Genetic Testing for GBA and LRRK2 Mutations: Is it Time for Routine Use?

In 1997, Mihael H. Polymeropoulos and colleagues announced to the world that a mutation in the α-synuclein (SNCA) gene was identified in families with Parkinson’s disease (PD). This discovery not only confirmed the role of genetics in the etiology of PD but also set the stage for future research in the field of genetics and underlying pathophysiological mechanisms in PD. Twenty-five years later, we have made many breakthroughs in understanding the genetics of PD and the mechanisms they bring about disease, which has led to the development of the first gene-targeted therapies. We are now in an exciting era, where several of these therapies are entering clinical trials, taking us a step closer to finally being able to offer our patients with PD hope of a causal treatment. Should these medical advancements prompt us clinicians taking care of patients with PD to offer routine genetic testing for glucocerebrosidase (GBA) and leucine-rich repeat kinase (LRRK2) pathogenic variants, as they are the most commonly known PD risk and causal variants and the most targeted in gene-specific interventional trials? At first sight, the answer to this question would be a no, because although there are various clinical trials ongoing, we currently have no US Food and Drug Administration (FDA)–approved gene-targeted therapy available, so genetic testing in the present climate would not affect management. However, in certain circumstances, our answer would be different, and we would offer genetic testing to our patients with PD. These circumstances would include an uncertain diagnosis with atypical features before more invasive investigations are considered, an earlier age of onset to help with prognosis, and counseling of patients and their families about disease progression, patients’ interests, and inclusion in clinical trials. Worldwide Mutation Frequencies of GBA and LRRK2

In 1997, Mihael H. Polymeropoulos and colleagues announced to the world that a mutation in the α-synuclein (SNCA) gene was identified in families with Parkinson's disease (PD). 1 This discovery not only confirmed the role of genetics in the etiology of PD but also set the stage for future research in the field of genetics and underlying pathophysiological mechanisms in PD. Twenty-five years later, we have made many breakthroughs in understanding the genetics of PD and the mechanisms they bring about disease, which has led to the development of the first gene-targeted therapies. We are now in an exciting era, where several of these therapies are entering clinical trials, taking us a step closer to finally being able to offer our patients with PD hope of a causal treatment. Should these medical advancements prompt us clinicians taking care of patients with PD to offer routine genetic testing for glucocerebrosidase (GBA) and leucine-rich repeat kinase (LRRK2) pathogenic variants, as they are the most commonly known PD risk and causal variants and the most targeted in gene-specific interventional trials?
At first sight, the answer to this question would be a no, because although there are various clinical trials ongoing, we currently have no US Food and Drug Administration (FDA)-approved gene-targeted therapy available, so genetic testing in the present climate would not affect management. However, in certain circumstances, our answer would be different, and we would offer genetic testing to our patients with PD. These circumstances would include an uncertain diagnosis with atypical features before more invasive investigations are considered, an earlier age of onset to help with prognosis, and counseling of patients and their families about disease progression, patients' interests, and inclusion in clinical trials.

Worldwide Mutation Frequencies of GBA and LRRK2
Pathogenic variants in the GBA and LRRK2 genes together account for most of the known strong genetic contributions to PD, with variants in LRRK2 being the most frequent cause 2 with an average mutation frequency of 3.1% 3 and variants in GBA being the most frequent risk factor 4 with a frequency of pathogenic variants of 8.5%. 3 However, the frequency of these variants differs in different ethnic groups. The G2019S variant is the most frequent LRRK2 pathogenic variant: It is found in 10% of familial PD cases, 1% to 2% of all PD cases, 5 3% in Europeans, 16% to 19% in Ashkenazi Jews, and up to 42% in African Arab-Berbers 6 and in most other populations investigated with the exception of several Asian populations. 7 Pathogenic GBA variants have been identified in various populations but are most frequently detected among people of Ashkenazi Jewish descent. 8 LRKK2 and GBA mutations bridge the etiological gap between sporadic and hereditary PD, being both risk and causal factors to variable degrees on a continuum of reduced penetrance of a causative variant and increased risk. This phenomenon has been confirmed in genome-wide association studies, cumulative burden tests, and in family studies for LRRK2. 9 GBA-PD is a non-Mendelian form of PD, but GBA mutations markedly increase the risk of developing PD. 10 A meta-analysis showed that there was a significant differential effect of severe versus mild GBA mutations on the risk and age at onset (AAO) of PD, and the odds ratio for PD ranged between 2.84 and 4.94 for mild GBA mutations and 9.92 and 21.29 for severe GBA mutations with a 5-year earlier AAO for severe mutations. 11

