Unrelated Bone Marrow Donor Registries Research Articles

139-P: HLA-A, -B, -C, -DRB1, -DQA1, -DQB1 AND -DPB1 ALLELE FREQUENCIES IN A SAUDI POPULATION USING NEXT GENERATION SEQUENCING TECHNIQUE

Next generation sequencing is a promising technique that can reveal the entire gene sequences and to the highest possible resolution without any phase ambiguities. We have employed this technique to investigate the frequencies of HLA-A, -B, -C, -DRB3/4/5, -DRB1, -DQA1, -DQB1 and -DPB1 in a Saudi cohort of healthy individuals. We employed Next Generation Sequencing using the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software to HLA genotype eight class I and class II loci. A total of 158 healthy Saudi adults were analyzed. The most frequent allele for HLA-A was HLA-A∗02:01:01:01 (13.6%); for HLA-B, HLA-B∗50:01:01 (15.8%); for HLA-C, HLA-C∗06:02:01:01 (18.7%); for HLA-DRB1, HLA-DRB1∗07:01:01:01 (26.6%); for HLA-DRB3/4/5, HLA-DRB4∗01:01:01:01 (44.0%); for HLA-DQA1, HLA- DQA1∗02:01(26.3%); for HLA-DQB1, HLA- DQB1∗02:01:01 (20.3%) and for HLA-DPB1, HLA- DPB1∗04:01:01 (33.2%). By studying Linkage Disequilibrium in this population the following two loci combinations were well represented; B∗50:01:01-C∗06:02:01:01; DRB1∗03:01:01:01-DQB1∗02:01:01; DRB1∗07:01:01:01-DQB1∗02:02. Two extended, conserved haplotypes were well represented in this population; A∗24:02:01:01-B∗08:01:01-C∗07:02:01:01- DRB1∗03:01:01:01-DRB3∗02:02:01-DQA1∗05:01:01- DQB1∗02:01:01-DPB1∗03:01:01 and A∗30:02:01-B∗53:01:01-C∗04:01:01:01-DRB1∗07: 01:01:01-DRB4∗01:01:01:01-DQA1∗02:01-DQB1∗03:03:02- DPB1∗04:01:01. Expected heterozygosity was significantly higher than observed. We have used a highly informative technique for HLA typing of a Saudi healthy cohort to establish allele and haplotype frequencies. This should be of great importance for population studies, disease associations and future planning of the unrelated bone marrow donor registry.

HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1 allele and haplotype frequencies in Saudis using next generation sequencing technique

Next generation sequencing (NGS) is a promising technique that can reveal the entire gene sequences and to the highest possible resolution without any phase ambiguities. We have used this technique to investigate the frequencies of HLA-A, -B, -C, -DRB1 and -DQB1 in a Saudi cohort of healthy individuals. We used NGS using the 454 genome sequence (GS) FLX System and Conexio assign atf 454 software to human leukocyte antigen (HLA) genotype eight class I and class II loci. A total of 158 healthy Saudi adults were analyzed. The most frequently observed allele for HLA-A was HLA-A*02:01:01:01 (13.6%); for HLA-B, HLA-B*50:01:01 (15.8%); for HLA-C, HLA-C*06:02:01:01 (18.7%); for HLA-DRB1, HLA-DRB1*07:01:01:01 (26.6%); and for HLA-DQB1, HLA-DQB1*02:01:01 (20.3%). The most common four loci haplotypes in the Saudi population were HLA-A*24:02:01:01-B*08:01:01-C*07:02:01:01-DRB1*03:01:01:01 and HLA-A*23:01:01-B*50:01:01-C*06:02:01:01-DRB1*07:01:01:01.. We have used a highly informative technique for HLA typing of a Saudi healthy cohort to establish allele and haplotype frequencies. These results should prove useful for population studies, disease associations and future planning of the unrelated bone marrow donor registry.

