DRB1 Loci Research Articles

CIITA gene variants are associated with rheumatoid arthritis in Scandinavian populations

Expression of the major autoimmune risk loci DRB1 and DQB1 is regulated by the class II MHC (major histocompatibility complex) transactivator (CIITA), making the CIITA gene a strong autoimmune risk locus candidate. A CIITA promoter single-nucleotide polymorphism (SNP), rs3087456 (-168 A/G), has indeed been associated with several autoimmune diseases, including rheumatoid arthritis (RA). Recently, an intronic SNP rs8048002 has been suggested as a better susceptibility marker in Addison's disease. Therefore, we tested both SNPs in a panel of autoimmune diseases, consisting of Norwegian patients with RA (n=819), juvenile idiopathic arthritis (JIA; n=524), or type 1 diabetes (T1D; n=1211), and 2149 controls. We also included an independent Swedish RA cohort (n=2503) and controls (n=1416). Both rs3087456 and rs8048002 were significantly associated with RA (combined Norwegian and Swedish patients P(corrected)=0.012 and P(corrected)=0.0016, respectively), but not with JIA or T1D. Meta-analysis of 16 RA cohorts confirmed rs3087456 with only marginal significance (P=0.016). However, results were stronger in the Scandinavian subgroup (4 cohorts, P=3.8 × 10(-4)), indicating a population-dependent effect. A similar pattern was observed in a meta-analysis of rs8048002. Our results support involvement of CIITA in RA, but imply that this is population dependent and that the aetiological variant is yet to be discovered.

Analysis of HLA-A, B, and DRB1 polymorphism and genetic distance in Han population living in Yantai and Weihai regions of Shandong province

To investigate allelic and haplotypic polymorphisms of human leukocyte antigen(HLA) genes at A, B and DRB1 loci in Yantai and Weihai Han population and analyze the genetic relationship between Yantai, Weihai Han population and other populations. A total of 4062 unrelated Han ethnic individual from Yantai and Weihai regions were genotyped by polymerase chain reaction-sequence specific olignucleotide probe(PCR-SSOP) for HLA-A, B and DRB1 loci. Allelic and haplotypic frequencies were estimated by maximum likelihood estimation method using Arlequin 3.5 software. Genetic distances were computed, and phylogenetic tree was constructed using Mega5.0 software. Respectively 18, 33 and 13 alleles were observed at HLA-A, B and DRB1 loci. The most frequent alleles were HLA-A*02(0.2935), HLA-B*15(0.1485) and HLA-DRB1*15(0.1621). And the most common three loci haplotype was A*30-B*13-DRB1*07(0.0649). A*33-B*58, A*66-DRB1*13 and B*08-DRB1*03 showed the strongest linkage disequilibrium. Yantai and Weihai Han population has the shortest genetic distance with Jilin Han population (0.0034). The HLA-A, B and DRB1 loci are highly polymorphic in Han population from Yantai and Weihai, and this population has closest relationship with Han population from Jilin province.

The Ever Elusive Permissive Mismatch

Unrelated donors are more commonly used than siblings for allogenic stem cell transplantation. However, despite almost 20 million donors and cords available worldwide, only two-thirds of the white population, and less of smaller ethnic minorities, can find a donor fully compatible for HLA-A, -B, -C, and -DRB1 loci, — such is the enormous population diversity at these loci. For the patients without such a match, clinicians struggle to pick the best possible donor among those who are mismatched. In this issue, Spellman et al. [1] evaluated an algorithm established by Elsner et al. [2] to estimate the allogenic reaction potential betweenmismatched individuals. TheHistoCheck program is available online at http://www.histocheck.de and can be used by anyone to calculate a dissimilarity score between two individuals mismatched for one HLA molecule [3]. The analysis used the National Marrow Donor Program transplantation outcome database previously reported by Lee et al. [4], and single locus mismatched pairs were scored by the HistoCheck algorithm. With the exception of neutrophil engraftment in a subset analysis, no association was found between the dissimilarity score and transplantation outcome. What is the lesson from this study? The principles used to calculate the dissimilarity score seem sound: the first is the functional relevance of mismatched amino acid residues within the HLA molecule. Amino acids that anchor the antigenic peptide or bind to the T cell antigen receptor score higher than others. The second principle assumes that disparities between amino acids that are structurally ‘‘similar,’’ as defined by Risler et al. [5], are better tolerated immunologically than dissimilar ones. By the Risler criteria, tyrosine and phenylalanine that differ only for a hydroxylic group have a score of 4 (similar), whereas tyrosine and serine that differ for an

Independent evolution of functional MHC class II DRB genes in New World bat species

