Genetic variation of FUT3 gene in the Han population from Northern China.

BACKGROUND AND OBJECTIVES: The FUT3 gene encodes α(1,3/1,4)-fucosyltransferase, which is a crucial enzyme in the synthesis of Lewis antigens. FUT3 gene variants show race-specific differences. In this study, we conducted a systematic sequence analysis of the FUT3 coding sequence. The objective was to explore genetic variations of the FUT3 gene within the Han population of Northern China. MATERIALS AND METHODS: A cohort of 313 blood donors was recruited for the study. The coding sequence of the FUT3 gene was amplified using polymerase chain reaction, followed by sequencing and haplotype construction. RESULTS: Twelve single nucleotide variations (SNVs) were identified within the coding sequence of the FUT3 gene. Notably, the c.59 T > G site exhibited the highest mutation frequency of 43.13%, followed by the c.508G > A and c.1067 T > A sites with mutation frequencies of 27.48% and 16.93%, respectively. Le was the most common haplotype, accounting for 67.57% of the cases, and Le/Le was the most common diplotype, accounting for 46.33% of the cases. The study also highlighted a significant difference in mutation frequencies of FUT3 gene between the Han Chinese of Northern China and the Dai of Xishuangbanna, China, but not the Han Chinese in Beijing in the North and the Southern Han Chinese, emphasising that Han Chinese in Northern China are genetically most distant from Europeans and closest to East Asians. CONCLUSIONS: Our study characterises FUT3 gene variations in Han Chinese from Northern China, and provides basic genetic data for genetics, forensic medicine, and genotyping of Lewis blood groups.

Accuracy of RHC/c genotyping in Chinese Han population.

BACKGROUND AND OBJECTIVES: The RHCE gene plays an important role in the complex and polymorphic Rh blood group system. RHCE genotyping holds significant clinical and transfusion-related implications. The objective of this study was to evaluate the accuracy of RHC/c genotyping in the Chinese Han population. MATERIALS AND METHODS: Blood samples were obtained from 653 Chinese Han blood donors. The serological RhD and RhCcEe types were determined using monoclonal antibodies. Subsequently, multiplex real-time polymerase chain reaction (PCR) analysis was performed for RHC and RHc genotyping. Additionally, exon 2 of RHCE and exon 1 of RHD were sequenced. RESULTS: The analysis in this study found 443 RhD-positive donors and 210 RhD-negative donors. Among the 653 total donors, discrepancies between the RHC genotyping results and the serological results were found in 37 individuals. Specifically, 6 false-positive RhC results in RhD-positive donors and 28 false-positive RhC results in RhD-negative donors were identified based on c.48C in RHCE exon 1. Additionally, 3 false-negative RhC results were observed in the RhD-positive donors due to a 109 bp insertion in RHCE intron 2. RHc typing demonstrated complete consistency between the real-time PCR and the serological results. CONCLUSION: In the Chinese Han population, RHC genotyping was reliable when consistent results were achieved by both c.48C-based and 109 bp insertion-based genotyping. Moreover, RHc genotyping based on c.203A and c.307C polymorphic loci demonstrated dependable performance.

RHC genotyping in Chinese Han population.

BACKGROUND: The Rh blood group system is characterized by its complexity and polymorphism, encompassing 56 different antigens. Accurately predicting the presence of the C antigen using genotyping methods has been challenging. The objective of this study was to evaluate the accuracy of various genotyping methods for predicting the Rh C and to identify a suitable method for the Chinese Han population. METHODS: In total, 317 donors, consisting 223 D+ (including 20 with the Del phenotype) and 94 D- were randomly selected. For RHC genotyping, 48C and 109bp insertion were detected on the Real-time PCR platform and -292 substitution was analyzed via restriction fragment length polymorphism (RFLP). Moreover, the promoter region of the RHCE gene was sequenced to search for other nucleotide substitutions between RHC and RHc. Agreement between prediction methods was evaluated using the Kappa statistic, and comparisons between methods were conducted via the χ2 test. RESULTS: The analysis revealed that the 48C allele, 109bp insertion, a specific pattern observed in RFLP results, and wild-type alleles of seven single nucleotide polymorphisms (SNPs) were in strong agreement with the Rh C, with Kappa coefficients exceeding 0.8. However, there were instances of false positives or false negatives (0.6% false negative rate for 109bp insertion and 5.4-8.2% false positive rates for other methods). The 109bp insertion method exhibited the highest accuracy in predicting the Rh C, at 99.4%, compared to other methods (P values≤0.001). Although no statistical differences were found among other methods for predicting Rh C (P values>0.05), the accuracies in descending order were 48C (94.6%) > rs586178 (92.7%) > rs4649082, rs2375313, rs2281179, rs2072933, rs2072932, and RFLP (92.4%) > rs2072931 (91.8%). CONCLUSIONS: None of the methods examined can independently and accurately predict the Rh C. However, the 109bp insertion test demonstrated the highest accuracy for predicting the Rh C in the Chinese Han population. Utilizing the 109bp insertion test in combination with other methods may enhance the accuracy of Rh C prediction.

