Diagnosis of Fanconi Anemia By Chromosome Breakage Tests Using 3 Different Scoring Systems and Whole Genome Sequencing Among Patients with Aplastic Anemia in Korean

BACKGROUD: Fanconi anemia (FA), an inherited bone marrow failure syndrome with impaired DNA repair system, is characterized by cytopenias, congenital abnormalities, and predisposition to malignancy as a consequence of chromosomal instability and hypersensitivity to DNA interstrand cross-linking agents. Differential diagnosis of FA and aplastic anemia requires integrated work-up including physical findings, bone marrow histologic findings and chromosome breakage test. Yet, there have been no consensus criteria for chromosome breakage test, which depend on each laboratory's own decision. The aim of our study was 1) to investigate the incidence of FA showing positive results for chromosome breakage test among patients diagnosed with aplastic anemia, and 2) to investigate the frequency of the gene mutations related to inherited bone marrow failure syndrome in patients with aplastic anemia. In addition to chromosome breakage test, we performed whole genome sequencing with bone marrow mononuclear cells in 18 pediatric patients with aplastic anemia whose bone marrow specimen was available.

METHOD: We reviewed total 79 chromosome breakage tests from 67 patients who had been on suspicion of aplastic anemia between May 2005 and April 2015. MMC and DEB stress test were performed at concentration of 50ng/mL and 100ng/mL both on peripheral blood of suspicious patients and normal controls, respectively. The scoring of chromosome breakages test was performed, based on widely used 3 different scoring systems: those proposed by Jean Soulier, Barch MJ, and Arleen D. Auerbach. In each cases, we applied 3 different scoring systems and compared the concordance rate. In 16 among 67 patients, we performed whole genome sequencing.

RESULTS: The median age of the pediatric patients was 11years (range, 7months - 19 years) and the male-to-female ratio was 1.39:1. Of 67 enrolled patients, 8 had been tested twice or 3 times because of ambiguous results. In these cases, we chose the last results. Five of 67 patients satisfied the all three criteria mentioned above, which shows 7.5% (5/67) of positive rates. Other 3 of 67 patients met only one or more of Soulier's prerequisites. Among those, one fulfilled both Barch MJ's system and Aeurbach's, and remain 2 patients were positive in each system, respectively. Mutation variants of BMF syndrome related genes were detected in 25% (4/16 patients); RPS19 (1 patient), PAX5 (1 patient), and FANC (3 patient). Inherited predisposition to myeloid leukemia related genes were detected in 56.3% (9/16 patients) and gene variants were MSH6 (5 patients), ATM (2 patients), PMS2 (1 patients), and MLH1 (1 patients). Coexisting somatic mutations of oncogene (ERB2) was detected in 6.3% (1/16 patients). Among 3 patients with fanconi anemia gene mutations, 2 patients showed positive results for chromosome breakage test and the other1patient showed negative results for chromosome breakage test.

CONCLUSION: The frequency of FA based on chromosome breakage test among patients with pancytopenia suspicious of aplastic anemia was 7.5% by Soulier's prerequisites, but 9.0% when based on either of 3 different criteria. Molecular testing can additionally detect FA in 4 (25.0%) among 16 patients showing negative result by chromosome breakage test. Our study shows it is necessary to standardize a diagnostic scoring system as well as to develop complementary molecular test for accurate diagnosis of FA.