Search

Aim HLA alleles are observed in specific haplotypes due to Linkage Disequilibrium (LD) between certain alleles. Recent improvements in Next-Generation Sequencing (NGS) technologies accommodate the tight linkage disequilibrium between genes as well as the high degree of polymorphism of the MHC. In order to assign HLA haplotypes at allele level (4-fields) in a Greek population, in the context of 17th IHWS, 25 families including parents and siblings (95 subjects), all of Greek origin, were typed at 11 loci (HLA-A*, -B*, -C*, -DRB1*, DRB3/4/5*, -DQA1*, -DQB1*, -DPA1* and -DPB1*) by NGS technology. Methods Instrument and Chemistry: Illumina MiSeq, v2 chemistry, 300 cycles (2X150) and standard flow cell size. Illumina TruSight HLA v2.0 and Omixon Holotype HLA kits were used. HLA haplotype count and frequency (%) were estimated according to the number of parental haplotypes identified in the whole cohort. Additionally, haplotype count in families (N = 25) and subjects (N = 95) were calculated. Results HLA-A*01:01:01:01-C*07:01:01:01-B*08:01:01:01-DRB3*01:01:02:01-HLA-DRB1*03:01:01:01/02-DQA1*05:01:01:02-DQB1*02:01:01-DPA1*01:03:01:02-DPB1*04:01:01:01/02 haplotype was found in 3 members of 2 families (haplotype frequency 2%). All other haplotypes were observed once, giving haplotype frequency = 1%. Conclusions It is confirmed that the HLA-A*01-Cw*07-B*08-DRB1*03:01-DQB1*02 haplotype, defined by low-resolution molecular techniques, is the most common in Greeks, as in all Caucasians. These results are useful as reference data (controls) in disease association studies as well as in population genetics.

Aim Genetic recombination occurs at specific hotspots within the MHC, leading to widely dispersed block-like structures; the alpha block containing HLA-A, beta block (HLA-C and HLA-B) and delta block (HLA-DR and HLA-DQ). Linkage disequilibrium (LD) is much stronger within than across these blocks. The HLA-A locus (most telomeric) has weaker LD with extended HLA haplotypes. Additionally, a recombination hotspot exists between HLA-DP genes and other class II loci, which leads to an HLA-DPB1 match rate of only 10–15% in 10/10 HLA-matched donor/recipient pairs. This report describes an extended 9 locus HLA haplotype found in 2 HCT and 1 kidney transplant recipients and their potential donors (8, 2 and 1 donors, respectively). Methods 1. Case review of 2 unrelated HCT recipients, both European Caucasian (CAU) and their related and unrelated potential donors. 2. Family study of HCT recipient and a sibling to identify the haplotype by segregation. 3. Determine frequency of observed haplotype in different populations through Haplostats.org (6 locus at 2 field resolution could yield higher estimate than the 9 locus 3 field resolution haplotype described here). 4. Searching lab database for incidence of this haplotype. Results All identified cases shared the following haplotype HLA-A*31:01:02∼C*07:02:01∼B*07:02:01∼DRB5*01:01:01∼DRB1*15:01:01∼DQA1*01:02:01∼DQB1*06:02:01∼DPA1*01:03:01∼DPB1*04:01:01. Although most alleles in this haplotype are fairly common, with HLA-A*31:01 least common (∼5%), Haplostats indicated that the haplotype HLA-A*31:01∼C*07:02∼B*07:02∼DRB5*01:01∼DRB1*15:01∼DQB1*06:02 ranks 61898th & 170477th in CAU & African Americans (AFA), respectively and is not seen in other populations. Searching our laboratory database for subjects A31 positive, 382 subjects out of 8340 typed between 2015 & 2017 were identified. Of those, 40 were positive for the haplotype HLA-A*31:01∼C*07:02∼B*07:02∼DRB5*01:01∼DRB1*15:01∼DQB1*06:02 and of these 17 were typed for HLA-DPB1. All were positive for the entire extended 9 locus haplotype and were either CAU (n = 6) or of unknown ethnicity (n = 11). Conclusions Although the 6 locus haplotype portion of this extended haplotype seems fairly uncommon, the extended 9 locus subset of that haplotype seems to be conserved especially in Caucasians.

