PCR-SSP Technique Research Articles

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.

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. 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. 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. 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.

A GE:-13 (GECT-) HETEROZIGOUS AND GE:-14 (GEAR) HOMOZIGOUS PHENOTYPE DETECTED BY RANDOM SCREENING FOR GE:-2 BLOOD DONORS IN BRAZIL

We report a rare case of GE:-13 and GE:-14 phenotype blood donor identified by random screening during blood bank routine. This is a descriptive work using immunohematology data from immunohematology reference laboratory (LRI) where samples were tested. A 29 year old blood donor, woman, self-declared as african american from the São Paulo city with no history of previous transfusions or pregnancies. Her blood was typed as O RhD+ (C-E+c+e+) and K- and the antibody screening was negative in gel cards. The phenotypes identified as Ge:-2,3 were confirmed by screening for GE:-2 blood group performed by hemagglutination in DG Gel cards using in house anti-Ge2 and others anti-Ge2 and anti-Ge3 from our inventory. Genomic DNA was isolated from donor whole blood and GE genotyping was performed by PCR-SSP techniques for analysis of exons 2, 3 and exon 4. The genetic variations that influence the expression of high-incidence antigens are deletions that involve exons 2, 3, 4 and the single nucleotide exchange (SNVs) in exons regions of the GYPC gene. These mutations, if not studied by molecular tests, may result in serological interpretation errors. Thus, sequencing by Sanger method of GE gene was performed and revealed a nucleotídeo change 59C>T (Pro20Leu in GPC) in Exon 2 in heterozygous and a nucleotide change 333A>C (non-coding) (Gly19Arg in GPD) in Exon 4 in homozigous state. No other nucleotide changes were identified. One of the criteria for AABB reference laboratory certification in immunohematology is the screening for blood donors with rare phenotypes. For this reason, donors from the Hospital Israelita Albert Einstein are tested with anti-Ge2 antiserum. The aim is detect the rare phenotype Ge:-2, for compatibility tests in patients with anti-Ge2 alloantibody. The antigens of the Gerbich System are located on glycophorins C (GPC), D (GPD) or both. There are eight highly prevalent antigens known as Ge2, Ge3, Ge4, GEPL, GEAT, GETI, GECT, GEAR. Polymorphisms that involve high incidence antigens such as deletions in introns and single nucleotide exchange (SNVs) in exons regions of the GYPC gene, if not studied by molecular tests, may result in serological interpretation errors. The fact of donor has a GECT allele (GE*01.-13) in heterozygosity does not imply the development of antibodies. The homozygous GEAR allele (GE*01-14) could result in the production of a rare anti-GEAR antibody in case of transfusion or pregnancy, however the clinical importance of this antibody is still unknown. Few articles report that these mutations can lead to loss of epitopes and the phenotypes can be interpreted as Ge:-2 or Ge:-3 in serological screening. This case showed that the molecular sequencing was crucial to determine the correct mutations present in the GYPC gene and consequently the correct deduction of the phenotype.

Polymorphismes 869C> T et 915 G>C du TGF-β1 dans la rétinopathie du diabète de type 1 chez la population algérienne

Diabetic retinopathy (DR) is characterized by chronic low-grade inflammation in which the effects of genetic factors is well established. The objective of our study is to explore an association of the 869C>T and 915G>C polymorphisms of the TGF-β1 gene with type 1 diabetic retinopathy in the Algerian population. A case-control study was carried out in which the SNPs 869C>T and 915G>C of the TGF-β1 gene were analysed by the PCR-SSP technique. We compared the distribution of allelic and genotypic frequencies between patients with and without retinopathy and looked for an association between these polymorphisms and certain clinical characteristics of and risk factors for diabetic retinopathy. A significant increase in the frequencies of the C allele (P=0.03) and GG genotype (P=0.007) of the 915 G>C polymorphism were found, respectively, in patients without and with retinopathy. However, no significant difference was found for allelic and genotypic frequencies of the 869C>T SNP (all P>0.05) or associations between genotypes and clinical characteristics or risk factors for DR. Our preliminary results suggest that the C allele of the 915 G>C polymorphism of TGF-β1 is protective against type 1 diabetic retinopathy in the Algerian population, while the GG genotype could confer susceptibility to it.

