Adaptation to HLA-associated immune pressure over the course of HIV infection and in circulating HIV-1 strains.

Adaptation to human leukocyte antigen (HLA)-associated immune pressure represents a major driver of human immunodeficiency virus (HIV) evolution at both the individual and population level. To date, there has been limited exploration of the impact of the initial cellular immune response in driving viral adaptation, the dynamics of these changes during infection and their effect on circulating transmitting viruses at the population level. Capturing detailed virological and immunological data from acute and early HIV infection is challenging as this commonly precedes the diagnosis of HIV infection, potentially by many years. In addition, rapid initiation of antiretroviral treatment following a diagnosis is the standard of care, and central to global efforts towards HIV elimination. Yet, acute untreated infection is the critical period in which the diversity of proviral reservoirs is first established within individuals, and associated with greater risk of onward transmissions in a population. Characterizing the viral adaptations evident in the earliest phases of infection, coinciding with the initial cellular immune responses is therefore relevant to understanding which changes are of greatest impact to HIV evolution at the population level. In this study, we utilized three separate cohorts to examine the initial CD8+ T cell immune response to HIV (cross-sectional acute infection cohort), track HIV evolution in response to CD8+ T cell-mediated immunity over time (longitudinal chronic infection cohort) and translate the impact of HLA-driven HIV evolution to the population level (cross-sectional HIV sequence data spanning 30 years). Using next generation viral sequencing and enzyme-linked immunospot interferon-gamma recall responses to peptides representing HLA class I-specific HIV T cell targets, we observed that CD8+ T cell responses can select viral adaptations prior to full antibody seroconversion. Using the longitudinal cohort, we uncover that viral adaptations have the propensity to be retained over time in a non-selective immune environment, which reflects the increasing proportion of pre-adapted HIV strains within the Western Australian population over an approximate 30-year period.

Long-Term Associations between Human Cytomegalovirus Antibody Levels with All-Cause Mortality and Cardiovascular Outcomes in an Australian Community-Based Cohort.

Human cytomegalovirus (HCMV) infection has been shown to increase the risk of cardiovascular events and all-cause death among individuals with clinically apparent cardiovascular disease (CVD). Whether this association exists in individuals with no history of CVD remains unclear. Serum levels of HCMV IgG antibody were measured using an ELISA in 2050 participants aged 40-80 years from the 1994/1995 Busselton Health Survey who did not have CVD at baseline. Outcomes were all-cause death, cardiovascular death, acute coronary syndrome (ACS) and major adverse coronary and cerebrovascular events (MACCE, composite of all-cause death, ACS, stroke and coronary artery revascularisation procedures). Cox proportional hazards regression analysis was used to investigate HCMV antibody levels as a predictor of death and cardiovascular outcomes during follow-up periods of 5, 10 and 20 years. At baseline, participants had a mean age of 56 years and 57% were female. During the 20-year follow-up, there were 448 (21.9%) deaths (including 152 from CVD), 139 (6.8%) participants had ACS and 575 (28.0%) had MACCE. In the fully adjusted model, levels of HCMV antibody at 20 years was associated with all-cause death (HR 1.04; 95% CI 1.00, 1.07, p = 0.037) but not with CVD death, ACS or MACCE. Levels of HCMV antibody are associated with all-cause death but not with cardiovascular outcomes in adults without pre-existing CVD.

A national study of the molecular epidemiology of HIV-1 in Australia 2005-2012.

