DRB Genes Research Articles

CLINICAL AND IMMUNOLOGICAL FEATURES OF KIDNEY TRANSPLANT RECIPIENTS WITH CYTOMEGALOVIRUS INFECTION MANIFESTATION IN THE EARLY POSTOPERATIVE PERIOD

Aim. To optimize the management of postoperative renal allograft recipients through the introduction of methods for predicting risk of manifestation of cytomegalovirus infection on the basis of a comprehensive assessment of the clinical and immunological status. Materials and methods. We retrospectively analyzed the medical records of 303 patients with end-stage renal disease, among them – were the recipients of renal allograft – 136, among whom 29 within 2 months after the operation had clinical signs of CMV infection. Assessable "CMV syndrome", laboratory evidence of CMV infection, the incidence of antigens (genes) of HLA A, B and DRB *1, calculated goodness of fi t χ 2 and relative risk RR, changes MCP-1 in urine. Results. In renal allograft recipients with clinical and laboratory evidence of CMV infection in the early postoperative period, signi fi cantly more ( χ 2 > 3,8) met antigen B35. A positive association with CMV infection was detected also for DRB1 * 08, B21, B22, B41, A24 (9), B51 (5), DRB1*14 and DRB1*15. Protective effects possessed antigens / alleles of genes A26 (10), B14, B38 (16) B61 (40) and DRB1*16. MCP-1 levels in this group of recipients were raised to 2174,7 ± 296,3 pg/ml with a strong negative correlation with the levels of urea and creatinine in serum (r = 0,9, p < 0.001). Conclusion. Immunological markers of risk manifestation of CMV infection in recipients of kidneys in the early postoperative period are: the carriage of В 35 и В 55,56(22), В 49(21), В 41, DRB1*08 и DRB1*15, an increase of levels of MCP-1 in urine without increasing the levels of urea and creatinine in the serum.

Whole-exome sequencing for the identification of susceptibility genes of Kashin-Beck disease.

ObjectiveTo identify and investigate the susceptibility genes of Kashin–Beck disease (KBD) in Chinese population.MethodsWhole-exome capturing and sequencing technology was used for the detection of genetic variations in 19 individuals from six families with high incidence of KBD. A total of 44 polymorphisms from 41 genes were genotyped from a total of 144 cases and 144 controls by using MassARRAY under the standard protocol from Sequenom. Association was applied on the data by using PLINK1.07.ResultsIn the sequencing stage, each sample showed approximately 70-fold coverage, thus covering more than 99% of the target regions. Among the single nucleotide polymorphisms (SNPs) used in the transmission disequilibrium test, 108 had a p-value of <0.01, whereas 1056 had a p-value of <0.05. Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis indicates that these SNPs focus on three major pathways: regulation of actin cytoskeleton, focal adhesion, and metabolic pathways. In the validation stage, single locus effects revealed that two of these polymorphisms (rs7745040 and rs9275295) in the human leukocyte antigen (HLA)-DRB1 gene and one polymorphism (rs9473132) in CD2-associated protein (CD2AP) gene have a significant statistical association with KBD.ConclusionsHLA-DRB1 and CD2AP gene were identified to be among the susceptibility genes of KBD, thus supporting the role of the autoimmune response in KBD and the possibility of shared etiology between osteoarthritis, rheumatoid arthritis, and KBD.

Positive selection on MHC class II DRB and DQB genes in the bank vole (Myodes glareolus).

The major histocompatibility complex (MHC) class IIB genes show considerable sequence similarity between loci. The MHC class II DQB and DRB genes are known to exhibit a high level of polymorphism, most likely maintained by parasite-mediated selection. Studies of the MHC in wild rodents have focused on DRB, whilst DQB has been given much less attention. Here, we characterised DQB genes in Swedish bank voles Myodes glareolus, using full-length transcripts. We then designed primers that specifically amplify exon 2 from DRB (202 bp) and DQB (205 bp) and investigated molecular signatures of natural selection on DRB and DQB alleles. The presence of two separate gene clusters was confirmed using BLASTN and phylogenetic analysis, where our seven transcripts clustered according to either DQB or DRB homologues. These gene clusters were again confirmed on exon 2 data from 454-amplicon sequencing. Our DRB primers amplify a similar number of alleles per individual as previously published DRB primers, though our reads are longer. Traditional d N/d S analyses of DRB sequences in the bank vole have not found a conclusive signal of positive selection. Using a more advanced substitution model (the Kumar method) we found positive selection in the peptide binding region (PBR) of both DRB and DQB genes. Maximum likelihood models of codon substitutions detected positively selected sites located in the PBR of both DQB and DRB. Interestingly, these analyses detected at least twice as many positively selected sites in DQB than DRB, suggesting that DQB has been under stronger positive selection than DRB over evolutionary time.