Comparative Analysis of a Developed Probe-Based Real-Time PCR and PCR-SSP for HLA-B*27 Detection among Thai Blood Donors

The human leukocyte antigen (HLA) class I gene, the B locus, allele 27, HLA-B*27 is one of the most fascinating risk factors that is strongly associated with developing spondyloarthropathies (SpA). HLA-B27 testing has been routinely available in the diagnosis of those diseases. This study aimed to develop a fluorogenic real-time PCR and to compare it with PCR-SSP to detect the HLA-B*27 allele among Thai blood donors. A total of 391 DNA samples were obtained from Thai blood donors at Thammasat University Hospital and tested for HLA-B*27 allele detection. A new real-time PCR was developed and validated to identify this allele and subsequently compared with those results tested with PCR-SSP. The sensitivity of detection was performed using known HLA-B*27-positive and -negative samples with concentrations ranging from 0.001 to 100 ng/µL. Additionally, HLA-B27 subtyping was performed by DNA sequencing containing second and third exons of this gene among all the HLA-B*27-positive donors. The validity of real-time PCR using known DNA controls and the results obtained by PCR-SSP techniques were in 100% concordance. The method was sensitive even at low DNA concentrations (1 ng/µL). Of 391 donors, 24 (6.14%; 95% CI, 3.97 - 9.00) were found to have the HLA-B*27 allele, while the remaining 367 (93.86%; 95% CI, 91.00 - 96.03) did not have this allele. Donors presented HLA-B*27-positive, HLA-B*27:06, the most common allele, followed by HLA-B*27:04, -B*27:05, and -B*27:07. HLA-B*27 using fluorogenic real-time qualitative PCR was found to be superior compared with that of PCR-SSP. The method is rapid, accurate, reliable, and sensitive for detection. In addition, this method provides convenience in the early treatment of SpA patients and relieves their suffering.

Development and implantation of PCR-SSP for the genotyping of JAK2 V617F mutation / Desenvolvimento e implantação de metodologia molecular baseada em PCR-SSP para genotipagem da mutação V617F de JAK2

The JAK2 protein promotes cells growth and proliferation, and mutations in the JAK2 gene can result in increase of the number of blood cells and in development of myeloproliferative neoplasms. This study aimed to develop and implement the PCR-SSP (Sequence-Specific Primer - Polymerase Chain Reaction) to detect the JAK2 V617F mutation. Therefore, a literature review about genotyping methodologies for this mutation was conducted and primers were based on the model proposed by Xavier (2009), tested and adjusted to the best amplification condition. The PCR-SSP technique was standardized and was effective for the detection of the JAK2 V617F mutation. As it is not expensive, the technique reduces costs and can be implanted even in small molecular biology laboratories, helping the diagnosis of patients with myeloproliferative diseases and favoring research related to this gene.

Establishment of an HLA laboratory in Madagascar: Technical and economic difficulties in developing country

BackgroundChronic renal failure is a worldwidepublic health issue. In Madagascar, this condition of heavy care is a medical challenge in a low-income country. In 2016, an estimated 8,000 - 10,000 people with kidney failure and only 1.26% have access to hemodialysis. About 30 patients were able to access live organ transplantation abroad.Our objectives are to set up an HLA histocompatibility laboratory providing HLA typing, search for anti-HLA antibodies, and crossmatch. Evaluate the available structures of the Immunology laboratory. Look for partner laboratories for more specialized research. Materials and methodsThe Joseph Ravoahangy Andrianavalona University Hospital Immunology Laboratory is equipped with a conventional PCR and real-time PCR platform, an ELISA immunoassay platform, and inverted fluorescence microscope. ResultsThe techniques to be implemented in Madagascar for the HLA Laboratory are the HLA typing of the donor and the recipient by the PCRSSP and microlymphocytotoxicity (LCT) technique. The search for anti-HLA antibodies will be done by ELISA and LCT and crossmatch in LCT.We have created cooperation with the Réunion France histocompatibility laboratory for the identification of recipient and donor HLA antibodies and crossmatch by Luminex. ConclusionThe presence of HLA platform in Madagascar will allow preparing the establishment of a future kidney transplant in the country but also for improved management of patients waiting on abroad transplant list in regards of donor selection and post-transplant monitoring.