Genes of the major histocompatibility complex (MHC) play a pivotal role in the vertebrate immune system and are attractive markers for functional, fitness-related, genetic variation. Although bats (Chiroptera) represent the second largest mammalian order and are prone to various emerging infectious diseases, little is known about MHC evolution in bats. In the present study, we examined expressed MHC class II DRB sequences (exons 1 to 4) of New World bat species, Saccopteryx bilineata, Carollia perspicillata, Noctilio albiventris and Noctilio leporinus (only exon 2). We found a wide range of copy number variation of DRB loci with one locus detected in the genus Noctilio and up to ten functional loci observed in S. bilineata. Sequence variation between alleles of the same taxa was high with evidence for positive selection. We found statistical support for recombination or gene conversion events among sequences within the same but not between bat species. Phylogenetic relationships among DRB alleles provided strong evidence for independent evolution of the functional MHC class II DRB genes in the three investigated species, either by recent gene duplication, or homogenization of duplicated loci by frequent gene conversion events. Phylogenetic analysis of all available chiropteran DRB exon 2 sequences confirmed their monophyletic origin within families, but revealed a possible trans-species mode of evolution pattern in congeneric bat species, e.g. within the genera Noctilio and Myotis. This is the first study investigating phylogenetic relationships of MHC genes within bats and therefore contributes to a better understanding of MHC evolution in one of the most dominant mammalian order.

HLA‐DRB1, ‐DQA1 and ‐DQB1 Alleles and Haplotypes in First‐Generation Pakistani Immigrants in Norway

Human leucocyte antigen (HLA) polymorphisms among immigrants from Pakistan have not been well investigated. Immigration to Norway started in the late 1960s for working purposes. From 1975, immigration was mainly for marriages and family reunion. When recruiting couples for a birth cohort study, we ended up with 65.5% of the 374 parents genotyped being closely related. This was also reflected in that 21% of newborns were homozygotes for their DRB1-DQA1-DQB1 genotype. For being able to study HLA class II genes frequencies among unrelated individuals, we had to exclude 195 of the parents from data analysis. High-resolution typing for the DRB1 locus, low/intermediate for the DQA1 locus and resolution genotyping for the DQB1 locus were performed in all the 179 parents and their newborns from the Punjab province of Pakistan. We identified 25 DRB1, nine DQA1 and 14 DRB1 alleles in the 179 unrelated parents included in our analysis. The most frequent alleles were DRB1*03:01:01 (15.9%) and DRB1*07:01:01 (15.9%), DQA1*01:03 (22.1%) and DQB1*02:01:01 (26.0%). Forty-one haplotypes were identified, including DRB1*13:02:01-DQA1*01:02-DQB1*06:03:01, not earlier reported. Supported by the few earlier reports on Pakistani groups living in Pakistan, it appears that alleles found among those living in Norway are of Indo-European or mixed ethnic origin. This study provides the first comprehensive report of HLA class II alleles and haplotypes in Norwegian Pakistani immigrants. When the unrelated parents were compared with all parents genotyped, there were, however, no significant differences in allele frequencies, confirming that consanguineous marriages are usual in Pakistan.

Spectrum of HLA associations: the case of medically refractory pediatric acute lymphoblastic leukemia.

Although studies of HLA and disease now date back some 50 years, a principled understanding of that relationship has been slow to emerge. Here, we examine the associations of three HLA loci with medically refractory pediatric acute lymphoblastic leukemia (pALL) patients in a case–control study involving 2,438 cases and 41,750 controls. An analysis of alleles from the class I loci, HLA-A and HLA-B, and the class II locus DRB1 illuminates a spectrum of extremely significant allelic associations conferring both predisposition and protection. Genotypes constructed from predisposing, protective, and neutral allelic categories point to an additive mode of disease causation. For all three loci, genotypes homozygous for predisposing alleles are at highest disease risk while the favorable effect of homozygous protective genotypes is less striking. Analysis of A–B and B–DRB1 haplotypes reveals locus-specific differences in disease effects, while that all three loci influence pALL; the influence of HLA-B is greater than that of HLA-A, and the predisposing effect of DRB1 exceeds that of HLA-B. We propose that the continuum in disease susceptibility suggests a system in which many alleles take part in disease predisposition based on differences in binding affinity to one or a few peptides of exogenous origin. This work provides evidence that an immune response mediated by alleles from several HLA loci plays a critical role in the pathogenesis of pALL, adding to the numerous studies pointing to a role for an infectious origin in pALL.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-012-0605-5) contains supplementary material, which is available to authorized users.

