PMC3996364_01
PMC3996364_02
Pedigree of the second patient (Case B), showing HLA-typing of patient, brother, and father. As expected the patient and his father were HLA haploidentical. The mother could not be tested due to early death.
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fig1
The HLA types of father and son, shown in Figure 1, revealed the existence of several informative regions; for instance, HLA-A*03 could be distinguished from A*02 and A*29 and B*44 could be distinguished from B*13 and B*51 by length discrepancies (http://www.ebi.ac.uk/imgt/hla/align.html).
Flowchart showing the two alternative analytical approaches to a patient with suspected HLA-LOH. The corresponding results are illustrated in Figure 3 (Case B).
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fig2
To attribute the allele discrepancy to the patient's leukemic blasts we confirmed testing in separated cells (Figures 2-4).  Figure 3(a) shows the RFQ analysis of a blood sample containing a mixed cell population of donor and leukemic cells.
Relative fluorescent quantification (RFQ) of HLA-DPB1 (Case B). (a) RFQ analysis of the patient's sample showed a chimeric allele pattern resulting from donor (artificially colored in red) and host cells. (b) Complement-dependent cytotoxicity removal of the paternal cells by anti-HLA antibodies (anti-HLA-A2 and anti-HLA-B13) revealed an imbalance in the number of DPB1 alleles in the purified host-derived cells, latter enriched for leukemic cells. This result indicated that approximately 50% of the leukemia cells lacked the maternal (mismatched) DPB1*04:01 allele. Complete donor cell removal was proven by disappearance of DPB1*17:01. (c) Blast enrichment by LAIP based (CD34+/CD56+) FACS-sorted cells from a bone marrow aspirate confirmed the imbalance of the DPB1 alleles again assigned to the leukemic cells. No donor DNA was detectable. (d) RFQ analysis of LAIP negative (CD34-/CD56-) cells showed balanced DPB1 alleles. No maternal DPB1*04:01 was detectable. (e) As another control, the HLA-identical brother of the patient was tested, as expected a balanced number of alleles were detected.
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fig3
To attribute the allele discrepancy to the patient's leukemic blasts we confirmed testing in separated cells (Figures 2-4).  Figure 3(a) shows the RFQ analysis of a blood sample containing a mixed cell population of donor and leukemic cells. The surviving cells were harvested, and their DNA was analyzed by RFQ (Figure 3(b)). The RFQ analysis of the CD34+/CD56+ cells, resembling enriched leukemic blasts, confirmed the previously obtained allele ratio as depicted in Figure 3(c). Likewise, CD34-/CD56- cells were donor derived (no maternal DPB1*04:01 allele) and showed equal peak heights (Figure 3(d)). As additional control, the patient's healthy HLA-identical brother was tested, showing as expected a balanced ratio between the DPB1 alleles (Figure 3(e)).
RFQ of HLA-B. Complement-dependent cytotoxicity donor-depleted cells of the second patient (Case B) were analyzed by RFQ which has taken advantage of an HLA-B length polymorphism in intron 4. The HLA-B fragments shown here (green filled peaks) have a size of 590 bp (B*44:03) and 601 bp (B*51:01). The fragments were amplified using the primers 5Bin3-389cons (TCC AGY ACT TCT GAG TCA CTT TAC) and 5' Nulight547-labeled 3Bin4-56 (CCT GAC CCT GCT GAA GGG CTC C). The table below the figure shows the results of three independent experiments, which give an average allele measure of 1.82. These data are in good agreement with the HLA-DPB1 results. Nearly 50% of the leukemic cells also lacked the HLA-B region. Thus, both the HLA-DPB1 and the HLA-B locus revealed repression of the maternal-derived allele and HLA-B*51:01 was underrepresented. The HLA-DPB1 amplification products are also depicted (blue filled peaks).
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fig4
To attribute the allele discrepancy to the patient's leukemic blasts we confirmed testing in separated cells (Figures 2-4).  Figure 3(a) shows the RFQ analysis of a blood sample containing a mixed cell population of donor and leukemic cells. In addition, further analysis of the CDC enriched cells confirmed a similar ratio for the HLA-B than for HLA-DPB1 region (Figure 4), proofing a broad imbalance in the allele number throughout the HLA region.
PMC3996364_03
Pedigree of the third patient (Case C). As shown by family HLA typing and patient's decent he must be positive for HLA-B*44:03. Actual typing revealed a loss of this allele. This discrepancy was explained by very high blast counts in the tested blood sample.
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fig5
By family typing and according to patient's ancestry (Figure 5), the patient should have exhibited the HLA allele B*44:03.
Allele loss in HLA-B visualized by RFQ (Case C). Due to the intron 4-length- polymorphism in HLA-B an amplicon of 595 bp (B*18:01) and 587 bp (B*44:03) was gained using the amplification primers 5Bin3-389cons (= 1319-42; TCC AGY ACT TCT GAG TCA CTT TAC) and 3Bin4-56 (= 1887-1908; CCT GAC CCT GCT GAA GGG CTC C). The HLA-B*44 allele is almost absent.
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fig6
In contrast, the HLA-B*44:03 showed only a very weak signal in RFQ analysis (Figure 6), confirmed by SSO and SSP-based HLA typing (data not shown) and resembling a homozygous genotype.