Effects of pulse repetition frequency on bubble cloud characteristics and ablation in single-cycle histotripsy.

Objective. Histotripsy is a cavitation-based ultrasound ablation method in development for multiple clinical applications. This work investigates the effects of pulse repetition frequency (PRF) on bubble cloud characteristics and ablative capabilities for histotripsy using single-cycle pulsing methods.Approach.Bubble clouds produced by a 500 kHz histotripsy system at PRFs from 0.1 to 1000 Hz were visualized using high-speed optical imaging in 1% agarose tissue phantoms at peak negative pressures,p-, of 2-36 MPa.Main results.Results showed a decrease in the cavitation cloud threshold with increasing PRF, ranging from 26.7 ± 0.5 MPa at 0.1 Hz to 15.0 ± 1.9 MPa at 1000 Hz. Bubble cloud analysis showed cavitation clouds generated at low PRFs (0.1-1 Hz) were characterized by consistently dense bubble clouds (41.7 ± 2.8 bubbles mm-2at 0.1 Hz), that closely matched regions of the focus above the histotripsy intrinsic threshold. Bubble clouds formed at higher PRFs measured lower cloud densities (23.1 ± 4.0 bubbles mm-2at 1000 Hz), with the lowest density measured for 10 Hz (8.8 ± 4.1 bubbles mm-2). Furthermore, higher PRFs showed increased pulse-to-pulse correlation, characteristic of cavitation memory effects; however, bubble clouds still filled the entire volume of the focus due to their initial density and enhanced bubble expansion from the restimulation of residual nuclei at the higher PRFs. Histotripsy ablation assessed through lesion analysis in red blood cell (RBC) phantoms showed higher PRFs generated lesions with lower adherence to the initial focal region compared to low PRF ablations; however, no trend of decreasing ablation efficiency with PRF was observed, with similar efficiencies observed for all the PRFs tested in this study.Significance.Notably, this result is different than what has previously been shown for shock-scattering histotripsy, which has shown decreased ablation efficiencies at higher PRFs. Overall, this study demonstrates the essential effects of PRF on single-cycle histotripsy procedures that should be considered to help guide future histotripsy pulsing strategies.

Bubble cloud characteristics and ablation efficiency in dual-frequency intrinsic threshold histotripsy.

Histotripsy is a non-thermal focused ultrasound ablation method that destroys tissue through the generation and activity of acoustic cavitation bubble clouds. Intrinsic threshold histotripsy uses single-cycle pulses to generate bubble clouds when the dominant negative pressure phase exceeds an intrinsic threshold of ∼25-30 MPa. The ablation efficiency is dependent upon the size and density of bubbles within the bubble cloud. This work investigates the effects of dual-frequency pulsing schemes on the bubble cloud behavior and ablation efficiency in intrinsic threshold histotripsy. A modular 500 kHz:3 MHz histotripsy transducer treated agarose phantoms using dual-frequency histotripsy pulses with a 1:1 pressure ratio from 500 kHz and 3 MHz frequency elements and varying arrival times for the 3 MHz pulse relative to the arrival of the 500 kHz pulse (-100 ns, 0 ns, and +100 ns). High-speed optical imaging captured cavitation effects to characterize bubble cloud and individual bubble dynamics. The effects of dual-frequency pulsing on lesion formation and ablation efficiency were also investigated in red blood cell (RBC) phantoms. Results showed that the single bubble and bubble cloud size for dual-frequency cases were intermediate to published results for the component single-frequencies of 500 kHz and 3 MHz. Additionally, bubble cloud size and dynamics were shown to be altered by the arrival time of the 3 MHz pulse with respect to the 500 kHz pulse, with more uniform cloud expansion and collapse observed for early (-100 ns) arrival. Finally, RBC phantom experiments showed that dual-frequency exposures were capable of generating precise lesions with smaller areas and higher ablation efficiencies than previously published results for 500 kHz or 3 MHz. Overall, results demonstrate dual-frequency histotripsy's ability to modulate bubble cloud size and dynamics can be leveraged to produce precise lesions at higher ablation efficiencies than previously observed for single-frequency pulsing.

