海南汉族RhD阴性个体RHD基因研究

目的探讨海南汉族Rh阴性献血者的RHD基因结构,为建立适合本群体的正确的RHD基因定型方法提供依据。方法采用常规盐水法和抗球蛋白法筛选RhD阴性献血者,通过吸收放散试验确定其中RhDel,并采用PCR SSP技术分析其RHD基因存在情况,对存在全部外显子的个体进一步检测RHD内含子2、10和RHDψ假基因。结果筛选获得的106名RhD阴性个体中,31例(29.25%)为RhDel,存在完整RHD基因;剩下75例中,67例(63.21%)完全缺失RHD基因,8例缺失部分RHD基因;存在全部外显子的31例RhDel个体均存在RHD内含子2、10而无RHDψ基因。另外,在1例ccdEe样本中检测到外显子1、3、4、6、7、9及10,仅缺少外显子5。结论海南汉族RhD阴性群体中存在高比率的RhDel,且所有RhDel样本均可检测到全部的RHD基因外显子;海南汉族真实RhD阴性个体的RHD基因呈多态性,缺失部分RHD基因的个体均未检测到RHD外显子5,提示对本群体而言,特异性扩增外显子5在RHD基因定型中具有重要意义。     

徐州地区汉族RhD阴性人群RHD基因多态性研究

目的：研究徐州地区汉族RhD阴性人群RHD基因多态性。方法采用常规血清学技术检测RhD抗原表型,采用抗球蛋白试验（IAT）确认结果。采用序列特异性引物-聚合酶链反应（SSP-PCR）方法检测RHD基因。结果110例RhD阴性个体中,SSP-PCR检测RHD基因结果为RHD 阳性基因携带者5例,RHD阴性基因携带者47例,RHD-CE（2-9）-D 基因携带者22例,DVI Ⅲ型基因携带者17例,弱D15基因携带者2例,DEL-1227A基因携带者17例。结论徐州地区汉族人群RhD阴性个体呈现RHD基因结构复杂的多态性,以变异体等位基因RH-CE（2-9）-D 基因、DVI Ⅲ型基因、DEL-1227A基因为主。     

Molecular background of D-negative phenotype in the Tunisian population

Background: Most studies of the molecular basis of Rhesus D‐negative phenotype have been conducted in Caucasian and African populations. A comprehensive survey of RHD alleles was lacking in people from North Africa (Tunisians, Moroccans and Algerians) which could be very efficient for managing donors and patients carrying an RHD molecular variant. We analyse the molecular background of D‐negative population in Tunisia in the present study. Materials and methods: Blood samples were collected from native Tunisians. A total of 448 D‐negative donors from different regions of Tunisia were analysed by RHD genotyping according to an adopted strategy using real‐time PCR, ASP‐PCR and sequencing. Results: Among the 448 D‐negative samples, 443 were phenotyped unequivocally as true D‐negative including three molecular backgrounds which were RHD gene deletion (n = 437), RHDψ pseudogene (n = 2) and RHD‐CE‐D hybrid gene (n = 4) with the respective frequencies of 0·9900, 0·0023 and 0·0046. The remaining five samples, in discordance with the serological results, were identified as two weak D type 11, one weak D type 29, one weak D type 4·0 and one DBT‐1 partial D. Conclusion: This study showed that the Tunisian population gets closer to Caucasians, given that the RHD gene deletion is the most prevalent cause of D‐negative phenotype, but it is slightly different by the presence of the RHDψ pseudogene which was found with a very low frequency compared with that described in the African population. Nevertheless, the relative occurrence of weak D variants among studied serologically D‐negative samples make necessary the adaptation of RHD genotyping strategy to the spectrum of prevalent alleles.     