INTRODUCTION: Rates of new HIV-1 diagnoses are increasing in Australia, with evidence of an increasing proportion of non-B HIV-1 subtypes reflecting a growing impact of migration and travel. The present study aims to define HIV-1 subtype diversity patterns and investigate possible HIV-1 transmission networks within Australia. METHODS: The Australian Molecular Epidemiology Network (AMEN) HIV collaborating sites in Western Australia, South Australia, Victoria, Queensland and western Sydney (New South Wales), provided baseline HIV-1 partial pol sequence, age and gender information for 4,873 patients who had genotypes performed during 2005-2012. HIV-1 phylogenetic analyses utilised MEGA V6, with a stringent classification of transmission pairs or clusters (bootstrap ≥98%, genetic distance ≤1.5% from at least one other sequence in the cluster). RESULTS: HIV-1 subtype B represented 74.5% of the 4,873 sequences (WA 59%, SA 68.4%, w-Syd 73.8%, Vic 75.6%, Qld 82.1%), with similar proportion of transmission pairs and clusters found in the B and non-B cohorts (23% vs 24.5% of sequences, p = 0.3). Significantly more subtype B clusters were comprised of ≥3 sequences compared with non-B clusters (45.0% vs 24.0%, p = 0.021) and significantly more subtype B pairs and clusters were male-only (88% compared to 53% CRF01_AE and 17% subtype C clusters). Factors associated with being in a cluster of any size included; being sequenced in a more recent time period (p<0.001), being younger (p<0.001), being male (p = 0.023) and having a B subtype (p = 0.02). Being in a larger cluster (>3) was associated with being sequenced in a more recent time period (p = 0.05) and being male (p = 0.008). CONCLUSION: This nationwide HIV-1 study of 4,873 patient sequences highlights the increased diversity of HIV-1 subtypes within the Australian epidemic, as well as differences in transmission networks associated with these HIV-1 subtypes. These findings provide epidemiological insights not readily available using standard surveillance methods and can inform the development of effective public health strategies in the current paradigm of HIV prevention in Australia.

Plasma CXCL10, sCD163 and sCD14 Levels Have Distinct Associations with Antiretroviral Treatment and Cardiovascular Disease Risk Factors.

We investigate the associations of three established plasma biomarkers in the context of HIV and treatment-related variables including a comprehensive cardiovascular disease risk assessment, within a large ambulatory HIV cohort. Patients were recruited in 2010 to form the Royal Perth Hospital HIV/CVD risk cohort. Plasma sCD14, sCD163 and CXCL10 levels were measured in 475 consecutive patients with documented CVD risk (age, ethnicity, gender, smoking, blood pressure, BMI, fasting metabolic profile) and HIV treatment history including immunological/virological outcomes. The biomarkers assessed showed distinct associations with virological response: CXCL10 strongly correlated with HIV-1 RNA (p<0.001), sCD163 was significantly reduced among 'aviraemic' patients only (p = 0.02), while sCD14 was unaffected by virological status under 10,000 copies/mL (p>0.2). Associations between higher sCD163 and protease inhibitor therapy (p = 0.05) and lower sCD14 with integrase inhibitor therapy (p = 0.02) were observed. Levels of sCD163 were also associated with CVD risk factors (age, ethnicity, HDL, BMI), with a favourable influence of Framingham score <10% (p = 0.04). Soluble CD14 levels were higher among smokers (p = 0.002), with no effect of other CVD risk factors, except age (p = 0.045). Our findings confirm CXCL10, sCD163 and sCD14 have distinct associations with different aspects of HIV infection and treatment. Levels of CXCL10 correlated with routinely monitored variables, sCD163 levels reflect a deeper level of virological suppression and influence of CVD risk factors, while sCD14 levels were not associated with routinely monitored variables, with evidence of specific effects of smoking and integrase inhibitor therapy warranting further investigation.

Longitudinal Trends in Western Australian HIV-1 Sequence Diversity and Viral Transmission Networks and Their Influence on Clinical Parameters: 2000-2014.

We examined baseline HIV-1 protease and reverse transcriptase sequences and HIV clinical parameters from 1,021 consecutive patients (814 male, 207 female) through the Royal Perth Hospital HIV service to investigate HIV-1 subtype diversity and local phylogenetic networks from 2000 to 2014. HIV-1 subtype B virus sequences were demonstrated in 619 (61%) of cases, with increasing non-B HIV-1 subtypes from 23.2% (2000-2003) to 48% (2008-2011) and 43% (2012-2014) (p < 0.001), including the CRF01_AE subtype [6.6% (2000-2003) to 21.5% (2008-2011)] and HIV-1 C subtype [9.5% (2000-2003) to 20.2% (2008-2011)]. More HIV-1 B subtypes were assigned to phylogenetic clusters compared to non-B subtypes (34% vs. 18%; p < 0.001), with larger clusters identified (cluster size >2: 135/211; 64% vs. 13/69; 19%; p = 0.001), including one cluster of 53 HIV-1 B subtype sequences that evolved from 2008 to 2014. Non-B subtype HIV-1 was associated with lower baseline CD4 T cell count (p = 0.005) but not plasma HIV-1 RNA levels (p = 0.31), suggesting relatively delayed diagnosis. Baseline viral load was strongly associated with calendar time [mean 18,620 copies/ml in 2000-2003; 75,858 copies/ml in 2012-2014 (p < 0.001)], and was also associated with larger phylogenetic clusters (size >2) in adjusted analyses (p = 0.03). This study identifies a number of temporal trends over the past 15 years, including an increasing prevalence of non-B subtype HIV-1 that highlights the growing influence of migration and travel on the Australian HIV-1 epidemic and the associated increased role of heterosexual HIV-1 transmission in this context. At the same time, these data indicate that local transmission within predominantly male networks remains a challenging issue for HIV-1 prevention.

