Differential serum microRNAs in premotor LRRK2 G2019S carriers from Parkinson's disease

Abstract

The LRRK2 G2019S pathogenic mutation causes LRRK2-associated Parkinson's disease (L2PD) with incomplete penetrance. LRRK2 non-manifesting carriers (L2NMC) are at PD high risk but predicting pheno-conversion is challenging given the lack of progression biomarkers. To investigate novel biomarkers for PD premotor stages, we performed a longitudinal microRNA (miRNA) assessment of serum samples from G2019S L2NMC followed-up over 8 years. Our cohort consisted of G2019S L2NMC stratified by dopamine transporter single-photon emission computed tomography (DaT-SPECT) into DaT-negative (n = 20) and DaT-positive L2NMC (n = 20), pheno-converted G2019S L2PD patients (n = 20), idiopathic PD (iPD) (n = 19), and controls (n = 40). We also screened a second cohort of L2PD patients (n = 19) and controls (n = 20) (Total n = 158). Compared to healthy controls, we identified eight deregulated miRNAs in DaT-negative L2NMC, six in DaT-positive L2NMC, and one in L2PD. Between groups, the highest miRNA differences, 24 candidate miRNAs, occurred between DaT-positive L2NMC and L2PD. Longitudinally, we found 11 common miRNAs with sustained variation in DaT-negative and DaT-positive L2NMCs compared to their baselines. Our study identifies novel miRNA alterations in premotor stages of PD co-occurring with progressive DaT-SPECT decline before motor manifestation, whose deregulation seems to attenuate after the diagnosis of L2PD. Moreover, we identified four miRNAs with relatively high discriminative ability (AUC = 0.82) between non-pheno-converted DaT-positive G2019S carriers and pheno-converted L2PD patients (miR-4505, miR-8069, miR-6125, and miR-451a), which hold potential as early progression biomarkers for PD.

Fig. 1. Workflow of the study and differential miRNA expression in DaT-negative L2NMC, DaT-positive L2NMC, L2PD patients, and iPD patients by genome-wide miRNA analysis in the discovery phase.

a Workflow of the study and number of participating subjects. b Volcano plot of differential miRNA expression between DaT-negative L2NMC and controls, c DaT-positive L2NMC and controls, d L2PD and controls, and e iPD and controls. Candidate differentially expressed miRNAs were defined as miRNAs with a fold-change above |1.5| and a p value below 0.05 under a two-tailed Student’s t test. Only names of the top-10 miRNAs are shown. Up-regulated and down-regulated DEmiR are respectively depicted in red and blue. DaT DaT-SPECT imaging, L2NMC LRRK2 non-manifesting carriers, L2PD LRRK2-associated PD patients, iPD idiopathic PD patients.

Fig. 2. Common and specific candidate DEmiR of DaT-negative L2NMC, DaT-positive L2NMC, L2PD, and iPD as compared to controls.

Venn diagram of a candidate DEmiR from the discovery analysis and b differentially expressed miRNAs (DEmiR) from the RT-qPCR miRNA assessment. DaT DaT-SPECT imaging, L2NMC LRRK2 non-manifesting carriers, L2PD LRRK2-associated PD patients, iPD idiopathic PD patients.

Fig. 3. Relative serum miRNA expression levels in DaT-negative L2NMC, DaT-positive L2NMC, L2PD, iPD and controls as assessed by RT-qPCR.

The central line of the boxes is plotted at the median, the boxes extend from the 25th to the 75th percentiles, and the whiskers extend to the minimum and maximum value. (*) depicts statistically significant DEmiR with fold-change difference above |1.5| and a multiple-test adjusted p value below 0.05 under two-tailed Student’s t test. DaT DaT-SPECT imaging, L2NMC LRRK2 non-manifesting carriers, L2PD LRRK2-associated PD patients, iPD idiopathic PD patients.

Fig. 4. Differential miRNA expression in successive G2019S LRRK2 progression stages and iPD.

Volcano plots showing miRNA expression differences between a DaT-negative and DaT-positive L2NMC, b DaT-positive L2NMC and L2PD, and c L2PD and iPD patients. DEmiR were defined as miRNAs with a fold-change above |1.5| and a p value below 0.05 under a two-tailed Student’s t test. Only names of the top-10 miRNAs are shown. Up-regulated and down-regulated DEmiRs are respectively depicted in red and blue. DaT DaT-SPECT imaging, L2NMC LRRK2 non-manifesting carriers, L2PD LRRK2-associated PD patients, iPD idiopathic PD patients.

Fig. 5. Area under the curve (AUC) analysis on a receiver operating characteristic (ROC) curve representing the discriminative ability of four miRNAs associated with pheno-conversion (miR-4505, miR-8069, miR-6125, and miR-451a) when comparing DaT-positive L2NMC non-pheno-converted after 8 years of follow-up (n = 16) vs. all pheno-converted subjects, i.e., L2PD discovery (n = 20) and validation (n = 19) sets, and DaT-positive L2NMC diagnosed with PD during the study (n = 4) (total n = 43).

DaT DaT-SPECT imaging, L2NMC LRRK2 non-manifesting carriers, L2PD LRRK2-associated PD patients.

Fig. 6. 8-year longitudinal follow-up of serum miRNA levels in DaT-negative and DaT-positive L2NMC groups with respect to their corresponding baselines.

Longitudinal fold-change values in a DaT-negative L2NMC and b DaT-positive L2NMC. DaT DaT-SPECT imaging, L2NMC LRRK2 non-manifesting carriers.