A multi-site study using high-resolution HLA genotyping by next generation sequencing.

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

High-resolution, high-throughput HLA genotyping by next-generation sequencing.

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome. Hematopoietic stem cell transplantation requires allele-level HLA typing at multiple loci to select the best matched unrelated donors for recipient patients. In current methods for HLA typing, both alleles of a heterozygote are amplified and typed or sequenced simultaneously, often making it difficult to unambiguously determine the sequence of the two alleles. Next-generation sequencing methods clonally propagate in parallel millions of single DNA molecules, which are then also sequenced in parallel. Recently, the read lengths obtainable by one such next-generation sequencing method (454 Life Sciences, Inc.) have increased to >250 nucleotides. These clonal read lengths make possible setting the phase of the linked polymorphisms within an exon and thus the unambiguous determination of the sequence of each HLA allele. Here we demonstrate this capacity as well as show that the throughput of the system is sufficiently high to enable a complete, 7-locus HLA class I and II typing for 24 or 48 individual DNAs in a single GS FLX sequencing run. Highly multiplexed amplicon sequencing is facilitated by the use of sample-specific internal sequence tags (multiplex identification tags or MIDs) in the primers that allow pooling of samples yet maintain the ability to assign sequences to specific individuals. We have incorporated an HLA typing software application developed by Conexio Genomics (Freemantle, Australia) that assigns HLA genotypes for these 7 loci (HLA-A, -B, -C, DRB1, DQA1, DQB1, DPB1), as well as for DRB3, DRB4, and DRB5 from 454 sequence data. The potential of this HLA sequencing system to analyze chimeric mixtures is demonstrated here by the detection of a rare HLA-B allele in a mixture of two homozygous cell lines (1/100), as well as by the detection of the rare nontransmitted maternal allele present in the blood of a severe combined immunodeficiency disease syndrome (SCIDS) patient.

Aortic morphology following endovascular repair of acute and chronic type B aortic dissection: implications for management.

OBJECTIVE: The study aimed to define early clinical outcomes, and medium term morphological changes, following endovascular treatment of acute (AAD) and chronic (CAD) Type B aortic dissections. MAIN OUTCOMES: The cohort comprised 78 patients who underwent endovascular repair for AAD (38) and CAD (40). Early and late clinical outcomes were prospectively recorded. All patients underwent serial follow up with CT scanning. False lumen thrombosis rates, true, false and total aortic short axis diameter were recorded at the mid point of the endograft and below this level in the thoracic aorta. The total maximum aortic diameter in the thoracic, abdominal aorta was quantified. RESULTS: The 30-d mortality was 2.6% in AAD and 7.5% in CAD. The 30-d stroke and paraplegia rates were 5.3% and 0% in AAD. There were no cases of stroke or paraplegia in patients with CAD. At 30 months follow up, the cumulative survival for the two groups was 93% for AAD and 66.5% for CAD (P=0.015, Kaplan Meier) and the cumulative re-intervention rate was 62% and 55% in AAD and CAD respectively (P=0.961, Kaplan-Meier). False lumen thrombosis rates were equivalent in the two groups and were higher at the level of the endograft than below this level (P<0.05). Aortic remodelling was greater in AAD, whereas the aortic dimensions after treatment of CAD remained relatively static. Up to 20% of patients in both groups demonstrated enlargement of the thoracic aorta. CONCLUSIONS: The data support the use of endovascular repair of the thoracic aorta in Type B aortic dissection. 30-d outcomes are acceptable. Patients with AAD demonstrate significant aortic remodelling whereas patients with CAD do not. This has significant implications for practice as patients with CAD must rely on maintenance of false lumen thrombosis to preserve the integrity of the endovascular repair.

Treatment response and durability of a double protease inhibitor therapy with saquinavir and ritonavir in an observational cohort of HIV-1-infected individuals.