Killer immunoglobulin like receptor gene profiling in Western Indian population

One hundred and sixty one healthy unrelated hematopoietic stem cell transplant donors from Western Indian were enrolled in this study. The study was initiated to observe the genotypic diversity of 16 killer immunoglobulin like receptor (KIR) genes in this population. KIR genotyping was carried out using the PCR-SSP technique. 56 KIR genotypes were observed in our population, where 15 genotypes were reported for the first time in any population. The KIR genotype data for the population can be accessed from Allele Frequencies Net Database under the population name “India Western KIR” and identifier “3570”.

Polymorphism of human platelet antigens in Tunisian population: Clinical and anthropological interests

Human Platelet Antigens (HPA) are of considerable interest in obstetric transfusion medicine and anthropological genetics. This study aims to provide clinicians with a detailed database of HPA antigenic variants, which allows them to estimate the probability of allo-immunisation of each antigen. In addition, it aims to make an interethnic comparison of the Tunisian population with other populations. The target population consists of 324healthy and unrelated Tunisian blood donors recruited from the National Blood Transfusion Center in Tunis. DNA extraction was performed by the Salting Out method and molecular genotyping was performed by the PCR-SSP technique. The statistical analysis was performed using two approaches: manual calculation and computerized calculation. Phylogenetic trees were constructed through the use of Standard Genetic Distances that were calculated from allelic frequencies. With the exception of the HPA-4 system, statistical analysis showed that all HPA systems are polymorphic especially the two systems HPA-3 and HPA-15. The inter-ethnic analysis showed that Tunisians are closer to North Africans and Caucasians than Sub-Saharan and Asian populations, which shows genetic mixing between Tunisians, Arabs, Europeans and Africans. The results of this study could be exploited to prepare a ready-to-use genotyping plate dedicated to HPA antigens, with the aim of ensuring better management, especially for polytransfused patients.

In-house HLA-B*27 optimization based on PCR-SSP technique and test kit development

AAmaç: Çalışmada Ankilozan spondilit ve diğer spondiloartropatilerin moleküler tanısında kullanılmak üzere güvenilir, ekonomik ve rutin kullanıma uygun laboratuvar yapımı (in-house) HLA-B*27 PCR-SSP tanı kiti geliştirilmesi amaçlanmıştır. Gereç ve Yöntem: Çalışmaya, ticari kitler kullanılarak HLA-B*27 pozitif olarak saptanan 119 ve negatif olarak saptanan 446 olmak üzere toplam 565 olgu dahil edildi. HLA-B*27 tipleme için sekansa spesifik primerler ve beta globin gen bölgesinden yararlanılarak dizayn edilen internal kontrol primer çifti kullanılarak PCR optimizasyonu gerçekleştirildi. Bulgular: HLA-B*27’si pozitif olan olgularda, %2’lik agaroz jelinde 273 bç internal kontrol bandının ilerisinde 149 bç’lik spesifik bant olmak üzere çift bant, B*27 negatif hastalarda ise sadece 273 bç’lik internal kontrol bandı saptandı. Ticari kitler kullanılarak HLA-B*27’si pozitif olarak saptanan 119 ve negatif olarak saptanan 446 bireyin sonuçları ile tarafımızdan geliştirilen PCR-SSP kiti kullanılarak elde edilen sonuçların tamamen uyumlu olması nedeniyle sensitivite ve spesifite %100 olarak belirlendi. Geliştirdiğimiz laboratuvar yapımı PCR-SSP kitinin maliyetinin, ticari kitlere göre en az 50 kat daha ucuz olduğu görüldü. Sonuç: Ticari kitler kullanılarak HLA-B*27’si pozitif olarak saptanan 119 ve negatif olarak saptanan 446 olgunun tamamında, geliştirdiğimiz yöntemle de birebir uyumlu sonuçlar elde edildi. Bu bulgular geliştirdiğimiz kitin güvenilir, ekonomik ve rutin kullanıma uygun olduğunu göstermektedir.

