Appropriate clinical use of human leukocyte antigen typing for coeliac disease: an Australasian perspective.

The past decade has seen human leukocyte antigen (HLA) typing emerge as a remarkably popular test for the diagnostic work-up of coeliac disease with high patient acceptance. Although limited in its positive predictive value for coeliac disease, the strong disease association with specific HLA genes imparts exceptional negative predictive value to HLA typing, enabling a negative result to exclude coeliac disease confidently. In response to mounting evidence that the clinical use and interpretation of HLA typing often deviates from best practice, this article outlines an evidence-based approach to guide clinically appropriate use of HLA typing, and establishes a reporting template for pathology providers to improve communication of results.

Common and well-documented HLA alleles: 2012 update to the CWD catalogue.

We have updated the catalogue of common and well-documented (CWD) human leukocyte antigen (HLA) alleles to reflect current understanding of the prevalence of specific allele sequences. The original CWD catalogue designated 721 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, and -DPB1 loci in IMGT (IMmunoGeneTics)/HLA Database release 2.15.0 as being CWD. The updated CWD catalogue designates 1122 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as being CWD, and represents 14.3% of the HLA alleles in IMGT/HLA Database release 3.9.0. In particular, we identified 415 of these alleles as being 'common' (having known frequencies) and 707 as being 'well-documented' on the basis of ~140,000 sequence-based typing observations and available HLA haplotype data. Using these allele prevalence data, we have also assigned CWD status to specific G and P designations. We identified 147/151 G groups and 290/415 P groups as being CWD. The CWD catalogue will be updated on a regular basis moving forward, and will incorporate changes to the IMGT/HLA Database as well as empirical data from the histocompatibility and immunogenetics community. This version 2.0.0 of the CWD catalogue is available online at cwd.immunogenomics.org, and will be integrated into the Allele Frequencies Net Database, the IMGT/HLA Database and National Marrow Donor Program's bioinformatics web pages.

A community standard for immunogenomic data reporting and analysis: proposal for a STrengthening the REporting of Immunogenomic Studies statement.

Modern high-throughput HLA and KIR typing technologies are generating a wealth of immunogenomic data with the potential to revolutionize the fields of histocompatibility and immune-related disease association and population genetic research, much as SNP-based approaches have revolutionized association research. The STrengthening the REporting of Genetic Association studies (STREGA) statement provides community-based data reporting and analysis standards for genomic disease-association studies, identifying specific areas in which adoption of reporting guidelines can improve the consistent interpretation of genetic studies. While aspects of STREGA can be applied to immunogenomic studies, HLA and KIR research requires additional consideration, as the high levels of polymorphism associated with immunogenomic data pose unique methodological and computational challenges to the synthesis of information across datasets. Here, we outline the principle challenges to consistency in immunogenomic studies, and propose that an immunogenomic-specific analog to the STREGA statement, a STrengthening the REporting of Immunogenomic Studies (STREIS) statement, be developed as part of the 16th International HLA and Immunogenetics Workshop. We propose that STREIS extends at least four of the 22 elements of the STREGA statement to specifically address issues pertinent to immunogenomic data: HLA and KIR nomenclature, data-validation, ambiguity resolution, and the analysis of highly polymorphic genetic systems. As with the STREGA guidelines, the intent behind STREIS is not to dictate the design of immunogenomic studies, but to ensure consistent and transparent reporting of research, facilitating the synthesis of HLA and KIR data across studies.

HLA-DRB1 associations with disease susceptibility and clinical course in Australians with multiple sclerosis.

Human leucocyte antigen (HLA)-DRB1*1501 and other class II alleles influence susceptibility to multiple sclerosis (MS), but their contribution if any to the clinical course of MS remains uncertain. Here, we have investigated DRB1 alleles in a large sample of 1230 Australian MS cases, with some enrichment for subjects with primary progressive (PPMS) disease (n = 246) and 1210 healthy controls. Using logistic regression, we found that DRB1*1501 was strongly associated with risk (P = 7 x 10(-45)), as expected, and after adjusting for DRB1*1501, a predisposing effect was also observed for DRB1*03 (P = 5 x 10(-7)). Individuals homozygous for either DRB1*15 or DRB1*03 were considerably more at risk of MS than heterozygotes and non-carriers. Both the DRB1*04 and the DRB1*01/DRB1*15 genotype combination, respectively, protected against PPMS in comparison to subjects with relapsing disease. Together, these data provide further evidence of heterogeneity at the DRB1 locus and confirm the importance of HLA variants in the phenotypic expression of MS.

