HLA-OLI: A new MHC class I pseudogene and HLA-Y are located on a 60 kb indel in the human MHC between HLA-W and HLA-J.

Analysis of publicly available whole-genome sequence data from the Human Pangenome Project and the 1000 Genomes Project has identified a DNA segment of approximately 60 kb in the major histocompatibility complex (MHC) between HLA-W and HLA-J that is present in some MHC haplotypes but not others. This DNA segment is largely repeat element-rich but includes the pseudogene HLA-Y, thus pinpointing the location of this pseudogene, and a new HLA class I sequence we have called HLA-OLI. HLA-OLI clusters phylogenetically with the HLA class I pseudogenes, HLA-P and HLA-W, and appears to have a similar genetic structure. The availability of whole-genome sequence data from diverse populations enables a detailed characterization of the MHC at the population level and will have implications for understanding MHC disease associations and the non-HLA MHC factors that impact unrelated hematopoietic cell transplant outcomes.

Investigation of MHC gamma block C4A and C4B polymorphisms in unrelated hematopoietic stem cell transplantation

ABSTRACT Background: Immunological life-threatening complications frequently occur in post-hematopoietic stem cell transplantation (HSCT), despite matching recipient and donor (R/D) pairs for classical human leukocyte antigens (HLA). Studies have shown that R/D non-HLA disparities within the major histocompatibility complex (MHC) are associated with adverse effects post-HSCT. Methods: We investigated the impact of mismatches of single-nucleotide polymorphisms (SNPs) in C4A/C4B genes, for showing the highest diversity in the MHC gamma block, on 238 patients who underwent HLA 10/10 unrelated donor (URD) HSCT. The endpoints were acute graft-versus-host disease (aGVHD), chronic graft-versus-host disease (cGVHD) and mortality. One hundred and twenty-nine R/D pairs had 23 C4-SNPs typed by PCR-SSP (Gamma-Type™v.1.0), and 109 R/D pairs had these 23 SNPs identified by next-generation sequencing (NGS) using the Illumina platform. Results: The percentage of patients who received HSC from HLA 10/10 donors with 1-7 mismatches was 42.9%. The R/D pairs were considered C4 mismatched when bearing at least one disparity. These mismatches were not found to be risk factors for aGVHD, cGVHD or mortality after unrelated HSCT when SNPs were analyzed together (matched or mm ≥ 1), independently or according to the percentage of incompatibilities (full match for 23 SNPs; 1-3 mm and >3 mm). An exception was the association between 1-3 mismatches at the composite of SNPs C13193/T14952/T19588 with the development of aGVHD (P = 0.012) and with grades III-IV of this disease (P = 0.004). Conclusion: Our data are not consistent with the hypothesis that disparities in C4A/C4B SNPs increase the risks of post-HSCT adverse effects for the endpoints investigated in this study.

Complete genetic sequence of 15 novel HLA-H alleles.

We describe the full gene sequences of 15 HLA-H alleles.

Investigation of MHC gamma block C4A and C4B polymorphisms in unrelated hematopoietic stem cell transplantation.

BACKGROUND: Immunological life-threatening complications frequently occur in post-hematopoietic stem cell transplantation (HSCT), despite matching recipient and donor (R/D) pairs for classical human leukocyte antigens (HLA). Studies have shown that R/D non-HLA disparities within the major histocompatibility complex (MHC) are associated with adverse effects post-HSCT. METHODS: We investigated the impact of mismatches of single-nucleotide polymorphisms (SNPs) in C4A/C4B genes, for showing the highest diversity in the MHC gamma block, on 238 patients who underwent HLA 10/10 unrelated donor (URD) HSCT. The endpoints were acute graft-versus-host disease (aGVHD), chronic graft-versus-host disease (cGVHD) and mortality. One hundred and twenty-nine R/D pairs had 23 C4-SNPs typed by PCR-SSP (Gamma-Type™v.1.0), and 109 R/D pairs had these 23 SNPs identified by next-generation sequencing (NGS) using the Illumina platform. RESULTS: The percentage of patients who received HSC from HLA 10/10 donors with 1-7 mismatches was 42.9%. The R/D pairs were considered C4mismatched when bearing at least one disparity. These mismatches were not found to be risk factors for aGVHD, cGVHD or mortality after unrelated HSCT when SNPs were analyzed together (matched or mm≥1), independently or according to the percentage of incompatibilities (full match for 23 SNPs; 1-3mm and >3mm). An exception was the association between 1-3 mismatches at the composite of SNPs C13193/T14952/T19588 with the development of aGVHD (P=0.012) and with grades III-IV of this disease (P=0.004). CONCLUSION: Our data are not consistent with the hypothesis that disparities in C4A/C4B SNPs increase the risks of post-HSCT adverse effects for the endpoints investigated in this study.