Considerable MHC Diversity Suggests That the Functional Extinction of Baiji Is Not Related to Population Genetic Collapse

To further extend our understanding of the mechanism causing the current nearly extinct status of the baiji (Lipotes vexillifer), one of the most critically endangered species in the world, genetic diversity at the major histocompatibility complex (MHC) class II DRB locus was investigated in the baiji. Nine highly divergent DRB alleles were identified in 17 samples, with an average of 28.4 (13.2%) nucleotide difference and 16.7 (23.5%) amino acid difference between alleles. The unexpectedly high levels of DRB allelic diversity in the baiji may partly be attributable to its evolutionary adaptations to the freshwater environment which is regarded to have a higher parasite diversity compared to the marine environment. In addition, balancing selection was found to be the main mechanisms in generating sequence diversity at baiji DRB gene. Considerable sequence variation at the adaptive MHC genes despite of significant loss of neutral genetic variation in baiji genome might suggest that intense selection has overpowered random genetic drift as the main evolutionary forces, which further suggested that the critically endangered or nearly extinct status of the baiji is not an outcome of genetic collapse.

The Genetic Polymorphisms of HLA Are Strongly Correlated with the Disease Severity after Hantaan Virus Infection in the Chinese Han Population

The polymorphism of human leukocyte antigen (HLA), which is a genetic factor that influences the progression of hemorrhagic fever with renal syndrome (HFRS) after Hantaan virus (HTNV) infection, was incompletely understood. In this case-control study, 76 HFRS patients and 370 healthy controls of the Chinese Han population were typed for the HLA-A, -B, and -DRB1 loci. The general variation at the HLA-DRB1 locus was associated with the onset of HFRS (P < 0.05). The increasing frequencies of HLA-DRB1∗09 and HLA-B∗46-DRB1∗09 in HFRS patients were observed as reproducing a previous study. Moreover, the HLA-B∗51-DRB1∗09 was susceptible to HFRS (P = 0.037; OR = 3.62; 95% CI: 1.00–13.18). The increasing frequencies of HLA-B∗46, HLA-B∗46-DRB1∗09, and HLA-B∗51-DRB1∗09 were observed almost in severe/critical HFRS patients. The mean level of maximum serum creatinine was higher in HLA-B∗46-DRB1∗09 (P = 0.011), HLA-B∗51-DRB1∗09 (P = 0.041), or HLA-B∗46 (P = 0.011) positive patients than that in the negative patients. These findings suggest that the allele HLA-B∗46 and haplotypes HLA-B∗46-DRB1∗09 and HLA-B∗51-DRB1∗09 in patients could contribute to a more severe degree of HFRS and more serious kidney injury, which improve our understanding of the HLA polymorphism for a different outcome of HTNV infection.

Association of HLA-DQB1*05:02 and DRB1*16 Alleles with Late-Onset, Nonthymomatous, AChR-Ab-Positive Myasthenia Gravis

An association of several HLA alleles with myasthenia gravis (MG) has been reported. Aim of this work was to analyze the HLA allele profile in a survey of 76 unselected Italian MG patients and in a subgroup characterized by disease onset after the age of 50 years, absence of thymoma, and presence of antiacetylcholine receptor antibodies. We defined this subgroup by the acronym LOAb. Typing was performed at low resolution for HLA-A, -B, and -DRB1 loci with sequence-specific oligonucleotide probe (PCR-SSO); at high resolution for HLA-DQB1 locus by PCR with sequence-specific primers (PCR-SSPS). HLA allele frequencies were compared with 100 healthy controls. No correlation was observed between MG and the studied HLA class I alleles. On the contrary, a strong positive association was found for the HLA class II alleles DQB1∗05:02 (P c = 0.00768) and DRB1∗16 (P c = 0.0211) in the LOAb subgroup (n = 27) of MG patients. Association between DQB1∗05:02 and some subtypes of MG has been previously reported but not in patients with the LOAb characteristics. Therefore, the HLA allele DQB1∗05:02 might be considered as a susceptibility marker for LOAb among Italians.

Sex-specific selection for MHC variability in Alpine chamois

BackgroundIn mammals, males typically have shorter lives than females. This difference is thought to be due to behavioural traits which enhance competitive abilities, and hence male reproductive success, but impair survival. Furthermore, in many species males usually show higher parasite burden than females. Consequently, the intensity of selection for genetic factors which reduce susceptibility to pathogens may differ between sexes. High variability at the major histocompatibility complex (MHC) genes is believed to be advantageous for detecting and combating the range of infectious agents present in the environment. Increased heterozygosity at these immune genes is expected to be important for individual longevity. However, whether males in natural populations benefit more from MHC heterozygosity than females has rarely been investigated. We investigated this question in a long-term study of free-living Alpine chamois (Rupicapra rupicapra), a polygynous mountain ungulate.ResultsHere we show that male chamois survive significantly (P = 0.022) longer if heterozygous at the MHC class II DRB locus, whereas females do not. Improved survival of males was not a result of heterozygote advantage per se, as background heterozygosity (estimated across twelve microsatellite loci) did not change significantly with age. Furthermore, reproductively active males depleted their body fat reserves earlier than females leading to significantly impaired survival rates in this sex (P < 0.008). This sex-difference was even more pronounced in areas affected by scabies, a severe parasitosis, as reproductively active males were less likely to survive than females. However, we did not find evidence for a survival advantage associated with specific MHC alleles in areas affected by scabies.ConclusionsIncreased MHC class II DRB heterozygosity with age in males, suggests that MHC heterozygous males survive longer than homozygotes. Reproductively active males appear to be less likely to survive than females most likely because of the energetic challenge of the winter rut, accompanied by earlier depletion of their body fat stores, and a generally higher parasite burden. This scenario renders the MHC-mediated immune response more important for males than for females, which implies a relatively stronger selection pressure on MHC genes in males than in females.