Frequencies of human platelet antigens (HPA-1, -2, -3, -4, and -5) among the Moroccan blood donors

Aims: Human platelet antigens (HPA) are involved in several clinical conditions, such as neonatal alloimmune thrombocytopenia (NAIT), platelet transfusion purpura (PTP), and refractoriness to platelet transfusion.The frequency of platelet antigens varies among populations. So far, typing of HPA systems has not been carried on Moroccan population. The frequencies of these antigens, their risk of alloimmunization, and their clinical implications and complications within Moroccan population are unknown. Our purpose is to define allele frequencies and genotypes in Moroccan population of the five HPA-1 to HPA-5 systems. Evaluate of the risk of anti-platelet alloimmunization among Moroccan blood donors, and estimate the mismatch probability of different platelet alloantigens, after random transfusions of platelet concentrates. Methods: The gene polymorphisms of HPA-1, -2, -3, -4, and -5 were determined by the PCRSSP technique on a DNA sample of 110 healthy Moroccan blood donors randomly chosen. Results: Alleles frequencies for the HPA systems were: HPA-1a: 0.704, HPA-2a: 0.709, HPA3a: 0.773, HPA-4a: 0.99, and HPA-5a: 0.760. The alleles were HPA-1b: 0.296, HPA-2b: 0.291, HPA-3b: 0.227, HPA-4b: 0.01, and HPA-5b: 0.240. The theoretical frequencies of descendants at risk of alloimmunization are ranged between 0.99% for HPA-4 to 20.76% for HPA-1. The estimated mismatch probability regarding platelet antigens HPA-1, -2, -3, -4, and –5 in Moroccan blood donors, after random platelet transfusion, varies from 1.96% for HPA-4, to 32.9% for HPA-1. Conclusion: Taking consideration of the previous studies, and our results, a clinical research associated with platelet disorders, such as: neonatal alloimmune thrombocytopenia (FNAIT), post transfusion purpura (PTP), and, multi-platelet transfusion refractoriness (MPR), are needed to ensure the proper diagnosis and the blood transfusion safety.

Red Cell Genotyping by Multiplex PCR Identifies Antigen-Matched Blood Units for Transfusion-Dependent Thai Patients

Background: Antigen-negative red cell transfusion is required for transfusion-dependent patients. We developed multiplex PCR for red cell genotyping and calculated the possibility of finding compatible predicted phenotypes in Thai blood donor populations according to red cell alloantibodies found among Thai patients. Methods: 600 DNA samples obtained from unrelated healthy central and northern Thai blood donors were tested with the newly developed multiplex PCR for FY*A, FY*B, JK*A, JK*B, RHCE*e, RHCE*E, DI*A and GYP*Hut, GYP*Mur, GYP*Hop, GYP*Bun, and GYP*HF allele detections. Additionally, the possibility of finding compatible predicted phenotypes in two Thai blood donor populations was calculated to estimate the minimal number of tests needed to provide compatible blood. Results: The validity of multiplex PCR using known DNA controls and the phenotyping and genotyping results obtained by serological and PCR-SSP techniques were in agreement. The possibility of finding at least one compatible blood unit for patients with multiple antibodies was comparable in Thai populations. Conclusions: The multiplex PCR for red cell genotyping simultaneously interprets 7 alleles and 1 hybrid GP group. Similar strategies can be applied in other populations depending on alloantibody frequencies in transfusion-dependent patients, especially in a country with limited resources.

