Effects of GYP mRNA alternative splicing on cell surface localization of MNS blood group glycoprotein GPA and GPB / 中国输血杂志

【Objective】 To analyze the polymorphisms of GYPA and GYPB mRNA spliceosomes associated with MNS blood group, and to explore the mechanism of subcellular localization of GPA and GPB protein isomerism encoded by various spliceosomes as well as the expression of MNS blood group antigen. 【Methods】 Ten blood samples of voluntary blood donors were randomly selected. The total mRNA of peripheral blood was extracted and reversed into cDNA. Nested PCR was used to amplify reading open frame of GYPA and GYPB gene, and sequencing was performed by Sanger. The base sequence obtained was compared with GYPA(NCBI: NM_002099) and GYPB(NCBI: Nm_002100.5). After the wild type and various splicing isomer of the open reading frame of GYPA and GYPB had been obtained, they were fused with the encoding gene of green fluorescent protein (GFP) by fusion PCR technology, then cloned and transfected into HEK293 cells for over expression. The subcellular localization of GPA-GFP and GPB-GFP fused fluorescent proteins was monitored by focusing laser scanning microscope. 【Results】 Exon-1 and Exon-2 were missing in GYPA mRNA of the 2 samples, and 2~26 amino acids were missing in the predicted GPA isomer, and the full length sequence of GYPB mRNA was complete. GYPA mRNA was intact in 6 samples, exon-2 was missing in GYPB mRNA, 13~45 amino acids were missing in the predicted GPB protein isomer, and other exon sequences were intact. One sample had intact GYPA mRNA, and 364~385 bases in exon-5 of GYPB mRNA were replaced by AG, indicating truncation of amino acid signal peptide. The GYP mRNA sequences of other samples were complete. The fluorescence signal of GP-GFP fusion protein showed that all GPA and GPB glycoprotein isomers, cloned according to various RNA splicing, could demonstrate the orientation distribution on the cell membrane surface, while some alternative splicing leaded to different degrees of protein dispersion in the cell, and affected the distribution speed and proportion of protein on the cell surface, which might be one of the reasons for the strength variation of MNS antigen. 【Conclusion】 The GYP mRNA spliceosome is obviously polymorphic, but the partial deletion of GYP mRNA fragment does not affect the localization and distribution of the protein isomers encoded by GYP mRNA on the cell surface, which can ensure the expression of MNS antigen characteristics.

Effect of platelet donation frequency on iron, cooper, and zinc content and superoxide dismutase activity / 中国输血杂志

【Objective】 To study the effects of platelets donation frequency on iron, copper, zinc content and superoxide dismutase(SOD) activity in plasma of blood donors. 【Methods】 128 apheresis platelet donors from August 25, 2020 to August 25, 2021 in our center were divided into 4 groups according to the frequency of platelet donation: first-time donors(n=30) were enrolled as group 1, and donors with 2 to 7 donations(n=23), 8 to 14 donations(n=29), 15 to 24 donations(n=46) within the previous period were group 2, group 3 and group 4. All these donors were males, with the average age of 42 ± 8.3, and had not donated whole blood in the past two years. Inductively coupled plasma mass spectrometry(ICP-MS) was used to detect the content of copper, iron and zinc in plasma of different groups of platelet donors. The SOD activity was detected by WST colorimetric kit. All data were statistically analyzed by SPSS 19.0 software. 【Results】 Significant differences in the content of iron and copper, but no in zinc, were noticed in donors of different groups(P0.05). There was no significant difference in zinc content between every two groups(P>0.05). The SOD inhibition rate of blood donors in different groups was not significantly different. 【Conclusion】 The content of plasma iron, copper, and zinc and the SOD activity were not significantly affected if platelet donations were less than 15 times within a year. For those donated platelets more than 15 times within a year, the content of iron was found to decrease and copper to increase. It is suggested that platelet donations more than 15 times is correlated with the content of iron and copper in plasma of blood donors. Therefore, the proportion of iron-rich food should be appropriately increased in the daily diet for high-frequency(≥15 times per year) apheresis platelet donors after blood donation.

