Ann Lab Med.  2023 Jan;43(1):127-131. 10.3343/alm.2023.43.1.127.

Validation of Pathogenicity of Gene Variants in Fanconi Anemia Using Patient-derived Dermal Fibroblasts

• Park SY
• Lee JM
• Kim MJ
• Chung NG
• Lee JB
• Kim Y
• Kim M

• KMID: 2551619
• DOI: http://doi.org/10.3343/alm.2023.43.1.127

Figure

• 

  Fig. 1 Allele-specific sequence analysis to identify novel FANCA variants in Korean patients with Fanconi anemia. (A, C) Schematic diagrams of mutant alleles of patient 1 (KFA5) and patient 2 (KFA6). (B, D) Sanger sequencing chromatograms of the patients and normal foreskin fibroblasts (BJ) from American Type Culture Collection, showing that each variant is located in different alleles.

• 

  Fig. 2 Molecular and cellular characterization of cell lines and complementation assay. (A) Western blot showing the absence of FANCA protein expression in both cell lines from patient 1 (KFA5) and patient 2 (KFA6), in contrast to the normal fibroblast cell line (BJ). (B) BJ, KFA5, and KFA6 cells were untreated or treated with 1 μM mitomycin C (MMC) for 24 h. Long-form FANCD2 (L-FANCD2) indicates monoubiquitinated FANCD2, and short-form FANCD2 (S-FANCD2) indicates non-ubiquitinated FANCD2. MMC-treated patients’ cells lack the L-FANCD2 bands, representing the FA pathway defect. (C) Patient cells (KFA5, KFA6) are sensitive to MMC treatment compared to the control cells (BJ). Triplicate cells were exposed to four MMC concentrations for 5 days. Percent survival was calculated by dividing the survival rate by that of untreated cells. (D) FANCA protein and L-FANCD2 bands appeared in patients’ cells transfected by FANCA cDNA (FA-A), but not by the empty vector (EV). (E, F) The complemented cell lines shows the successful restoration of function. (G) Immunofluorescence reveals FANCD2 focus formation in the complemented cell lines. (H) Flow cytometry showing cell cycle recovery in the complemented cell lines after MMC treatment.

Reference

1. Auerbach AD. 2009; Fanconi anemia and its diagnosis. Mutat Res. 668:4–10. DOI: 10.1016/j.mrfmmm.2009.01.013. PMID: 19622403. PMCID: PMC2742943.

2. Niedernhofer LJ, Lalai AS, Hoeijmakers JH. 2005; Fanconi anemia (cross)linked to DNA repair. Cell. 123:1191–8. DOI: 10.1016/j.cell.2005.12.009. PMID: 16377561.

3. Knipscheer P, Räschle M, Smogorzewska A, Enoiu M, Ho TV, Schärer OD, et al. 2009; The Fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair. Science. 326:1698–701. DOI: 10.1126/science.1182372. PMID: 19965384. PMCID: PMC2909596.

4. Ceccaldi R, Sarangi P, D'Andrea AD. 2016; The Fanconi anaemia pathway: new players and new functions. Nat Rev Mol Cell Biol. 17:337–49. DOI: 10.1038/nrm.2016.48. PMID: 27145721.

5. Oostra AB, Nieuwint AW, Joenje H, De Winter JP. 2012; Diagnosis of fanconi anemia: chromosomal breakage analysis. Anemia. 2012:238731. DOI: 10.1155/2012/238731. PMID: 22693659. PMCID: PMC3368163.

6. Matsushita N, Kitao H, Ishiai M, Nagashima N, Hirano S, Okawa K, et al. 2005; A FancD2-monoubiquitin fusion reveals hidden functions of Fanconi anemia core complex in DNA repair. Mol Cell. 19:841–7. DOI: 10.1016/j.molcel.2005.08.018. PMID: 16168378.

7. Lee HJ, Park S, Kang HJ, Jun JK, Lee JA, Lee DS, et al. 2012; A case report of Fanconi anemia diagnosed by genetic testing followed by prenatal diagnosis. Ann Lab Med. 32:380–4. DOI: 10.3343/alm.2012.32.5.380. PMID: 22950077. PMCID: PMC3427829.

8. The Rockefeller University. Fanconi Anemia Mutation Database. http://www.rockefeller.edu/fanconi/. Updated on August 2022.

9. De Rocco D, Bottega R, Cappelli E, Cavani S, Criscuolo M, Nicchia E, et al. 2014; Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology. Haematologica. 99:1022. DOI: 10.3324/haematol.2014.104224. PMID: 24584348. PMCID: PMC4040906.