Human Leukocyte Antigen Analysis Research Articles

THU671 Genes Encoding Adaptive Immune Receptor Repertoire Might Be Associated With The Risk Of Graves’ Disease And Antithyroid Drug-induced Agranulocytosis

Abstract Disclosure: C. Huang: None. S. Yang: None. S. Lai: None. H. Chen: None. P. Chen: None. Graves' disease (GD) is an autoimmune disorder characterized by the immune system attacking the thyroid gland cells, leading to hyperthyroidism. One serious adverse effect that can occur in GD patients receiving antithyroid drugs, such as thionamides, is antithyroid drug-induced agranulocytosis (TiA), which affects 0.1% to 0.4% of patients. While previous research has shown that specific human leukocyte antigen (HLA) genes/alleles are associated with GD or TiA, the role of the genes encoding adaptive immune receptor repertoire (gAIRR), including the B cell receptor and T cell receptor genes, in GD or TiA has not been explored. This study aims to investigate whether gAIRR has a genetic association with GD or TiA. We enrolled 128 GD patients, 19 TiA patients, and 104 controls, and conducted gAIRR genotyping using gAIRR Suite, a probe capture-based targeted sequencing and analysis platform. Our results identified several gAIRR alleles that were more prevalent in GD cases than in controls, including IGLV1-41*02N (a novel allele with the sequence closest to IGLV1-41*02, 20.3% in GD patients vs. 7.7% in controls, p=0.008), IGHV1-69*04N (12.5% vs. 3.8%, p=0.02), TRGV4*01 (46.9% vs. 31.7%, p=0.022), and several other alleles (such as TRBV6-6*03, IGKV1/OR22-5*01N, TRVB12-2*01N, TRAV8-4*01N, IGHV3-71*02, IGHV2-70*04N, etc.) We also found several gAIRR alleles that were less prevalent in GD cases than in controls, including IGLV5-45*03N, IGLV2-14*04, IGLV(V)-66*01, IGLV3-13*01N, TRBV20/OR9-2*02N, TRBD2*01, TRAV6*02, among others. Regarding the TiA cohort (compared to GD patients without TiA), some gAIRR alleles showed frequencies difference, but did not reach statistical significance. Overall, our findings provide supporting evidence that gAIRR genes/alleles might be associated with the development of GD or TiA. Future studies with larger sample sizes are necessary. Furthermore, a joint analysis of HLA and gAIRR genotypes might provide valuable insight into the pathogenesis of GD and TiA. Presentation: Thursday, June 15, 2023

B-253 Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing

Abstract Background High-resolution human leukocyte antigen (HLA) typing is a crucial process in transplant matching. Sequencing-based typing (SBT) has been the gold standard method used in HLA-based clinical applications. Recently, next-generation sequencing (NGS) has been adopted in clinical laboratories for HLA typing. NGS HLA typing assays cover whole or near-whole target HLA genes, allowing the detection of genetic variants outside of key exons. Multiplex amplicon-based NGS is an efficient high-resolution HLA typing method with minimal ambiguity and a high throughput. Here, we report the performance and validation of NGS for HLA typing and compare the results obtained using NGS with those of SBT to identify any discrepancies between the 2 methods. Methods A total of 237 samples from 2019 to July 2022 were collected from Chang Gung Memorial Hospital, including 42 samples previously subjected to SBT sequencing for 4-digit HLA analysis. NGS was performed for 5 whole genes (HLA-A/B/C/DQA1/DPA1) and 3 near-whole genes (HLA-DRB1/DQB1/DPB1; excluding exon 1 and part of intron 1) by using the AllType NGS kit (One Lambda), Illumina MiSeq platform, and Type Steam Visual NGS analysis software (One Lambda). Results Our validation process comprised 34 NGS runs and 237 samples. In total, 42 samples were analyzed with an overall accuracy and precision of 100% and 100% reproducibility. The overall ambiguity rate was 0% in HLA-A/B/C compared with 56.9% for SBT. Two discrepant alleles of HLA-A and DRB1 were noted in NGS and Sanger sequencing. In accordance with additional SBT exon analyses, one of the results was corrected according to the NGS results. Conclusion NGS HLA typing is a high-throughput approach with high accuracy and precision and a low ambiguity and is promising for use in clinical HLA laboratories.

