Study On Association Between Human Endogenous Retrovirus And Sporadic Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal illness characterized by the progressive loss of spinal and cortical motor neurons. Due to the degeneration of upper and lower motor neurons, the ALS patients end with respiratory paralysis, even respiratory failure and death usually in the prevalence of three to five years. Despite clinical associations, past approaches to identify a definite causative viral agent in ALS have been unsuccessful, although there are claims of association with polioviruses, enterovirus and retroviruses. About more than 90% are sporadic ALS patients, it is only 10% of all the ALS patients familial form. Approximately 20% of familial ALS is caused by mutations in the superoxide dismutase (SOD1) gene. But the pathogenesis for sporadic ALS is still unknown, there is no valid remedy for this disease.1. Objective and MethodsOur experiment utilized the brain tissue of ALS patients and non-ALS control individuals, which were obtained from the Johns Hopkins School of Medicine Brain Bank, and we studied the association between HERV and sporadic ALS.First the total RNA from the brain samples were purified, and then the level of HERV-K pol gene transcripts were determined by the method of Real-Time PCR. Then the target DNA were amplified through regular PT-PCR, and cloned into the TA cloning vector pGM-T. Competent DH5αE. coli was transformed with the recombinant plasmid and the clones were randomly picked from each patient sample. In order to avoid the false positive, clones were identified by specific PCR, and then the plasmids were digested with EcoRI enzyme. Then the purified positive plasmids from the positive bacteria colony were sequenced. The cDNA sequence data were translated into amino acid sequences using ExPASy platform and both the nucleotides and amino acid sequences were aligned and compared with that of HERV-K 10 using BioEdit software.2. Results HERV-K pol mRNA expressed in both ALS and non-ALS autopsy brain samples with no significant differences, but detection rate in ALS brain sample (25/29)is higher than non-ALS(9/14). Compared with the HERV-K10 sequence, alterations occurred in clones from both ALS and non-ALS brain samples. The sequence polymorphism of HERV-K pol sequence in RT region from ALS clones result in the motif LPQG alterations, the most conserved region, some clones showed either the composition or the ORF shift mutation in LPQG. And there were also some mutations in compositions of another conserved motif YIDD/YTDD which are highly related with the enzymatic activity of RT. So there were different level changes in the expression of HERV-K pol sequence in ALS. But in non-ALS, a few premature stop codons were detected and there was no mutation found in YIDD/YTDD.3. ConclusionIn summary, it is more general that the mutations of HERV-K pol sequence occur in sporadic ALS. Variations in motif LPQG and YIDD/YTDD suggest that the HERV-K pol sequence may play important influences in the pathogenesis of ALS.