Associations of HLA gene with leukemia in 1186 cases

The purpose of this study was to evaluate the potential associations between HLA-A, B, DRB1 gene and leukemia. A total of 1186 leukemic patients, including 326 patients with acute lymphoblastic leukemia (ALL), 545 patients with acute myeloid leukemia (AML), 315 patients with chronic myeloid leukemia (CML), and 1234 healthy unrelated donors were typed and were compared in a single centre by using same technique, then the Bonferroni correction method was used to correct the Type I error. The results indicated that as compared with the control,the frequency of HLA-DRB1(*)09 in ALL group significantly decreased (10.87% versus 16.08%; Pc = 0.014, OR = 0.637, 95% CI = 0.487-0.834), while in comparison with control, the frequency of HLA-B(*)18 in CML group was significantly higher (1.28% vs 0.20%; Pc = 0.039, OR = 6.336, 95% CI = 2.066-19.434). The positive and negative relation may exist between certain HLA molecules and leukemia. The negative relation between HLA-DRB1(*)09 and ALL indicated that DRB1*09 might play an important role by a restricted T-cell immune response in the early leukemogenic events, whereas the positive relation between HLA-B(*)18 and CML suggests that the B(*)18 molecules may not actively present leukemia-specific antigens resulting in immune escape. It is concluded that these findings can contribute to developing more appropriate method in leukemic immunotherapy.

Comparative pharmacogenetics of multiple sclerosis: IFN-β versus glatiramer acetate.

Various diseases require the selection of preferable treatment out of available alternatives. Multiple sclerosis (MS), an autoimmune inflammatory/neurodegenerative disease of the CNS, requires long-term medication with either specific disease-modifying therapy (DMT) - IFN-β or glatiramer acetate (GA) - which remain the only first-line DMTs in all countries. A significant share of MS patients are resistant to treatment with one or the other DMT; therefore, the earliest choice of preferable DMT is of particular importance. A number of conventional pharmacogenetic studies performed up to the present day have identified the treatment-sensitive genetic biomarkers that might be specific for the particular drug; however, the suitable biomarkers for selection of one or another first-line DMT are remained to be found. Comparative pharmacogenetic analysis may allow the identification of the discriminative genetic biomarkers, which may be more informative for an a priori DMT choice than those found in conventional pharmacogenetic studies. The search for discriminative markers of preferable first-line DMT, which differ in carriage between IFN-β responders and GA responders as well as between IFN-β nonresponders and GA nonresponders, has been performed in 253 IFN-β-treated MS patients and 285 GA-treated MS patients. A bioinformatics algorithm for identification of composite biomarkers (allelic sets) was applied on a unified set of immune-response genes, which are relevant for IFN-β and/or GA modes of action, and identical clinical criteria of treatment response. We found the range of discriminative markers, which include polymorphic variants of CCR5, IFNAR1, TGFB1, DRB1 or CTLA4 genes, in different combinations. Every allelic set includes the CCR5 genetic variant, which probably suggests its crucial role in the modulation of the DMT response. Special attention should be given to the (CCR5*d+ IFNAR1*G) discriminative combination, which clearly points towards IFN-β treatment choice for carriers of this combination. As a whole the comparative approach provides an option for the identification of prognostic composite biomarkers for a preferable medication among available alternatives.

The great diversity of major histocompatibility complex class II genes in Philippine native cattle

Bovine leukocyte antigens (BoLA) are extensively used as markers for bovine disease and immunological traits. However, none of the BoLA genes in Southeast Asian breeds have been characterized by polymerase chain reaction (PCR)-sequence-based typing (SBT). Therefore, we sequenced exon 2 of the BoLA class II DRB3 gene from 1120 individual cows belonging to the Holstein, Sahiwal, Simbrah, Jersey, Brahman, and Philippine native breeds using PCR-SBT. Several cross-breeds were also examined. BoLA-DRB3 PCR-SBT identified 78 previously reported alleles and five novel alleles. The number of BoLA-DRB3 alleles identified in each breed from the Philippines was higher (71 in Philippine native cattle, 58 in Brahman, 46 in Holstein×Sahiwal, and 57 in Philippine native×Brahman) than that identified in breeds from other countries (e.g., 23 alleles in Japanese Black and 35 in Bolivian Yacumeño cattle). A phylogenetic tree based on the DA distance calculated from the BoLA-DRB3 allele frequency showed that Philippine native cattle from different Philippine islands are closely related, and all of them are closely similar to Philippine Brahman cattle but not to native Japanese and Latin American breeds. Furthermore, the BoLA-DRB3 allele frequency in Philippine native cattle from Luzon Island, located in the Northern Philippines was different from that in cattle from Iloilo, Bohol, and Leyte Islands, which are located in the Southern Philippines. Therefore, we conclude that Philippine native cattle can be divided into two populations, North and South areas. Moreover, a neutrality test revealed that Philippine native cattle from Leyte showed significantly greater genetic diversity, which may be maintained by balancing selection. This study shows that Asian breeds have high levels of BoLA-DRB3 polymorphism. This finding, especially the identification of five novel BoLA-DRB3 alleles, will be helpful for future SBT studies of BoLA-DRB3 alleles in East Asian cattle.