Probability of Cavitation in a Custom Iron-Based Coupling Medium for Transcranial Magnetic Resonance-Guided Focused Ultrasound Procedures.

OBJECTIVE: A coupling bath of circulating, chilled, degassed water is essential to safe and precise acoustic transmittance during transcranial magnetic resonance-guided focused ultrasound (tMRgFUS) procedures, but the circulating water impairs the critical real-time magnetic resonance imaging (MRI). An iron-based coupling medium (IBCM) using iron oxide nanoparticles previously developed by our group increased the relaxivity of the coupling bath such that it appears to be invisible on MRI compared with degassed water. However, the nanoparticles also reduced the pressure threshold for cavitation. To address this concern for prefocal cavitation, our group recently developed an IBCM of electrosterically stabilized and aggregation-resistant poly(methacrylic acid)-coated iron oxide nanoparticles (PMAA-FeOX) with a similar capability to reduce the MR signal of degassed water. This study examines the effect of the PMAA-FeOX IBCM on the cavitation threshold. METHODS: Increasing concentrations of PMAA-FeOX nanoparticles in degassed, deionized water were placed at the focus of two different transducers to assess low and high duty-cycle pulsing parameters which are representative of two modes of focused ultrasound being investigated for tMRgFUS. Passive cavitation detection and high-speed optical imaging were used to measure cavitation threshold pressures. RESULTS: The mean cavitation threshold was determined in both cases to be indistinguishable from the degassed water control, between 6-8 MPa for high duty-cycle pulsing (CW) and between 25.5-26.5 MPa for very low duty-cycle pulsing. CONCLUSION: The findings of this study indicate that an IBCM of PMAA-FeOX nanoparticles is a possible solution to reducing MRI interference from the coupling bath without increasing the risk of prefocal cavitation.

Bubble Cloud Characteristics and Ablation Efficiency in Dual-Frequency Intrinsic Threshold Histotripsy.

Histotripsy is a non-thermal focused ultrasound ablation method that destroys tissue through the generation and activity of acoustic cavitation bubble clouds. Intrinsic threshold histotripsy uses single-cycle pulses to generate bubble clouds when the dominant negative pressure phase exceeds an intrinsic threshold of ~25-30 MPa. The ablation efficiency is dependent upon the size and density of bubbles within the bubble cloud. This work investigates the effects of dual-frequency pulsing schemes on the bubble cloud behavior and ablation efficiency in intrinsic threshold histotripsy. A modular 500 kHz:3 MHz histotripsy transducer treated agarose phantoms using dual-frequency histotripsy pulses with a 1:1 pressure ratio from 500 kHz and 3 MHz frequency elements and varying arrival times for the 3 MHz pulse relative to the arrival of the 500 kHz pulse (-100 ns, 0 ns, and +100 ns). High-speed optical imaging captured cavitation effects to characterize bubble cloud and individual bubble dynamics. The effects of dual-frequency pulsing on lesion formation and ablation efficiency were also investigated in red blood cell (RBC) phantoms. Results showed that the single bubble and bubble cloud size for dual-frequency cases were intermediate to published results for the component single frequencies of 500 kHz and 3 MHz. Additionally, bubble cloud size and dynamics were shown to be altered by the arrival time of the 3 MHz pulse with respect to the 500 kHz pulse, with more uniform cloud expansion and collapse observed for early (-100 ns) arrival. Finally, RBC phantom experiments showed that dual-frequency exposures were capable of generating precise lesions with smaller areas and higher ablation efficiencies than previously published results for 500 kHz or 3 MHz. Overall, results demonstrate dual-frequency histotripsy's ability to modulate bubble cloud size and dynamics can be leveraged to produce precise lesions at higher ablation efficiencies than previously observed for single-frequency pulsing.

Investigation of Shared Genetic Risk Factors Between Parkinson's Disease and Cancers.

