Von Willebrand disease (VWD) is caused by either quantitative or qualitative von Willebrand (VWF) defects and is the commonest inherited bleeding disorder with an estimated prevalence of about 1% in the general population. According to several guidelines, patients with a mild quantitative reduction in VWF (30-50 IU/dL) should be labeled as "low VWF". Quantitatively VWF defects account for almost 75% of all cases with VWD and among them, low VWF seems to be the most common form. Studies on patients with VWD reported only around 50% VWF mutations in low VWF cases indicating that some possible genes outside of the VWF gene may be responsible for the low VWF levels. To date, using genome-wide association study (GWAS) more than 19 non-VWF loci (such as ABO blood group system, Stabilin 2, Scavenger Receptor Class A Member 5, C-Type Lectin Domain Family 4 Member M, etc.) were identified to be associated with VWF levels. The identified genes are related to different mechanisms of the VWF life-cycle such as synthesis, secretion, glycosylation, or clearance. Despite the importance of the genetic background of low VWF levels for understanding its etiology, this issue is not well investigated yet. Thus the Low VWF Milan Cohort (LOVMIC) Study is designed to address some unanswered questions in patients with low VWF.

Despite the absence of mutation in the VWF gene in a significant number of individuals with reduced VWF levels and also the lack of knowledge for the responsible mechanisms, this study sought to determine the following goals:

• Evaluation of the genetic background of low VWF level dilemma and identifying the gene (s) outside of the VWF gene that is associated with decreased VWF levels.
• Evaluating the correlation between candidate variants and patients' bleeding manifestations.

Study design:

Non-pharmacological Interventional National Monocentric Study. Promoter: Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Coordinating center and patient recruitment unit: Department of General Medicine - Hemostasis and Thrombosis - Angelo Bianchi Bonomi Hemophilia and Thrombosis Center

Setting: outpatients' clinic

The study population will be selected from the referral adult patients/healthy controls to the A. Bianchi Bonomi Hemophilia and Thrombosis Center in Fondazione IRCCS Ca' Granda Maggiore Policlinic hospital.

Recruiting method:

Selected patients (base on the previous laboratory results) will be invited to participate in the study by physicians at the center through phone calls. Also, normal controls (age- and sex-matched with patients) will be enrolled in the study. Data regarding the healthy controls will be obtained either from the available public database or obtained by evaluation of collected samples from the normal subjects who have been selected by the A. Bianchi Bonomi Hemophilia and Thrombosis Center.

Enrolment, visit, and blood samples collection:

Following the agreement of patients for participating in the study and signing the informed consent, 3 tubes (each 3.5 ml) of the blood sample will be collected for performing VWD-related laboratory tests (VWF antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), Factor VIII clotting assay (FVIII:C)) and Whole-exome sequencing (WES). In addition, a routinely clinical examination will be done by specialized physicians, according to a Case Report Form (CRF) to collect the data regarding age, sex, blood group, and clinical manifestations including the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT).

Genetic analysis:

• Genomic DNA will be extracted using the automated instrument from QIAGEN available in the central genetic laboratory at the Fondazione IRCCS Ca' Granda Maggiore Policlinic hospital
• WES will be performed on all samples using NextSeq 2000 instrument in the central genetic laboratory at the Fondazione IRCCS Ca' Granda Maggiore Policlinic hospital.
• Data will be analyzed following the same strategy in both cases and controls. First, the VWF gene will be evaluated. Then, the analysis will be extended to the other genes that were previously described as related to VWF level variations. Lastly, exome data will be considered.
• The association between VWF levels and candidate variants will be assessed.
• All analyses will be performed considering variants' minor allele frequency (MAF), age, sex, ABO to control for confounding.

Inclusion Criteria for patients:

• Adult patients who were diagnosed with "low VWF" and have VWF:Ag and/or VWF:RCo between 30-50 IU/dL with a ratio of VWF:RCo/VWF:Ag > 0.6.
• Subjects who have given informed consent to participate in the study according to the Declaration of Helsinki

Inclusion Criteria for healthy controls:

• Healthy subjects with no known bleeding disorders and with negative thrombophilia screening results
• Subjects who have given informed consent to participate in the study according to the Declaration of Helsinki

Exclusion Criteria:

• Pregnant women
• Patients with acquired von Willebrand disease syndrome

All data, including exposure and outcomes, will be collected based on a Case report form (CRF) and thereafter they will be reported on a computer database. Each process to promote data quality will be guaranteed. In particular, data insertion and correction will be performed by the principal investigator (PI) or by those investigators designated by the PI and all data will be securely stored to ensure complete pseudonymization. The insertion of all the data in the CRF and the computer database will be performed correctly and accurately according to the source documentation (medical reports and records). In case of a lack of data, and accurate motivation will be provided (not available/not requested from the patient).