Distribution of KIR/HLA alleles among ethnic Han Chinese patients with hepatocellular carcinoma from southern China / 中华医学遗传学杂志

OBJECTIVE@#To assess the association of KIR/HLA alleles with hepatocellular carcinoma (HCC) and hepatitis B virus (HBV) infection among ethnic Han Chinese patients from southern China.@*METHODS@#For 95 patients with HCC and 171 healthy controls, the genotype of HLA-C alleles was determined with a PCR sequence-specific oligonucleotides typing method on an Illumina GenDx NGSgo platform. Genotypes comprised of HLA-C and KIR gene alleles were also subjected to statistical analysis.@*RESULTS@#In total 16 KIR genes (2DL2, 2DS2, 2DS3, 2DS5, 3DS1, 2DS1, 2DL5, 2DS4, 3DL1, 3DP1, 2DL3, 2DP1, 3DL3, 2DL1, 3DL2 and 2DL4) were discovered in the two groups. The frequencies of KIR2DL3 alleles and combinational genotypes of KIR2DL3/HLA-C1C2 were significantly lower in the patient group compared with the controls (0.9368 vs. 0.9883, χ²>3.84; P3.84; P<0.05, RR = 0.03). The frequency of HLA-C2C2 genotype of the patient group was significantly lower than that of the controls (0.0316 vs. 0.2690, P<0.05, RR = 0.09), while the frequency of HLA-C1C2 genotype was significantly higher than that of the controls (0.2316 vs. 0.0058, P<0.05, RR = 51.23).@*CONCLUSION@#Above results suggested that the KIR2DL3 allele is associated with lower risk for HCC. There may be individual difference in patients with HCC and HBV infection but various combinations of KIR/HLA alleles.

Effect analysis of improving the HIV positive detection rate by single sample nucleic acid amplification test in Shenzhen / 国际检验医学杂志

Objective To investigate the effect of improving the human immunodeficiency virus (HIV)posi-tive detection rate by single sample nucleic acid amplification test (SS-NAT) in Shenzhen ,and to explore the effect of SS-NAT on reducing the risk of HIV infection in transfusion .Methods 269 228 blood samples were performed parallel detection by SS-NAT (Procleix Tigris ) and two kinds of enzyme-linked immuno sorbent assay(ELISA)reagents ,and then the samples with nonreactive by ELISA and reactive by SS-NAT were tested by HIV identification assay .The blood donors with reactive HIV identification assay were made tracing tests . All the samples with reactive by ELISA or HIV identification assay were sent to the Shenzhen Center for Dis-ease Control and Prevention (CDC) for Western Blot (WB) diagnostic tests .Results The samples with reac-tive by the third generation ELISA reagents ,the fourth generation ELISA reagents ,both ELISA reagents and SS-NAT were 188 ,340 ,422 and 103 ,which reactive rate was 0 .698‰(188/269 228) ,1 .263‰(340/269 228) , 1 .567‰(422/269 228) and 0 .383‰(103/269 228) ,respectively .We found four samples with nonreactive by ELISA but reactive by SS-NAT .The four donors were found HIV reactive by both ELISA and SS-NAT after tracing .All the samples with reactive by ELISA or HIV identification assay were sent to CDC for confirmatory tests and 103 of them were positive .The positive detection rate of transfusion-transmissible HIV infection af-ter ELISA detection was 1∶67 307(4/269 228) .Conclusion The application of SS-NAT in blood screening can improve the HIV positive detection rate ,shorten window period of HIV detection and reduce residual risk of transfusion-transmissible HIV infection ,and then blood safety can be effectively improved .