Research of HLA II Class DRB1, DQA1, DQB1 Genetic Markers in Patients with HIV Infection and AIDS

Aims: To find out the most frequent associations of the HLA II class loci DRB1*, DQA1*, DQB1* with the HIV/AIDS infection. Place and Duration of Study: The study took place in The Laboratory of Clinical Immunology and Immunogenetics (LCII) of Riga Stradiņs University (RSU), Riga, Latvia, Riga Eastern Clinical University Hospital, “Infectology Centre of Latvia”, between May 1991 and December 2004. Original Research Article British Journal of Medicine & Medical Research, 4(17): 4482-4500, 2014 4483 Methodology: We analysed the medical documentation of 2500 patients and included 1180 (888 men, 292 women, 185 of them in AIDS phase) HIV infected patients. Genomic DNA was extracted from the blood with phenol-chloroform extraction method. Lowresolution HLA typing for HLADRB1*; DQB1*; DQA1* was performed by polymerise chain reaction (PCR) with amplification with sequence-specific primers (SSP). PCR products were separated on 3% agarose, the amplified bands were visualized, and the HLA-DRB1; DQA1; DQB1 type was deduced. Results: Genetic markers of immunologic alleles upon development of HIV infection – HLA-DRB1*03(17:01); 05(11:01); 07:01; HLA-DQA1*01:01; 02:01; 03:01; 06:01; HLADQB1* 03:02; 05:01; 03:03; 03:04, as well as resistance markers connected with slow development of HIV infection – HLA-DRB1*01:01; 04:01; 06(13:01); HLA-DQA1*01:03; 04:01; 05:01; HLA-DQB1*03:01; 03:03; 04:01-2; 06:01; 06:02-8 are located in different groups of patients. High risk markers in case of HIV infection development belonging to the following groups of alleles: HLA-DRB1*03(17:01), DRB1*05(11:01), DQA1*01:01; 03:01 un DQB1*05:01; 03:02, as well as three-loci haplotypes HLA-DRB1*03(17:01)/ DQB1*05:01/ DQA1*01:01; HLA-DRB1*05(11:01)/DQB1*03:01/ DQA1 *05:01; DRB1*01:01/DQB1*03:02/ DQA1*03:01 and DRB1*01:01/DQB1*05:01/DQA1*01:01 are determined. Resistance to HIV infection development forms in the following groups of alleles: HLA-DRB1*01:01; 06(13:01), HLA-DQB1* 03:01; 06:02-8; HLA-DQA1*01:02; 01:03, as well as in haplotypes HLADRB1*01:01/DQB1*06:02-8/DQA1*01:02;HLADRB1*06(13:01)/DQB1*06:02-8/DQA1*01:02; HLADRB1*01:01/DQB1*03:01/DQA1*01:02; and HLA-DRB1*06(13:01)/DQB1*06:02-8/ DQA1*01:02 in different groups of HIV/AIDS patients. Conclusion: The prevalence of genes DRB1; DQA1; DQB1 and DRB1-DQA-DQB1 combinations in the five groups of HIV infected patients have been established. Comparative analysis was performed also in the group of healthy donors (control group). The role of the main histocompatibility complex has been established, it enables marker functions and that can be used in the additional prognostic diagnostics in case of HIV infection. The obtained results testify that upon the identification of HIV genes it is possible to understand the molecular mechanisms in case of progression of AIDS syndrome complex; this possibly can be beneficial for the determination of the clinical results of infected patients.

