Whole exome sequencing and rare variant association study to identify genetic modifiers, KLF1 mutations, and a novel double mutation in Thai patients with hemoglobin E/beta-thalassemia.

OBJECTIVES: Clinical manifestations of patients with Hemoglobin E/beta-thalassemia vary from mild to severe phenotypes despite exhibiting the same genotype. Studies have partially identified genetic modifiers. We aimed to study the association between rare variants in protein-coding regions and clinical severity in Thai patients. METHODS: From April to November 2018, a case-control study was conducted based on clinical information and DNA samples collected from Thai patients with hemoglobin E/beta-thalassemia over the age of four years. Cases were patients with severe symptoms, while patients with mild symptoms acted as controls. Whole exome sequencing and rare variant association study were used to analyze the data. RESULTS: All 338 unrelated patients were classified into 165 severe and 173 mild cases. Genotypes comprised 81.4% of hemoglobin E/beta-thalassemia, 2.7% of homozygous or compound heterozygous beta-thalassemia, and 0.3% of (뫧)0 thalassemia Hb E while 15.7% of samples were not classified as beta-thalassemia. A novel cis heterozygotes of IVS I-7 (A > T) and codon 26 (G > A) was identified. Six genes (COL4A3, DLK1, FAM186A, PZP, THPO, and TRIM51) showed the strongest associations with severity (observed p-values of <0.05; significance lost after correction for multiplicity). Among known modifiers, KLF1 variants were found in four mild patients and one severe patient. CONCLUSION: No rare variants were identified as contributors to the clinical heterogeneity of hemoglobin E/beta-thalassemia. KLF1 mutations are potential genetic modifiers. Studies to identify genetic factors are still important and helpful for predicting severity and developing targeted therapy.

Targeting acute myeloid cell surface using a recombinant antibody isolated from whole-cell biopanning of a phage display human scFv antibody library.

To discover new therapeutic antibodies for treatment of acute myeloid leukemia (AML) without the requirement of a known antigen, a human single-chain variable fragment (scFv) library was used to isolate novel antibody fragments recognizing HL-60 AML cells. After three rounds of biopanning, scFv-expressing phages were selected to test for binding to the target cell by flow cytometry. The clone with highest binding specificity to HL-60 cells (designated y1HL63D6) was further investigated. Fluorescent staining indicated that y1HL63D6 scFv bound to a target located on the cell surface. Whole immunoglobulin, IgG-y1HL63D6 was then generated and tested for the binding against bone marrow mononuclear cells (BMMCs) from AML patients. Significantly higher fluorescent signals were observed for some patient samples when compared to normal BMMCs or non-AML patients' BMMCs. Next, the IgG-y1HL63D6 format was tested for antibody-dependent cell cytotoxicity (ADCC). The results demonstrated that IgG-y1HL63D6 but not the control antibody, trastuzumab, could mediate specific killing of HL-60 target cells. In conclusion, our results indicate that specific antibodies can be isolated by biopanning whole cells with a non-immunized human scFv antibody phage display library and that the isolated antibody against HL-60 cells showed therapeutic potential.