OBJECTIVE: To evaluate treatment response, durability and tolerance of a four-drug regimen including saquinavir and ritonavir in combination with either zidovudine/lamivudine or stavudine/lamivudine. DESIGN: Observational cohort of HIV-positive individuals. METHODS: Viral load, CD4+ and CD8+ T lymphocyte counts were assessed at intervals of 1-3 months in subjects commencing therapy between July 1996 and November 1996. Adverse events were evaluated as well as risk factors for therapeutic failures. RESULTS: A group of 56 male patients were included and followed for 48 weeks. Of these, 66% had already taken a protease inhibitor. Viral load dropped by a median 1.98 log10 HIV RNA copies/ml from baseline (interquartile range: 1.49-2.46) and became undetectable (< 400 copies/ml) in 68% of patients. Response varied: 9% were non-responders (HIV RNA reduction < 0.5 log10 copies/ml) and 23% were incomplete responders (nadir of HIV RNA > 400 copies/ml). After 48 weeks, viral load remained undetectable in 49%. Median CD4+ T lymphocyte count increased from 191 x 10(6) to 418 x 10(6) cells/l (range, 241-537 x 10(6) cells/l). Although protease inhibitor and nucleoside pretreatment selected for drug-resistant viral mutants, only the protease inhibitor experience was identified as a risk factor for therapeutic failure. Adverse events occurred in 73% of patients and led to a change of therapy in 9%. CONCLUSION: Despite advanced HIV disease and pretreatment with multiple antiretroviral drugs, a strong initial treatment response to this drug regimen was observed. However, virological failure occurred in 51% of patients after 48 weeks and frequent adverse events complicated therapy.

A randomised, open-label comparison of three highly active antiretroviral therapy regimens including two nucleoside analogues and indinavir for previously untreated HIV-1 infection: the OzCombo1 study.

BACKGROUND: Highly active antiretroviral therapy (HAART) including two nucleoside analogues and a potent protease inhibitor is standard of care initial therapy for HIV-infected adults. The best-tolerated and most potent initial HAART regimen is unknown and was investigated in this study. METHODS: One hundred and nine HIV-infected adults with no prior antiretroviral therapy, and CD4 lymphocyte counts < 500 x 10(6) cells/l or plasma HIV RNA > 30,000 copies/ml were randomized to zidovudine-lamivudine-indinavir (ZDV-3TC-IDV), stavudine-lamivudine-indinavir (d4T-3TC-IDV) or stavudine-didanosine-indinavir (d4T-ddI-IDV) for 52 weeks. The primary endpoints were plasma HIV RNA and drug-related adverse events. Other assessments were overall safety, adherence and adverse events, CD4 lymphocyte counts, cutaneous delayed type hypersensitivity (DTH) responses and quality of life (Euroqol). RESULTS: Only 58% patients had HIV RNA < 50 copies/ml plasma at 12 months, with no significant difference between the three regimes (P = 0.34). Drug-related adverse events sufficiently severe to warrant drug discontinuation were less common (P = 0.06) in patients receiving d4T-3TC-IDV (18%) than in those receiving ZDV-3TC-IDV (34%) or d4T-ddI-IDV (41%). The percentages of patients who remained on their assigned therapy with plasma HIV RNA < 50 copies/ml at 52 weeks were 60% with d4T-3TC-IDV, 53% with ZDV-3TC-IDV and 35% with d4T-ddI-IDV. Virological failure at 52 weeks was more likely in those whose adherence was estimated to be < 100% in the first 4 weeks of therapy (P = 0.02), but not in those who developed grade 3 or 4 drug-related adverse events. At 52 weeks, the mean CD4 lymphocyte count increase was 200 x 10(6) cells/l with only 7% of patients having counts lower than at baseline; DTH responses improved but remained clinically impaired in most patients. Quality of life improved significantly in all groups. CONCLUSIONS: Initial HAART regimens including IDV failed to suppress plasma HIV RNA to < 50 copies/ml in > 40% patients after only 12 months of therapy although there was significant overall improvement immunologically and in quality of life. The type of dual nucleoside combination used was less important in predicting virological failure than was imperfect adherence early in therapy. Consideration should be given to modifying a HAART regimen relatively early in non-adherent patients.

Population frequencies and putative haplotypes of the killer cell immunoglobulin-like receptor sequences and evidence for recombination.