P078 The relationship between MiHAs compatibility and HLA matching degree of related donor and recipient pairs in allogeneic hematopoietic stem cell transplantation

Aim This study is aimed at investigating the relationship between minor histocompatibility antigens (MiHAs) compatibility and human leucocyte antigens (HLA) matching degree of related donor and recipient pairs in allogeneic hematopoietic stem cell transplantation (allo–HSCT). Methods The 1116 samples is consisted of 439 leukemia patients and 677 related potential donors including 229 HLA-matched, 245 HLA-semi-matched and 204 HLA-unmatched groups. PCR-SSP technique was used to genotype the 14 autosomal MiHAs coding genes. Typing data were analyzed for allelic, genotypic and phenotypic frequencies of the 14 MiHAs. The disparity rate of MiHAs in HLA-matched, HLA-semi-matched and HLA-unmatched groups were analyzed respectively. Linkage analysis was performed on PECAM-1A (codon 125) – PECAM-1A (codon 563)-PECAM-1A (codon 670) haplotypes. Results The disparity rates of HA-1,HA-2,HA-8,ACC-1, PECAM-1 (codon 125) V,PECAM-1 (codon 563) N,PECAM-1 (codon 670) G,CD62 (codon 206) and CD62 (codon 213) were observed significantly lower in HLA-matched group than those in HLA-semi-matched group ( P P P P P ≅ 1). Conclusion This is the first time to report the positive correlation between MiHAs matching degree and HLA compatibility of donor-recipient pairs, which provided important data to select best suitable donor for a recipient in allo-HSCT. Keywords: allo-HSCT; MiHAs; HLA; GVHD.

P106 Killer immunoglobilun-like receptors (KIR) BB haplotype confers susceptibility to acute childhood lymphoblastic leukemia (ALL) and 2DS2, 2DS3 and 2DS4 activating genes are of low risk in mexican children

Aim NK cells have the ability to destroy leukemic cells when lacking the ligand for the corresponding inhibitory KIRs. It is documented, that a strong GVHD effect mediated by alloreactive NK cells, results in a reduced risk for relapse in acute myeloid leukemia, and it depends of the inhibitory and stimulatory signals mediated through their KIR ligands. We looked for KIR gene frequencies and haplotypes in ALL Mexican Mestizo children. Methods KIR and HLA genotyping was performed in 66 ALL patients and 357 controls, all of them, Mexican Mestizos. The presence of 14 KIR genes was detected using a PCR-SSP technique with four multiplex reactions. HLA typing was done using a Luminex PCR-SSOP method. Frequency (%) for KIR genes was determined by direct counting. Genotypes were assigned according to www.allelefrequencies.net. A and B haplotypes were deduced from the genotype data. The gene content and haplotype frequencies were compared between patients and controls, using the Chi 2 test with the SPSS17 software. Results We found a significant decrease of 2DS4 (54.5% vs. 72.8%; OR = 0.45; CI = 0.26–0.77; p = 0.003), 2DL2 (21.2% vs. 42.9%; OR = 0.36; CI = 0.19–0.67; p = 0.001), 2DS2 (19.7% vs. 44.8%; OR = 0.302; CI = 0.16–0.57; p p = 0.001) in ALL children. Framework genes were present in all individuals (2DL4, 3DL2 and 3DL3). The frequency of AA genotype was significantly decreased in the cases vs. controls (31.9% vs. 45.5%; OR = 0.56; CI = 0.33–0.96; p = 0.03). Conclusions A low risk was shown for ALL, if carrying 2DS4, 2DL2, 2DS2 or 2DS3 in Mexicans. We also found that the AA haplotype is protective and contains the 2DS4 activating gene. Controversial results exist in ALL depending on ethnicity. In Canadians, a reduced risk exists when activating KIR genes are present, while in Germans no association was shown; AA was not associated in non-hispanics, while an increased risk was observed in Hispanics. Thus, it is crucial to analyze specific ethnic groups, since Hispanics include many different racial backgrounds. In our study, which is the first one done in Mexicans with ALL, only Mestizos were included. Our results are consistent with data demonstrating that KIR genes play an important role in disease survival. Therefore, more studies of KIR genes and ALL must be done in non-Caucasians.

HLA-A68 and HLA-B15 alleles correlate with poor immune response among AIDS patients on combined antiretroviral therapy

Around 15–30% of AIDS patients fail to recover their CD4+ T cell levels following combined antiretroviral therapy despite successful inhibition of HIV-1 replication. The exact reasons for this immune recovery failure are not completely understood. HLA alleles are among the candidate that may explain this failure. A total of 65 adult AIDS patients, with viral load of <50 copies per ml were investigated. Viral load and CD4 T cells counts were performed following standard techniques. HLA genotyping was performed using PCR-SSP technique. The Statistical Package for Social Sciences (SPSS version 19) was used for data processing and analysis. A significantly higher proportion of poor immune responders were carrying HLA-A68 (4.8% compared to 25.0%, P=0.025) and HLA-B15 (2.4% compared to 20.8%, P=0.023). The etiological fraction (Efe%) among carriers of HLA-A68 was 57.89% (95% CI=26.79, 75.79) and was 61.35% (95% CI=35.33, 76.91) among carriers of HLA-B15.

