Identification of a new HLA-B*57 allele, HLA-B*57:71, by next-generation sequencing.

The sequence of a novel allele, HLA-B*57:71, differs from HLA-B*57:01:01 by three-nucleotide exchanges in exon 2.

Next-generation sequencing can reveal in vitro-generated PCR crossover products: some artifactual sequences correspond to HLA alleles in the IMGT/HLA database.

The high-resolution human leukocyte antigen (HLA) genotyping assay that we developed using 454 sequencing and Conexio software uses generic polymerase chain reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing property of the 454 system, were generally observed at a read depth of 5%-10% of the true alleles. They usually contained at least one mismatch with the IMGT/HLA database, and consequently, were easily recognizable and did not cause a problem for HLA genotyping. Sometimes, however, these artifactual sequences matched a rare allele and the automatic genotype assignment was incorrect. These observations raised two issues: (1) could PCR conditions be modified to reduce such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA database be artifacts rather than true alleles? Because PCR crossing over occurs during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, crossover products were detected. In 33% of the cases, these hybrid sequences corresponded to named alleles. With amplification for only 28 cycles, these artifactual sequences were not detectable. To investigate whether some rare alleles in the IMGT/HLA database might be due to PCR artifacts, we analyzed four samples obtained from the investigators who submitted the sequences. In three cases, the sequences were generated from true alleles. In one case, our 454 sequencing revealed an error in the previously submitted sequence.

High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ System for simplified amplicon library preparation.

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome; distinguishing the thousands of HLA alleles is challenging. Next generation sequencing of exonic amplicons with the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides high resolution, high throughput HLA genotyping for eight class I and class II loci. HLA typing of potential donors for unrelated bone marrow donor registries typically uses a subset of these loci at high sample throughput and low cost per sample. The Fluidigm Access Array System enables the incorporation of 48 different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples with up to 48 different primer pairs in a microfluidic device generating 2304 parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. During genomic PCR, in this 4-primer system, the outer set of primers containing the MID and the 454 adaptor sequences are incorporated into an amplicon generated by the inner HLA target-specific primers each containing a common sequence tag at the 5' end of the forward and reverse primers. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX System, followed by genotyping with Conexio software. We have genotyped 192 samples with 100% concordance to known genotypes using 8 primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per amplicon was obtained. We have also genotyped 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon.

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.

Multi-locus HLA class I and II allele and haplotype associations with follicular lymphoma.

Follicular lymphoma (FL) is an indolent, sometimes, fatal disease characterized by recurrence at progressively shorter intervals and is frequently refractive to therapy. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the human leukocyte antigen (HLA) region on chromosome 6p21.32-33 that are statistically significantly associated with FL risk. Low to medium resolution typing of single or multiple HLA genes has provided an incomplete picture of the total genetic risk imparted by this highly variable region. To gain further insight into the role of HLA alleles in lymphomagenesis and to investigate the independence of validated SNPs and HLA alleles with FL risk, high-resolution HLA typing was conducted using next-generation sequencing in 222 non-Hispanic White FL cases and 220 matched controls from a larger San Francisco Bay Area population-based case-control study of lymphoma. A novel protective association was found between the DPB1*03:01 allele and FL risk [odds ratio (OR) = 0.39, 95% confidence interval (CI) = 0.21-0.68]. Extended haplotypes DRB1*01:01-DQA1*01:01-DQB1*05:01 (OR = 2.01, 95% CI = 1.22-3.38) and DRB1*15-DQA1*01-DQB1*06 (OR = 0.55, 95% CI = 0.36-0.82) also influenced FL risk. Moreover, DRB1*15-DQA1*01-DQB1*06 was highly correlated with an established FL risk locus, rs2647012. These results provide further insight into the critical roles of HLA alleles and SNPs in FL pathogenesis that involve multi-locus effects across the HLA region.

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.

'I am part of the community but...' The changing context of rural living for persons with advanced cancer and their families.