BACKGROUND: The killer cell immunoglobulin-like receptors (KIR) are a family of receptors expressed on natural killer (NK) cells and some T cells. Class I HLA molecules on target cells are the ligands for the KIR receptors. The number of KIR genes has been reported to vary between individuals, resulting in different KIR haplotypes. There is little published data on the frequency of each KIR gene and the linkage disequilibrium between the genes. Because there is evidence that NK cells may be involved in bone marrow transplant rejection, we have determined the KIR gene frequencies and possible haplotypic arrangements by linkage disequilibrium analysis in an Australian population. METHODS: Controls, patients with leukemia, and unrelated bone marrow donor-recipient pairs were typed for the presence of 11 KIR genes by polymerase chain reaction-sequence specific priming. RESULTS: Ninety percent of the population was found to have a sufficient number and variety of KIR genes to detect any mismatch of HLA-A, -B, and -C alleles on NK target cells. The 11 KIR genes could be divided into two groups based on linkage disequilibrium between pairs of genes. Evidence for a recombination within the KIR gene complex is presented. CONCLUSION: Typing for the presence of particular KIR genes may be indicated for bone marrow donor-recipient pairs for whom a class I HLA mismatch is unavoidable.

Unrelated donors selected prospectively by block-matching have superior bone marrow transplant outcome.

Previous retrospective studies have demonstrated improved outcome in patients whose donors were matched for non-HLA markers in the MHC as well as for HLA genes. Forty patients receiving transplants from unrelated donors were typed prospectively for HLA and non-HLA markers. Non-HLA markers near HLA-B (beta-block markers) and in the DRB1 introns (delta-block markers) were used to assess MHC match between donors and recipient. Patients whose donors were matched at the beta- and delta-blocks had improved event free survival (63%) compared to patients whose donors were mismatched at one or both blocks (25%) (p < 0.05). Patients whose donors were matched at the beta-block had significantly less severe acute graft versus host disease (p < 0.05). In order to investigate the basis for improved outcome block matching was correlated with HLA matching as determined by DNA sequencing. Beta-block matching was highly correlated with matching for exons 2 and 3 of HLA-B but less so for HLA-C. Delta-block matching was highly correlated with matching for exon 2 of HLA DRB1. It is concluded that matching for non-HLA markers in the MHC improves matching for HLA genes. Further studies are required to determine whether matching for non-HLA markers improves outcome to a greater extent than matching for the HLA genes alone.

A quality control program for crossmatching procedures for solid organ transplantation. The participating laboratories of the Australasian and South Asian Tissue Typing Association.

The National Kidney Matching Scheme (NKMS) allows matching and allocation of donor organs throughout Australia. Sera from potential recipients are distributed to each interstate tissue typing laboratory on a monthly basis for crossmatching in the event of a cadaver donor. Therefore, it is essential there is consensus for results between these laboratories in order for donated organs to be allocated appropriately. A quality control program conducted under the auspices of ASEATTA was undertaken for (1) panel reactive antibody characterization; (2) routine T lymphocyte crossmatching; and (3) characterization of antibody isotype by DTT treatment. These aims were achieved by distribution of (1) six sera for the determination of PRA activity; (2) 20 scrambled trays including replicate dilutions of a strongly positive lymphocytotoxic serum, high titer monoclonal antibody and negative sera and; (3) 20 trays containing sera with IgG and/or IgM antibodies. These were then evaluated by each laboratory on a panel of T cells. There was concordance between laboratories for PRA levels and antibody characterization. However, there was considerable variation in crossmatch sensitivity and reproducibility, several laboratories had carryover and others could not detect weak IgM antibodies. These results demonstrate the utility and need for ongoing crossmatch exchange programs, particularly for laboratories participating in organ exchange programs.

Structure and polymorphism of two stress-activated protein kinase genes centromeric of the MHC: SAPK2a and SAPK4.

As MHC genes are potent determinants of susceptibility to immunopathological diseases, the mapping of SAPK2a (CSBP) and SAPK4 to chromosome 6p 21.2-21.3 suggested that these genes may mediate the effects of the MHC on disease. Here we describe the genomic structure and localisation of both genes approximately 2.3Mb centromeric of HLA-DP. Examination of the complete coding region and selected intronic regions of SAPK2a and SAPK4 from 22 human EBV-transformed B-cell lines of different MHC haplotypes and racial background revealed complete sequence conservation. There were no notable differences in levels of expression of SAPK2a and SAPK4 mRNA in cell lines of different MHC haplotypes or racial origin. Examination of the SAPK2a and SAPK4 sequences from two chimpanzees revealed 3 nucleotide differences between human and chimpanzee in each gene resulting in only one amino acid change in SAPK4, and 6 nucleotide substitutions plus 2 deletions in 600bp of intronic sequence from SAPK4. This highlights the selective pressure placed on these genes to maintain their protein sequence, but does not favour a role in genetic regulation of disease or provide evidence of linkage disequilibrium with the MHC.

