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The ubiquitin-proteasome system mediates the degradation of a wide variety of proteins. Proteasome dysfunction is associated with neurodegenerative diseases and neurodevelopmental disorders in humans. Here we identified mutations in PSMC5, an AAA ATPase subunit of the proteasome 19S regulatory particle, in individuals with neurodevelopmental disorders, which were initially considered as variants of unknown significance. We have now found heterozygotes with the following mutations: P320R (6 individuals), R325W, Q160A, and one nonsense mutation at Q69. We focused on understanding the functional consequence of PSMC5 insufficiency and the P320R mutation in cells and found that both impair proteasome function and activate apoptosis. Interestingly, the P320R mutation impairs proteasome function by weakening the association between the 19S regulatory particle and the 20S core particle. Our study supports that proteasome dysfunction is the pathogenic cause of neurodevelopmental disorders in individuals carrying PSMC5 variants., (© The Author(s) 2024. Published by Oxford University Press.)

Objective: To analyze the clinical and laboratory abnormalities of two patients with α1-antitrypsin (α1-AT) Pittsburgh in a family and review the literatures. Methods: Both plasma clotting time and factor activities were performed using clotting or substrate methods. Platelet aggregation was evaluated using an optical aggregometer. The serum protein electrophoresis was performed on Sebia HYDRASYS by using Agarose gel. The exons of α1-AT were amplified by using polymerase chain reaction (PCR) and then sequenced and compared with NCBI GenBank records. Results: The proband had several ruptures of corpus luteum and bleeding after operation, while her daughter had no bleeding history. Both of them showed prolonged coagulation tests which could not be corrected by mixing with the normal plasma. They also showed low levels of plasma coagulation factors, undetected protein C and S activity and abnormal bands of α1-globulin. The results of gene sequencing demonstrated that they were heterozygous for g.T17132G (p.Met358Arg) mutation of α1-antitrypsin gene (NG_008290.1) . Conclusions: Comparing with the data of previously reported cases, our results confirmed the obvious abnormality of coagulation test and the discrepancy of bleeding tendency of α1-antitrypsin Pittsburgh patients, and suggested that the rupture of corpus luteum would be a specific characteristic in women of child-bearing age.

This study looked into a family involving a rare mother-child ABO blood type inconsistency and explored its genetic and molecular basis. In the family, the mother had type AB blood and the father was blood type B and they gave birth to a baby of blood type O. Their blood types were phenotypically identified by using different techniques, including micro-column gel test, immune inhibition test, absorption and elution tests. The sequences of all 7 exons of ABO allele from the core family members were determined by using PCR and clone-based sequencing. The loci of mutated gene were compared against normal human genes. The result showed that the mother's erythrocytes were agglutinable with monoclonal anti-A antibody (2+) and had agglutination reaction with anti-B antibody (4+). The mother's serum registered agglutination action with standard blood type A cells. The findings showed an ABO inconsistency. When domestic antibodies were used, the mother's erythrocytes yielded agglutination reaction with humanized anti-B serum (4+) and anti-B monoclonal antibody but were non-agglutinable with humanized anti-A serum and anti-A monoclonal antibody. Upon absorption and elution, the titer of anit-A antibody was 128 both before and after the absorption test, with no significant difference found between pre- and post-absorption values. Our results confirmed that the mother's allelic gene was type B and contained type A. The father's blood type was type B, and son's blood type was type O. Clone-based sequencing revealed that the mother carried a heterozygous gene of B101.01 (ntA640→G)/O01, which contained an M214→V mutation that could express a weak expression of antigen A, resulting in blood type AB. However, their son did not have the M214→V mutation, which yielded a false ABO-inconsistency between him and his mother. We were led to conclude that type B gene with a M214→V mutation can encode both antigen B and weak antigen B that can lead to false ABO-inconsistencies.

