OBJECTIVE: To explore the serological characteristics and bioinformatics analysis results of 4 blood donors with RHCE*cE(281C, 282T) variant allele. METHODS: A total of 4 non-related blood donors with RHCE*cE (281C, 282T) variant allele (donors 1-4) were selected as the study objects. They donated blood at Shenzhen Blood Center from January 2022 to June 2023. The 4 blood donors were all Han. And 5 mL elbow venous blood was collected from these 4 blood donors. Regular serological assaying with 4 kinds of monoclonal antibody reagents was used for determination of the RhCcEe type. The nucleotide sequences of all 10 exons and adjacent flanking intron regions of RHCE gene in these 4 donors were analyzed by Sanger sequencing, and the full-length haplotype analysis of RHCE gene was performed by using the single-molecule real-time sequencing (SMRT) third-generation technology. DeepTMHMM software was used to analyze the structure of protein transmembrane region of wild type and variant RhCcEe protein and predict the location of amino acid substitution. The effects of mutations on RhCcEe protein function were analyzed using PolyPhen-2, SIFT and Mutation Taster bioinformatics software. Robetta and Swiss-PdbViewer v4.1.0 were used for modeling the tertiary structures of RhCcEe to analyze the difference between wild type and variant RhCcEe protein. The mutation was rated according to the standards and guidelines for the classification of genetic variants of the American College of Medical Genetics and Genomics (ACMG). This study has been approved by the Medical Ethics Committee of Shenzhen Blood Center (Approval No. SZBCMEC-2022-024). RESULTS: The RhCcEe phenotypes of the 4 blood donors were CCEweake by serological assaying. The RhE antigen were weakly expressed form 0 to 3+. The analysis of RHCE gene sequence indicated that all the 4 donors with RHCE*cE (281C, 282T) allele. The mutation caused the substitution of a single amino acid in the RhCcEe protein (p.Leu94 Pro) and the amino acid substitution was located in the transmembrane α3 chain resulted in significant changes in the 3D structure of the extracellular region of RhCcEe protein. The substitution was predicted to be "Probably damaging", "Damaging" and "Polymorphism" by PolyPhen-2, SIFT and Mutation Taster bioinformatics software. According to the guidelines of ACMG, the variant was rated to be likely pathogenic. CONCLUSION The RHCE*cE (281C, 282T) variant allele was first found in the Han Chinese population. The serological data of this allele were enriched. It provides an important guarantee for the safety of blood transfusion. Bioinformatics analysis provided evidences for further study of the structure and functions of RhCcEe protein.
Humans ; Blood Donors ; Computational Biology/methods* ; Alleles ; Rh-Hr Blood-Group System/genetics* ; Male ; Female ; Adult ; Exons

OBJECTIVE: To carry out serological and molecular tests on 12 blood donors and family members of one proband with discrepancy results for ABO serological typing. METHODS: Twelve blood donors with ABO discrepancies identified by the Blood Center of Shaanxi Province from March 2015 to December 2023 and family members of one proband were selected as the study subjects. Serological blood typing was carried out to determine their blood phenotype. ABO genotype of the samples was determined by direct sequencing of amplicons of exons 1 to 7 and cloning sequencing of amplicons of exons 6 and 7. This study has been approved by the Ethics Committee of Blood Center of Shaanxi Province (202328). RESULTS: Serological results showed that 5 samples were Aweak, 4 samples were Aweak with anti-A1 antibody, and 3 samples were AweakB with anti-A1. Direct sequencing and cloning sequencing results showed that all 12 samples had the haplotype ABO*A1.01/c.389T>C, and family studies showed that the allele could be stably inherited. Glycosyltransferase activity in the plasma was decreased in all samples. CONCLUSION The c.389T>C variant of the ABO*A1.01 allele can alter the encoded amino acid p.Leu130Pro, which weakens the activity of A glycosyltransferase, ultimately leading to the weak expression of A antigen.
Humans ; ABO Blood-Group System/genetics* ; Blood Donors ; Alleles ; Male ; Female ; Exons ; Genotype ; China ; Adult ; Base Sequence ; Haplotypes

