Human Leukocyte Antigen Alleles (HLA-A, HLA-B, and HLA-DRB1) are associated with Acute Lymphoblastic Leukemia (ALL) : A Case-Control Study in a Sample of Iranian Population.

OBJECTIVE: This study seeks to elucidate the association between HLA-A, HLA-B, and HLA-DRB1 alleles and their relative risk contributions to ALL within an Iranian cohort. METHODS: Utilizing a robust case-control design, this research involved 71 ALL patients and 71 age and sex-matched healthy individuals. Genotyping of specified HLA alleles was performed using the advanced PCR-SSP technique. RESULTS: Our findings reveal a marked increase in the prevalence of the HLA-DRB1*04 allele among patients diagnosed with ALL compared to the control group (P<0.027). Conversely, the alleles HLA-A*26 (P=0.025), HLA-A*33 (P=0.020), and HLA-DRB1*03 (P=0.035) were observed at significantly reduced frequencies within the patient population. CONCLUSION: Our findings highlight HLA-DRB1*04 as a potential genetic marker for increased susceptibility to ALL, while HLA-A*26, HLA-A*33, and HLA-DRB1*03 emerge as protective factors.

HLA class II polymorphisms as prognostic biomarkers for right and left-sided colon cancer.

BACKGROUND: Colon cancer (CC) is one of the most common malignancies worldwide. Characterization of new prognostic biomarkers for right-sided CC (RCC) and left-sided CC (LCC) may contribute to improving early detection. An association of human leukocyte antigens class II (HLA-II) with the predisposition to CC was suggested. AIM OF THE STUDY: We evaluated the association of DRB1 and DQB1 with the risk of LCC and RCC. PATIENTS AND METHODS: Our study comprised 93 CC patients and 100 healthy controls. Genotyping of HLA class II alleles were performed by the Polymerase Chain Reaction Sequence-Specific Primers (PCR-SSP). RESULTS: DRB1*03 was positively associated with CC. In contrast, DRB1*11, DRB1*13, DQB1*03, and DQB1*05 were negatively linked to CC. Haplotype analysis revealed that DRB1*04-DQB1*04 and DRB1*09-DQB1*02 were positive, while DRB1*01-DQB1*05, DRB1*04-DQB1*03, DRB1*07-DQB1*02, DRB1*11-DQB1*03, DRB1*11-DQB1*05, and DRB1*13-DQB1*06 were negatively associated with CC. For sigmoid CC, DRB1*13, DRB1*11, and DQB1*05 were negative, while DRB1*04-DQB1*02, and DRB1*07-DQB1*03 were positively associated. DRB1*03 and DRB1*04-DQB1*04 were positive, while DRB1*11 and DQB1*03 were negatively linked to RCC. According to the LCC, DRB1*07, DRB1*11, DQB1*03, DQB1*05, and DRB1*07-DQB1*02 were negative. In contrast, DRB1*09-DQB1*02 was positively associated with LCC. Stratified analysis revealed that DRB1*11 is associated with higher risk of metastasis in CC and sigmoid CC, and tolerance to treatment in RCC. DQB1*03 was associated with lymph-node invasion in CC. CONCLUSION: DRB1 and DQB1 polymorphisms could be used as future biomarkers for the early detection of subjects at a higher risk of developing RCC and LCC, metastasis in sigmoid CC, and tolerance to treatment in RCC.

Fully genotyping and screening of clinically important blood-group antigens by MALDI TOF mass spectrometry.

Several molecular biology methods are available for high-throughput blood typing. In this study, we aimed to build a high-throughput blood-group genetic screening system for high-frequency blood-group antigen-negative rare-blood groups in donors and patients. The amplification primers for all blood-type gene fragments involving the selected alleles were designed for detection. Single-base extend primers were also designed based on specific loci. DNA fragments were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) for the last nucleotide identification of amplification products in the extend step. The accuracy was verified by known samples. Thirty-six random samples were detected by serological tests and sequencing to verify the system stability. After verification, according to the collected known rare-blood-type samples, all the alleles designed to be detected matched with the validated single-nucleotide polymorphisms. The verification tests showed that all genotyping results of the random samples were in accordance with the findings of serotyping and sequencing. Then, 1258 random donor samples were screened by the built typing system after the verification. Three Fy(a-) and four s- were screened out in 1258 random blood samples. The multiple polymerase chain reaction-based MS detection system can be used in rare-blood-type screening with good accuracy and stability.