Recombination in human leukocyte antigen region in two Asian Indian families

BACKGROUND:Recombination (crossing over) may generate novel haplotypes that can be beneficial to a population against recently introduced pathogens. It may lead to the generation of new alleles.SETTINGS AND DESIGN:A prospective study at a tertiary care centre.AIM:To report two rare cases of crossing over in HLA region.MATERIALS AND METHODS:Tissue-typing was done by sequence specific primers (SSP) for DR locus and by both SSP and serology for Class I which was reconfirmed on fresh samples.RESULTS:In one patient crossing over had taken place in the region of A locus resulting in inheritance of A*01 instead of expected A*11. In second family crossing over had taken place in region of DRB1 locus and the sibling inherited DRB1*08 instead of DRB1*10.CONCLUSIONS:Possibility of recombination must be considered when interpreting implausible tissue-typing results of families worked up for BMT.

Investigation of Caucasian rheumatoid arthritis susceptibility loci in African patients with the same disease

IntroductionThe largest genetic risk to develop rheumatoid arthritis (RA) arises from a group of alleles of the HLA DRB1 locus ('shared epitope', SE). Over 30 non-HLA single nucleotide polymorphisms (SNPs) predisposing to disease have been identified in Caucasians, but they have never been investigated in West/Central Africa. We previously reported a lower prevalence of the SE in RA patients in Cameroon compared to European patients and aimed in the present study to investigate the contribution of Caucasian non-HLA RA SNPs to disease susceptibility in Black Africans.MethodsRA cases and controls from Cameroon were genotyped for Caucasian RA susceptibility SNPs using Sequenom MassArray technology. Genotype data were also available for 5024 UK cases and 4281 UK controls and for 119 Yoruba individuals in Ibadan, Nigeria (YRI, HapMap). A Caucasian aggregate genetic-risk score (GRS) was calculated as the sum of the weighted risk-allele counts.ResultsAfter genotyping quality control procedures were performed, data on 28 Caucasian non-HLA susceptibility SNPs were available in 43 Cameroonian RA cases and 44 controls. The minor allele frequencies (MAF) were tightly correlated between Cameroonian controls and YRI individuals (correlation coefficient 93.8%, p = 1.7E-13), and they were pooled together. There was no correlation between MAF of UK and African controls; 13 markers differed by more than 20%. The MAF for markers at PTPN22, IL2RA, FCGR2A and IL2/IL21 was below 2% in Africans. The GRS showed a strong association with RA in the UK. However, the GRS did not predict RA in Africans (OR = 0.71, 95% CI 0.29 - 1.74, p = 0.456). Random sampling from the UK cohort showed that this difference in association is unlikely to be explained by small sample size or chance, but is statistically significant with p<0.001.ConclusionsThe MAFs of non-HLA Caucasian RA susceptibility SNPs are different between Caucasians and Africans, and several polymorphisms are barely detectable in West/Central Africa. The genetic risk of developing RA conferred by a set of 28 Caucasian susceptibility SNPs is significantly different between the UK and Africa with p<0.001. Taken together, these observations strengthen the hypothesis that the genetic architecture of RA susceptibility is different in different ethnic backgrounds.

