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Clinical evolution, genetic landscape and trajectories of clonal hematopoiesis in SAMD9/SAMD9L syndromes

A Publisher Correction to this article was published on 19 November 2021

This article has been updated

Abstract

Germline SAMD9 and SAMD9L mutations (SAMD9/9Lmut) predispose to myelodysplastic syndromes (MDS) with propensity for somatic rescue. In this study, we investigated a clinically annotated pediatric MDS cohort (n = 669) to define the prevalence, genetic landscape, phenotype, therapy outcome and clonal architecture of SAMD9/9L syndromes. In consecutively diagnosed MDS, germline SAMD9/9Lmut accounted for 8% and were mutually exclusive with GATA2 mutations present in 7% of the cohort. Among SAMD9/9Lmut cases, refractory cytopenia was the most prevalent MDS subtype (90%); acquired monosomy 7 was present in 38%; constitutional abnormalities were noted in 57%; and immune dysfunction was present in 28%. The clinical outcome was independent of germline mutations. In total, 67 patients had 58 distinct germline SAMD9/9Lmut clustering to protein middle regions. Despite inconclusive in silico prediction, 94% of SAMD9/9Lmut suppressed HEK293 cell growth, and mutations expressed in CD34+ cells induced overt cell death. Furthermore, we found that 61% of SAMD9/9Lmut patients underwent somatic genetic rescue (SGR) resulting in clonal hematopoiesis, of which 95% was maladaptive (monosomy 7 ± cancer mutations), and 51% had adaptive nature (revertant UPD7q, somatic SAMD9/9Lmut). Finally, bone marrow single-cell DNA sequencing revealed multiple competing SGR events in individual patients. Our findings demonstrate that SGR is common in SAMD9/9Lmut MDS and exemplify the exceptional plasticity of hematopoiesis in children.

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Fig. 1: Study cohorts, prevalence of germline SAMD9, SAMD9L and GATA2 mutations and overall survival.
Fig. 2: Mutational landscape in SAMD9 and SAMD9L genes.
Fig. 3: Assessment of SAMD9 and SAMD9L mutations.
Fig. 4: Clonal events in patients with MDS with germline SAMD9 and SAMD9L mutations.
Fig. 5: Trajectories of clonal hematopoiesis arising from germline SAMD9 and SAMD9L mutations.

Data availability

All data are available in the manuscript or in the Supplementary Information. Population MAF of studied variants are from the gnomAD v2.1.1 aggregation database (https://gnomad.broadinstitute.org/). The Python-based Mosaic package for analysis of scDNA-seq is available at GitHub (https://github.com/MissionBio/mosaic). Raw sequence datasets have been deposited at the European Genome-Phenome Archive (http://www.ebi.ac.uk/ega/) hosted by the European Bioinformatics Institute under accession numbers EGAS00001005431 (targeted panel sequencing), EGAS00001005432 (WES) and EGAS00001005433 (scDNA-seq). The detailed clinical annotation of the SAMD9/9Lmut cohort is provided in Supplementary Tables 14. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the Deutsche Krebshilfe Max-Eder-Nachwuchsgruppenprogramm 70109005 (to M.W.W.), ERA PerMED GATA2-HuMo German Federal Ministry of Education and Research (BMBF) 2018-123/01KU1904 (to M.W.W.), German Cancer Consortium DKTK (to M.W.W. and C.M.N.), Fritz-Thyssen Foundation 10.17.1.026MN (to M.W.W.), Deutsche Kinderkrebsstifung 2017.03 (to M.W.W.), BMBF MyPred 01GM1911A (to M.W.W., M.E., C.M.N., G.G., B.S. and C.F.), José Carreras Leukämie-Stiftung (to V.B.P.), DFG SFB1160 (to M.K.), AIRC (Associazione Italiana Ricerca sul Cancro) Special Program Metastatic disease: the key unmet need in oncology 5 per mille 2018, project code 21147 (to F.L.), Cancer Center Core Grant (CA021765, to St. Jude) and Cooperative Centers of Excellence in Hematology NIDDK U54 grant (DK106829 to Fred Hutchinson Cancer Research Center). S.S.S. is a previous recipient of a Spemann Graduate School of Biology and Medicine scholarship. We thank S. Krueger, C. Jaeger, S. Zolles, S. Hollander, M. Teller and A.-R. Kaya for excellent laboratory assistance; A. Breier, A. Fischer, W. Truckenmueller, M. Siskou-Zwecker, A. Gebert, M. Boerries and H. Busch for data management (all University of Freiburg); D. Cullins (St. Jude) for FACS services; and P. Mitra for technical support. We extend great appreciation to M. Weiss and J. Crispino (St. Jude) for valuable and constructive discussions. We also acknowledge the Hilda Biobank Freiburg and Genomics Core Facility at the German Cancer Research Center/DKFZ for specimen processing. Patient care within the EWOG-MDS consortium would not have been possible without the continuous effort of the National Reference Pathologists, National Reference Cytogeneticists, physicians, nurses and other staff of pediatric oncology units and transplant centers in all 17 participating countries (www.ewog-mds-saa.org).