OBJECTIVE: To assess the value of combined detection strategies using multiple technologies for the genetic testing and prenatal diagnosis for pedigrees affected with Duchenne muscular dystrophy (DMD) for optimizing genetic counseling and reproductive guidance. METHODS: This study has involved 142 subjects from 65 suspected DMD families who had visited the First Affiliated Hospital of Chongqing Medical University from January 2018 to December 2023. A combination of multiple ligation-dependent probe amplification (MLPA), quantitative fluorescence PCR, and next-generation sequencing (NGS) was used. After confirming the genetic diagnosis of the probands, prenatal diagnosis was provided for carrier mothers. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: 2021-264). RESULTS: Among the 142 subjects tested, 73 cases of large deletions/duplications and 15 cases of small variants of the DMD gene were detected. The hotspot regions for the variants were exons 45 to 55. A total of 41 variant types were identified, of which 3 were previously unreported. In 19 families with suspected patients, 7 exonic deletions, 2 exonic duplications, and 3 small variants were identified. Prenatal diagnosis was performed on 48 fetuses from 46 families, revealing 16 affected male fetuses (including 12 with deletion variants, 2 with duplication variants, and 2 with small variants). Seven carrier females were identified among the 16 female fetuses (including 6 with deletions and 1 with duplication). Among the couples with an affected fetus, 16 had opted to terminate the pregnancy, while the parents of 32 fetuses had chosen to continue with the pregnancy. In families undergoing prenatal diagnosis, 53 (79.1%) pregnant women and their family members were found to carry mutations of the DMD gene. CONCLUSION The combined detection strategy of MLPA, qPCR, and NGS can encompass large deletions/duplications and small variants of the DMD gene, providing timely and accurate prenatal diagnosis for families affected by DMD. In conjunction with genetic counseling, this can effectively reduce the risk of producing affected offspring, which is crucial for the prevention and control of this disease.
Humans ; Muscular Dystrophy, Duchenne/diagnosis* ; Prenatal Diagnosis/methods* ; Female ; Male ; Pregnancy ; Pedigree ; Genetic Testing/methods* ; Dystrophin/genetics* ; Adult ; Genetic Counseling ; High-Throughput Nucleotide Sequencing/methods* ; Exons

OBJECTIVE: To analyze the phenotype and genotype of a family with hereditary coagulation factor Ⅹ (FⅩ) deficiency and preliminarily explore its molecular pathogenesis. METHODS: A hereditary FⅩ deficiency pedigree presented at the First Affiliated Hospital of Wenzhou Medical University on August 13, 2024 was selected as the study subject. Coagulation parameters of the proband and her family members (7 individuals from 3 generations) were measured using a one-stage clotting assay. All of the 8 exons and flanking sequences of the F10 gene were amplified by PCR and directly sequenced. Bioinformatics software was used to analyze the functional impact and pathogenicity of the variant proteins, as well as the spatial conformational changes and evolutionary conservation of the mutation sites. This study has been approved by the Medical Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics No.: KY2022-R193). RESULTS: The proband exhibited significantly abnormal prothrombin time (PT, 33.3 s), activated partial thromboplastin time (APTT, 47.7 s), and FⅩ activity (FⅩ:C, 3%), while other coagulation parameters remained normal. The plasma thromboplastin generation test (PTGT) demonstrated that the proband and her children had lower thromboplastin generation levels compared with the healthy control group, and the proband's thromboplastin generation capacity was more severely impaired. Genetic analysis revealed that the proband, her daughter, and grandson have all harbored a heterozygous missense variant c.212T>C (p.Phe71Ser) in exon 2 of the F10 gene, which was located in the β-sheet core region of the Gla domain. The variant has altered surrounding hydrogen bonds and disrupted calcium-binding sites. Additionally, the proband, her son, and granddaughter have all carried a heterozygous missense variant c.1270G>T (p.Val424Phe) in exon 8, which increased the side-chain volume, leading to steric hindrance in the catalytic domain and impaired coagulation function. Bioinformatics analysis confirmed that both p.Phe71Ser and p.Val424Phe were pathogenic variants, with Phe71 and Val424 being highly conserved residues. CONCLUSION The reduced FⅩ levels in this hereditary FⅩ-deficient family may be attributed to the heterozygous missense variants c.212T>C (p.Phe71Ser) in the exon 2 and c.1270G>T (p.Val424Phe) in the exon 8 of the F10 gene.
Humans ; Female ; Male ; Pedigree ; Adult ; Heterozygote ; Mutation ; Middle Aged ; Factor X/genetics* ; Exons ; Factor X Deficiency/genetics*

