Metabolomic profiling of cerebrospinal fluid to improve diagnosis and treatment monitoring of patients with inborn or acquired errors of metabolism

Summary
The objective of this project is to develop new tests, using modern technology, to improve our diagnostic service. Our diagnostic laboratory is an NHS national referral centre for patients suspected of having abnormalities in brain metabolism, particularly with regards to neurotransmitter and vitamin metabolism (see below). The sample type that we use for diagnosis is cerebrospinal fluid (CSF). We receive around 1000 CSF samples per year. Many of these samples are from young children with suspected genetic disorders affecting brain metabolism, causing them to have clinical symptoms such as movement disorders, developmental delay or epileptic seizures. We also receive CSF samples from older patients for treatment monitoring or to identify potentially treatable symptoms in patients with neuroinflammation, movement disorders (such as Parkinson's disease) or neurodegeneration.
Cerebrospinal fluid (often abbreviated as CSF) is the fluid that surrounds the brain and spinal cord. Nutrients from the blood are transported into the CSF via the blood-brain barrier. The CSF then takes these nutrients to brain cells and removes waste products from the brain to be transported back across the blood-brain barrier and either be recycled or excreted from the body. In contrast to blood, CSF is clear in colour and has little or no cells. This makes it a nice clean matrix for looking at brain metabolism. The small molecules that we measure in body fluids, such as blood and CSF, are known as metabolites and help us to understand how the organs of the body are using and recycling nutrients from the diet. Studying the metabolites can also help us to identify and diagnose disorders where the body is not using or recycling these nutrients effectively.
Our current CSF service results in a definitive diagnosis in only around 5% of patients. However, almost 50% have abnormalities of unknown significance in our current tests and this is likely to be higher if more brain metabolites could be measured. These patients are likely to have an undiagnosed neurometabolic condition. The aim of this project is to use modern techniques to increase the number of metabolites that we can measure so that we can diagnose this group of currently undiagnosed patients.
We currently have four tests that measure metabolites in CSF. These tests are based on 30 year old technology but are very reliable and we have many years of experience of interpreting the results from these tests. These tests measure neurotransmitter metabolites and vitamins/co-factors that are required for neurotransmitter synthesis and many other essential chemicals in the brain. With these four tests we currently measure 10 metabolites, in total, in CSF.
The new tests that we will develop will use mass spectrometry, a modern ultra-sensitive and rapid technique. This technology will allow us to increase the number of metabolites that we can measure from 10 currently to over 30. Although mass spectrometers are capable of measuring many more metabolites, we initially intend to focus on a smaller number (around 30) and expand as required. This is so that we can focus on specific pathways that are hypothesised to be affected in our group of patients. This will also make it easier to ensure that the method is robust, reproducible and accurate.
At the moment, our diagnostic assays are very good at highlighting defects in brain neurotransmitter or vitamin metabolism. However, due to the limited number of metabolites that we measure, we can not always define the exact cause and functional effect of the defect. A modern mass spectrometry method will give us much greater insight into brain metabolism and allow us to characterise and diagnose currently undiagnosed disorders. It will also allow us to suggest and monitor treatments better. This will in turn improve the lives of children and families affected by these devastating but often treatable diseases affecting brain metabolism.

Our unit analyses around 1000 CSF samples each year. Our current CSF service results in a definitive diagnosis in about 5% of cases. Many of these disorders respond well to treatment (e.g. tyrosine hydroxylase deficiency, vitamin B6 responsive epilepsies, folate deficiencies, pterin defects) and early diagnosis makes a huge difference to families affected by these disorders. However, almost 50% of samples show metabolic abnormalities of unknown significance. If more CSF metabolites could be measured, the number of observed abnormalities is likely to be higher. Many of these patients with metabolic abnormalities could have a treatable but undiagnosed neurometabolic condition. With extra resources to support research, there is huge potential to identify and understand novel and known defects affecting brain and CSF metabolism. This will also help to inform treatment.

The aim of this project is to use modern techniques to increase our diagnostic repertoire to help diagnose this group of currently undiagnosed patients. We propose to develop robust and precise, multi-analyte (metabolomics) mass spectrometry assays to investigate inborn and acquired disorders of dopamine, serotonin, vitamin and antioxidant metabolism. These assays will also measure metabolites related to immune activation (neopterin and kynurenine pathway metabolites). These assays will improve on the speed and depth of current HPLC assays and will be able to measure multiple metabolites in small sample volumes. These assays will initially be used for the diagnosis and treatment monitoring of children with neurometabolic conditions but will also be applicable to neurological disorders in adults such as Parkinson's disease (dopamine deficiency), Alzheimer's disease (abnormal 1-carbon metabolism), depression (serotonin dysfunction), motor neurone disease (antioxidant dysfunction) and multiple sclerosis (immune activation).