Genotypic diversity of the Killer Cell Immunoglobulin-like Receptors (KIR) and their HLA class I Ligands in a Saudi population.

Killer Cell Immunoglobulin-like Receptors (KIR) have been used as good markers for the study of genetic predisposition in many diseases and in human genetic population dynamics. In this context, we have investigated the genetic diversity of KIR genes and their main HLA class I ligands in Saudi population and compared the data with other studies of neighboring populations. One hundred and fourteen randomly selected healthy Saudi subjects were genotyped for the presence or absence of 16 KIR genes and their HLA-C1, -C2, -Bw4Thr80 and Bw4Ile80 groups, using a PCR-SSP technique. The results show the occurrence of the framework genes (3DL2, 3DL3 and 2DL4) and the pseudogenes (2DP1 and 3DP1) at highest frequencies. All inhibitory KIR (iKIR) genes appeared at higher frequencies than activating genes (aKIR), except for 2DS4 with a frequency of 90.35%. A total of 55 different genotypes were observed appearing at different frequencies, where 12 are considered novel. Two haplotypes were characterized, AA and Bx (BB and AB), which were observed in 24.5% and 75.5% respectively of the studied group. The frequencies of iKIR + HLA associations were found to be much higher than aKIR + HLA. KIR genes frequencies in the Saudi population are comparable with other Middle Eastern and North African populations.

The likelihood ratio and frequency of DQ2/DQ8 haplotypes in Iranian patients with celiac disease.

The aim of this study was to evaluate the likelihood ratio and frequency of DQ2 and DQ8 in Iranian patients with celiac disease (CD). The HLA DQ2 and HLA DQ8 are the important mediators in the development of celiac disease. A few studies evaluated the frequency of HLA DQ2 and HLA DQ8 haplotypes among the Iranian population with low sample size. In this cross-sectional study, to predict HLA-DQ2 and DQ8 haplotypes, 141(73 male, 78 female) confirmed CD patients compared to 151 healthy controls were enrolled into this study during 2013-2014. HLA DQ2/ DQ8 haplotypes was determined in cases and controls using PCR-SSP technique. DQ2 and DQ8 were positive in 80% (n=111) and 49% (n= 69) of CD patients and 36% (n=61) and 13% (n=21) of control group respectively. Moreover, 32% (n=45) of CD patients and 5.3% (n=8) of the control group were carrier of both haplotypes. In the case group about one-third of patients (32.2%) were positive for carrying both DQ2 and DQ8 heterodimers while only 5.3% (n=8) of the control group were carrier. In addition, the positive likelihood ratio of DQ2 and DQ8 were 1.74 (CI: 1.4- 2.1), and 2.6 (CI: 1.8- 2.7), respectively. The result of this study showed that the frequency of DQ8 among our population is higher than those reported by European countries, but it is close to those founded in South America and Middle East. This result suggests that the higher prevalence of HLA DQ8 pattern in Iranian CD patients is similar to non-European patients.

Neonatal Alloimmune Neutropenia Due to Maternal Alloimmunization Against Human Neutrophil Alloantigen-1 and -3