BACKGROUND: Epidemiological studies that examined the association between Parkinson's disease (PD) and cancers led to inconsistent results, but they face a number of methodological difficulties. OBJECTIVE: We used results from genome-wide association studies (GWASs) to study the genetic correlation between PD and different cancers to identify common genetic risk factors. METHODS: We used individual data for participants of European ancestry from the Courage-PD (Comprehensive Unbiased Risk Factor Assessment for Genetics and Environment in Parkinson's Disease; PD, N = 16,519) and EPITHYR (differentiated thyroid cancer, N = 3527) consortia and summary statistics of GWASs from iPDGC (International Parkinson Disease Genomics Consortium; PD, N = 482,730), Melanoma Meta-Analysis Consortium (MMAC), Breast Cancer Association Consortium (breast cancer), the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (prostate cancer), International Lung Cancer Consortium (lung cancer), and Ovarian Cancer Association Consortium (ovarian cancer) (N comprised between 36,017 and 228,951 for cancer GWASs). We estimated the genetic correlation between PD and cancers using linkage disequilibrium score regression. We studied the association between PD and polymorphisms associated with cancers, and vice versa, using cross-phenotypes polygenic risk score (PRS) analyses. RESULTS: We confirmed a previously reported positive genetic correlation of PD with melanoma (Gcorr = 0.16 [0.04; 0.28]) and reported an additional significant positive correlation of PD with prostate cancer (Gcorr = 0.11 [0.03; 0.19]). There was a significant inverse association between the PRS for ovarian cancer and PD (odds ratio [OR] = 0.89 [0.84; 0.94]). Conversely, the PRS of PD was positively associated with breast cancer (OR = 1.08 [1.06; 1.10]) and inversely associated with ovarian cancer (OR = 0.95 [0.91; 0.99]). The association between PD and ovarian cancer was mostly driven by rs183211 located in an intron of the NSF gene (17q21.31). CONCLUSIONS: We show evidence in favor of a contribution of pleiotropic genes to the association between PD and specific cancers. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Iron-based coupling media for MRI-guided ultrasound surgery.

PURPOSE: In this study, we examine the effects of a recently developed, iron-based coupling medium (IBCM) on guidance magnetic resonance (MR) scans during transcranial, magnetic-resonance-guided, focused ultrasound surgery (tMRgFUS) procedures. More specifically, this study tests the hypotheses that the use of the IBCM will (a) not adversely affect image quality, (b) remove aliasing from small field-of-view scans, and (c) decouple image quality from the motion state of the coupling fluid. METHODS: An IBCM, whose chemical synthesis and characterization are reported elsewhere, was used as a coupling medium during tMRgFUS procedures on gel phantoms. Guidance magnetization-prepared rapid-gradient-echo (MP-RAGE), TSE, and GRE scans were acquired with fields of view of 28 and 18 cm. Experiments were repeated with the IBCM in several distinct flow states. GRE scans were used to estimate temperature time courses as a gel target was insonated. IBCM performance was measured by computing (i) the root mean square difference (RMSD) of TSE and GRE pixel values acquired using water and the IBCM, relative to the use of water; (ii) through-time temperature uncertainty for GRE scans; and (iii) Bland-Altman analysis of the temperature time courses. Finally, guidance TSE and GRE scans of a human volunteer were acquired during a separate sham tMRgFUS procedure. As a control, all experiments were repeated using a water coupling medium. RESULTS: Use of the IBCM reduced RMSD in TSE scans by a factor of 4 or more for all fields of view and nonstationary motion states, but did not reduce RMSD estimates in MP-RAGE scans. With the coupling media in a stationary state, the IBCM altered estimates of temperature uncertainty relative to the use of water by less than 0.2°C. However, with a high flow state, the IBCM reduced temperature uncertainties by the statistically significant amounts (at the 0.01 level) of 0.5°C (28 cm field of view) and 5°C (18 cm field of view). Bland-Altman analyses found a 0.1°C ± 0.5°C difference between temperature estimates acquired using water and the IBCM as coupling media. Finally, scans of a human volunteer using the IBCM indicate more conspicuous grey/white matter contrast, a reduction in aliasing, and a less than 0.2°C change in temperature uncertainty. CONCLUSIONS: The use of an IBCM during tMRgFUS procedures does not adversely affect image quality for TSE and GRE scans, can decouple image quality from the motion state of the coupling fluid, and can remove aliasing from scans where the field of view is set to be much smaller than the spatial extent of the coupling fluid.