Replication of KIAA0350, IL2RA, RPL5 and CD58 as multiple sclerosis susceptibility genes in Australians.

A recent genome-wide association study (GWAS) conducted by the International Multiple Sclerosis Genetics Consortium (IMSGC) identified a number of putative MS susceptibility genes. Here we have performed a replication study in 1134 Australian MS cases and 1265 controls for 17 risk-associated single nucleotide polymorphisms (SNPs) reported by the IMSGC. Of 16 SNPs that passed quality control filters, four, each corresponding to a different non-human leukocyte antigen (HLA) gene, were associated with disease susceptibility: KIAA0350 (rs6498169) P=0.001, IL2RA (rs2104286) P=0.033, RPL5 (rs6604026) P=0.041 and CD58 (rs12044852) P=0.042. There was no association (P=0.58) between rs6897932 in the IL7R gene and the risk of MS. No interactions were detected between the replicated IMSGC SNPs and HLA-DRB1*15, gender, disease course, disease progression or age-at-onset. We used a novel Bayesian approach to estimate the extent to which our data increased or decreased evidence for association with the six most-associated IMSGC loci. These analyses indicated that even modest P-values, such as those reported here, can contribute markedly to the posterior probability of 'true' association in replication studies. In conclusion, these data provide support for the involvement of four non-HLA genes in the pathogenesis of MS, and combined with previous data, increase to genome-wide significance (P=3 x 10(-8)) evidence of an association between KIAA0350 and risk of disease.

Resolving incomplete single nucleotide polymorphism tagging of HLA-DQ2.2 for coeliac disease genotyping using digital droplet PCR.

A hallmark of coeliac disease (CD) is the exceptionally strong genetic association with HLA-DQ2.5, DQ8, and DQ2.2. HLA typing provides information on CD risk important to both clinicians and researchers. A method that enables simple and fast detection of all CD risk genotypes is particularly desirable for the study of large populations. Single nucleotide polymorphism (SNP)-based HLA typing can detect the CD risk genotypes by detecting a combination of six SNPs but this approach can struggle to resolve HLA-DQ2.2, seen in 4% of European CD patients, because of the low resolution of one negatively predicting SNP. We sought to optimise SNP-based HLA typing by harnessing the additional resolution of digital droplet PCR to resolve HLA-DQ2.2. Here we test this two-step approach in an unselected sample of Mexican DNA and compare its accuracy to DNA typed using traditional exon detection. The addition of digital droplet PCR for samples requiring negative prediction of HLA-DQ2.2 enabled HLA-DQ2.2 to be accurately typed. This technique is a simple addition to a SNP-based typing strategy and enables comprehensive definition of all at-risk HLA genotypes in CD in a timely and cost-effective manner.

Calculation of relative differences in the binding free energies of HIV1 protease inhibitors: a thermodynamic cycle perturbation approach.

An iterative computer-assisted drug design (CADD) method that combines molecular mechanics, dynamics, thermodynamic cycle perturbation (TCP) calculations, molecular design, synthesis, and biochemical testing of peptidomimetic inhibitors and crystallographic structure determination of the protein-inhibitor complexes has been successfully applied to the design of novel inhibitors for the HIV1 protease. The first "designer" compound in this series (I) was designed by replacing the C-terminal Val-Val methyl ester of a known hydroxyethylene inhibitor with a diphenhydramine amide derivative in which two phenyl groups fill the p2' and p3' side-chain binding pockets in the HIV1 protease. Subsequent testing showed modest inhibition (Ki = 1.67 microM). Concurrently, molecular mechanics calculations on designed analogs indicated the feasibility of replacement of a phenyl ring with an indole ring (II). Synthesis and biochemical testing resulted in better inhibition potency for II. X-ray crystal structure determination of HIV1 protease complexed with I and II provided structural information for subsequent design and TCP calculations. A TCP protocol was established and validated for the mutation of I-->II. TCP results showed a net gain of 2.1 (+/- 0.9) kcal/mol in replacing II with I, which agreed with experimental result within an error margin of 0.8 kcal/mol. TCP calculations for six other mutations (I-->III, II-->III, IV, V, VI, and VII) were performed prior to synthesis and testing. These results allowed for the prioritization of design ideas for synthesis. In all cases where experimental results are available, TCP calculations showed good agreement. These results demonstrate that the TCP approach can be used with medicinal chemistry and crystallography for screening the proposed derivatives of a lead compound prior to synthesis, thus potentially reducing the time for the discovery of new drugs.