Next-generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing.

Human leukocyte antigen (HLA) typing has been a challenge for more than 50 years. Current methods (Sanger sequencing, sequence-specific primers [SSP], sequence-specific oligonucleotide probes [SSOP]) continue to generate ambiguities that are time-consuming and expensive to resolve. However, next-generation sequencing (NGS) overcomes ambiguity through the combination of clonal amplification, which provides on-phase sequence and a high level of parallelism, whereby millions of sequencing reads are produced enabling an expansion of the HLA regions sequenced. We explored HLA typing using NGS through a three-step process. First, HLA-A, -B, -C, -DRB1, and -DQB1 were amplified with long-range PCR. Subsequently, amplicons were sequenced using the 454 GS-FLX platform. Finally, sequencing data were analyzed with Assign-NG software. In a single experiment, four individual samples and two mixtures were sequenced producing >75 Mb of sequence from >300,000 individual sequence reads (average length, 244 b). The reads were aligned and covered 100% of the regions amplified. Allele assignment was 100% concordant with the known HLA alleles of our samples. Our results suggest this method can be a useful tool for complete genomic characterization of new HLA alleles and for completion of sequence for existing, partially sequenced alleles. NGS can provide complete, unambiguous, high-resolution HLA typing; however, further evaluation is needed to explore the feasibility of its routine use.

14th International HLA and Immunogenetics Workshop: report from the sequencing-based typing group.

Sequencing-Based Typing (SBT) provides the golden standard for the identification of polymorphism among the human leukocyte antigen (HLA) alleles. Several SBT approaches have been published. Updated protocols for HLA-DRB and -DQB were presented and an approach for DQA, covering all exons, validated. The highest level of allele typing can be realized with strategies for resolving allele and genotype ambiguities. Bioinformatics provides the tools for exchange and sharing data with a community standard in XML format.

Pilot study: assessment of interlaboratory variability of sequencing-based typing DNA sequence data quality.

This pilot study showed that variability in the sequence quality scores exists within and between laboratories performing human leukocyte antigen typing by sequence-based typing (SBT). It also showed that the base call score (BCS) system in Assign-SBT is a useful tool for comparing sequence quality between laboratories.

Sequencing-based typing identifies novel alleles due to single nucleotide polymorphisms in 'conserved' regions.

The Royal Perth Hospital laboratory has been using sequencing-based typing for all HLA loci since 2002. In the period to October 2005, approximately 12,000 HLA A and HLA B, 5000 HLA C and DQB1, and 17,000 DRB1 requests have been processed. Twenty nine novel alleles have been identified in that time. These comprise 10 HLA-A (including one null allele), five HLA-B, six HLA-C, six DRB1 (including a null allele), and one DQB1 novel allele. (At the time of identifying the DRB1 null allele, there were no other reported examples.) In addition, we have seen one example of a blast-specific HLA-A null allele. One HLA-A allele (HLA-A*0264) and one HLA-B allele (HLA-B*400104) were subsequently identified in other laboratories and submitted to the international ImMunoGeneTics project (IMGT) database.

Identification of a new allele in a Sicilian individual: HLA-DPB1*0302.

We report here the identification and characterization of a novel human leucocyte antigen (HLA)-DPB1 allele that was subsequently named HLA-DPB1*0302 by the WHO Nomenclature Committee. HLA-DPB1*0302 was identified in a single Sicilian individual by a combination of sequence-specific primers, reverse line sequence-specific oligonucleotide probing and DNA sequencing-based typing. The DPB1*0302 allele is most similar to the DPB1*3101 allele, differing by a single mismatch at nucleotide position 301 (T to G).

Does a central MHC gene in linkage disequilibrium with HLA-DRB1*0401 affect susceptibility to type 1 diabetes?

