Validation of two commercial real-time PCR assays for rapid screening of the HLA-B*57:01 allele in the HIV clinical laboratory.

The pharmacogenetics approach to screen for the presence of the HLA-B*57:01 allele in HIV-1 infected patients is mandatory to prevent the potential development of hypersensitivity reaction to abacavir treatment. Given the limitations of current genotype methodologies, commercial real-time PCR assays were specifically developed for this purpose, but have not been sufficiently validated and are still not widely used. Here, in the context of the HIV laboratory, we assessed the ability of two commercial kits, the LightSNiP rs2395029 HPC5 assay (TIB Molbiol) and the DuplicαReal-TimeHLA-B*5701 Genotyping kit (Euroclone), to retrospectively detect HLA-B*57:01 positive and negative samples of Israeli HIV-1 infected patients. The LightSNiP rs2395029 HPC5 assay had false-positive results, whereas the DuplicαReal-Time HLA-B*5701 Genotyping kit was highly accurate and could be readily implemented into clinical practice. It is hoped that this study will facilitate the assessment of additional commercial kits for HLA-B*57:01 detection and expand their use in the clinical laboratory. Such studies can likely help the use of abacavir treatment in HIV-1 infected patients.

A novel HLA-B allele, HLA-B*35:279, identified by sequencing-based typing in a Czech patient.

The identification of a novel HLA-B*35:279 allele in a Czech patient is described. This allele is identical to the B*35:03:01 variant except the G/A nucleotide exchange at position 652 of the HLA-B gene that corresponds to the amino acid substitution from valine to isoleucine in alpha 3 domain of the HLA-B antigen.

A novel HLA-B*40 allele, B*40:01:40, identified in a Chinese individual.

A new allele, officially named B*40:01:40, was detected in a Chinese individual by sequence-based typing (SBT). The new allele differs from B*40:01:01 by a single nucleotide exchange at position 99 in codon 9, which results in synonymous substitution and seems not to compromise the HLA complex and T-cell receptor interaction.

Detection of HLA-B*57:01 by real-time PCR: implementation into routine clinical practice and additional validation data.

AIM: HLA-B*57:01 status needs to be determined before initiating abacavir therapy. We developed a pharmacogenetic real-time (Q)-PCR screening test using two sets of sequence specific primers. This test has been implemented into routine clinical practice. MATERIALS & METHODS: HIV-infected patients admitted at our University Hospital were thus genotyped using the above mentioned test. A panel of 80 DNA samples with a known genotype were used to characterize Q-PCR conditions using different master mixes. RESULTS: A total of 353 patients were genotyped, detecting 15 (4.25%) HLA-B*57:01 positive carriers. Among the negative patients, 17.2% were treated with abacavir without any hypersensitivity reaction. Using different Q-PCR master mixes, significantly lower cutoff Ct values were found, thus new analytical settings are provided. CONCLUSION: The pharmacogenetic test developed in our laboratory for the fast screening of HLA-B*57:01 can be successfully implemented into routine clinical practice. All 16 sequences (including an additional six) currently known for the HLA-B*57:01 allele are detected by sequence specific primers used in this test. The Brilliant II SYBR(®) Green QPCR MM (Stratagene) can safely replace the master mix originally used to develop the test.

Next-generation HLA sequencing using the 454 GS FLX system.

Next-generation sequencing (NGS) of HLA class I and II loci (HLA-A, HLA-B, HLA-C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPB1) is described here in detail using the 454 Life Sciences GS FLX System and Titanium chemistry. An overview of the protocol with our experience on sequence performance efficiencies, read depth and ambiguity analyses using the GS FLX System are also presented. A total of 14 HLA primer pairs with multiplex identifiers (MIDs) are used in clonal, amplicon-based pyrosequencing of up to 44 samples per plate using the GS FLX. Genotype assignment and ambiguity reduction -analysis is performed using Conexio Assign ATF 454 software. Clonal NGS gives a significant reduction in genotyping ambiguity during analysis of the highly complex HLA system.

Sequence-based typing identified a new HLA-B*40 allele, HLA-B*40:124:02.

The sequence of a novel allele, HLA-B*40:124:02, differs from HLA-B*40:42 by three-nucleotide exchanges in exon 3.