INTRODUCTION: Older rural persons who are receiving palliative care experience multiple co-existing transitions that can be distressing. These transitions do not occur in a vacuum, but occur in a context that reflects the uniqueness of rural living and the complexities of end of life in rural settings. The context or situation (geographical, physical, and social) in which an experience occurs influences the way people view and interpret the world around them; this contextual perspective contributes greatly to perceptions held by rural residents. PURPOSE: The purpose of this study was to explore the context in which older rural patients receiving palliative care and their families experience transitions. Following a study of the transition experiences of older rural palliative patients, an in-depth interpretive description analysis was conducted specific to the context in which the participants' transitions occurred. METHODS: Twenty-seven open-ended, individual, audio-taped, qualitative interviews were conducted and 4 focus group discussions were held to gather data. Individual audio-taped interviews were conducted with six older rural persons with advanced cancer and 10 bereaved (post-death) family caregivers. Four focus groups were conducted with 12 palliative care healthcare professionals. Participants were recruited from 3 rural health regions in a western Canadian province classified as one of the most 'rural' Canadian provinces. All interviews were transcribed verbatim, coded, and analyzed using Thorne's interpretive description qualitative approach. RESULTS: From the data analysis four themes emerged: (1) community connectedness/isolation; (2) lack of accessibility to care; (3) communication and information issues; and (4) independence/dependence. Participants described feelings of being connected to the community at the same time as they also reported feeling isolated. They described their value of independence at the same time as finding themselves becoming increasingly dependent on others. At times this value of independence interfered with their seeking and accessing needed health or supportive care. They perceived their lack of access to health care resulted in little or no choice in where they die. CONCLUSIONS: These findings reveal that the rural context has a major impact on the types of community support and healthcare services needed by older persons with advanced disease and their families. With advanced disease, the participants' sense of solitude became one of isolation, and with increasing dependence on others, they needed more connection and support from others. The findings reflected a more complex view of rural aging and dying than has been cited in the literature to date. This study suggests there is a need to renegotiate community supports and the independence available to persons with advanced disease as they undergo multiple transitions near the end of life.

Palliation of dyspnoea in advanced COPD: revisiting a role for opioids.

Chronic obstructive pulmonary disease (COPD) will be the third leading cause of death worldwide by 2020. The burdens of this increasingly prevalent illness borne by patients, their family caregivers and the healthcare system are substantial. Dyspnoea as the predominant symptom becomes increasingly difficult to palliate as COPD progresses through advanced stages and, for 50% of patients, can become refractory to conventional treatment. This narrative review focuses on the potential role for carefully initiated and titrated opioids in the management of dyspnoea for patients with advanced COPD who are not yet in a terminal stage, yet struggle with symptoms that reflect underlying mechanisms of dyspnoea that lend themselves to this approach. The many barriers that currently exist to the provision of opioids in this setting are addressed, and recommendations are provided for an approach that should engender confidence among patients, their caregivers and the physicians who treat them.

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.

Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing.

Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing.

Assessing the psychosocial consequences of epilepsy: a community-based study.

BACKGROUND: Few studies have measured, using validated scales, the psychosocial handicap of epilepsy in a general practice setting. AIM: To assess the prevalence of psychosocial problems associated with epilepsy. METHOD: A survey was undertaken of 309 subjects, with one or more non-febrile epileptic seizures, drawn from two general practices in the United Kingdom (UK). The outcome measures were the Subjective Handicap of Epilepsy Scale (SHE), the SF-36, and the Hospital Anxiety and Depression scale (HAD). RESULTS: One-third of persons with active epilepsy were significantly handicapped by their condition. The severity of subjective handicap was related to seizure frequency and to the duration of remission of seizures. Between one-third and one-half of subjects scored as 'cases' on the HAD scale and on the mental health subscale of the SF-36. Only one-third of the psychiatric morbidity revealed by the questionnaires had been recognized by the general practitioner (GP). Scores on the SF-36 indicated that people with active seizures perceived themselves as significantly less healthy than those in remission, and that, for persons in remission, drug treatment had a detrimental effect on certain aspects of well-being. CONCLUSIONS: The occurrence of seizures, even at low frequencies, is associated with psychosocial handicap, and this may remain covert in general practice.

Trials of an inactivated equid herpesvirus 1 vaccine: challenge with a subtype 1 virus.

Vaccinated yearlings , two-year-old and in-foal pony mares with appropriate controls were exposed to aerosols of a subtype 1 virus one to two months after two or three vaccinations; all became infected. No obvious differences in the febrile responses, clinical signs and subsequent abortions were found between vaccinated and control mares. All vaccinated yearlings and two-year-old ponies developed a febrile respiratory disease but this was less severe than that suffered by the controls and the amounts and duration of virus shedding were reduced.

Field and laboratory studies of equine influenza viruses isolated in 1979.

Experimental ponies developed signs of disease four days after the intranasal instillation of A/England 1/79 equine influenza virus and virus was recovered from the nasopharynx from the second to the ninth day. No significant antigenic difference was found between the virus and the prototype A/Miami 1/63 virus, using post infection ferret and chicken sera and post vaccination pony sera. No antigenic differences were found between four viruses isolated between January and July 1979, although some differences were found in their ability to detect haemagglutination inhibiting antibody in convalescent horse sera.