Introduction: Neonatal alloimmune neutropenia (NAN) is a potentially lethal disorder that results from maternal alloimmunization to human neutrophil antigens (HNAs) present in the fetus. The alloantibodies more frequently involved in NAN are against the HNA-1 and -2 systems; however HNA-3, HNA-4 and HNA-5 systems have also been associated with cases of NAN. In this study we examined the frequency of neonatal neutropenia and investigated the presence of maternal anti-HNA-1, anti-HNA-3 and anti-HLA alloantibodies when neonatal neutropenia and maternal-fetal incompatibility to HNA-1 and HNA-3 systems were observed. Furthermore, we investigated the association between neutrophil alloimmunization and HLA-DRB1 alleles.Methods: A cross sectional study included samples from 10,000 unselected neonates born in 4 obstetric units in Sao Paulo City (Brazil). Neonatal neutropenia was defined as neutrophil count <1.5x109/L in cord blood. Genotyping studies were performed in 88 neutropenic newborns and their mothers (83 mothers with 3 pairs of twins and 1 triplet) by PCR-SSP technique for the detection of HNA-1a, -1b and -1c alleles and by PCR-RFLP technique for the detection of HNA-3a and -3b alleles; the PCR-RFLP technique amplifies the sequence for rs2288904 and the amplified product was digested with enzyme Taqα1 specific to nucleotide guanine that codifies the HNA-3a antigen (Lopes et al., Transfusion 2014;54(6):1619-21.). Serologic studies for detecting maternal HNA alloantibodies were performed in maternal-fetal incompatibility cases by granulocyte agglutination test (GAT) that was done in duplicate using a specific panel of donors previously genotyped for HNA-1 and HNA-3 systems. Anti-HLA-I/II antibodies were investigated in maternal serum, and all 83 mothers were genotyped for the HLA-DRB1 gene. Two control groups included mothers with maternal-fetal incompatibility but without alloantibodies, and healthy individuals.Results: Neonatal neutropenia was identified in 88 of 10,000 (0.9%) newborns in the studied population. Genotyping studies revealed 39/88 (44.3%) maternal-fetal HNA incompatibilities corresponding to 28/88 (31.8%) for HNA-1 and 13/88 (14.8%) for HNA-3. In 12/28 (42.9%) cases we found incompatibility for the HNA-1a allele; in 9/28 (32.1%) for HNA-1b; in 4/28 (14.3%) for HNA-1c; in 1/28 (3.6%) for both HNA-1a and -1c; and in 2/28 (7.1%) for both HNA-1b and -1c alleles. In all neutropenic cases related to HNA-3 system mothers were typed as HNA-3a/a and neonates as HNA-3a/b. Using the GAT, HNA alloantibodies were found in 21/39 (53.8%) mothers including 16/28 (57.2%) anti-HNA-1 and 5/13 (38.5%) anti-HNA-3b. The specificity of HNA-1 alloantibodies was confirmed in 9 cases for anti-HNA-1a and in 7 for anti-HNA-1b. Anti-HLA antibodies were detected in 6/16 GAT(+) maternal serum containing anti-HNA-1 and in 2/5 GAT(+) with anti-HNA-3. Two maternal-fetal incompatibility cases occurred concomitantly for HNA-1 and HNA-3 systems; however, only anti-HNA-3b alloantibodies could be identified using the panel of donors. HLA-DRB1 genotyping showed an increased frequency of HLA-DRB1*01 and HLA-DRB1*10 alleles in mothers with HNA incompatibility when anti-HNA were not found. A statistically significant difference was observed between these mothers with HNA incompatibility not alloimmunized and the healthy individuals (HLA-DRB1*01 p=0.0433; HLA-DRB1*10 p=0.0208).Conclusions: The observed frequency of neonatal neutropenia in Brazilians of 0.9% is similar to those described in North Americans and Europeans with a comparable positive serology rate of 53.8% more frequently due to the presence of anti-HNA-1 (76.2%). As for the best of our knowledge this is the first study reporting the presence of anti-HNA-3b alloantibodies in newborns with NAN (23.8%). The frequencies of the HLA-DRB1*01 and HLA-DRB1*10 alleles were increased in mothers with HNA incompatibility without HNA alloantibodies suggesting that such HLA-DRB1 alleles may confer protection against neutrophil alloimmunization in cases of maternal-fetal incompatibility for HNA systems involved with NAN. DisclosuresNo relevant conflicts of interest to declare.

