Gain-of-function SAMD9L mutations cause a syndrome of cytopenia, immunodeficiency, MDS, and neurological symptoms

Abstract

Several monogenic causes of familial myelodysplastic syndrome (MDS) have recently been identified. We studied 2 families with cytopenia, predisposition to MDS with chromosome 7 aberrations, immunodeficiency, and progressive cerebellar dysfunction. Genetic studies uncovered heterozygous missense mutations in SAMD9L, a tumor suppressor gene located on chromosome arm 7q. Consistent with a gain-of-function effect, ectopic expression of the 2 identified SAMD9L mutants decreased cell proliferation relative to wild-type protein. Of the 10 individuals identified who were heterozygous for either SAMD9L mutation, 3 developed MDS upon loss of the mutated SAMD9L allele following intracellular infections associated with myeloid, B-, and natural killer (NK)-cell deficiency. Five other individuals, 3 with spontaneously resolved cytopenic episodes in infancy, harbored hematopoietic revertant mosaicism by uniparental disomy of 7q, with loss of the mutated allele or additional in cisSAMD9L truncating mutations. Examination of 1 individual indicated that somatic reversions were postnatally selected. Somatic mutations were tracked to CD34⁺ hematopoietic progenitor cell populations, being further enriched in B and NK cells. Stimulation of these cell types with interferon (IFN)-α or IFN-γ induced SAMD9L expression. Clinically, revertant mosaicism was associated with milder disease, yet neurological manifestations persisted in 3 individuals. Two carriers also harbored a rare, in trans germ line SAMD9L missense loss-of-function variant, potentially counteracting the SAMD9L mutation. Our results demonstrate that gain-of-function mutations in the tumor suppressor SAMD9L cause cytopenia, immunodeficiency, variable neurological presentation, and predisposition to MDS with -7/del(7q), whereas hematopoietic revertant mosaicism commonly ameliorated clinical manifestations. The findings suggest a role for SAMD9L in regulating IFN-driven, demand-adapted hematopoiesis.

Figure 1.

Cellular deficiencies and neurological manifestations in family 1. (A) Peripheral blood hemoglobin (Hb), platelet, neutrophil (Neutro.), and monocyte (Mono.) counts as well as (B) lymphocyte subset counts in F1:III-1 and F1:I-3, who subsequently were diagnosed with childhood and adult MDS, respectively. Each patient was repeatedly assessed, with individual measurements denoted by circles. Gray boxes depict age-related reference values. (C) Plots show perforin versus CD56 expression among gated CD3⁻ lymphocytes, as assessed by flow cytometry. (D) Bar graph depicts the relative median fluorescence intensity (R-MFI) of CD107a, perforin, granzyme A (Gzm A), and granzyme B (Gzm B), as indicated, in gated CD3⁻CD56^dim NK cells. (E) Bar graph depicts the frequency of exocytosing CD3⁻CD56^dim NK cells, as evaluated by induction of surface CD107a expression (ΔCD107a), by target cells, as indicated. (F) Sagittal magnetic resonance image of F1:III-1 revealing bilateral white substance changes and cerebellar degeneration. Ctrl, control; Trp, transport control.

Figure 2.