Non-invasive foetal RHD genotyping via real-time PCR of foetal DNA from Chinese RhD-negative maternal plasma

Background  A majority of studies predicting the foetal RhD blood group in free foetal DNA from RhD-negative maternal plasma have been conducted in Caucasian populations, whereas limited data have been accumulated for Asian populations. In this study, we assessed the feasibility of prenatal genotyping of RHD in RhD-negative Chinese pregnant women.
Materials and methods  Cell-free plasma DNA was extracted from 78 RhD-negative Chinese women carrying a singleton foetus (gestation between 14 and 40 weeks). Foetal DNA was confirmed by testing SRY or nine different polymorphic STR loci in the maternal plasma and buffy coat. Foetal RHD exons 5, 7 and 10 and intron 4 were successfully amplified with RQ-PCR. The RHD 1227A allele was examined in all RhD-positive individuals. The foetal RHD genotyping results were compared with the infant cord blood serological analysis.
Results  Among the 78 specimens, RHD genotyping results of 70 cases were in complete concordance with serological results from foetal umbilical cord blood. Sixty of these cases were identified as RhD-positive, and 10 cases were typed as RhD-negative. In addition, five cases were 'false-positives', while three cases were considered inconclusive. The detection rate was 89·7% (70/78). In four of the five 'false-positive' cases, the RhDel phenotype was assessed by detecting the RHD 1227A allele. Thus, this method yielded a 94·9% (74/78) accuracy rate.
Conclusions  The correct foetal RhD phenotype may be accurately predicted from RhD-negative maternal plasma in Chinese subjects. The RHD 1227A allele proved to be an important genetic marker in the RhDel Chinese population.     

福建省RhD阴性个体RHD基因多态性

为了探讨RhD阴性福建个体的RHD基因结构，设计14对序列特异性引物，应用PCR—SSP技术检测104名福建省RhD阴性献血者的RH基因型，并对部分样本进行吸收放散试验，同时对2例携带RHD基因RhD阴性样本进行DNA序列测定。结果显示，61．54％阴性福建个体完全缺失RHD基因（RHD^-／RHD^-），25．97％RhD携带RHD1227A等位基因（其中62．96％为RHD^＋／RHD^-杂合子，37．04％为RHD^＋／RHD^＋纯合子），8．65％携带RHD-CE（2—9）-D等位基因，1．92％携带RHD710delC等位基因；多数RHD基因缺失存在于dce单倍体中，发现6例RHD基因缺失存在于dCe单倍体（RH基因型为dce／dCe）中，2例存在于dcE单倍体（RH基因型为dce／dcE）中；同时检测8个RHD基因外显予以及RHD1227A等位基因，以预测RhD表型的准确性明显高于单独检测某个RHD基因外显子的准确性（Χ^2=24．43，P〈0.005）。结论：RhD阴性福建个体RHD基因具有多态性；在中国用RHD基因分型完全替代血清学检测RhD表型的条件尚未成熟。     

Systematic RHD genotyping in Brazilians reveals a high frequency of partial D in transfused patients serologically typed as weak D

BackgroundThe discrimination between weak D types and partial D can be of clinical importance because carriers of partial D antigen may develop anti-D when transfused with D-positive red blood cell units. The aim of this study was to determine by molecular analysis the type of D variants among Brazilian patients requiring transfusions with serologic weak D phenotypes.Material and methodsSamples from 87 patients (53 with sickle cell disease, 10 with thalassemia and 24 with myelodysplastic syndrome), serologic typed as weak D by manual tube indirect antiglobulin test or gel test were first RHD genotyped by using the RHD BeadChip Kit (BioArray, Immucor). Sanger sequencing was performed when necessary.ResultsRHD molecular analysis revealed 32 (36.8 %) variant RHD alleles encoding weak D phenotypes and 55 (63.2 %) alleles encoding partial D antigens. RHD variant alleles were present in the homozygous state or as a single RHD allele, one variant RHD allele associated with the RHDΨ allele, or two different variant RHD alleles in compound heterozygosity with each other in 70 patients, 4 patients and 13 patients, respectively. Alloanti-D was found in 9 (16.4 %) cases with RHD alleles predicting a partial D.DiscussionThe frequency of partial D was higher than weak D types in Brazilian patients serologically typed as weak D, showing the importance to differentiate weak D types and partial D in transfused patients to establish a transfusion policy recommendation.     