OUTCOME of Unrelated DONOR BONE MARROW TRANSPLANTATION for THALASSEMIA MAJOR PATIENTS

Abstract 149At present, allogeneic bone marrow transplantation (BMT) is still recognized as the only treatment potentially able to cure a large proportion of patients with thalassemia major. For many years, the only donor employed was an HLA-identical sibling. Unfortunately, at least 70% of patients who might benefit from BMT lack a compatible sibling. With the establishment of bone marrow donor registries, including now more than 15 million volunteers world-wide, many patients are able to locate a suitable unrelated donor. Aim of this study was to evaluate the outcome of patients transplanted from an unrelated volunteer, selected using stringent criteria of compatibility after high-resolution molecular typing of HLA loci, and to identify factors with a prognostic relevance. We analyzed 122 thalassemia patients (96 children and 26 adults, age range at time of BMT 1–35 years, median 10,5) given the allograft in one of 4 Italian Centers participating to this study. Seventy-one patients were males and 51 females; 13 patients had positive Hepatitis C Virus (HCV) serology. Prior to transplantation, all patients underwent a complete check-up and children were assigned to one of 3 classes of risk according to the criteria proposed the Pesaro group. In detail, 39 pediatric patients belonged to the class I, 41 to the class II (23) and 16 to the class III of the Pesaro classification. In 105 donor/recipient pairs were matched for the HLA-A, -B, -C, -DRB1 loci; 17 donor/recipient pairs had a disparity at the HLA-C locus, but both HLA-C molecules pertained to the same HLA-C ligand group. The patients were prepared for the allograft using a myeloablative conditioning regimen based on the combination of busulfan/treosulfan (BU/TREO), thiotepa (TT) and either cyclophosphamide (CY, 54 patients), or fludarabine (FLU, 68 patients). All patients received fresh, unmanipulated bone marrow cells, the median dose of nucleated cells infused being 5×108/Kg recipient b.w. (range 1.4–15). Graft-versus-host disease (GvHD) prophylaxis was homogeneous in all patients and consisted of cyclosporine-A, short-term methotrexate (15 mg/m2 on day +1 and 10 mg/m2 on days +3, +6 and + 11) and anti-thymocyte globulin (ATG, 2.5 mg/Kg from day –4 to –2). Twenty patients died and 16 patients had either primary (11 patients) or secondary (5 patients) graft failure, the 5-year probability of overall survival (OS), and thalassemia-free survival (TFS) being 84% (95% Confidence Interval, CI, 76–90) and 75% (95% CI, 66–81) respectively. Acute and chronic GvHD were the main causes of death accounting for 6 and 4 fatal events respectively. Thirty-four patients at risk (28%) developed grade II-IV acute GvHD with 16 of them (13%) experiencing grade III-IV acute GvHD. Fourteen patients at risk (13%) had chronic GvHD, which was limited in 8 cases and extensive in 6. The probability of survival was not influenced by the occurrence of graft failure which was responsible of only 2 of the 20 fatal events. The chance of being alive for children belonging to class I, II and III of the Pesaro classification was 97% (95% CI, 83–100), 85% (95% CI, 70–93) and 78% (95% CI, 51–93), respectively, while that of adults was 65% (95% CI, 44–80) (p<0.01). Occurrence of both grade III-IV acute and chronic GvHD, older patient’s age, positive HCV serology and adult/class III group of risk predicted a poor survival in univariate analysis; however, in multivariate analysis, only occurrence of grade III-IV acute GvHD was associated with a poor outcome (Hazard ratio, 10.4, 95% CI 3.19 – 34.06, P=0.001). Altogether, these data indicate that, provided that selection of the donor is based on stringent criteria of HLA-compatibility, transplantation from unrelated volunteers is able to cure a large proportion of patients with patients with thalassemia major, the results being comparable to those obtained when the donor is an HLA-compatible sibling. Prevention of severe acute GvHD is able to optimize the outcome of patients with thalassemia major transplanted from an unrelated volunteer. Disclosures:No relevant conflicts of interest to declare.

Fludarabine-Based Conditioning for Allogeneic Marrow Transplantation From Unrelated Donors in Severe Aplastic Anemia (SAA): Serious and Unexpected Adverse Events in Pre-Defined Cyclophosphamide (CY) Dose Levels