Úlcera genital aguda no relacionada con enfermedad de transmisión sexual / Acute genital ulcer unrelated to a sexually transmitted disease

No disponible

Behçet disease presenting with frosted branch angiitis.

The authors present a case of Behçet disease presenting with frosted branch angiitis. Frosted branch angiitis is a rare clinical finding and there are only two reported cases in the literature associated with Behçet disease.

Cost-effectiveness analysis of HLA-B*5701 typing in the prevention of hypersensitivity to abacavir in HIV+ patients in Spain

Introduction Approximately 4% to 8% of patients with HIV-1 treated with abacavir present a hypersensitivity reaction (HSR). Various studies have shown a direct association between human leukocyte antigen (HLA)-B*5701 and HSR to abacavir. The objective of this study was to analyze whether systematic HLA-B*5701 testing to prevent HSR in patients treated with abacavir is a cost-effective option for the Spanish National Health System .Methods An analytical decision-making model was constructed as a decision tree model for a simulated cohort of 1000 HIV patients to evaluate whether HLA-B*5701 testing to prevent HSR to abacavir was cost effective compared with not performing the test. The parameters included in the model and the use of healthcare resources should the patient develop HSR were taken from the PREDICT-1 study and the opinion of clinical experts. The principal result obtained was the incremental cost per HSR avoided. The time horizon of the analysis was 6 months. All costs were expressed in 2008 Euros. Results The analysis showed that the total direct healthcare costs per patient were €1344 and €1322 with and without HLA-B*5701 testing respectively, and that 36 cases of HSR were prevented per 1000 screened patients. These results yielded a cost per HSR avoided of €630. The sensitivity analysis showed that the results were sensitive to the cost of the test, with an economic saving of €102 or a cost-effectiveness ratio of €4234. Conclusions The model predicts that generalized use of the HLA-B*5701 test before prescribing abacavir in HIV+ patients could represent an economic saving or a limited additional cost for the National Health System which may be counterbalanced by the benefits in terms of a lower incidence of HSR (AU)

Introducción Aproximadamente el 4–8% de los pacientes con VIH-1 tratados con abacavir presentan una reacción de hipersensibilidad (RHS). Diversos estudios han mostrado que existe una asociación directa entre el antígeno leucocitario humano (HLA)-B*5701 y la RHS a abacavir. El objetivo del presente estudio ha sido analizar si la realización sistemática del test HLA-B*5701 para prevenir la RHS en los pacientes tratados con abacavir es una opción coste-efectiva para el Sistema Nacional de Salud (SNS) español. Métodos Se realizó un modelo analítico de decisiones mediante un modelo de árbol de decisión para simular una cohorte de 1.000 pacientes con VIH en el que se comparó si la realización del test HLA-B*5701 para prevenir la RHS al tratamiento con abacavir era una opción coste-efectiva versus no realizar el test. Los parámetros introducidos en el modelo así como el uso de recursos sanitarios en caso de que el paciente desarrollase una RHS provenían del estudio PREDICT-1 y de la opinión de expertos clínicos. El resultado principal del studio fue el coste incremental por RHS evitada. El horizonte temporal del análisis fue de 6 meses. Todos los costes se expresaron en euros del año 2008.Resultados El análisis demostró que los costes sanitarios directos totales por paciente fueron 1.344 € y 1.322 € al realizar o no el test HLA-B*5701, respectivamente, evitando unos 36 casos de RHS por cada 1.000 pacientes cribados. Estos resultados dieron lugar a una razón de coste por RHS evitada de 630 €. El análisis de sensibilidad mostró que los resultados fueron sensibles al coste del test produciendo desde un ahorro económico de 102 € hasta una razón coste-efectividad de 4.234 €. Conclusiones (..) (AU)

Correlación, en pacientes infectados por el VIH-1 y previamente tratados, entre la reacción de hipersensibilidad a abacavir y el alelo HLA-B*5701 / Correlation, in previously treated HIV-1 positive patients, between hypersensitivity reaction to abacavir and the presence of the HLA-B*5701 allele