Elevated plasma soluble CD14 and skewed CD16+ monocyte distribution persist despite normalisation of soluble CD163 and CXCL10 by effective HIV therapy: a changing paradigm for routine HIV laboratory monitoring?

OBJECTIVE: We investigated plasma and flow cytometric biomarkers of monocyte status that have been associated with prognostic utility in HIV infection and other chronic inflammatory diseases, comparing 81 HIV+ individuals with a range of treatment outcomes to a group of 21 healthy control blood donors. Our aim is to develop and optimise monocyte assays that combine biological relevance, clinical utility, and ease of adoption into routine HIV laboratory practice. DESIGN: Cross-sectional evaluation of concurrent plasma and whole blood samples. METHODS: A flow cytometry protocol was developed comprising single-tube CD45, CD14, CD16, CD64, CD163, CD143 analysis with appropriately matched isotype controls. Plasma levels of soluble CD14 (sCD14), soluble CD163 (sCD163) and CXCL10 were measured by ELISA. RESULTS: HIV status was associated with significantly increased expression of CD64, CD143 and CD163 on CD16+ monocytes, irrespective of the virological response to HIV therapy. Plasma levels of sCD14, sCD163 and CXCL10 were also significantly elevated in association with viremic HIV infection. Plasma sCD163 and CXCL10 levels were restored to healthy control levels by effective antiretroviral therapy while sCD14 levels remained elevated despite virological suppression (p<0.001). CONCLUSIONS: Flow cytometric and plasma biomarkers of monocyte activation indicate an ongoing systemic inflammatory response to HIV infection, characterised by persistent alterations of CD16+ monocyte expression profiles and elevated sCD14 levels, that are not corrected by antiretroviral therapy and likely to be prognostically significant. In contrast, sCD163 and CXCL10 levels declined on antiretroviral therapy, suggesting multiple activation pathways revealed by these biomarkers. Incorporation of these assays into routine clinical care is feasible and warrants further consideration, particularly in light of emerging therapeutic strategies that specifically target innate immune activation in HIV infection.

Establishment of gene copy number-specific normal ranges for serum C4 and its utility for interpretation in patients with chronically low serum C4 concentrations.

OBJECTIVE: To establish gene copy number (GCN)-specific normal ranges for serum C4 genes and to determine their utility with respect to the interpretation of chronically low serum C4 concentrations in patients with clinically quiescent systemic lupus erythematosus (SLE). METHODS: C4 serum concentrations were estimated by automated turbidimetry, and C4 GCNs were determined using the TaqMan real-time polymerase chain reaction (PCR) analysis in 184 unselected individuals and in 10 patients with type 1 diabetes mellitus (DM) who were selected for the presence of only 2 copies of the C4 gene. C4 GCNs were also determined in 11 patients with clinically quiescent SLE who had chronically low serum C4 concentrations. RESULTS: A total of 33% of the variation in serum C4 concentrations could be accounted for by both C4A and C4B GCNs (R(2) = 0.30, P ≤ 0.0001). There was a median of 2 gene copies at the C4A locus (53.8%) and 2 at the C4B locus (58.7%). The median total number of C4 genes was 4 (55.4%). C4 GCN-specific normal ranges were established. A chronically low serum C4 concentration was explained by a low C4 GCN in 3 of 11 patients tested. CONCLUSION: This study establishes the feasibility of establishing C4 GCN-specific normal ranges using the TaqMan real-time PCR assay. Chronically low serum C4 concentrations in SLE patients are sometimes explained by low C4 GCNs.

