Atopic dermatitis (AD) is a common immune-medicated skin disease. Previous studies have explored the relationship between Human Leukocyte Antigen (HLA) allelic variation and AD with conflicting ...results. The aim was to examine HLA Class I genetic variation, specifically peptide binding groove variation, and associations with AD. A case-control study was designed to evaluate HLA class I allelic variation and binding pocket polymorphisms, using next generation sequencing on 464 subjects with AD and 388 without AD. Logistic regression was used to evaluate associations with AD by estimating odds ratios (95% confidence intervals). Significant associations were noted with susceptibility to AD (B*53:01) and protection from AD (A*01:01, A*02:01, B*07:02 and C*07:02). Evaluation of polymorphic residues in Class I binding pockets revealed six amino acid residues conferring protection against AD: A9F (HLA-A, position 9, phenylalanine) pocket B/C, A97I pocket C/E, A152V pocket E, A156R pocket D/E, B163E pocket A and C116S pocket F. These findings demonstrate that specific HLA class I components are associated with susceptibility or protection from AD. Individual amino acid residues are relevant to protection from AD and set the foundation for evaluating potential HLA Class I molecules in complex with peptides/antigens that may initiate or interfere with T-cell responses.
This study presents performance specifications of an in‐house developed human leukocyte antigen (HLA) typing assay using next‐generation sequencing (NGS) on the Illumina MiSeq platform. A total of ...253 samples, previously characterized for HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1 were included in this study, which were typed at high‐resolution using a combination of Sanger sequencing, sequence‐specific primer (SSP) and sequence‐specific oligonucleotide probe (SSOP) technologies and recorded at the two‐field level. Samples were selected with alleles that cover a high percentage of HLA specificities in each of five different race/ethnic groups: European, African‐American, Asian Pacific Islander, Hispanic and Native American. Sequencing data were analyzed by two software programs, Omixon's target and GenDx's NGSengine. A number of metrics including allele balance, sensitivity, specificity, precision, accuracy and remaining ambiguity were assessed. Data analyzed by the two software systems are shown independently. The majority of alleles were identical in the exonic sequences (third field) with both programs for HLA‐A, ‐B, ‐C and ‐DQB1 in 97.7% of allele determinations. Among the remaining discrepant genotype calls at least one of the analysis programs agreed with the reference typing. Upon additional manual analysis 100% of the 2530 alleles were concordant with the reference HLA genotypes; the remaining ambiguities did not exceed 0.8%. The results demonstrate the feasibility and significant benefit of HLA typing by NGS as this technology is highly accurate, eliminates virtually all ambiguities, provides complete sequencing information for the length of the HLA gene and forms the basis for utilizing a single methodology for HLA typing in the immunogenetics labs.
Summary
Human leucocyte antigens (HLA) typing has been a challenge due to extreme polymorphism of the HLA genes and limitations of the current technologies and protocols used for their ...characterization. Recently, next‐generation sequencing techniques have been shown to be a well‐suited technology for the complete characterization of the HLA genes. However, a comprehensive assessment of the different platforms for HLA typing, describing the limitations and advantages of each of them, has not been presented. We have compared the Ion Torrent Personal Genome Machine (PGM) and Illumina MiSeq, currently the two most frequently used platforms for diagnostic applications, for a number of metrics including total output, quality score per position across the reads and error rates after alignment which can all affect the accuracy of HLA genotyping. For this purpose, we have used one homozygous and three heterozygous well‐characterized samples, at HLA‐A, HLA‐B, HLA‐C, HLA‐DRB1 and HLA‐DQB1. The total output of bases produced by the MiSeq was higher, and they have higher quality scores and a lower overall error rate than the PGM. The MiSeq also has a higher fidelity when sequencing through homopolymer regions up to 9 bp in length. The need to set phase between distant polymorphic sites was more readily achieved with MiSeq using paired‐end sequencing of fragments that are longer than those obtained with PGM. Additionally, we have assessed the workflows of the different platforms for complexity of sample preparation, sequencer operation and turnaround time. The effects of data quality and quantity can impact the genotyping results; having an adequate amount of good quality data to analyse will be imperative for confident HLA genotyping. The overall turnaround time can be very comparable between the two platforms; however, the complexity of sample preparation is higher with PGM, while the actual sequencing time is longer with MiSeq.
Summary
Many genes related to innate and adaptive immunity reside within the major histocompatibility complex (MHC) and have been associated with a multitude of complex, immune‐related disorders. ...Despite years of genetic study, this region has seen few causative determinants discovered for immune‐mediated diseases. Reported associations have been curated in various databases including the Genetic Association Database, NCBI database of clinically relevant variants (ClinVar) and the Human Gene Mutation Database and together capture genetic associations and annotated pathogenic loci within the MHC and across the genome for a variety of complex, immune‐mediated diseases. A review of these three distinct databases reveals disparate annotations between associated genes and pathogenic loci, alluding to the polygenic, multifactorial nature of immune‐mediated diseases and the pleiotropic character of genes within the MHC. The technical limitations and inherent biases imposed by current approaches and technologies in studying the MHC create a strong case for the need to perform targeted deep sequencing of the MHC and other immunologically relevant loci in order to fully elucidate and study the causative elements of complex immune‐mediated diseases.
