Construction and application of a multiplex PCR system for genotyping of human red blood cell antigens / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>A reliable method for genotyping blood group antigens Dib, k, Jsb1910 and Jsb2019 was developed. Through screening for rare blood types, the National Rare Blood Bank of China may be enriched.</p><p><b>METHODS</b>The controls for allele detection of blood groups Dib, k, Jsb1910 and Jsb2019 were prepared via polymerase chain reaction (PCR)-mediated gene site-directed mutagenesis (SDM) technique. Sequence-specific primers were designed according to known single nucleotide polymorphism (SNP) sites of alleles of blood groups antigens Dib, k, Jsb1910 and Jsb2019, a multiplex PCR system was developed by optimizing PCR reaction system. And 4190 random healthy donors samples were screened for the blood group antigens.</p><p><b>RESULTS</b>Using SDM technique, controls for alleles in blood group Dib, k, Jsb1910 and Jsb2019 were successfully generated. And a multiplex PCR system for genotyping above blood groups was developed. After verification, the system has performed with good stability and reproducibility. Two Di (b-) samples have been discovered from 4190 samples, no k- and Js(b-) sample was found.</p><p><b>CONCLUSION</b>Multiplex PCR features rapid detection, high throughput and low cost, and can be used for screening for donors of rare blood types. Information of donors may be registered in a database, which in turn can help those with rare blood types or require long-term blood transfusion to obtain matched blood, thereby reduce the adverse reactions of blood transfusion.</p>

The establishment of the controls for blood group genotyping and the application in the screening of three rare blood groups / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish the controls for allele detection of blood groups s and Ok(a). A multiplex PCR method for the detection of three blood group antigens Fy(a), s and Ok(a) was developed and used to investigate the distribution of these blood groups in Chinese random blood donors.</p><p><b>METHODS</b>Polymerase chain reaction (PCR)-based, gene site-directed mutagenesis (SDM) technique were used to make site-directed mutagenesis for the single nucleotide polymorphism (SNP) sites of the blood group alleles (the 153 C/T point mutation of the GYPB gene, and the 274 G/A point mutation of the BSG gene) as controls for allele detection. Sequence specific primers were designed according to the SNP sites of alleles of blood group antigens Fy(a), s and Ok(a). A multiplex PCR system was developed and 438 random donor samples were screened for the blood group antigens Fy(a), s and Ok(a).</p><p><b>RESULTS</b>The controls for alleles in blood groups s and Ok(a) were successfully made with the SDM technique, a multiplex PCR system was set up and successfully used to analyze the genotypes of three blood group antigens Fy(a), s and Ok(a). Two Fy(a-) samples were detected in the 438 samples, no s- and Ok(a-) sample was found.</p><p><b>CONCLUSION</b>The PCR-based SDM technique can be used to obtain the unavailable controls in blood group genotyping. The multiplex PCR technique established in this study is an efficient genotyping method for blood groups Fy(a), s and Ok(a).</p>

Short tandem repeat panel settled for quantitative chimerism analysis following allogeneic stem cell transplantation and its application / 中国实验血液学杂志

To evaluate the roles of 8 short tandem repeats (STR) loci as STR panel in quantitative analysis of chimerism following transplantation, the primers were synthesized and marked with different dyes for D3S3045, D4S2366, D4S2639, D5S818, D13S317, D18S1002, D20S481 and D22S689. The blood samples of 15 cases received allogeneic stem cell transplantation were collected before and after transplantation, then DNA was extracted and amplified with these primers, and was further analysed under ABI Genetic Analyser 3100 to select suitable informative STR locus. Donor/recipient dilution series were prepared to get standard curves in selected loci, the DNAs extracted at different days after transplantation were used to quantitatively analyze the chimerism in patients according to the values of peak area or peak height of fluorescent signals. The standard curves can be used to calculate the chimerism by plotting the respective R/D quotient value against the percentage of recipient DNA. The results indicated that the calculated chimerism was in concordance with the donor/recipient dilution. The STR panel succeeded in identifying at least one informative marker and quantitative monitoring the chimerism after HSCT in 15 donor-recipient pairs and a relapsed case was diagnosed. It is concluded that the STR panel and its detection method can accurately and quantitatively monitor the chimerism after allogeneic HSCT, which is more economical and flexible than using commercial kits.

Analysis of Kell blood group system using polymerase chain reaction-restriction fragment-single strand conformation polymorphism combined with heteroduplex in Chinese / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the polymorphism of Kell blood group system in Chinese and to find a suitable method for large scale screening.</p><p><b>METHODS</b>An analysis method of polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) combined with heteroduplex was established to detect abnormal sample in KEL exon 7-9 area, then sequencing was used to find out the mutation site.</p><p><b>RESULTS</b>Two mutations were found from 500 samples: 966G > A mutation in exon 9 and C > A mutation in 67th site of intron 7, both with no amino acid change. The mutation rate was 4/1000. No mutation was found as missed in using PCR-RF-SSCP combined with heteroduplex.</p><p><b>CONCLUSION</b>PCR-RF-SSCP combined with heteroduplex is confirmed as an effective, economical and simple method, it is quite suitable for large scale population screening study with unclear gene background and unavailable positive controls. Since there is special polymorphism for Kell blood group system in Chinese, further study is needed.</p>