Definition and Clinical Evaluation for Trimethoprim-Sulfamethoxazole Severe Acute Respiratory Failure.

Trimethoprim-sulfamethoxazole (TMP-SMX)-associated severe acute respiratory distress syndrome (ARDS) has gone underrecognized. We propose the first disease definition and clinical evaluation for a novel adverse drug reaction (ADR) based on a series of recently identified rare cases of life-threatening ADRs. A retrospective study was conducted. All medical records were evaluated. Available pathology samples were sent to Massachusetts General for clinical consultation. Blood samples from surviving patients were obtained and human leukocyte antigen (HLA) analysis was performed by the Children's Mercy Hospital Genomic Center and Vanderbilt University Medical Center. U.S. ICUs, 1996-2021. Nineteen young patients (10-37) were identified. Patients were previously healthy, with no preexisting pulmonary disease, no other cause for respiratory failure, and no chronic history of smoking/vaping. None. Through our retrospective analysis, we analyzed clinical characteristics associated with TMP-SMX. Pathology samples were reviewed, and HLA analysis was performed on available samples by the study team or as standard of care at treatment hospitals in some cases. In 19 critically ill patients, we identified a pattern of severe respiratory failure requiring ICU admission, mechanical ventilation, and frequent extracorporeal membrane oxygenation use. We describe the first three-part clinical diagnosis and evaluation strategy: 1) Clinical definition: Unexplained severe respiratory failure in a patient receiving greater than or equal to 6 days of TMP-SMX at treatment dose (not prophylaxis). TMP-SMX ARDS is a diagnosis of exclusion. 2) Genetic association: One hundred percent of currently available TMP-SMX respiratory failure patient genomic data, ( n = 11) have been carriers of both HLA-B*07:02 and HLA-C*07:02 alleles. HLA allele evaluation could be considered in patients with suspected TMP-SMX respiratory failure. 3) Lung pathology: A unique pulmonary pathologic pattern of lung injury termed diffuse alveolar injury with delayed epithelialization has been observed in these cases. In suspected cases, surgical lung biopsy early in the clinical course could be considered. TMP-SMX is a commonly prescribed antibiotic. However, we find it imperative to share this relatively rare but life-threatening condition with clinicians as the mortality rate approaches 40%.

Race/ethnicity-stratified fine-mapping of the MHC locus reveals genetic variants associated with late-onset asthma.

Introduction: Asthma is a chronic disease of the airways that impairs normal breathing. The etiology of asthma is complex and involves multiple factors, including the environment and genetics, especially the distinct genetic architecture associated with ancestry. Compared to early-onset asthma, little is known about genetic predisposition to late-onset asthma. We investigated the race/ethnicity-specific relationship among genetic variants within the major histocompatibility complex (MHC) region and late-onset asthma in a North Carolina-based multiracial cohort of adults. Methods: We stratified all analyses by self-reported race (i.e., White and Black) and adjusted all regression models for age, sex, and ancestry. We conducted association tests within the MHC region and performed fine-mapping analyses conditioned on the race/ethnicity-specific lead variant using whole-genome sequencing (WGS) data. We applied computational methods to infer human leukocyte antigen (HLA) alleles and residues at amino acid positions. We replicated findings in the UK Biobank. Results: The lead signals, rs9265901 on the 5' end of HLA-B, rs55888430 on HLA-DOB, and rs117953947 on HCG17, were significantly associated with late-onset asthma in all, White, and Black participants, respectively (OR = 1.73, 95%CI: 1.31 to 2.14, p = 3.62 × 10-5; OR = 3.05, 95%CI: 1.86 to 4.98, p = 8.85 × 10-6; OR = 19.5, 95%CI: 4.37 to 87.2, p = 9.97 × 10-5, respectively). For the HLA analysis, HLA-B*40:02 and HLA-DRB1*04:05, HLA-B*40:02, HLA-C*04:01, and HLA-DRB1*04:05, and HLA-DRB1*03:01 and HLA-DQB1 were significantly associated with late-onset asthma in all, White, and Black participants. Conclusion: Multiple genetic variants within the MHC region were significantly associated with late-onset asthma, and the associations were significantly different by race/ethnicity group.