Polymorphisms of MICA gene and their linkage disequilibrium with HLA-B among ethnic Han Chinese from Shenzhen / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the distribution of MICA alleles among ethnic Han Chinese blood donors from Shenzhen and their linkage disequilibrium with HLA-B gene.</p><p><b>METHODS</b>For 143 randomly selected blood donors, the MICA and HLA-B alleles were determined with a PCR-sequence based typing (SBT) method. Allelic frequency, haplotypic diversity and linkage disequilibrium were analyzed with a Pypop software.</p><p><b>RESULTS</b>Thirteen MICA and 35 HLA-B alleles were identified among the 143 blood donors, among which MICA*008:01 had the highest frequency (76/286), whilst MICA*008:01-HLA-B*40:01 and MICA*010-HLA-B*46:01 were the most common haplotypes. No novel allele was identified.</p><p><b>CONCLUSION</b>The allele frequencies, haplotype diversities and linkage disequilibrium parameters under a high resolution can facilitate further studies and applications of the MICA and HLA-B genes.</p>

Establishment of a quality control system for HLA allele typing and its key points / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To list the key points for quality control during HLA-A, B, C, DRB1 and DQB1 allele typing by taking consideration of hardware, software and experimental procedures.</p><p><b>METHODS</b>A total of 10 167 samples from randomly selected healthy blood donors and donor-recipient pairs from Shenzhen were typed for exons 2-4 of HLA-A, B, C, exon 2 of HLA-DRB1, and exons 2 and 3 of HLA-DQB1 by PCR- sequence-based typing. For 56 cases whose forward and reverse sequences were inconsistent, the samples were re-checked by a PCR-sequence specific oligonucleotide probe method. Novel alleles not included in the IMGT/HLA database were cloned and sequenced using in-house primers.</p><p><b>RESULTS</b>Eight novel HLA alleles were identified. A table for key positions of single nucleotide polymorphisms (SNPs) were generated, which summarized the key points for quality control during HLA-A, B, C, DRB1 and DQB1 allele typing. Among the listed SNPs, 3 were located at the HLA-A locus, 8 were at the HLA-B locus, 6 were at the C locus, 6 were at the DQB1 locus, and 4 were at the DRB1 locus. To ensure the quality control, an unique sample number for DNA transferring tubes in the process of experiment should be considered.</p><p><b>CONCLUSION</b>A protocol for quality control should be enforced by checking all of the key points. The SNPs and critical control points of the alleles should be examined to ensure the accuracy of HLA typing results.</p>

Genetic polymorphisms of KIR2DS4 gene among ethnic Hans from southern China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study genetic polymorphisms of the KIR2DS4 gene among ethnic Hans from southern China.</p><p><b>METHODS</b>Genomic DNA was isolated from 306 unrelated individuals and amplified with KIR2DS4-specific PCR primers. KIR2DS4-positive samples were genotyped for the entire coding sequence by sequencing-based typing (SBT). Assignment of allelic genotypes was accomplished by using Assign 3.5 software. For samples with inconclusive SBT results, RT-PCR products covering the entire coding sequence of the KIR2DS4 gene were subjected to cloning and haplotype sequencing.</p><p><b>RESULTS</b>Among all tested samples, 297 were demonstrated to have carried the KIR2DS4 framework gene. For KIR2DS4-positive samples subjected to SBT for the entire coding sequences, no background was observed with the obtained sequences. Three of the seven identified alleles were of novel types, which were officially named by the KIR subcommittee of the World Health Organization Nomenclature Committee for Factors of HLA System. The observed frequencies for the 7 alleles were KIR2DS4*00101 (78.8%), *003 (10.5%), *004 (16.0%), *010 (23.2%), *017 (0.3%), *00105 (0.3%) and *018 (0.7%), respectively. Allele KIR2DS4*007 was not found. The overall frequency for normal cell-surface expression KIR2DS4 alleles including 2DS4*00101, *017 and *00105 was 79.4%, and that for non cell-surface expression alleles including 2DS4*003, *004, *010 and *018 was 50.4%. The ratio between the two was 1.6:1.</p><p><b>CONCLUSION</b>The present study has elucidated the allelic diversity of KIR2DS4 among ethnic Hans from southern China, which may provide valuable data for transplantation as well as studies on KIR-associated disease and evolution.</p>

Identification of genomic full-length sequence of human leukocyte antigen-E and its two novel alleles / 中国组织工程研究