Abstract 3009▪FN2▪This icon denotes a clinically relevant abstract Objective:To determine toxicity and efficacy of adding fludarabine (FLU) to a regimen of total body irradiation (TBI), anti-thymocyte globulin (ATG) and cyclophosphamide (CY), with de-escalation of the CY dose. The goal is to minimize CY-related toxicities while preserving (or improving) engraftment and survival in patients with SAA receiving an unrelated donor marrow transplant. Patients and Methods:Since May 2006, the Blood and Marrow Transplant Clinical Trials Network (BMT CTN), sponsored by the NHLBI and NCI, has conducted a prospective Phase I/II clinical trial of unrelated donor marrow transplantation in SAA (BMT CTN 0301). Patients with SAA are eligible if they are < 65 years, have adequate organ function, and have an available unrelated marrow donor matched 7 of 8 HLA-A, B, C, DRB1 loci. Patients with Fanconi anemia and other marrow failure syndromes are excluded. All patients receive TBI 200 cGy, ATG (either thymoglobulin: 3 mg/kg IV or ATGAM 30 mg/kg IV daily × 3, days –4 to –2), FLU (30 mg/m2 IV daily × 4, days –5 to –2). The Phase I portion of the trial sequentially tested four CY dose levels: 150 mg/kg (administered days –4 to –2); 100 mg/kg (days –3 to –2); 50 mg/kg (day –2); and, 0 mg/kg, and allowed enrollment of up to six patients at each CY dose level unless toxicity or graft failure boundaries were crossed. In the Phase II portion of the trial, patients enroll onto the optimal CY dose level, which is chosen using adaptive Bayesian criteria to rank desirability of the CY doses. All patients are followed for two years after transplantation. Early stopping guidelines are used to monitor for graft failure and early transplant-related mortality through day 100. Results:Twenty-one patients accrued to the Phase I portion of the trial. CY dose level 0 mg/kg was closed after all three enrolled patients developed secondary graft failure. All received a second allograft. One patient remains alive at 23 months after the first transplantation. The other two died from adult respiratory distress syndrome (ARDS) at 114 days after the first transplant and idiopathic pneumonia at 200 days after the first transplant. Review of Phase I results for the other three dose levels suggested CY dose 150 mg/kg as the optimal dose level for Phase II testing. However, after an additional eight patients were treated at this dose, the level was closed to further accrual because of excess toxicity. Seven of the 14 patients receiving 150 mg/kg of CY (and 7 of the last 8 enrolled) died. Causes of death were cardiac/pulmonary/multi-organ failure (n=4), ARDS (n=2), and parainfluenza virus type 3 pneumonia (n=1). Bayesian evaluation of the two remaining dose levels indicates very similar desirability scores and accrual continues at both the CY 100 mg/kg and 50 mg/kg levels. As of July 15, 2011, a total of 61 patients have been enrolled, 17 on the two closed levels, 33 on the 100 mg/kg level and 11 on the 50 mg/kg level. Conclusions:Early analysis of this trial, made necessary by these unexpected severe adverse events, revealed two important findings among patients receiving low-dose TBI, ATG, and FLU at the doses outlined: 1) CY dose 0 mg/kg is associated with higher than expected graft failure; and, 2) CY dose 150 mg/kg is associated with excess transplant-related toxicity. Neither CY dose level should be tested further in the context of the regimen used in this trial. To date, the two intermediate CY dose levels (100 mg/kg and 50 mg/kg) have not crossed the graft failure or fatality stopping boundaries and accrual is in progress. Disclosures:Pulsipher:Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Duration of Unrelated Donor Search for Hematopoietic Stem Cell Transplantation

Abstract 2046▪▪This icon denotes a clinically relevant abstract Objective:Donor search is a major challenge once the indication for allogeneic hematopoietic stem cell transplantation (HCT) has been established. For those patients who lack an HLA-identical sibling, unrelated donor search (UDS) needs to be initiated. Only if UDS is not successful, alternative stem cell sources like cord blood (CB) or alternative concepts like haploidentical HCT have to be considered. Since UDS is a time consuming process it is important to assess the probability to find a matched unrelated donor (UD) during prolonged UDS. Yet, very few data on the required times and the success rates of unrelated donor searches are available. Furthermore, information on the probability to find a donor within short time is crucial in the context of early allogeneic transplantation including HCT in aplasia after induction chemotherapy in AML or for the interpretation and planning of clinical trials employing donor versus no donor comparisons. Therefore, we have retrospectively analyzed the duration of UDS for a large cohort of patients. Methods:All unrelated donor searches of one large search unit, which were initiated between January 2004 and July 2010 were analyzed retrospectively. Throughout that period blood from all donor-recipient pairs has been typed for HLA-A, -B, -C, -DRB1 and –DQB1 at the allele level (4 digits). A full match was defined by HLA-identity for HLA-A, -B, -C and DRB1 (8/8), while a partial match was defined as a single mismatch in the HLA-A, -B, -C or DRB1 locus without additional DQB1 mismatch (9/10). The duration of UDS was calculated from the date of data entry to the date of receipt of confirmatory typing (CT) of the first 8/8 HLA-matched donor or the first 9/10 HLA-matched donor. When given as reasons for the termination of the search, poor clinical condition or death of a patient were considered as competing events. Unsuccessful searches were censored at the day of the receipt of the last typing request or when UDS was stopped for other reasons. Cumulative incidences of successful UDS were calculated with competing event statistics. Approximative 95% confidence intervals are provided for point estimates. Results:In the respective time period 852 UDS comprising 8477 donor requests were performed. In the Caucasian population, an HLA compatible UD (8/8) has been found for 29% (95% CI, 26% to 32%) of the patients within two weeks, 54% (95% CI, 51% to 58%) of patients within 4 weeks, and 61% (95% CI, 57% to 64%) of patients within 6 weeks (Figure 1). In high risk diseases and high urgent indications for allogeneic transplantation one antigen or allele mismatch may be acceptable. Therefore, we calculated the probabilities to find an HLA matched UD (8/8) or a partially matched UD (9/10) within defined times. The resulting probabilities of finding an acceptable donor who met these criteria for Caucasian patients were 35% (95% CI, 32% to 39%), 66% (95% CI, 63% to 70%) and 75% (95% CI, 72% to 78%) at two, four, and six weeks respectively. After six weeks of UDS the probability of finding a donor declined. Between the six week landmark and the 12 week landmark 8/8 HLA matched unrelated donors have been identified only for additional 4% of the patients. Extending UDS up to one year resulted in additional 3% HLA matched donors (8/8). The incidence of the competing events (death or deteriorated condition of the patient) was 10% (95% CI, 6.6% to 13.8%) at one year when the aim was to find an 8/8 HLA matched UD. Eight UDS (1%) were started for non-Caucasian patients. Only for one of the eight non-Caucasian patients an HLA-compatible donor (8/8) was found. Conclusions:For Caucasian patients HLA compatible UDs (8/8) can be identified in our population for 61% of the patients within six weeks. If donors with one mismatch (9/10) are accepted the probability increases to 75% at that time. Beyond that landmark, the probability of finding donors for the remaining patients declines steeply and alternatives like haploidentical or cord blood transplantation should be considered. Since an HLA matched donor can be identified for 29% of the patients within two weeks, UDS is a valid option even for very high urgent searches like in the context of allogeneic HCT in aplasia. Unrelated donor searches for patients with non-Caucasian ancestry are much less successful. Further analyses are planned in order to define the probability to find a donor related to the haplotype frequency of the patient. [Display omitted] Disclosures:Off Label Use: pentostatin is not licensed for use in acute GvHD.