Analysis of Single Nucleotide Polymorphisms in the Gamma Block of the Major Histocompatibility Complex in Association with Clinical Outcomes of Hematopoietic Cell Transplantation: A Center for International Blood and Marrow Transplant Research Study.

HLA haplotype mismatches have been associated with an elevated risk of acute graft-versus-host disease (aGVHD) in patients undergoing HLA-matched unrelated donor (URD) hematopoietic cell transplantation (HCT). The gamma block (GB) is located in the central MHC region between beta and delta blocks (encoding HLA-B and -C and HLA-DQ and -DR antigens, respectively) and contains numerous inflammatory and immune regulatory genes, including Bf, C2, and C4 genes. A single-center study showed that mismatches in SNPs c.2918+98G, c.3316C, and c.4385C in the GB block (C4 SNPs) were associated with higher risk of grade III-IV aGVHD. We investigated the association of GB SNP (GBS) mismatches with outcomes after 10/10 and 9/10 URD HCT (n = 714). The primary outcome was acute GVHD. Overall survival, disease-free survival, transplantation-related mortality, relapse, chronic GVHD, and engraftment were also analyzed. DNA samples were GBS genotyped by identifying 338 SNPs across 20 kb using the Illumina NGS platform. The overall 100-day incidence of aGVHD grade II-IV and II-IV were 41% and 17%, respectively. The overall incidence of matching at all GBSs tested and at the C4 SNPs were 23% and 81%, respectively. Neither being matched across all GB SNPs tested (versus mismatched) nor having a higher number of GBS mismatches was associated with transplantation outcomes. There was no association between C4 SNP mismatches and outcomes except for an unexpected significant association between having 2 C4 SNP mismatches and a higher hazard ratio (HR) for relapse (association seen in 15 patients only; HR, 3.38, 95% confidence interval, 1.75 to 6.53; P = .0003). These data do not support the hypothesis that mismatching at GB is associated with outcomes after HCT.

Haplotyping the human leukocyte antigen system from single chromosomes.

We describe a method for determining the parental HLA haplotypes of a single individual without recourse to conventional segregation genetics. Blood samples were cultured to identify and sort chromosome 6 by bivariate flow cytometry. Single chromosome 6 amplification products were confirmed with a single nucleotide polymorphism (SNP) array and verified by deep sequencing to enable assignment of both alleles at the HLA loci, defining the two haplotypes. This study exemplifies a rapid and efficient method of haplotyping that can be applied to any chromosome pair, or indeed all chromosome pairs, using a single sorting operation. The method represents a cost-effective approach to complete phasing of SNPs, which will facilitate a deeper understanding of the links between SNPs, gene regulation and protein function.

An Observational Study of 2,248 Patients Presenting With Headache, Suggestive of Subarachnoid Hemorrhage, Who Received Lumbar Punctures Following Normal Computed Tomography of the Head.

OBJECTIVES: The objective was to determine the incidence of subarachnoid hemorrhage (SAH) diagnosed by lumbar puncture (LP) when the head computed tomography (CT) was reported as demonstrating no subarachnoid blood. METHODS: Data were obtained on patients who received LP to diagnose or exclude SAH attending six hospitals over 5 years. Subsequent investigations and outcomes were reviewed in all patients with LPs that did not exclude SAH. RESULTS: A total of 2,248 patients were included. A total of 1,898 LPs were suitable for biochemical analysis, of which 92 (4.8%) were positive for blood, suggesting SAH; 1,507 (79.4%) were negative; and 299 (15.6%) were inconclusive. Of the 92 patients with positive cerebrospinal fluid analysis, eight patients (0.4%) had aneurysms on further imaging, and one had a carotid cavernous fistula. CONCLUSIONS: In patients presenting to the emergency department with acute severe headache, LP to diagnose or exclude SAH after negative head CT has a very low diagnostic yield, due to low prevalence of the disease and uninterpretable or inconclusive samples. A clinical decision rule may improve diagnostic yield by selecting patients requiring further evaluation with LP following nondiagnostic or normal noncontrast CT brain imaging.

Capacity building and predictors of success for HIV-1 drug resistance testing in the Asia-Pacific region and Africa.