Expanding Our Knowledge of the Immunogenetic Characteristics of Anti-LGI1 Encephalitis—A Study of an Israeli Cohort Suggests Additional Significant HLA Associations With DQ Alleles

Objective Exploring the clinical characteristics and HLA associations of patients with anti-leucine–rich glioma-inactivated 1 encephalitis (LGI1E) from a large single center in Israel Background Anti-LGI1E is one of the most commonly diagnosed antibody-associated encephalitic syndromes in adults. Recent studies of various populations reveal significant associations with specific Human Leukocyte Antigen (HLA) genes. We examined the clinical characteristics and HLA associations of a cohort of Israeli patients. Design/Methods Seventeen consecutive anti-LGI1E patients diagnosed at Tel Aviv Sourasky Medical Center between the years 2011-2018 were included. HLA typing was performed using NGS methodology at the tissue typing laboratory of Sheba Medical Center and compared to data from the "Ezer Mizion" Bone Marrow Donor Registry, containing over 1,000,000 samples. Results Our cohort displayed a male predominance and median age of onset in the 7th decade, as previously reported. All patients responded to immunotherapy, though residual damage was not uncommon (23% with MRS >1). HLA analysis revealed overexpression of DRB1*07:01 (OR 13, CI 0.6 p < 1.e-10) and DRB1*04:02 (OR 12, CI-0.6 p < 1.e-10), as previously reported, as well as of the DQ alleles DQB1*02:02 (OR 12, CI 0.6 p < 1.e-10), DQB1*03:03 (OR 27, CI 0.9 p < 1.e-10), previously attributed to linkage disequilibrium (LD) with the mentioned DR alleles. An additional allele overexpressed among our patients was the DQB1*03:02 allele (OR 12, CI 0.6 p < 1.e-10), which appeared in complete LD with DRB1*04:02. Linkage disequilibrium analysis performed on patients and controls suggests these DR-DQ associations are unique to anti-LGI1E patients. In silico predictions performed for the overexpressed DQ alleles reveal them to be strong binders of LGI1 derived peptides, and suggest a correlation between peptide binding sites of paired DR-DQ alleles. Conclusions Our findings shed additional light on the complex role of immunogenetics in the pathogenesis of anti-LGI1E, implying the possible relevance of certain DQ alleles as well as DR-DQ interactions.

HLA analysis of immune checkpoint inhibitor-induced and idiopathic isolated ACTH deficiency.

Isolated adrenocorticotropic hormone deficiency is a rare disease; however, since immune check point inhibitors (ICIs) have become widely used, many more cases have been reported. In this study, we compared the human leukocyte antigen (HLA) signatures between ICI-induced isolated adrenocorticotropic hormone deficiency (IAD) and idiopathic IAD (IIAD). Clinical features and HLA frequencies were compared among 13 patients with ICI-induced IAD, 8 patients with IIAD, and healthy controls. HLA frequencies of healthy controls were adopted from a HLA database of Japanese population. Age and body mass index were higher, while the rate of weight loss was lower, in patients with ICI-induced IAD than in those with IIAD. No HLA alleles had a significantly higher frequency in patients with ICI-induced IAD than in healthy controls, whereas the frequencies of HLA-DRB1*09:01, HLA-DQA1*03:02, and DQB1*03:03 were significantly higher in patients with IIAD than in healthy controls. ICI-induced IAD and IIAD were different in terms of HLA frequencies. There were no specific HLAs related to ICI-induced IAD, whereas several HLAs in strong linkage disequilibrium were associated with IIAD. This might suggest that the two diseases have different pathological mechanisms. HLAs unique to IIAD may be helpful in predicting its pathophysiology.

HLA3D: an integrated structure-based computational toolkit for immunotherapy.

