[Genetic analysis of a Chinese pedigree affected with Hereditary coagulation factor Ⅴ deficiency due to compound heterozygous variants IVS24+3A>T and p.Asp2222Gly].

OBJECTIVE: To explore the clinical phenotype and genetic etiology for a Chinese pedigree affected with Hereditary coagulation factor Ⅴ deficiency (FⅤD). METHODS: A 47-year-old female who visited the First Affiliated Hospital of Wenzhou Medical University in July 2025 and her family members (husband, mother, younger brother and younger brother's wife, elder sister, second elder sister, daughter, son, elder niece, and second niece) were selected as study subjects. Clinical data and family history were retrospectively collected. Peripheral blood samples were collected from the proband and her family members. Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), coagulation factor Ⅱ activity (FⅡ:C), coagulation factor Ⅶ activity (FⅦ:C), coagulation factor Ⅷ activity (FⅧ:C), coagulation factor Ⅹ activity (FⅩ:C), and FⅤ activity (FⅤ:C) were measured in all participants using the one-stage clotting assay. FⅤ antigen (FⅤ:Ag) levels in plasma were determined with enzyme-linked immunosorbent assay (ELISA). Genomic DNA was extracted from peripheral blood samples of all participants, and the coding regions of the F5 gene were amplified by PCR and subjected to Sanger sequencing. Candidate variants were verified with multiple online tools to predict their pathogenicity and impact on splicing. Based on guidelines formulated by the American College of Medical Genetics and Genomics (ACMG), the pathogenicity of candidate variants was evaluated. Coagulation function of all participants was evaluated using thrombin generation assay and thromboelastography. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: KY2022-R193). RESULTS: The proband's peripheral blood PT and APTT were 25.6 s and 58.3 s, respectively, while FⅤ:C and FⅤ:Ag were 7% and 6%, respectively, which was in keeping with a phenotype of type I FⅤD. Her brother also showed significantly decreased FⅤ:C and FⅤ:Ag levels. The proband's mother, eldest sister, second sister, daughter, son, eldest niece, second niece had FⅤ:C and FⅤ:Ag approximately 50% of those of normal controls, whereas her husband and sister-in-law showed no obvious abnormality in such indices. Genetic testing revealed that the proband and her brother both harbored compound heterozygous variants of the F5 gene, namely c.6528+3A>T (IVS24+3A>T) and c.6665A>G (p.Asp2222Gly), for which other family members with reduced FⅤ:C and FⅤ:Ag were heterozygous carriers, and those with normal coagulation indices were of the wild-type. Bioinformatics analyses using multiple software indicated that the IVS24+3A>T variant is located at a splice donor site and can significantly weaken the strength of the splice site, potentially leading to exon skipping. Based on the ACMG guidelines, the novel variant is classified as likely pathogenic (PM2_Moderate+PM3_Moderate+PP1_Supporting+PP3_Supporting+PP4_Supporting). Results of thrombin generation assay indicated reduced thrombin generation capacity in the proband, whilst thromboelastography results showed abnormal coagulation characteristics manifested as prolonged coagulation initiation time. Ultimately, both the proband and her younger brother were diagnosed with hereditary type I FⅤD. CONCLUSION: The IVS24+3A>T and p.Asp2222Gly compound heterozygous variants of the F5 gene probably underlay the pathogenesis of FⅤD in this pedigree. Above finding has enriched the mutational spectrum of the F5 gene and provided a basis for genetic counseling and molecular diagnosis.