[Compound heterozygous plasminogen mutations causing hereditary plasminogen deficiency: a family study and...

Objective: To investigate the molecular mechanisms underlying compound heterozygous mutations in a patient with hereditary plasminogen (PLG) deficiency. Methods: The proband presented to the First Affiliated Hospital of Wenzhou Medical University with a 2-day history of left-sided limb weakness. Plasminogen activity (PLG∶A) and plasminogen antigen (PLG∶Ag) were measured by chromogenic substrate and enzyme-linked immunosorbent assays, respectively, in the proband and family members (eight individuals across three generations). Sanger sequencing was performed to identify the PLG mutation sites. Bioinformatic analyses were conducted to assess evolutionary conservation and to predict pathogenicity of the mutation sites. Mutant protein models were constructed to examine mutation-induced structural changes. Recombinant plasmid expression vectors were constructed, and in vitro expression of recombinant PLG protein was studied using quantitative real-time PCR (qRT-PCR), ELISA, and Western blot analysis. Results: The proband's PLG∶A was 27% (reference range, 80%-120%) and PLG∶Ag was 103% (reference range, 50%-150%), consistent with type Ⅱ plasminogen deficiency. Genetic analysis revealed compound heterozygous missense mutations in the proband: c.1702G>A (p. Gly568Arg) in exon 14 and c.1858G>A (p. Ala620Thr) in exon 15. The c.1702G>A site is highly conserved across seven species, and is predicted to be pathogenic by bioinformatic tools. Protein modeling showed that p. Gly568Arg introduces a longer side chain and forms a new hydrogen bond with Leu686. In vitro expression showed that neither mutation caused abnormalities in PLG transcript levels, protein expression, or secretion; however, the PLG∶A/PLG∶Ag ratios in the culture supernatants were significantly lower than wild-type for both variants (Ala620Thr: 0.598 ± 0.114 vs 1.000, P=0.013; Gly568Arg: 0.412 ± 0.079 vs 1.000, P=0.022) . Conclusion: The heterozygous missense mutations p.Gly568Arg and p. Ala620Thr are associated with decreased PLG∶A in the family proband and may cause functional impairment by altering protein conformation.