HLA-B*58 and HLA-B*27 Play a Role in the Development of Acute Leukemia: A Case Control Study.

BACKGROUND: Acute leukemia (AL) constitutes a group of malignant hematological diseases with multifactor origins. Some human leukocyte alleles (HLA) may be important genetic risk factors for development of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). It is still unknown whether there is a relationship between ALL and AML with some alleles of the major histocompatibility complex. Our study looks specifically at western and southwest Algerian populations. METHOD: Using the polymerase chain reaction with the sequence specific probe (PCR- SSP) method, we investigated the relationship of HLA-B alleles in 163 Algerian AL patients and 293 controls from the same ethnic origin. The study ran from 2013 - 2020. RESULTS: Allele frequencies of HLA-B*27 and HLA-B*58 was higher in AL patients compared with control individuals; p=0.05 and p=0.03 respectively. Interestingly, all patients carrying HLA-B*27 allele and 88% of patients carrying HLA-B*58 allele had AML. However, there were no significant differences when we compared these results with the rest of AL group (HLA-B*X allele) (p=0.387). Response to induction chemotherapy treatment were comparable between the two patient groups 67% and 65% (p=0.978) respectively. CONCLUSION: These results suggest that the HLA-B*27 and HLA-B*58, may be factors predisposing individuals to acute leukemia, in west and southwest Algerian patients. A large-scale study is still needed to confirm these findings.

Development of cost-effective and fast KIR genotyping by multiplex PCR-SSP.

Killer-cell immunoglobulin-like receptors (KIR) control natural killer (NK) cell functions by recognizing HLA molecules and modulating the activity of NK cells. The KIR gene cluster contains polymorphic and highly homologous genes. Diversity of the KIR region is achieved through differences in gene content, allelic polymorphism, and gene copy number, which result in unrelated individuals having different KIR genotypes and individualized immune responses that are relevant to multiple aspects of human health and disease. Therefore, KIR genotyping is increasingly used in epidemiological studies. Here, we developed multiplex polymerase chain reaction with sequence-specific primers (PCR-SSP) to compensate for the shortcomings of the conventional PCR-SSP method, which is most commonly used for KIR analysis. Multiplex PCR-SSP method involves six multiplex reactions that detect 16 KIR genes and distinguish variant types of some KIR genes by adding two reactions. The assay was evaluated in a blind survey using a panel of 40 reference DNA standards from the UCLA KIR Exchange Program. The results are 100% concordant with the genotype determined using Luminex-based reverse sequence-specific oligonucleotide typing systems. Additionally, we investigated the currently known 16 KIR genes and their common variants in 120 unrelated Korean individuals. The results were consistent with the KIR genotype previously reported by Hwang et al. This multiplex PCR-SSP is an efficient method for analyzing KIR genotypes in both small- and large-scale studies with minimal labor, reagents, and DNA. Furthermore, by providing a better definition of KIR polymorphisms it can contribute to developments in immunogenetics.

Allelic frequency variation of ACKR1 in three Algerian populations: Zenata, Reguibat, and Oran.