BACKGROUND: Human leukocyte antigen-E (HLA-E) is one of non-classical HLA class I genes. Up to now, the polymorphism analysis is mainly aimed at the variation in exon 3 of HLA-E, which determines HLA-E*01:01 or HLA-E*01:03. However, the identification of the full-length HLA-E and its novel alleles is rare reported.OBJECTIVE: To establish the method of identification of HLA-E genomic full-length sequence, and to identify its novel alleles in healthy blood donors in Shenzhen, China.METHODS: Peripheral blood DNA samples were extracted from the subjects, and the amplified primers and sequencing primers in conserved regions were designed according to the sequences of HLA-E published in the IMGT/HLA database.A high-fidelity reaction system was used to amplify the genomic full-length of HLA-E, followed by sequencing,assembling, confirming and typing.RESULTS AND CONCLUSION: Herein, we successfully established the method for amplifying genomic full-length sequence and sequence-based typing. Two novel HLA-E alleles were nominated by WHO HLA Nomenclature committee as HLA-E*01:01:01:06 and HLA-E*01:01:01:07. Compared with the most related allele HLA-E*01:01:01:01,HLA-E*01:01:01:06 had one nucleotide change at nt-26(G->T) in 5'-promoter, and HLA-E*01:01:01:07 had one nucleotide change at nt3345(T->C) in 3'-UTR. The polymorphism data of genomic full-length HLA-E in Chinese individuals need to be filled, and the method we developed here supplies the key technique for the further studies.

Accurate determination of HLA ambiguous results based on group-specific haploid full-length sequencing / 中国组织工程研究

BACKGROUND: Due to the polymorphism of HLA, a large number of ambiguities have been generated by conventional HLA typing techniques, and confirmed stereotypes of ambiguous results based on group-specific haploid full-length typing are rarely reported.OBJECTIVE: To analyze the accuracy of HLA-typing ambigulity based on group-specific haploid full-length sequencing. METHODS: The low-resolution results were used as the starting point for two ambiguous samples. Sanger sequencing (PCR-SBT) based on haploid full-length was performed after group-specific amplification. RESULTS AND CONCLUSION: One case showed a new A*02:03:01 allele, which was found a mutation in NT817 from C to T in comparison with A*11:01:01:01. The other case indicated another new C*07:02:01:01, which was found a mutation in NT879 from A to G in comparison with C*08:01:01. In conclusion, these results indicate that the group-specific haploid full-length sequencing method can be used to accurately classify HLA alleles and to discover new alleles.

Cloning and sequencing of KIR2DL1 framework gene cDNA and identification of a novel allele / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop an assay for cDNA cloning and haplotype sequencing of KIR2DL1 framework gene and determine the genotype of an ethnic Han from southern China.</p><p><b>METHODS</b>Total RNA was isolated from peripheral blood sample, and complementary DNA (cDNA) transcript was synthesized by RT-PCR. The entire coding sequence of the KIR2DL1 framework gene was amplified with a pair of KIR2DL1-specific PCR primers. The PCR products with a length of approximately 1.2 kb were then subjected to cloning and haplotype sequencing.</p><p><b>RESULTS</b>A specific target fragment of the KIR2DL1 framework gene was obtained. Following allele separation, a wild-type KIR2DL1*00302 allele and a novel variant allele, KIR2DL1*031, were identified. Sequence alignment with KIR2DL1 alleles from the IPD-KIR Database showed that the novel allele KIR2DL1*031 has differed from the closest allele KIR2DL1*00302 by a non-synonymous mutation at CDS nt 188A>G (codon 42 GAG>GGG) in exon 4, which has caused an amino acid change Glu42Gly. The sequence of the novel allele KIR2DL1*031 was submitted to GenBank under the accession number KP025960 and to the IPD-KIR Database under the submission number IWS40001982. A name KIR2DL1*031 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee.</p><p><b>CONCLUSION</b>An assay for cDNA cloning and haplotype sequencing of KIR2DL1 has been established, which has a broad applications in KIR studies at allelic level.</p>