Study on the molecular background of Del phenotype in Chinese population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To elucidate the molecular background of Del phenotype in the Chinese population and explore new Del alleles.</p><p><b>METHODS</b>Five hundred and fifteen RhD negative blood samples was tested by Rh typing test, indirect antiglobulin test and adsorption and elution assay to screen the Del phenotype. DNA of all the Del samples was analysed by multiplex polymerase chain reaction (MPX PCR) for the presence of RHD and by sequence-specific primer polymerase chain reaction (PCR-SSP) for Del alleles: RHD 1227A and RHD 885T. Samples which showed the negative result by PCR-SSP, were additionally analysed by genomic DNA sequencing and cDNA sequencing.</p><p><b>RESULTS</b>Seventy-nine Del samples were found by adsorption and elution assay. All these samples had RHD exons 3, 4, 5, 6, 7 and 9. Except 4 Del samples, other 75 Del samples carried the RHD 1227A allele. None of the samples had the RHD 885T allele. Four novel RHD alleles were found in these four Del sample. There were RHD 3G-->A (GenBank DQ310735), RHD 28C-->T, RHD 53T-->C (GenBank DQ451877,DQ451878), RHD 251T-->C (GenBank DQ310734).</p><p><b>CONCLUSION</b>fnRh blood group system is very complex. New D variation phenotypes and new RHD alleles may be discovered ceaselessly.</p>

Molecular study on CisAB and B(A) blood group in Chinese individuals / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>This is a study on some ABO subgroup samples which show discordant results of serological and molecular blood typing, the aim is to clarify their true ABO type by means of nucleotide analysis on exons 6 and 7 of their ABO gene.</p><p><b>METHODS</b>Absorb-elution test and family investigation were conducted to study 7 samples which were involved in ABO grouping discrepancies. Duplex polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method was used to identify their ABO genotypes. PCR products of exons 6 and 7 were cloned and sequenced.</p><p><b>RESULTS</b>All the 7 ABO subgroup samples with the discordant results of serological and molecular blood typing were found to have the normal O gene. Four out of them were typed as ABsub by serology, they were all of the A*102/O genotype. Sequencing analysis found all their A gene having the nt467 (C-->T) and nt803 (G-->C) mutation by comparison with the A*101 allele, i.e. their real type should be CisAB/O. Three out of 7 were typed as AsubB by serology and as BO by genotype; and point mutation was detected in all of their B gene. One of them had the nt700 (C-->G) mutation, the other 2 unrelated individuals had the novel nt640 (A-->G) mutation in their B alleles.</p><p><b>CONCLUSION</b>Through nucleotide analysis, 7 samples have been typed as AB subgroup in serology with the normal O gene, their real ABO type being CisAB in 4 cases and B(A) in 3 cases. At the same time, a kind of novel B (A)640 allele has been uncovered in this study.</p>

Analysis on FUT1 and FUT2 gene of 10 para-Bombay individuals in China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>This is a study on the allele composing of ABO, FUT1 and FUT2 gene loci of 10 para-Bombay individuals in China.</p><p><b>METHODS</b>Ten samples coming from different districts of China were suspected of para-Bombay phenotype by primary serology tests. Routine and absorb-elution tests were conducted to identify their ABO type, and duplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied to getting their ABO genotype. Most of them were submitted to a test of their Lewis type as well. Then through direct DNA sequencing with PCR products of FUT1 and FUT2 genes, the genotypes of their H and SE gene loci were analyzed.</p><p><b>RESULTS</b>It can be confirmed that the 10 samples are para-Bombay. All of their ABO genotypes are consistent with the serological absorb-elution results and the substances detected results in saliva. Seven out of 10 have recessive homozygous gene at their H locus. Each phenotype of h1h1 (nt547-552Deltaag), h2h2 (nt880-882Deltatt) and h4h4 (nt35 t-->c) are ascertained in 2 individuals; moreover, h3h3 (nt 658 c-->t) is identified in one individual. The rest are hh heterozygous individuals: one is h3/h(new-1); the other is h2/h(new-2); the last one is h1/h2. The h(new-1) (nt586 c-->t) allele has a point mutation at nt 586 C to T, which leads a nonsense mutation Gln(CAG) to stop (TAG).The second h (new-2) (nt328 g-->a) has an nt328 G to A missense mutation,which leads Ala (GCC),was replaced by Thr (ACC) at 110 amino acid position. All the 10 samples have Se (nt357 c-->t) synonymous mutation. One Bm(h) (B/O) individual with h4h4 phenotype has a Se(w)(nt357 c-->t; nt385 a-->t) allele, whose Lewis type is Le(a+b+). Moreover, the authors detected a (nt716 g-->a) mutation in two samples' Se gene.</p><p><b>CONCLUSION</b>Four kinds of known h alleles (h1-h4), 2 kinds of novel non-functional FUT1 alleles, a Se(w) allele, and a novel SeG716A polymorphism in Chinese para-Bombay individuals were detected. At the same time, the authors noticed that all the 10 samples have the nt357 c-->t mutation in their FUT2 gene.</p>