A rare sequence variant in intron 1 of THAP1 is associated with primary dystonia

Abstract

Although coding variants in THAP1 have been causally associated with primary dystonia, the contribution of noncoding variants remains uncertain. Herein, we examine a previously identified Intron 1 variant (c.71+9C>A, rs200209986). Among 1672 subjects with mainly adult-onset primary dystonia, 12 harbored the variant in contrast to 1/1574 controls (P < 0.01). Dystonia classification included cervical dystonia (N = 3), laryngeal dystonia (adductor subtype, N = 3), jaw-opening oromandibular dystonia (N = 1), blepharospasm (N = 2), and unclassified (N = 3). Age of dystonia onset ranged from 25 to 69 years (mean = 54 years). In comparison to controls with no identified THAP1 sequence variants, the c.71+9C>A variant was associated with an elevated ratio of Isoform 1 (NM_018105) to Isoform 2 (NM_199003) in leukocytes. In silico and minigene analyses indicated that c.71+9C>A alters THAP1 splicing. Lymphoblastoid cells harboring the c.71+9C>A variant showed extensive apoptosis with relatively fewer cells in the G2 phase of the cell cycle. Differentially expressed genes from lymphoblastoid cells revealed that the c.71+9C>A variant exerts effects on DNA synthesis, cell growth and proliferation, cell survival, and cytotoxicity. In aggregate, these data indicate that THAP1 c.71+9C>A is a risk factor for adult-onset primary dystonia.

Keywords: DYT6; Dystonia; THAP1; intronic variant; minigene assay.

Figure 1

Ingenuity interactome analysis of upregulated genes.

Figure 2

Ingenuity interactome analysis of downregulated genes.

Figure 3

Minigene assay of THAP1 splicing. (A) Segments of THAP1 were cloned into the multiple cloning site (MCS) of the pET01 exon trap vector, which contains a short stretch of a eukaryotic phosphatase gene (E) 3′ to the eukaryotic strong long terminal repeater promoter of the Rous Sarcoma Virus (RSV). A second segment of E is followed by a polyadenylation site (pA). (B) The results of three wild-type and three c.71+9C>A transfections showed that the C>A mutation exerted significant effects on splicing patterns in HEK293 cells (*P < 0.05, for all).

Figure 4

Cell-cycle analysis of lymphoblastoid cell lines. Data from representative normal and c.71+9C>A cells are shown. In comparison to normal cells, the proportion of c.71+9C>A cells in apoptotic phase is increased and G2 phase is decreased.