Short Tandem Repeats in disputed parentage analysis: a current approach
D. Kouniaki (Athens, GR)
Short Tandem Repeats (STRs) and Humsan Leukocyte Antigens (HLA) constitute genetic systems widely used in disputed parentage analysis. The aim of this study was to evaluate the HLA polymorphism contribution in parentage cases investigation. A total of 489 DNA samples (147 trio/24 duo parentage cases) were analyzed. Genomic DNA was isolated from whole blood or buccal swabs. Analysis was performed using 16 STR loci typing by DNA sequencing and HLA typing (LR and HR) by PCR-SSP/SSOP. Power of Exclusion (PE), Random Man Not Excluded (RMNE), Combined Parentage Index (CPI), and Probability of Parentage (W) values were calculated using allele frequency of Caucasoid (STRs) and Greek (HLA) population databases. Out of 171 cases, 42 (30 trios/12 duos) were excluded by HLA & STRs and 128 (116 trios/12 duos) were not excluded by both approaches. In one case, where the two alleged fathers were relatives, there was exclusion by STRs, but not by HLA alleles. The low W rates (0.9870-0.9999) using HLA typing were increased up to 0.9999999 using STRs. The CPI was ranged from 15.173:1 (duo) to 6.76x1013:1 (trio) with statistically significant difference between trios and duo, RMNE (from 1.46x10-4 to 8.83x10-12), while PE was estimated up to 0.999999 using STRs. Additionally, STRs mutations were observed in seven cases: SE33, D10S1248, vWA, D12S391(2), D2S1338, D21S11 and 1 Null allele (SE33), in equal number of parentage disputes cases (8/129, 6.20%), (7 trios/1 duo) without excluding relation by blood. In mutation cases (ratio of paternal versus maternal 7:1) the low W as determined by STRs typing (0.9972-0.9999) was increased up to 0.9999999 using both systems. In conclusion, STR-genotyping is the current approach used in parentage or kinship cases. However, the combination of two systems (HLA & STRs) diminishes the possibility of false exclusion due to STRs mutations, and minimizes the risk of wrong inclusion due to the absence of mother’s genotype.
Report of a false negative amplification shown to be a new null allele variant
J. Granemo (Stockholm, SE)
This case study highlights the complementary strength of different molecular techniques used in the HLA field. Three different techniques and cooperation between three different labs allowed this novel null allele to be characterized. A complaint was received by Olerup SSP regarding a false negative reaction for A*01:04N in one of Olerup SSP’s HLA typing kits. The customer tested the sample as A*01:04N using Sanger sequencing. A new SSP primer targeting A*01:04N was designed for confirmatory and investigational purposes. This primer failed to amplify the sample, but amplified another allele, A*24:11N, identical to A*01:04N at the target motif. Primer functionality was then confirmed using an A*01:04N reference sample. As it still was not possible to achieve a positive amplification with the customer’s sample, it was sequenced again by another lab. The two sequencing attempts indicated a C insertion in the c.621-627 poly C region and an A629G substitution in line with an A*01:04N / A*33:01:01:01 result. However, the SSP results from Olerup did not amplify the customer’s questionable sample but amplified other A*01:04N samples and alleles with identical motifs. A new primer was designed to amplify a theoretical new allele with a C insertion at position c.627_628insC and a A629G substitution. This new primer amplified the customer’s sample but not A*01:04N and identical A*24:11N samples, supporting the theory of a new allele. This was confirmed by Pacific Biosciences' SMRT Sequencing. The sample was shown to be A*01:01:01:01 and A*33:01:01:NEW, with a c.627_628insC insertion in codon 186, exon 4. This frameshift creates a premature stop codon 196, and it was determined to be a novel null allele variant. The novel A*33 null allele has been reported to EMBL with accession number LT707076.