Objective: To investigate the effect of epithermal growth factor receptor (EGFR) expression and K-ras, B-raf and PIK3CA mutation status on the radiosensitivity of human colorectal carcinoma (CRC) cell lines in vitro., Methods: Real-time RT-PCR was used to measure EGFR mRNA expression in nine human CRC cell lines, and K-ras, B-raf and PIK3CA mutation status of each CRC cell line was also identified respectively. After treatment with irradiation at graded dose, the cell viability was measured by clonogenic survival assay. The rate of cell apoptosis and cell cycle distribution were tested by flow cytometry. The cell morphology was observed with hoechst 33258 staining to analyze the correlation between EGFR mRNA expression and radiosensitivity of CRC cell lines., Results: A positive correlation between EGFR mRNA expression and survival fraction of 2 Gy(SF2) was observed (r=0.717, P=0.030). Association was also identified between the mutation status of PIK3CA and radiosensitivity (t=2.401, P=0.047), while mutation status of K-ras and B-raf was not associated with radiosensitivity. At 48-hour after exposing to irradiation, the apoptosis rate of radiosensitive cell line (HCT116) was significantly increased in a dose-dependent manner (P<0.05), while the apoptosis rate of radioresistant cell line (HT29) was significantly increased only when radiation dose increased to 6 Gy. The ratio of G0/G1 phase was reduced significantly with the increase of radiation dose in radiosensitive cell line (HCT116, P<0.05), while this trend was not observed in radioresistant cell line (HT29, P>0.05)., Conclusions: Over-expression of EGFR mRNA is correlated to radioresistance of human CRC cell lines, and mutation status of PIK3CA is closely related with radiosensitivity of CRC cells. The inhibition of apoptosis and G0/G1 arrest may induce the radioresistance of CRC cell lines.

Objective: To investigate the clinical manifestation and gene mutation in four Chinese pedigrees with the congenital coagulation factor VII deficiency., Methods: Prothrombin time (PT), activated partial thromboplastin time, thrombin time and plasma fibrinogen were measured using STAGO STA-R automatic coagulation analyzer, and the coagulation activity of factor VII (FVII:C) was determined by a PT-based one stage method, and factor VII antigen (FVII:Ag) level by a sandwich enzyme-linked immunoabsorbsent assay. All exons, exon-intron boundaries and 3',5'untranslated regions of the FVII gene from the genomic DNA of the probands and their families were amplified by PCR, and then sequenced., Results: PT was significantly prolonged, and FVII:C and FVII:Ag were decreased and the following mutations were identified in the four probands: a homozygous transversion of 18041 T→G resulting in His408→Gln substitution in exon 8 in proband 1, a homozygous double nucleotide deletion, del CT (5078 - 5079) in exon 1 in proband 2, a double heterozygous of IVS6-1G→A and Gln426→stop in proband 3, and a double heterozygous of IVS6-1G→A and Arg364Gln in prohand 4., Conclusion: Two missense mutations, His408Gln, Arg364Gln and one nonsense, Gln426stop in the catalytic domain of FVII and one double nucleotide deletion, del CT (5078 - 5079) in exon 1 and one splicesome mutation, IVS6-1G→A in intron 6 were separately identified in four Chinese pedigrees with inherited coagulation factor VII deficiency. The Gln426stop and IVS6-1G→A were first identified in the world and the homozygous del CT (5078 - 5079) and His408Gln were first found in China.

Objective: To identify gene mutations involved in five cases of inherited factor V (FV) deficiency., Methods: Activity of FV was determined by one-stage clotting assay using FV-deficiency plasma, and FV antigen by an ELISA assay. All the exons and exon-intron boundaries of FV gene were amplified by PCR and then DNA sequencing. Restriction enzyme analysis was used to analyze the probands, their family members and healthy volunteers., Results: Both activity and antigen of FV in the 5 patients were extremely lower compared with that of normal mixed plasma. Six mutations were identified in these 5 patients, G69969T (G2079V), C45533T (R712Ter), C46796T (R1133Ter), G45366A (C656Y), C46253T (R952C) and G16088C (D68H), the latter three were novel mutations reported for the first time and the C46253T (R952C) was the first missense mutation reported in B domain. The result of sequencing or restriction enzyme analysis showed that the three novel missense mutations were not caused by single nucleotide polymorphisms., Conclusion: Gene mutations in 5 type I inherited FV deficiency of patients including 2 nonsense mutations and 4 missense mutations identified which led to the instability of FV protein and the reducing of FV: Ag in the plasma.