A Rapid Method for Retroviral Mediated Subtyping of Complementation Group in Fanconi Anemia Patients.
Authors: Chandra Saurabh , Levran Orna , Jurickova Ingrid , Mass Chiel , Kapur Rick , Hanenberg Helmut , Auerbach Arleen D. , Williams David A. 
Published: October 2019

Fanconi anemia (FA) is a rare autosomal recessive disorder that results from mutations in at least eleven different genes (A, B, C, D1, D2, E, F, G, I, J, L). Recent studies have demonstrated that clinical progression of the disease may be influenced by inter- and intra-genic variations, underlining the importance of subtyping complementation groups. Previously we have utilized retroviral mediated gene transfer of FA cDNAs and subsequent analysis for the correction of hypersensitivity to Mitomycin C, to develop a valid method for subtyping of groups (Hanenberg et al., 2002). However, for lymphoblastic cell lines (LCL), G418 selection was required to compensate for low transduction efficiencies achieved. In the present study we have utilized RD114/GALV psuedotyped (high titer stable producer clones) bicistronic retroviral vectors that coexpress EGFP. This results in higher transduction efficiencies in LCLs (20–70% for C, F, G and 10–20 % for A vector) and allows for specific analysis of transduced EGFP-positive cells within the bulk cultures, thus obviating the need for selection. In addition, the assay relies on the correction of G2/M arrest that is analyzed by FLOW, further reducing the assay time. After transduction with cDNA-containing vectors for groups A, C, F and G, cells are exposed for 48 hours to the alkylating agent Melphalan, stained with Hoechst 33342 dye and cell cycle analysis for FA-specific G2/M arrest is performed by flow cytometry. The percentage of EGFP-positive cells in the G2/M phase of cell cycle for each vector is calculated and compared using the MODFIT software. A complementation group is identified when correction in the G2/M arrest occurs. Results obtained with the assay matched the complementation group known for 12 control LCLs tested. We report here the results obtained for 39 FA patients with unknown complementation groups using this new assay. Of the 39 cell lines tested, complementation group was identified for 20 lines [FA-A(17), FA-C(1), FA-F(1), FA-G(1)], while 15 lines were typed as Non A/C/F/G. Cells from 4 patients did not undergo G2/M arrest. Mutation analysis by direct sequencing of genomic DNA after DHPLC completed for 11 patients thus far, has detected a mutation in one or both alleles confirming the results of the rapid complementation assay in every case (Table). For the 15 cell lines typed as Non A/C/F/G, additional studies including vectors for the FA genes FANCD2, FANCE, FANCL are underway.

In summary, we have developed a rapid and efficient method for the identification of complementation groups in FA patients that also serves as a guide for quick mutational analysis. This methodology can also be applied to myeloid progenitor cells as well as fibroblasts, an advantage in patients with lymphoid mosaicism.

Table 1. Mutational analysis of cell lines identified by cell cycle analysis

Cell Line Ref # Cell cycle analysis Mutation 1(paternal) Mutation 2 (maternal) RA2593 A c.1471-?_2151+?del c.1471-?_2151+?del RA2483 A c.1901-?_2014+?del RA2625 A c.3239G>T(p.R1080L) RA2644 A c.2806G>A(p.E936K) c.2806G>A(p.E936K) RA2685 A c.2678G>A(p.W893X) RA2698 A c.337_338delTC RA2704 A c.3514-?_3626+?del c.3520_3522delTGG RA2724 C c.322delG c.711+4A>T(IVS4+4A>T) RA2191 A c.3520_3522dellTGG RA2709 F c.484-485delCT c.219delG RA2727 G c.908T>C c.908T>C