Holotype HLA™ automated on TBG DX-A™ and Sequenced using MiniSeq
T. Vágó (Budapest, HU)
Next Generation Sequencing (NGS) is a powerful new method for HLA genotyping. Currently, the rate of NGS adoption by HLA laboratories is rapidly increasing driven by the accuracy, resolution, reproducibility and high-throughput nature of the technology. The elimination of human and random error is a desirable attribute of any diagnostic test and the implementation of a liquid handling system can significantly reduce both sources of error in an NGS workflow. Holotype HLA is a commercially available HLA genotyping product developed in a clinical lab with clinical routine in mind. With Holotype HLA one can easily scale up to 96 samples at 11 loci on a single MiSeq run. Due to its flexibility, it is compatible with multiple liquid handling systems, such as the TBG DX-A, which is a small footprint robot capable of reducing the hands-on time for technicians for both pre- and post-PCR steps in the Holotype workflow. Together, Holotype HLA and DX-A provides an ideal solution for labs who wish to fully automate their HLA genotyping workflow, reducing variation and human error, thus increasing the specificity, sensitivity and accuracy of the genotyping. The DX-A reduces the total hands-on time of the Holotype workflow to 40 minutes. An Illumina’s MiniSeq system was used to test a set of 192 samples. In this study, the PCR setup, amplicon quantitation and normalisation, and library preparation were automated on the DX-A for HLA-A, -B, -C (class I) and HLA-DRB1, -DQA1, -DQB1, DPB1 (class II) loci for each sample. In each setup, 24 samples were prepared on the robot, then four runs of 24 samples were collected together for each sequencing run on the MiniSeq. Two MiniSeq runs have been performed to sequence 96 samples for 7 loci per run. The MiniSeq results were compared to known genotyping calls previously determined from Sanger-based sequencing data. All MiniSeq data was analysed using Omixon HLA Twin. Here we present the results of our validation of Holotype HLA automated on DX-A and sequenced on the MiniSeq system - an accurate, reproducible and high-throughput system for HLA typing in less than 48 hours from gDNA to genotype.
Calreticulin mutations in Bulgarian MPN patients – frequency and functional implication
M. Ivanova (Sofia, BG)
Somatic mutations in JAK2, MPL and CALR are recurrently identified in most cases of Philadelphia chromosome negative myeloproliferative neoplasms (MPNs). Reports on CALR mutations in south-eastern populations are relatively limited. We applied four molecular genetic methods for identification of CALR exon 9 mutations, including high resolution melt (HRM) analysis, Sanger sequencing, semi-conductor target genes sequencing and whole exome sequencing. A total of 78 patients with myeloid malignancies were included in the study. We identified 14 CALR exon 9 mutated cases out of 78 studied patients with myeloid malignancies. All mutated patients were diagnosed with MPN being either PMF (n = 7) or ET (n = 7). Nine cases had type 1 mutations and five cases had type 2 mutations. CALR exon 9, MPL exon 10 and JAK2 p. V617F were mutually exclusive. There were no statistically significant differences in the hematological parameters between the cases with CALR and JAK2 or MPL mutations. Notably, all four techniques were fully concordant in the detection of CALR mutations and in this study we demonstrated their utility. All mutations observed in exon 9 lead to a shift in the open reading frame and expression of peptides with a novel mutant C terminus. We applied further a straightforward bioinformatic approach (PONDR-FIT, RONN and peptide charge calculator) to address the mechanisms through which CALR mutations promote neoplastic transformation. We observed structural and electrochemical properties of mutated C termini of CALR probably leading to the loss of Ca2+ binding activity and therefore contributing significantly to the pathogenetic mechanisms of these mutations. However, as CALR is a protein with versatile cellular functions, this might be just one of several mechanisms. This will hopefully lead to innovative therapies for this subset of MPN patients.
Haplotype of HLA-B*13:01/HLA-B*15:02 and Dapsone-induced drug hypersensitivity syndrome in Thai
C. SUKASEM (Bangkok, TH)
Dapsone is the 5th most common cause of the drug reaction of eosinophilia and systemic symptoms (DRESS) and the 20th most common cause of drug-induced Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) in Thailand. To investigate the association of HLA genotypes with dapsone-induced hypersensitivity syndrome (DHS) in Thai non-leprosy patients. We prospectively enrolled 35 subjects (13 DHS and 22 dapsone tolerant controls) from the Thai Severe Cutaneous Adverse Drug Reaction (THAI-SCAR) project. In addition, data for a general Thai population was obtained from 986 subjects undergoing HLA-B genotyping through the Laboratory for Pharmacogenomics, Somdech Phra Debaratana Medical Center (SDMC), Ramathibodi Hospital. The HLA-B genotypes were determined by the reverse polymerase chain reaction sequence-specific oligonucleotides probe (PCR-SSOs). The HLA-B*13:01 allele was significantly associated with DHS sufferers compared to tolerant controls with a odds ratio of (OR) 33.33 (95% CI 4.77-232.79, p-value = 0.0001) in patients and OR=21.19 (95% CI 5.76-78.00, p-value = 0.0001) in the Thai population. In addition, HLA-B*15:02 was significantly associated with dapsone-induced SCARs when compared with dapsone-tolerant controls (OR=13.13; 95% CI 1.32-130.43, p-value = 0.0185) and in the general Thai population (OR=3.30; 95% CI 1.07 - 10.22, p-value = 0.0450). The haplotype of HLA-B*13:01/HLA-B*15:02 were only found in DHS (n = 3/13, 23.08 %). There were significantly associated with DHS by OR 73.65, p-value = 0.0001. This study demonstrated the association between HLA-B*13:01/HLA-B*15:02 and DHS in Thai non-leprosy patients. Moreover these results suggest this haplotype screening may be a useful genetic marker for prevention of DHS in Thai population.