Risk-assessment scores, prevention strategies, and the incidence of pressure ulcers among the elderly in four Canadian health-care facilities.

The accurate prediction of pressure ulcer (PU) development among hospitalized elderly patients is a complex endeavour. A prospective, longitudinal, cohort study of 330 patients over age 65 in 2 Canadian tertiary-care teaching hospitals and 2 long-term-care facilities examined the association between risk-assessment scores, prevention strategies, and PU incidence. The overall PU incidence rate was 9.7%, with half of the subjects who developed a PU doing so in the first week of hospitalization. The incidence rate for "at risk" patients (10.1%) was similar to the rate for "not at risk" patients (9.3%). The number of prevention strategies used was related to risk-assessment scores and to PU development. Paradoxically, the incidence rate increased with the number of prevention strategies employed. The total risk-assessment score that appeared to have the best balance of sensitivity (69%) and specificity (55%) was 19. Four of the 6 risk-assessment subscales were associated with PU development. Logistic regression modelling confirmed the univariate results that the number of prevention strategies used was the best single predictor of PU development. The data confirm that predicting PU development for individual patients is difficult at best. Results suggest that use of a risk-assessment scale alone is not sufficient to accurately predict PU development. The clinical judgement and experience of nurses are required in providing supplementary information to standard measurement instruments.

Pressure ulcer risk assessment tools. What's new for gerontological nurses.

Impaired skin integrity is one of the most common and most costly problems facing the institutionalized elderly. Risk assessment tools provide the nurse with an efficient and systematic means of identifying those clients at high risk for pressure ulcer development. Pressure ulcer risk assessment tools are usually based on a combination of the following variables: mobility, continence, mental status, activity, nutrition, or physical status. Further testing and comparison of the various pressure ulcer risk assessment tools in the elderly population is needed to establish relative scientific and practical merit.

Long-term risk of developing epilepsy after febrile seizures: a prospective cohort study.

OBJECTIVE: We report the prospective follow-up of a cohort of people from the onset of febrile seizures for a median of 24 years to estimate the long-term risk of developing epilepsy. METHODS: The National General Practice Study of Epilepsy is a large prospective community study of 1,195 people with a first suspected seizure followed from the 1980s, of whom 220 (18%) had febrile seizures. Standardized incidence ratios (SIRs) with 95% confidence intervals (CIs) for subsequent epilepsy were calculated in 5-year age bands. RESULTS: Follow-up information was obtained for 181 (83%) people with a mean follow-up for the whole cohort of 21.6 (SD 6.0) years. Of these, 175 (97%) were seizure-free in the preceding 5 years, whereas 171 (94%) were seizure-free and off antiepileptic drugs. Six percent developed epilepsy, but the risk of developing epilepsy in the cohort over the whole follow-up period was almost 10 times that of the general population (SIR 9.7, 95% CI 5.7-16.4). The SIR was significantly elevated in the 0- to 14-year age groups but not in the 15- to 19-year age group (SIR 4.5, 95% CI 0.6-32.1). CONCLUSION: The risk of developing epilepsy in people who had febrile seizures seems to decrease with time. Further long-term studies are needed to confirm this.

Alpha-1 antitrypsin deficiency targeted testing and augmentation therapy: a Canadian Thoracic Society clinical practice guideline.

Alpha-1 antitrypsin (A1AT) functions primarily to inhibit neutrophil elastase, and deficiency predisposes individuals to the development of chronic obstructive pulmonary disease (COPD). Severe A1AT deficiency occurs in one in 5000 to one in 5500 of the North American population. While the exact prevalence of A1AT deficiency in patients with diagnosed COPD is not known, results from small studies provide estimates of 1% to 5%. The present document updates a previous Canadian Thoracic Society position statement from 2001, and was initiated because of lack of consensus and understanding of appropriate patients suitable for targeted testing for A1AT deficiency, and for the use of A1AT augmentation therapy. Using revised guideline development methodology, the present clinical practice guideline document systematically reviews the published literature and provides an evidence-based update. The evidence supports the practice that targeted testing for A1AT deficiency be considered in individuals with COPD diagnosed before 65 years of age or with a smoking history of <20 pack years. The evidence also supports consideration of A1AT augmentation therapy in nonsmoking or exsmoking patients with COPD (forced expiratory volume in 1 s of 25% to 80% predicted) attributable to emphysema and documented A1AT deficiency (level ≤11 µmol/L) who are receiving optimal pharmacological and nonpharmacological therapies (including comprehensive case management and pulmonary rehabilitation) because of benefits in computed tomography scan lung density and mortality.

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.