Comparison of Unrelated Cord Blood Transplantation and HLA-Mismatched Unrelated Bone Marrow Transplantation for Adults with Leukemia

Recent advances in unrelated cord blood transplantation (UCBT) and high-resolution typing of human leukocyte antigen (HLA) from an unrelated donor have increased choices in alternative donor/stem cell source selection. We assessed HLA-mismatched locus-specific comparison of the outcomes of 351 single-unit UCB and 1,028 unrelated bone marrow (UBM) adult recipients 16 years old or older at the time of transplantation who received first stem cell transplantation with myeloablative conditioning for acute leukemia or myelodysplastic syndromes. With adjusted analyses, HLA 0 to 2 mismatched UCBT showed similar overall mortality (relative risk [RR] = 0.85, 95% confidence interval [CI], 0.68-1.06; P = .149) compared with that of single-HLA-DRB1-mismatched UBMT. UCBT showed inferior neutrophil recovery (RR = 0.50, 95% CI, 0.42-0.60; P < .001), lower risk of acute graft-versus-host disease (RR = 0.55, 95% CI, 0.42-0.72; P < .001), and lower risk of transplantation-related mortality (RR = 0.68, 95% CI, 0.50-0.92; P = .011) compared with single-HLA-DRB1-mismatched UBMT. No significant difference was observed for risk of relapse (RR = 1.28, 95% CI, 0.93-1.76; P = .125). HLA 0 to 2 antigen-mismatched UCBT is a reasonable second alternative donor/stem cell source with a survival outcome similar to that of single-HLA-DRB1-mismatched or other 7 of 8 UBMT.

Race-specific type 1 diabetes risk of HLA-DR7 haplotypes.

The aim of this study was to test the hypothesis that closely related human leukocyte antigen (HLA) haplotypes containing the DRB1*07:01 gene ['DR7' (DRB1*07:01-DQA1*02:01-DQB1*02:01g or DRB1*07:01-DQA1*03:01-DQB1*02:01g) haplotypes] derived from European and African populations differ in their genetic susceptibility for type 1 diabetes (T1D) depending on the DQ-α molecule present. A combined total of 98 African American T1D patients from the Type 1 Diabetes Genetics Consortium and from Children's Hospital and Research Center Oakland were genotyped for the HLA class II loci DRB1, DQA1, and DQB1. DNA samples extracted from newborn blood spot cards from African Americans born in California (n = 947) were used as a population-based control group. Among African American cases, the European-derived DRB1*07:01-DQA1*02:01-DQB1*02:01g haplotype was protective for T1D risk (odds ratio (OR) = 0.34; 95% confidence interval (CI) 0.14-0.78; P < 0.011), but the African-derived DRB1*07:01-DQA1*03:01-DQB1*02:01g haplotype increased T1D risk (OR = 3.96; 95% CI 1.94-8.08; P < 5.5E-05). The effect of DRB1*07:01-DQB1*02:01g on T1D susceptibility depends on the DQA1 allele. DRB1*07:01-DQA1*02:01-DQB1*02:01g is protective for T1D; however, the presence of DQA1*03:01 on the DRB1*07:01-DQB1*02:01g haplotype not only renders the DR7 haplotype not protective but also creates a haplotype with significant T1D risk. These data underscore the importance of assessing genetic effects within ethnic context.