Contributions of vitamin D response elements and HLA promoters to multiple sclerosis risk.

OBJECTIVE: The identification of a vitamin D-responsive (VDRE) motif within the HLA-DRB1*15:01 promoter region provides an attractive explanation for the combined effects of HLA-DR inheritance and vitamin D exposure on multiple sclerosis (MS) risk. We therefore sought to incorporate HLA-DRB1 promoter variation, including the VDRE motif, in an assessment of HLA-DRB1-associated MS risk. METHODS: We utilized 32 homozygous HLA cell lines (covering 17 DRB1 alleles) and 53 heterozygote MS samples (20 DRB1 alleles) for HLA-DRB1 promoter sequencing. The influence of HLA-DRB1 variation on MS risk was then assessed among 466 MS cases and 498 controls. RESULTS: The majority of HLA*DRB1 alleles (including HLA-DRB1*15:01) express the functional VDRE motif, apart from HLA-DRB1*04, *07, and *09 alleles that comprise the HLA-DR53 serologic group. Allele-specific variation within functional X-box and Y-box motifs was also associated with serologically defined HLA-DR haplotypes. Incorporating these results in an analysis of MS risk, we identified a strong protective effect of HLA-DRB1*04, *07, and *09 (DR53) alleles (p = 10(-12)) and elevated risk associated with DRB1*15 and *16 (DR51) and *08 (DR8) alleles (p < 10(-18)). CONCLUSIONS: HLA-DRB1 groups corresponding to serologic HLA-DR profiles as well as promoter polymorphism haplotypes effectively stratified MS risk over an 11-fold range, suggesting functional relationships between risk-modifying HLA-DRB1 alleles. An independent contribution of VDRE motif variation to increase MS risk was not discernible, although vitamin D-dependent regulation of HLA-DR expression may still play an important role given that HLA-DRB1*04/*07/*09 (DR53) alleles that express the "nonresponsive" VDRE motif were associated with significantly reduced risk of MS.

Methods for diagnostic HLA typing in disease association and drug hypersensitivity.

This chapter describes the application of diagnostic HLA typing for disease association and five methods used for specific HLA genotypes. The methods utilise a combination of polymerase chain reaction (PCR) amplification to detect sequence polymorphism by the presence or absence of amplification, nucleotide sequencing of the PCR product, and hybridisation of the PCR product with labelled probes. The probes are specific for sequence polymorphism associated with the genotype and are attached to either a Micro Bead or a Solid Phase. In addition, the detection of single nucleotide polymorphism(s) which "tag" for the genotype using a real-time PCR is described.

Molecular analysis of complement component C4 gene copy number.

Classical, alternative, or lectin pathways may activate the complement system cascade. The classical pathway includes the C4 protein and functions in the prevention of immune complex precipitation and in clearance of immune complexes.Two isotypes of C4-C4A and C4B-are coded by genes located at two loci within the major histocompatibility complex (MHC) on chromosome 6. While these isotypes share over 99% amino acid sequence homology, five nucleotide differences located in exon 26 are responsible for major structural and functional differences between the C4 isotypes.C4A and C4B are highly polymorphic with over 40 alleles, gene duplications, and "null alleles". C4 genes may be short (14.6 kb) or long (21 kb), due to the absence or presence of an endogenous retroviral sequence-HERV-K(C4)-in intron 9, respectively. The C4 gene copy number (GCN) can vary from 1-3 per haplotype or 2-6 per diploid genome. The variation in GCN leads to a range of C4 plasma protein concentrations among healthy subjects. In subjects with equal numbers of C4 genes, subjects with short genes have C4 plasma levels relatively higher than subjects with long genes.Variation of the C4 GCN, the gene size (long or short) and the C4 isotypes (C4A and C4B) may also lead to susceptibility to autoimmune disease. Therefore, in subjects with autoimmune disease, a low serum C4 level may be due to ongoing disease activity associated with complement activation and consumption or it may be due to genetic factors. Distinguishing between these will have clinical implications.Exact determination of GCN can be difficult, at least in part due to the high degree of homology between C4A and C4B and a variety of techniques has been described. This chapter describes a quantitative TaqMan real-time PCR (qPCR) copy number assay, based on our laboratory experience using this assay.