A meta-analysis of MHC diversity in Pan troglodytes verus

We performed a meta-analysis of the Major Histocompatibility Complex (MHC) diversity for locus A, B, C, DQB1, DPB1 and DRB1 in Chimpanzees by selecting cohorts of animals for which MHC genotypes were available in publications and by extracting DNA sequences available on the IPD-MHC database. The MHC genes play a key role in the immune responses and are located on the short arm of chromosome 6. In humans, this system, also called HLA, is one of the most polymorphic regions of the genome and is consequently very informative to study peopling history. The MHC region has been subdivided in different classes according to the functional and structural characteristics of the genes. For Class I, chimpanzees possess single A, B and C loci orthologous to the human MHC loci A, B and C, respectively. They also possess an additional class I locus, AL, with low polymorphism, poorly expressed and found only on the haplotypes of 50% of Pan troglodytes individuals and not observed in Pan paniscus. As AL sequences are very similar to chimpanzee and human A sequences, this locus is estimated to have diverged from A more than 20 mya (Adams and Parham, Species-specific evolution of MHC class I genes in the higher primates, Immunol Rev, 2001, 183: 41-64). The Class II region is subdivided into DR, DQ and DP loci each containing A and B genes. The DR locus of catarrhines may display allelic polymorphism, configuration polymorphism and gene-related transcription levels (Doxiadis et al., DR haplotype diversity of the cynomolgus macaque as defined by its transcriptome, Immunogenetics, 2012, 64: 31-37). Different numbers and/or combinations of DRB genes have been defined: human DRB has five configurations with DRB1 displaying abundant allelic variation; chimpanzees have nine configurations with or without DRB1 and moderate allelic variation. Like in humans, the DQ locus of chimpanzees contains two sets of gene pairs: DQA1-DQB1 and DQA2-DQB2. In chimpanzees both DQA1 and DQB1 are polymorphic (Bontrop et al., Comparative genetics of MHC polymorphisms in different primate species: Duplications and deletions, Hum Immunol, 2006, 67: 388-397) with DQA1 lineages shared between humans and chimpanzees. Similarly, the DP locus contains two sets of gene pairs: DPA1-DPB1 and DPA2-DPB2 but the latter are inactive. The region is similar in humans and chimpanzees and is stable (Bontrop et al., 2006). In the present study, we analyze the MHC molecular diversity in Chimpanzees at six different loci based on all data currently available on the IPD-MHC database and we discuss the results in relation to human HLA variation. In addition, we investigate which peculiar evolutionary forces could have acted at different loci given the genetic profile observed in populations. This gives us an original focus on MHC evolution and its mechanisms and helps us to understand the specificities of the human and chimpanzee lineages, respectively.

Decoding multiple sclerosis: an update on genomics and future directions

A wealth of data confirms that genetic variation is an important determinant of multiple sclerosis (MS) risk. Population, family and molecular studies provide strong empirical support for a polygenic model of inheritance, driven primarily by allelic variants relatively common in the general population. The major histocompatibility complex (MHC) in chromosome 6p21.3 represents by far the strongest MS susceptibility locus genome-wide and was unambiguously identified in all studied populations. The primary signal arises from the HLA–DRB1 gene in the Class II segment of the locus, with hierarchical allelic and haplotypic effects. Independent protective signals in the telomeric Class I region of the locus have been described as well. Over the last 6 years, large multicenter DNA collections have thrived and the development of new laboratory and analytical approaches has matured at a remarkable pace, allowing pursuit of comprehensive ‘agnostic’ genome-wide association studies to identify and characterise the non-MHC genetic component of MS. Taken together, the results have provided unambiguous evidence for the association of over 100 non-MHC loci with disease susceptibility. Follow-up experiments refined some of the association signals (IL2RA and CD58), identified gene–gene interactions (HLA–DRB1/EVI5) and revealed mechanistic insights into the functional consequences of the identified gene variants, most notably an increase in the soluble to membrane-bound ratio for IL-7, IL-2 and TNF receptors and a tyrosine–protein kinase 2-mediated immune deviation. These results significantly broaden our understanding of disease pathogenesis and permit, for the first time, modeling an individual’s disease risk within the context of his or her familial history. Progress in identifying additional risk alleles is likely to be rapid in the near future. Although the effect of any given predisposing variant is modest, the possibility exists that multifaceted gene–gene and/or gene–environment interactions could substantially increase the contribution of some variants to the overall genetic risk. In addition, susceptibility genes may be subject to epigenetic modifications, which greatly increase the complexity of MS inheritance. Despite these remarkable advances, the knowledge of MS genetics remains incomplete. For example, a key but unresolved question is whether genetic variants influence disease trajectory. Ongoing efforts to fully characterize the repertoire of genes that predispose to MS and modulate its presentation is discussed. Functional characterization of even a moderate genetic effect on MS pathogenesis by a known gene or group of genes can assist in elucidating fundamental mechanisms of disease expression and yield important therapeutic opportunities.