Penetrance and Risk
"What is my risk of developing PD?" asked a young, healthy, asymptomatic lady who tested positive for a pathogenic LRRK2 variant and whose father, with PD, had tested positive earlier for this variant. The incomplete penetrance and degree of conferred risk of PD in LRRK2 and GBA mutation carriers make it difficult to predict the development of disease in asymptomatic carriers and should be considered when counseling patients with PD and their families. When neuroprotective therapies become available, we must learn more about the penetrance of PD in these mutation carriers and their predictive value. The only way we can achieve this goal is through widespread genotyping and deep phenotyping of patients with PD and the investigation of genetic modifiers. The French PD Genetic Study group estimated that PD penetrance in GBA mutation carriers was 29.7% at 80 years using a dominant model, 12 which is strikingly similar to the penetrance of the LRRK2 G2019S variant 13 and may posit GBA as an autosomal dominant condition with reduced penetrance.
There are 2 groups to consider when discussing routine genetic testing: people with PD and people at risk of developing PD with a positive family history. In both groups, we would argue against routine testing, and this will only change if genetargeted therapy is available in the PD group and if neuroprotective treatment is available in the group at risk for PD. With this in mind, are there any benefits, and what are the drawbacks of testing unaffected family members? The knowledge of one's mutation carrier status can assist with reproductive decisions, counseling about their risk of disease development, and the identification of individuals for neuroprotective trials when they are available. But this will come at a price, as insurance may not cover the costs of genetic testing and counseling as the result may not be an actionable finding. The other important drawbacks of genetic testing in this group are the negative social, psychological, and economic implications, as asymptomatic mutation carriers may be denied life insurance or future employment opportunities.

Clinical Clues and Consequences
Are there clinical clues that can distinguish LRRK2-PD and GBA-PD from idiopathic PD (iPD) ?
LRRK2-PD 14 (MDS gene) and GBA-PD 15 can present very similar to iPD except for a slightly earlier AAO in some patients, with mean AAOs of 59.4 years in LRRK2 variant carriers and 55.8 years in GBA variant carriers. 3 However, with GBA-PD, the phenotype is largely dependent on the severity of the variant type, with severe variant carriers displaying a more severe phenotype than mild variant carriers and iPD. This severe phenotype in GBA-PD includes an earlier AAO, more severe cognitive decline, more frequently developed Lewy body dementia, rapid motor progression, and reduced survival. 16 Are there important clinical consequences of LRRK2-PD and GBA-PD that would make routine genetic testing justifiable? Because LRRK2-PD and GBA-PD can look similar to iPD and their presentation can be equally heterogeneous, no single clinical sign can adequately indicate a particular gene mutation. Thus, the only way to definitively rule out or rule in a genetic cause is by genetic testing, and this is also the only strategy to identify participants for clinical trials. Consider the following scenario: a 50-year-old man presents to a neurology clinic, and he has mild motor features of PD with normal cognition and a negative family history. He could have an LRRK2 or GBA pathogenic variant or just have iPD. Although routine testing for LRRK2 pathogenic variants does not significantly alter clinical management, testing for the type of GBA variant a patient has can affect the way we plan management and counsel the patients and their families already today. A patient diagnosed with GBA-PD is often afraid of developing dementia; however, the risk of dementia and development of Lewy body dementia is higher with severe GBA mutations, 17 but even then, the patient may not develop cognitive impairment. On the other hand, many patients with iPD eventually develop dementia, 18 but carrying a severe GBA mutation does put the patient in a higher risk group for developing dementia, and genetic testing may be beneficial in these cases to better inform counseling and prognosis despite the aforementioned uncertainties in an individual patient. In the same way, there is evidence that shows cognitive impairment worsened after deep brain stimulation (DBS) in patients with GBA mutations, 19-21 but many of these studies involved a small number of GBA carriers and did not always specify which GBA variant was associated with this outcome. Therefore, the detection of a GBA mutation in a patient being considered for DBS should not be used as a strict exclusion criterion, and more largescale studies investigating the differential effect of GBA mutation types and DBS are needed before a firm conclusion can be drawn about DBS in these patients.

