Description

The PFOCUS study is a prospective, case-control observational investigation designed to explore the biological, vascular, and genetic factors associated with the coexistence of migraine with aura (MA) and patent foramen ovale (PFO). Epidemiological evidence indicates that individuals with MA have an increased risk of ischemic stroke even in the absence of traditional vascular risk factors. In parallel, a higher prevalence of PFO has been consistently reported in patients with MA, suggesting a potential role of PFO as a modifier of cerebrovascular risk. The mechanisms underlying this association are not fully understood and may involve factors beyond paradoxical embolism, including vascular dysfunction, altered cerebral hemodynamics, platelet activation, and genetic susceptibility.

The primary objective of the study is to compare the distribution of NOTCH3 gene variants and single nucleotide polymorphisms (SNPs) between MA patients with PFO (PFO+) and MA patients without PFO (PFO-). NOTCH3 encodes a receptor involved in vascular smooth muscle cell differentiation and small-vessel integrity, and pathogenic variants are known to cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Variants in this gene have also been associated with migraine phenotypes and alterations in cerebral small-vessel function. Given the role of Notch signaling pathways in cardiac septation and closure of the fetal foramen ovale, genetic variation in this pathway may contribute to both MA susceptibility and persistence of PFO.

Secondary objectives include: (1) the assessment of variants and SNPs in additional genes involved in Notch signaling, cardiac development, vascular regulation, and PFO heritability; (2) evaluation of the relationship between genetic variants and the degree of right-to-left shunt; (3) comparison of cerebral microembolic signals between PFO+ and PFO- participants using automated transcranial Doppler detection; (4) assessment of cerebrovascular reactivity (CVR) and dynamic cerebral autoregulation (dCA) using standardized neurosonological protocols; and (5) comparison of platelet function profiles between groups.

Each participant undergoes a single study visit. Data collection includes a structured clinical and demographic assessment, detailed characterization of migraine phenotype (including aura characteristics, attack frequency, and coexistence of migraine without aura), and review of available brain magnetic resonance imaging (MRI) findings when present. The neurosonological assessment includes continuous transcranial Doppler monitoring for detection of microembolic signals, evaluation of CVR and cerebral autoregulation using breath-hold and hyperventilation maneuvers with transfer function analysis, and contrast-enhanced right-to-left shunt detection using agitated saline. Peripheral blood samples are collected for genetic analysis and platelet function testing. Whole-exome sequencing is performed, with subsequent analysis focused on a predefined panel of genes, including but not limited to NOTCH3, NOTCH1, JAG1, and HTRA1. Platelet aggregation studies are conducted using standardized laboratory methods.

The planned sample size is 240 participants, equally distributed between the PFO+ and PFO- groups. This sample size is based on the expected prevalence of NOTCH3 SNPs and anticipated differences between groups. Statistical analyses include comparison of allele and genotype frequencies, assessment of Hardy-Weinberg equilibrium, linkage disequilibrium analyses, and multivariable comparisons of neurosonological and platelet function parameters.

By integrating genetic, neurosonological, and hematological data, the PFOCUS study aims to characterize potential biological differences between MA patients with and without PFO and to contribute to a better understanding of migraine-related vascular vulnerability.