An approach to rapid estimation of relative binding affinities of enzyme inhibitors: application to peptidomimetic inhibitors of the human immunodeficiency virus type 1 protease.

This report describes a method for rapid assessment of the binding affinities of a series of analogous ligands to an enzyme. This approach is based on two variables (scores), representing (i) the enthalpy of binding and (ii) the strength of hydrophobic interaction. The method is then used to evaluate the binding of 11 different peptidomimetic inhibitors to the HIV-1 protease. Three-dimensional structures of these enzyme-inhibitor complexes are modeled based on the crystal structures of HIV-1 protease complexes with the known inhibitors. These structures are minimized using the AMBER force field, and the scores of binding enthalpy for each of the ligands are calculated. A second score to represent the hydrophobic interaction between a pair of atoms uses an exponential function of distance between the atoms and the product of their atomic hydrophobicity constants. This exponential function is used to assess the hydrophobic interaction energy between an enzyme and its inhibitor and also to compute and display a 'molecular hydrophobicity map' as a 3D visualization tool. These methods are then applied to obtain trends in relative binding affinities of pairs of analogous inhibitors. Calculated scores agree well with corresponding results from thermodynamic cycle perturbation (TCP) simulations as well as experimental binding data. Since the proposed calculations are computationally cheaper and faster than TCP calculations, it is suggested that these scores can form the basis for rapid, preliminary theoretical screening of proposed derivatives of an inhibitor prior to TCP analysis, synthesis, and testing.

Crystal-structure-based design and synthesis of novel C-terminal inhibitors of HIV protease.

The X-ray crystal-structure-based design, synthesis, computational evaluation, and activity of a novel class of HIV protease inhibitors are described. The initial lead compounds 2 and 3 were designed by modeling replacement groups for the C-terminal Val-Val-OCH3 of a known hydroxyethylene inhibitor into the active site of the reported crystal structure of HIV protease complexed with MVT-101. The lead compound 2 was found to be a modest inhibitor with a Ki = 1.67 microM. The X-ray crystal structure of compound 2 complexed with HIV protease was solved and used for subsequent design. The lead compound 3 was found to be a more potent inhibitor with Ki = 0.2 microM, and the structure of it complexed with HIV protease was also solved and used for subsequent design. Modification of both the C-terminus and N-terminus of indole 3 resulted in compounds with Ki = 30 nM. Using the crystal structure of compounds 2 and 3 with HIV protease as a starting point, the thermodynamic cycle perturbation molecular dynamics method was applied to a select group of compounds in order to test the accuracy of this type of computation within a series of closely related compounds.

Synthesis and biological evaluation of novel 2,6-diaminobenz[cd]indole inhibitors of thymidylate synthase using the protein structure as a guide.

The design, synthesis, and biochemical and biological evaluations of a novel series of 2,6-diaminobenz[cd]indole-containing inhibitors of human thymidylate synthase (TS) are described. The compounds are characterized by having either a pyridine or pyridazine ring in place of the (phenylsulfonyl)morpholinyl group of the known inhibitor N6-[4-(morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[ cd]indole glucuronate (i). Active compounds from this series showed human TS inhibition constants below the 10 nM level and were potent, selective submicromolar antitumor agents in cell culture. The compounds were synthesized by reductive alkylation of a substituted 6-aminobenz[cd]indole or reductive cyclization of a substituted 1-cyano-8-nitronaphthalene.

Crystal-structure-based design and synthesis of benz[cd]indole-containing inhibitors of thymidylate synthase.

The X-ray crystal-structure-based design, synthesis, and biological activity of a novel family of benz[cd]indole-containing inhibitors of thymidylate synthase (TS) are described. The structure-activity of the lead compound was studied by conceptually dividing the molecule into four regions and independently optimizing the substituents for each region. Combination of favored substituents for each region led to inhibitors with Ki's against the human enzyme in the range of 10-20 nM. Thymidine shift experiments suggested that the cytotoxic properties of the best enzyme inhibitors were due to TS targeting in cells. The inhibitors were synthesized from substituted 6-aminobenz[cd]indol-2(1H)-ones by alkylation with both a simple alkyl group and a substituted benzylic portion. The 2,6-diaminobenz[cd]indoles were prepared from the corresponding lactams by conversion to the thiolactam, alkylation to the methylated thiolactam, and then displacement with a substituted or unsubstituted amine.