Gene-Targeted Therapies
Insight into the pathophysiological pathways involved in the genetic forms of PD 2 can provide important therapeutic targets for gene-targeted therapies. Of all the monogenic forms of PD, LRRK2 and GBA mutations are the most advanced targets in several trials because they are considerably more common than the other monogenic forms of PD attributed to SNCA, PINK1, Parkin, and DJ1 mutations, for which it will be difficult to achieve an adequately powered clinical trial. 22 Widespread genetic testing of patients with PD can help stratify appropriate candidates for these gene-targeted trials. Currently, this is only possible in the realm of large research projects such as the Global Genetics Parkinson Project (GP2) Monogenic hub 23 that will identify monogenic forms of PD globally, the PD GENEration study (https://www.parkinson.org/PDGENEration; NCT04057794) that provides free CLIA-certified genetic testing and counseling to patients with PD, or industry-sponsored interventional studies 3 that offer genetic testing to prospective participants. The best time to test disease-modifying treatments would be in presymptomatic mutation carriers; however, this would entail screening the general population, which would be a major challenge. 2 One way around this would be to test and include relatives of LRRK2 and GBA-PD in clinical trials, but the incomplete and variable penetrance of these mutations would also pose a major hurdle.
There are 2 main gene-targeted approaches to LRRK2. LRRK2-PD pathogenic variants cause a toxic gain-of-function increase in LRRK2 kinase activity, which has been targeted by the development of kinase inhibitors. 2 A total of 2 LRRK2 kinase inhibitors, DNL201 (NCT03710707) and DNL151 (NCT04056689), will soon enter late-stage clinical trials, with DNL151 being advanced first (Denali Therapeutics, South San Francisco, CA). There are other inhibitors in preclinical phases and include MLi-2, PF-06685360, 24 and EB-42168, a selective kinase inhibitor that may avoid the peripheral toxic effects of the nonselective inhibitors. 25 The second approach is antisense oligonucleotides (ASOs) to reduce LRRK2 mRNA transmission. A mouse PD model 26 demonstrated that LRRK2 ASOs injected into the brain reduce LRRK2 protein levels and the formation of α-synuclein inclusions. A phase I trial is underway to assess the safety, tolerability, and pharmacokinetics of intrathecal administration of an ASO called BIIB094 that targets LRRK2 mRNA expression in patients with PD (clinicaltrials.gov, NCT03976349). For GBA-PD, there are 3 gene-targeted approaches. 27 The first approach is glucocerebrosidase substrate reduction using the glucosylceramide synthase inhibitor, venglusat, to decrease α-synuclein expression (MOVES-PD). The second approach is gene therapy via a viral vector that can penetrate the blood-brain barrier and increase Glucocerebrosidase (GCase) levels in the brain (AAVa-PR001A). The third approach enhances GCase activity in the brain by using a repurposed drug called ambroxol (NCT02941822, NCT02914366), a mucolytic used for cough. A small, uncontrolled study confirmed the safety, tolerability, and ability of ambroxol to penetrate the cerebrospinal fluid (CSF) of patients with PD and increase CSF GCase protein levels in patients with PD with a GBA pathogenic variant and those without. 28 Important challenges for these genotype-driven trials are the lack of routine genetic testing, problems with interpreting the test results, lack of availability of biomarkers to guide treatment responses, and comedication with dopaminergic agents. 29