Two conserved HLA haplotypes (HLA-A∗11:127N-B∗54:01-DRB1∗04:05 and HLA-A∗11:01-B∗40:221-C∗03:04-DRB1∗14:54-DQB1∗05:02) observed in the Taiwanese population

ObjectiveHLA-A∗11:127N and HLA-B∗40:221 are two rarely observed alleles in the HLA-A locus and in HLA-B locus, respectively. The objective of this study is to report the two plausible deduced HLA haplotypes in association with HLA-A∗11:127N and HLA-B∗40:221 in unrelated Taiwanese bone marrow stem cell donors. Materials and MethodsA sequence-based typing method was used to confirm the low incidence alleles observed. Polymerase chain reaction was carried out to amplify exons 2, 3, and 4 of the HLA-A, -B, and -C loci with group-specific primer sets. Amplicons were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit in both directions according to the manufacturer's protocol. ResultsWe deduced the two probable HLA haplotypes in association with A∗11:127N and B∗40:221 as HLA-A∗11:127N-B∗54:01-DRB1∗04:05 and HLA-A∗11:01-B∗40:221-C∗03:04-DRB1∗14:54-DQB1∗05:02, respectively. ConclusionInformation on the two deduced HLA haplotypes associated with the rare A∗11:127N and B∗40:221 alleles that we report here is valuable for HLA tissue typing laboratories for reference purposes and for stem cell transplantation donor search coordinators to determine the likelihood of finding compatible donors in unrelated bone marrow donor registries for patients bearing these two uncommon HLA alleles. Because A∗11:127N and B∗40:221 have so far been found only in the Taiwanese population, we think the haplotypes that we report here are most likely conserved in Taiwanese.

1025-LB

Aim Next generation sequencing of HLA exonic amplicons with the 454 Life Sciences GS FLX System and Conexio Assign TM -ATF software provides high resolution, high throughput HLA genotyping for 8 class I and class II loci (Bentley et al., 2009 1 , Holcomb et al., 2011 2 ). HLA typing of potential donors for Unrelated Bone Marrow Donor Registries requires using a subset of these loci at high sample throughput and low cost per sample. To maximize the throughput, we have incorporated the Fluidigm ® Access Array TM system to simplify amplicon library preparation and allow the efficient introduction of MIDs (Multiplex Identifiers) or barcodes. Methods For this application, a streamlined amplicon sequencing workflow employing many different Multiplex Identifiers (MIDs) enables multiplexed sequencing of a large number of samples, allowing researchers to meet these cost targets. The Fluidigm ® 48 x 48 Access Array TM system and the “4 primer” approach provides an efficient way to achieve parallel semi-automated genomic PCRs with simultaneous incorporation of 48 different MIDs corresponding to 48 genomic DNA samples, and up to 48 different primer pairs, making possible 2,304 reactions in one amplification run. Minimal volumes (calculated to be about 45% less cost per run) of reagents are used in this micro- fluidics-based system. During genomic PCR, the outer set of primers containing the MIDs and the 454 adaptor sequences are incorporated into the amplicons generated by the inner set of HLA specific primers containing a complementary “universal” Fluidigm tag. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 GS FLX System, as well as genotyping with Conexio Assign TM -ATF software. Results Using the Access Array system, we have successfully (100% concordance with known genotypes) genotyped 192 samples with 8 primer pairs (covering exons 2 and 3 in HLA-A, B, C and Exon 2 in DRB1, DRB3/4/5 and DQB1) using 96 MIDs per region in a single GS FLX run on a 2 region PicoTiterPlate™ (PTP) and 96 samples using 48 MIDs per region in a 4 region PTP. An average of 166 and 137 sequence reads per amplicon were recovered respectively. We have also genotyped, in a single run, 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 175 sequence reads per amplicon at 100% concordance. The system reduces the overall time for the entire process (192 samples) from 8 days for manual processing to about 4 days with 1 FTE.

Probable HLA haplotypes in association with the uncommon HLA-C*03:36, -C*03:56, and -C*03:86 alleles in a Taiwanese population

ObjectiveHLA-C*03:36, -C*03:56 and -C*03:86 are three human leukocyte antigen type C (HLA-C) locus alleles that are rarely observed in the Taiwanese population. The objective of this study is to report the three plausible deduced HLA haplotypes in association with C*03:36, C*03:56, and C*03:86 in Taiwanese unrelated bone marrow stem cell donors. Materials and MethodsThe sequence-based typing method was used to confirm the low-incidence alleles observed. Polymerase chain reaction was carried out to amplify exons 2 and 3 of the HLA-C locus with group-specific primer sets. Amplicons were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit in both directions according to the manufacturer's protocol. ResultsWe confirmed the DNA sequences of C*03:36, C*03:56, and C*03:86 and deduced the three plausible HLA haplotypes in association with C*03:36 (A*33:03-B*58:01-C*03:36-DRB1*01:01), C*03:56 (A*02:01-B*48:01-C*03:56-DRB1*12:02), and C*03:86 (A*02-B*35-C*03:86-DRB1*09:01) in unrelated Taiwanese bone marrow stem cell donors. ConclusionThe three uncommon HLA-C haplotypes that we provide here are valuable for HLA tissue-typing laboratories for reference purposes and for stem cell transplantation donor search coordinators to determine the likelihood of finding compatible donors in unrelated bone marrow donor registries for patients bearing these three uncommon HLA-C locus alleles.

