Resequencing of four novel alleles with nanopore technology.

Long nanopore reads allow phasing of heterozygous positions but show limitations in sequencing of homopolymers.

Anti-HLA alloantibodies of the IgA isotype in re-transplant candidates part II: Correlation with graft survival.

We reported previously on the widespread occurrence of anti-HLA alloantibodies of the IgA isotype (anti-HLA IgA) in the sera of solid-organ re-transplantation (re-tx) candidates (Arnold et al., ). Specifically focussing on kidney re-tx patients, we now extended our earlier findings by examining the impact of the presence and donor specificity of anti-HLA IgA on graft survival. We observed frequent concurrence of anti-HLA IgA and anti-HLA IgG in 27% of our multicenter collective of 694 kidney re-tx patients. This subgroup displayed significantly reduced graft survival as evidenced by the median time to first dialysis after transplantation (TTD 77 months) compared to patients carrying either anti-HLA IgG or IgA (TTD 102 and 94 months, respectively). In addition, donor specificity of anti-HLA IgA had a significant negative impact on graft survival (TTD 74 months) in our study. Taken together, our data strongly indicate that presence of anti-HLA IgA, in particular in conjunction with anti-HLA-IgG, in sera of kidney re-tx patients is associated with negative transplantation outcome.

Influence of photodynamic therapy on peripheral immune cell populations and cytokine concentrations in head and neck cancer.

BACKGROUND: Photodynamic therapy (PDT) represents a palliative treatment option for a selected group of patients with head and neck squamous cell carcinoma (HNSCC). PDT induces a local inflammatory reaction with the potential to initiate antitumor immune responses. However, the systemic impact on peripheral immune cells has not been described so far. METHODS: HNSCC patients (n=9) were treated with PDT in a palliative setting. All patients had previously undergone several oncologic treatment regimens. Blood samples were taken before, during and after PDT. Age-matched healthy donors served as control group (NC, n=15). The frequency and absolute number of T- and B-lymphocytes, CD4+CD39+ regulatory T-cells (Treg) and NK-cells were measured by 10-color flow cytometry. Serum concentrations of T cell related cytokine panel, including HMGB1, IL-6, IL-10 and perforin were measured by bead array and ELISA. RESULTS: In heavily pretreated HNSCC patients, the number and frequency of Treg and NK-cells were increased as compared to NC. PDT induced a further increase of the frequency of Treg and NK-cells in the peripheral blood. Additionally, the serum concentrations of HMGB1, IL-6 and IL-10 showed a significant elevation after treatment with simultaneously decreased perforin levels. CONCLUSION: Although PDT is a local treatment regimen, a systemic inflammatory response with altered peripheral immune cell populations and cytokine concentrations is visible. The increased Treg and NK cell numbers after PDT support the hypothesis that PDT may successfully be combined with NK cell or T cell activating immune checkpoint modulators in HNSCC patients to improve HNSCC specific immunity.

HLA typing by next-generation sequencing - getting closer to reality.

Next generation sequencing (NGS) denotes novel sequencing technologies that enable the generation of a large number of clonal sequences in a single sequencing run. NGS was initially introduced for whole genome sequencing and for quantitation of viral variants or genetic mutations in tumor tissues; more recently, the potential for high resolution HLA typing and high throughput analyses has been explored. It became clear that the complexity of the HLA system implicates new challenges, especially for bioinformatics. From an economical point of view, NGS is becoming increasingly attractive for HLA typing laboratories currently relying on Sanger based sequencing. Realizing the full potential of NGS will require the development of specifically adapted typing strategies and software algorithms. In the present review, three laboratories that were among the first to perform HLA-typing using different NGS platforms, the Roche 454, the Illumina Miseq and the Ion Torrent system, respectively, give an overview of these applications and point out advantages and limitations.

16(th) IHIW: anti-HLA alloantibodies of the of IgA isotype in re-transplant candidates.

In this multicentre study, sera from 803 retransplant candidates, including 775 kidney transplant recipients, were analysed with regard to the presence and specificity of anti-HLA alloantibodies of the IgA isotype using a modified microsphere-based platform. Of the kidney recipients, nearly one-third (n = 237, 31%) had IgA alloantibodies. Mostly, these antibodies were found in sera that also harboured IgG alloantibodies that could be found in a total of 572 (74%) of patients. Interestingly, IgA anti-HLA antibodies were preferentially targeting HLA class I antigens in contrast to those of the IgG isotype, which targeted mostly both HLA class I and II antigens. Donor specificity of the IgA alloantibodies could be established for over half of the 237 patients with IgA alloantibodies (n = 124, 52%). A further 58 patients had specificities against HLA-C or HLA-DP, for which no information regarding donor typing was available. In summary, these data showed in a large cohort of retransplant candidates that IgA alloantibodies occur in about one-third of patients, about half of these antibodies being donor specific.

