Blood, Vol. 109, Issue 12, 5234-5237, June 15, 2007
Perforin gene mutations in patients with acquired aplastic anemia
Blood Solomou et al. 109: 5234
Supplemental materials for: Solomou et al, Vol 109, Issue 12, 5234-5237
Patients and Controls
After informed consent was acquired according to protocols approved by the Institutional Review Board of the National Heart, Lung and Blood Institute, blood samples were obtained from 75 unrelated patients with acquired aplastic anemia who were treated in the Hematology Branch. The diagnosis of aplastic anemia was based on the bone marrow and blood-count criteria of the International Agranulocytosis and Aplastic Anemia study. Marrow cytogenetics were normal in all cases and Fanconi anemia was excluded by chromosome breakage studies in children and younger adults. Of the 75 patients examined, 33 were female and 42 were male. Race or ethnic background, as reported by the patients, was as follows: white, 40 patients (53.3%); black, 16 patients (21.3%); Hispanic, 11 patients (14.6%); and Asian, 8 patients (10.6%). Buccal mucosa specimens were used to confirm the germ-line origin of the mutations in four patients that we found to carry a mutation in the PRF1 gene. None of the patients had a posthepatitis aplasia; none of the patients had an active infection at sampling. Re-sequence analysis was performed in both the SNP500 Cancer set and the Human Genomic Diversity Panel (HGDP)10,11. The SNP500Cancer set includes 102 subjects from four major, self-described ethnic U.S.A. groups (Caucasians, Hispanics, African Americans, and Pacific Rim). HGDP consists of 1036 individuals from 52 separate populations. The HGDP can be categorized into seven ‘continental’ geographic regions: Africans, Europeans, Asians, Western Asians, Oceanians, and Native Americans, with each group containing 7, 8, 3, 9, 18, 2, and 5 representative groups, respectively. In addition, 18 anonymous healthy Caucasian subjects were sequenced. In total, sequence analysis was attempted for more than 2300 chromosomes with completion rates for each amplicon above 96%.
Characteristics of patients with perforin mutations
The index case, patient A, was a previously healthy United States Army Special Forces sergeant who became ill in Iraq, presenting with fever and pancytopenia. Bone marrow examination revealed profound hemophagocytosis and a hypocellular bone marrow. A diagnosis of aplastic anemia was made and immunosuppressive treatment was initiated. Six months later, he had not shown an adequate hematologic response to treatment, remaining transfusion-dependent, although his absolute neutrophil count had risen between 0.4 and 0.5|×|109/L. Matched unrelated bone marrow transplantation was undertaken, and at the present time, day +170 post-transplant, he has an absolute neutrophil count between 1.6 and 1.75|×|109/L, hemoglobin 100 g/L, and platelets of 40|×|109/L; chronic graft versus host disease is a major transplant-related complication. Patient A was examined for PRF1 mutations at diagnosis before receiving any treatment. The same mutation in exon 2 (91AmtV) and the same single nucleotide polymorphism in exon 3 (300HmtH) were also identified in another two unrelated patients (B and C). Both are older than patient A; both showed only transient responses to immunosuppressive treatment, as they relapsed and became cyclosporine-dependent. As of this writing, patient B has absolute neutrophil counts between 1.3|×|109/L and 1.5|×|109/L, hemoglobin around 110g/L, and platelets of 110|×|109/L. Patient C has absolute neutrophil counts between 1.9|×|109/L and 2.05|×|109/L, hemoglobin around 120 g/L, and platelets of 95|×|109/L. Patient B’s bone marrow examination showed hemophagocytosis at diagnosis. Both patients were examined (for PRF1 mutations) after immunosuppressive treatment. Patient D was diagnosed with severe aplastic anemia two years ago, has not responded to 3 courses of immunosuppressive treatment (horse antithymocyte globulin, thymoglobulin, and alemtuzumab),1 and remained erythrocyte and platelet transfusion-dependent. He had very high serum levels of ferritin and ultimately developed congestive heart failure (CHF), but there were no other laboratory findings of FHLH. MRI did not show iron deposition. Bone marrow examination revealed profound hemophagocytosis. He died 2 months after the onset of CHF of cardiac complications. Patient D was examined for PRF1 mutations 7 months after the completion of 3 courses of immunosuppressive treatment. Patient E, a 21-year old Jamaican man, was diagnosed with severe aplastic anemia by bone marrow examination that showed cellularity less than 5% and mild hemophagocytosis (Figure 1). He has failed three courses of immunosuppressive treatment (horse antithymocyte globulin, thymoglobulin, and alemtuzumab) and remains transfusion-dependent with an absolute neutrophil count less than 0.5|×|109/L. Sequencing analysis of PRF1 gene (following treatment with horse antithymocyte globulin and thymoglobulin and also 4 months after alemtuzumab) revealed a heterozygous mutation in exon 2: in codon 4, with an arginine residue replaced by histidine (4RmtH). The germ-line origin of PRF1 mutations was established by their detection in DNA extracted from buccal mucosa specimens obtained from four out of four patients (A, B, C, and D).