Genome-wide Association and Meta-analysis of Age at Onset in Parkinson Disease: Evidence From the COURAGE-PD Consortium.

BACKGROUND AND OBJECTIVES: Considerable heterogeneity exists in the literature concerning genetic determinants of the age at onset (AAO) of Parkinson disease (PD), which could be attributed to a lack of well-powered replication cohorts. The previous largest genome-wide association studies (GWAS) identified SNCA and TMEM175 loci on chromosome (Chr) 4 with a significant influence on the AAO of PD; these have not been independently replicated. This study aims to conduct a meta-analysis of GWAS of PD AAO and validate previously observed findings in worldwide populations. METHODS: A meta-analysis was performed on PD AAO GWAS of 30 populations of predominantly European ancestry from the Comprehensive Unbiased Risk Factor Assessment for Genetics and Environment in Parkinson's Disease (COURAGE-PD) Consortium. This was followed by combining our study with the largest publicly available European ancestry dataset compiled by the International Parkinson Disease Genomics Consortium (IPDGC). RESULTS: The COURAGE-PD Consortium included a cohort of 8,535 patients with PD (91.9%: Europeans and 9.1%: East Asians). The average AAO in the COURAGE-PD dataset was 58.9 years (SD = 11.6), with an underrepresentation of females (40.2%). The heritability estimate for AAO in COURAGE-PD was 0.083 (SE = 0.057). None of the loci reached genome-wide significance (p < 5 × 10-8). Nevertheless, the COURAGE-PD dataset confirmed the role of the previously published TMEM175 variant as a genetic determinant of the AAO of PD with Bonferroni-corrected nominal levels of significance (p < 0.025): (rs34311866: ß(SE)COURAGE = 0.477(0.203), p COURAGE = 0.0185). The subsequent meta-analysis of COURAGE-PD and IPDGC datasets (Ntotal = 25,950) led to the identification of 2 genome-wide significant association signals on Chr 4, including the previously reported SNCA locus (rs983361: ß(SE)COURAGE+IPDGC = 0.720(0.122), p COURAGE+IPDGC = 3.13 × 10-9) and a novel BST1 locus (rs4698412: ß(SE)COURAGE+IPDGC = -0.526(0.096), p COURAGE+IPDGC = 4.41 × 10-8). DISCUSSION: Our study further refines the genetic architecture of Chr 4 underlying the AAO of the PD phenotype through the identification of BST1 as a novel AAO PD locus. These findings open a new direction for the development of treatments to delay the onset of PD.

The Interaction between HLA-DRB1 and Smoking in Parkinson's Disease Revisited.

BACKGROUND: Two studies that examined the interaction between HLA-DRB1 and smoking in Parkinson's disease (PD) yielded findings in opposite directions. OBJECTIVE: To perform a large-scale independent replication of the HLA-DRB1 × smoking interaction. METHODS: We genotyped 182 single nucleotide polymorphism (SNPs) associated with smoking initiation in 12 424 cases and 9480 controls to perform a Mendelian randomization (MR) analysis in strata defined by HLA-DRB1. RESULTS: At the amino acid level, a valine at position 11 (V11) in HLA-DRB1 displayed the strongest association with PD. MR showed an inverse association between genetically predicted smoking initiation and PD only in absence of V11 (odds ratio, 0.74, 95% confidence interval, 0.59-0.93, PInteraction  = 0.028). In silico predictions of the influence of V11 and smoking-induced modifications of α-synuclein on binding affinity showed findings consistent with this interaction pattern. CONCLUSIONS: Despite being one of the most robust findings in PD research, the mechanisms underlying the inverse association between smoking and PD remain unknown. Our findings may help better understand this association. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Dairy Intake and Parkinson's Disease: A Mendelian Randomization Study.