Analysis for 2 samples with HLA-DQB1 allele dropout at exon 2 in sequence-based typing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the reason for HLA-DQB1 allele dropout during routine sequence-based typing(SBT) in order to improve the accuracy of typing.</p><p><b>METHODS</b>Two thousand samples derived from HLA high-resolution typing laboratory were typed for HLA-DQB1 locus using an AlleleSEQR HLA-DQB1 SBT kit. Non-conclusive results and "abnormal" sequencing samples were retyped using a LABType rSSO HD HLA-DQB1 kit and further analyzed with both sequence-specific primers and group-specific primers and sequenced for haplotype analysis.</p><p><b>RESULTS</b>Among the 2000 samples, 2 samples with no conclusive result were identified. The heterozygosity was confirmed with both the LAB Type SSO HD HLA-DQB1 kit and PCR-SBT in house method. Subsequent HLA-DQB1 cloning and haplotype sequencing have elucidated that HLA-DQB1*02:02 dropped out at exon 2 for the first sample and HLA-DQB1*02:01:01 dropped out at exon 2 for the second sample during PCR amplification. No novel nucleotide mutation was found.</p><p><b>CONCLUSION</b>Our results indicated that preferential amplification at exon 2 of DQB1 may result in allele dropout in exon 2 sequences during HLA-DQB1 SBT test. This may provide useful information for HLA genotyping.</p>

Sequence analysis of a novel allele HLA-C*01:78 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the genetic basis for a novel allele HLA-C*01:78.</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood using a QIAGEN quick DNA extraction kit. The regions encompassing HLA-C from exon 1 to intron 3 and intron 3 to 3'UTR were amplified and cloned using a cloning sequencing kit in order to split the two alleles apart. Selected clones were sequenced to include exons 2 to 4.</p><p><b>RESULTS</b>Sequencing results have indicated the HLA-C alleles of the proband to be a novel C*03:04 allele. The sequence has been submitted to GenBank (KF049216). BLAST analysis has confirmed the novel allele to have one nucleotide difference as C*01:03 at genomic nt316C>A (codon 82CGC>AGC) in exon 2, which has resulted in replacement of one amino acid (82R>S).</p><p><b>CONCLUSION</b>The novel allele has been officially named as C*01:78 by the WHO Nomenclature Committee. The HLA allele type of the proband was therefore A*02:07, 24:02; B*40:01, 46:01; C*01:78, 03:04; DQB1*05:02, 05:02; DRB1*16:02, 16:02.</p>

Sequencing analysis of exons 1,5, 6, 7 of HLA-Cw gene located outside of the routine testing region and its application in clinical matching / 中华检验医学杂志

Objective To study the molecular genetic polymorphism of exons 1,5, 6, 7 of HLA-C gene in Chinese population and evaluate the significance of additional sequencing based typing at exons 1,5, 6, 7 of HLA-Cw gene in clinical HLA matching, Methods A total of 324 individuals were typed at exons 2,3, 4 of HLA-C gene by sequence-based typing. If ambiguities appeared outside of exons 2 -4, we designed a total of 5 in-house sequencing primers and optimized the sequencing reaction, additional sequencing based typing at exons 1,5, 6, 7 was performed to solove the emerging ambiguities. Results In the three hundred and twenty-four samples typed by PCR-SBT at exons 2, 3 and 4 of HLA-Cw gene, 23.8 % (77/324) of the typed samples were assigned the conclusive genotype in four digital level 76. 2% (247/324) of the typed samples were given with the ambiguous allele combination results, in which 73 kinds of ambiguous allele combinations were detected. Increasing the additional sequencing analysis at exons 1, 5, 6, 7 of HIA-C gene, ten frequent ambiguities including Cw* 030201/030202, Cw* 070201/0750, Cw* 040101/0409N/0430, Cw* 0403/0409N/0430, Cw* 080101/0822 could be distinguished. ConclusionsIncreasing the sequencing anlysis at exons 1, 5, 6 and 7 of HLA-Cw gene will help to make clear the ambiguous SBT results and also improve the accuracy of HLA-Cw typing. It shows important significance in clinical histoeompatibility matching.