We previously showed that germline SAMD9 and SAMD9L (SAMD9/9L) disorders are the most common predisposition to childhood myelodysplastic syndromes with bone marrow failure (BMF). Heterozygous mutations in both genes are growth inhibitory and undergo negative selection by acquiring compensatory rescue events in blood. Here, we describe the first report of SAMD9L genetic rescue occurring during embryonal development and resulting in germline triple-allelic mosaicism (1 wildtype (WT) and 2 mutant alleles), where only one mutant allele was transmitted to each of the two diseased children. In a family of 2 affected siblings with hypocellular BMF, the Index case carried germline heterozygous SAMD9L V1512M mutation and her brother was heterozygous for V1512L mutation. The clinical presentation for Index with V1512M was more severe compared to her brother with V1512L mutation. Genetic assessment of the parents showed the asymptomatic mother to be a triple-allelic mosaic, carrying WT SAMD9L allele at ~50% frequency in all tissues, while both V1512M and V1512L mutant alleles “competed” in their allelic distribution depending on the tissue origin. Single-cell DNA sequencing on her peripheral blood (PB) revealed 3 independent diploid clones: V1512M in 14%, V1512L in 68%, and WT (due to revertant UPD7q) in 18% of cells. Because her parents were SAMD9L WT, one of the mutations likely arose de novo and underwent failed embryonic rescue attempt leading to a second mutation. Towards studying the effect on hematopoiesis in vitro, we knocked in V1512M and V1512L mutations in inducible pluripotent stem cells (iPSC). For in vivo functional validation, we created constitutive mouse models with ortholog mutations (V1507M and V1507L). Mutant hematopoietic progenitor cells from iPSC had severely compromised proliferative capacity and yielded fewer erythroid and myeloid cells. This effect was more severe in V1512M vs. V1512L mutants. The divergent mutational phenotypes were also replicated in our mouse models: two-thirds (13/19) of the heterozygous V1507M (V1507Mhet) pups died before 4 weeks of age. In contrast, V1507Lhet mice had overall survival equal to WT mice, while one-third of homozygous V1507L (V1507Lhom) mice showed decreased survival post 19 weeks of age. At baseline, V1507Mhet mice showed severe growth retardation with multisystemic issues, which were absent in the V1507L mutant models. PB cytopenia (anemia, B-cell lymphopenia) though observed in both V1507Mhet and V1507Lhom mice, the degree of severity was high in the V1507Mhet than in the V1507Lhom model. Meanwhile, V1507Lhet mice had blood counts similar to WT. In summary, V1512M compared to V1512L mutation resulted in a more severe phenotype in all analyzed model systems. This observation showed how different mutational permutations of the same amino acid can exert divergent phenotypic effects in SAMD9L BMF disorder. Therefore, together with our genetic analysis, we could postulate that the V1512L mutation arose as a failed rescue attempt of V1512M during embryogenesis.

Citation Format: Sushree S Sahoo, Charnise Goodings, Shondra M. Pruett-Miller, Maria A Lillo, Lei Han, Baranda Hansen, Ti-Cheng Chang, Tim Lammens, Mattias Hofmans, Marta Derecka, Barbara De Moerloose, Marcin W Wlodarski. Divergent effects of MDS predisposing SAMD9L point mutations, in humans, mice, and cellular models [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A51.