BACKGROUND: Previous prospective studies highlighted dairy intake as a risk factor for Parkinson's disease (PD), particularly in men. It is unclear whether this association is causal or explained by reverse causation or confounding. OBJECTIVE: The aim is to examine the association between genetically predicted dairy intake and PD using two-sample Mendelian randomization (MR). METHODS: We genotyped a well-established instrumental variable for dairy intake located in the lactase gene (rs4988235) within the Courage-PD consortium (23 studies; 9823 patients and 8376 controls of European ancestry). RESULTS: Based on a dominant model, there was an association between genetic predisposition toward higher dairy intake and PD (odds ratio [OR] per one serving per day = 1.70, 95% confidence interval = 1.12-2.60, P = 0.013) that was restricted to men (OR = 2.50 [1.37-4.56], P = 0.003; P-difference with women = 0.029). CONCLUSIONS: Using MR, our findings provide further support for a causal relationship between dairy intake and higher PD risk, not biased by confounding or reverse causation. Further studies are needed to elucidate the underlying mechanisms. © 2022 International Parkinson and Movement Disorder Society.

Particle-Mediated Histotripsy for the Targeted Treatment of Intraluminal Biofilms in Catheter-Based Medical Devices.

Objective. This paper is an initial work towards developing particle-mediated histotripsy (PMH) as a novel method of treating catheter-based medical device (CBMD) intraluminal biofilms. Impact Statement. CBMDs commonly become infected with bacterial biofilms leading to medical device failure, infection, and adverse patient outcomes. Introduction. Histotripsy is a noninvasive focused ultrasound ablation method that was recently proposed as a novel method to remove intraluminal biofilms. Here, we explore the potential of combining histotripsy with acoustically active particles to develop a PMH approach that can noninvasively remove biofilms without the need for high acoustic pressures or real-time image guidance for targeting. Methods. Histotripsy cavitation thresholds in catheters containing either gas-filled microbubbles (MBs) or fluid-filled nanocones (NCs) were determined. The ability of these particles to sustain cavitation over multiple ultrasound pulses was tested after a series of histotripsy exposures. Next, the ability of PMH to generate selective intraluminal cavitation without generating extraluminal cavitation was tested. Finally, the biofilm ablation and bactericidal capabilities of PMH were tested using both MBs and NCs. Results. PMH significantly reduced the histotripsy cavitation threshold, allowing for selective luminal cavitation for both MBs and NCs. Results further showed PMH successfully removed intraluminal biofilms in Tygon catheters. Finally, results from bactericidal experiments showed minimal reduction in bacteria viability. Conclusion. The results of this study demonstrate the potential for PMH to provide a new modality for removing bacterial biofilms from CBMDs and suggest that additional work is warranted to develop histotripsy and PMH for treatment of CBMD intraluminal biofilms.

Mendelian Randomisation Study of Smoking, Alcohol, and Coffee Drinking in Relation to Parkinson's Disease.

BACKGROUND: Previous studies showed that lifestyle behaviors (cigarette smoking, alcohol, coffee) are inversely associated with Parkinson's disease (PD). The prodromal phase of PD raises the possibility that these associations may be explained by reverse causation. OBJECTIVE: To examine associations of lifestyle behaviors with PD using two-sample Mendelian randomisation (MR) and the potential for survival and incidence-prevalence biases. METHODS: We used summary statistics from publicly available studies to estimate the association of genetic polymorphisms with lifestyle behaviors, and from Courage-PD (7,369 cases, 7,018 controls; European ancestry) to estimate the association of these variants with PD. We used the inverse-variance weighted method to compute odds ratios (ORIVW) of PD and 95%confidence intervals (CI). Significance was determined using a Bonferroni-corrected significance threshold (p = 0.017). RESULTS: We found a significant inverse association between smoking initiation and PD (ORIVW per 1-SD increase in the prevalence of ever smoking = 0.74, 95%CI = 0.60-0.93, p = 0.009) without significant directional pleiotropy. Associations in participants ≤67 years old and cases with disease duration ≤7 years were of a similar size. No significant associations were observed for alcohol and coffee drinking. In reverse MR, genetic liability toward PD was not associated with smoking or coffee drinking but was positively associated with alcohol drinking. CONCLUSION: Our findings are in favor of an inverse association between smoking and PD that is not explained by reverse causation, confounding, and survival or incidence-prevalence biases. Genetic liability toward PD was positively associated with alcohol drinking. Conclusions on the association of alcohol and coffee drinking with PD are hampered by insufficient statistical power.