Distribution of killer cell immunoglobulin-like receptor genes in population of Vojvodina, Serbia

Background: Killer cell immunoglobulin-like receptors (KIRs) are glycoproteins regulating the response of natural killer (NK) cells and a few sub-sets of T-cells. The KIR gene frequencies and genotype content vary considerably among different ethnic groups.Aim: The aim of this study was to analyse KIR gene polymorphism in the population of Vojvodina and to compare it with selected worldwide populations.Subjects and methods: The studied sample consists of 134 healthy unrelated individuals, residents of different geographical regions of Vojvodina. DNA samples isolated from peripheral blood leukocytes by the silica-based extraction method were used in reverse PCR-SSO and PCR-SSP technique to detect the presence and absence of KIR genes.Results: All 16 KIR genes, a total of 37 different KIR genotypes, were observed in the Vojvodina population with the presence of framework and pseudogenes in all individuals. The neighbour-joining phylogenetic tree shows that the Vojvodina population is in the same cluster with Croatians, Turkish, Russians, Czechs, Irish, Italians, French, Macedonians and Polish. The Vojvodina population shows polymorphism of the KIR gene family present in other European and European-derived populations studied previously.Conclusion: The present study may serve as a reference for comparisons in further anthropological and disease association studies and also provide more informative data valuable for donor search strategy in haematopoietic stem cell transplantation.

The involvement of the HLA-DQB1 alleles in the risk and the severity of Iranian coeliac disease patients.

Coeliac disease (CD) is a highly prevalent autoimmune disorder that is triggered by the ingestion of wheat gluten and related proteins in genetically susceptible individuals. The CD is associated with human leucocyte antigen (HLA) genes particularly with HLA-DQ alleles encoding HLA-DQ2 and DQ8 proteins. To define risk and severity alleles for CD, a total of 120 definite CD patients and 100 healthy controls were genotyped for HLA-DQB1 gene. HLA-DQB1 genotyping was performed in all patients and controls using PCR-SSP technique, and to evaluate the clinical relevance of testing for HLA-DQB1 and determining absolute risk of disease, prevalence-corrected positive predictive value and prevalence-corrected negative predictive value (PcPPV and PcNPV) were calculated. Our results for a first time show that DQB1*02:00 and DQB1*03:02 alleles and DQB1*02:01/03:02 genotype very significantly associated with increased risk of patients with CD, and DQB1*03:01,4 allele provides protection against CD in Iranian patients. Furthermore, the PcPPV for DQB*02:01 and 03:02 alleles in CD were 0.014 and 0.012, respectively, and the highest absolute risk presented by DQB*0201/0302 genotype (PcPPV = 0.079) and 98% of patients with CD carried DQB1*02:01/x or DQB1*03:02/x genotype. The results also clearly demonstrated that the DQB1*02:01 allele significantly associated with severity of CD, while DQB1*03:02 allele associated with mild form of CD. These results suggest that clinically suspected individuals for CD and first-degree relatives of patients with CD to be screened for HLA-DQB*0201 and DQB*0302 alleles for possible early diagnosis and treatments.

The L-selectin Phe206Leu gene polymorphism is not associated with visceral leishmaniasis.

Previous studies revealed that selectins play key roles in homing of immune cells to inflamed tissues and lymphatic organs. L-selectins are expressed on immune cells and interact with P and E selectins to homing to the tissues, hence, the polymorphisms within the gene of L-selectins may are associated with alteration in its expression. Thus, the current cross-sectional analytical study has been designed to investigate the polymorphisms within L-selectin gene and their relation with visceral leishmaniasis (VL). This study was performed on 194 samples during 2004-2012.The PCR-SSP and immunoflorescence techniques were used to evaluate the L-selectins polymorphism and anti-Leishmania antibody titration, respectively, in 56, 74 and 64 seropositive VL patients (group 1), seropositive healthy controls (group 2) and seronegative healthy controls (group 3). The results showed that the genotypes (P=0.711) and alleles (P=0.679) within L-selectins gene (A/C) was not differ between groups. Our results also demonstrated that the genotypes within L-selectins in group 1 (P=0.807) and 2 (P=0.441) were not associated with the titration of anti-leishmania antibody. The results identified that the polymorphisms within L-selectins gene were not associated with VL and it may be concluded that these genotypes and alleles are unable to affect immune responses in VL patients.