assessments, we sought congruence of the results to identify linkage to specific genomic regions. Given that the disorder is complex in expression and in inheritance, LOD scores achieving classical significance in excess of 3.0 were not achieved. Lower LOD scores that are viewed as suggestive of linkage are frequently used in genome linkage studies for human polygenic traits and disorders [37
] as well as complex disease in horses [40
]. The complex nature of epilepsy in the dog would also suggest a liberal interpretation of potential linkage indicators is appropriate. By applying numerous assessments to confirm suggestive LOD scores, the likelihood of identifying false linkage was reduced.
Within the population studied, males and females were equally affected and the age at onset was, for the vast majority of dogs, prior to 6 years of age. Dogs in which the first seizure was reported at older ages may reflect a different form of epilepsy representing different mutations, or quite possibly earlier seizure episodes that went unnoticed by owners. The dataset demonstrated that although the risk of expressing epilepsy changes with age, the risk of expressing epilepsy drops precipitously with increasing age. Specifically, epilepsy, like many disorders would be expected to have a significant age at onset component. Any linkage analysis should therefore accommodate this anticipated effect. Yet as the counts in the supplemental table reveal, there was a dichotomous nature to age at onset in the present data suggesting age at onset was accounted for by the trait of epilepsy. Indeed, the polyserial correlation of age of observation and epilepsy status was -1.0 (computed with package polycor in R) [26
]. The linkage analysis for age of onset measured as the Martingale residual yielded results nearly identical to the binary trait of epilepsy without any age adjustment, as should be expected if age of observation and epilepsy status were so closely related.
In some cases single LOD scores were potentially interesting but unsupported by adjacent markers, multipoint, or Fisher exact test. Based upon multiple assessments of linkage and association, six genomic regions on four chromosomes were found to tentatively harbor QTL associated with epilepsy in the Belgian shepherd. The linkage blocks, although large, perhaps represent the best available evaluation using a linkage association based on microsatellites and their incumbent limitations. For example, 13% of the microsatellites specifically chosen for their allele diversity were uninformative for use in the BT and BS populations. In contrast, microsatellites seem to be very useful in the exclusion of potential candidate genes [41
]. If applied to the present study, numerous chromosomes can be excluded from further consideration as harboring major QTL associated with epilepsy.
The rather limited utility of the sib-pair analysis was not unexpected given the design of the experiment as a familial linkage study and the incomplete availability of littermates from a given breeding. However, all the data taken together do provide compelling evidence of suggestive QTLs in influencing the expression of epilepsy in the Belgian shepherd dog. From the present study microsatellites appear to lack resolution to clearly identify possible causal mutations for a complex, variable phenotypic disorder. Fine mapping of these regions will require greater saturation of markers to generate greater haplotype diversity using the genetic structure of the dog [42
There are numerous positional candidate genes, based upon their involvement in human epilepsy, possible for these six regions. Syntenic to CFA 2 are the human genes BAFME2 (Benign adult familial myoclonic epilepsy 2) [43
] and EIG5 (Epilepsy, idiopathic generalized, susceptibility to, 5) [44
]. Candidate genes on CFA 6 based on human studies would be KCTD7, a potassium channel associated with progressive myoclonic epilepsy [45
] and EIM [46
]. There are 51 identified genes or regions associated with human epilepsy on human chromosome 6 which represents a great deal of synteny with CFA 12; many of these genes/regions are syntenic to the QTL suggested in the present study. A recent characterization of CLN8, a gene involved in human progressive epilepsy and found on CFA 37, was found to be mutated in English Setter dogs having heritable neurodegenerative disease [47
]. While CLN8 is not a good candidate gene for the phenotype of the idiopathic epilepsy seen in the Belgian breeds, it does underscore the generalizability of the human and canine gene discovery as it relates to epilepsy. However, the plethora of epilepsy associated genes identified in human families, indicates that caution needs to be applied when investigating candidate genes and that a more precise localization would be a more prudent approach for polygenic disorders.
The absence of robust association may be due to the polygenic nature of the disease as noted before. Further aspects that confound the study of epilepsy are the accuracy of seizure characterization, the subtle differences in seizure presentation that while appearing to be common in terms of expression represent differences in underlying genetic causality, and the classification as idiopathic epilepsy in the absence of a positive diagnosis. Recent reports of canine epilepsy suggest that the genetics involved in seizures may be distinctly different within the different lines of a single breed [11
]. Additionally, some disorders described as polygenic are the result of copy number variations [48
] and the approach of linkage mapping used in the present study would not identify copy number variability. Another consideration in complex diseases is the concept of "common disease/common variant" hypothesis whereby it is assumed that a common complex disease is caused by few but common genetic variants. However, it is also likely that a particular combination of relatively rare alleles could also evoke the disease [49
]. If the latter is the case for canine epilepsy then the classical linkage studies may be insufficient to detect the genomic regions.