Effects of frequency on bubble-cloud behavior and ablation efficiency in intrinsic threshold histotripsy.

Objective.Histotripsy is a non-thermal focused ultrasound ablation method that destroys tissue through the generation of a cavitation bubble cloud. Previous work studying intrinsic threshold histotripsy has shown that dense bubble clouds can be formed by a single-cycle pulse when the negative pressure exceeds an intrinsic threshold of ∼25-30 MPa, with the ablation efficiency dependent upon the size and density of bubbles within the cloud. This work investigates the effects of frequency on bubble-cloud behavior and ablation efficiency in intrinsic threshold histotripsy.Approach.A modular transducer was used to expose agarose tissue phantoms to 500 kHz, 1 MHz, or 3 MHz, histotripsy pulses. Optical imaging was used to measure the bubble-cloud dimensions, bubble density, and bubble size. The effects of frequency on ablation efficiency were also investigated by applying histotripsy to red blood cell (RBC) phantoms.Main results.Results revealed that the bubble-cloud size closely matched theoretical predictions for all frequencies. The bubble density, which is a measure of the number of bubbles per unit area, was shown to increase with increasing frequency while the size of individual bubbles within the cloud decreased at higher frequencies. Finally, RBC phantom experiments showed decreasing ablation efficiency with increasing frequency.Significance.Overall, results demonstrate the effects of frequency on histotripsy bubble-cloud behavior and show that lower frequency generates more efficient tissue ablation, primarily due to enhanced bubble expansion.

Focused Ultrasound Biofilm Ablation: Investigation of Histotripsy for the Treatment of Catheter-Associated Urinary Tract Infections (CAUTIs).

Urinary catheters often become contaminated with biofilms, resulting in catheter-associated urinary tract infections (CAUTIs) that adversely affect patient outcomes. Histotripsy is a noninvasive focused ultrasound therapy previously developed for the noninvasive ablation of cancerous tumors and soft tissues. Histotripsy has also previously shown the ability to treat biofilms on glass slides and surgical meshes. Here, we investigate the potential of histotripsy for the treatment of CAUTIs for the first time in vitro. Clinically relevant catheter materials (Tygon, Silicone, and latex catheter mimics) and commonly used clinical catheters were tested to determine the feasibility of producing luminal histotripsy bubble clouds. A Pseudomonas aeruginosa (strain PA14) biofilm model was developed and tested to produce luminal biofilms in an in vitro Tygon catheter mimic. This model was treated with histotripsy to determine the ability to remove a luminal biofilm. Finally, the bactericidal effects of histotripsy were tested by treating PA14 suspended inside the Tygon catheter mimic. Results showed that histotripsy produced precise luminal cavitation within all tested catheter mimics and clinical catheters. Histotripsy treatment of a PA14 biofilm with histotripsy reduced luminal biofilm OD590 signal down to background levels. Further, the treatment of suspended PA14 in Luria-Bertani (LB) showed a 3.45 ± 0.11 log10 reduction in CFU/mL after six histotripsy scans across the catheter mimics. Overall, the results of this study demonstrate the potential of histotripsy to provide a new modality for removing bacterial biofilms from catheter-based medical devices and suggest that additional work is warranted to investigate histotripsy for the treatment of CAUTIs and other biomaterial-associated infections.

Bubble Cloud Behavior and Ablation Capacity for Histotripsy Generated from Intrinsic or Artificial Cavitation Nuclei.