Subtypes of HLA-DR4 are associated with susceptibility or protection against type 1 diabetes (T1DM). We addressed whether this reflects linkage disequilibrium with the true susceptibility locus by studying broader MHC haplotypes marked by alleles of HLA-B, IKBL (adjacent to TNFA) and complement C4. The study used a largely Caucasian cohort from Western Australia. HLA-DRB1*0401 and HLA-DRB1*0405 marked susceptibility to T1DM. In Caucasians, DRB1*0401 occurs predominantly in the 44.1 ancestral haplotype (AH; HLA-A2,B44, DRB1*0401,DQB1*0301) and the 62.1AH (HLA-A2,B15(62),DRB1*0401,DQB1*0302). HLA-B15 marked susceptibility and HLA-B44 marked with resistance to T1DM in patients and controls preselected for HLA-DRB1*0401. A gene between TNFA and HLA-B on the 8.1AH (HLA-A1,B8,;DR3,DQ2) modifies the effects of the class II alleles. Here, alleles characteristic of the 62.1AH (C4B3, IKBL+446*T and HLA-A2,B15) were screened in donors preselected for HLA-DRB1*0401. C4B3 was associated with diabetes, consistent with a diabetes gene telomeric of MHC class II. However, increases in carriage of IKBL+446*T and HLA-A2,B15 were marginal, as too few control subjects were available with the diabetogenic alleles. However, with these tools, selection of HLA-DRB1*0401, DQB1*0302 donors who are positive and negative for C4B3 will allow bidirectional mapping of diabetes genes in the central MHC.

HLA-B*27 typing by sequence specific amplification without DNA extraction.

HLA-B27 appears to play a direct role in the pathogenesis of ankylosing spondylitis and almost all patients with this disease have HLA-B27. Therefore, a diagnosis of ankylosing spondylitis can virtually be excluded in the absence of HLA-B27. Many techniques have been used for HLA-B*27 typing. Of these, molecular methods are the most sensitive and specific but require extracted DNA as the testing material. A technique where HLA-B*27 is amplified directly from whole blood using sequence specific primers has been developed. This technique uses small sample volumes, is not restricted by choice of anticoagulant or sample age up to at least six weeks, and can be applied to other clinical polymerase chain reaction based procedures.

Assign 2.0: software for the analysis of Phred quality values for quality control of HLA sequencing-based typing.

As improvements to DNA sequencing technology have resulted in increasing the throughput of DNA sequencing, the bottleneck for high throughput DNA sequencing-based typing (SBT) has shifted to sequence analysis, genotyping and quality control (QC). Consistent high-quality DNA sequence is required in order to reduce manual verification and editing of sequence electropherograms. However, identifying systematic changes in quality is difficult to achieve without the aid of sophisticated sequence analysis programs dedicated to this purpose. We describe a computer software program called Assign 2.0, which integrates sequence QC analysis and genotyping in order to facilitate high-throughput SBT. Assign 2.0 performs an analysis of Phred quality values in order to produce quality scores for a sample and a sequencing run. This enables sample-to-sample and run-to-run QC monitoring and provides a mechanism for the comparison of sequence quality between various genes, various reagents and various protocols with the aim of improving the overall quality of DNA sequence data. This, in turn, will result in reducing sequence analysis as a bottleneck for high-throughput SBT.

HLA-DRB1 DNA sequencing based typing: an approach suitable for high throughput typing including unrelated bone marrow registry donors.

The HLA-DRB1 sequencing based typing strategies reported to date require separate amplifications of each sample with a series of group-specific primers followed by sequencing of any resulting polymerase chain reaction (PCR) products. Whilst this results in high resolution typing in the majority of cases, a number of unnecessary amplifications are performed. We report here a novel approach where amplification of the second exon of HLA-DRB1 is performed in a single tube for all alleles. Retrospective analysis of 642 consecutive Western Australian unrelated bone marrow registry donors has shown that this approach results in unambiguous typings in 71.1% of cases. Ambiguities can be readily resolved if necessary with a single additional sequencing reaction on the original PCR product.

Increasing right hand dominance with age on a motor skill task.

A standardized pegboard task was used to investigate changes in manipulative skill as a function of age in 119 right-handed subjects. The typical pattern of cognitive impairment in old age indicates a relative preservation of functions which depend on the integrity of the left hemisphere. In accord with these observations, we predicted that, with increasing age, right hand motor skills would be better preserved than left hand skills. We found this on initial exposure to the task (P less than 0.01); however, the phenomenon was masked by practice, because older subjects (over 60 years of age) derived more improvement to their left hand motor skill, as a result of practice, than they did to their right hand skill (P less than 0.05). The asymmetrical effects of ageing on motor skill may be relevant to the increasing prevalence of emotional lability and neurosis in the elderly, since emotional control is thought to be dependent on right hemisphere mechanisms.