TNFA promoter alleles--frequencies and linkage with classical HLA genes in a South German Caucasian population.

The tumor necrosis factor alpha (TNFA) promoter region exhibits several polymorphisms, which have been hypothesized to influence gene expression, thereby associating positively or negatively with inflammatory conditions. Many studies have focused on single nucleotide polymorphisms (SNPs) taking not into account additive or inverse effects between different SNPs. We typed 1,021 healthy Caucasian volunteer stem cell donors for their TNFA promoter as well as their HLA-A,-C,-B,-DRB1 loci. Using statistical methods, we reconstructed TNFA promoter alleles and analyzed their frequency and linkage with HLA genes. We show that the number of TNFA promoter alleles frequent enough to be analyzed in clinical studies is limited and that a strong linkage with classical HLA genes is present, especially for the extended HLA-haplotype HLA-A*01:01/HLA-C*07:01/HLA-B*08:01/TNFA promoter allele 3/HLA*DRB1*03:01. Taking into account SNP frequency information, it is possible to quite accurately deduce TNFA promoter alleles by generic Sanger sequencing, obviating the need for elaborating allele-specific sequencing. This information may enable investigators to consider the complete TNFA regulatory region in a phase-separated manner in contrast to previous approaches examining only one or few isolated SNPs.

Cytokine genetic profile in Whipple's disease.

Whipple's disease (WD) is a very rare chronic systemic condition characterised by a Th2/T regulatory (Treg) dysregulated immune response versus Tropheryma whipplei, a bacterium widely diffuse in the environment. To investigate whether this Th2/Treg polarised response has a genetic background, we investigated the Th1, Th2, Th17 and Treg cytokine genetic profile of 133 patients with WD. Thanks to the European Consortium on WD (QLG1-CT-2002-01049), the polymorphism of 13 cytokine genes was analysed in 111 German and 22 Italian patients using the polymerase chain reaction with sequence-specific primers (PCR-SSP) technique. The frequencies of the genotypes, haplotypes and functional phenotypes were compared with those obtained in 201 German and 140 Italian controls. Clinical heterogeneity was also considered. Functionally, WD patients may be considered as low producers of TGF-ß1, having an increased frequency of the genotype TGF-ß1+869C/C,+915C/C [12.3 % vs. 3.81 %, odds ratio (OR) = 4.131, p = 0.0002] and high secretors of IL-4, carrying the genotype IL-4-590T/T (5.34 % vs. 1.17 %, OR = 5.09, p = 0.0096). No significant association was found between cytokine polymorphism and clinical variability. Analogously to the recent cellular findings of a Th2/Treg polarised response, we showed that the cytokine genetic profile of WD patients is skewed toward a Th2 and Treg response. This was similar in both German and Italian populations. However, the significant deviations versus the controls are poorer than that expected on the basis of these recent cellular findings.

Influence of cytokine gene polymorphisms on graft rejection in Turkish patients with renal transplants from living related donors.

BACKGROUND: Certain cytokine gene polymorphisms (CGPs) have been shown to be associated with renal transplant rejection episodes or graft outcomes. We sought to evaluate the relationships between gene polymorphisms and acute rejection episodes (RG, n = 19) versus stable graft function (NRG, n = 71) in transplant recipients compared with healthy control subjects (HCG, n = 150). The follow-up time period was 18 months. Using polymerase chain reaction sequence-specific primers with the Heidelberg kit we genotyped 22 single nucleotide polymorphisms distributed across 13 cytokine and cytokine receptor genes. RESULTS: Interleukin (IL)-2 TT/GT haplotype was found in 36.8% of RG patients and 6.7% of HCG but not among the NRG (P < .0001; .0007). The IL-2 GG/TT haplotype was observed among 13 NRG and nine HCG patients (P = .007); the IL-2 GG/GG haplotype, 18.7% HCG and 4.2% NRG patients (P = .0033); and the IL-2 TT/TT haplotype, five NRG and eight HCG patients, but none of the RG cohort (P > .05). The transforming-growth factor-beta 1 CG/CC haplotype was noted in 15 NRG (21.1%) and four HCG but no RG patients (P < .0001). The IL-2 +166 GT genotype was detected in 36.8% of RG, 8.5% of NRG, and 14.7% of HCG patients (P = .005, .0244). The IL-2 -330 GG genotype was demonstrated in 32 healthy controls and three nonrejection transplant patients (P = .0007). Significant differences were concluded between NRG and HCG for IL-6 565 AG, IL-1beta -511 TT and +3962 CC/CT/TT genotypes. DISCUSSION: We observed significant differences among the frequencies of IL-2 gene polymorphisms among RG and NRG subjects, which agreed with previous clinical, but not in vitro studies.