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.

Routine HLA‐B genotyping with PCR‐sequence‐specific oligonucleotides detects a B*52 variant (B*5206)

A new human leukocyte antigen (HLA)-B allele was found during routine typing of samples for a German unrelated bone marrow donor registry, the "Aktion Knochenmarkspende Bayern". After first interpretation of data of two independent low-resolution sequence-specific oligonucleotide typing tests, a B*51 variant was suggested. Further analysis via sequence-based typing identified the sequence as new B*52 allele. This new allele officially assigned as B*5206 differs from HLA-B*520102 by one nucleotide exchange in exon 2. The mutation is located at nucleotide position 274, at which a cytosine is substituted by a thymine leading to an amino acid change at protein position 67 from serine (TCC) to phenylalanine (TTC).

A new HLA-B allele, B*1565, identified in three unrelated samples.

Anew human leukocyte antigen-B allele, B*1565, has been identified during routine typing of cord blood samples. Subsequently, two individuals from the same family as the first cord blood sample plus two unrelated Australian Bone Marrow Donor Registry samples have been found to carry this novel allele.

Ten years of unrelated bone marrow transplantation in Hong Kong.

The Hong Kong Marrow Match Foundation was established as a charity in 1991 to set up and operate an unrelated bone marrow donor registry for the largely Chinese population of Hong Kong and to assist patients requiring bone marrow transplantation to locate donors in Hong Kong or elsewhere. In the 10 years that followed, the registry has recruited over 42000 predominantly Chinese donors and has provided donors for 150 patients in Hong Kong and overseas requiring transplantation. This report describes the current status of the Foundation.

Identification of new DRB1*07 (DRB1*0703), DRB1*08 (DRB1*0817) and two DRB3* (DRB3*0302 and DRB3*01014) alleles.

We report here the identification of four novel DRB alleles using a reverse hybridization (CANTYPE) assay. Molecular cloning and sequencing confirmed the initial unusual hybridization patterns. All four new alleles were detected during routine HLA typing for the Canadian Unrelated Bone Marrow Donor Registry. DRB1*0703 is identical to DRB1*0701 except for a single nucleotide substitution (AGA-->AGT), changing codon 29 from Arg to Ser, a so far undetected DRB polymorphism. DRB1*0817 differs from DRB1*0801 by a single nucleotide substitution (TAC-->TTC), changing codon 47 from Tyr to Phe. This polymorphism has not, until now, been identified in DRB1*08 alleles. Compared with DRB3*0301, DRB3*0302 contains a single nucleotide substitution (TAC-->CAC) at codon 30, changing the encoded Tyr to His. This polymorphism is typical for DRB3*02 alleles. DRB3*01014 is identical to DRB3*0101 except for a single silent nucleotide substitution (GGG-->GGA) at codon 84. This polymorphism has previously only been described for the DRB1*15012 allele. DRB1*0817, DRB3*0302 and DRB3*01014 may have arisen from gene conversion, but DRB1*0703 most likely was generated by a point mutation event. The DRB3*0302 allele was detected in two unrelated subjects, while the other three have each only been detected once.

A three-step allele-level DRB1-DRB3-DRB4-DRB5 genotyping assay using polymerase chain reaction with immobilized sequence-specific oligoprobes.