INTRODUCTION: The discovery of the Duffy antigen is of great significance, given its essential role in immune response and various physiological processes. Genetic mutations in the Duffy gene not only affect antigen expression but also result in different antigen types. This underscores the importance of genetic characterization for clinical studies and exploring genetic diversity within the population. This study primarily aims to genetically characterize the Duffy blood group within three Algerian populations: the Zenata, Reguibat, and Oran populations. METHODS: The genetic polymorphism of the Duffy erythrocyte group was examined, focusing on five allelic versions of the ACKR1 locus: FY*01, FY*02, FY*X, and silent alleles FY*01 N.01 and FY*02 N.01. A total of 223 Algerian individuals, including 90 from the Oran population, 66 from the Zenata population, and 67 from the Reguibat population, were analyzed using the polymerase chain reaction with sequence-specific primer (PCR-SSP) method. The results revealed the presence of the silent alleles (FY*01 N.01 and FY*02 N.01) in all three populations, with a total frequency of 78.03% in the Zenata population. Additionally, the FY*X allele was exclusively detected in the Reguibat population, with a frequency of 0.75% CONCLUSION: This study provides valuable insights into the allele and genotypic frequencies of the Duffy system in the Zenata, Reguibat and Oranpopulations, contributing to our understanding of the genetic history and origins of the Algerian population. Further research incorporating additional genetic markers and establishing a comprehensive database would enhance our knowledge in this area.

A novel pyrosequencing strategy for RHD zygosity for predicting risk of hemolytic disease of the fetus and newborn.

OBJECTIVE: The aim of this study was the development of an accurate and quantitative pyrosequence (PSQ) method for paternal RHD zygosity detection to help risk management of hemolytic disease of the fetus and newborn (HDFN). METHODS: Blood samples from 96 individuals were genotyped for RHD zygosity using pyrosequencing assay. To validate the accuracy of pyrosequencing results, all the samples were then detected by the mismatch polymerase chain reaction with sequence-specific primers (PCR-SSP) method and Sanger DNA sequencing. Serological tests were performed to assess RhD phenotypes. RESULTS: Serological results revealed that 36 cases were RhD-positive and 60 cases were RhD-negative. The concordance rate between pyrosequencing assay and mismatch PCR-SSP assay was 94.8% (91/96). There were 5 discordant results between pyrosequencing and the mismatch PCR-SSP assay. Sanger sequencing confirmed that the pyrosequencing assay correctly assigned zygosity for the 5 samples. CONCLUSION: This DNA pyrosequencing method accurately detect RHD zygosity and will help risk management of pregnancies that are at risk of HDFN.

Establishment of Q-PCR method for qualitative testing of the presence or absence of KIR genes / 中国输血杂志

【Objective】 To establish a method for qualitative detection of the presence or absence of all KIR genes by quantitative polymerase chain reaction(Q-PCR). 【Methods】 Based on the polymorphism of high-resolution level KIR alleles in Chinese population and the IPD-KIR database, KIR gene-specific primers were designed to amplify all the 16 KIR genes and 2DS4-Normal and 2DS4-Deleted subtypes by Q-PCR. Meanwhile, one negative control and one positive control specific amplifying human growth hormone (HGH) gene fragment were set to monitor the false positive and false negative results in PCR amplification, respectively. A total of 302 samples with known KIR genotype previously identified by KIR PCR-SSP commercial kit were randomly selected for blind inspection to verify the reliability of KIR Q-PCR method established by authors. 【Results】 The results of 300 samples detected by our KIR Q-PCR method were consistent with the known results, but two samples showed inconsistent results. One sample was negative for 2DS5 by Q-PCR but positive by PCR-SSP, another sample was positive for 2DS1 by Q-PCR but negative by PCR-SSP. The two doubtful samples were genotyped by sequencing-based typing (PCR-SBT) for 2DS5 and 2DS1, respectively. PCR-SBT results confirmed that the results of Q-PCR test was correct. 【Conclusion】 The KIR Q-PCR method established in this paper can provide accurate and reliable results for testing the presence or absence of KIR genes.