High-resolution HLA-DRB1 genotyping in an Australian inclusion body myositis (s-IBM) cohort: an analysis of disease-associated alleles and diplotypes.

We performed high-resolution (4-digit) HLA-DRB1 genotyping in an Australian cohort of 105s-IBM patients and 189 controls. Our findings showed that whilst the strongest association was with the HLA-DRB1*03:01 allele and the HLA-DRB1*03:01/*01:01 diplotype, HLA-DRB1*01:01 and HLA-DRB1*13:01 are also risk alleles. A number of other alleles, HLA-DRB1*04:01, *04:04, *07:01, *09:01, *11:01 and *15:01, as well as the HLA-DRB1*03:01/*04:01 and HLA-DRB1*03:01/*07:01 diplotypes were reduced in s-IBM cases and may be protective. The HLA-DRB1*03:01 and HLA-DRB1*13:01 alleles also appear to have an influence on the age at onset of the disease and severity of muscle weakness. Our findings indicate that the influence of HLA-DRB1 in s-IBM is complex and that epistatic interactions at the HLA-DRB1 locus contribute both to disease susceptibility and to the clinical phenotype.

Frequency of autoantibodies and correlation with HLA-DRB1 genotype in sporadic inclusion body myositis (s-IBM): a population control study.

The frequency of myositis-associated and myositis-specific autoantibodies (AAb) was compared in 51 s-IBM patients and a population control group. Non-organ specific AAb (ANA, anti-Ro52, anti-Ro60, anti-La, anti-RNP) but not anti-thyroid peroxidase, anti-tissue transglutaminase or myositis-specific antibodies, were more frequent in s-IBM patients, and 14/51 (27%) had another autoimmune disease (Sjögren's syndrome, thyroiditis, psoriasis, vitiligo). The presence of AAb did not correlate with carriage of HLA-DRB1*0301, but there was a negative correlation between ANA/anti-Ro52 and carriage of HLA-DRB1*1301. The findings in this cohort confirm that patients with sIBM do not show evidence of a muscle-specific humoral immune process but have an increased frequency of non-organ specific AAb and other autoimmune disorders.

Spinal cord involvement in multiple sclerosis: a correlative MRI and high-resolution HLA-DRB1 genotyping study.

BACKGROUND: There have been few magnetic resonance imaging (MRI) studies of the spinal cord in large multiple sclerosis (MS) patient cohorts and little is known about correlations between cord lesions and human leukocyte antigen (HLA) alleles. OBJECTIVE: To investigate the spectrum of MRI changes in the spinal cord in MS and associations with the HLA-DRB1 genotype. METHODS: Two hundred and fifty two consecutive MS patients from the Perth Demyelinating Diseases Database had MRI of the spinal cord and brain and high-resolution HLA-DRB1 genotyping. The numbers, locations, shape and segmental extent of cord lesions were analysed and were correlated with carriage of individual HLA-DRB1 alleles and diplotypes. RESULTS: Focal cord lesions were present in 82.9% of cases, with numbers being maximal in the cervical cord and increasing with disease duration. Focal lesions were usually round or oval in shape but in 35% of cases subpial wedge-shaped lesions were present. Diffuse cord involvement was present in 10% of cases and correlated with carriage of HLA-DRB1*1501 and with higher disability. Carriage of the minor allele HLA-DRB1*0701 was significantly associated with numbers of wedge-shaped lesions and lesions in the cervical cord, while HLA-DRB1*1104 and DRB1*0103 were significantly associated respectively with higher and lower numbers of thoracic cord lesions. HLA-DRB1*1501 and the HLA-DRB1*11 sub-alleles DRB1*1101 and DRB1*1104 were significantly associated with the segmental length of cord lesions. CONCLUSIONS: Our study is the first to investigate the frequency of subpial wedge-shaped lesions in the cord in vivo and has provided preliminary evidence that HLA-DRB1 alleles may play a role in determining the severity and extent of spinal cord involvement in MS.

