Effect of HLA-A, -B functional epitope mismatch on platelet transfusions in patients with hematological diseases / 中国输血杂志

【Objective】 To investigate the impact of human leukocyte antigen (HLA) functional epitope mismatch (EM) on the efficacy of platelet transfusion in patients with hematological diseases. 【Methods】 HLA genotyping was performed on platelet donors and patients with hematological diseases who applied for platelet serological cross-matching and HLA antigen matching from June 2021 to June 2023 by PCR-SBT method. HLA platelet matching was based on the principle of CREG to select donors for patients. HLA Matchmaker 4.0 software was used to analyze donor-recipient HLA EM information. The expression level and gene distribution of related HLA functional epitope (Eplet) were searched from the international HLA Epitope registry website (www.Epregistry.com.br). Retrospective analysis was conducted on clinical platelet transfusion data to evaluate the impact of HLA EM on platelet transfusion effectiveness. 【Results】 Platelet transfusion efficacy showed no correlation with gender and age, but it was associated with platelet matching strategy. When the total number of HLA EMs was less than 20, a lower total number of donor-recipient HLA EMs resulted in higher platelet transfusion efficiency (χ2=19.311, P=0.001) and higher average value of 24 h corrected count increment (CCI)(F=7.737, P<0.001). The total number of donor-recipient HLA EMs had negative correlation with actual 24 h CCI (Rho=-0.322, P<0.001). Further statistical analysis revealed that 17 Eplets were related to the effectiveness of platelet transfusion. The locus distribution of 17 Eplets might be unique to HLA-A (17.6%) or -B (64.7%) or shared between HLA-A and -B (17.6%), and its expression may be high (58.8%) or intermediate (41.2%). 【Conclusion】 The total number of donor-recipient HLA EMs is a crucial factor influencing platelet transfusion effectiveness, and several HLA Eplets associated with this effectiveness have been identified.

Molecular genetic analysis of two individuals with weak D variant of the Rh blood type / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular basis of two individuals with weak D variant of the Rh blood type.@*METHODS@#Routine serological testing was carried out to detect the D, C, c, E and e antigens of the Rh blood group. The D antigen was further detected with an indirect antiglobulin test. The presence of Rhesus box was detected by PCR to determine the homozygosity of the RHD gene.@*RESULTS@#Both samples were determined as weak D phenotype by the indirect antiglobulin test. DNA sequencing revealed that case 1 harbored a heterozygous 208C>T variant in exon 2 and a heterozygous 1227G>A variant in exon 9; while case 2 harbored homozygous 779A>G variants of exon 5 of the RHD gene. Case 1 was determined as RHD+/RHD+, while case 2 was determined as RHD+/RHD-. The two samples were respectively named as weak D type 122 and weak D type 149 based on the rules of Rhesus Base Nomenclature.@*CONCLUSION@#D negative blood donors should subject to indirect antiglobulin testing and molecular analysis for safer transfusion.

Identification of a novel FUT1 allele of para-Bombay phenotype / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular basis for an individual with para-Bombay phenotype of the H blood group.@*METHODS@#Intron 5 to 3'-UTR of the ABO gene and exon 4 of the FUT1 gene were amplified with PCR and subjected to direct sequencing. Mutations of the FUT1 gene were identified by TOPO cloning sequencing.@*RESULTS@#Direct sequencing showed that her ABO genotype was B101/O01. TOPO cloning sequencing found that this individual had three mutations of the FUT1 gene, including an heterozygous AG deletion (CAGAGAG→CAGAG) at position 547 to 552, and two C→T mutations at positions 35 (C35T) and 293 (C293T) on the other homologous chromosome. The two alleles comprised a new recombination of mutations c.35T>C and c.293C>T, and the sequence has been submitted to NCBI (No. MG597611).@*CONCLUSION@#A novel combination of FUT1 alleles with c.35 C>T and c.293C>T has been identified in an individual with para-Bombay phenotype.

A case with a novel weak D type / 中华医学遗传学杂志

OBJECTIVE@#To report on a novel weak D type identified in a Chinese individual.@*METHODS@#Peripheral blood sample was collected for a voluntary blood donor with weakened expression of D antigen. Routine serological testing was carried out to determine the D, C, c, E and e antigens of the Rh blood group. A D-screening kit was used to analyze the RhD epitopes. The 10 exons and flanking intronic regions of the RHD gene were sequenced. The zygosity of RHD was determined with a sequence-specific primer PCR method.@*RESULTS@#A novel RHD allele, RHD (1022T>A), was found in the subject with a weak D phenotype. Serological testing of the RhD epitopes has coined with the weak D phenotype.@*CONCLUSION@#A novel weak D allele has been identified in Chinese population.