Protein structure-based design, synthesis, and biological evaluation of 5-thia-2,6-diamino-4(3H)-oxopyrimidines: potent inhibitors of glycinamide ribonucleotide transformylase with potent cell growth inhibition.

The design, synthesis, biochemical, and biological evaluation of a novel series of 5-thia-2,6-diamino-4(3H)-oxopyrimidine inhibitors of glycinamide ribonucleotide transformylase (GART) are described. The compounds were designed using the X-ray crystal structure of human GART. The monocyclic 5-thiapyrimidinones were synthesized by coupling an alkyl thiol with 5-bromo-2, 6-diamino-4(3H)-pyrimidinone, 20. The bicyclic compounds were prepared in both racemic and diastereomerically pure forms using two distinct synthetic routes. The compounds were found to have human GART KiS ranging from 30 microM to 2 nM. The compounds inhibited the growth of both L1210 and CCRF-CEM cells in culture with potencies down to the low nanomolar range and were found to be selective for the de novo purine biosynthesis pathway. The most potent inhibitors had 2,5-disubstituted thiophene rings attached to the glutamate moiety. Placement of a methyl substituent at the 4-position of the thiophene ring to give compounds 10, 18, and 19 resulted in inhibitors with significantly decreased mFBP affinity.

Structure-based design of lipophilic quinazoline inhibitors of thymidylate synthase.

To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.

Structure-based design of substituted diphenyl sulfones and sulfoxides as lipophilic inhibitors of thymidylate synthase.

Six new diphenyl sulfoxide and five new diphenyl sulfones were designed, synthesized, and tested for their inhibition of human and Escherichia coli thymidylate synthase (TS) and of the growth of cells in tissue culture. The best sulfoxide inhibitor of human TS was 3-chloro-N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4- (phenylsulfinyl)-N-(prop-2-ynyl)-aniline (7c) that had a Ki of 27 nM. No sulfone improved on TS inhibition by the previously reported 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2- ynylamino)phenyl phenyl sulfone (Ki = 12 nM). Nevertheless, one sulfone, 4-((2-chlorophenyl)sulfonyl)-N-((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)-N-(prop-2-ynyl)aniline, was selected, on the basis of its inhibition of both TS and cell growth, for antitumor testing; it gave a 61% increase in life span to mice bearing the thymidino kinase-deficient L5178Y (TK-) lymphoma. A crystal structure of N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4-((2- methylphenyl)sulfinyl)-N-(prop-2-ynyl)aniline complexed with E. coli TS was solved and revealed selective binding of one sulfoxide enantiomer. AMBER calculations showed that the enantioselection was due to asymmetric electrostatic effects at the mouth of the active site. In contrast, a similar crystal structure of the sulfoxide 7c, along with AMBER calculations, indicated that both enantiomers bound, but with different affinities. The side chain of Phe176 shifted in order to structurally accommodate the chlorine of the more weakly bound enantiomer.

Polymorphism in the regulatory regions of the HLA-DPB1 gene.

Nucleotide sequencing of approximately 400 basepairs upstream from exon 1 of the DPB1 gene and sequence specific oligonucleotide hybridisation identified eight nucleotide positions to be polymorphic which were in linkage disequilibrium (LD) with DPA1 and DPB1 alleles. Substitutions at two sites (-230 and -224) formed three genotypes (DP-PRO1-3). DP-PRO 1 was the most common genotype and was in LD with DPA1*0103, *0202 and DPB1*0401, *0501. DP-PRO 2 was observed in LD with DPB1*02012, *1601, *1701 and DPA1*0104. DP-PRO3 was in LD with DPB1*0901, *1001 and DPA1*0201. Electrophoretic Mobility Shift Assays (EMSA) performed with restriction enzyme fragments showed substitutions at -230 and -224 not to be involved in binding nuclear proteins. Six substitutions were found on a single genotype (DP-PRO4) which was observed in seven samples; 67% of DP-PRO4 inferred haplotypes were HLA-A2-B46, DRB1*0901, DQB1*03032, DPA1*0401, DPB1*1301. Three of the substitutions occurred in conserved regulatory region boxes, W', X and Y, and three in the signal and leader sequences. EMSA competitive binding assays performed with oligonucleotide probes for the substitutions showed no difference in binding affinity for W' and X probes. The DP-PRO4 Y box had a decreased nuclear protein binding affinity compared to DP-PRO1-3. Whether the sum of the differences in DP-PRO4 relate to a change in the cell surface expression of HLA-DP is yet to be determined.