Clinical profile and HLA-DRB1 genotype of late onset multiple sclerosis in Western Australia.

We aimed to characterize the clinical profile and human leukocyte antigen (HLA)-DRB1 genotype of patients with late onset multiple sclerosis (LOMS) in Western Australia. The clinical features, laboratory studies and HLA-DRB1 alleles were analysed in patients with multiple sclerosis (MS) with onset over 50years of age and compared with 100 patients with early onset MS (EOMS). Of a cohort of 829 patients with MS, 73 (8.8%) presented at over 50years of age, including 14 (1.7%) over 60years. Patients with LOMS had a lower female to male ratio, more frequent initial motor dysfunction, less frequent sensory symptoms and optic neuritis, a more frequent primary-progressive course and shorter time to reach Extended Disability Status Scale (EDSS) scores of 3.0 and 6.0. More LOMS patients were initially misdiagnosed compared to patients with EOMS. HLA-DRB1 *1501 was strongly associated with both LOMS and EOMS compared to the Control subjects, while HLA-DRB1 *0801 was over-represented in patients with LOMS. We concluded that patients with LOMS have a different clinical profile when compared to those with EOMS. Carriers of HLA-DRB1 *0801 may be more prone to develop MS at a later age.

Influence of HLA-DRB1 allele heterogeneity on disease risk and clinical course in a West Australian MS cohort: a high-resolution genotyping study.

BACKGROUND: Previous studies on the influence of HLA-DRB1 alleles on multiple sclerosis (MS) susceptibility and clinical course have mostly employed the 2-point genotyping method. OBJECTIVE: To assess the influence of HLA-DRB1 alleles and allele interactions on disease risk and clinical course in a large West Australian MS patient cohort using high-resolution genotyping. METHODS: Four digit HLA-DRB1 genotyping was performed on a group of 466 clinically definite or probable MS patients from the Perth Demyelinating Diseases Database and 189 healthy Caucasian controls from the Busselton Community Health Study. RESULTS: In addition to the known risk allele HLA-DRB1*1501, evidence of increased susceptibility to MS was found for three additional alleles, DRB1*0405, DRB1*1104 and DRB1*1303, though the power was insufficient to sustain significance for these when crudely Bonferroni corrected over all alleles considered. DRB1*0701 was found to be protective even after correction for multiple comparisons. In addition we found evidence that the DRB1*04 sub-allele HLA-DRB1*0407 and HLA-DRB1*0901 may be protective. Among the diplotypes, the highest estimated risk was in HLA-DRB1*1501/*0801 heterozygotes and DRB1*1501 homozygotes and the lowest in HLA-DRB1*0701/*0101 heterozygotes. There was no significant gender association with HLA-DRB1*1501 overall, but the HLA-DRB1*1501/*1104 risk genotype was significantly associated with female gender. HLA-DRB1*1501 was the strongest risk allele in both primary progressive MS and relapsing-remitting MS. CONCLUSION: Our results demonstrate the advantages of high-resolution HLA genotyping in recognizing risk-modifying alleles and allele combinations in this patient cohort and in recognizing the differential effects of HLA-DRB1*04 and DRB1*11 sub-alleles.

Low level of systemic autoimmunity in Western Australian multiple sclerosis patients.

Previous autoantibody (AAb) studies in multiple sclerosis MS have produced conflicting results. The objective of this study was to determine AAb frequency and association with the HLA-DRB1 genotype. Antinuclear antibody, antithyroid peroxidase and anti-aquaporin-4 assays and HLA-DRB1 genotyping were performed in 198 MS patients and 188 controls. There were no significant differences in AAb frequency or titres between MS and control subjects. AQP4-IgG was not found in any MS patients. There was no correlation between AAbs and HLA-DRB1 alleles. In conclusion, this study failed to confirm previous reports of increased AAbs in MS or to show an association between HLA-DRB1 genotype and the presence of AAbs.

Presence of CSF oligoclonal bands (OCB) is associated with the HLA-DRB1 genotype in a West Australian multiple sclerosis cohort.