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M.W.W., S.S.S. and C.M.N. designed the research. V.B.P., S.S.S., C.G., A.S., D.L., P.K.P., J.W. and R.D.-D. performed genomic studies and analyzed data. S.S.S., E.K., A.S. and C.G. performed functional studies. M.W.W., R.K.V., P.N., M.E., B.S. and C.M.N. analyzed and interpreted clinical data. P.N. performed clinical statistical analysis. M.D., J.S., F.L., R.M., M.S., B.D.M., A.C., K.K., D.T., H.H., J.B., K.J., M.U., S.P., O.P.S., O.F., S.B., V.H., I.B., S.S.-F., M.R.N., M.G.S., B.B., P.L., P.B., R.B., I.M., M.H.A., R.M., A.S., G.C., S.H., G.G., A.Y.N., M.C., M.E., C.F., B.S., C.M.N. and M.W.W. were involved in patient care, testing and data presentation. S.S.S., M.W.W. and C.M.N. wrote the manuscript. All authors contributed to the manuscript and approved its final version.

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Correspondence to Marcin W. Wlodarski.

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Competing interests

J.B. declares personal fees, advisory board/steering committee honorarium and/or non-financial support from Novartis, Pfizer, Kite and Janssen Pharma. P.B. declares research grants from Neovii, Reimser and Medac to his affiliated institution; is on the advisory board for Novartis, Cellgene, Amgen, Medac and Servier, both for personal and on behalf of his affiliated institution; and is part of the speaker’s bureau for Miltenyi, Jazz, Reimser, Novartis and Amgen, on behalf of his affiliated institution. Also, P.B. holds patents and receives royalties from Medac. R.D.-D. is employed by Mission Bio and owns equity in Mission Bio.

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Peer review information Nature Medicine thanks Valeria Santini and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Joao Monteiro was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Constitutional abnormalities in patients with germline SAMD9/9L mutations.

Frequency of all (pie charts) and individual (bar graphs) non-hematopoietic abnormalities in n = 67 SAMD9/9Lmut patients (n = 39 SAMD9mut and n = 29 SAMD9Lmut). Detailed phenotypic findings are outlined in Supplemental Table 4.

Extended Data Fig. 2 Computational assessment of germline SAMD9/9L and GATA2 mutations.

Rank scores of GATA2, SAMD9, and SAMD9L population variants (gnomAD) or patient-specific germline mutations (MUT) was assessed using 10 in silico algorithms: REVEL, CADD, DANN, M_CAP, FATHMM, SIFT, Polyphen2 HDIV, Polyphen2 HVAR, Mutation Taster and Mutation Assessor. The Y-axis depicts the rank scores (0, benign; 1, pathogenic). Analyzed number of gnomAD variants: GATA2 n = 217, SAMD9 n = 767, SAMD9L n = 716 and patient germline mutations from this study: GATA2 n = 19, SAMD9 n = 33, SAMD9L n = 20. (a) Box plots depicting the minimum, lower quartile, median, upper quartile and maximum of each analyzed data set, p-values are calculated by comparing the medians of each data set using Kruskal-Wallis one-way analysis of variance. The significant p-values (<0.05) are bolded. (b) Density plot showing the distribution of rank scores from gnomAD and patient missense variants across GATA2, SAMD9 and SAMD9L genes.

Extended Data Fig. 3 Functional consequence and in silico discordance of SAMD9/9L mutations.

Representative Western blot showing the protein levels 24 h after transfection with plasmid expressing SAMD9 wildtype (WT), SAMD9 n = 5 patient mutations (left), and SAMD9L wildtype (WT) with SAMD9L n = 5 patient mutations (right). The mock control was HEK293 transfected with empty vector. (b) Effect of co-occurring germline and second-site (somatic) SAMD9/9Lmut on HEK293 cell proliferation when expressed either alone or in cis from single constructs. Mean ± SEM from n = 3 independent experiments performed in triplicate are presented. P-values using paired t-test are shown for each double mutant in comparison to the respective germline SAMD9/9Lmut. (c) Comparative evaluation of pathogenicity of 49 germline SAMD9/9L mutations by CADD prediction and HEK293 growth assay.

Source data

Extended Data Fig. 4 Transduction of CD34 + cells with SAMD9/9L mutant lentiviruses.

(a) RT-PCR confirming the overexpression of SAMD9 wildtype, SAMD9 E974K, SAMD9L wildtype and SAMD9L V1512M lentiviruses compared to empty control in sorted GFP positive CD34 + cells 24 h post transduction. Mean ± SEM from n = 2 independent experiments are presented. SAMD9 and SAMD9L expression data is normalized to GAPDH, and untransduced CD34 + sample is set to 1. (b) Flow cytometry plots outlining staining strategy with Annexin V-APC and DAPI in GFP positive CD34 + cells transduced with lentiviral constructs (top: SAMD9L, bottom SAMD9). Corresponding data are shown in main manuscript Fig. 3d.

Extended Data Fig. 5 Association of germline SAMD9/9L mutation allelic frequency with karyotype.

