Next-generation sequencing (NGS) has now been established, and widely recognized, to be the preferred choice for human leukocyte antigen (HLA) typing. This transformation is based upon the many scientific, operational and economic benefits this technology affords. In this report, we review the major advantages, existing limitations and significant promise derived from adopting this technology in immunogenetics.Significant benefits have emerged from the usage of NGS in a relatively short period, whereby we realize that this technology not only helps addressing the technical and operational problems we have had with the legacy methods for HLA typing, but equally important, it also allows for creative applications in stem cell and organ transplantation, new ways to investigate associations of the major histocompatibility complex (MHC) with many diseases and enhance our understanding regarding the MHC and non-MHC genomic interactions. The emerging picture is one of significant benefits in the diagnostic sphere of immunogenetics and transplantation and one of interconnectivity, integrating the many biological pathways controlled and affected by this unique genomic region.NGS has revolutionized the science and practice of immunogenetics. In this article, we identify the still unresolved issues, the current benefits to transplantation and the potential for dissecting the complexity of the MHC, one of the most fascinating regions of the human genome. Using current trends, an attempt is made to predict future directions and outcomes.
Diabetes impacts approximately 200 million people worldwide, of whom approximately 10% are affected by type 1 diabetes (T1D). The application of genome-wide association studies (GWAS) has robustly revealed dozens of genetic contributors to the pathogenesis of T1D, with the most recent meta-analysis identifying in excess of 40 loci. To identify additional genetic loci for T1D susceptibility, we examined associations in the largest meta-analysis to date between the disease and ∼2.54 million SNPs in a combined cohort of 9,934 cases and 16,956 controls. Targeted follow-up of 53 SNPs in 1,120 affected trios uncovered three new loci associated with T1D that reached genome-wide significance. The most significantly associated SNP (rs539514, P = 5.66×10−11) resides in an intronic region of the LMO7 (LIM domain only 7) gene on 13q22. The second most significantly associated SNP (rs478222, P = 3.50×10−9) resides in an intronic region of the EFR3B (protein EFR3 homolog B) gene on 2p23; however, the region of linkage disequilibrium is approximately 800 kb and harbors additional multiple genes, including NCOA1, C2orf79, CENPO, ADCY3, DNAJC27, POMC, and DNMT3A. The third most significantly associated SNP (rs924043, P = 8.06×10−9) lies in an intergenic region on 6q27, where the region of association is approximately 900 kb and harbors multiple genes including WDR27, C6orf120, PHF10, TCTE3, C6orf208, LOC154449, DLL1, FAM120B, PSMB1, TBP, and PCD2. These latest associated regions add to the growing repertoire of gene networks predisposing to T1D.
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.
Evaluation of human histocompatibility leukocyte antigen (HLA) class II genes in 54 cases of tuberculoid leprosy (TL) and 44 controls has shown a positive association with HLA-DRB1 alleles that contain Arg13 or Arg70-Arg71. Among TL patients, 87% carry specific alleles of DRB1 Arg13 or Arg70-Arg71 as compared to 43% among controls (p = 5 x 10(-6)) conferring a relative risk of 8.8. Thus, susceptibility to TL involves three critical amino acid positions of the beta chain, the side chains of which, when modeled on the DR1 crystal structure, line a pocket (pocket 4) accommodating the side chain of a bound peptide. This study suggests that disease susceptibility may be determined by the independent contribution of polymorphic residues participating in the formation of a functional arrangement (i.e., pocket) within the binding cleft of an HLA molecule.
Recombinant human interleukin 2 (rIL-2) drives the proliferation of the cloned murine T-helper line L2. The initial G1 activation occurs during the first 20 hr after stimulation, with DNA synthesis (S phase) beginning approximately 20 hr after rIL-2 stimulation. Three patterns of protein synthesis were observed during G1 activation. Type I proteins (e.g., p72 and p66) were synthesized at near maximal rates as early as 4 hr after stimulation, with little change in rates of synthesis through the G1 to S phase transition. Type II proteins (e.g., p52 and p36) were detectable early after stimulation, but their rates of synthesis continued to increase throughout G1 activation, becoming maximal 24-28 hr after stimulation. Type III proteins (e.g., p93, p89, and p63) were synthesized maximally 4 or 8 hr after rIL-2 stimulation, then their rates of synthesis declined markedly to prestimulation levels. Type II proteins, p52 and p36, were shown to be correlated with cell proliferation, since their rates of synthesis were maximal while L2 cells were proliferating and declined as the cells returned to a quiescent state. The potassium channel blocker quinine inhibited cell growth and the synthesis of p52 and p36 when added 0 or 2 hr after rIL-2 stimulation but not when added 6 hr after rIL-2 stimulation. Thus, a quinine-sensitive event occurring in L2 cells between 2 and 6 hr after rIL-2 stimulation is necessary for synthesis of type II proteins, DNA synthesis, and cell proliferation.