Studies on the effect of the p.Arg352Gln mutation on Bw07 transferase / 中华检验医学杂志

Objective:To investigate the molecular biological mechanism of Bw07 allele and its transferase alteration carried by a proband of ABw07 subtype.Methods:A 2-year-old male child was selected as the research object. The peripheral blood of the proband and his parents was identified for ABO blood type by the test tube method, and the ABO subgroup PCR-SSP detection and ABO gene sequencing were performed on the three individuals to determine their blood type genotypes. Finally, the effect of the p.Arg352Gln mutation on Bw07 transferase was verified by virtual mutation, DUET structure prediction, molecular dynamics analysis, and in vitro cellular experiments.Results:The serological phenotypes of the proband and his mother were ABw and Bw, respectively, while his father was normal A. The ABO subgroup PCR-SSP assay identified the three genotypes as Bw07/A, Bw07/O, and A/A, respectively.Sanger sequencing further verified that the proband and his mother carried the Bw07 gene, and virtual mutation showed that the intermolecular forces were weakened by the R352Q mutation. DUET predicted that this p.Arg352Gln mutation could affect the thermodynamic stability of Bw07 transferase. Molecular dynamics analysis confirmed that the alteration of thermodynamic stability was mainly related to the appearance of large fluctuations in the amino acid backbone atoms in the 125-133, 193-198 and 336-354 regions, and in vitro cellular experiments further verified the weakened antigen synthesis of Bw07 transferase.Conclusion:The formation of the ABw07 phenotype is associated with the mutation of the highly conserved Arg352 to Gln in Bw07 transferase.

Application of a simplified PCR-SSP method to detect A4GALT*01 and A4GALT*02 typing among Thai blood donors.

OBJECTIVES: An intronic A4GALT single nucleotide variant, rs5751348:G>T, P2 or A4GALT*02 allele has a lower level of the enzyme-encoding A4GALT transcripts than the P1 individuals. Here, we first develop and validate a simple inhouse PCR-SSP method to detect A4GALT*01 and A4GALT*02 alleles, and second, apply this method to compare the allele frequencies between Thai and other populations. MATERIAL AND METHODS: The conventional test tube technique was used to detect the P1 antigen in 222 blood samples from Thai blood donors at Thammasat University Hospital. A PCR-SSP method was optimized and validated for reproducibility and specificity to identify these alleles and was subsequently tested on 1,840 DNA samples of unknown phenotypes obtained from central, northern and southern Thais. In addition, allele frequencies of central Thais were compared with those of other populations. RESULTS: In the tested cohort (n = 222), P1 and P2 phenotypes were typed in 26.13 and 73.87% of donors, respectively. The developed PCR-SSP was successfully optimized, and the outcomes were consistent with those of serological phenotyping and DNA sequencing results, demonstrating its validity for predicting P1/P2 phenotype. For central, northern and southern Thais, the A4GALT*01 frequency was 0.1579 (430/2,724), 0.1183 (71/600), and 0.2575 (206/800), whereas the A4GALT*02 frequency was 0.8421 (2,294/2,724), 0.8817 (529/600), and 0.7425 (594/800), respectively. Their observed frequencies among central Thais significantly differed from those in other populations (p < 0.05). CONCLUSION: Our study has successfully developed a simple, precise, and reliable method to genotype A4GALT*01 and A4GALT*02 using inhouse developed PCR-SSP for predicting P1/P2 status.

Tumor Necrosis Factor-α (-308G>A) Gene Polymorphism and Its Association with Asthma and Atopy Status.

Asthma is one of the most prevalent chronic lung diseases that afflict genetically predisposed individuals. Certain cytokine gene polymorphisms have been associated with asthma. Tumor necrosis factor-alpha (TNF-α) is a potent inflammatory cytokine that can modulate nonspecific inflammation to influence asthma. This study aimed to define the relationship between the TNF gene polymorphism at position -308 and asthma susceptibility, as well as atopic and non-atopic asthma. Using polymerase chain reaction with sequence-specific primers, we investigated genotype frequencies and alleles of a polymorphic gene coding for TNF-α in 86 pediatric patients with asthma and 470 healthy controls of the same race. Seventy-four patients underwent a skin prick test. The homozygous AA variant (-308, rs1800629) was the most common genotype among patients, accounting for 63.3% of all cases. In contrast, homozygous GG (-308) was significantly less prevalent in the patient group compared to the control group. TNF A (-308) allele frequency was 85.5% among asthma patients and 16.6% among healthy controls. The genotype and allele frequencies of TNF (-308 A>G, rs1800629) did not differ between atopic and non-atopic asthma. In conclusion, TNF (-308) AA and AG genotypes are associated with asthma susceptibility in Iranian children, although there was no significant difference in polymorphism between atopic and non-atopic asthma and no difference in asthma severity groups.