Identification of a novel HLA-DRB3 allele
S. Ulrich (Graz, AT)
We describe the identification of the new HLA-DRB3*02:61Q allele during routine confirmatory human leukocyte antigen (HLA) typing of a volunteer bone marrow donor. Initial typing of HLA-DRB3 locus by SSP (Olerup SSP AB, Sweden) showed an unusual reaction pattern detecting the rare alleles DRB3*02:37/51/52 by analysis in SCORE Standard Version (Helmberg, Austria) with IPD-IMGT/HLA Database Release 3.25.0. To verify this result sequence based typing of DRB3 exon 2 was performed using Protrans S1 HLA DRB3 kit (Protrans GmbH, Germany) following manufacturer`s protocol. Subsequent allele assignment with SBTengine (Genome Diagnostics B.V, Utrecht, Netherlands) identified the new allele. Compared to the common DRB3*02:02:01 allele, the new sequence results from a continuous deletion of 3 nucleotides in exon 2 between nucleotide positions 187 to 196. The nucleotide stretch from 188 to 195 of DRB3*02:02:01 represents an incomplete triplet repeat of AGG (AGG AGG AG), thus any deletion of a triplet within this section delivers an identical sequence. The consequence of this deleted triplet is the removal of one glutamic acid in the protein sequence. According to these facts expression of the allele on the cell surface is questionable.
The sequence was submitted to GenBank (accession number BankIt1966791 Seq1 KY099331) and to the IPD-IMGT/HLA Database (accession number HWS10027219). The new allele was officially named DRB3*02:61Q by the World Health Organization (WHO) Nomenclature Committee in December 2016.
Frequency of HLA-B*57:01 allele in HIV infected patients and screening for Abacavir therapy
S. Giuliodori (Parma, IT)
Abacavir (ABC) is an anti-retroviral drug recommended for Human Immunodeficiency Virus (HIV) infected individuals. During ABC therapy hypersensitivity reactions (HSR) can occur approximately 4-9% of the patients. The development of HSR to ABC is strongly associated with the presence of HLA-B*57:01. Its frequency varies in different ethnic populations at about 5% in caucasian populations, while other populations, especially Africans, have a lower allele frequencies. This study was designed to establish HLA-B*57:01 frequency in our population of HIV infected patients to establish the effectiveness of prospective HLA-B*57:01 screening. A total of 212 HIV-infected patients, from January 2013 to July 2016, were admitted to the prospective HLA-B*57:01 screening for the prevention of ABC-correlated HSR. 210 healthy blood donors were used as a control group. Patients mean age was 40 years (range 14-74); 67 females (32%) and 145 males (68%), 53% (113 patients) were of Italian origin. Control group mean age was 48, (range 21-73), 111 females (53%) and 99 males (47%). HLA-B low resolution genotyping was performed using SSO-PCR (HistoSpot, BAG) and/or SSP-PCR (One Lambda). In HLA-B*57 positive samples we searched for the presence of HLA-B*57:01 allele using high-resolution (HR) technique, such as SSO-PCR (HistoSpot 4D, BAG) and/or SSP-PCR (Olerup). Among 212 HIV-infected patients, 15 (7%) were carriers of HLA-B*57 allele; 5 of Italian origin and 10 of African origin. HR typing allowed us to identify HLA-B*57:01 allele among 5 Italian patients, but not in any African patients. HLA-B*57:01 allele frequency was 4.42% in Italian HIV infected patients and 5.24% in the control population. HLA-B*57:01 allele frequency in HIV-infected local patients was similar in Italian population and controls (about 5%), similar to caucasians. Since the clinical importance of HSR to ABC treatment in HIV infected patients and the frequency of HLA-B*57:01 carriers reported in this study, we suggest the preventive use of genetic screening in Italian population before ABC treatment.