Novel missense mutation c.797T>C (p.Met266Thr) gives rise to the rare B(A) phenotype in a Chinese family.

BACKGROUND AND OBJECTIVES: B(A) phenotype is usually formed by nucleotide mutations in the ABO*B.01 allele, with their products exhibiting glycosyltransferases (GTs) A and B overlapping functionality. We herein report a B(A) allele found in a Chinese family. MATERIALS AND METHODS: The entire ABO genes of the probands, including flanking regulatory regions, were sequenced through PacBio third-generation long-read single-molecule real-time sequencing. 3D molecular models of the wild-type and mutant GTB were generated using the DynaMut web server. The effect of the mutation on the enzyme function was predicted by PROVEAN and PolyPhen2. The predictions of stability changes were performed using DynaMut and SNPeffect. RESULTS: Based on serological and sequencing features, we concluded the two probands as possible cases of the B(A) phenotype. Crystallization analysis showed that Thr266 substitution does not disrupt the hydrogen bonds. However, some changes in interatomic contacts, such as loss of ionic interactions and hydrophobic contacts, and addition of weak hydrogen bonds, may have affected protein stability to some extent. This mutation was predicted to have a benign effect on enzyme function and slightly reduce protein stability. CONCLUSION: The probands had the same novel B(A) allele with a c.797T>C (p.Met266Thr) mutation on the ABO*B.01 backbone.

A novel allele of FUT2 gene containing a deletion of nine bases (c.461_469delGGACCTTCT) in a Chinese Han blood donor.

BACKGROUND AND OBJECTIVES: The FUT2 gene is responsible for the synthesis of the H antigen in body secretions. It is highly polymorphic and population specific. We investigated the FUT2 gene polymorphism in Chinese blood donors and found a novel deletion mutation in one non-secretor individual. This study aimed to identify mutation(s) responsible for a non-secretor phenotype. MATERIALS AND METHODS: The Lewis blood group of a Chinese Han blood donor was typed using the standard serological technique and the FUT2 gene of the sample was analysed by Sanger sequencing. Clone sequencing was performed for determining the haplotype of the FUT2 gene. Bioinformatics tools were used for predicting the effect of the deletion on the FUT2 gene. RESULTS: A novel nine-base deletion (c.461_469delGGACCTTCT) in the FUT2 gene was identified in a Chinese Han blood donor. Two haplotypes Se390,418 and se204,249,461_469del,772,993 were determined by clone sequencing. According to the prediction of bioinformatics tools, the mutation at c.461_469delGGACCTTCT might not influence the activity of the Se enzyme. CONCLUSION: We identified a new FUT2 mutation, the deletion of nine bases (c.461_469delGGACCTTCT), in a Chinese Han blood donor. This deletion was reported for the first time.

Polymorphisms in the promoter regions of RHD and RHCE genes in the Chinese Han population.

BACKGROUND AND OBJECTIVES: The Rh blood group system is the most polymorphic human blood group system. Previous studies have investigated variants in the RHD and RHCE promoter. The relevance of these variants to the Chinese Han population is further clarified in this study. MATERIALS AND METHODS: In total, 317 donors (223 Rh D-positive [D+], including 20 Del and 94 Rh D-negative [D-]) were randomly selected. The promoter regions and exon 1 of RHD and RHCE were amplified through polymerase chain reaction (PCR) whose products were directly sequenced using forward and reverse primers. RESULTS: Expected PCR products of the RHD promoter and exon 1 were amplified in 223 D+ individuals, including 20 Del individuals, and were absent in 81 of 94 D- individuals. Expected PCR products of RHCE were observed in all donors. Two single nucleotide variants (SNVs) were observed in the RHD promoter region. Moreover, 11 SNVs were observed in the promoter and exon 1 of RHCE. rs4649082, rs2375313, rs2281179, rs2072933, rs2072932, rs2072931 and rs586178 with strong linkage disequilibria were significantly different between the D+ and D- groups. [A;C] was the most common haplotype in the RHD promoter (NC_000001.11:g.[-1033A>G;-831C>T]). [G;T;T;A;T;A;C;G;A;C;G] was the most predominant haplotype in both total and D- groups. In D+ individuals, [A;C;T;G;C;G;C;G;C;C;C] was the most frequent haplotype in the RHCE promoter (NC_000001.11:g.[-1080A>G;-958C>T;-390T>C;-378G>A;-369C>T;-296G>A;-144C>G;-132G>A;-122C>A;28C>T;48C>G]). CONCLUSION: We speculate that the SNVs/haplotypes found in this article cannot significantly affect gene expression. The present study findings should help elucidate the molecular basis of the polymorphic expression of RHD and RHCE promoter regions.

Genetic analysis of 42 Y-STR loci in Han and Manchu populations from the three northeastern provinces in China.

BACKGROUND: Y-STR polymorphisms are useful in tracing genealogy and understanding human origins and migration history. This study aimed to fill a knowledge gap in the genetic diversity, structure, and haplogroup distribution of the Han and Manchu populations from the three northeastern provinces in China (Liaoning, Jilin, and Heilongjiang). METHODS: A total of 1,048 blood samples were collected from unrelated males residing in Dalian. Genotyping was performed using the AGCU Y37 + 5 Amplification Kit, and the genotype data were analyzed to determine allele and haplotype frequencies, genetic and haplotype diversity, discrimination capacity, and haplotype match probability. Population pairwise genetic distances (Fst) were calculated to compare the genetic relationships among Han and Manchu populations from Northeast China and other 23 populations using 27 Yfiler Plus loci set. Multi-dimensional scaling and phylogenetic analysis were employed to visualize the genetic relationships among the 27 populations. Moreover, haplogroups were predicted based on 27 Yfiler Plus loci set. RESULTS: The Han populations from Northeast China exhibited genetic affinities with both Han populations from the Central Plain and the Sichuan Qiang population, despite considerable geographical distances. Conversely, the Manchu population displayed a relatively large genetic distance from other populations. The haplogroup analysis revealed the prevalence of haplogroups E1b1b, O1b, O2, and Q in the studied populations, with variations observed among different ethnic groups. CONCLUSION: The study contributes to our understanding of genetic diversity and history of the Han and Manchu populations in Northeast China, the genetic relationships between populations, and the intricate processes of migration, intermarriage, and cultural integration that have shaped the region's genetic landscape.

The differential panorama of clinical features of lupus erythematosus patients with different onset ages: a cross-sectional multicenter study from China.

OBJECTIVES: This study aims to compare the differences among patients of different onset ages in various subtypes of lupus erythematosus (LE) and to draw a panorama of the clinical characteristics of patients with different onset ages. METHOD: Subjects were recruited from the Lupus Erythematosus Multicenter Case-control Study in Chinese populations (LEMCSC), grouped by the age of onset (childhood-onset: onset < 18 years, adult-onset: onset 18-50 years, late-onset: onset > 50 years). The data collected included demographic characteristics, LE-related systemic involvement, LE-related mucocutaneous manifestations, and laboratory results. All included patients were assigned into three groups: systemic LE (SLE) group (with systemic involvement, with or without mucocutaneous lesions), cutaneous LE (CLE) group (patients who were accompanied by any type of LE-specific cutaneous manifestations), and isolated cutaneous LE (iCLE) group (patients who were in CLE group without systemic involvements). Data were analyzed using R version 4.0.3. RESULTS: A total of 2097 patients were involved, including 1865 with SLE and 232 with iCLE. We also identified 1648 patients with CLE among them, as there was some overlap between the SLE population and CLE population (patients with SLE and LE-specific cutaneous manifestations). Later-onset lupus patients seemed to be less female predominance (p < 0.001) and have less systemic involvement (except arthritis), lower positive rates of autoimmune antibodies, less ACLE, and more DLE. Moreover, childhood-onset SLE patients presented a higher risk of LE family history (p = 0.002, vs adult-onset SLE). In contrast to other LE-nonspecific manifestations, the self-reported photosensitivity history decreased with the age of onset in SLE patients (51.8%, 43.4%, and 39.1%, respectively) but increased in iCLE patients (42.4%, 64.9%, and 89.2%, respectively). There was also a gradual increase in self-reported photosensitivity from SLE, CLE, to iCLE in both adult-onset and late-onset lupus patients. CONCLUSIONS: A negative correlation was suggested between the age of onset and the likelihood of systemic involvement, except for arthritis. As the age of onset increases, patients have a greater propensity to exhibit DLE compared to ACLE. Moreover, the presence of rapid response photodermatitis (i.e., self-reported photosensitivity) was associated with a lower rate of systemic involvement. TRIAL REGISTRATION: This study was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR2100048939) on July 19, 2021, retrospectively registered. Key Points • We confirmed some phenomena that have been found in patients with SLE, such as the highest proportion of females of reproductive age, the higher risk of LE family history in childhood-onset SLE patients, and the less self-reported photosensitivity in the late-onset SLE group. We also compared the similarities and differences of these phenomena in patients with CLE or iCLE for the first time. • In patients with SLE, the proportion of females peaked in adult-onset patients, but this phenomenon disappeared in iCLE patients: the female-male ratio tends to decrease from childhood-onset iCLE, adult-onset iCLE, to late-onset iCLE. • Patients with early-onset lupus are more likely to have acute cutaneous lupus erythematosus (ACLE), and patients with late-onset lupus are more likely to have discoid lupus erythematosus (DLE). • In contrast to other LE-nonspecific manifestations, the incidence of rapid response photodermatitis (i.e., self-reported photosensitivity) decreased with the age of onset in SLE patients but increased with the age of onset in iCLE patients.