Identification of a novel Ax allele of the ABO blood group / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular basis for an individual with Ax28 phenotype of the ABO subtype.@*METHODS@#The ABO group antigens on red blood cells of the proband were identified by monoclonal antibodies. The ABO antibody in serum was detected by standard A, B, O cells. Exons 1 to 7 of the ABO gene were respectively amplified by PCR and directly sequenced. Amplicons for exons 5 to 7 were also sequenced after cloning.@*RESULTS@#Weakened A antigen was detected on red blood cells from the proband. Both anti-A and anti-B antibodies were detected in the serum. Heterozygous 261G/del was detected in exon 6, while heterozygous 467C/T and 830T/C were detected in exon 7 by direct DNA sequencing. After cloning and sequencing, two alleles (O01 and Ax28) were obtained. Compared with A102, the sequence of Ax28 contained one nucleotide changes (T to C) at position 830, which resulted in amino acid change (Val to Ala) at position 277.@*CONCLUSION@#The novel mutation c.830T>C of the galactosaminyltransferase gene may give rise to the Ax28 phenotype.

Identification of a novel Bx allele of the ABO blood group / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify a novel Bx13 allele.</p><p><b>METHODS</b>Serological characteristics was determined with standard serological methods. All of the seven exons and flanking regions of the ABO gene were analyzed with PCR and direct sequencing. The amplicon of exon 7 was also cloned and sequenced.</p><p><b>RESULTS</b>The individual was determined as with a rare Bx phenotype by serological tests. Direct DNA sequencing showed that the individual was heterozygous for the B/O01 allele, while there was a novel 893C>T mutation in the B101 allele, which has led to an amino acid substitution Ala298Val in the α,3-D-galactosyl-transferase. The mutation was not found among 100 randomly selected blood donors.</p><p><b>CONCLUSION</b>A novel Bx13 allele has been identified. Substitution of amino acid in the conserved region of the enzyme may reduce the activity of α,3-D-galactosyl-transferase.</p>

Molecular genetic analysis of four cases with weak D variant of Rh blood type / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular basis of 4 cases with weak D variant of Rh blood type.</p><p><b>METHODS</b>Routine serological testing was applied to determine the D, C, c, E and e antigens of the Rh blood group. The D antigen was further detected with an indirect antiglobulin test. RHD zygosity was detected by sequence-specific primer PCR method. All exons and flanking intron regions of the RHD gene were sequenced.</p><p><b>RESULTS</b>The samples were determined as weak D phenotype by serological testing. DNA sequencing showed that the 4 cases were heterozygous for 17C>T mutation in exon 1, 29G>C mutation in exon 1, 1212C>A mutation in exon 9, and IVS4+5G>A mutation in intron 4 of the RHD gene, respectively. According to the rule of Rhesus Base Nomenclature, the 4 samples were respectively named as weak D type 31, weak D type 71, weak D type 72, and weak D type 82.</p><p><b>CONCLUSION</b>Serological and molecular testing for the weak D can facilitate in-depth understanding of its immunology and genetics, and provide guidance for clinical blood transfusion and prevention of hemolytic disease in newborns.</p>

Study of molecular mechanism for a blood sample with A3 phenotype / 中华医学遗传学杂志

OBJECTIVE To explore the molecular mechanism for a blood sample with mixed-field hemagglutination upon determination of ABO blood group. METHODS Serological techniques were employed to identify the erythrocyte phenotype. The A and B antigens were detected by flow cytometry. The preliminary genotype of ABO gene was assayed with sequence-specific primer-polymerase chain reaction (PCR-SSP). Exons 6 and 7 of the ABO gene were amplified with PCR and analyzed by direct sequencing. Haplotypes of the ABO gene were analyzed by cloning sequencing as well. RESULTS The serological reaction pattern has supported an O phenotype when all the tubes were centrifuged for the first time. However, a mixed-field hemagglutination of red blood cells (RBCs) with anti-A antibodies was present after the tube was centrifuged five times later. A antigens were detected on the surface of partial red blood cells of the sample by flow cytometry. PCR- SSP results have shown that the preliminary ABO genotype was A/O. Analysis of the fragments of exons 6 and 7 of the ABO gene has indicated that heterozygosis lied as follows: 261G/A, 425T/T, 467C/T, 646A/T, 681A/G, 745C/T, 771C/T, 829A/G, conjecturing the genotype to be A307/O02, which was confirmed by haplotype sequence analysis. Compared with A101 allele, A307 allele has two missense mutations, 467C> T and 745C> T, which have resulted in substitutions Pro156Leu and Arg249Trp in the A glycosyltransferase polypeptide chain. CONCLUSION A variant allele (A307) has been identified for the first time in mainland China, which is responsible for the formation of A3 phenotype.