Introducción: La reacción de hipersensibilidad a abacavir (un potente inhibidor de la transcriptasa inversa) es un efecto adverso importante que limita su uso en la terapia antirretroviral y precisa un elevado grado de vigilancia clínica. Determinados haplotipos de las proteínas del complejo principal de histocompatibilidad (HLA-B*5701) predicen, de forma muy significativa, el riesgo de hipersensibilidad a este fármaco. El objetivo del estudio es identificar los casos que, después de desarrollar una probable reacción de hipersensibilidad a abacavir, presentaban el alelo HLA-B*5701. Métodos: Se ha realizado un estudio retrospectivo a todos los pacientes adultos infectados por el virus de la inmunodeficiencia humana 1 (VIH-1) que recibieron tratamiento con abacavir entre enero de 2000 y diciembre de 2007, en el Departamento 6 de la Agencia Valenciana de Salud. Se recogieron los efectos adversos desarrollados por los pacientes para identificar los casos con probable reacción de hipersensibilidad diagnosticada clínicamente. Finalmente, se realizó la tipificación de HLA-B*5701 a estos 39 pacientes. Resultados: En total, 323 pacientes recibieron tratamiento con abacavir entre 2000 y 2007. Se retiró el tratamiento por reacción de hipersensibilidad a 39 pacientes (12,1 %); 9 (23,1 %) de ellos resultaron HLA-B*5701 positivo; 8 pacientes manifestaron exantema y únicamente se observó positividad en un paciente con síntomas gastrointestinales y fiebre. Conclusiones: La realización del test genético HLA-B*5701 podría ser favorable para la práctica clínica habitual, ya que evita errores en el diagnóstico de la reacción de hipersensibilidad y permite interpretar los síntomas con más seguridad (AU)

Introduction: Hypersensitivity reaction to abacavir (a powerful inverse transcriptase inhibitor) is a serious adverse effect that limits its use in antiretroviral treatment and requires a high level of clinical surveillance. Certain haplotypes of the primary histocompatibility complex proteins (HLA-B*5701) are very significant predictors of the risk of hypersensitivity to this drug. The purpose of this study is to identify the cases where a probable hypersensitivity reaction to abacavir presented the HLA-B*5701 allele. Method: A retrospective study was conducted in all HIV-1 positive adult patients infected treated with abacavir between January 2000 and December 2007, in Department 6 of the Agencia Valenciana de Salud (Valencia Health Agency). The adverse effects developed by the patients were collected to determine which cases presented a probable clinically diagnosed hypersensitivity reaction. Finally, these 39 patients were screened for HLA-B*5701. Results: In total, 323 patients were treated with abacavir between 2000 and 2007. The treatment was discontinued in 12.1 % (n = 39 patients) presenting a hypersensitivity reaction. Nine (23.1 %) of these were HLA-B*5701 positive. Eight patients presented skin rash and positivity was observed in only single patient with gastrointestinal symptoms and fever. Conclusions: The administration of the HLA-B*5701 gene test may be of benefit in clinical practice, because it prevents diagnostic errors of the hypersensitivity reaction and enables more accurate interpretation of the symptoms (AU)

A new HLA-B*44 variant, B*4453, identified by haplotype-specific extraction and sequencing-based typing.

We report the identification of a novel HLA B*44 allele, officially named B*4453, found in an Austrian patient and his two sisters.

[Identifying and sequence analysis of HLA-B*2736].

An unknown HLA-B allele which was similar to HLA-B*270401 was detected by FLOW-SSOPCR-SSP and heterozygous sequence-based typing (SBT) in Chinese Han individual. Its anomalous patterns suggested the possible presence of new allele. Amplifying exon 2-5(include intron 2-4) of the HLA-B*27 allele separately by using allele-specific primers and sequencing in both directions. Identifying the difference between the novel B*27 allele and B*270401. The sequence of novel B*27 from exon 2 to partial exon 5 is 1 815 bp. There are 10 nt changes from B*270401 in exon 3-4, at nt634where A-->C(codon130 AGC-->CGC, 130 S-->R); nt670 where A-->T (codon142 ACC-->TCC, 142 T-->S); nt683 where G-->T (codon146 TGG-->TTG, 146 W-->L); nt698 where A-->T (codon151 GAG-->GTG, 151 E-->V); nt774 where G-->C (codon176 GAG-->GAC, 176 E-->D); nt776 where C-->A (codon177 ACG-->AAG, 177 T-->K); nt781 where C-->G (codon179 CAG-->GAG, 179Q-->E); nt789 where G-->T (codon181 GCG-->GCT) resulting no coding change; nt1438 where C-->T (codon206 GGC-->GGT) resulting no coding change; nt1449 where G-->C (codon210 GGG-->GCG, 210G-->A). In IMGT/HLA database, only three alleles (B*270502/2706/2732) have sequences of introns. The same sequence in intron 2 showed homology between the novel HLA-B*27 allele and B*2706, but their homology could not be supported in intron 3-4. Comparing the sequence of the novel B*27 allele in intron 3 and 4 with B*27 group, it showed there are three mutations at nt106 C-->G, nt179 G-->A, nt536 G-->A and one deletion at nt168 in intron 3 and one mutations at nt82 T-->C in intron 4, but the sequence of the novel B*27 allele in intron 3 and 4 was all the same to B*070201. The sequence was submitted to Gen-Bank and the accession number was DQ915176. The allele has been confirmed as an extension of B*2736 by the WHO Nomenclature committee in November 2006.