OBJECTIVE: To analyze the RHD genotyping and sequencing results of RhD serology negative samples in the clinic, and to further explore the laboratory methods for RhD detection, in order to provide a basis for clinical precision blood transfusion. METHODS: A total of 27 200 whole blood samples were screened for RhD blood group antigen using microcolumn gel card method.Serologic RhD-negative confirmation tests were performed on blood samples that were negative for RhD on initial screening using three different clonal strains of IgG anti-D reagents. The 10 exons of the RHD gene on chromosome 1 were also analyzed by PCR-SSP to determine RHD genotyping.When the PCR-SSP method did not yield definitive results, the RHD gene of the sample was analyzed by the third-generation sequencing. RESULTS: The results of the initial screening test by the microcolumn gel card method showed that 136 of the 27 200 samples were RhD-negative, of which 86 underwent RhD-negative confirmation testing and RHD genotyping, 88.37% (76/86 cases) of the RhD-negative confirmation test results were negative for the three anti-D reagents, and the results of RHD genotyping showed that 67.44% (58/86 cases) of the cases had a complete deletion of 10 exons, and the remaining 28 cases were RHD*711delC (1 case), RHD*D-CE(1-9)-D (1 case), RHD*D-CE(2-9-)D (2 cases), RHD*D-CE(3-9)-D (4 cases), RHD*DEL1 (c.1227G >A) mutation (16 cases), RHD*weak partial 15(845G >A) mutation (3 cases), and a mutation of c.165C >T base was found in 1 sample by three-generation sequencing. CONCLUSION RHD genotype testing of samples that are serologically negative for RhD antigen shows that some of the samples have RHD gene variants, not all of which are total deletions of RHD, suggesting that there are some limitations of the serologic method for RhD detection. Due to the polymorphism of the RHD gene structure, different RhD variants present different serologic features, which need to be further detected in combination with molecular biology testing, especially for the identification of Asian-type DELs, which is important for clinical precision blood transfusion.
Humans ; Rh-Hr Blood-Group System/genetics* ; Genotype ; Polymerase Chain Reaction ; Exons ; Blood Grouping and Crossmatching

OBJECTIVE: To analyze the B_el subtype gene mutation and its genetic mechanism in a family line. METHODS: ABO blood groups were identified by serologic tests. ABO genotyping was performed by polymerase chain reaction with sequence-specific primer (PCR-SSP). Sanger sequencing was performed on exons 1-7 of the ABO gene, the flanking intronic region, and exon 7 of the single strand of the gene confirmed the mutation site location. Missense3D software was used to predict the protein structure alteration caused by this mutation. RESULTS: Conventional serologic tests failed to detect erythrocyte B antigen in the proband and her three family members, and only trace amounts of B antigen expression could be detected by the absorption-dispersal test. DNA analysis showed that, on the basis of the normal ABO gene, there was a G>T substitution in the position of exon 7, position 803, which resulted in the change of amino acid 268 from Gly to Val. Further single-stranded sequencing analysis showed that the mutation site was located in the B gene. CONCLUSION In this family line, the proband, her father, her son, and her daughter all have reduced B type glycosyltransferase activity due to the new point mutation (c.803G>T) in exon 7 of the B gene, and the B antigen can only be detected by the absorption-dispersal method, and the point mutation can be stably inherited by offspring.
Point Mutation ; Alleles ; ABO Blood-Group System/genetics* ; Exons ; Introns ; Genotype ; Humans ; Male ; Female ; Glycosyltransferases/genetics*

OBJECTIVE: To investigate the molecular mechanism of weak D phenotype in a Chinese family. METHODS: Routine Rh typing tests were performed first, and RHD exons 1-10 of the proband and his family members were sequenced by first-generation sequencing. RHD zygosity was also determined. Third-generation sequencing was used to analyze the haplotypes of the RHD gene. RESULTS: The proband showed a weak D serological phenotype. First-generation sequencing revealed a c.787G>A point mutation in exon 5. The family pedigree investigation showed that the proband and his younger sister had the same serological phenotype and molecular mechanism. His father carried this gene mutation, while his mother and younger brother were normal. Hybrid box was not detected, suggesting that all the family members did not have a haplotype with a complete deletion of the RHD gene. The results of third-generation sequencing showed that the proband and his sister inherited the weak D allele from their father and the non-functional allele RHD -CE(3-9)-D from their mother, respectively. CONCLUSION Third-generation sequencing technology enables haplotype analysis of the RHD gene and can detect complex genotypes such as genetic exchanges between RHD and RHCE combined with other mutations.
Female ; Humans ; Male ; Alleles ; Exons ; Genotype ; Haplotypes ; High-Throughput Nucleotide Sequencing ; Pedigree ; Phenotype ; Rh-Hr Blood-Group System/genetics* ; East Asian People/genetics*