Human T-cell-mediated autoimmune diseases are genetically linked to particular alleles of MHC class II genes. Susceptibility to pemphigus vulgaris (PV), an autoimmune disease of the skin, is linked ...to a rare subtype of HLA-DR4 (DRB1*0402, 1 of 22 known DR4 subtypes). The PV-linked DR4 subtype differs from a rheumatoid arthritis-associated DR4 subtype (DRB1*0404) only at three residues (DRβ 67, 70, and 71). The disease is caused by autoantibodies against desmoglein 3 (DG), and T cells are thought to trigger the autoantibody production against this keratinocyte adhesion molecule. Based on the DRB1*0402 binding motif, seven candidate peptides of the DG autoantigen were identified. T cells from four PV patients with active disease responded to one of these DG peptides (residues 190-204); two patients also responded to DG-(206-220). T-cell clones specific for DG-(190-204) secreted high levels of interleukins 4 and 10, indicating that they may be important in triggering the production of DG-specific autoantibodies. The DG-(190-204) peptide was presented by the disease-linked DRB1*0402 molecule but not by other DR4 subtypes. Site-directed mutagenesis of DRB1*0402 demonstrated that selective presentation of DG-(190-204), which carries a positive charge at the P4 position, was due to the negatively charged residues of the P4 pocket (DRβ 70 and 71). DRβ 71 has a negative charge in DRB1*0402 but a positive charge in other DR4 subtypes, including the DR4 subtypes linked to rheumatoid arthritis. The charge of the P4 pocket in the DR4 peptide binding site therefore appears to be a critical determinant of MHC-linked susceptibility to PV and rheumatoid arthritis.
Guillain-Barré syndrome (GBS), an acute, immune-mediated paralytic disorder affecting the peripheral nervous system, is the most common cause of acute flaccid paralysis in the post-polio era. GBS is ...classified into several subtypes based on clinical and pathologic criteria, with acute inflammatory demyelinating polyneuropathy (AIDP) and acute motor axonal neuropathy (AMAN) being the most common forms observed. To better understand the pathogenesis of GBS and host susceptibility to developing the disease, the distribution of HLA class II Ags along with the seroreactivity to Campylobacter jejuni were investigated in a population of GBS patients from northern China. Using DNA-based typing methods, 47 patients with AMAN, 25 patients with AIDP, and 97 healthy controls were studied for the distribution of class II alleles. We found that the DQ beta RLD(55-57)/ED(70-71) and DR beta E(9)V(11)H(13) epitopes were associated with susceptibility to AIDP (p = 0.009 and p = 0.004, respectively), and the DQ beta RPD(55-57) epitope was associated with protection (p = 0.05) from AIDP. These DQ beta/DR beta positional residues are a part of pockets 4 (DQ beta 70, 71, DR beta 13), 6 (DR beta 11), and 9 (DQ beta 56, 57, DR beta 9); have been demonstrated to be important in peptide binding and T cell recognition; and are associated with other diseases that have a pathoimmunological basis. Class II HLA associations were not identified with AMAN, suggesting a different immunological mechanism of disease induction in the two forms of GBS. These findings provide immunogenetic evidence for differentiating the two disease entities (AMAN and AIDP) and focuses our attention on particular DR beta/DQ beta residues that may be instrumental in understanding the pathophysiology of AIDP.
Summary
We have recently described the presence of perivascular CD3+ CD45RO+ T cells infiltrating the brains of children with AIDS. To determine whether these infiltrates contain oligoclonal ...populations of T cells, we amplified by PCR β‐chain T‐cell receptor (TCR) transcripts from autopsy brains of four paediatric patients with AIDS. The amplified transcripts were cloned and sequenced. Sequence analysis of the β‐chain TCR transcripts from all four patients revealed multiple identical copies of TCR β‐chain transcripts, suggesting the presence of oligoclonal populations of T‐cells. These TCR transcripts were novel. The presence of oligoclonal populations of T cells in the brains of these four paediatric patients with AIDS suggests that these T cells have undergone antigen‐driven proliferation and clonal expansion very likely in situ, in the brains of these AIDS patients, in response to viral or self‐antigens. Although the specificity of the clonally expanded β‐chain TCR transcripts remains to be elucidated, none of the β‐chain TCR transcripts identified in this study were identical to those specific for HIV‐1 antigens that are currently reported in the GENBANK/EMBL databases. Certain common CDR3 motifs were observed in brain‐infiltrating T cells within and between certain patients. Large proportions (24 of 61; 39%) of β‐chain TCR clones from one patient (NP95‐73) and 2 of 27 (7%) of another patient (NP95‐184‐O) exhibited substantial CDR3 homology to myelin basic protein (MBP)‐specific TCR derived from normal donors or TCR expressed in the brain of patients with multiple sclerosis (MS) or with viral encephalitis. These two patients (NP95‐73 and NP95‐184‐O) also shared HLA class II with the normal donors and the MS patients who expressed these homologous TCR. Pathologic examination at autopsy of the brains revealed the presence of myelin pallor only in patient NP95‐73. T‐cell clones identified in the brain of patients NP95‐73 and NP95‐184‐O may recognize MBP or another CNS self antigen and this recognition may be restricted by either DRB1*15 or DQB1*0602 specificities.
