Platelet Studies to Unravel Defective Pathways in Neurodevelopmental Disorders

Background: Next generation sequencing has opened new horizons for hitherto unexplained rare diseases. But with the discovery of novel candidate genes, it is becoming increasingly difficult to pin-point the cellular pathways modified by these genes, especially for neurodevelopmental disorders that are very heterogenous and typically lack patient-derived cell models. Platelets are easily accessible cells and share molecular players that regulate granules, receptors, calcium-dependent activation and cell junctions with neurons. The serotonin pathway, frequently studied in Autism Spectrum Disorder (ASD) also plays an important role in platelet-based hemostasis, as serotonin is stored in the platelet dense granules and is released upon activation. We have used platelets to study ASD and other developmental disorders to gain insights in underlying pathophysiological mechanisms (Bijl et al, Molecular Autism, 2015 and Goubau et al, Dev Med Child Neurol, 2014).

Objectives: We here present ‘platelets as model cell to study neuropathology’ with the description of a patient with a severe neurodevelopmental disorder caused by recessive variants in SLC18A2 that codes for the vesicular monoamine transporter 2 (VMAT2) protein. VMAT2 is expected to transport dopamine, serotonin and other monoamines from the cell cytosol into synaptic granules. Its exact biological role in granules is still largely unexplored.

Methods: Our patient was included in the National Institute for Health Research (NIHR) BioResource - Rare Disease study using whole genome sequencing (WGS) for diagnosis of unexplained inherited disorders. Platelet granules were evaluated using functional assays and electron microscopy (EM) and a sensitive ELISA was conducted to measure serotonin levels in platelet extracts

Results: Our patient from consanguineous parents presented with severe psychomotor developmental delay, hypotonia, mental retardation, uncontrolled movements, gastrointestinal problems, and died at the age of 5.5 years due to pulmonary complications. Metabolic testing, brain MRI, ECG and cerebrospinal fluid analysis were normal. Though the patient had no hemostatic dysfunction, platelet functional tests were performed. This showed reduced aggregation responses to epinephrine and ADP and a mildly abnormal ATP secretion after ADP stimulation indicative of a dense granule secretion defect. Platelet EM analysis further revealed reduced numbers of dense granules with abnormal content and the presence of immature membrane complexes. WGS identified a novel homozygous variant c.946C>G in SLC18A2 that results in p.P316A in VMAT2. Both parents were heterozygous carriers. VMAT2 is highly expressed in platelets compared to other blood cell types but its role in dense granules was not studied. ELISA measurements of serotonin levels in platelet extracts showed almost undetectable levels for the patient while the parents have comparable levels to platelets from unrelated healthy controls. This, together with the EM observation indicates a defect in VMAT2 function in the patient, abolishing the serotonin transport into platelet dense granules

Conclusions: This is the first report of defective platelet dense granule morphology, function and content in a patient with a mutation in VMAT2. It shows that defective monoamine transport in neurons can be studied using mirrored defective serotonin transport in platelets and stresses the possibility of using platelet studies to gain insight into the pathophysiology of neurological disorders