OBJECTIVE: Serological and molecular biological analysis of a B(A) subtype family was carried out to explore the underlying mechanism of B(A) subtype and clinical safe blood transfusion strategies. METHODS: The ABO blood type of the proband and her four family members were identified by serological methods, and serological experiments such as anti-H, anti-A1 and absorption-elution tests was added. In addition, the exons 6 and 7 of the ABO gene were sequenced by PCR-SSP (polymerase chain reaction - sequence specific primer). RESULTS: The serological results showed that the agglutination intensity of the proband, her mother and her maternal grandmother was imbalanced during forward typing, showing weak A and strong B antigens, and there were strong H antigens and their intensity were higher than that of normal B type. The results of reverse typing indicated the presence of weak anti-A1 antibodies, and human anti-A was positive in the absorption-elution test. Genetic sequencing revealed a characteristic mutation of c.700 C>G in all three individuals. The sequencing results showed that the proband was B(A)02/B01, her mother was B(A)02/O02, and her maternal grandmother was B(A)02/O01 . According to the compatibility principle, 1.5 units of type O washed red blood cells were transfused intraoperatively, resulting in no adverse reactions. CONCLUSION The c.700 C > G mutation on exon 7 is the molecular basis for the formation of B(A)02, and pedigree analysis shows that the B(A)02 allele was inherited from the proband's maternal grandmother to the proband's mother and then to the proband, showing a stable cis-inheritance pattern rather than a spontaneous mutation. For patients with B(A)02 subtype, type O washed red blood cells and type AB plasma can be transfused according to the principle of compatibility.
Humans ; ABO Blood-Group System/genetics* ; Female ; Blood Transfusion ; Blood Grouping and Crossmatching ; Pedigree ; Male ; Mutation ; Adult ; Exons

OBJECTIVE: To study the characteristics of a novel variant of the α-1,3-N-acetylgalactosaminyltransferase gene in a family through serological and gene sequence analyses of a proband with ABO subtype and her family members. METHODS: Blood samples of the proband and four family members were collected. The ABO phenotypes were detected by serological methods, and the ABO blood group genotyping was performed by fluorescence PCR. Direct sequencing was carried out for exons 1-7 of the ABO gene in the proband and family members, and cloning sequencing was conducted for exons 6 and 7. RESULTS: The serological test showed that the blood group phenotype of the proband was Ael type, and the ABO blood group genotyping result was A/O. Sequencing results indicated that on the basis of the ABO*A1.01 sequence, there were simultaneous variations of c.467C>T and c.664G>A in exon 7 of the A allele, which belonged to a novel variation of the A allele and had been registered in GenBank with the accession number MZ076784.1. Family investigation revealed that the proband, her son and granddaughter all had this novel variation. CONCLUSION On the basis of the ABO*A1.01 sequence, the new variation of the combination of c.467C>T and c.664G>A in exon 7 is a heritable variation. It is speculated that this variation is the cause of the weakened expression of the A antigen.
Humans ; N-Acetylgalactosaminyltransferases/genetics* ; ABO Blood-Group System/genetics* ; Pedigree ; Female ; Genotype ; Male ; Exons ; Alleles ; Phenotype

OBJECTIVE: The molecular biology of alleles of ABO blood group A_el and B_el subtype from two samples was analyzed to explore the effect of mutations on the structure of glycosyltransferase. METHODS: The ABO phenotypes were identified by serological techniques, then exons 6 and 7 of ABO gene were amplified and sequenced, combined with haplotype analysis to determine the genotypes. Finally, homology modeling of the mutated A/B glycosyltransferase were conducted by Modeller software and the effect of mutations on the spatial structure was analyzed by PyMol software. RESULTS: The serological phenotypes of the two samples were A_el and B_el, and their genotypes were ABO*AW.37/ABO*O.01.01 and ABO*BEL.03/ABO*O.01.01, respectively. The three-dimensional structure modeling of the protein showed that, compared to the wild-type glycosyltransferase, two hydrogen bonds between the side chain of p.Glu314 and surrounding amino acid disappeared in the p.Lys314Glu mutant GTA; the hydrogen bonds between the side chain of p.Trp168 and surrounding amino acid also disappeared, and the hydrogen bond between the main chain of p.Trp168 and p.Gly165 was shortened to 3.3 Å in the p.Arg168Trp mutant GTB. CONCLUSION Mutations in exon 7 of ABO gene c.940A>G and c.502C>T are keys to the formation of AW.37 and BEL.03 alleles, resulting in decreased expression of A and B antigens, respectively.
ABO Blood-Group System/classification* ; Humans ; Genotype ; Mutation ; Alleles ; Glycosyltransferases/genetics* ; Exons ; Haplotypes ; Phenotype ; Models, Molecular