Figure 2.
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.

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.

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