Variant allelic frequencies of germline SAMD9/9L mutations in n = 65 patients either with monosomy 7 (-7), n = 37 or normal, karyotype, n = 28. The median allelic frequency of each group is marked, and the p-value is calculated using unpaired t-test.

Extended Data Fig. 6 Loss of heterozygosity (LOH) plots for chromosome 7 and the association of -7 with somatic events.

Allelic frequency of all informative chromosome 7 variants detected by bone marrow whole exome sequencing (variant allele frequency (VAF) between >5% and <95%) plotted for all sequenced SAMD9/9Lmut cases. The karyotypes at time of sequencing are shown, including monosomy 7 (-7), normal karyotype (NK), and uniparental isodisomy of 7q (UPD7q). (b) Chromosome 7 plots for patients with UPD7q. Two types of loss of heterozygosity (LOH) detected in patient D1297 are indicated by blue (-7) and orange (-7 & UPD7q) arrows. (c) Proportion of somatic events in SAMD9/9Lmut patients. Stacked bars demonstrate the percentage of SAMD9/9Lmut patients with different somatic events (UPD7q, somatic second-site SAMD9/9Lmut and somatic cancer gene mutations) within the -7 (blue) and other than -7 karyotype (grey). Within each bar the absolute number of patients for each group is shown.

Extended Data Fig. 7 Age at diagnosis in SAMD9/9Lmut patients with UPD7q.

Patients with detected uniparental isodisomy 7q (UPD7q), n = 7, had a diagnosis (Dx) at a younger age of median 2.1, range 0.5–7.7 years) in comparison to n = 59 patients without detectable UPD7q (median 9.4, range 0.1–18.1 years). The p-value is calculated using unpaired t-test with Welch’s correction (due to unequal cohort size). Dot plots represent the mean ± SD.

Extended Data Fig. 8 Clonal architecture and chromosome 7 copy number inferred from single-cell DNA sequencing (scDNAseq).

Pattern of clonal evolution (created with BioRender.com) and visualization of genotypes of individual clones constructed from high quality single cells (allelic dropout rate <0.9) detected in 3 patients with SAMD9 (a, b) or SAMD9L (c) germline mutations. Mutational phylogeny was inferred from scDNAseq data using Tapestri Insights v2.2 and Mosaic packages. Root denotes the total number of cells analyzed for each sample and bolded circle symbolizes ancestral clone with germline SAMD9/9Lmut. Percentage and number of single cells appear within colored circles; native state hematopoiesis (grey), second-site SAMD9L mutation (blue), UPD7q (green), -7 and -7 with somatic cancer mutations (both red). Variant allele frequency (VAF) from bulk sequencing is shown for reference. Panels (a) and (c) exemplify patients with branching evolution of independent benign and malignant SGR events arising from germline SAMD9/9Lmut hematopoiesis. Panel (b) depicts the linear evolution of malignant -7 clone with SETBP1/ASXL1 mutation to acquire an additional MYB M375_I376dup mutation. Lower part of panels ac depict the genotype annotation of the observed individual clones (shown above each genotype plot). Selected variants flanking SAMD9 or SAMD9L have either wildtype (WT), heterozygous (HET) or homozygous (HOM) genotype states. Right of lower panels show normalized amplicon read distribution of informative variants from scDNAseq, with red line marking the diploid state referenced from diploid cells.

Extended Data Fig. 9 SAMD9/9Lmut patients with stable disease or remission.

Timeline depicting the clinical course in n = 10 SAMD9/9Lmut patients with a follow up longer than 1 year who had stable disease (defined as no need for transfusions or therapeutic interventions, and no infections) or natural hematopoietic improvement. All patients presented with refractory cytopenia of childhood (RCC). The upper panel (black box) shows n = 5 patients with stable disease course characterized by chronic cytopenia, normal karyotype (NK); n = 4 had SAMD9mut. Two patients underwent immunosuppressive therapy (IST). Lower panel (green box) shows cases with spontaneous hematological improvement; 2 SAMD9Lmut patients with previous monosomy 7 (-7) had normalization of karyotype and complete blood counts (CBC).

Extended Data Fig. 10 Age at diagnosis in SAMD9/9Lmut patients according to outcomes.

Patients in remission group (n = 5) were diagnosed at a younger age in comparison to stable disease (n = 6) or high-risk/progression (n = 21) groups. The p-value was calculated using unpaired t-test with Welch’s correction.. The dot plots represent the mean ± SD. Group were defined as follows: Remission - long-lasting resolution of hematologic symptoms; Stable disease - prolonged chronic cytopenia (>1 year) without the need for therapies; High-risk/progressed - advanced MDS, somatic cancer mutations, and disease progression (progression of karyotype from normal to abnormal or MDS subtype from refractory cytopenia of childhood (RCC) to MDS with excess blast (MDS-EB)).

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Unprocessed western blots with relevant samples lanes marked within a box

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Sahoo, S.S., Pastor, V.B., Goodings, C. et al. Clinical evolution, genetic landscape and trajectories of clonal hematopoiesis in SAMD9/SAMD9L syndromes. Nat Med 27, 1806–1817 (2021). https://doi.org/10.1038/s41591-021-01511-6

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