BACKGROUND: The TREAT Asia Quality Assessment Scheme (TAQAS) was developed as a quality assessment programme through expert education and training, for laboratories in the Asia-Pacific and Africa that perform HIV drug-resistance (HIVDR) genotyping. We evaluated the programme performance and factors associated with high-quality HIVDR genotyping. METHODS: Laboratories used their standard protocols to test panels of human immunodeficiency virus (HIV)-positive plasma samples or electropherograms. Protocols were documented and performance was evaluated according to a newly developed scoring system, agreement with panel-specific consensus sequence, and detection of drug-resistance mutations (DRMs) and mixtures of wild-type and resistant virus (mixtures). High-quality performance was defined as detection of ≥95% DRMs. RESULTS: Over 4.5 years, 23 participating laboratories in 13 countries tested 45 samples (30 HIV-1 subtype B; 15 non-B subtypes) in nine panels. Median detection of DRMs was 88-98% in plasma panels and 90-97% in electropherogram panels. Laboratories were supported to amend and improve their test outcomes as appropriate. Three laboratories that detected <80% DRMs in early panels demonstrated subsequent improvement. Sample complexity factors - number of DRMs (p<0.001) and number of DRMs as mixtures (p<0.001); and laboratory performance factors - detection of mixtures (p<0.001) and agreement with consensus sequence (p<0.001), were associated with high performance; sample format (plasma or electropherogram), subtype and genotyping protocol were not. CONCLUSION: High-quality HIVDR genotyping was achieved in the TAQAS collaborative laboratory network. Sample complexity and detection of mixtures were associated with performance quality. Laboratories conducting HIVDR genotyping are encouraged to participate in quality assessment programmes.

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.

Data analysis of HLA sequencing using Assign-SBT v3.6+ from Conexio.

DNA Sequencing is now a standard frontline high-throughput HLA typing procedure with some unrelated bone marrow donor registry typing laboratories performing tens of thousands of tests per year. The advantage of DNA sequencing is that, by definition, sequencing directly identifies all bases in the DNA template. Alternative molecular-based assays such as the use of sequence-specific PCR primers (PCR-SSP) and oligonucleotide probes (PCR-SSO) provide information only for those regions to which the oligos are designed and no information is obtained for the regions between primers and probes.The era of routine high-throughput sequencing-based typing (SBT) was made possible by the development of locus-specific PCR-based assays and the development of the HLA sequencing-based typing software, Assign-SBT v3.2.7 by Conexio Genomics. A single PCR per locus simplified the template preparation stage of the test and Assign-SBT simplified the sequence analysis and allele assignment stage. Together these developments dramatically simplified the SBT procedure, making SBT cost effective.This chapter provides a comprehensive description of Assign-SBT sequence analysis software for use in a HLA typing laboratory.

TREAT Asia Quality Assessment Scheme (TAQAS) to standardize the outcome of HIV genotypic resistance testing in a group of Asian laboratories.

The TREAT Asia (Therapeutics, Research, Education, and AIDS Training in Asia) Network is building capacity for Human Immunodeficiency Virus Type-1 (HIV-1) drug resistance testing in the region. The objective of the TREAT Asia Quality Assessment Scheme - designated TAQAS - is to standardize HIV-1 genotypic resistance testing (HIV genotyping) among laboratories to permit rigorous comparison of results from different clinics and testing centres. TAQAS has evaluated three panels of HIV-1-positive plasma from clinical material or low-passage, culture supernatant for up to 10 Asian laboratories. Laboratory participants used their standard protocols to perform HIV genotyping. Assessment was in comparison to a target genotype derived from all participants and the reference laboratory's result. Agreement between most participants at the edited nucleotide sequence level was high (>98%). Most participants performed to the reference laboratory standard in detection of drug resistance mutations (DRMs). However, there was variation in the detection of nucleotide mixtures (0-83%) and a significant correlation with the detection of DRMs (p<0.01). Interpretation of antiretroviral resistance showed approximately 70% agreement among participants when different interpretation systems were used but >90% agreement with a common interpretation system, within the Stanford University Drug Resistance Database. Using the principles of external quality assessment and a reference laboratory, TAQAS has demonstrated high quality HIV genotyping results from Asian laboratories.

Spatial distribution of mosquito larvae and the potential for targeted larval control in The Gambia.

We examined the distribution of aquatic stages of malaria vectors in a 400-km(2) area in rural Gambia to assess the practicality of targeting larval control. During the rainy season, the peak period of malaria transmission, breeding sites were 70% more likely to have anopheline larvae in the floodplain of the Gambia River than upland sites (P < 0.001). However, mosquitoes were found in some examples of all habitats, apart from moving water. Habitats most often colonized by anopheline larvae were the largest water bodies, situated near the landward edge of the flood-plain, where culicine larvae were present. In the wet season, 49% of sites had anophelines versus 19% in the dry season (P < 0.001). Larval control targeted at specific habitats is unlikely to be successful in this setting. Nonetheless, larval control initiated at the end of the dry season and run throughout the rainy season could help reduce transmission.

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.

The septic shock associated HSPA1B1267 polymorphism influences production of HSPA1A and HSPA1B.