Influence of cytokine gene polymorphisms on graft rejection in Turkish patients with renal transplants from living related donors.

INTRODUCTION: Certain cytokine gene polymorphisms (CGPs) have been shown to be associated with renal transplant rejection or graft outcomes. We sought to evaluate the relationship between gene polymorphisms in patients with acute rejection episodes (rejection group, RG, n = 19) versus those with stable graft function (nonrejection group, NRG, n = 71) in comparison with healthy control subjects (HCG, n = 150). The follow-up time period was 18 months. In the present study, 22 single nucleotide polymorphisms distributed across 13 cytokine and cytokine receptor genes were genotyped by polymerase chain reaction sequence-specific primers (PCR-SSP) using the Heidelberg kit. RESULTS: The interleukin-2 (IL-2) TT/GT haplotype was observed among 36.8% of patients in the RG and 6.7% of those in the HCG but not in any NRG patient (P < .0001; .0007). The IL-2 GG/TT haplotype was observed among 13 NRG and 9 HCG patients (P = .007). The IL-2 GG/GG haplotype was noted in 18.7% of HCG and 4.2% of NRG patients (P = .0033) and the IL-2 TT/TT haplotype in 5 and 8 patients of NRG and HCG, respectively, but not in any RG patient (P > .05). The transforming growth factor beta1 CG/CC haplotype was noted in 15 NRG (21.1%) and 4 HCG patients but not any RG (P < .0001). The IL-2 + 166 GT genotype was detected in 36.8% of RG, 8.5% of NRG, and 14.7% of HCG patients (P = .005, .0244). The IL-2 -330 GG genotype was demonstrated in 32 healthy controls and 3 nonrejection transplant patients (P = .0007). Significant differences were found between NRG and HCG for IL-6 565 AG, IL-1 beta -511 TT and +3962 CC/CT/TT genotypes. DISCUSSION: We observed significant differences among the frequencies of IL-2 gene polymorphisms between the RG and the NRG, which were consistent with previous clinical but not in vitro studies.

HLA-E polymorphisms in hematopoietic stem cell transplantation.

Human leukocyte antigen (HLA)-E is an inhibitory ligand of natural killer cells and γ/δ T-cells. Differential expression of HLA-E alleles on the cell surface has been reported to influence outcome of hematopoietic stem cell transplantation (HSCT). We performed HLA-E genotyping in 116 HSCT patients and their HLA-matched unrelated donors. The impact of HLA-E genotypes on patient's overall survival (OS), disease free survival (DFS), cumulative incidences for relapse, transplant-related mortality (TRM) and acute graft vs host disease (aGvHD) was assessed. Neither univariate nor multivariate analysis showed any influence of HLA-E polymorphisms on the investigated endpoints of HSCT in our cohort. We could not confirm any of the previous observations in our cohort and consider it unlikely that HLA-E polymorphisms affect outcome of HSCT.

One amino acid change located in the conserved region of the alpha 1 domain specifies the novel HLA-C*07:147 allele.

The novel HLA-C allele HLA-C*07:147 contains one nucleotide substitution in exon 2 leading to an amino acid change in the alpha 1 domain from phenylalanine to leucine.

Sequence-based typing of major histocompatibility complex class I chain-related gene A alleles by use of exons 2-5 information.