MotivationThe human major histocompatibility complex (MHC), also known as human leukocyte antigen (HLA), plays an important role in the adaptive immune system by presenting non-self-peptides to T cell receptors. The MHC region has been shown to be associated with a variety of diseases, including autoimmune diseases, organ transplantation and tumours. However, structural analytic tools of HLA are still sparse compared to the number of identified HLA alleles, which hinders the disclosure of its pathogenic mechanism.ResultTo provide an integrative analysis of HLA, we first collected 1296 amino acid sequences, 256 protein data bank structures, 120 000 frequency data of HLA alleles in different populations, 73 000 publications and 39 000 disease-associated single nucleotide polymorphism sites, as well as 212 modelled HLA heterodimer structures. Then, we put forward two new strategies for building up a toolkit for transplantation and tumour immunotherapy, designing risk alignment pipeline and antigenic peptide prediction pipeline by integrating different resources and bioinformatic tools. By integrating 100 000 calculated HLA conformation difference and online tools, risk alignment pipeline provides users with the functions of structural alignment, sequence alignment, residue visualization and risk report generation of mismatched HLA molecules. For tumour antigen prediction, we first predicted 370 000 immunogenic peptides based on the affinity between peptides and MHC to generate the neoantigen catalogue for 11 common tumours. We then designed an antigenic peptide prediction pipeline to provide the functions of mutation prediction, peptide prediction, immunogenicity assessment and docking simulation. We also present a case study of hepatitis B virus mutations associated with liver cancer that demonstrates the high legitimacy of our antigenic peptide prediction process. HLA3D, including different HLA analytic tools and the prediction pipelines, is available at http://www.hla3d.cn/.

Genetic Determinants of Antibody-Mediated Immune Responses to Infectious Diseases Agents: A Genome-Wide and HLA Association Study.

BackgroundInfectious diseases are causally related to a large array of noncommunicable diseases (NCDs). Identifying genetic determinants of infections and antibody-mediated immune responses may shed light on this relationship and provide therapeutic targets for drug and vaccine development.MethodsWe used the UK biobank cohort of up to 10 000 serological measurements of infectious diseases and genome-wide genotyping. We used data on 13 pathogens to define 46 phenotypes: 15 seropositivity case–control phenotypes and 31 quantitative antibody measurement phenotypes. For each of these, we performed genome-wide association studies (GWAS) using the fastGWA linear mixed model package and human leukocyte antigen (HLA) classical allele and amino acid residue associations analyses using Lasso regression for variable selection.ResultsWe included a total of 8735 individuals for case–control phenotypes, and an average (range) of 4286 (276–8555) samples per quantitative analysis. Fourteen of the GWAS yielded a genome-wide significant (P < 5 ×10-8) locus at the major histocompatibility complex (MHC) on chromosome 6. Outside the MHC, we found a total of 60 loci, multiple associated with Epstein-Barr virus (EBV)–related NCDs (eg, RASA3, MED12L, and IRF4). FUT2 was also identified as an important gene for polyomaviridae. HLA analysis highlighted the importance of DRB1*09:01, DQB1*02:01, DQA1*01:02, and DQA1*03:01 in EBV serologies and of DRB1*15:01 in polyomaviridae.ConclusionsWe have identified multiple genetic variants associated with antibody immune response to 13 infections, many of which are biologically plausible therapeutic or vaccine targets. This may help prioritize future research and drug development.

Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing.

The human immune system relies on highly complex and diverse transcripts and the proteins they encode. These include transcripts encoding human leukocyte antigen (HLA) receptors as well as B cell and T cell receptors (BCR and TCR). Determining which alleles an individual possesses for each HLA gene (high-resolution HLA typing) is essential to establish donor–recipient compatibility in organ and bone marrow transplantations. In turn, the repertoires of millions of unique BCR and TCR transcripts in each individual carry a vast amount of health-relevant information. Both short-read RNA-seq-based HLA typing and BCR/TCR repertoire sequencing (AIRR-seq) currently rely on our incomplete knowledge of the genetic diversity at HLA and BCR/TCR loci. Here, we generated over 10,000,000 full-length cDNA sequences at a median accuracy of 97.9% using our nanopore sequencing-based Rolling Circle Amplification to Concatemeric Consensus (R2C2) protocol. We used this data set to (1) show that deep and accurate full-length cDNA sequencing can be used to provide isoform-level transcriptome analysis for more than 9000 loci, (2) generate accurate sequences of HLA alleles, and (3) extract detailed AIRR data for the analysis of the adaptive immune system. The HLA and AIRR analysis approaches we introduce here are untargeted and therefore do not require prior knowledge of the composition or genetic diversity of HLA and BCR/TCR loci.