The study described here examined the effects of cavitation nuclei characteristics on histotripsy. High-speed optical imaging was used to compare bubble cloud behavior and ablation capacity for histotripsy generated from intrinsic and artificial cavitation nuclei (gas-filled microbubbles, fluid-filled nanocones). Results showed a significant decrease in the cavitation threshold for microbubbles and nanocones compared with intrinsic-nuclei controls, with predictable and well-defined bubble clouds generated in all cases. Red blood cell experiments showed complete ablations for intrinsic and nanocone phantoms, but only partial ablation in microbubble phantoms. Results also revealed a lower rate of ablation in artificial-nuclei phantoms because of reduced bubble expansion (and corresponding decreases in stress and strain). Overall, this study demonstrates the potential of using artificial nuclei to reduce the histotripsy cavitation threshold while highlighting differences in the bubble cloud behavior and ablation capacity that need to be considered in the future development of these approaches.

Frequency of Loss of Function Variants in LRRK2 in Parkinson Disease.

Importance: Pathogenic variants in LRRK2 are a relatively common genetic cause of Parkinson disease (PD). Currently, the molecular mechanism underlying disease is unknown, and gain and loss of function (LOF) models of pathogenesis have been postulated. LRRK2 variants are reported to result in enhanced phosphorylation of substrates and increased cell death. However, the double knockout of Lrrk2 and its homologue Lrrk1 results in neurodegeneration in a mouse model, suggesting that disease may occur by LOF. Because LRRK2 inhibitors are currently in development as potential disease-modifying treatments in PD, it is critical to determine whether LOF variants in LRRK2 increase or decrease the risk of PD. Objective: To determine whether LRRK1 and LRRK2 LOF variants contribute to the risk of developing PD. Design, Setting, and Participants: To determine the prevailing mechanism of LRRK2-mediated disease in human populations, next-generation sequencing data from a large case-control cohort (>23 000 individuals) was analyzed for LOF variants in LRRK1 and LRRK2. Data were generated at 5 different sites and 5 different data sets, including cases with clinically diagnosed PD and neurologically normal control individuals. Data were collected from 2012 through 2017. Main Outcomes and Measures: Frequencies of LRRK1 and LRRK2 LOF variants present in the general population and compared between cases and controls. Results: Among 11 095 cases with PD and 12 615 controls, LRRK1 LOF variants were identified in 0.205% of cases and 0.139% of controls (odds ratio, 1.48; SE, 0.571; 95% CI, 0.45-4.44; P = .49) and LRRK2 LOF variants were found in 0.117% of cases and 0.087% of controls (odds ratio, 1.48; SE, 0.431; 95% CI, 0.63-3.50; P = .36). All association tests suggested lack of association between LRRK1 or LRRK2 variants and PD. Further analysis of lymphoblastoid cell lines from several heterozygous LOF variant carriers found that, as expected, LRRK2 protein levels are reduced by approximately half compared with wild-type alleles. Conclusions and Relevance: Together these findings indicate that haploinsufficiency of LRRK1 or LRRK2 is neither a cause of nor protective against PD. Furthermore, these results suggest that kinase inhibition or allele-specific targeting of mutant LRRK2 remain viable therapeutic strategies in PD.

Insufficient evidence for pathogenicity of SNCA His50Gln (H50Q) in Parkinson's disease.

SNCA missense mutations are a rare cause of autosomal dominant Parkinson's disease (PD). To date, 6 missense mutations in SNCA have been nominated as causal. Here, we assess the frequency of these 6 mutations in public population databases and PD case-control data sets to determine their true pathogenicity. We found that 1 of the 6 reported SNCA mutations, His50Gln, was consistently identified in large population databases, and no enrichment was evident in PD cases compared to controls. These results suggest that His50Gln is probably not a pathogenic variant. This information is important to provide counseling for His50Gln carriers and has implications for the interpretation of His50Gln α-synuclein functional investigations.

Genetics of early-onset Parkinson's disease in Finland: exome sequencing and genome-wide association study.

