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>

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>

Differentially expressed genes between a fertile patient and an infertile patient in a large Chinese androgen insensitivity syndrome pedigree / 中国医学科学院学报

<p><b>OBJECTIVE</b>To screen and identify differentially expressed genes between a fertile patient and another infertile patient who belonged to a large Chinese pedigree affected with androgen insensitivity syndrome (AIS).</p><p><b>METHODS</b>We constructed the forward and reversed subtracted libraries using genital skin fibroblasts (GSF), which were obtained from the fertile patient MJ and infertile patient ZGJ, as tester respectively. Candidate clones were screened with colony in situ hybridization, dot blot, and Southern blot analysis step by step and conformed with Northern blot analysis. The potential positive clones were sequenced and the homology of the sequences was analyzed.</p><p><b>RESULTS</b>The forward and reversed subtracted libraries containing differentially expressed pattern of two GSF cell lines were constructed. Two positive clones identified by Northern blot were obtained in the reversed subtracted library. Eleven candidate clones from the two libraries that failed to hybridize with both RNA populations were obtained simultaneously, which might represent differentially expressed low abundance transcripts. Sequencing results and homology analysis demonstrated that the two positive clones were significantly homologous with the genes of autotaxin-t and calcium binding protein calcyclin (S100A6), respectively.</p><p><b>CONCLUSIONS</b>Two positive clones and eleven clones showing no hybridization signals may represent differentially expressed genes between the two GSFs. This finding may be useful to elucidate the molecular mechanisms leading to phenotypic variation and preserved fertility of the AIS pedigree.</p>