Cytokines and soluble human leukocyte antigen G marrow stroma levels and survival in childhood T-cell acute lymphoblastic leukemia
N. Lucena-Silva (Recife, BR)
Leukemic cells can induce defective expression of soluble factors and can change marrow cytokine profile, leading to aberrant cell signaling, cell fixation and cell proliferation in bone marrow. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disorder which accounts for 15% of pediatric ALL. To evaluate the contribution of immunological factors on T-ALL survival, we measured Th1, Th2, Th17 cytokines and soluble human leukocyte antigen G (sHLA-G) levels in bone marrow from 32 Brazilian children at diagnosis (D0), after induction (D19) and after consolidation (D49) of the chemotherapy phase. Data analyses were performed using the Wilcoxon and Friedman tests. Correlations were evaluated by the Pearson coefficient and reciprocal influences of the analyzed factors were determined by multivariate analysis using Pearson linear regression. Survival analyses were performed by the Kaplan-Meier method using log-rank test. TNF, IL-10 and IL-6 marrow levels were increased at diagnosis compared to the D19 and D49. IL-10 marrow levels < 4.57 pg/mL at diagnosis were associated with increased survival rates. In this group, a positive correlation was observed between IL-2 and IL-17 levels. Increased survival rate was also associated with IFN-γ marrow levels < 1.17 pg/mL at consolidation phase. In this group, we observed a positive correlation between IL-4 and IL-2 as well IL-4 and IL-17 levels. In contrast, a worse survival rate was associated with IL-2, IL-4 and IL-10 levels imbalance. In addition, increased sHLA-G levels at diagnosis were associated with increased leukocyte count, a well-known factor for poor prognosis.
A simple and fast method for enrichment of lymphocyte subsets for complement-dependent cytotoxicity assays
M. Könn (Bergisch-Gladbach, DE)
Serological cross-match analysis such as complement-dependent cytotoxicity (CDC) assay is routinely done before solid organ transplantation to detect donor-specific antibodies that may lead to graft rejection or dysfunction. This analysis is based on isolated donor lymphocyte subpopulations and recipient serum. Enrichment of cells for CDC can be exceedingly time consuming or result in co-enrichment of non-target or dead cells. We aimed to develop a fast and convenient method for enrichment of donor lymphocyte subsets directly from whole blood or spleen cell suspensions.
Using MACSxpress® technology, untouched human leukocyte subsets can be isolated within only 20 minutes from up to 30 mL of anticoagulated whole blood. For the enrichment of B, T cells, or a combination of both cell types, anticoagulated whole blood or cell suspensions obtained from rinsed donor spleens were incubated with the respective bead cocktails for 5 min at room temperature. Then the open tube was placed in the magnetic field of a MACSxpress Separator for 15 min. With the tube inside the magnetic field, the supernatant containing the enriched target cells was collected and transferred into a new tube. Magnetically labeled non-target cells as well as aggregated erythrocytes were retained in the tube. Purity and viability of magnetically enriched cell populations were determined by flow cytometric analysis and functionality shown in CDC assays. MACSxpress-enriched lymphocyte subsets (B/T/BT cells) from whole blood had average purities of 94/96/98 % with yields of 70/54/40 % (n=17/19/21) respectively. Viability analysis of enriched fractions showed that the frequency of dead cells was below 5% for all samples. Cell enrichment from spleen single cell suspensions resulted in purities of 97/96/98 % and recoveries of 27/32/29 % (n=5) (B/T/BT cells) respectively. Viability of the enriched fractions varied due to the age of the samples but was never below 90%. CDC analysis of positive and negative control sera with MACSxpress enriched cells showed no influence of fluorescent readout by the enrichment process.
Using MACSxpress technology, lymphocytes for crossmatch analysis can be enriched within 20 minutes from whole blood or spleen cell suspensions without the need of expensive lab equipment or preparation of PBMC by density gradient centrifugation.