Evaluating seroprevalence to circumsporozoite protein to estimate exposure to three species of Plasmodium in the Brazilian Amazon

BackgroundBrazil has seen a great decline in malaria and the country is moving towards elimination. However, for eventual elimination, the control program needs efficient tools in order to monitor malaria exposure and transmission. In this study, we aimed to evaluate whether seroprevalence to the circumsporozoite protein (CSP) is a good tool for monitoring the exposure to and/or evaluating the burden and distribution of Plasmodium species in the Brazilian Amazon.MethodsCross-sectional surveys were conducted in a rural area of Porto Velho, Rondônia state. Parasite infection was detected by microscopy and polymerase chain reaction. Antibodies to the sporozoite CSP repeats of Plasmodium vivax, P. falciparum, and P. malariae (PvCS, PfCS, and PmCS) were detected using the enzyme-linked immunosorbent assay technique. Human leukocyte antigen (HLA)-DRB1 and DQB1 genes were typed using Luminex® xMAP® technology.ResultsThe prevalence of immunoglobulin G against P. vivax CSP peptide (62%) was higher than P. falciparum (49%) and P. malariae (46%) CSP peptide. Most of the studied individuals had antibodies to at least one of the three peptides (72%), 34% had antibodies to all three peptides and 28% were non-responders. Although the majority of the population was not infected at the time of the survey, 74.3% of parasite-negative individuals had antibodies to at least one of the CSPs. Importantly, among individuals carrying the haplotypes DRB1*04~DQB1*03, there was a significantly higher frequency of PfCS responders, and DRB1*16~DQB1*03 haplotype for PvCS and PfCS responders. In contrast, HLA-DRB1*01 and HLA-DQB1*05 allelic groups were associated with a lack of antibodies to P. vivax and P. falciparum CSP repeats, and the haplotype DRB1*01~DQB1*05 was also associated with non-responders, including non-responders to P. malariae.ConclusionsOur results show that in low transmission settings, naturally acquired antibody responses against the CSP repeats of P. vivax, P. falciparum, and P. malariae in a single cross-sectional study may not represent a valuable marker for monitoring recent malaria exposure, especially in an area with a high prevalence of P. vivax. Furthermore, HLA class II molecules play an important role in antibody response and require further study with a larger sample size. It will be of interest to consider HLA analysis when using serosurveillance to monitor malaria exposure among genetically diverse populations.

PyHLA: tests for the association between HLA alleles and diseases

BackgroundRecently, several tools have been designed for human leukocyte antigen (HLA) typing using single nucleotide polymorphism (SNP) array and next-generation sequencing (NGS) data. These tools provide high-throughput and cost-effective approaches for identifying HLA types. Therefore, tools for downstream association analysis are highly desirable. Although several tools have been designed for multi-allelic marker association analysis, they were designed only for microsatellite markers and do not scale well with increasing data volumes, or they were designed for large-scale data but provided a limited number of tests.ResultsWe have developed a Python package called PyHLA, which implements several methods for HLA association analysis, to fill the gap. PyHLA is a tailor-made, easy to use, and flexible tool designed specifically for the association analysis of the HLA types imputed from genome-wide genotyping and NGS data. PyHLA provides functions for association analysis, zygosity tests, and interaction tests between HLA alleles and diseases. Monte Carlo permutation and several methods for multiple testing corrections have also been implemented.ConclusionsPyHLA provides a convenient and powerful tool for HLA analysis. Existing methods have been integrated and desired methods have been added in PyHLA. Furthermore, PyHLA is applicable to small and large sample sizes and can finish the analysis in a timely manner on a personal computer with different platforms. PyHLA is implemented in Python. PyHLA is a free, open source software distributed under the GPLv2 license. The source code, tutorial, and examples are available at https://github.com/felixfan/PyHLA.

Relation between HLA genes, human skin volatiles and attractiveness of humans to malaria mosquitoes