A three-step reverse hybridization assay for allele level-resolution DRB1-DRB3-DRB4-DRB5 genotyping is described. Samples are initially amplified using a generic primer pair for all DRB1-DRB3-DRB4-DRB5 alleles and PCR products are hybridized to generic typing membranes. An intermediate-resolution level genotyping is obtained at this point. Depending on the phenotype, samples are then subjected to a DR1, DR2, DR4, DR52A, DRB3 and/or DRB5 type-specific amplification and hybridization. A third step, involving sequence-specific PCR followed by type-specific hybridization, is only performed to solve certain DR4 and DR52A heterozygous combinations. The assay allows 100% allele level-resolution DRB genotyping. Hybridization membranes contain panels of SSO probes that were optimized to all react specifically under identical stringency conditions. A computer program was written to assist in analysis of the hybridization patterns. The assay was throughly evaluated and has been used to type over 10,000 donors from the Canadian Unrelated Bone Marrow Donor Registry (UBMDR) at allele level-resolution. This method proved to be flexible, easy to update for newly described alleles, easy to perform, fast, and safe. It is also reliable and specific, as 9 novel DRB alleles so far have been detected as aberrant hybridization patterns.

Strategies to recruit plateletpheresis donors from a registry of HLA-typed, unrelated, bone marrow donors.

Rising demand for single-donor platelet components--from random donors, to maintain platelet inventories, or from HLA-compatible donors, to support alloimmune platelet-refractory patients--necessitated increasing the size of a community plateletpheresis donor registry. This study compares two strategies for recruiting whole-blood donors into a plateletpheresis program. The whole-blood donors who were asked to participate in this study had recently joined an unrelated bone marrow donor registry and had been HLA-typed as part of that process. An in-person recruitment strategy, which was time-intensive for the apheresis donor coordinator, served as the standard. A by-mail strategy involved the mailing of recruitment materials to marrow-donor registry participants. Marrow-donor registry participants were approached about apheresis participation after they had indicated an interest in the plateletpheresis program by returning a tear-off section of an informational brochure that was sent to them along with their marrow-donor registry materials. A total of 852 marrow-donor registry participants were randomly assigned to one of two recruitment strategies, and the recruitment rates were the same (46%) for both methods. In addition, levels of apheresis participation and attrition rates of donors recruited by either strategy were comparable. Thus, the simple strategy of mailing information about a plateletpheresis program is a very cost-effective method of recruiting donors.

Twelve HLA-DR6-related DRB1 alleles and associated DR, DQ haplotypes in traditional Australians and other populations of Asia-Oceania.

The relative distributions of 12 HLA-DR6-related HLA-DRB1 alleles in indigenous populations of Australia, Melanesia, Polynesia, Micronesia, and northern and southern China have been determined by analysis of nucleotide sequence polymorphisms in 364 examples of HLA-DR6 positive chromosomes. Oligonucleotide hybridizations of polymerase chain reaction products of HLA-DQA1, DQB1, DRB1 and DRB3 genes generated 24 HLA-DR6-related haplotypes. The study aimed to determine the regional distribution of DR,DQ haplotypes associated with three novel HLA-DR6 alleles, namely DRB1*1408, 1409, and 1410, known to occur in Australian Aborigines, to gain further insights into the molecular phylogeny of these alleles. DRB1*1408 was the most common HLA-DR6 subtype in Oceania, although it was not detected in Chinese. In Australian Aborigines and Papua New Guinean highlanders, DRB1*1408 was associated with DRB3*0202, while in Polynesians and Micronesians it was associated with DRB3*0101. The different haplotype arrangements, together with the near absence of DRB1*1408 in coastal Melanesians, suggest the possibility that two independent mutations have generated DRB1*1408 in Australia and Oceania. DRB1*1409 and 1410 alleles were confined to Australian Aborigines, while DRB1*1407 was found exclusively in Melanesians; DRB1*1401 was the only HLA-DR6 allele represented in all study populations. The population-specific HLA-DR6 alleles and haplotypes have important implications for unrelated bone-marrow donor registries in Australia and Oceania.

The first‐year costs of establishing an unrelated bone marrow donor registry

The cost of therapies like bone marrow transplantation has been an important consideration for several decades. Bone marrow transplantation is becoming increasingly accepted as an effective treatment for hematologic disorders, including acute nonlymphocytic leukemia. To find suitable donors, bone marrow donor registries are being developed. The first-year costs of establishing an unrelated bone marrow donor registry are reported here. First-year costs are largely due to personnel costs and HLA typing charges. The cost per registrant decreases over time, but further decreases due to economies of scale are limited by the continued fixed requirement for HLA typing. Data are presented by separating costs into six unique categories, thereby allowing other blood centers to estimate start-up costs based on our experience.