A Novel Homozygous Canonical Splice-Donor VWF Variant Causing Type 3 von Willebrand Disease in an Iranian Patient: Case Report

Technical Summary: A Novel Homozygous Canonical Splice-Donor VWF Variant Causing Type 3 von Willebrand Disease

Problem Statement
Type 3 von Willebrand disease (VWD) is the most severe form of VWD, characterized by a near-complete deficiency of von Willebrand factor (VWF) and secondary reduction in Factor VIII (FVIII). It is typically inherited in an autosomal recessive pattern and caused by biallelic loss-of-function variants, including nonsense, frameshift, and canonical splice-site mutations. While the clinical diagnosis relies on hemostatic laboratory testing, molecular characterization is essential for confirming the diagnosis, clarifying inheritance patterns, and facilitating genetic counseling. In populations with high rates of consanguinity, such as Iran, the burden of severe autosomal recessive disorders is elevated. However, the mutational spectrum of the VWF gene in Iranian patients remains incompletely defined, and novel pathogenic variants require identification to expand the database of disease-causing mutations.

Methodology
The study presents a case report of a 16-year-old Iranian female with a severe bleeding phenotype. The investigation followed a multi-step diagnostic workflow:

1. Clinical and Laboratory Evaluation: The patient underwent comprehensive hemostatic testing, including VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), Factor VIII activity, coagulation screening (PT, aPTT), and platelet function assays (Ristocetin-induced platelet aggregation).
2. Genomic Analysis: Whole-exome sequencing (WES) was performed on the proband using the Twist Comprehensive Exome Panel and Illumina NovaSeq 6000 platform. Data were aligned to GRCh38/hg38, and variants were called using GATK Best Practices.
3. Variant Prioritization and Annotation: Candidate variants were filtered based on allele frequency, zygosity, and predicted molecular consequence. Annotations were performed using Ensembl VEP, gnomAD, ClinVar, HGMD, and other databases. In silico tools (CADD, MutationTaster, GERP++, MaxEntScan, SpliceAI) were utilized to predict functional impact and splicing disruption.
4. Validation and Segregation: The identified variant was confirmed via bidirectional Sanger sequencing. Segregation analysis was performed on both parents to determine the inheritance pattern.
5. Classification: The variant was classified according to the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines, incorporating ClinGen recommendations for the PVS1 criterion.

Key Results

• Clinical Phenotype: The patient presented with recurrent mucocutaneous bleeding (epistaxis, gingival bleeding, soft-tissue hematomas) since infancy. Laboratory results showed markedly reduced VWF:Ag (4%), VWF:RCo (3%), and FVIII activity (4%), with a prolonged aPTT (75 s) that corrected on mixing study. Ristocetin-induced platelet aggregation was absent. These findings were consistent with Type 3 VWD.
• Genetic Finding: WES identified a homozygous canonical splice-donor variant in the VWF gene: NM_000552.5:c.2186+1G>A. This variant affects the invariant guanine at the +1 position of the donor splice site of intron 16.
• Variant Characteristics:
  • Novelty: The variant was absent from gnomAD v4.1, ClinVar, HGMD, and the published literature at the time of analysis.
  • In Silico Prediction: Tools predicted a disease-causing effect (MutationTaster probability 1.0), strong evolutionary constraint (GERP++ score 4.77), and marked disruption of the native donor splice site (SpliceAI delta-score ≥0.5).
  • Segregation: Sanger sequencing confirmed the proband was homozygous for the variant, while both parents were heterozygous carriers, consistent with autosomal recessive inheritance in a consanguineous family (first cousins once removed).
• Classification: The variant was classified as Pathogenic based on the ACMG/AMP framework:
  • PVS1 (Very Strong): Affects the invariant +1 nucleotide of a canonical donor splice site in a gene where loss of function is a known mechanism for Type 3 VWD.
  • PM2_Supporting: Absent from population databases.
  • PP4: The patient's phenotype (severe bleeding, profoundly low VWF/FVIII) is highly specific for Type 3 VWD.

Significance and Claims
The paper claims that this report identifies a previously unreported pathogenic variant in the VWF gene, expanding the mutational spectrum of Type 3 VWD, particularly within the Iranian population. The authors highlight the following points:

• Diagnostic Value: The case demonstrates the utility of molecular diagnosis in confirming Type 3 VWD, especially when clinical and laboratory findings are severe but a specific genetic etiology is unknown.
• Genetic Counseling: Identifying the specific variant and confirming the autosomal recessive inheritance pattern facilitates carrier detection, cascade testing for at-risk relatives, and informed genetic counseling regarding reproductive options.
• Population Genetics: The finding aligns with the observation that consanguinity in the Greater Middle East contributes to a higher prevalence of homozygous loss-of-function variants in severe recessive disorders.
• Limitations: The authors modestly note that the study lacks RNA-based functional analysis to definitively confirm the specific splicing consequence (e.g., exon skipping vs. intron retention) and that VWF multimer analysis was not performed due to the extremely low antigen levels.

In conclusion, the study provides a molecularly confirmed case of Type 3 VWD caused by a novel homozygous splice-site variant, reinforcing the importance of genetic testing in the management of severe inherited bleeding disorders.