False Homozygosity Results in HLA Genotyping due to Loss of Chromosome 6 in a Patient with Acute Lymphoblastic Leukemia

Loss of heterozygosity (LOH) in chromosome 6p has been reported in a number of tumors and some hematologic malignancies, including ALL. LOH in chromosome 6p, on which the HLA genes are located, can give rise to false homozygosity results in HLA genotyping of patients with hematologic malignancies. Here we report false homozygosity results in HLA genotyping due to the loss of whole chromosome 6 in the neoplastic cells of a patient with ALL. A 33-yr-old Korean female patient was admitted for the evaluation of leukocytosis detected during a workup for headache. Her initial white blood cell count was 336.9×109/L with 84% of blasts in the differential count. Precursor-B lymphoblastic leukemia was diagnosed from a subsequent bone marrow study. HLA high-resolution genotyping of the patient was requested at the time of diagnosis for possible hematopoietic stem cell transplantation. Homozygosity results (A*02:01, B*54:01, C*08:01, DQB1*04:01) were obtained, except for the DRB1 locus (DRB1*04:05, DRB1*11:01), in sequence-based typing. Conventional karyotyping of bone marrow metaphase cells revealed chromosomal abnormalities, with loss of multiple chromosomes including chromosome 6, and reduplication of the remaining chromosomes: 29,X,+X,+8,inv(9)(p11q13),+10,+14,+18,+21[15]/58,idemX2[3]/46,XX,inv(9)[2]. LOH at the HLA region was suspected and HLA genotyping was repeated with the peripheral blood in remission state after induction chemotherapy. All 5 HLA loci were typed as heterozygous (A*02:01, A*02:06, B*40:01, B*54:01, C*03:04, C*08:01, DRB1*04:05, DRB1*11:01, DQB1*03:01, DQB1*04:01). To avoid false HLA typing results in patients with hematologic malignancies, clinicians, as well as laboratory personnel, need to be aware of such problems and take appropriate precautions.

The distribution of HLA-A, -B, and -DRB1 alleles and haplotypes in inhabitants of Guizhou Province of China

The present study was aimed to analyze the frequencies of human leukocyte antigen ( HLA)-A, -B, and - DRB1 alleles and A-B-DRB1, A-B, A-DRB1 and B-DRB1 haplotypes in inhabitants of Guizhou province, China. All samples were typed in the HLA-A,-B, and - DRB1 loci using the polymerase chain reaction-reverse sequence specific oligonucleotide probe (PCR-rSSOP) method and HLA polymorphisms were analyzed. A total of 18 HLA-A, 31 HLA-B, and 13 HLA-DRB1 alleles were found in the Guizhou population. The first two frequent alleles in the HLA-A, -B, and - DRB1 loci were A*11(30.72%) and A*02(30.65%), B*40(16.27%) and B*46(16.27%), and DRB1*09(15.91%) and DRB1*15(13.51%), respectively. The most common haplotype was A*02-B*46- DRB1*09(5.59%) in A-B-DRB1, A*02-B*46(11.73%) in A-B, B*46- DRB1*09(7.49%) in B-DRB1, and A*02- DRB1*09(8.08%) in A-DRB1. Some haplotypes with strong linkage disequilibrium (LD) were found not only in the common haplotypes, such as A*33-B*58, B*30-DRB1*07, and B*33-DRB1*03, but also in the rare haplotypes, such as A*01-B*37, B*37-DRB1*10, and A*01-DRB1*10. Guizhou inhabitants shared some characteristics of the Southern Chinese population but also had their own unique features. Overall, HLA polymorphism in Guizhou population was more consistent with that of Chengdu population than that of other populations in China.

Pregnancy, Microchimerism, and the Maternal Grandmother

BackgroundA woman of reproductive age often harbors a small number of foreign cells, referred to as microchimerism: a preexisting population of cells acquired during fetal life from her own mother, and newly acquired populations from her pregnancies. An intriguing question is whether the population of cells from her own mother can influence either maternal health during pregnancy and/or the next generation (grandchildren).Methodology/Principal FindingsMicrochimerism from a woman's (i.e. proband's) own mother (mother-of-the-proband, MP) was studied in peripheral blood samples from women followed longitudinally during pregnancy who were confirmed to have uncomplicated obstetric outcomes. Women with preeclampsia were studied at the time of diagnosis and comparison made to women with healthy pregnancies matched for parity and gestational age. Participants and family members were HLA-genotyped for DRB1, DQA1, and DQB1 loci. An HLA polymorphism unique to the woman's mother was identified, and a panel of HLA-specific quantitative PCR assays was employed to identify and quantify microchimerism. Microchimerism from the MP was identified during normal, uncomplicated pregnancy, with a peak concentration in the third trimester. The likelihood of detection increased with advancing gestational age. For each advancing trimester, there was a 12.7-fold increase in the probability of detecting microchimerism relative to the prior trimester, 95% confidence intervals 3.2, 50.3, p<0.001. None of the women with preeclampsia, compared with 30% of matched healthy women, had microchimerism (p = 0.03).Conclusions/SignificanceThese results show that microchimerism from a woman's own mother is detectable in normal pregnancy and diminished in preeclampsia, supporting the previously unexplored hypothesis that MP microchimerism may be a marker reflecting healthy maternal adaptation to pregnancy.