TAF1 Transcripts and Neurofilament Light Chain as Biomarkers for X-linked Dystonia-Parkinsonism

Abstract

Background: X-linked dystonia-parkinsonism is a rare neurological disease endemic to the Philippines. Dystonic symptoms appear in males at the mean age of 40 years and progress to parkinsonism with degenerative pathology in the striatum. A retrotransposon inserted in intron 32 of the TAF1 gene leads to alternative splicing in the region and a reduction of the full-length mRNA transcript.

Objectives: The objective of this study was to discover cell-based and biofluid-based biomarkers for X-linked dystonia-parkinsonism.

Methods: RNA from patient-derived neural progenitor cells and their secreted extracellular vesicles were used to screen for dysregulation of TAF1 expression. Droplet-digital polymerase chain reaction was used to quantify the expression of TAF1 mRNA fragments 5' and 3' to the retrotransposon insertion and the disease-specific splice variant TAF1-32i in whole-blood RNA. Plasma levels of neurofilament light chain were measured using single-molecule array.

Results: In neural progenitor cells and their extracellular vesicles, we confirmed that the TAF1-3'/5' ratio was lower in patient samples, whereas TAF1-32i expression is higher relative to controls. In whole-blood RNA, both TAF1-3'/5' ratio and TAF1-32i expression can differentiate patient (n = 44) from control samples (n = 18) with high accuracy. Neurofilament light chain plasma levels were significantly elevated in patients (n = 43) compared with both carriers (n = 16) and controls (n = 21), with area under the curve of 0.79.

Conclusions: TAF1 dysregulation in blood serves as a disease-specific biomarker that could be used as a readout for monitoring therapies targeting TAF1 splicing. Neurofilament light chain could be used in monitoring neurodegeneration and disease progression in patients. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Keywords: TAF1; XDP; biomarkers; extracellular vesicles; neurofilament light chain.

FIG. 1

TAF1‐3′/5′ ratio expression in NPCs, NPC EVs, and peripheral blood. (A) RT‐qPCR expression of TAF1‐3′/5′ ratio in XDP (n = 8) and control (n = 6) NPCs. (B) Droplet‐digital polymerase chain reaction expression of TAF1‐3′/5′ ratio in XDP (n = 8) and control (n = 5) EVs isolated from NPCs in culture. (C) Droplet‐digital polymerase chain reaction expression of TAF1‐3′/5′ ratio in patients with XDP (n = 43), female carriers (n = 17), and controls (n = 18) in whole‐blood RNA. Each dot represents a unique sample. Mean and standard error are represented. (D) Schematic for TAF1‐5′ primers relative to TAF1 exons 2 and 3 and TAF1‐3′ primers relative to the SVA and TAF1 exons 32 and 33. (E) Receiver operating characteristic analysis of TAF1‐3′/5′ ratio in whole‐blood RNA in XDP versus control samples. (F) Receiver operating characteristic analysis of TAF1‐3′/5′ ratio in whole‐blood RNA in XDP versus female carrier samples. AUC, area under the curve; CI, confidence interval; EV, extracellular vesicle; NPCs, neural progenitor cells; XDP, X‐linked dystonia‐parkinsonism. *P < 0.05, **P < 0.01, ****P < 0.0001.

FIG. 2

TAF1‐32i expression in NPCs, NPC EVs, and peripheral blood. (A) RT‐PCR expression of TAF1‐32i in XDP (n = 8) and control (n = 5) NPCs. (B) Droplet‐digital polymerase chain reaction expression of TAF1‐32i in XDP (n = 8) and control (n = 5) EVs isolated from NPCs in culture. (C) Droplet‐digital polymerase chain reaction expression of TAF1‐32i ratio in XDP (n = 40), female carriers (n = 17), and controls (n = 18) in whole‐blood RNA. Dotted line shown at y = 5 to separate background expression seen in all 17 controls, in 8 XDP samples, and 3 carrier samples (y < 5). Each dot represents a unique sample. Means with standard errors are represented. Data are logarithmically transformed. (D) Schematic for TAF1‐32i and TAF1‐32i preamplification primers (TAF1‐E32i‐nest) relative to the SVA and TAF1 exons 32 and 32i. (E) receiver operating characteristic analysis of TAF1‐32i expression in whole‐blood RNA in XDP versus control samples. (F) Receiver operating characteristic analysis of TAF1‐32i expression in whole‐blood RNA in female carrier versus control samples. AUC, area under the curve; CI, confidence interval; EV, extracellular vesicle; NPCs, neural progenitor cells; XDP, X‐linked dystonia‐parkinsonism. ****P < 0.0001.

FIG. 3

NfL in plasma. (A) NfL protein concentration (pg/ml) in plasma from patients with XDP (n = 43), female carriers (n = 16), and healthy controls (n = 21) using SiMoA. Medians with 95% CIs are represented. (B) Receiver operating characteristic analysis of plasma NfL in patients with XDP versus female carriers and controls. (C) Subgroup analysis of plasma NfL in patients with XDP according to the type of symptom, if any, at disease onset. (D) Subgroup analysis of plasma NfL in female carriers according to the type of symptom at presentation, if any. (E) Subgroup analysis of plasma NfL in controls according to the type of symptom at presentation, if any. Medians with 95% CIs represented. AUC, area under the curve; CI, confidence interval; NfL, neurofilament light chain; XDP, X‐linked dystonia‐parkinsonism. *P < 0.05, **P < 0.01, ***P < 0.001.