山东地区汉族RhD阴性献血者RHD基因的纯合性分析

目的：Rhesus血型（简称Rh血型）因其抗原众多、变种各异和临床疾病相关性而备受重视。该血型系统中与临床疾病关联最密切的抗原即RhD抗原具有较高的免疫原性。编码RhD抗原的RHD基因两侧各有一个序列高度相似的Rh盒子基因,RhD阴性即是由上、下游Rh盒子基因之间的基因重组引起。分析RhD阴性孕妇丈夫RHD基因的纯合性可预测胎儿患新生儿溶血病的几率。本研究的目的是分析山东地区汉族人RhD阴性表型形成的分子机制,并对Rh盒子基因的扩增产物进行分析以确定RHD基因的纯合性。方法：74例RhD阴性献血者的DNA样品首先进行多重聚合酶链反应-序列特异性引物（PCR-SSP）分析。然后对Rh盒子基因进行PCR基因扩增,其扩增产物采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法进行RHD基因的纯合性测定。结果：46例（62%）样品在多重PCR-SSP分析中显示缺失RHD基因,在PCR-RFLP分析中显示为纯合的RHD基因阴性。22例（30%）样品显示存在RHD基因,其中19例显示为杂合的RHD基因,3例显示为纯合的RHD基因。5例（7%）样品缺失RHD基因,但PCR-RFLP分析显示存在一个RHD基因,进一步的分析表明它们至少存在RHD基因第1和10外显子。1例（1%）样品显示存在RHD基因,但缺失第6外显子。结论：HD 基因缺失是引起中国汉族人RhD阴性表型形成的主要分子机制。RhD阴性个体主要表现为纯合的RHD基因阴性,少部分RhD阴性个体存在杂合的RHD基因和纯合的RHD基因。     

Comparison of PCR methods for detecting fetal RhDin maternal plasma

Background: Aim of this study was to establish the method yielding the highest sensitivity routinely used to determine fetal RhD type and gender from maternal cell‐free plasma DNA in different periods of gestation. Methods: Plasma DNA concentrations were measured from 46 pregnant women in different gestational periods and tested for RhD using three different PCR methods on exon 7: Thermal Cycler, Taqman method on LightCycler, and melting curve analysis on LightCycler. In addition, fetal gender was determined by PCR. Cell‐free plasma DNA was measured in 100 healthy volunteers as a reference group. Results: The mean value of cell‐free plasma DNA in the reference group was 10.9 pg/µL mean, (standard deviation (SD): 3.66) in 50 healthy women and 12.7 pg/µL (SD: 8.2) in 50 healthy men. In the firsttrimester of pregnancy cell‐free plasmaDNA was 14.9 pg/µL mean, (SD: 4.2), in the second trimester 15.4 pg/µL mean, (SD: 4.96), and the maximum was achieved in the third trimester of pregnancy 15.6 pg/µl mean, (SD: 6.49). TaqMan probes had the same accuracy, when compared with Thermal Cycler technology (46 samples, 6 failures). Using real‐time PCR with melting curve analysis 12 of 17 samples were correctly tested. Gender determination was correctly in 41 of 46 samples. Conclusion: RhD determinations with TaqMan and Thermal Cycler technology are useful methods for fetal RhD prediction. To increase the accuracy of RhD determination it is necessary to test on other exons in addition. J. Clin. Lab. Anal. 23:24–28, 2009. © 2009 Wiley‐Liss, Inc.     

中国人RHD基因合子型检测分析

目的判定中国人群中RHD基因是否是纯合子。方法从上海地区正常献血者中选取RhD阳性、RhD阴性、Del和其它D变异型样本共50份,采用PCR-RFLP方法扩增Rhesus盒子判断RHD合子型,同时相对实时定量real-time quantitative(RQ)PCR扩增检测RHD基因第5、7外显子的特征区域。结果9份样本在2种方法的检测结果中出现矛盾。进一步对RHD基因的特异性序列进行PCR-SSP及克隆测序分析,证实这9份样本中,4份是DⅥⅢ变异型,2份是Del与RHD711del C等位基因杂合子,另外3份是携带有中国RhD阴性人群中较常见的RHD-CE(2—9)-D RH阴性等位基因。结论中国人Rh血型系统遗传背景较白种人复杂,RHD基因存在较多的变异情况。仅使用单一方法判断RHD合子型及RhD血清学表型容易产生误判,而从Rhesus盒子和RHD基因不同外显子2个角度进行综合判断,可以减少误判。     