The polymorphic MICA (major histocompatibility complex class I chain-related gene A) (Gene ID: 100507436) gene products are a ligand of the activating natural killer cell receptor, NKG2D. Their clinical importance spans from solid organ transplantation to bone marrow transplantation and disease susceptibility. Typing of MICA genes by sequencing is hampered by an exon 5 short tandem repeat, the definition of which is critical for the final allelic and functional assignment. We present a novel sequencing approach, which uses group-specific (7T/8T) exon 5 polymerase chain reactions (PCRs) and facilitates hemizygous exon 5 MICA-PCR in approximately 70% of the tested individuals. With this method we typed the International Histocompatibility Workshop Group MICA reference panel (40 cell lines) as well as 110 healthy South German blood donors. All ambiguities, with the exception of MICA*008:01/008:04 (synonymous substitution in exon 1) and MICA*009:01/049 (nonsynonymous substitution in exon 6), could be resolved with our method. Analysis of Hardy-Weinberg equilibrium for our cohort showed no significant difference between expected and observed frequencies of MICA alleles (P = 0.6142). The three most frequent alleles in our blood donor cohort were MICA*008:01/008:04 (40.5%), MICA*002:01 (13.2%), and MICA*009:01/049 (8.6%). The 7T polymorphism was observed in 67.7% and the 8T polymorphism in 32.3% of our blood donor cohort. Individuals (24.5%) tested were homozygous. The approach described in this paper is suitable for accurate sequencing of large sample numbers, including direct readout of exon 5 sequences. It is compatible with laboratory automation and commercial human leukocyte antigen analysis software tools. It may therefore be applied in large clinical trials.

A new polymorphic position in exon 2 shows the novel allele HLA-DPB1*123:01.

Introduction of a novel human leukocyte antigen-DPB1 allele, DPB1*123:01, which featured one nucleotide mismatch in comparison with DPB1*02:01:02.

The frequency of DRB1*1454 in South German Caucasians.

Human leukocyte antigen (HLA)-DRB1*1454 differs from HLA-DRB1*1401 in only one position in exon 3. Before description of this polymorphism both alleles were routinely typed as HLA-DRB1*1401. This prompted our study on relative frequency of these alleles. We determined relative frequencies of DRB1*1454 and DRB1*1401 by sequence-based tying (SBT) in 106 samples and found DRB1*1454 in 87.9% and DRB1*1401 in 12.1% of previously DRB1*1401/54 ambiguous tested individuals. Population frequencies were estimated using data from the local bone marrow donor database. Phenotype and genotype frequency of DRB1*1454 and DRB1*1401 were 5.592%, 0.2828, and 0.773%, 0.00391, respectively. The corresponding figures for DRB1*1404 were 0.238% and 0.00119. Other DRB1*14 alleles were very rare. The most frequent haplotype was DRB1*1454-DRB3*0202-DQB1*0503. Although DRB1*1454 is almost exclusively associated with DRB3*0202, DRB1*1401 is linked with either DRB3*0201 or DRB3*0202. HLA-DRB1*1454 is the most common DRB1*14 allele among German Caucasians and should be considered as a preferred allele over DRB1*1401.

Prediction of duration and success rate of unrelated hematopoietic stem cell donor searches based on the patient's HLA-DRB1 allele and DRB1-DQB1 haplotype frequencies.

Rapid identification of a matched unrelated donor is essential for patients in need of hematopoietic SCT. We carried out a retrospective evaluation of 549 unrelated donor searches (UDSs), which were completed in 2005 for 23 German transplant centers. On the basis of the patient's HLA-DRB1 allele and DRB1-DQB1 haplotype frequencies, UDSs were divided into four groups with different search success probability predictions. For 90.5% of the patients, an acceptable HLA-matched, and for 61.6% an HLA-A-B-Cw-DRB1-DQB1-identical (10/10 matched) unrelated donor was found. The median search duration was 22 days. In the groups with high (n = 318), medium (n = 157), low (n = 56) and very low (n = 18) UDS success probability, an acceptable donor was found for 99.1, 86.6, 75.0 and 22.2% of the patients, and a 10/10-matched donor was found for 78.3, 49.7, 17.9 and 4.5% of the patients, respectively. The median search duration was 20, 27, 45 and 477 days in the groups with high, medium, low and very low probability, respectively. The search success rate and duration can be predicted on the basis of the patient's HLA-DRB1 allele and HLA-DRB1-DQB1 haplotype frequencies. An unrelated donor can be found for most of the patients, even if the indication for transplantation is urgent.

HLA-Cw*0740, a new allele mistyped by generic sequencing and identified by allelic separation.