131-P: THOUSAND GENOMES AND HLA TYPING BY NGS: HIDDEN TREASURES IN PUBLIC SHORT READ DATA

Aim One of the important goals of the 1000 Genomes project was to find common mutation in diverse populations with the help of next generation sequencing. Earlier, the HapMap project with similar goals made it possible not only to map frequent mutations but there are already publications about sequencing based HLA typing using NGS. Methods We are presenting a sufficiently fast algorithm using 1KG Illumina data to obtain HLA types for HLA-A, B, C, DRB1 and DQB1 genes. For validation the results of Sanger Capillary sequencing based HLA typingwas used for over thousand Coriell samples. Results According to our results it is possible to determine the correct HLA types from public 1KG whole-exome Illumina data with 90% or higher concordance if proper quality check measures are applied. We are also presenting the possible causes of mistyping. Conclusions The method opens perspectives for typing other systems like MICA, MICB and KIR.

128-P: EVALUATION OF LONG READ SEQUENCING CHEMISTRY FOR NEXT GENERATION SEQUENCING ON THE PGM PLATFORM

Next generation sequencing (NGS) technologies offer several advantages over current HLA genotyping methods including complete coverage of gene sequences and lower per sample costs due to the massively parallel sequencing of millions of fragments in a single run. Additionally, clonal amplification of template fragments during the template preparation process can enable cis/trans ambiguity resolution. Many common cis/trans ambiguities require read lengths over 300bases. We are developing an HLA typing assay on the Ion Torrent Personal Genome Machine® (PGM) platform and have compared the evolution of sequencing chemistries for this semiconductor sequencing platform. Long range PCR strategies were used to compare different read length reagents and protocols. Over 20 different samples have been re-sequenced for the HLA-A,B,C,DRB1 genes to evaluate the scalability of the platform. Sequence reads covering each gene were analyzed using in-house software tools and assessed for accuracy, read length, and cis/trans ambiguity resolution. The latest protocols provide superior results for all metrics. The most challenging typings were selected to compare sequencing chemistry for cis/trans phasing over exon2 and exon3 of class I genes. The 200bp Ion Torrent reagents show the longest perfect match read of 252 bases and a modal read length of 200 bases. More recent protocols produce long reads over 450 bases with modal read lengths over 350 bases. Phase ambiguity resolution was accomplished with both chemistries but the longer read methods produced more contiguous reads over the cis/trans region. The raw sequence accuracy improved from 96.6% to 97.2% using the updated protocols. The scalability of the PGM platform has been demonstrated by producing over double the sequence output while maintaining a high degree of raw read accuracy as protocols and reagents evolve. The current PGM protocols combined with full gene sequencing offers absolute HLA typing resolution in a single run. Zhao: Life Technologies: Employee. Bialozynski: Life Technologies: Employee. Shi: Life Technologies: Employee. Radick: Life Technologies: Employee. Conradson: Life Technologies: Employee. Dinauer: Life Technologies: Employee.

178-P: A CASE OF AN UNUSUAL DRB1/DRB3/DRB5 RECOMBINATION

Aim Disease association testing was requested on a 46 year old female. Class II HLA typing was performed. The initial results showed a homozygous DRB1∗15 and homozygous DRB3∗03, with DQB1∗05 and DQB1∗06. The common association between DRB1∗15 and DRB5 was absent. Methods Medium resolution was performed using the Luminex SSO Thermofisher method, high resolution SSP Olerup and Thermofisher, and SBT Life Technologies. Results The SSO test was repeated with the same results. Follow-up included high resolution SSP HLA typing with confirmation by SBT techniques. Patient’s typing: HLA DRB1∗15:01, -; DRB5∗Blank, DRB3∗03:01; DQB1∗05:01, ∗06:02. DRB1∗13 was not detected by SSP. DRB5 was not detected by PCR amplification using DRB group specific primers (SeCore sequencing kit, Life Technologies). Using NMDP haplostats, analysis with the full 2011 dataset, there is no hit for haplotypes of DR∗15:01, DRB3∗03:01, DQB1∗06:02 or DQB1∗05:01.[ Table 1 ] Conclusions We were unable to obtain DNA from a first degree relative to check for possible segregation. There was no known hematological malignancy in this patient. We hypothesized that a rare recombination has possibly occurred within the DR-DQ cluster on both paternal and maternal chromosomes which led to deletion of DRB5 commonly associated with DRB∗15 in one chromosome, (previously reported in Schatz et al, Human Immunology, 2005 and in Nesci et al, Tissue Antigens, 1997), and deletion of DRB1 gene that is commonly associated with DRB3 on the second chromosome.

