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Objective: To analyze the phenotype and genetic variant in a pedigree affected with inherited protein C (PC) deficiency., Methods: The proband and her family members (7 individuals from 3 generations) were tested for plasma protein C activity (PC:A), protein C antigen (PC:Ag) content and other coagulation indicators. All of the 9 exons and flanking sequences of the proband's PROC gene were amplified by PCR and sequenced. Suspected variants were verified by reverse sequencing of the proband and her family members. Bioinformatic software was used to analyze the pathogenicity and conservation of the variant site. Swiss-PdbViewer was used to analyze the three-dimensional model and the interaction with the mutant amino acid., Results: The PC:A and PC:Ag of the proband, her grandmother, father and elder brother were decreased to 55%, 52%, 48%, 51% and 53%, 55%, 50%, 56%, respectively. Genetic analysis showed that the four individuals have all carried heterozygous c.1318C>T (p.Arg398Cys) missense mutation in exon 9 of the PROC gene. The score of MutationTaster was 0.991, PROVEAN was -3.72, and FATHMM was -2.49, all predicted it to be a harmful mutation. Phylogenetic analysis also showed that Arg398 was weakly conservative among homologous species. Protein model analysis showed that, in the wild type, Arg398 can form a hydrogen bond with Glu341 and Lys395 respectively, when it was mutated to Cys398, the hydrogen bond with Glu341 disappears and an additional hydrogen bond was formed with Lys395, which has changed the spatial structure of the protein., Conclusion: The heterozygous missense mutation c.1318C>T (p.Arg398Cys) of the PROC gene probably underlay the decreased PC:A and PC:Ag in this pedigree.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common X-linked enzymopathies caused by G6PD gene variant. We aimed to provide the characteristics of G6PD deficiency and G6PD gene variant distribution in a large Chinese newborn screening population. We investigated the prevalence of G6PD in China from 2013 to 2017. Then, we examined G6PD activity and G6PD gene in representative Chinese birth cohort to explore the distribution of G6PD gene variant in 2016. We then performed multicolor melting curve analysis to classify G6PD gene variants in 10,357 neonates with activity-confirmed G6PD deficiency, and DNA Sanger sequencing for G6PD coding exons if hot site variants were not found. The screened population, organizations, and provinces of G6PD deficiency were increased from 2013 to 2017 in China. The top five frequency of G6PD gene variants were c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, and c.871G>A and varied in different provinces, with regional and ethnic features, and four pathogenic variant sites (c.152C>T, c.290A>T, c.697G>C, and c.1285A>G) were first reported. G6PD deficiency mainly occurs in South China, and the frequency of G6PD gene variant varies in different regions and ethnicities., (© 2019 Wiley Periodicals, Inc.)

Objective: To analyze the phenotype and F5 gene variant in a pedigree affected with hereditary coagulation factor V (FV) deficiency., Methods: All of the exons, flanking sequences, and 5' and 3' untranslated regions of the F5 gene were subjected to PCR and direct sequencing. Suspected variant sites were confirmed by clone sequencing. Influence of the variants was predicted by using software including ClustalX and Mutation Taster., Results: The prothrombin time (PT) and activated partial thromboplastin time (APTT) of the proband were prolonged to 20.3 s and 59.2 s, respectively, while FV activity (FV:C) and FV antigen (FV:Ag) were reduced by 13% and 17%, respectively. The FV:C and FV:Ag of his father, sister and daughter were decreased to 35%, 37%, 29% and 42%, 46%, 35%, respectively. The proband was found to carry a heterozygous c.2851delT variant in exon 13 of the F5 gene, which caused a frameshift and resulted in a truncated protein (p.Ser923LeufsX8). In addition, a heterozygous c.1538G to A (p.Arg485Lys) variant was found in exon 10. The father, sister and daughter of the proband all carried the p.Ser923LeufsX8 variant, while his mother and son carried the heterozygous p.Arg485Lys polymorphism. His younger brother and wife were of the wild type. Bioinformatic analysis showed that p.Ser923 was highly conserved across various species. Mutation Taster scored 1.00 for the p.Ser923LeufsX8 variant, and the result has predicted a corresponding disease., Conclusion: A heterozygous deletional mutation c.2851delT in exon 13 of the F5 gene and a heterozygous c.1538G to A polymorphism harbored by the proband may be associated with the decreased FV level in this pedigree.

Objective: To explore the molecular pathogenesis and clinical phenotypes in 10 probands with inherited fibrinogen (Fg) deficiency., Methods: The diagnosis of hereditary Fg deficiency was validated by prothrombin time (PT), thrombin time (TT), Fg activity (Fg:C) and Fg antigen (Fg:Ag) in plasma. All of the exons and their flanking sequences of the Fg gene were analyzed by direct sequencing. Detected mutations were confirmed by reverse sequencing., Results: The ranges of Fg:C and Fg:Ag in the 10 probands were 0.52-0.91 g/L and 0.62-2.98 g/L, respectively. Five of the probands had type I disorders, and 5 had type II disorders. Seven point mutations were identified, among which 6 have located in the D region. γThr277Arg, γAsp316His, γTrp208Leu and Lys232Thr were novel mutations, and αArg19Ser was first reported in Chinese. Four probands had the same mutation site (γArg275). As to the clinical manifestation, probands with type I disorders were asymptomatic or with mild or medium symptoms, while those belonged to type II disorders had moderate or serious symptoms. Two probands have carried an Arg275Cys mutation but had different clinical manifestations., Conclusion: Mutations of the Fg gene seem to aggregate to the D region of FGG in our region, and Arg275 is a common mutation. However, no correlation has been found between the mutation site and clinical manifestations.

Objective: To identify the genetic mutation underlying congenital hypofibrinogenamia in a Chinese pedigree., Methods: Standard coagulation tests including the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), plasminogen activity (PLG:A), D-Dimer (DD) and fibrin degradation products (FDP) were tested with fresh plasma using a STA-R analyzer. The activity of fibrinogen (Fg:C) and fibrinogen antigen (Fg:Ag) were measured respectively with the Clauss method and immunoturbidimetry. All exons and exon-intron boundaries of the fibrinogen Aα-, Bβ-, and γ-chain genes (FGA, FGB and FGG) were amplified by PCR followed by direct sequencing. Suspected mutation was confirmed by reverse sequencing and analyzed with a Swiss-PdbViewer., Results: The PT level in the proband was normal, while the APTT and TT were slightly prolonged. The functional and antigen fibrinogen levels were both significantly reduced (0.91 g/L and 0.95 g/L, respectively). Similar abnormalities were also found in her father, elder sister, son and niece. The coagulant parameters of her mother were all within the normal range. Genetic analysis has reveled a heterozygous A>C change at nucleotide 5864 in exon 7 of γ gene in the proband, predicting a novel Lys232Thr mutation. The proband's father, elder sister, son and niece were all carriers of the same mutation. Protein model analysis indicated that the Lys232Thr mutation did not disrupt the native network of hydrogen bonds, but has changed the mutual electrostatic forces, resulting in increased instability of the protein., Conclusion: The heterozygous Lys232Thr mutation identified in the FGG gene probably underlies the hypofibrinogenemia in this pedigree.