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SAMD9L-related familial MDS
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Figure 6. Functional evaluation of SAMD9L mutations. (A,B) The effect of SAMD9L mutations on cell proliferation was assessed by dye dilution assays. 293FT cells were transiently transfected with TFP-SAMD9L wild type (WT), the disease-associated mutations p.R986C and p.V1512M, and the protective variant p.T233N previ- ously reported by Tesi et al.20 (A) Histograms depict the dye levels in transfected cells. Dye levels were monitored in TFP-transfected cells (filled gray histograms) and compared to cells expressing uniformly intermediate levels of TFP-SAMD9L wild-type (blue histograms) or variants (red/orange lines), as indicated. A single represen- tative experiment is shown. (B) Cumulative summary of three independent experiments on inhibition of cell proliferation associated with indicated TFP-SAMD9L muta- tions. Values (mean ± SD) are calculated based on a scale defined by 0 (dye levels in TFP-transfected cells) and -1 (dye levels in cells transfected with TFP-SAMD9L wild-type). Unpaired t-test, two tailed: *P<0.05; ** P<0.005; ***P<0.001.
Both genes can be upregulated by type I and II interfer- ons20,31-33 and by this might suppress inflammatory path- ways and exert anti-viral properties.31,34 Although there was no evidence for a defined immunodeficiency in our cohort, three of the seven patients experienced recurrent respiratory infections with or without cytopenias before they developed RCC/-7.
Further evidence supports that SAMD9/SAMD9L genes act as tumor suppressors as their inactivation is associated with increased cellular proliferation, i.e. in normophosphatemic familiar tumoral calcinosis (SAMD9), in hepatitis B virus-associated hepatocellular carcinoma (SAMD9L), in MDS/acute myeloid leukemia with microdeletion in 7q21 (both genes), and in Samd9l- haploinsufficient mice. Based on the observations in this murine model, the cytokine-receptor complexes cannot be properly degraded due to impaired endosomal func- tion in SAMD9L-haploinsufficient cells, which results in constitutive intracellular signaling with prolonged cell survival.18 Moreover, Samd9l+/- and Samd9l-/- mice develop MDS with normo/hypercellular bone marrow, drawing a parallel to human SAMD9L-related MDS in which the initial marrow hypocellularity associated with the “toxic” mutation is restored upon the loss of the mutant SAMD9L allele. This loss is accomplished through -7, del(7q) or UPD7q, and leads to a proliferative advantage with clonal expansion. In our cohort, although initially all patients presented with hypocellular marrow, longer observation periods in P1, P2, P4, and P7 revealed succes- sive normalization of marrow cell content associated with increasing -7/del(7q) clone or the appearance of UPD7q. In P1, the leukemic progression was further aggravated by the accumulation of typical MDS driver mutations in SETBP1, KRAS and EZH2 within the -7 clone. P5 also demonstrated an acquired splice site muta-
tion in RUNX1, a known leukemic driver gene. Notably, typical adult MDS driver mutations (i.e., TET2, DNMT3A, and IDH1/2) were not encountered in our cohort. This is in line with previously published findings discussing SETBP1, RAS pathway mutations and RUNX1 (identified in our SAMD9L-mutated patients) as recur- rent drivers of pediatric MDS.10 Building on our observa- tions we propose the following mechanism of MDS evo- lution in SAMD9L disease: the bone marrow attempts to circumvent the toxicity of the constitutional SAMD9L mutation and selects for fitter, yet premalignant - 7/del(7q) clones (with only one wild-type SAMD9L copy), or benign clones with truncated SAMD9L (Figure 3). Over time the resulting haploinsufficiency of tumor supressor genes on 7q (e.g. EZH2 or CUX1) in all patients likely provides the first step towards progression. Finally, additional driver somatic mutations might be encoun- tered in some but not all patients.
Somatic revertant mosaicism has been reported in inher- ited bone marrow failure syndromes with hypocellular bone marrow, including telomeropathy with germline mutations in TERT,35 and Fanconi anemia in which mosaicism in blood occurs at rates of up to 140 times high- er than in the general population.36,37 However, in general, revertant mosaicism is a rare facet to clonal hematopoiesis because spontaneous correction of the pathogenic allele is a random event. In our study, we report two patients (P2 and P7) who presented with RCC/-7 at young age and demonstrated complete hematopoietic remission with nor- mal cytogenetics throughout an observation period rang- ing from 16 to 20 years. The clinical picture of these patients fits the previously described transient monosomy 7 syndrome; to our knowledge eight patients with primary MDS with transient -7 or del(7q) have been reported in the literature.14-16,38-40 Their ages at diagnosis ranged from 8
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