Nucleotide Sequencing
Polymerase-chain reaction (PCR) amplification of exons 2 and 3 of the gene encoding perforin (PRF1) was performed with DNA samples extracted from peripheral blood or buccal smear cells from patients and healthy controls as previously described.12 Sequencing products were analyzed in an automated genetic-sequencer analyzer (ABI Prism 3100 and 3730X, Applied Biosystems). All sequences were determined bidirectionally, and patients’ mutations were confirmed by two separate PCR amplification products from chronologically different specimens obtained from the same patient. The BLAST program (National Center for Biotechnology Information) was used to compare the obtained sequences to the reported gene structure index (GI: M28393).
Western Blot Analysis
Cytoplasmic protein extracts (10 µg/lane) from purified T cells, isolated by magnetic depletion of non-T cells using MACS Pan T cell Isolation Kit II, were resolved in 12% Tris-glycine SDS gels as previously described.2 Immunoblot was performed with primary anti-PRF1 delta-G9 monoclonal antibody (BD Biosciences). Actin antibody was used as control to show equal loading of the lanes. Immunoblots were performed in patients that we identified to carry PRF1 mutations, and in another seven patients, not carrying mutations, that were randomly chosen from all the patients that we analyzed for PRF1 mutations. Eight healthy volunteers (previously examined and found not to carry any PRF1 mutations or polymorphisms) served as controls.
Confocal Microscopy
CD8+ T cells were isolated by magnetic depletion of non-T cells (MACS CD8+ T cells Isolation Kit II) based on the manufacturer’s instructions and as previously described.2 CD8+ T cells were cultured in PHA (2 µg/mL) for one day followed by IL-2 (20 IU/mL) and PHA (1 µg/mL) for 5 days.13 Cells were collected and fixed on glass cover slips previously coated with poly-L-lysine (Sigma). Cells were stained with anti-cathepsin D (Upstate Biotechnology, NY) and anti-perforin (BD Pharmingen) antibodies, followed by Texas-Red- and FITC-conjugated secondary antibodies, respectively (Jackson Immuno-Research Laboratories, PA). Immunofluorescence was examined by confocal laser microscopy with a Zeiss 510 confocal system equipped with UV-Vis lasers (Carl Zeiss Inc, Jena, Germany). Images were acquired sequentially using a 488-nm laser line and emission between 505 and 580 nm for FITC, and differential interference contrast (DIC) and a 561-nm laser line and emission over 585nm for Texas Red. High-resolution (100nm/pixel) images were obtained with a 63×, 1.2-numerical-aperture C-Apochromat water immersion objective and were deconvolved using Huygens software (SVI Hilversum, Netherlands). Overlay images were assembled, enlarged and cropped using Imaris 5.0 software form Bitplane AG (Zurich, Switzerland) and montaged using Photoshop 7.0.2 software (Adobe sytems).
Cytotoxicity
NK cells were isolated from PBMC by magnetic depletion of non-NK cells (MACS NK cell Isolation Kit II) based on the manufacturer’s instructions. Cytotoxic activity from NK cells from patients A, D, E (all three carrying PRF1 mutations) and from another two patients (randomly chosen, with no mutations or polymorphisms identified in these patients) was compared to that from healthy controls in a standard 4 hr Cr51-release assay against K562 cells, as previously described.13