Several genes and risk factors are associated with Parkinson's disease (PD). Although many of the genetic markers belong to a common pathway, a unifying pathogenetic mechanism is yet to be found. Also, missing heritability analyses have estimated that only part of the genetic influence contributing to PD has been found. Here, we carried out whole-exome sequencing (WES) on 438 Finnish patients with early-onset PD. We also reanalyzed previous data from genome-wide association studies (GWAS) on the same cohort. Variants in the CEL gene/locus were associated with PD in both GWAS and WES analysis. Exome-wide gene-based association tests also identified the MPHOSPH10, TAS2R19, and SERPINA1 genes in the discovery data set (p < 2.5E-6). MPHOSPH10 had estimated odds ratio (OR) of 1.53, and the rs141620200 variant in SERPINA1 had OR of 1.27. We identified several candidate genes, but further investigation is required to determine the role of these genes in PD.

NeuroX, a fast and efficient genotyping platform for investigation of neurodegenerative diseases.

Our objective was to design a genotyping platform that would allow rapid genetic characterization of samples in the context of genetic mutations and risk factors associated with common neurodegenerative diseases. The platform needed to be relatively affordable, rapid to deploy, and use a common and accessible technology. Central to this project, we wanted to make the content of the platform open to any investigator without restriction. In designing this array we prioritized a number of types of genetic variability for inclusion, such as known risk alleles, disease-causing mutations, putative risk alleles, and other functionally important variants. The array was primarily designed to allow rapid screening of samples for disease-causing mutations and large population studies of risk factors. Notably, an explicit aim was to make this array widely available to facilitate data sharing across and within diseases. The resulting array, NeuroX, is a remarkably cost and time effective solution for high-quality genotyping. NeuroX comprises a backbone of standard Illumina exome content of approximately 240,000 variants, and over 24,000 custom content variants focusing on neurologic diseases. Data are generated at approximately $50-$60 per sample using a 12-sample format chip and regular Infinium infrastructure; thus, genotyping is rapid and accessible to many investigators. Here, we describe the design of NeuroX, discuss the utility of NeuroX in the analyses of rare and common risk variants, and present quality control metrics and a brief primer for the analysis of NeuroX derived data.

Large-scale meta-analysis of genome-wide association data identifies six new risk loci for Parkinson's disease.

We conducted a meta-analysis of Parkinson's disease genome-wide association studies using a common set of 7,893,274 variants across 13,708 cases and 95,282 controls. Twenty-six loci were identified as having genome-wide significant association; these and 6 additional previously reported loci were then tested in an independent set of 5,353 cases and 5,551 controls. Of the 32 tested SNPs, 24 replicated, including 6 newly identified loci. Conditional analyses within loci showed that four loci, including GBA, GAK-DGKQ, SNCA and the HLA region, contain a secondary independent risk variant. In total, we identified and replicated 28 independent risk variants for Parkinson's disease across 24 loci. Although the effect of each individual locus was small, risk profile analysis showed substantial cumulative risk in a comparison of the highest and lowest quintiles of genetic risk (odds ratio (OR) = 3.31, 95% confidence interval (CI) = 2.55-4.30; P = 2 × 10(-16)). We also show six risk loci associated with proximal gene expression or DNA methylation.

Assessment of Parkinson's disease risk loci in Greece.

Genome-wide association studies (GWAS) have been shown to be a powerful approach to identify risk loci for neurodegenerative diseases. Recent GWAS in Parkinson's disease (PD) have been successful in identifying numerous risk variants pointing to novel pathways potentially implicated in the pathogenesis of PD. Contributing to these GWAS efforts, we performed genotyping of previously identified risk alleles in PD patients and control subjects from Greece. We showed that previously published risk profiles for Northern European and American populations are also applicable to the Greek population. In addition, although our study was largely underpowered to detect individual associations, we replicated 5 of 32 previously published risk variants with nominal p values <0.05. Genome-wide complex trait analysis revealed that known risk loci explain disease risk in 1.27% of Greek PD patients. Collectively, these results indicate that there is likely a substantial genetic component to PD in Greece, similarly to other worldwide populations, that remains to be discovered.