OBJECTIVE: To identify a functional polymorphic site(s) within HSPA1A and/or HSPA1B which is in linkage disequilibrium with the silent mutation HSPA1B1267A>G and explains its association with septic shock. SUBJECTS: The promoter region of HSPA1A and HSPA1B was sequenced in 100 healthy whites. Stimulation experiments were performed on 36 healthy subjects. MEASUREMENTS AND RESULTS: Sequencing the HSPA1A and HSPA1B promoter regions (approx. 500 bp upstream of the translation start site) identified ten novel and three known polymorphisms. Mononuclear cells were stimulated with lipopolysaccharide (10 microg/ml) for 4 and 8 h, and mRNA levels measured by reverse transcriptase polymerase chain reaction. Two polymorphisms, HSPA1A-27G>C and HSPA1A-327A>C, were found to be in strong linkage with HSPA1B1267A>G but, as with HSPA1B1267A>G, were not associated with stimulated mRNA levels. However, HSPA1B-179C>T, which is also in linkage with HSPA1B1267, was associated with stimulated HSPA1A and HSPA1B mRNA levels. Individuals homozygous for the C allele of HSPA1B-179C>T were associated with lower HSPA1A and HSPA1B mRNA levels than HSPA1B-179CT after 8 h lipopolysaccharide stimulation. CONCLUSIONS: HSPA1B-179C>T is in linkage disequilibrium with HSPA1B1267A>G and is associated with variable production of HSPA1B and HSPA1A production. This suggests that HSPA1B-179C>T affects HSP70 production and is a key determinant of individual susceptibility to a variety of infectious and inflammatory diseases.

Intrafamilial transmission of HIV-1 infection from individuals with unrecognized HIV-1 infection.

OBJECTIVE: To describe the clinical, epidemiological and molecular evidence for transmission of HIV-1 infection from a person with unrecognized HIV infection to a family member in two unconnected families where the route of transmission could not be conclusively determined. DESIGN: Case studies, molecular analysis of viral strains and a clinical and laboratory investigation of risk factors for transmission. SETTING: State referral centres for HIV/AIDS in two Australian teaching hospitals. RESULTS: Previously unrecognized HIV-1 infection was diagnosed in two unconnected females following blood donation in different Australian cities. Initially, no source of infection was identified but subsequently HIV-1 infection was diagnosed in the sister of one case and the adult son of the other. Using nucleic acid-based methods, it was demonstrated that one index case and her sister were infected with highly homologous 'Russian-type' HIV-1 subtype A, and the other index case and her son were infected with highly homologous HIV-1 subtype E (CRF01_AE). Sexual history taking from the sister and the son of the respective index cases revealed prior sexual partners from geographical areas in which the corresponding subtypes are known to be prevalent. Extensive history taking, cross-validated by independent reviewers, found no evidence whatsoever that any form of sexual contact or known blood contact could explain the HIV-1 infection in the two index cases. However, there was evidence that some form of domestic contact involving unperceived blood transfer may have occurred. CONCLUSION: Intra-familial transmission of HIV-1 infection should be considered when a source of HIV-1 infection cannot be determined.

Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study.

BACKGROUND: A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities. OBJECTIVES: A collaborative study was undertaken to evaluate intra-assay precision and between laboratory concordance of measurements of mitochondrial DNA quantity, as a component of a comprehensive quality assurance project. STUDY DESIGN: Four laboratories were asked to measure and report mitochondrial DNA and nuclear DNA genome copy number, as well as mitochondrial DNA copy number/cell, for 17 coded aliquots of DNA derived from serial dilutions of pooled DNA from a lymphoblastoid cell line. Samples included masked replicates and five standards. All samples had similar mitochondrial DNA/nuclear DNA ratios. Precision within laboratories was assessed by determining the coefficient of variation of replicates. Concordance between laboratories was assessed by determining the average coefficient of variation of the mean replicate values for each sample. The effect of standardising the assay for these three measurements was also assessed for laboratories A, B and C. RESULTS: Measurements of mitochondrial DNA and nuclear DNA content for replicate samples varied by an average of less than 6% (based on log(10) values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log(10) values, 32% non-logged values), with no improvement of precision after standardisation. Standardisation did significantly improve the concordance of results for measurements of mitochondrial DNA content and mitochondrial DNA/cell. Non-standardised measurements of mitochondrial DNA content for the same sample set varied by 19% between laboratories (based on log(10) values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log(10) values, 54% non-logged values). There was no significant improvement for concordance of measures of nuclear DNA content after standardisation, with results varying by 4.56% between laboratories (based on log(10) values, 45% non-logged values) before standardisation, and by 2.49% (based on log(10) values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log(10) values) before and by 56% (non-logged, 13% log(10) values) after standardisation. CONCLUSION: All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.