Identification and characterization of mutations in FANCL gene: a second case of Fanconi anemia belonging to FA-L complementation group

Abstract

Fanconi anemia (FA) is a rare autosomal recessive or X-linked disorder characterized by aplastic anemia, cancer susceptibility and cellular sensitivity to DNA crosslinking agents. Eight FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL and FANCM) and three non-FA proteins (FAAP100, FAAP24 and HES1) form an FA nuclear core complex, which is required for monoubiquitination of the FANCD2-FANCI dimer upon DNA damage. FANCL possesses a PHD/RING-finger domain and is a putative E3 ubiquitin ligase subunit of the core complex. In this study, we report an FA patient with an unusual presentation belonging to the FA-L complementation group. The patient lacks an obvious FA phenotype except for the presence of a café-au-lait spot, mild hypocellularity and a family history of leukemia. The molecular diagnosis and identification of the FA subgroup was achieved by FA complementation assay. We identified bi-allelic novel mutations in the FANCL gene and functionally characterized them. To the best of our knowledge, this is the second reported case belonging to the FA-L complementation group.

Figure 1. Cellular defects observed in RA3056 cells were complemented by FANCL

A. Cell cycle analysis of wild-type cells (HCKE), FA-L (EUFA868), patient-derived LCLs (RA3056) and RA3056 cells expressing functional FANCL. The RA3056 cells showed a higher number of G2/M arrested cells, and this defect was corrected by expressing functional FANCL. The number of cells arrested in G2 is denoted as a percentage above the graphs. B. Immunoblot analysis showing reduced FANCD2 monoubiquitination in RA3056 cells compared to that of wild-type (HCKE) cells. C. Survival curve showing MMC sensitivity of wild-type (HCKE), FA-L (EUFA868), RA3056 and RA3056 stably expressing functional FANCL (RA3056+HF-FANCL). Note that the RA3056 cells are more sensitive compared to wild-type cells, but they show more resistance compared to FA-L cells, a defect which is corrected by expressing functional FANCL. D. Bar diagram showing chromosome breakage analysis data of EUFA868 and RA3056 cells with or without functional FANCL correction. While the RA3056 cells showed higher chromosome breakage, this was corrected by FANCL.

Figure 2. Mutations observed in FANCL gene

A. Schematic diagram of FANCL cDNA showing WD-40 repeats and PHD/RING-finger domains. The positions of the mutations are marked by upward arrows. The numbers in the boxes represent exon numbers. B. Sequence chromatogram showing the altered region in genomic DNA and cloned cDNA obtained from RA3056 cells. The site of mutations is indicated by a downward arrow. The duplication is underlined. C. The altered sequence of FANCL protein caused by mutation.

Figure 3. Effect of mutations on the cellular phenotype

EUFA868 cells were transduced with virus carrying functional FANCL (HF-FANCL), FANCL with 3-bp deletion (HF-FANCLdelTAT) or 4-bp insertion (HF-FANCLdupAATT). A. Cell cycle analysis shows that the 3-bp deletion failed to complement these cells and that the 4-bp duplication showed partial restoration of this defect. B. Immunoblot analysis showing FANCD2 monoubiquitination. The FANCD2-L form was completely absent in cells transduced with 3-bp deletion, but the 4-bp duplication showed FANCD2-L form that was similar in intensity toRA3056 cells. C. Survival curve showing MMC sensitivity. The cells expressing 3-bp del mutant show similar sensitivity to EUFA868 cells, and the cells expressing 4-bp duplication show partial sensitivity. D. Bar diagram showing chromosome breakage analysis data.

Figure 4

A. Immunoblot analysis showing FANCD2 monoubiquitination in HeLa S3 cells transduced with vector alone, HF-FANCL or HF-FANCLdelTAT. Over expression of 3-bp deletion does not abolish FANCD2-L band. B. Immunoblot showing immunoprecipitated complex from FA-L or HeLa S3 cells transduced with vector alone, HF-FANCL, HF-FANCLdelTAT or HF-FANCLdupAATT and probed with FANCA, FANCG, FANCL, FANCM and FAAP100 antibodies. The 3-bp del shows negligible or weak interaction, and the 4 bp duplication shows weak to moderate interaction compared to wild-type FANCL with core complex proteins.