Identification of cattle carrying alleles associated with resistance and susceptibility to the Bovine Leukemia Virus progression by real-time PCR

Previous studies have shown a significant association between polymorphisms of the BoLA DRB3 gene and Bovine Leukemia Virus (BLV) infection profile. The presence of allele ∗1501 has been associated with high proviral load in peripheral blood while allele ∗0902 has been associated with low proviral load. The purpose of this study was to develop allele-specific real-time PCRs to identify cattle carrying alleles associated with resistance (BoLA DRB3∗0902) or susceptibility (BoLA DRB3∗1501) to the BLV progression. Specific primers were designed and differential amplification was carried out by real-time PCR and monitored by SYBR® Green dye in DNA samples from peripheral blood. Conditions were also adjusted for traditional PCR amplification (end point amplification). These methods are rapid, simple and suitable for high throughput screening, and could aid in marker-assisted selection of BLV-resistant and susceptible cattle.

The importance of HLA DRB1 gene allele to clinical features and disability in patients with multiple sclerosis in Lithuania.

BackgroundThe association of HLA DRB1 alleles with susceptibility to multiple sclerosis (MS) has been consistently reported although its effect on the clinical features and disability is still unclear probably due to diversity in ethnicity and geographic location of the studied populations. The aim of the present study was to investigate the influence of HLA DRB1 alleles on the clinical features and disability of the patients with MS in Lithuania.MethodsThis was a prospective study of 120 patients with MS. HLA DRB1 alleles were genotyped using the polymerase chain reaction.ResultsThe first symptoms of MS in patients with HLA DRB1*15 allele manifested at younger age than in those without this allele (28.32 +/− 5.49 yrs vs. 30.94 +/− 8.43 yrs, respectively, p = 0.043). HLA DRB1*08 allele was more prevalent among relapsing-remitting (RR) MS patients than among patients with progressive course of MS (25.0% vs. 8.3%, respectively, chi^2 = 6.000, p = 0.05). MS patients with this allele had lower relapse rate than those without this allele (1.00 +/− 0.97 and 1.44 +/− 0.85, respectively, p = 0.043). Degree of disability during the last visit was lower among the patients with HLA DRB1*08 allele (EDSS score 3.15 +/− 1.95 vs. 4.49 +/− 1.96, p = 0.006), and higher among those with HLA DRB1*15 allele (EDSS score 4.60 +/− 2.10 vs.4.05 +/− 1.94, p = 0.047) compared to patients without these alleles but there were no significant associations between these alleles and the duration of the disease to disability. HLA DRB1*08 allele (OR = 0.18, 95% CI 0,039-0,8, p = 0.029) was demonstradet to be independent factor to take a longer time to reach an EDSS of 6, while HLA DRB1*01 allele (OR = 5.92, 95% CI 1,30-26,8, p = 0.021) was related in a shorter time to reach and EDSS of 6. Patients with HLA DRB1*08 allele had lower IgG index compared to patients without this allele (0.58 +/− 0.17 and 0.73 +/− 0.31, respectively, p = 0.04), and HLA DRB1*15 allele was more often found among MS patients with oligoclonal bands (OCBs) in cerebrospinal fluid than among those without OCBs (OR 2.3, CI 95% 1.017-5.301; p = 0.043).ConclusionsHLA DRB1*15 allele was related with an earlier manifestation of the first MS symptoms, progressive course of the disease and higher degree of disability. HLA DRB1*08 allele was more prevalent among the RR MS patients and was associated with the lower rate of relapse, degree of disability and IgG index.

Association of BoLA DRB3 alleles with variability in immune response among the crossbred cattle vaccinated for foot-and-mouth disease (FMD)