Chemical cues are considered to be the most important cues for mosquitoes to find their hosts and humans can be ranked for attractiveness to mosquitoes based on the chemical cues they emit. Human leukocyte antigen (HLA) genes are considered to be involved in the regulation of human body odor and may therefore affect human attractiveness to mosquitoes, and hence, affect the force of malaria transmission. In the present study the correlations between HLA profiles, human skin volatiles and human attractiveness to the malaria mosquito Anopheles gambiae Giles sensu stricto were examined.Skin emanations of 48 volunteers were collected by rubbing a foot over glass beads. Previously the attractiveness of these emanations to An. gambiae was determined. In this study, the chemical composition of these emanations was determined by gas chromatography–mass spectroscopy (GC–MS) and blood samples of all volunteers were taken for HLA analysis. Hierarchical cluster analysis (HCA), partial least squares discriminant analysis (PLS-DA), Fisher’s exact test and random forest regression were used to test for correlations between individuals classified as either highly or poorly attractive to mosquitoes and their HLA profile and volatile composition.HLA profiling suggests that people carrying HLA gene Cw∗07 are more attractive to mosquitoes. GC–MS revealed that limonene, 2-phenylethanol and 2-ethyl-1-hexanol were associated with individuals that were poorly attractive to An.gambiae and lactic acid, 2-methylbutanoic acid, tetradecanoic acid and octanal with individuals that were highly attractive. Such compounds offer potential for disruption of mosquito behavior in malaria intervention programs.

Cancerous HLA class I expression and regulatory T cell infiltration in gastric cancer.

Since antitumor immune reactions between tumors and intratumoral immunocytes have been verified in several human tumors, immunological therapeutic strategies must be considered to obtain the proper efficacy of tumor shrinkage under these conditions. Human leukocyte antigen (HLA) class I expression in cancer cells and degree of infiltration of regulatory T cells (Tregs) in the stroma have been regarded as important markers of antitumor immune reactions in the context of independent immunological mechanisms. In the current study, we investigated HLA class I expression and Treg cells infiltration in gastric cancer and discussed the clinical implications of this combinatory analysis in gastric cancer. A total of 141 gastric cancer patients who received R0 gastrectomy at Kagoshima University Hospital were studied. Immunohistochemically, in 141 gastric cancer patients, HLA class I expression and Treg cell infiltration in cancerous tissue were evaluated using HLA class I (EMR8-5) and forkhead box p3 (FOXP3) monoclonal antibodies. The correlation between clinical factors and tumor-infiltrating Treg cells was analyzed. HLA class I expression was positively associated with depth of tumor invasion (P<0.05). Infiltration of Foxp3-positive cells did not correlate with any clinicopathological markers. HLA class I expression had no association with Treg cell infiltration (r=0.04). A better postoperative outcome was associated with fewer numbers of Treg infiltration (P=0.034). A combination of HLA and Treg analysis may lead to a more accurate prediction of postoperative outcome (P=0.02). Two different antitumor immunological markers, Treg infiltration and HLA class I expression, affected clinicopathological factors in gastric cancer by different mechanisms. Thus, an immunological combination of HLA class I expression and Treg cell infiltration may more accurately predict postoperative outcome. Immunological balance needs to be restored after evaluation of each immunological deficit in gastric cancer.

Sweet’s syndrome in a neonate with non-B54 types of human leukocyte antigen

Sweet's syndrome (acute febrile neutrophilic dermatosis) is characterized by fever, polymorphonuclear leukocytosis of blood, painful plaques on the limbs, face and neck, and histologically a dense dermal infiltration with mature neutrophils. Sweet's syndrome is often a complication of hematologic malignant disease or drug-induced sensitivity reactions and has a significant susceptibility correlated with certain human leukocyte antigen (HLA). A 5-week-old Japanese girl with Sweet's syndrome confirmed by skin biopsy was successfully treated and HLA analysis was performed. The patient was one of the youngest patients reported with Sweet's syndrome, suggesting the importance of the genetic background. Although the HLA types of the patient did not have B54, which was reported as a significant susceptibility correlation, structural analysis of the patient's HLAs suggested a similar possible motif for the bound peptides. Studies on the HLA bound peptides and HLA structural analysis for patients with Sweet's syndrome would be valuable for understanding the molecular mechanism of the pathogenesis.

HLA-DRB1*0102 is associated with TINU syndrome and bilateral, sudden-onset anterior uveitis but not with interstitial nephritis alone

BackgroundTubulointerstitial nephritis and uveitis (TINU) syndrome is a rare form of uveitis. Previously, the authors had demonstrated a strong association of human leukocyte antigen (HLA) DRB1*0102 with TINU. Here, the...

Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient

BackgroundNeural stem cells are currently being investigated as potential therapies for neurodegenerative diseases, stroke, and trauma. However, concerns have been raised over the safety of this experimental therapeutic approach, including, for example, whether there is the potential for tumors to develop from transplanted stem cells.Methods and FindingsA boy with ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human fetal neural stem cells. Four years after the first treatment he was diagnosed with a multifocal brain tumor. The biopsied tumor was diagnosed as a glioneuronal neoplasm. We compared the tumor cells and the patient's peripheral blood cells by fluorescent in situ hybridization using X and Y chromosome probes, by PCR for the amelogenin gene X- and Y-specific alleles, by MassArray for the ATM patient specific mutation and for several SNPs, by PCR for polymorphic microsatellites, and by human leukocyte antigen (HLA) typing. Molecular and cytogenetic studies showed that the tumor was of nonhost origin suggesting it was derived from the transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor is derived from at least two donors.ConclusionsThis is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor-derived brain tumor. Further work is urgently needed to assess the safety of these therapies.

The analysis of the entire HLA, partial non‐HLA and HPV for Chinese women with cervical cancer

Human leukocyte antigen (HLA) alleles have been associated with an increased or decreased risk of developing cervical cancer through infection with oncogenic forms of the human papillomavirus (HPV). The aim of this study was to analyze the interactions between HPV and HLA, particularly of the entire HLA and partial non-HLA regions, in invasive cervical cancers. The results showed the distribution of HPV genotypes among patients with cervical cancer in this group: the overall prevalence of HPV DNA was 91.3% (274/300). A total of 16 HPV genotypes, HPV16, 18, 58, 33, 81, 31, 68, 39, 67, 6, 11, 26, 52, 62, 66, and 70, were detected in patients with cervical cancer. Among the cervical cancer specimens, 68.7% were positive for HPV16. Genotyping was performed with 112 microsatellite markers spanning the entire HLA region and partial region of chromosome 6q15-16.3. D6S2766 and D6S2764, which are located near to the region containing the HLA-DPB genes, were negatively related with cervical cancer (OR for the D6S2766-195 allele carriers = 0.50), and positively related with cervical cancer (OR for the D6S2764-209 allele carriers = 2.44), respectively. Significant association was found for D6S1043 (allele 132) with HPV16-positive cervical cancer (OR = 4.52). There was an increased risk for carriers of alleles 112 and 132 at the D6S1043 locus, in particular for homozygotes. The present findings indicate that the antigenic presentation of HPV-derived peptides and non-HLA regions may be involved in the pathogenesis of HPV-associated cervical cancer.

SKDM human leukocyte antigen (HLA) tool: A comprehensive HLA and disease associations analysis software

The immensely polymorphic and gene-rich landscape of the major histocompatibility complex on chromosome 6 necessitates a thorough and consistent investigation of its constituting elements. The human leukocyte antigens (HLAs) are an example of such polymorphic elements, implicated in many immune-based diseases. So far, analyses of HLA molecules in the context of diseases have been ad hoc, frequently incomplete, and extremely cumbersome. SKDM provides a comprehensive and automated workflow for detecting and dissecting HLA associations in diseases. We created a Java application to consistently perform our proposed method of analysis of HLAs in case-control datasets. The SKDM HLA tool can test for HLA allele differences between two populations and, by retrieving amino acid sequences, evaluates each polymorphic amino acid residue or a pocket of amino acids as an independent variant. Once primary associations are identified, the program examines zygosity and tests for strongest association, interaction, and linkage disequilibrium among amino acid epitopes of the same HLA molecule or between HLA isotypes. A summary of the analysis is output in plain language. The software and a user's manual are freely available at http://sourceforge.net/projects/skdm.