Human leukocyte antigen (HLA)-Cw*0740 differs from HLA-Cw*070101 by one nucleotide substitution at codon 73 (GCT>ACT).

Distribution of 22 cytokine gene polymorphisms in the healthy Czech population.

Cytokine gene polymorphisms (CGP) have been implicated in the pathogenesis of immune-mediated diseases including transplant complications via their effect on cytokine production and regulation. This study aimed to determine population frequencies of selected cytokine single nucleotide polymorphisms in the healthy Czech population and compare them with the data from other selected European populations. CGP were genotyped by polymerase chain reaction with sequence-specific primers (PCR-SSP) using the Heidelberg kit in 120 unrelated Czech healthy individuals. Chi-squared analysis was used to test for a deviation from Hardy-Weinberg equilibrium. Allelic and genotype frequencies and carriage rates were determined for 22 CGP located within 13 cytokine genes in total. The frequencies observed in this study were similar to those available from the other two geographically close Central European centres, but they differed for several CGP from the data reported in south European populations. The data on the distribution of 22 CGP in the healthy Czech population reported here may be utilized to investigate possible associations of CGP with diseases or transplantation outcome.

A new PCR-SSP typing method for six single-nucleotide polymorphisms impairing the blood-clotting cascade as well as T-cell stimulation.

Single-nucleotide polymorphisms (SNPs) within the genes of factor V (FV) (G1691A; exon 10), prothrombin (FII) (G20210A; 3'untranslated - region) and methylenetetrahydrofolate reductase (MTHFR) (C677T; exon 4) are associated with hypercoagulability, and systematic screening of individuals being at higher risk of thrombosis has been suggested. SNPs in the 2q33 region within the genes of CD28 (+17T/C; intron 3) and CTLA4 (-318C/T; promoter and +49A/G; exon 1) are likely to affect T-cell proliferation and antigen presentation signaling, which may lead to altered sensitivity of allograft or self-tissue recognition and affect the incidence of autoimmune diseases. We developed primers that allow specific amplification of these six SNPs at test conditions identical with those used for HLA typing with the CTS PCR-SSP reagents. One hundred ninety-six healthy German Caucasian individuals were tested for the six SNPs. The genotype frequencies for all SNPs were in Hardy-Weinberg equilibrium. There was no significant difference in the distribution of genotypes when compared to other published studies in which these SNPs were tested. The described PCR-SSP method can be used to screen large numbers of patients for these SNPs.

Description and characterization of two new HLA alleles, B*4051 and DRB1*1364, identified by sequence-based typing.

Human leukocyte antigen (HLA)-B and HLA-DRB1 typing in two female individuals revealed reaction patterns that did not correspond to any known HLA-B specificity and appeared to identify a very rare HLA-DRB1 allele, respectively. Sequence-based analysis of these samples revealed two new HLA alleles, one similar to B*4023 and the other to DRB1*1308. The new HLA-B allele, which was assigned the name HLA-B*4051, could have been generated by a double crossing over recombination between B*4001 and B*1401 or 1402, whereas DRB1*1364, the new DRB1 allele, could have been generated either by a double crossing over recombination between DRB1*1308 and DRB1*1201, 1202, or 1203 or by two independent crossing over events between DRB1*1401, DRB1*1201, 1202, or 1203 and DRB1*1301.

Identification of two new HLA alleles: B*3546* and B*5611*. How reliable are the published HLA-B intron 2 sequences?

Polymerase chain reaction-sequence-specific primer (PCR-SSP) typing for human leukocyte antigen (HLA)-B in a male 25-year-old Caucasian individual of Iranian origin and in a 42-year-old German Caucasian bone marrow donor revealed reaction patterns that did not agree with any known HLA specificity, thus suggesting in both cases the existence of a novel allele. Sequence-based typing (SBT) after allelic separation revealed the sequences of the new alleles HLA-B*5611 and B*3546. The sequence patterns of both new alleles might have been generated as the results of double crossing over, possibly over several generations. During the analysis of the HLA-B*3546 intron 2 sequence for possible crossing over points, a base insert, an additional G after position 700, was found. This insert was analyzed using SBT and PCR-SSP and was found to be present not only in all samples carrying B*35, but also in all HLA-B specificities tested. It appears that all known HLA-B alleles may contain a G insert at position 700 of intron 2, and that the published intron 2 sequence alignments of the HLA-B locus may contain errors at this position.