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Sharma, Nutan

Background: Mutations in the GCH1 gene are associated with childhood onset, dopa-responsive dystonia (DRD). Correct diagnosis of DRD is crucial, given the potential for complete recovery once treated with L-dopa. The majority of DRD associated mutations lie within the coding region of the GCH1 gene, but three additional single nucleotide sequence substitutions have been reported within the 5’ untranslated (5’UTR) region of the mRNA. The biologic significance of these 5’UTR GCH1 sequence substitutions has not been analyzed. Methodology/Principal Findings Luciferase reporter assays, quantitative real time PCR and RNA decay assays, combined with bioinformatics, revealed a pathogenic 5’UTR GCH1 substitution. The +142C>T single nucleotide 5’UTR substitution that segregates with affected status in DRD patients, substantially attenuates translation without altering RNA expression levels or stability. The +142C>T substitution disrupts translation most likely by creating an upstream initiation start codon (uAUG) and an upstream open reading frame (uORF). Conclusions/Significance: This is the first GCH1 regulatory substitution reported to act at a post-transcriptional level, increasing the list of genetic diseases caused by abnormal translation and reaffirming the importance of investigating potential regulatory substitutions in genetic diseases.

X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease associated with an antisense insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This unique insertion coincides with six additional noncoding sequence changes in TAF1, the gene that encodes TATA-binding protein–associated factor-1, which appear to be inherited together as an identical haplotype in all reported cases. Here we examined the sequence of this SVA in XDP patients (n = 140) and detected polymorphic variation in the length of a hexanucleotide repeat domain, (CCCTCT)n. The number of repeats in these cases ranged from 35 to 52 and showed a highly significant inverse correlation with age at disease onset. Because other SVAs exhibit intrinsic promoter activity that depends in part on the hexameric domain, we assayed the transcriptional regulatory effects of varying hexameric lengths found in the unique XDP SVA retrotransposon using luciferase reporter constructs. When inserted sense or antisense to the luciferase reading frame, the XDP variants repressed or enhanced transcription, respectively, to an extent that appeared to vary with length of the hexamer. Further in silico analysis of this SVA sequence revealed multiple motifs predicted to form G-quadruplexes, with the greatest potential detected for the hexameric repeat domain. These data directly link sequence variation within the XDP-specific SVA sequence to phenotypic variability in clinical disease manifestation and provide insight into potential mechanisms by which this intronic retroelement may induce transcriptional interference in TAF1 expression.