HLA-Cw8 primarily associated with hypersensitivity to nevirapine

We read with interest the report by Littera et al. [1] about human leukocyte antigen (HLA)-dependent hypersensitivity to nevirapine in Sardinian HIV patients. The authors state that high levels of genetic homogeneity and linkage disequilibrium make the Sardinian population particularly suitable for genetic association studies, and they observed a statistically significant association between a nevirapine-hypersensitivity reaction and the HLA-Cw*0802-B*1402 haplotype. In the Sardinian population, however, HLA-Cw*0802 and B*1402 are in such strong linkage disequilibrium that they could not establish which one of these two alleles is primarily associated with the hypersensitivity reaction to nevirapine. Considering that HLA-B14(65) can not be found in the Japanese population, it might be helpful to analyse the patients in our clinic for a determination of the primarily associated HLA allele [2–5]. In our outpatient clinic, a total of 326 HIV-1-infected individuals (309 were Japanese) had given written informed consent for HLA analysis and the study of its association with HIV-1 disease progression and drug-induced adverse events. High resolution typing of the alleles at the HLA-A, HLA-B, HLA-Cw, HLA-DRB1, and HLA-DQB1 loci had been performed by polymerase chain reaction amplification using sequence-specific primers in all of them. The allele frequency of HLA-Cw8 and HLA-B14 was 13 and 0%, respectively, which is compatible with previous reports of HLA frequency in the Japanese population [2–5]. Forty-three of the analysed patients were on nevirapine treatment or had a history of nevirapine treatment. One of them died of malignant lymphoma 4 weeks after the introduction of nevirapine-containing treatment. In another patient, nevirapine-containing treatment was terminated 17 days after initiation because of granulocytopenia probably induced by co-administered zidovudine. These two patients were excluded from further analysis and the remaining 41 patients were divided into two groups; a nevirapine-hypersensitive group and a nevirapine-tolerant group (Table 1). The nevirapine-hypersensitive group included 11 patients who experienced extensive skin rash (accompanied by fever > 38°C in three) and one patient with chronic hepatitis C who developed nevirapine-induced hepatotoxicity with aspartate aminotransferase/alanine aminotransferase values three times above the baseline. The nevirapine-tolerant group included 29 others who had been treated with nevirapine for a period of more than 6 months and did not develop any hypersensitive reaction [1]. There were no significant differences in age, sex, ethnicity, weight, HIV-1 viral load, CD4 and CD8 cell counts between the two groups (Fisher's exact test for dichotomous variables, Student's t-test for continuous variables). The frequency of HLA-Cw8-positive patients in the nevirapine-hypersensitive group was 42%, which was significantly higher than those of the nevirapine-tolerant group (10%) and the general Japanese population (9–14%) [2–5]. In the nevirapine-hypersensitive group, four patients including one who developed hepatotoxicity had HLA-Cw*0801 and one had HLA-Cw*0803. In the nevirapine-tolerant group, three patients had HLA-Cw*0801. HLA-Cw*0802 was not identified in the patients we analysed. There was no significant difference in the frequency of the other HLA alleles between the two groups.Table 1: Demographics and immunological variables in the nevirapine-hypersensitive group and nevirapine-tolerant group.Considering our data together with that of Littera et al. [1], HLA-Cw8 antigen rather than specific alleles of other genes linked with HLA-Cw*0801 or HLA-Cw*0802 may be primarily associated with a nevirapine-hypersensitivity reaction. Nevirapine or nevirapine metabolite coupled with HLA-Cw8 antigen may be expressed on the cell surface and may induce hypersensitive reactions including skin rash and hepatotoxicities. We totally agree with Littera et al. [1] that a careful choice of drugs in susceptible patients identified by HLA typing would considerably reduce the risk of severe and sometimes life-threatening hypersensitive reactions.

A High Degree of HLA Disparity Arises From Limited Allelic Diversity: Analysis of 1775 Unrelated Bone Marrow Transplant Donor-Recipient Pairs

The allelic diversity and associated human leukocyte antigen (HLA) disparity of 1775 bone marrow recipients and their unrelated donors, matched for six of six (1361/1775,77%), five of six (397/1775, 22%), or four of six (17/1775, 1%) HLA-A, -B, -DR antigens, were retrospectively evaluated. The comprehensive HLA analysis included the class I (A, B, C) and II (DRB1, DQA1, DQB1, DPA1, DPB1) loci. Most (>66%) of the predominantly Caucasian study population carried one or two of five to seven common alleles at each HLA locus. In spite of this limited diversity, 29% of the six of six antigen-matched transplants carried allele mismatches at HLA-A, -B, and/or -DRB1, and 92% carried at least one allele mismatch at one of the eight HLA loci tested. Of the 968 HLA-A,-B,-DRB1 allele-matched pairs, 89% carried mismatches at other HLA loci, predominantly at DP loci. The substantially greater than expected HLA allelic disparity between donor and recipient suggests extensive haplotypic diversity and underscores the importance of enhancing approaches to mitigate the deleterious effect of HLA mismatches.