Genotyping Approach to Predict Coa and Cob Antigens in Thai Blood Donor Populations.

Purpose: Coa and Cob antigens of the Colton (CO) blood group system are implicated in acute and delayed hemolytic transfusion reactions (HTRs). Owing to the inadequate supply of specific antiserum, data on CO phenotypes remain limited. This study aimed to develop genotyping methods to predict Coa and Cob antigens and to estimate transfusion-induced alloimmunization risks in three Thai blood donor populations. Materials and Methods: The study included 2451 blood samples from unrelated healthy Thai blood donors obtained from central, northern, and southern Thailand. DNA sequencing was used to determine the CO*A and CO*B alleles. In-house PCR with sequence-specific primers (PCR-SSP) and high-resolution melting curve (HRM) assays were performed and genotyping results were compared using DNA sequencing. CO*A and CO*B allele frequencies among Thais were determined using PCR-SSP and their frequencies were compared with other populations. The risks of Coa and Cob transfusion-induced alloimmunization among Thai donor populations were calculated. Results: The validated genotyping results by PCR-SSP and HRM assays agreed with DNA sequencing. The CO*A/CO*A was the most common (100.0, 100.0, and 99.3%), followed by CO*A/CO*B (0.0, 0.0, and 0.7%) among central, northern and southern Thais. Homozygous CO*B/CO*B was not found. The CO*A and CO*B allele frequencies among central Thais significantly differed compared among southern Thais (p < 0.01) but not among northern Thais. Those allele frequencies among Thais were similar to those of Taiwanese, Chinese and Malay-Malaysian populations but not to South Asian, Southeast Asian, Korean, Japanese, Filipino, French Basque, and Maltese populations (p < 0.01). A higher risk of anti-Cob production rather than anti-Coa production was particularly noted in the southern Thai population. Conclusion: This study constitutes the first to determine CO*A and CO*B genotypes using PCR-SSP and HRM assays among Thais and this finding would be beneficial in predicting alloimmunization risk and providing safe transfusions among Thais.

The impact of killer cell immunoglobulin-like receptor (KIR) genes and human leukocyte antigen (HLA) class I ligands on predisposition or protection against prostate cancer.

Natural killer (NK) cell development largely depends on killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I ligands. In the current study, we investigated the role of KIR genes, HLA ligands, and KIR-HLA combinations in vulnerability or protection against prostate cancer (PC). To analyze the frequency of 16 KIR genes and 5 HLA ligands, polymerase chain reaction with sequence-specific primers (PCR-SSP) was conducted in 150 PC patients and 200 healthy controls (CNs). KIR2DL5 (p = 0.0346, OR = 0.606, CI = 0.3916-0.9336), KIR2DS5 (p = 0.0227, OR = 0.587, CI = 0.3793-0.9139), HLA-B Bw4Thr80 (p = 0.0401, OR = 0.3552, CI = 0.1466-0.9059), HLA Bw4 (p = 0.0190, OR = 0.4744, CI = 0.2656-0.8521), and T4 gene cluster (including KIR2DS5-2DL5-3DS1-2DS1 genes) (p = 0.0194, OR = 0.5575, CI = 0.3449-0.8938) had a lower frequency in the PC patients compared to the control group. Moreover, a lower frequency of the genotypes contacting activating KIR (aKIR) > inhibitory KIR (iKIR) (p = 0.0298, OR = 0.5291, CI = 0.3056-0.9174) and iKIR + HLA < aKIR + HLA (p = 0.0183, OR = 0.2197, CI = 0.0672-0.7001) in PC patients compared to the CNs implies a protective role for aKIR genes. In the case of KIR-HLA interactions, we detected a significant association between KIR3DS1+ + HLA-A Bw4+ (p = 0.0113, OR = 0.5093, CI = 0.3124-0.8416) and KIR3DL1- + HLA-A Bw4+ (p = 0.0306, OR = 0.1153, CI = 0.0106-0.6537) combinations and resistance to prostate cancer. In contrast, the presence of KIR3DL1 in the absence of HLA-A Bw4 (p = 0.0040, OR = 2.00, CI = 1.264-3.111), HLA Bw4 (p = 0.0296, OR = 2.066, CI = 1.094-3.906), and HLA-Bw4Thr80 (p = 0.0071, OR = 2.505, CI = 1.319-4.703) genes probably predisposes to prostate cancer. Carrying the CxT4 genotype in PC patients was positively associated with lower tumor grades (Gleason score ≤ 6) (p = 0.0331, OR = 3.290, and CI = 1.181-8.395). Altogether, our data suggest a protective role for aKIRs, HLA-B Bw4Thr80, and HLA Bw4 ligands as well as a predisposing role for certain KIR-HLA combinations in prostate cancer. The findings of this study offer new insight into the population's risk assessment for prostate cancer in men. Additionally, predicting immunotherapy response based on KIR-HLA combinations aids in implementing the most effective therapeutic approach in the early stages of the disease.

