The PFOCUS Study aims to clarify the relationship between patent foramen ovale (PFO) and migraine with aura (MA). Patients diagnosed with MA according to ICHD-3 criteria will be classified as cases (PFO+) or controls (PFO-) based on the presence or absence of a right-to-left shunt detected by transcranial Doppler or a previously documented PFO. The study will enroll 240 adult participants over 36 months.

The primary objective is to determine whether genetic variants differ significantly between patients with MA with and without PFO. Secondary objectives include assessing cerebral blood flow regulation and platelet function in patients with MA with and without PFO.

Participants will undergo a single study visit including the collection of demographic and clinical data and a comprehensive neurosonological assessment. This will include microembolic signal counts, cerebrovascular reactivity testing, and a contrast-enhanced transcranial Doppler study for shunt detection. Blood samples will be collected for whole-exome sequencing (with a focus on NOTCH3 and an expanded gene panel) and for platelet aggregometry.

The study is expected to provide new insights into the biological mechanisms linking PFO and MA-potentially involving endothelial dysfunction, altered vascular smooth muscle responses, or platelet activation-thereby informing prevention strategies and guiding future research.

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 patients with MA and PFO (PFO+) and patients with MA 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) assessing variants and SNPs in additional genes involved in NOTCH signaling, cardiac development, vascular regulation, and PFO heritability; (2) evaluating the relationship between genetic variants and the degree of right-to-left shunt; (3) comparing microembolic signals (MES) between PFO+ and PFO- participants using automated transcranial Doppler detection; (4) assessing cerebrovascular reactivity (CVR) and dynamic cerebral autoregulation (dCA) using standardized neurosonological protocols; and (5) comparing platelet function profiles between groups.

Each participant will undergo a single study visit. Data collection will include 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 available. The neurosonological assessment will include continuous transcranial Doppler monitoring for MES detection, evaluation of CVR using breath-hold and hyperventilation maneuvers, and assessment of cerebral autoregulation using transfer function analysis, as well as contrast-enhanced detection of right-to-left shunt using agitated saline. Peripheral blood samples will be collected for genetic analysis and platelet function testing. Whole-exome sequencing will be performed, with subsequent analyses focused on a predefined panel of genes, including but not limited to NOTCH3, NOTCH1, JAG1, and HTRA1. Platelet aggregation studies will be 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 the anticipated between-group differences. Statistical analyses will include comparisons 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 patients with MA with and without PFO and to contribute to a better understanding of migraine-related vascular vulnerability.

Inclusion Criteria:

• Diagnosis of migraine with aura (according to Chapter 1.2 of the International Classification of Headache Disorders, 3rd edition [ICHD-3])
• Age ≥ 18 years
• Both sexes
• Obtained written informed consent to participate in the study

Exclusion Criteria:

• History of congenital heart disease

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