Polymorphism of bovine leukocyte antigen (BoLA) DRB3 gene is being intensively investigated for potential association with economically important diseases of cattle. Accordingly, we investigated the association of DRB3 Exon 2 polymorphism as evidenced by the variation in the binding pockets with variability in immune response to inactivated trivalent (O, A and Asia1) foot and mouth disease virus (FMDV) vaccine in a closed population of crossbred cattle. Antibody titer of ⩾1.8 was set as the cut off value to distinguish the protected (⩾1.8) and unprotected (<1.8) animals. Eleven different alleles of over 3% frequency were detected in the population. We found that DRB3 alleles ∗0201, ∗0801 and ∗1501 always ranked high for protective immune response whereas alleles ∗0701, ∗1103 and ∗1101 consistently ranked low for unprotected immune response for all the three serotypes. Rank correlation of DRB3 alleles among the three serotypes was positive, high in magnitude and statistically significant (P<0.05). Logistic regression analysis revealed that odds of protection from the vaccine were highest for all the three serotypes if allele ∗1501 was present and strengthened the results of allele ranking. Predicted amino acid substitution in the peptide binding pockets revealed that all the important sites had high Wu–Kabat index. Similarly, specific residues in pockets were crucial for immune response to FMD vaccine. There were specific substitutions in un-protected alleles such as absence of acidic amino acids substituted by basic amino acid at β71, presence of non-polar cysteine or basic histidine at β30 and presence of polar tyrosine at β37. From the observations, we hypothesize that the substitutions lead to unique conformational changes in the protein products of the studied alleles that would associate with the protective or unprotective antibody response to FMDV vaccine. The knowledge has potential implications in future selection programs if integrated with the complete BoLA haplotype details and production traits of the herd.

Structural and Dynamical Insights on HLA-DR2 Complexes That Confer Susceptibility to Multiple Sclerosis in Sardinia: A Molecular Dynamics Simulation Study

Sardinia is a major Island in the Mediterranean with a high incidence of multiple sclerosis, a chronic autoimmune inflammatory disease of the central nervous system. Disease susceptibility in Sardinian population has been associated with five alleles of major histocompatibility complex (MHC) class II DRB1 gene. We performed 120 ns of molecular dynamics simulation on one predisposing and one protective alleles, unbound and in complex with the two relevant peptides: Myelin Basic Protein and Epstein Barr Virus derived peptide. In particular we focused on the MHC peptide binding groove dynamics. The predisposing allele was found to form a stable complex with both the peptides, while the protective allele displayed stability only when bound with myelin peptide. The local flexibility of the MHC was probed dividing the binding groove into four compartments covering the well known peptide anchoring pockets. The predisposing allele in the first half cleft exhibits a narrower and more rigid groove conformation in the presence of myelin peptide. The protective allele shows a similar behavior, while in the second half cleft it displays a narrower and more flexible groove conformation in the presence of viral peptide. We further characterized these dynamical differences by evaluating H-bonds, hydrophobic and stacking interaction networks, finding striking similarities with super-type patterns emerging in other autoimmune diseases. The protective allele shows a defined preferential binding to myelin peptide, as confirmed by binding free energy calculations. All together, we believe the presented molecular analysis could help to design experimental assays, supports the molecular mimicry hypothesis and suggests that propensity to multiple sclerosis in Sardinia could be partly linked to distinct peptide-MHC interaction and binding characteristics of the antigen presentation mechanism.

The intellectual disability of trisomy 21: differences in gene expression in a case series of patients with lower and higher IQ.

Trisomy 21 (T21), or Down syndrome (DS), is the most frequent and recognizable cause of intellectual disabilities. The level of disability, as evaluated by the intelligence quotient (IQ) test, varies considerably between patients independent of other factors. To determine the genetic or molecular basis of this difference, a high throughput transcriptomic analysis was performed on twenty T21 patients with high and low IQ, and 10 healthy controls using Digital Gene Expression. More than 90 millions of tags were sequenced in the three libraries. A total of 80 genes of potential interest were selected for the qPCR experiment validation, and three housekeeping genes were used for normalizing purposes. HLA DQA1 and HLA DRB1 were significantly downregulated among the patients with a low IQ, the values found in the healthy controls being intermediate between those noted in the IQ+ and IQ- T21 patients. Interestingly, the intergenic region between these genes contains a binding sequence for the CCCTC-binding factor, or CTCF, and cohesin (a multisubunit complex), both of which are essential for expression of HLA DQA1 and HLA DRB1 and numerous other genes. Our results might lead to the discovery of genes, or genetic markers, that are directly involved in several phenotypes of DS and, eventually, to the identification of potential targets for therapeutic interventions.

Characterization of bovine MHC DRB3 diversity in Latin American Creole cattle breeds