Aim Family studies are the gold standard for studying HLA haplotype segregation. This information is critical when studying haplotype associations among populations where few members of the population have been typed, rendering bioinformatics algorithms less reliable. Here, we characterize HLA haplotypes in 5 Jamaican families identified through the Caribbean Bone Marrow Registry. Methods Twenty Jamaican individuals of 5 families were tested by NGS HLA genotypes at loci A, B, C, DRB345, DRB1, DQA1, DQB1, DPA1 & DPB1. We initially attempted full gene sequencing but amplification failed repeatedly due to suboptimal DNA quality from buccal swabs. Typing was performed using Scisco Genomics kit on the MiSeq. Identified haplotypes were compared to haplotype frequency ranking among the 5 major US populations (Caucasians (CAU), African Americans (AFA), Hispanics (HIS), Asian Pacific Islanders (API), and Native Americans (NAM)) using the NMDP Haplostats. Haplostats provides data on 6-locus 2-field resolution, which could give a higher estimate than the 9-locus 3-field haplotypes described here. Results Typing was successful in all individuals and all loci except for failed DPB1 typing on 2 subjects due to low DNA quantity. Analysis of 20 members identified 17 unique haplotypes. The most common haplotype among CAU, HIS, and NAM and 2nd most common in AFA (Haplotype A in Table 1) was observed in 3 families. Another haplotype in 2 families (B) was uncommon among CAU, HIS & AFA (ranked 20466, 23219 & 8463, respectively) and not reported in other populations. Overall observed haplotypes were more common in AFA than other population and 2 haplotypes (C & D) were seen only in AFA. One haplotype (E) was seen only in API (ranked 5660). One haplotype (F) was not reported for any population. Conclusions Although the number of tested families is small, this data suggest that the Jamaican population is unique and diverse, and supports the proposition of establishing a local/regional BMT registry to maximize the chances of identifying matched donors.

Aim Analysis of circulating donor derived cell-free DNA (ccfDNA) promises a non-invasive method of assessing allograft rejection and possibly eliminating the need for surgical biopsies. In this study we compared the concentration and purity of ccfDNA in specimens collected in ACD (BD Vacutainer) tubes and Cell-Free DNA BCT (Streck) tubes and processed after different storage times. Methods Blood samples from 4 healthy donors were drawn into collection tubes (3 tubes each for analysis at different time points). Samples were stored at room temperature and processed at 18, 24, and 72 h. The samples were processed using Promega LV ccfDNA Custom kit according to the manufacturer protocol. Tubes were centrifuged at 2000 g for 10 min and an average of 4 ml of plasma was separated from the cells. Samples were extracted on the Maxwell RSC, and eluted in a volume of 65 ul. Samples were measured by Qubit (Thermo Fisher) & ProNex DNA QC Assay (Promega) to determine DNA concentration. The level of genomic DNA (gDNA) contamination was determined by the ProNex Assay and confirmed by the TapeStation D1000 High Sensitivity kit (Agilent Technologies). The ProNex Assay uses a multiplexed probe-based qPCR assay to detect quantity and quality of genomic DNA. It detects 75 bp, 150 bp and 300 bp genomic DNA sequences. The presence of 300 bp sequences indicates contamination with gDNA. Results DNA concentration by the Qubit showed a wide range of quantity. The quantity of 75 bp and 150 bp DNA fragments as shown by the qPCR assay indicate the amount of ccfDNA in each sample. The ratios of smaller fragments to 300 bp in each sample indicate the level of gDNA contamination. The Streck specimens had consistently high ratios up to 72 h. The ACD specimens had much lower ratios, decreasing by 72 h. The presence of gDNA contamination was confirmed by TapeStation traces and one such example at 72 h is shown below. Conclusions ACD tubes consistently exhibited higher gDNA contamination, as reflected by lower ratios in qPCR, which increased over time. Download : Download high-res image (190KB) Download : Download full-size image

Aim Chromosomal abnormality which may down regulate the human leukocyte antigen (HLA) on chromosome 6 may result in inaccurate homozygous typing of a patient and thus adversely affect donor and recipient matching and transplant outcome. We present a case of a 60 year old female with AML who had chromosomal abnormality in tumor cells. Methods HLA typing was performed by SSP, rSSO microarray and SBT. Short Tandem Repeat (STR) was performed using primers targeting 10 genetic loci. Results Initial typing on patient’s blood revealed the following: SSP method: A∗32, weak bands for A∗2; B∗51, weak bands of B∗44; C∗01, weak band of C∗12, DRB1∗1301, weak band of 07:01; DRB3, weak band of DRB4; DQB1∗06:03, weak band of 02:02. rSSO (class I only): A∗02:XX, 32:XX; B∗51:XX, C∗01:XX. SBT (class I only): A∗32:01, 32:01; B∗51:01, 51:01; C∗01:01, 01:01. Since SSP showed presence of a second haplotype with weak bands, and rSSO detected two A locus antigens, HLA typing was questionable and thought that could be due to transfusion or chromosomal abnormality. HLA typing was repeated using patient’s buccal swab. Results of both SSP and SBT confirmed heterozygosity (mentioned above) with strong bands in all loci in SSP and amplification of both haplotypes in SBT. rSSO typing was not performed on buccal swab. Short tandem repeat (STR) profiles of blood and buccal swabs samples showed identical amplification for 10 primers used. Conclusions The above results show the loss of HLA genes on chromosome 6 in an AML patient. This abnormality was not included in 10 short tandem repeat we tested for. In this case, SSP method showed some weak amplification and helped us to address the possibility of chromosomal abnormality. HLA typing of buccal swab, blood at the time of remission or confirmatory typing must be considered to accurately type leukemia patients with possible chromosomal abnormality.