Study of molecular mechanism and antigen expression of CisAB01 blood group / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular mechanism of CisAB01 subtype in the ABO blood group system, and to investigate the expression of A and B antigens in red blood cells (RBCs).</p><p><b>METHODS</b>For 5 unrelated individuals with the CisAB phenotype, the molecular basis for the blood type was studied with serological assay, DNA sequencing and haplotype analysis. Bioinformatics analysis was carried out to investigate the changes in structure and function of relevant enzymes. Expression of A and B antigens in RBCs of CisAB01 was detected by flow cytometry.</p><p><b>RESULTS</b>All of the 5 samples were found to have a CisAB01 subtype. The underlying mutations, 467C>T and 803G>C in exon 7, have resulted in replacement of amino acid P156L and G268A. The mean fluorescence intensity (MFI) of A antigen in CisAB01 cases was 135, while the control group was 172. The B antigens in CisAB01 cases (MFI=38) showed significant decrease in MFI compared with the control group (MFI=164).</p><p><b>CONCLUSION</b>803G>C mutation of the ABO gene probably underlies the CisAB01 subtype. Fluorescence intensity of A antigens in CisAB01 subtype cases is slightly lower than the normal type, while the B antigen was significantly lower.</p>

Development of a multiplex polymerase chain reaction system for screening low-frequency blood group antigens K and Ytb / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>A multiplex PCR system for screening rare blood group antigens K and Yt(b) was constructed to study the distribution of the two blood groups in a Uygur population in Xinjiang, China.</p><p><b>METHODS</b>Sequence-specific primers (SSP) were designed based on single nucleotide polymorphism sites of KEL and ACHE alleles encoding the two blood group antigens. The system was designed for simultaneously detecting the two antigens by optimizing the PCR reaction. Three hundred and sixty-two randomly selected healthy individuals were screened. Products of PCR were further analyzed for heterozygosity.</p><p><b>RESULTS</b>The system was set up successfully. No KK sample was identified and 9 K+ k+ , 41 Yt (a+ b+ ), 4 Yt (a- b+ ) were found among the 362 samples.</p><p><b>CONCLUSION</b>The established PCR-SSP based multiple PCR system is efficient to screen the rare blood group antigens K and Yt(b). The information of rare blood donors obtained from the screening can be used to improve the capability of compatible transfusion.</p>

Application of multiplex PCR for the screening of genotyping system for the rare blood groups Fy(a-), s-,k-,Di(b-) and Js(b-) / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen rare blood groups Fy(a-), s-, k-, Di(b-) and Js(b-) in an ethnic Zhuang population.</p><p><b>METHODS</b>Sequence-specific primers were designed based on single nucleotide polymorphism (SNP) sites of blood group antigens Fy(b) and s. A specific multiplex PCR system I was established. Multiplex PCR system II was applied to detect alleles antigens Di(b), k, Js(b)1910 and Js(b) 2019 at the same time. The two systems was were used to screen for rare blood group antigens in 4490 randomly selected healthy donors of Guangxi Zhuang ethnic origin.</p><p><b>RESULTS</b>We successfully made the multiplex PCR system I. We detected the rare blood group antigens using the two PCR system. There are five Fy(a-), three s(-), two Di(b-) in 4490 Guangxi zhuang random samples. The multiplex PCR system I has achieved good accuracy and stability. With multiplex PCR systems I and II, 4490 samples were screened. Five Fy(a-), three s(-) and two Di(b-) samples were discovered.</p><p><b>CONCLUSION</b>Multiplex PCR is an effective methods, which can be used for high throughput screening of rare blood groups. The rare blood types of Guangxi Zhuang ethnic origin obtained through the screening can provide valuable information for compatible blood transfusion. Through screening we obtained precious rare blood type materials which can be used to improve the capability of compatible infusion and reduce the transfusion reactions.</p>