The identification of causative genes associated with a disease is a complex task requiring gathering of genome-wide information. The assessment of the expression of thousands of genes simultaneously may pinpoint the metabolic pathways affected by causative mutation(s) in order to focus on a small set of target genes.
We have tried to assess the metabolic pathways affected by the spastic paresis in Romagnola cattle breed, which presents this disease at quite a high frequency , using microarrays. The cDNA microarray we used in this study contains a brain library and, even if carrying an incomplete transcriptome representation, it has proven to be a valuable tool allowing us to add useful and new information to a poorly studied disease. Today, it is possible to prepare custom microarrays , while direct sequencing of transcripts by high-throughput sequencing technologies (RNA-Seq) would probably make possible to expand microarray investigation. However by using microarray tool, we analyzed 15 K genes with the aim to acquire knowledge of a genetic disease for which the current literature only provides scant results.
Results on metabolic pathways support an alteration of the Neurodegenerative Diseases and Nervous system C2 classes with the most frequently associated differentially expressed genes in the BSP (BAD, NEFH, NDUFB2, DERL1, NEFL). Moreover, the ATP6V0E1 gene, encoding for a lysosomal H + -transporting ATPase, showed the strongest signal (FC 81.29) on microarrays and together with NDUFB2, is involved in intracellular processes and is linked to Oxidative Phosphorylation pathway. PRKCG is a member of the protein kinase C (PKC) gene family, whose members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signalling pathways. Defects in this protein have been associated with neurodegenerative disorder spinocerebellar ataxia-14 .
Four calcium binding proteins involved in Calcium Signalling pathway, S100A12, S100A11, S100A8 and STK25, were weakly over-expressed in the affected animals. In Chianina cattle, a missense mutation in exon 6 (c.491G > A) of the bovine ATP2A1/SERCA1 gene, was implicated in Congenital Pseudomyotonia [24, 25], a disease very similar to BSP and paralleled to human Brody disease by Drögemüller and collaborators, . The same gene has been implicated also in Pseudomyotonia in Dutch Improved Red and White cross-breed . We tested this mutation in our diseased animals and we did not find any carrier nevertheless Congenital Pseudomyotonia shares many symptoms with BSP. Therefore, even if BSP does not result associated to the mutations causing Congenital Pseudomyotonia reported in other cattle breeds, our data suggest the involvement of the calcium signalling proteins and two C3 classes, the Neurotrophin signalling pathway and Amyotrophic lateral sclerosis.
Charlier and coll  identified a bovine disorder named CMD2, reminiscent of congenital myoclonus in Hereford cattle, and reported a mutation in the glycine transporter GlyT2 (SLC6A5) gene already associated to human hyperexplexia . Recently, SLC6A5 was also associated to hyperexplexia in Irish wolfhounds . BSP shows many similarities with human hyperexplexia (OMIM 149400), an autosomal disease that can be caused by mutations in the genes encoding the alpha-1 subunit of the glycine receptor (GLRA1), the presynaptic glycine transporter-2 (SLC6A5), the beta-subunit of the glycine receptor (GLRB), the postsynaptic glycinergic proteins GPHN and ARHGEF9[32–39]. Mutations in the latter gene cause hyperexplexia with epilepsy (OMIM 300607). In a previous study, we observed a number of mutations in GLRA1 and GLRB in Romagnola cattle , but none resulted significantly involved in the disease.
Our microarray results suggest also a defective glycinergic synaptic transmission in Romagnola BSP. The expression of the Rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA) gene, of two neurotransmitter transporters (SLC41A3 and SLC25A20), of the organic anion transporter SLCO1A2 resulted altered in affected animals. In affected samples, we observed the over-expression of two genes involved in defective glycinergic synaptic transmission, GRINA and SLC6A9. This latter gene, encoding GlyT1, was recently implicated in defective glycinergic synaptic transmission in Zebrafish . GFAP, encoding glial fibrillary acidic protein, linked to expression of the glutamate transporter Glt1 (SLC1A2) in Alexander disease, was also over-expressed in affected animals (FC 2.60). Mutations in NR4A2 (FC 3.06), a nuclear receptor family protein, have been associated with disorders related to dopaminergic dysfunction . Most transporters involved in the drug disposal, characterized by broad substrate specificities and accepting structurally unrelated compounds, include members of SLC family. The involvement of glycinergic proteins seems supported also at phenotypic level: the cholecystokinin (CCK) is involved in the neuroactive ligand-receptor interaction pathway. The release of CCK (ACBP) is linked to GABA and sensitive to clonazepam . These features are found also in human hyperexplexia and are in agreement with the differential expression of some GABA neurotransmitter transporters of the solute carrier family emerging from our analysis.