High-resolution HLA-DRB1 genotyping was performed in 97 OCB-positive and 68 OCB-negative cases with demyelinating disease to determine the influence of HLA-DRB1 alleles on the presence of OCB in a West Australian multiple sclerosis (MS) cohort. Carriage of the HLA-DRB1*1501 allele was associated with both OCB-positive and OCB-negative MS compared with controls, but more strongly with the OCB-positive group, and increased the likelihood of having OCB 2.1-fold with evidence of a dominant dose-effect. The HLA-DRB1*0301 allele was negatively correlated with OCB, with all homozygotes OCB-negative, suggesting a possible recessive protective effect of HLA-DRB1*0301. There was no significant correlation between OCB and the DRB1*04 alleles which have been associated with OCB-negative MS in previous Swedish and Japanese studies. Evidence of allelic interactions was found with HLA-DRB1*1501/*1301 heterozygotes having a reduced frequency of OCB and HLA-DRB1*0301/*0401 heterozygotes all being OCB-negative. These findings confirm the strong association between HLA-DRB1*1501 and OCB which has been found in other populations but indicate that the influence of other HLA-DRB1 alleles varies in different populations. Our study is the first to show that HLA-DRB1 allele interactions and dose-effects influence the frequency of OCB.

HLA-DRB1 allele heterogeneity influences multiple sclerosis severity as well as risk in Western Australia.

Susceptibility to multiple sclerosis (MS) has been consistently associated with the Human Leukocyte Antigen (HLA)-DRB11501 genotype, however effects on disease severity and clinical outcome have varied in different populations. We present the results of a high-resolution HLA-DRB1 genotyping and genotype-phenotype correlation study in a large West Australian MS cohort. Our findings indicate that in this population, which is of largely Anglo-Celtic and Northern European origin, HLA-DRB11501 is not only a strong determinant of disease risk but may also be associated with disease severity as measured by the Multiple Sclerosis Severity Score (MSSS), with the MSSS increasing by an estimated 0.51 per DRB11501 allele. We also found evidence that the HLA-DRB11201 allele is associated with less severe disease.

Modifying effects of HLA-DRB1 allele interactions on age at onset of multiple sclerosis in Western Australia.

The contribution of genetic factors to the age at onset in multiple sclerosis is poorly understood. Our objective was to investigate the disease modifying effects of HLA-DRB1 alleles and allele interactions on age at onset of multiple sclerosis. High-resolution four-digit HLA-DRB1 genotyping was performed in a cohort of 461 multiple sclerosis patients from the Perth Demyelinating Diseases Database. Carriage of the HLA-DRB1*1501 risk allele was not significantly associated with age at onset but HLA-DRB1*0801 was associated with a later onset of the disease. The HLA-DRB1*0401 allele was associated with a reduced age at onset when combined with DRB1*1501 but may delay age at onset when combined with DRB1*0801. These findings indicate that epistatic interactions at the HLA-DRB1 locus have significant modifying effects on age at onset of multiple sclerosis and demonstrate the value of high-resolution genotyping in detecting such associations.

Clinical applications of whole-blood PCR with real-time instrumentation.

BACKGROUND: As the genetic basis of many human diseases is being discovered, there is increasing need for the detection of single-nucleotide polymorphisms/mutations in medical laboratories. We describe an innovative approach that combines PCR amplification directly on whole blood and real-time detection PCR technology (WB-RTD PCR). METHODS: We compared WB-RTD PCR with the method for extracted DNA-RTD PCR for the detection of mutations in the prothrombin (n = 94), factor V Leiden (n = 49), and hemochromatosis (n = 22) genes. Mutation detection on the Roche LightCycler was based on use of fluorescence resonance energy transfer (FRET) probes and melting curve analysis. We also compared the WB-RTD PCR on the LightCycler and the ABI Prismtrade mark 7700 sequence detection system with minor groove- binding nonfluorescent quencher probes. RESULTS: We obtained complete concordance between both methods in assigning genotypes. We also demonstrated that the WB-RTD PCR method can be performed on real-time PCR instruments from Applied Biosystems and the LightCycler. Omission of the need for DNA extraction and gel electrophoresis allowed substantial labor and cost savings with this method. CONCLUSION: This approach has applications for testing other medically relevant single-nucleotide polymorphisms.