Expression, purification and preliminary X-ray crystallographic analysis of the human major histocompatibility antigen HLA-B*1402 in complex with a viral peptide and with a self-peptide.

The product of the human major histocompatibility (HLA) class I allele HLA-B*1402 only differs from that of allele HLA-B*1403 at amino-acid position 156 of the heavy chain (Leu in HLA-B*1402 and Arg in HLA-B*1403). However, both subtypes are known to be differentially associated with the inflammatory rheumatic disease ankylosing spondylitis (AS) in black populations in Cameroon and Togo. HLA-B*1402 is not associated with AS, in contrast to HLA-B*1403, which is associated with this disease in the Togolese population. The products of these alleles can present peptides with Arg at position 2, a feature shared by a small group of other HLA-B antigens, including HLA-B*2705, the prototypical AS-associated subtype. Complexes of HLA-B*1402 with a viral peptide (RRRWRRLTV, termed pLMP2) and a self-peptide (IRAAPPPLF, termed pCatA) were prepared and were crystallized using polyethylene glycol as precipitant. The complexes crystallized in space groups P2(1) (pLMP2) and P2(1)2(1)2(1) (pCatA) and diffracted synchrotron radiation to 2.55 and 1.86 A resolution, respectively. Unambiguous solutions for both data sets were obtained by molecular replacement using a peptide-complexed HLA-B*2705 molecule (PDB code 1jge) as a search model.

Sequencing of a novel HLA-A allele, A*6836, showing a Bw4 epitope.

A novel A*6836 allele was completely characterized by sequence-based typing (SBT) in a cord blood sample from an Ecuadorian donor. A*6836 discloses six clustered amino acid residue changes at the alpha-1 domain, codons 76-83, regarding its closer A*680102 allele. Therefore, A*6836 would be a new human leukocyte antigen (HLA)-A molecule showing a Bw4-epitope.

Identification of a novel HLA-B*44 variant (B*4441) in three unrelated Caucasian individuals.

Here, we report the identification of a new human leukocyte antigen (HLA)-B*44 allele found almost simultaneous in three DNA samples which were part of routine bone marrow donor typing by order of the German registry 'Aktion Knochenmarkspende Bayern'. The samples appeared noticeable in different polymerase chain reactions using sequence-specific primers (PCR-SSP) or sequence-specific oligonucleotides (PCR-SSO). Sequence-based typing revealed a novel allele officially designated as B*4441*. This sequence differs from HLA-B*44020101/4427 by two nucleotide positions at the beginning of exon 3: by position 353 (T to C) and by position 355 (A to C). These differences in sequence result in deviant amino acids at codon 94 (Ile94Thr) and codon 95 (Ile95Leu).

HLA class I epitopes: A and B loci.

After our initial report on the HLA class I epitopes, we continue to demonstrate the power of the HLA recombinant single antigens in identifying the specificities of mouse monoclonal antibodies and alloantibodies that were absorbed to and eluted from single HLA antigens expressed by recombinant HLA single antigen cell lines (rHLA cell lines). We have expanded the list of HLA class I epitopes to 94, including the 58 reported earlier. Groups of as many as 58 HLA antigens can apparently share a single epitope. All positive antigens, identified by a mAb or an eluted alloantibody, shared unique amino acids (aa) at one to four positions on the alpha chain of the HLA antigen. The shared aa's were considered a distinguishing characteristic of the epitope.