Spinal fluid marker could help identify type 1 narcolepsy: Study

Testing cerebrospinal fluid for fragments of the signaling molecule hypocretin-1 could help identify narcolepsy type 1 (NT1) and, potentially, track the disease’s progression more consistently than current diagnostic methods, a study found.

Part of differentiating NT1 from narcolepsy type 2 (NT2) or other sleep disorders involves testing for hypocretin-1 (Hcrt-1). However, existing tests have several limitations. The research team identified a small fragment of Hcrt-1, Hcrt-1(1-16), as a possible alternative to the full-length molecule.

“Hypocretin-1[1-16] is significantly reduced in patients with narcolepsy type 1 and appears to serve as a possible marker to discriminate NT1 from controls,” the researchers wrote. However, they said, the method will require further validation before it can be used for standard clinical diagnosis.

The study, “Hypocretin-1/ Orexin-A fragment1-16 as a potential surrogate marker for diagnosing narcolepsy type 1,” was published in Sleep.

In NT1, mistaken immune activity reduces levels of Hcrt-1, also called orexin-A, a signaling molecule that helps regulate sleep. This causes sleep disturbances and excessive daytime sleepiness, along with other narcolepsy symptoms. NT2 is related but has less clear autoimmune origins and slightly different symptoms.

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Limitations of current testing

Diagnosing narcolepsy typically involves a series of sleep and neurological tests. Testing cerebrospinal fluid (CSF), the liquid that surrounds the brain and spinal cord, for Hcrt-1 can then help distinguish NT1 from NT2. People with NT1 generally have lower CSF Hcrt-1 levels than healthy people.

Radioimmunoassay (RIA) testing is the standard method for measuring Hcrt-1 levels. This technique uses antibodies and radioactive labels to quantify levels of the molecule. However, “there is a current debate about the significance of intermediate Hcrt-1 levels and the cut-off determination using the RIA method for the diagnosis of NT1,” the researchers wrote.

RIA has other limitations. As it can’t measure Hcrt-1 precisely enough, this technique does not enable assessing NT1 progression over time. And the label may bind to fragments of molecules other than Hcrt-1, making the results more difficult to interpret.

In the study, the team first assessed a previously proposed alternative method to identify Hcrt-1 without RIA. They used liquid chromatography-mass spectrometry (LC-MS) in samples from 10 participants with NT1 and 21 controls. LC-MS separates the components of a substance and analyzes them based on their molecular weights.

When comparing CSF samples from people with NT1 and healthy controls using this method, the researchers saw overlapping yet significantly lower Hcrt-1 levels in the NT1 group than in controls. That indicates “full-length Hcrt-1 seems not to be the ideal target for mass spectrometric quantification,” they wrote.

They tried using the same method to detect a related molecule, Hcrt-2, but couldn’t detect Hcrt-2 at all with LC-MS.

For the second part of their experiment, the researchers closely assessed which molecules and fragments current RIA techniques capture most. The test strongly identified a fragment of Hcrt-1, called Hcrt-1(1-16). The team hypothesized that Hcrt-1(1-16) could therefore be a marker of Hcrt-1.

They tested this hypothesis using an advanced type of LC-MS and two different methods for preparing CSF samples for analysis. One of these purification techniques showed significant differences in Hcrt-1 levels between the NT1 and control samples, whereas the other did not. However, regardless of the preparation, Hcrt-1(1-16) levels were significantly lower in people with NT1 than in controls.

This consistency across methodologies “strengthens its potential role as a new surrogate marker for hypocretin-1 for targeted quantification,” the scientists wrote. Measuring Hcrt-1(1-16) with advanced LC-MS could theoretically be a useful way to diagnose and track NT1, they said.

The study was small, with four NT1 participants and four control participants contributing samples to the final part. Replicating and validating these results in a larger group would help strengthen the findings, the researchers wrote.

While distinguishing people with NT1 from healthy controls is important, a useful diagnostic test would also be able to distinguish NT1 from NT2 or other sleep disorders, the researchers said. Future trials could compare Hcrt-1(1-16) levels across these diseases to determine how specific the findings are to NT1.