Analysis of RHD genes in Taiwanese RhD-negative donors by the multiplex PCR method

The determination of the RhD phenotype is important in transfusion medicine. However, due to the complexity of D antigen expression, the routine serological method cannot differentiate all RhD variants. In addition, the induction of the anti-D antibody is still the major cause of severe hemolytic disease of the newborn (HDN). Therefore, it is important to understand RHD gene profiles. To analyze the RHD gene profiles of Taiwanese RhD-negative donors, the multiplex PCR method was applied to amplify RHD specific exons 3, 4, 5, 7, and 9. Based on the PCR results, the 156 RhD-negative donors were divided into 12 groups according to the different expression patterns of the RHD gene. These 12 groups were further divided into three categories: type I=Rh D(el) (21.8%); type II = partial D, containing some exons (9.0%); and type III = true RhD-negative (69.2%). The results indicated that 21.8% of RhD-negative donors in Taiwan were RhD(el), and 9% carried a part of the RHD gene. Six defined RhD variants were found in this study: four R(O) (Har), one D(Va), and two D(IVb). However, no true RhD-negative or RhD(el) donor with the CcdEe phenotype was found in this analysis.     

RHD基因的克隆及其在K562细胞中的表达

本研究目的在于克隆人类红细胞RHD基因，构建RhD表达载体，观察其在K562细胞中的表达。从脐血网织红细胞中提取总RNA，采用逆转录聚合酶链反应扩增RHD／CE基因，扩增产物通过TA连接克隆到pGEM-T质粒，采用测序方法筛选多个克隆获得RHD基因。将获得的RHD基因进一步亚克隆构建pcDNA3．1(-)表达载体。重组表达载体通过superfect试剂盒转染K562细胞，最后观察K562细胞中RHD基因的转录和表达。结果表明：成功分离克隆到RHD基因，构建的pcDNA3.1(-)重组表达载体经酶切和测序证实RHD cDNA序列及插入方向正确。转染后的K562细胞内有相应的mRNA转录，细胞表面有RhD抗原表达。结论：RHD cDNA转染的K562细胞能表达RhD抗原，该表达体系有助于进一步研究各种RhD变异型的分子基础。     

Patterns of Fetal Growth in a Rural Indian Cohort and Comparison With a Western European Population

Objective. The purpose of this study was to describe fetal size on sonography in a rural Indian population and compare it with those in European and urban Indian populations. Methods . Participants were from the Pune Maternal Nutrition Study of India. Fetal growth curves were constructed from serial ultrasound scans at approximately 18, 30, and 36 weeks' gestation in 653 singleton pregnancies. Measurements included femur length (FL), abdominal circumference (AC), biparietal diameter (BPD), and occipitofrontal diameter, from which head circumference (HC) was estimated. Measurements were compared with data from a large population‐based study in France and a study of urban mothers in Vellore, south India. Results. Fetal AC and BPD were smaller than the French reference at 18 weeks' gestation (?1.38 and ?1.30 SD, respectively), whereas FL and HC were more comparable (?0.77 and ?0.59 SD). The deficit remained similar at 36 weeks for AC (?0.97 SD), FL (?0.43 SD), and HC (?0.52 SD) and increased for BPD (?2.3 SD). Sonography at 18 weeks underestimated gestational age compared with the last menstrual period date by a median of ?1.4 (interquartile range, ?4.6, 1.8) days. The Pune fetuses were smaller, even at the first scan, than the urban Vellore sample. Conclusions. Fetal size was smaller in a rural Indian population than in European and urban Indian populations, even in mid pregnancy. The deficit varied for different fetal measurements; it was greatest for AC and BPD and least for FL and HC.