Heterozygous SAMD9L gain-of-function mutations associated with cytopenia, susceptibility to MDS with chromosome 7 aberrations, immunodeficiency, and ataxia. (A) Pedigree of family 1. Segregation of the SAMD9L c.2956C>T mutation and the rare SAMD9L c.698C>A variant is shown. Filled quadrants depict the clinical manifestations, as illustrated in the legend. Individuals with somatic in vivo reversion are indicated by asterisk (*) showing UPD(7q), or paragraph symbol (^¶) showing second-site mutation. Genotypes are indicated with a vertical line (|) or forward slash (/), depending on whether phase information is known or unknown, respectively. (B) Sanger traces from family 1 for the SAMD9L c.2956C>T mutation (arrow). Individuals I-3, III-1, III-2, II-4, I-4 are shown. (C) Pedigree of family 2. Segregation of the SAMD9L c.2672T>C mutation is shown. Individuals with somatic in vivo reversion are indicated by asterisk (*) showing UPD(7q), or paragraph symbol (^¶), showing second-site mutation. (D) Sanger traces from family 2 for the c.2672T>C mutation (arrow). Individuals II-4, I-2, II-1, and II-2 are shown. (E-F) Multispecies evolutionary conservation of the amino acid residues (E) Ile891 and (F) Arg986 mutated in family 2 and family 1, respectively. The sequence alignment was performed with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). The asterisk (*) indicates positions which have a single, fully conserved residue; the colon (:) and period (.) indicate conservation between groups of strongly similar properties, scoring >0.5 in the Gonnet PAM 250 matrix and ≤0.5 in the Gonnet PAM 250 matrix, respectively. (G-I) 293FT cells were transiently transfected (Tx) with TFP-SAMD9L wild-type (WT) or patient-derived mutants, as indicated. (G) Western blots of recombinant SAMD9L variant expression, as indicated. (H-I) Cell proliferation in 293FT cells assessed by dye dilution assays. CellTrace (Thermo Fisher Scientific) dye-labeled 293FT cells were transfected with TFP or the TFP-SAMDL variants indicated, cultured for 72 hours, and analyzed by flow cytometry. (H) Effect of proliferation/microtube inhibitor nocodazole (10 µg/mL) on 293FT cell proliferation. (I) Dye dilution assays in TFP-SAMD9L–transfected 293FT cells. Dye levels were monitored in TFP⁻ cells (filled gray histograms) and compared with cells expressing uniformly intermediate levels of TFP-SAMD9L variants, as indicated. (G-I) Single, representative experiments of 5 experiments are shown. Impair, impairment.

Figure 3.

Loss of the SAMD9L gain-of-function–mutated allele in myelodysplastic cells. (A) Microarray-based comparative genomic hybridization of bone marrow–derived DNA of F1:I-3 showing duplication of 1q (in red) and deletion of 7q (in green), consistent with the der(1;7)(q10;p10) finding by karyotype. (B) Fluorescence in situ hybridization analysis with chromosome 7 painting reveals monosomy 7 in bone marrow cells from F2:II-4. (C) Frequency of SAMD9L c.2956C>T mutation relative to wild type assessed by mutation-specific droplet-dPCR in fibroblasts and serial bone marrow samples of F1:I-3. (D) Frequency of SAMD9L c.2672T>C mutation relative to wild type assessed by mutation-specific dPCR in DNA derived from MDS, peripheral blood, and fibroblasts of F2:II-4. For each sample, the data displayed is the combination of at least 2 dPCR chips. Error bars show 95% confidence levels.

Figure 4.

Somatic revertant mosaicism of the SAMD9L mutations. (A-B) Quantification of the frequency of the (A) SAMD9L c.2956C>T in family 1 and (B) SAMD9L c.2672T>C in family 2 relative to the wild-type allele in peripheral blood–derived DNA, as assessed by mutation-specific dPCR assay. (C) Additional quantification of the frequency of the SAMD9L c.2956C>T in DNA from a Guthrie card dried blood spot and a buccal swab from F1:III-2. For each sample, values represent the mean of at least 2 chips. Error bars denote 95% confidence levels. (D) Allele frequency of phased heterozygous variants on chromosome 7 of F1:III-2, as calculated from the allelic read-depth from WES data. The variants are color-coded on the basis of the parental origin, as indicated. The gray vertical lines indicate the centromere of chromosome 7, and the dashed line indicates the position of the SAMD9L c.2956C>T mutation. (E) Log R ratio of intensity signal and B-allele frequency for SNP on chromosome 7 of F2:II-1, as determined by SNP array. The dashed vertical line indicates the position of the SAMD9L c.2972T>C mutation. (F) Sanger traces from members of family 2, as indicated, for the SAMD9L c.2302A>T nonsense mutation identified in F2:I-2 (arrow). (G) Schematic representation of the genotype of PCR-derived SAMD9L clones spanning the SAMD9L c.2302A>T and c.2972T>C mutations. Open circles represent wild-type (wt) nucleotide sequence, whereas filled circles represent mutated (mut) nucleotide sequence.