Human neutrophil antigen-1, -3, -4, and -5 allele and genotype frequencies in the Croatian blood donor population and their clinical significance.

OBJECTIVES: Human neutrophil antigens (HNAs) and antibodies play an important role in allo- and autoimmunity associated with immune neutropenia and transfusion reactions. The aim of this study was to determine the HNA-1, -3, -4 and -5 allele and genotype frequencies in the Croatian blood donor population to assess the role of HNA-1, -3, -4, and -5 alleles in the development of neonatal alloimmune neutropenia and antibody-mediated transfusion-related acute lung injury. MATERIAL AND METHODS: A total of 371 blood samples from unselected healthy blood donors were analyzed. Samples from all 371 donors were genotyped for HNA-1, samples from 160 donors were genotyped for HNA-3, and samples from 142 donors were genotyped for HNA-4 and HNA-5 using the polymerase chain reaction with sequence-specific primers (PCR-SSP) method. RESULTS: The frequencies of the FCGR3B*01, FCGR3B*02 and FCGR3B*03 HNA-1 alleles were 0.393, 0.607 and 0.022, and of the SLC44A2*01 and SLC44A2*02 HNA-3 alleles 0.781 and 0.219, respectively. The frequencies of the ITGAM*01 and ITGAM*02 HNA-4 alleles were 0.796 and 0.204, and of the ITGAL*01 and ITGAL*02 HNA-5 alleles 0.718 and 0.282, respectively. CONCLUSION: These are the first results on the HNA allele and genotype frequencies in the Croatian blood donor population. We observed no deviations from previous reports on Caucasian populations. Determination of the HNA antigen frequencies in the population is important to estimate the risk of alloimmunization to HNA, especially the risk of fetal-maternal incompatibility and alloantibody production by transfusion of the HNA incompatible blood components.

Concordance of identifying the presence or absence of KIR genes by Flow-rSSO and PCR-SBT methods: a comparative study / 中国输血杂志

【Objective】 To study the concordance of identifying the presence or absence of KIR genes using flow reverse sequence-specific oligonucleotide probe (Flow-rSSO) hybridization and sequencing based typing-PCR (PCR-SBT) methods. 【Methods】 A total number of 131 cases of DNA samples from Han population were subjected to identify the presence or absence of all 16 KIR genes by Flow-rSSO method, and then sequenced at coding sequence for all 14 functional KIR genes using our in-house KIR PCR-SBT assay. The concordance of identifying the presence or absence of all functional KIR genes by Flow-rSSO and PCR-SBT was analyzed. Samples with inconsistent initial results were re-tested using the Flow-rSSO commercial kits with different Lot number, and further tested using the PCR-SSP commercial kit. 【Results】 The presence or absence of 14 functional KIR genes for 129 of 131 samples were completely in accordance via the PCR-SBT and Flow-rSSO methods. Two samples, one with 3DL1 negative, the other with both 2DS3 and 2DS5 negative initially-identified by Flow-rSSO, were actually all positive tested by PCR-SBT. Further retest by Flow-rSSO commercial kits with different Lot number and PCR-SSP commercial kit indicated that the two samples were all positive, which agreed well with PCR-SBT results. 【Conclusion】 In this paper, the initial test results of the presence or absence of KIR genes identified by Flow-rSSO for 2 samples were wrong, which indicated the importance of carrying out the quality control for reagents in KIR gene testing.

Development of label-free electrochemical impedance spectroscopy for the detection of HLA-B*15:02 and HLA-B*15:21 for the prevention of carbamazepine-induced Stevens-Johnson syndrome.

BACKGROUND: Carbamazepine (CBZ) is an FDA-approved anticonvulsant that is widely used to treat epilepsy, bipolar disorder, trigeminal neuralgia and chronic pain. Several studies have reported a strong association between HLA-B*15:02 and carbamazepine-induced Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN). However, the HLA-B75 serotype (HLA-B*15:02, HLA-B*15:08, HLA-B*15:11 and HLA-B*15:21) has been found in patients with carbamazepine-induced SJS/TEN. METHODS: This study aimed to develop label-free electrochemical impedance spectroscopy (EIS) for the detection of HLA-B*15:02 and HLA-B*15:21 after PCR-SSP amplification. A total of 208 DNA samples were tested. The impedance was measured and compared to standard gel electrophoresis. RESULTS: The developed label-free EIS identified HLA-B*15:02 and HLA-B*15:21 alleles with 100% sensitivity (95% CI: 86.773%-100.000%) and 95.05% specificity (95% CI: 90.821%-97.714%), comparable to commercial DMSc 15:02 detection kits. CONCLUSIONS: We successfully developed a novel PCR-SSP associated with signal impedance changes to detect the HLA-B*15:02 allele and HLA-B*15:21 without downstream amplicon size analysis that is suitable for screening individuals before indication of CBZ therapy.

Contribution of HLA class II genes, DRB4*01:01, DRB1*07:01, and DQB1*03:03:2 to clinical features of Vitiligo disease in Iranian population.

BACKGROUND: Vitiligo is a multifactorial depigmentation condition, which is due to skin melanocyte destruction. Increased expression of HLA class II genes in patients with pre-lesions of Vitiligo suggests a crucial role for the participation of immune response in Vitiligo development. Recent studies progressively focused on HLA-DRB1 and DQB1 genes. In this study, we have evaluated the association and role of HLA-DRB4*01:01, -DRB1*07:01, and -DQB1*03:03:2 genes in different clinical subtypes of Vitiligo in the Iranian population. METHODS: First, Genomic DNA from peripheral blood of 125 unrelated Vitiligo patients and 100 unrelated healthy controls were extracted through the salting-out method. Then, HLA class II genotyping was performed using the sequence-specific primer PCR method. Finally, the clinical relevance of the testing for these genotypes was evaluated by applying the PcPPV (prevalence-corrected positive predictive value) formula. RESULTS: Our results indicated the positive associations of DRB4*01:01 and DRB1*07:01 allelic genes with early-onset Vitiligo (p = 0.024 and 0.022, respectively). DRB4*01:01 also showed strong protection against late-onset Vitiligo (p = 0.0016, RR = 0.360). Moreover, our data revealed that the DRB1*07:01 increases the susceptibility to Sporadic Vitiligo (p = 0.030, RR = 1.702). Furthermore, our findings proposed that elevated vulnerability of Vitiligo patients due to DRB4*01:01 and DRB1*07:01 alleles maybe is correlated with the presence of amino acid Arginine at position 71 at pocket 4 on the antigen-binding site of the HLA-DRB1 receptor. CONCLUSION: Our findings on different subtypes of Vitiligo suggest that, despite a more apparent autoimmune involvement, a non-autoimmune nature for the etiology of Vitiligo should also be considered.