This study combines two independent domains of science, the high throughput DNA sequencing capabilities of Genomics and complexity theory from Physics, to assess the information encoded by the ...different genomic segments of exonic, intronic and intergenic regions of the Major Histocompatibility Complex (MHC) and identify possible interactive relationships. The dynamic and non-extensive statistical characteristics of two well characterized MHC sequences from the homozygous cell lines, PGF and COX, in addition to two other genomic regions of comparable size, used as controls, have been studied using the reconstructed phase space theorem and the non-extensive statistical theory of Tsallis. The results reveal similar non-linear dynamical behavior as far as complexity and self-organization features. In particular, the low-dimensional deterministic nonlinear chaotic and non-extensive statistical character of the DNA sequences was verified with strong multifractal characteristics and long-range correlations. The nonlinear indices repeatedly verified that MHC sequences, whether exonic, intronic or intergenic include varying levels of information and reveal an interaction of the genes with intergenic regions, whereby the lower the number of genes in a region, the less the complexity and information content of the intergenic region. Finally we showed the significance of the intergenic region in the production of the DNA dynamics. The findings reveal interesting content information in all three genomic elements and interactive relationships of the genes with the intergenic regions. The results most likely are relevant to the whole genome and not only to the MHC. These findings are consistent with the ENCODE project, which has now established that the non-coding regions of the genome remain to be of relevance, as they are functionally important and play a significant role in the regulation of expression of genes and coordination of the many biological processes of the cell.
•The estimation of Tsallis q-triplet were found to deviate from unity in both cases indicating non-Gaussian Boltzmann–Gibbs statistics.•Our results indicate the usability of Tsallis theory for the discrimination of DNA regions.•We find that the information of the number of genes/exonic regions is interacting with the information level of the intergenic regions.•MHC sequences, whether exonic, intronic or intergenic include varying levels of information. These regions most likely interact with each other.•We showed the Long-range correlations and the memory character of the DNA sequence.
We analyze 4 Mb sequences of the Major Histocompatibility Complex (MHC), which is a DNA segment on chromosome 6 with high gene density, controlling many immunological functions and associated with ...many diseases. The analysis is based on modern theoretical and mathematical tools of complexity theory, such as nonlinear time series analysis and Tsallis non-extensive statistics. The results revealed that the DNA complexity and self-organization can be related to fractional dynamical nonlinear processes with low-dimensional deterministic chaotic and non-extensive statistical character, which generate the DNA sequences under the extremization of Tsallis q-entropy principle. While it still remains an open question as to whether the DNA walk is a fractional Brownian motion (FBM), a static anomalous diffusion process or a non-Gaussian dynamical fractional anomalous diffusion process, the results of this study testify for the latter, providing also a possible explanation for the previously observed long-range power law correlations of nucleotides, as well as the long-range correlation properties of coding and non-coding sequences present in DNA sequences.
•We uncovered the DNA complexity and self-organization in DNA sequences of MHC.•We showed the extremization of Tsallis q-entropy.•We showed the strong nonlinearity of the DNA dynamics.•The DNA dynamics is a non-Gaussian dynamical fractional anomalous diffusion process.•We showed the long-range correlations and the memory character of the DNA sequence.
Guillain-Barré syndrome in northern China occurs in two forms: acute inflammatory demyelinating polyneuropathy (AIDP) and acute motor axonal neuropathy (AMAN). AMAN and AIDP have an immunologic ...basis, and some cases are associated with preceding Campylobacter jejuni infection. The distribution of allelic forms of the histocompatibility genes HLA-DPB1, DQB1, DRB1, DRB3, DRB4, and DRB5 was examined by DNA-based technology in 34 control, 12 AIDP, and 31 AMAN cases. In AIDP patients, the DRB1*1301 allele showed a significant increase (18% vs. 0%, P = .055). In AMAN patients, alleles DRB1*1301–03 and DRB1*1312, taken collectively, were increased (19% vs. 0%, P = .009), but by itself, the DRB1*1301 allele was not increased, as in AIDP patients. With a larger number of persons, more definitive statements will be possible; however, the differential distribution of DR13 allelic forms between AIDP and AMAN cases may suggest that there are different immunologic mechanisms operating at the molecular level of these diseases.
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