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for diseases and immunological traits. However, none of the highly adapted Latin American Creole breeds have been characterized for BoLA gene polymorphism by high resolution typing methods. In this work, we sequenced exon 2 of the BoLA class II DRB3 gene from 179 cattle (113 Bolivian Yacumeño cattle and 66 Colombian Hartón del Valle cattle breeds) using a polymerase chain reaction sequence-based typing (PCR-SBT) method. We identified 36 previously reported alleles and three novel alleles. Thirty-five (32 reported and three new) and 24 alleles (22 reported and two new) were detected in Yacumeño and Hartón del Valle breeds, respectively. Interestingly, Latin American Creole cattle showed a high degree of gene diversity despite their small population sizes, and 10 alleles including three new alleles were found only in these two Creole breeds. We next compared the degree of genetic variability at the population and sequence levels and the genetic distance in the two breeds with those previously reported in five other breeds: Holstein, Japanese Shorthorn, Japanese Black, Jersey, and Hanwoo. Both Creole breeds presented gene diversity higher than 0.90, a nucleotide diversity higher than 0.07, and mean number of pairwise differences higher than 19, indicating that Creole cattle had similar genetic diversity at BoLA-DRB3 to the other breeds. A neutrality test showed that the high degree of genetic variability may be maintained by balancing selection. The FST index and the exact G test showed significant differences across all cattle populations (FST=0.0478; p<0.001). Results from the principal components analysis and the phylogenetic tree showed that Yacumeño and Hartón del Valle breeds were closely related to each other. Collectively, our results suggest that the high level of genetic diversity could be explained by the multiple origins of the Creole germplasm (European, African and Indicus), and this diversity might be maintained by balancing selection.

Associations of human leukocyte antigen-G with resistance and susceptibility to HIV-1 infection in the Pumwani sex worker cohort

To determine the association between human leukocyte antigens (HLA)-G genotypes and resistance or susceptibility to HIV-1. A group of sex workers in Pumwani, Kenya can be epidemiologically defined as resistant to HIV-1 infection despite frequent exposure and provide an example of natural protective immunity. HLA class I and II molecules have been shown to be associated with resistance/susceptibility to infection in this cohort. HLA-G is a nonclassical class I allele that is primarily involved in mucosal and inflammatory response, which is of interest in HIV-1 resistance. In this study, we used a sequence-based typing method to genotype HLA-G for 667 women enrolled in this cohort and examined the influence of HLA-G genotypes on resistance or susceptibility to HIV-1 infection. The G*01 : 01:01 genotype was significantly enriched in the HIV-1-resistant women [P = 0.002, Odds ratio: 2.11, 95% confidence interval (CI): 0.259-0.976], whereas the G*01 : 04:04 genotype was significantly associated with susceptibility to HIV-1 infection (P = 0.039, OR:0.502, 95% CI:0.259-0.976). Kaplan-Meier survival analysis correlated with these results. G*01 : 01:01 genotype was associated with significantly lower rate of seroconversion (P = 0.001). Whereas, G*01 : 04:04 genotype was significantly associated with an increased rate of seroconversion (P = 0.013). The associations of these HLA-G alleles are independent of other HLA class I and II alleles identified in this population. Our study showed that specific HLA-G alleles are associated with resistance or susceptibility to HIV-1 acquisition in this high-risk population. Further studies are needed to understand its functional significance in HIV-1 transmission.

NRAMP1, VDR, HLA-DRB1, and HLA-DQB1 Gene Polymorphisms in Susceptibility to Tuberculosis among the Chinese Kazakh Population: A Case-Control Study

Background. To explore the potential role of natural-resistance-associated macrophage protein 1 (NRAMP1) gene, vitamin D receptor (VDR) gene, (human leukocyte antigen, (HLA-DRB1) HLA) -DRB1 gene, and HLA-DQB1 gene polymorphisms in susceptibility to tuberculosis (TB) in the Chinese Kazakh population. Methods. A case-control study was performed on the Chinese Kazak population. Genetic polymorphisms of NRAMP1 gene (3′UTR) and VDR gene (TaqI and FokI) were analysed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing analysis in TB patients and healthy controls. Genetic polymorphisms of HLA-DRB1 gene and HLA-DQB1 gene in the two groups were detected with polymerase chain reaction-sequence-specific primers (PCR-SSPs) technique and sequencing analysis. Results. There was statistically significant difference in the 3′UTR polymorphism between the TB patients and healthy controls in the Chinese Kazak population (P = 0.002; OR = 1.859; 95% CI = 1.182–2.926). Significant difference was observed in the FokI polymorphism between the TB patients and healthy controls (P = 0.001; OR = 1.530; 95% CI = 1.007–2.325). It does not disclose any significant association between the disease and TaqI (P > 0.05). Alleles HLA-DRB1∗04 and HLA-DQB1∗0201 occurred more frequently in patients than in controls (P = 0.011 and 0.002; OR = 1.889 and 1.802; 95% CI = 1.153–3.095 and 1.230–2.639, resp.). Conclusions. Polymorphisms in the NRAMP1 gene, VDR gene, HLA-DRB1 gene, and HLA-DQB1 gene are statistically associated with susceptibility to TB in the Chinese Kazakh population.