Figure 5.

Functional evaluation of disease-modifying and somatic reversion SAMD9L variants. (A-C) 293FT cells were transiently transfected (Tx) with TFP-SAMD9L wild-type (WT) or potentially disease-modifying p.Thr233Asn variant, as indicated. (A) Western blots of recombinant SAMD9L variant expression, as indicated. (B) Cell proliferation in 293FT cells assessed by dye dilution assays of TFP-SAMD9L–transfected 293FT cells. Dye levels were monitored in TFP⁻ cells (filled gray histograms) and compared with cells expressing uniformly intermediate levels of TFP-SAMD9L variants, as indicated. A single representative experiment is shown. (C) Cumulative data from independent experiments on growth inhibition associated with specific TFP-SAMD9L variants, as indicated. According to the index, 0 denotes growth of vector-transfected cells, whereas −1 approximates TFP-SAMD9L wild-type transfected cells. Significance was determined by 1-way analysis of variance (*P < .05; **P < .005; ***P < .0005. (D) Overview of SAMD9L structure, including SAM domain (blue). Positions of identified disease-associated germ line SAMD9L gain-of-function mutations (red), germ line loss-of-function variants (blue), and somatic reversion mutations (green) are indicated. Disease-associated germ line SAMD9L mutations reported by Chen and colleagues are included (red).

Figure 6.

Frequency of SAMD9L mutation in hematopoietic stem, progenitor, and differentiated cell populations and interferon-stimulated expression of SAMDL. (A) Quantification of the frequency of the SAMD9L c.2956C>T in distinct bone marrow–derived HSPC populations from F1:II-2, as indicated. (B-C) Quantification of the frequency of the SAMD9L (B) c.2956C>T and (C) c.2672T>C mutations in specific peripheral blood–derived immune cell populations from F1:III-2 and F2:II-1, respectively, as indicated. For each sample, values represent the mean of at least 2 chips. Error bars denote 95% confidence levels. (D-F) Evaluation of SAMD9L and SAMD9 expression in (D) bone marrow-derived CD34⁺ HSPCs, (E) peripheral blood–derived NK cells, and (F) fibroblasts stimulated with interferon (IFN)-α or IFN-γ, as indicated. (G) Hypothetical model of the pathophysiology of germ line SAMD9L gain-of-function (GoF) mutations in relation to HSPC proliferation and differentiation. Healthy individuals, with 2 wild-type SAMD9L copies (top panel), have (1) normal, steady-state hematopoiesis, and (2) increased cellular output upon infection-induced, demand-adapted hematopoiesis. In contrast, carriers of heterozygous SAMD9L GoF mutations (middle panel) may (1) display grossly normal (and perhaps subclinical) hematopoiesis for some time, but (2) experience cytopenias and immunodeficiency upon infection early in life. In this setting, interferons can promote SAMD9L expression, with SAMD9L GoF mutants acting as potent suppressors of cell proliferation, dramatically impairing hematopoiesis and immunity. The ensuing hematopoietic crisis can facilitate (3) selection and expansion of revertant mutants, by UPD(7q), SAMD9L loss-of-function (LoF) mutations in cis, or monosomy 7. Whereas UPD(7q) and in cis SAMD9L LoF mutations can support clonal hematopoiesis and recovery from cytopenia, monosomy 7 is associated with development of MDS. Finally, carriers of combined SAMD9L GoF mutation and rare LoF variants in trans (bottom panel) are asymptomatic, suggesting they have normal (1) steady-state and (2) demand-adapted hematopoiesis. As such, pathogenic effects of SAMD9L GoF mutations may be balanced by SAMD9L LoF mutations. Mono, monocyte; Neu, neutrophil.