Matching for HLA DPA1 and DPB1 alleles in unrelated bone marrow transplantation.

The impact of donor-recipient DPA1 and DPB1 matching was examined in 122 unrelated bone marrow transplant pairs. All pairs were serologically matched at the time of transplantation for HLA class I and II and a majority also DRB1 allele matched. Retrospective A, B, C, DRB1, DQA1, DQB1 in addition to DPA1 and DPB1 allele matching was performed by molecular techniques. The percentage of pairs that were allele matched was as follows; HLA-A = 91% (n = 80), HLA-B = 94% (n = 80), HLA-C = 78% (n = 80), HLA-DRB1 = 96% (n = 122), HLA-DQA1 = 99% (n = 80), HLA-DQB1 = 92% (n = 122). 92 recipient/donor pairs with informative clinical data were available for analysis. DPA1 identity (no incompatibility in either direction) was observed in 57% and DPA1 compatibility in 76% of pairs with no apparent beneficial effect of matching on patient survival or Graft Versus Host Disease (GVHD). DPB1 identity was observed in 11% and compatibility in 27% of pairs. A significant improvement in patient survival was observed in DPB1 matched compared to one DPB1 mismatch (p < 0.01) and combined one and two DPB1 mismatched transplants (p = 0.03). This beneficial effect remained when allele mismatches at HLA-A, B, C, DRB1, DQA1, DQB1 were excluded (p = 0.05, p = 0.03, respectively). There was a significant association of increased frequency of severe GVHD (grades III-IV) compared to mild GVHD (grades I-II) with DPB1 mismatched transplants compared to DPB1 matched transplants (p = 0.04). In DPB1 mismatched transplants an association between patient survival and matching for individual DPB1 polymorphic regions was not observed; however in the HLA-A, B, DRB1, DQA1, DQB1 allele matched transplants a non significant increase in the frequency of Grade IV GVHD was observed in recipients who were negative compared to those who were positive for DPB1 alleles coding for glutamic acid at position 69.

HLA antigens and age at diagnosis of insulin-dependent diabetes mellitus.

IDDM results from the immune-mediated destruction of pancreatic islet beta cells. Clinicopathologic heterogeneity in IDDM is reflected in part by the wide age range over which the onset of clinical symptoms can occur, after months to years of subclinical "insulitis." Because MHC genes play a critical role in immune function we studied their possible contribution to IDDM heterogeneity by analyzing HLA profiles of 194 IDDM patients in relation to their age at diagnosis. Restriction of HLA-DR heterogeneity was observed in patients diagnosed before age 21 years. Frequencies of DR3 and DR3/4 were highest in the < or = 6-year-old age group and thereafter declined with increasing age at diagnosis. In contrast, the frequency of DR4 remained increased up to age 30 years at diagnosis. DR7, normally considered to be a neutral allele, was like DR2 and DR5, significantly decreased in patients diagnosed before age 21 years. The A30-B18-DR3 haplotype was significantly increased in the < or = 6-year-old age group, A1-B8-DR3 was increased in the > or = 31-year-old group. B62-DR4 was increased only in the > 12-year-old age group. In DR4 patients the frequency of DQ8 was increased across all age groups. A sex difference was observed in those diagnosed at < or = 12 years of age, with an excess of females in the DR3+/DR4- group and males in the DR3-/DR4+ group. An association of DPB1 with IDDM was revealed by an increased frequency overall of DPB1*0301 and/or DPB1*0401, being more pronounced in patients diagnosed at > 20 years of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Higher body mass index in adults at diagnosis of the slowly progressive form of type 1 diabetes mellitus is associated with lower risk HLA genes.

We hypothesised that higher body weight, a proposed risk factor for type 1 diabetes mellitus, would be associated with increased penetrance of lower risk genes. In adults at diagnosis of the slowly progressive form of type 1 diabetes mellitus we found that higher body mass index was associated with the absence of the highest risk HLA genes.