1.Clinical Laboratory, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou People’s Hospital, Wenzhou 325000, China; 2.Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China
WANG Haijian,ZHENG Shuang,YU Xiaomin, et al. A single case gene mutation analysis of hypodysfibrinogenaemia associated with heterozygous mutation
in fibrinogen α Chain (IVS2+1G>T)[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2024, 54(7): 598-602, 封三.
Abstract：Objective: To investigate the molecular pathogenesis of hypodysfibrinogenaemia in a clinically discovered proband. Methods: A male proband who was admitted to the Wenzhou People’s Hospital on February
17, 2023 due to “right hydronephrosis and abnormal preoperative coagulation function” was selected as the study subject. The exons and flanking sequences of FGA, FGB, and FGG were PCR amplified to identify mutation sites through forward and reverse sequencing. Fibrinogen was purified from plasma using saturated ammonium sulfate for analysis of fibrinogen aggregation rate, coagulation rate, and SDS-PAGE. Wild and mutant pcDNA3.1-minigene vectors containing exons 1 to 4 sequences were constructed and transfected into HEK293T cells. Total mRNA was extracted using trizol and mutations on splice-site were confirmed using RT-PCR, Nest PCR, and q-PCR. Results: The proband exhibited a heterozygous splice-site mutation (IVS2+1G>T/c.180+1G>T) in FGA intron 2, which led to a significant decrease in the maximum rate of fibrinogen aggregation (t=443.069, P<0.001),the difference in absorbance value (t=112.317, P<0.001), and the coagulation rate (t=65.147, P<0.001) of the proband.SDS-PAGE analysis revealed the presence of FGA fibrin mutant chains in plasma. Following the construction of the minigene vector and transient transfection into HEK293T cells, RT-PCR, Nested PCR, and q-PCR analyses revealed that the mutation would disrupt the normal splicing of FGA gene mRNA. This disruption led to the deletion of 126 base pairs from the FGA exon 2 sequence during translation. Conclusion: Following the mutation rating guidelines of the American College of Medical Genetics and Genomics (ACMG) and considering the combination of supporting evidence (PVS1+PS3+PP4), the FGA IVS2+1G>T mutation variant was classified as a pathogenic mutation. The FGA IVS2+1G>T mutation is responsible for hypodysfibrinogenaemia, causing abnormal release of FGA into the blood with a deletion of 42 amino acids. However, it does not impact the assembly and secretion of fibrinogen.