Clinicians' Versus Patients' Views
With the advent of gene-specific clinical trials, are clinicians and patients with PD and their families ready for routine genetic testing as a standard of care, and are their knowledge, interests, and attitudes toward genetic testing in PD aligned? In 2019, 178 movement disorder specialists at 146 clinical sites in Canada and the United States completed a questionnaire evaluating their current practice, knowledge, attitudes, and barriers to genetic testing in PD. 30 Of the respondents, 41% had not referred any PD patient for genetic testing, and the most common reason included cost or lack of insurance coverage, lack of perceived clinical utility, and lack of confidence in their genetic knowledge. These are major barriers that need to be overcome before genetic testing is routine in patients with PD. When clinicians' attitudes toward general screening of the Ashkenazi Jewish population for the breast cancer (BRCA1/2) gene and LRRK2 mutations were compared, 31 52% were in favor of BRCA screening, being an actionable finding, but 86% opposed LRRK2 screening, which currently has no specific treatment available. However, this will likely change should a gene-targeted therapy for LRRK2 become available and may even have a diseasemodifying effect. Genetic testing raises social and ethical concerns. Many clinicians are concerned about the implications of the genetic test results for their patients and families and need to consider their patients' attitudes and beliefs, 30 including any potential stigma attached to a genetic diagnosis. But are patients with PD interested in genetic testing? A study in the United States assessing the knowledge, attitudes, and interests of patients with PD in genetic testing 32 found that they were interested in genetics and genetic testing but lacked the associated genetics knowledge and overestimated their risk. Another study compared the knowledge and attitudes toward genetic testing in PD in American and Asian populations 33 and reported that the American patients with PD had a higher level of knowledge of PD genetics, and this was associated with a more positive attitude toward genetic testing. Hence, an essential step in preparation for routine genetic testing in PD is to improve the genetics knowledge of both clinicians and patients.

The Choice and Cost of Genetic Testing and Who Should Pay for It
Genetic tests available for PD include targeted testing in which a single or few variants in (a) gene(s) are tested, multigene panels in which multiple PD genes are tested by next-generation sequencing together with deletion/duplication analysis offered by commercial companies, and direct-to-consumer testing. 34 However, few laboratories have testing for LRRK2 and GBA mutations on the same panel. A potential solution would be to create a high-quality sequencing panel covering both the LRRK2 and GBA genes that is easily accessible and low cost.
As genetic testing is evolving, the costs are declining. In countries where health insurance is standard, different insurance companies offer different coverage depending on a patient's/relative's personal risk and family history. Genetic testing and counseling are billed separately, so overall costs are greater with counseling included. Patients or relatives may have to pay out of pocket or get payment plans with the genetic laboratories. When genetargeted therapy is available, insurance companies will have an incentive to pay for genetic testing as there will be an actionable finding. There are a few research studies 34 that offer free genetic testing AE counseling to patients with PD, such as Parkinson's Progression Markers Initiative and PDGENEration (Parkinson's Disease Genetic registry), sponsored by The Michael J. Fox Foundation and the Parkinson's Foundation, respectively. In addition, several pharmaceutic or genetic testing companies offer free genetic testing to patients with PD with no obligation to commit to the ensuing trial. A combination of industry and research would be the best option for the payment of genetic testing to patients with PD and populations where these clinical trials are available.
In the hypothetical situation that a gene-targeted therapy is FDA approved and available, how would our approach to genetic testing in PD change? We would recommend initiating genetic testing using a stepwise approach, where we first test patients for the specific pathogenic variant(s) or gene(s) that the therapy is directed against and where this therapy is available. Table 1 compares the opportunities and challenges of genetic testing in the clinical versus research setting and when genetargeted treatment is available. At present, we are just seeing the tip of the iceberg of patients with PD with a genetic cause, whereas the vast majority of patients do not have access to genetic testing, so the genetic diagnoses of many patients are buried. The more imminent future may hold gene-targeted treatments that may also become an option for neuroprotective treatment in the more distant future, each requiring different testing approaches. As gene-targeted therapies become available, genetic testing may become routine in patients with PD, but it is likely that these treatments will be expensive and accessible to only a minority of patients with PD, and we will have to overcome the same problem as with access to genetic testing.
In conclusion, the multitude of clinical trials ongoing infuses hope of a gene-targeted therapy being available to patients with PD soon. However, we do not feel it is time to offer genetic testing routinely until such gene-targeted treatment is available to patients with PD and neuroprotective therapy is available to asymptomatic mutation carriers. We would recommend genetic testing in patients with PD with atypical features, early age of onset, significant family history, and in the research setting for inclusion in clinical trials.