Constitutional SAMD9L mutations cause familial myelodysplastic syndrome and transient monosomy 7

Abstract

Familial myelodysplastic syndromes arise from haploinsufficiency of genes involved in hematopoiesis and are primarily associated with early-onset disease. Here we describe a familial syndrome in seven patients from four unrelated pedigrees presenting with myelodysplastic syndrome and loss of chromosome 7/7q. Their median age at diagnosis was 2.1 years (range, 1-42). All patients presented with thrombocytopenia with or without additional cytopenias and a hypocellular marrow without an increase of blasts. Genomic studies identified constitutional mutations (p.H880Q, p.R986H, p.R986C and p.V1512M) in the SAMD9L gene on 7q21, with decreased allele frequency in hematopoiesis. The non-random loss of mutated SAMD9L alleles was attained via monosomy 7, deletion 7q, UPD7q, or acquired truncating SAMD9L variants p.R1188X and p.S1317RfsX21. Incomplete penetrance was noted in 30% (3/10) of mutation carriers. Long-term observation revealed divergent outcomes with either progression to leukemia and/or accumulation of driver mutations (n=2), persistent monosomy 7 (n=4), and transient monosomy 7 followed by spontaneous recovery with SAMD9L-wildtype UPD7q (n=2). Dysmorphic features or neurological symptoms were absent in our patients, pointing to the notion that myelodysplasia with monosomy 7 can be a sole manifestation of SAMD9L disease. Collectively, our results define a new subtype of familial myelodysplastic syndrome and provide an explanation for the phenomenon of transient monosomy 7. Registered at: www.clinicaltrials.gov; #NCT00047268.

Figure 1.

Germline SAMD9L mutations in pedigrees with familial myelodysplastic syndrome. (A) Identification of four pedigrees with MDS and monosomy 7 harboring germline heterozygous SAMD9L mutations: p.V1512M (pedigree I), p.R986H (pedigree II) and p.R986C (pedigree III), p.H880Q (pedigree IV), and somatic mutations: p.R1188X (P1) and p.S1317RfsX21 (P7). Dotted symbols indicate healthy mutation carriers. Sanger sequencing on DNA extracted from hair follicles (HR) confirmed the germline status of mutations as visualized in electropherograms. Sequencing in P1 was performed on peripheral blood granulocytes (GR) revealing a minor mutational peak, corresponding to a variant allelic frequency of 8.3% by whole exome sequencing. In pedigree III, the mutation in P5 was confirmed in fibroblast (FB) DNA, while for P6 and remaining family members whole blood (WB) was analyzed. In pedigree IV other family members were not tested (n.t.). (B) SAMD9L and SAMD9 gene orientation on 7q22 in reverse strand direction (3′–5′). The SAMD9L protein is coded by one exon and contains two known functional sites: N-terminal sterile alpha motif (SAM) and nuclear localization sequence (NLS). Four germline and two somatic (*) mutations were identified in SAMD9L. Germline missense mutations are evolutionarily highly conserved. (C) TA cloning of the double mutated SAMD9L region of P1 revealed cis-configuration of mutations p.V1512M (germline) and p.R1188X (somatic) in ten out of 172 clones tested.

Figure 2.

Bone marrow findings in P1 and P2 at different timepoints during the course of the disease. Hematoxylin and eosin staining of bone marrow (BM) at diagnosis of RCC in P1 showing dysplastic granulopoiesis with hypergranulation and a pseudo-Pelger cell (top left), myeloblast and dysplastic eosinophil (top right). BM at diagnosis in P2 (synchronous with monosomy 7) showing hypergranulation and vacuolization in myelocytes, and dysplastic erythropoiesis with double nuclei (bottom left). Normal BM morphology in P2, 15 years after initial BM confirming spontaneous phenotype reversion (bottom right).

Figure 3.

Mechanisms of clonal escape from SAMD9L germline mutations. Multiple mechanisms of clonal escape from damaging germline missense SAMD9L mutations are observed and lead to complete (monosomy 7) or partial (deletion 7q) loss of chromosome 7 with decreasing mutant SAMD9L allele (red circles), both situations can lead to MDS development; UPD7q and truncating somatic SAMD9L mutations (green circles), which have a benign outcome and contribute to normal hematopoiesis. Multiple clonal outcomes can occur in a single patient.

Figure 4.

Loss of mutated SAMD9L allele due to genomic deletion or mitotic recombination. Variant allelic frequency (VAF) scores for chromosome 7 in P1 and P2. single nucleotide polymorphisms and Indels detected using whole exome sequencing (~4000 variants with a VAF score >5% and <95%), show a complete loss of chromosome 7 in P1, as the VAF scores are either low or high. P2, unlike P1 demonstrates a partial loss of the chromosome 7 after position 7q11.22 towards the q terminal site. The read depth of the single nucleotide polymorphisms for P2 was maintained throughout for chromosome 7 with no loss thus confirming that loss of heterozygosity is due to UPD and not −7q. Whole exome sequencing VAF values are marked by a star within the graph. VAF: variant allelic frequency; UPD: uniparental isodisomy. Blue line: centromere; red line: SAMD9L gene position; yellow dotted line: start of UPD. For P7, targeted next-generation sequencing identified 14 informative (heterozygous) polymorphisms located on chromosome 7q with an average depth of 1036 reads (Online Supplementary Table S1). Single nucleotide polymorphisms are represented in a VAF graph depicting the skewing of heterozytosity towards one allelle occurring after position g.66098482 (rs3764903).

Figure 5.

Clonal evolution and spontaneous reversion due to UPD7q. Clonal evolution model in P2 (D154) depicting disease history during an observation period of 20 years. At diagnosis, initial bone marrow harbored monosomy 7 (77% by fluorescence in situ hybridization and 51% by metaphase karyotyping). Blood counts normalized 3.7 years later and since then P2 maintained normal complete blood counts until last follow-up at the age of 22 years. From the age of 12 years, repeated yearly bone marrow examinations revealed normocellular hematopoiesis with no dysplasia and normal cytogenetics. Bone marrow collected at the age of 17 years (*) was subjected to whole exome and targeted deep sequencing. Germline heterozygous SAMD9L mutation p.V1512M was detected at a variant allelic frequency (VAF) of ~20%, corresponding to a clonal size of ~40% in a diploid chromosome 7 background. Concurrently, a spontaneous genetic correction of the SAMD9L locus occurred resulting from uniparental isodisomy (UPD)7q of paternal origin. This self-corrected clone occurred either initially (dotted line) or after termination of monosomy 7 and contributed to normal hematopoiesis. Abbreviations: Dx, diagnosis; pat, paternal origin; mat, maternal origin; UPD; uniparental isodisomy, LFU; last follow-up.

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 previously reported by Tesi et al. (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 representative experiment is shown. (B) Cumulative summary of three independent experiments on inhibition of cell proliferation associated with indicated TFP-SAMD9L mutations. 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.