Panoramic view of clinical features of lupus erythematosus: a cross-sectional multicentre study from China.

OBJECTIVE: Lupus erythematosus (LE) is a complicated disease with highly heterogeneous clinical manifestations. Previous studies have rarely included all subgroups of patients with lupus and have overlooked the importance of the cutaneous manifestations thereof. We aimed to compare the demographic and clinical differences among patients with different subtypes of lupus. METHODS: This is the first real-world study with a relatively large sample size that simultaneously includes patients with isolated cutaneous lupus erythematosus (iCLE) and SLE. All samples were obtained from the Lupus Erythematosus Multicenter Case-control Study in Chinese populations (LEMCSC) (registration number: ChiCTR2100048939). Comparative analyses between different LE subgroups were performed. RESULTS: A total of 2097 patients with lupus were included, with 1865 patients with SLE, 1648 with cutaneous lupus erythematosus (CLE), and 232 with iCLE. Among the patients with CLE, 1330 had acute cutaneous lupus erythematosus (ACLE); 160 had subacute cutaneous lupus erythematosus (SCLE); and 546 had chronic cutaneous lupus erythematosus (CCLE). The study included a relatively large number of patients with CCLE subtypes, including 311 with discoid lupus erythematosus (DLE), 262 with chilblain lupus erythematosus (CHLE) and 45 with lupus erythematosus profundus (LEP). Demographic characteristics, systemic involvement, mucocutaneous manifestations and autoantibodies were significantly different among the groups. CONCLUSIONS: CLE and iCLE are two distinct disease states, and the selection of broad or narrow CLE definitions should be emphasised in scientific reports. LE-non-specific cutaneous lesions imply more severity, while self-reported photosensitivity and LE-specific cutaneous manifestations imply milder severity. Generalised ACLE appears to be a more severe state than localised ACLE, and CHLE appears to be more severe than DLE. Anti-Sjögren's syndrome-related antigen B (SSB) antibodies have higher specific directivity than anti-Sjögren's syndrome-related antigen A (SSA) antibodies for SCLE lesions. Anti-double-stranded DNA antibodies have a higher co-occurrence with ACLE and a lower co-occurrence with SCLE and CCLE. Compared with DLE, CHLE has significantly higher positive rates of anti-SSA/Ro60 (71%) and anti-SSA/Ro52 (42.4%) antibodies, whereas LEP is associated with a higher positive rate of antinucleosome antibodies (31.1%).

Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing.

OBJECTIVES: To assess the efficacy of Y-chromosome mini-STR-based next-generation sequencing (NGS) for non-invasive prenatal paternity testing (NIPPT). METHODS: DNA was extracted from the plasma of 24 pregnant women, and cell-free fetal DNA (cffDNA) haplotyping was performed at 12 Y-chromosome mini-STR loci using the Illumina NextSeq 500 system. The cffDNA haplotype was validated by the paternal haplotype. Subsequentlly, the paternity testing parameters were attributed to each case quantitatively. RESULTS: The biological relationship between the alleged fathers and infants in all 24 family cases were confirmed by capillary electrophoresis (CE). The Y-chromosome mini-STR haplotypes of all 14 male cffDNA were obtained by NGS without any missing loci. The alleles of cffDNA and paternal genomic DNA were matched in 13 cases, and a mismatched allele was detected at the DYS393 locus in one case and considered as mutation. No allele was detected in the 10 female cffDNA. The combined paternity index (CPI) and probability of paternity calculation was based on 6 loci Y-haplotype distributions of a local population. The probability of paternity was 98.2699-99.8828% for the cases without mutation, and 14.8719% for the case harboring mutation. CONCLUSIONS: Our proof-of-concept study demonstrated that Y-chromosome mini-STR can be used for NGS-based NIPPT with high accuracy in real cases, and is a promising tool for familial searching, paternity exclusion and sex selection in forensic and medical applications.

Association between the Genetic Polymorphisms of CCL2, CCL5, CCL8, CCR2, and CCR5 with Chronic Hepatitis C Virus Infection in the Chinese Han Population.

Hepatitis C virus (HCV) infection is a global public health burden. Chronic HCV infection leads to the development of fibrosis, cirrhosis, liver cancer, and liver failure over time. A total of 250 patients with chronic HCV infection and 299 healthy blood donors were recruited. Sixteen candidate single nucleotide polymorphisms (SNPs) in chemokine (C-C motif) ligand 2 (CCL2), CCL5, CCL8, C-C chemokine receptor 2 (CCR2), and CCR5 were genotyped in all participants. The rs1024610 AA genotype was significantly associated with decreased susceptibility to chronic HCV infection. Aspartate aminotransferase (AST) levels, AST/platelet ratio index, and the fibrosis 4 score were significantly lower in the CCL2 rs1024610 T allele and haplotype ATGC carriers. Moreover, expression levels of collagen IV (C-IV) and laminin (LN) were significantly higher in patients with the CCL5 rs2280788 C allele compared to the non-carriers. Similarly, the expression levels of C-IV, LN, and hyaluronic acid were significantly higher in patients with the CCL5 haplotype, TGCT. No significant differences were identified between the SNPs/haplotypes and plasma levels of CCL2, CCL5, CCL8, CCR2, and CCR5 in the healthy controls, and the rs1024610 allele alteration had no effect on CCL2 promoter activity. This is the first study to report an association between CCL2 rs1024610 and the risk of chronic HCV infection in the Chinese Han population. rs1024610 and ATGC haplotype in CCL2 were reasonable candidate markers of liver abnormalities. rs2280788 and TGCT haplotype in CCL5 are likely to play a significant role in liver fibrosis during chronic HCV infection.

Association of the 3' untranslated region polymorphisms of HLA-G with susceptibility to chronic hepatitis C virus infection in the Chinese population.

Hepatitis C virus (HCV) infection is a global health problem. Several previous studies have addressed the role of host single-nucleotide polymorphisms (SNPs) in HCV infection. SNPs in the regulatory region of the human leukocyte antigen G (HLA-G) gene play an important role in several diseases. The objective of this study is to determine the association of HLA-G 3'untranslated region (UTR) polymorphisms with the susceptibility to chronic hepatitis C infection in the Chinese population. HLA-G 3' UTR polymorphisms, which include 14-bp Ins/Del (rs371194629), +3003T/C (rs1707), +3010C/G (rs1710), +3027 A/C (rs17179101), +3035C/T (rs17179108), +3142 G/C (rs1063320), +3187 A/G (rs9380142) and + 3196C/G (rs1610696), were analyzed in 246 patients with chronic hepatitis C infection and 294 healthy individuals. The alleles, genotypes, and haplotypes were compared between chronic hepatitis C-infected subjects and controls using chi-square tests and logistic regression models. After a correction of multiple comparisons by the false discovery rate (FDR), the allele frequency of + 3196C, genotype frequencies of + 3187 AA and + 3196CC and frequency of the UTR-3 haplotype were significantly higher in the patients than in the control group (P < 0.05), whereas the frequencies of UTR-1 and UTR-2 haplotypes were significantly lower in the patients than in the control group (P < 0.05). After a correction of multiple comparisons by FDR, UTR-2 and UTR-3 maintained significant associations with chronic hepatitis C. This study indicates that HLA-G 3'UTR polymorphisms are associated with the susceptibility to chronic hepatitis C infection in the Chinese population. HLA-G 3'UTR may play an important role in risk modulation toward HCV infection.

Comprehensive analysis of epigenetic modifications and immune-cell infiltration in tissues from patients with systemic lupus erythematosus.

Aim: To explore potential abnormal epigenetic modifications and immune-cell infiltration in tissues from systemic lupus erythematosus (SLE) patients. Materials & methods: To utilize bioinformatics analysis and 'wet lab' methods to identify and verify differentially expressed genes in multiple targeted organs in SLE. Results: Seven key genes, IFI44, IFI44L, IFIT1, IFIT3, PLSCR1, RSAD2 and OAS2, which are regulated by epigenetics and may be involved in the pathogenesis of SLE, are identified by combined long noncoding RNA-miRNA-mRNA network analysis and DNA methylation analysis. The results of quantitative reverse transcription PCR, immunohistochemistry and DNA methylation analysis confirmed the potential of these genes as biomarkers. Conclusion: This study reveals the potential mechanisms in SLE from epigenetic modifications and immune-cell infiltration, providing diagnostic biomarkers and therapeutic targets for SLE.

Epigenetic regulation of melanogenesis.

Melanogenesis is a complex process in which melanin is synthesized in melanocytes and transported to keratinocytes, which involves multiple genes and signaling pathways. Epigenetics refers to the potential genetic changes that affect gene expression without involving changes in the original sequence of DNA nucleotides. DNA methylation regulates the expression of key genes such as tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome tautomerase (DCT) and microphthalmia-associated transcription factor (MITF), as well as paracrine factors such as stem cell factor (SCF) and endothelin-1 (ET-1) in melanogenesis. Potential DNA methylation sites are present in the genes of melanogenesis-related signaling pathways such as "Wnt", "PI3K/Akt/CREB" and "MAPK". H3K27 acetylation is abundant in melanogenesis-related genes. Both the upstream activation and downstream regulation of MITF depend on histone acetyltransferase CBP/p300, and pH-induced H3K27 acetylation may be the amplifying mechanism of MITF's effect. HDAC1 and HDAC10 catalyze histone deacetylation of melanogenesis-related gene promoters. Chromatin remodelers SWI/SNF complex and ISWI complex use the energy of ATP hydrolysis to rearrange nucleosomes, while their active subunits BRG1, BRM and BPTF, act as activators and cofactors of MITF. MicroRNAs (miRNAs) can directly target a large number of melanogenesis-related genes, while long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) regulate melanogenesis in a variety of ways. Interactions exist among the epigenetic mechanisms of melanogenesis. For example, the methyl CpG binding domain protein 2 (MeCP2) links DNA methylation, histone deacetylation, and histone methylation. Epigenetic-based therapy provides novel opportunities for treating dermatoses that are caused by pigmentation disturbances. This review summarizes the epigenetic regulation mechanisms of melanogenesis, and examines the pathogenesis and treatment of epigenetics in pigmentation disorders.

Genotyping of RHD c.1227G>A allele by melting curve analysis.

BACKGROUND: DEL is the weakest known D-positive phenotype and is detectable only by adsorption and elution tests. RHD c.1227G>A is an important marker for DEL phenotype in East Asians. The aim of this study was to develop a method for RHD c.1227G>A genotyping by single-tube PCR with melting curve analysis. METHODS: Two GC-rich tails of different lengths were attached to the 5'-end of allele-specific primers for RHD 1227G and 1227A alleles, such that RHD c.1227G>A could be distinguished by the melting temperature. A total of 145 D-negative Chinese Han blood donors were genotyped for RHD c.1227G>A by melting curve analysis, conventional polymerase chain reaction with sequence-specific primers (PCR-SSP), and sequencing. RESULTS: In 143 subjects (143/145, 98.6%), PCR-SSP and melting curve analysis produced consistent results with RHD exon 9 sequencings. Two samples were genotyped as RHD 1227G/A by PCR-SSP, but as RHD 1227A/A or A/- by melting curve analysis. These two samples were confirmed to be RHD 1227A/A or A/-. Based on RHD exon 9 sequencing, the accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the melting curve analysis for detecting both RHD 1227A and 1227G were all 100%. In contrast, the accuracy, specificity and positive predictive value of PCR-SSP for RHD 1227G detection were 98.62%, 98.21% and 94.29%, respectively, which were lower than those observed with the melting curve analysis. CONCLUSION: Melting curve analysis for RHD c.1227G>A genotyping is a simple, rapid, and reliable method, superior to conventional PCR-SSP.