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Now, Dr. Heim we've talked in the past on this podcast about RT Quic specifically for alpha [00:01:00] synuclein on skin biopsies. That was episode 46, if our listeners want to listen to that as well. Today we're going to be talking about this in the context of cerebrospinal fluid. Can you just briefly tell us what is RT Quic and you also cover another technique called protein misfolding cyclic amplification, PMCA. How are they the same and how are they different?
[00:01:25] Dr. Beatrice Heim: Thank you for the invitation. So basically RT Quic and PMCA are two distinct laboratory techniques to detect and amplify misfolded proteins with key differences in term of their methodology. While RT Quick is based on the ability of misfolded proteins to induce the conversation of normal soluble proteins into misfolded aggregated forms, PMCA involves cycles of incubation and sonication to [00:02:00] promote the conversion.
Furthermore, while RT Quic promotes real time monitoring of the protein aggregation, PMCA typically requires additional steps for protein detection.
[00:02:13] Dr. Sara Schaefer: Alright. So do I take that to mean that PMCA might be a longer and perhaps more labor intensive way of detecting aggregates?
[00:02:23] Dr. Beatrice Heim: Correct. So the time required for PMCA is significantly longer with lasting about 13 to 15 days compared to RT Quic which has a duration of one to five days.
[00:02:36] Dr. Sara Schaefer: It's quick! Sorry, I couldn't resist. Okay. What the reasoning behind the systematic review and how did it come about?
[00:02:44] Dr. Beatrice Heim: So synucleinopathies are neurodegenerative disorders associated with misfolding and aggregation of alpha synuclein. It's crucial that early in the disease course we have biomarkers [00:03:00] and validated tools to differentiate between Parkinson's disease and other, maybe atypical forms of Parkinsonian disorders.
As alpha synuclein shows a prion like self propagation spreading from neuron to neuron all over the brain. This has enabled some development of amplification assays synuclein amplification assays, which can detect alpha synuclein in different bio samples. So to date there are, you know, these two assays with RT Quicc and PMCA, primarily used in CSF to differentiate between patients with synucleinopathies and non synucleinopathies
and we did this review in order to discriminate the sensitivity and specificity of these assays.
[00:03:53] Dr. Sara Schaefer: And we mentioned that this was a systematic review briefly. How were the studies selected in your [00:04:00] review?
[00:04:01] Dr. Beatrice Heim: We selected studies including patients with synucleinopathies, with Parkinson's disease, with dementia with Lewy body, but also with MSA and prodromal patients patients with RBD, with pure autonomic failure, and also non manifesting carriers, for instance, of a genetic variant causing possibly PD like LRRK2.
[00:04:26] Dr. Sara Schaefer: And as with all systemic reviews, the variability across studies is a big factor in interpretation of the pooled data. What kinds of things did you consider here, and how did you attempt to reconcile the studies?
[00:04:41] Dr. Beatrice Heim: Yeah. Study heterogeneity is indeed a common challenge in systematic reviews and meta-analysis and can make the interpretation of pooled data more complex. In our review, we performed several subgroup analysis focusing on different population [00:05:00] types for the diagnostic value of alpha synuclein seeding amplification assays.
And furthermore, we perform the quality assessment based on the quarters.
[00:05:11] Dr. Sara Schaefer: And what were the results of your study?
[00:05:15] Dr. Beatrice Heim: So in total, we included 22 studies, including over 1, 800 patients with synucleinopathies and a control group of about nearly 1, 400 patients. And what we found is that as well, RT Quic, but also PMCA showed a high pooled sensitivity and specificity to distinguish, to distinguish patients with synucleinopathies versus patients with non synucleinopathies
[00:05:48] Dr. Sara Schaefer: Now, let's dig into a couple of specific populations that you discuss in your paper. You discuss LRRK2 and multiple system atrophy or [00:06:00] MSA patients. Can you talk about the results in these populations and how you interpret them?
[00:06:06] Dr. Beatrice Heim: Well, overall, CSF alpha synuclein assays have shown inconsistent results in patients with MSA so far, with detection rates varying between under 10 percent and 35%. So, that is using PMCA as a seed amplification assay showed higher diagnostic performance than RT Quic. One possible explanation for this observed discrepancy could be the use of different reaction buffers between these two methods, but also maybe possible structural differences in the alpha synuclein strains of MSA patients. Furthermore, it is not quite clear if there are differences between MSA patients with the Parkinsonian variant and the cerebellar variant. And[00:07:00] furthermore, we also included, as you mentioned or as I mentioned before, patient with non manifested genetic variant with LRRK2, and also showing that these patients might be negative in the alpha synuclein assays, which could be possibly due to the lacking of an alpha synuclein pathology in this, in this population.
[00:07:25] Dr. Sara Schaefer: It's interesting to think about LRRK2, you know, more and more, we're thinking about how this one, you know, class of genetic mutations can lead to different pathological issues, tauopathies and synucleinopathies. And it's just so interesting to see this play out in these types of studies. What do you find as the differences in sensitivities between RT Quic and PMCA in general?
You mentioned that PMCA was of higher sensitivity in MSA patients specifically. Did you [00:08:00] find any differences in the rest of the cohorts or based on your data? Do you think that one might be preferred or over the other, or does it depend on the population or you don't have enough data to make a determination?
[00:08:15] Dr. Beatrice Heim: Yeah, it's, it's, it's still very few data to discriminate between these two methods accordingly. Because for instance, in MSA patients, PMCA showed a higher sensitivity than RT Quic, but there are more studies performing RT Quic in patients with Parkinson's syndromes. So, as our results demonstrate that both seeding amplifications represent a highly sensitive method for differentiating synucleinopathies both have their pros and cons, maybe also based on population type, also on timely basis, as I just mentioned, PMCA [00:09:00] does require more time than RT Quic. So, here further studies are needed to, to compare between these two methods.
[00:09:10] Dr. Sara Schaefer: And finally, where do you see this information influencing clinical and or research practices down the road? What are the implications for reliable in vivo biomarkers in these various nucleonopathies? How do you see this going forward?
[00:09:27] Dr. Beatrice Heim: Yes, we and also other meta analyses have already shown we could clearly demonstrate the high diagnostic performance of this seeding amplification assays in the CSF. So for, at least for differentiating synucleinopathies with Lewy body So maybe one should consider this these tests when framing supporting criteria for the clinical diagnosis of PD or DLB in the future.
[00:09:59] Dr. Sara Schaefer: [00:10:00] And of course, On the research side of things, it may be difficult, or may be helpful to categorize patients pathologically before using certain medications that might interrupt synuclein aggregation or things like that, especially if things like the genetic mutation don't give you that information as we see in the LARC2 patients, right?
[00:10:28] Dr. Beatrice Heim: Yeah, yeah, correct, especially when thinking about disease modifying therapists, this would be an option.
[00:10:36] Dr. Sara Schaefer: We're getting there! Alright. Well, thank you for taking the time today and congratulations on being awarded the review article of the year. Looks like a lot of work and an excellent paper.
[00:10:50] Dr. Beatrice Heim: Thank you.