Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

TLDR

Somatic mosaicism has been previously observed in the lymphocyte population of patients with Fanconi anemia. The study aimed to determine the cellular origin of genotypic reversion in an FA patient carrying a 2815–2816ins19 FANCA mutation with known lymphocyte mosaicism. Researchers isolated individual peripheral blood T‑cell colonies and marrow progenitor colonies, performed DNA analysis to detect the FANCA exon 29 mutation, and used interphase FISH with an MLL probe to identify a partial 11q deletion exclusively in colonies harboring the mutation. The data show that spontaneous back mutation leading to genotypic reversion originates in a lymphohematopoietic stem cell, that pancytopenia continues to progress despite reversion, and that a clonal cytogenetic abnormality arises in nonrevertant cells, indicating that ex vivo gene transfer may not provide lifelong stable hematopoiesis in FA.

Abstract

Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815–2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein–Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA.