Study On Polymorphism Of SMP1 Gene In Chinese Hans And Tibetans

BackgroundThe Rh blood group system was discovered by Levine and Stetson in 1940. The Rh system is one of the clinically important blood groups, whose importance is secondary to the ABO blood group system, because it can induce Rh-incompatible hemolytic transfusion reactions, hemolytic disease of newborn (HDN), and autoimmune hemolytic anemia. Researchs on structure and function of RH gene have advanced since cDNA of RHD and RHCE allele was cloned in 1990. Over 180 RH alleles of which were 120 RHD alleles and 60 RHCE alleles were detected with genotyping technology. Domestic and international research showed that RH gene presents rich ethnic diversity whose polymorphism existed not only in coding region but also in non-coding region.The non-coding region of Rh gene mainly included richly polymorphic promoter, intron, flanking sequence and spacer, which of above regions were important for RH gene to regulate gene expression. The spacer between RHD and RHCE gene involves the downstream box and a SMP1 gene.3'UTR in exon 7 of the SMP1 gene known as TMEM50A (transmembrane protein 50A) was partial overlap with exon 10 of RHCE gene. Speculated from the location that the polymorphism of SMP1 gene is closely linked with speical RH haplotype, and relatively functional mutation may be related to selective pressure of special RH haplotype. Recent researchs focus on coding region rather than on non-coding region, furthermore, there are few studies on SMP1 gene, and researchs on Tibetan SMP1 gene has not been reported.Comparative study on SMP1 gene between Chinese Hans and Tibetans was helpful to provide insight into RH gene structure and ethnic genetic differences, to explore the functional mechanism of RH gene in evolution, recombination and mutation, as well as to reveal genetic background of RHD and RHCE polymorphism. ObjectiveTo research comparatively on gene structure and polymorphism of SMP1 gene between RHDand RHCEgene in Chinse Hans and Tibetans, and to establish specific detection for SMP1 gene; To explore the functional mechanism of SMP1 polymorphism in evolution, recombination and mutation of RH gene, as well as to study comparatively on ethnic background of SMP1 polymorphism.Methords1. Rh antigens were detected with micro-column gel technology and monoclonal and multiclonal D, C, c, E, e antibodies. Negative results from routine salt agglutination were confirmed with IAT, and Del types were detected with absorption/elution test.2. SMP1 gene were amplified by polymerase chain reaction (PCR) with specific primers designed according to reported RH gene (BN000065.1 and NC₀00001) and SMP1 gene (NT₀04610.19) sequences from GeneBank, and amplicons were sequenced after being purified.3. Sequence and structure of SMP1 gene were analyzed with DNAMAN5.2.9 sof tware and REPEATMASKER WEB SERVER (http://ftp. genome. washington. edu/cgi-bi n/RepeatMasker)4.1351T>C polymorphism of SMP1 gene was detected with PCR-SSP whose primers were directed to 1351T>C polymorphism.Results1.6 phenotypes out of 182 cases of random unrelative Guangdong Hans were detected, which were 104 cases of DCCee(57.1%),52 cases of DCcEe (28.6%), 11 cases of DCcee (6.1%),9 cases of DccEE(5.0%),5 cases of DccEe (2.7%) and 1 cases of dccee (0.5%) respectively.2.5 phenotypes out of 50 cases of random unrelative Tibetans were detected, which were 27 cases of DCcEe(54.0%),14 cases of DCCee(28.0%),4 cases of DccEe (8.0%),4 cases of DccEE(8.0%),1 cases of DCcee(2.0%) respectively, and none of RhD(-) cases was detected in Tibetans.3. Hans and Tibetans are different in Rh phenotype distribution. The DCCee phenotype frequency (57.1%) in hans was higher than that (28.0%) in Tibetans (x2=20.65,P<0.05), while DCcEe (54.0%) frequency in Tibetans was higher than that (28.6%) in Hans (x2=11.29, P<0.05).4. The whole length of SMP1 gene was 24066 bp in Hans and Tibetans, and composed of 7 exons and 6 introns. The length of exon 1-7 of SMP1 gene was 159bp,106bp,113bp,68bp,93bp,61bp and 1702bp respectively, while the length of intron 1-7 of SMP1 gene was 2017bp,2381bp,8553bp,1188bp,3818bp and 3807bp respectively.5. The SMP1 gene comprised A:6631bp (27.5%), T:7390bp (30.7%), C:5026bp (20.9%), G:5019bp (20.9%), and 3 A-T rich regions as well as 34 Alu (SINE/Alu) sequence were identified.6. Polymorphism was not found in exon 1-6 of SMP1 gene, while 1351T>C and 1726G>A polymorphism was found in exon7.7. Analysis of 1351T>C from 181 cases of RhD(+) samples,2 cases of RhD(-) samples and 3 cases of weak D samples in the Hans demonstrated that there existed 66 cases of T/C heterozygotes,106 cases of CC and 14 cases of TT homozygotes, while there had 32 cases of T/C heterozygotes,14 cases of CC and 11 cases of TT homozygotes out of 50 cases of RhD(+) and 7 cases of RhD(-) in the Tibetans.8. There existed significantly different (P<0.05) between the Hans and the Tibeans in the distribution of 1351T>C polymorlphism.Conclusion1. The distribution of Rh phenotypes in Chinses Hans and Tibetans not only had the same feature but also existed own characteristic.2. The SMP1 gene showed conserved structure i n Hans and Tibetans. Polymorphism was not found in exon 1-6 of SMP1 gene, while 1351T>C and 1726G>A polymorphism was detected in exon7.3. The methods for detecting the exon of SMP1 gene and 1351T>C polymorphism were set up successfully and that could be used to research Chinses SMP1 gene structure.4. There existed significantly different between the Hans and Tibetans in the 1351T>C polymorphism.