High resolution mapping of a DNA locus genetically linked to febrile seizures

Abstract

Epilepsy is regarded as a diverse family of disorders having in common an abnormally increased predisposition to seizures. Approximately 70 per cent of all patients with epilepsy lack an obvious symptomatic cause, and therefore are presumed to have a predominantly genetic basis for their condition. To date, a number of mutations in ion channel and neuroreceptor constituent genes have been identified in some syndromes with clear Mendelian inheritance. Moreover, various studies have shown that epilepsy syndromes tend to aggregate in families. The main aims of this study were to narrow down a previously identified linkage region on chromosome 20 by performing a linkage study using short tandem repeat markers, and to identify and sequence two genes found within this region which could be predisposing the family to the familial febrile seizures phenotype. This study was carried out on a Maltese three generation family having seven members affected by febrile seizures. All thirteen family members were genotyped for seven short tandem repeat markers found in the linkage region on chromosome 20 previously identified in a study conducted by Cassar in 2008. These markers were spaced at approximately 2cM from each other, using the deCODE map as a reference. Following genotyping, linkage analysis was performed and the two candidate genes chosen from the newly narrowed region were NKAIN4 and HRH3. Primer design for the exon/intron junctions of these two genes was carried out using NCBI's Primer-Blast designing tool and the respective reference sequences. Samples from an affected individual and a non-affected individual were sequenced. Subsequently the sequences obtained were first compared to each other and then to their reference sequences to determine any significant differences.

Analysis of DNA from affected family members reduced the previously identified linkage region to about 5cM on chromosome 20q13.3. A maximum multipoint LOD score of 2.67 was obtained when 0.9 penetrance was used. This score is suggestive evidence for linkage. The identified locus has never been associated to familial febrile seizures syndrome before. This region spans a total of 84 genes, of which NKAIN4 and HRH3 were selected for further analysis. These two genes have been previously associated to epilepsy or seizures and are both expressed in the central nervous system. In fact, five different single nucleotide polymorphisms were found on analysis of the sequences obtained from the affected and non-affected individuals. These variations were characterised using the relevant online databases, however for various reasons none of these point mutations seemed to be immediately implicated in the formation of the febrile seizures observed in the family. Even though four of the observed single nucleotide polymorphisms in NKAIN4 were not found in the exons of the gene, this is not conclusive evidence that they do not confer a disease risk to the family. A literature review shows that heterogeneity in untranslated regions of a gene is likely to have an impact on protein expression in an individual, possibly leading to disease. This difference in protein expression may happen through various mechanisms, namely polymorphisms in micro RNA targets and disruption of splicing, leading to unstable mRNAs and defective protein structure. This project has identified a novel locus associated to the familial febrile seizures syndrome in a Maltese family and contributes to a better understanding of the condition.

Discovery of gene mutations that predispose to epilepsies have led to the possibility of using gene therapy in the hope of overcoming common problems observed with the currently available drug therapy, especially in cases of pharmacoresistance. It is evident that the knowledge gained through such genetic studies can be applied to the search for improved antiepileptic drugs.