Genetic background of RhD negative blood donors / 中国输血杂志

【Objective】 To analyze the genetic background of RhD-negative blood donors by detecting RHD and RHCE genes of those donors. 【Methods】 From March 2021 to May 2022, the blood samples of RhD-negative blood donors, who had been screened out by RhD primary screening and confirmatory experiments in the Yaan Blood Center, were firstly identified whether the RHD allele was completely deleted, then whether there were deletions in 10 exons of non-RHD allele complete deletion samples, finally, the remaining samples without RHD alleles and exon deletions were further analyzed by DNA sequencing. RHCE gene was detected by SSP-PCR method. 【Results】 Among the RHD gene test results of 104 RhD-negative samples, 65 cases were completely deleted (d/d), 33 were RHD partially deleted (one allele deletion), and 6 were without RHD gene deletion. The RHD alleles of 33 samples with partial deletion were detected by 10 exons, 13 had partial exon deletion, with genotype as RHD*D-CE(3-9)-D/d and phenotype as RhD negativity, and the remaining 20 samples had no exon deletion. The exon sequencing results of the non-deletion samples showed RHD*1227A/RHD*1227A in 6 samples, RHD*1227A/d in 19, RHD*3A/d in 1; both of the last two were considered Del by ISBT. The RHCE gene test results showed that all cc genotype blood donors were RhD true negative, while Del blood donors had no cc genotype. 【Conclusion】 Through the genetic background study of RhD negative blood donors, it is found that there is a high proportion of Del with weak expression of RhD antigen, whether this blood type affects clinical blood safety needs further researches.

Molecular basis of Del phenotype in RhD negative population of Zhongshan / 中国输血杂志

【Objective】 To conduct serological and molecular study of Del type in RhD-negative donor population in Zhongshan area, so as to improve the diagnosis of Del type. 【Methods】 A total of 102 initially RhD-negative samples, collected from December 2017 to February 2019, were classified by RHCE and PCR-SSP genotyping. And 95 cases of truly negative RhD were confirmed by IAT, 28 cases of Del type were identified by absorption and elution test. The phenotype and genotyping characteristics of Del type in Zhongshan area were summarized based on domestic data of relative literature. 【Results】 Among 102 initially RhD-negative samples by serological test, 95 were truely RhD-negative, 28 were DELRHD 1227A without any other Del allele. Among them, RHCE antigen type were Ccee in 20(71.4%) cases, CCee in 8(28.6%), with no difference in comparison with other regions in China. The frequency of Del in RhD-negative blood donors was 29.5% (28/95), with difference between Shanghai, Taiwan, and Fuzhou, but no difference between Nanchang, Zhejiang, and Wuhan. 【Conclusion】 The study showed that the Del phenotype was closely related to Ce haplotype, and has no difference with other regions in China. The frequency of Del type in RhD negative donors was 29.5%, with regional differences. RHD1227A was the main allele of Del.

Predominant frequency of HLA-B*27 in patients with ankylosing spondylitis in southeastern China.

OBJECTIVES: This study was to investigate the polymorphism and distribution of alleles of HLA-B*27 in patients with ankylosing spondylitis (AS) in Han population of southeastern China. METHODS: A total of 89 peripheral blood samples from southeastern Chinese Han patients with AS that diagnosed according to Modified New York criteria were subtyped using the high-resolution PCR-SSP.Exon 2-3 of HLA-B*27 gene was amplified and sequenced to further confirm the HLA-B*27 subtype. RESULTS: The frequency of HLA-B*27 was 99.87% in AS patients. Three subtypes, HLA-B*2704, HLA-B*2705, and HLA-B*2706 were identified. The frequencies for these three alleles were HLA-B*2704 in 84/88 (95.46%), HLA-B*2705 in 3/88(3.41%), and HLA-B*2706 in 1/88 (1.13%) of the HLA-B*27 positive patients, respectively. CONCLUSIONS: Our study shows that HLA-B*2704 has an overwhelming frequency in southeastern Chinese Han AS patients. A combined analysis including previous studies of HLA-B*27-subtype distributions in Chinese Han populations showed that HLA-B*2704 may originate from the southern Han and then migrate and spread to the northern areas, and HLA-B*2705 show the opposite result.