Figure 6

Molecular diagnosis and mechanism. Sequence analysis of CSF2RA exon 3 in a control subject (Figure. 6 A, upper panel), in one of the parents (Figure. 6 A, middle panel), and in the proband (Figure. 6 A, lower panel). In peripheral blood leukocytes genomic DNA, exons 3-13 of the CSF2RA gene were amplified and the PCR products were sequenced. The electropherograms illustrate the c.74 C > A substitution (boxed) in heterozygous (middle panel) and in homozygous form (lower panel), which leads to the replacement of a serine (TCG) by a premature stop codon (TAG) at amino acid position 25. Family tree shows consanguinity (Figure. 6 B). Flow cytometric analysis of peripheral blood cells demonstrates the absence of the alpha-chain (Figure. 6 C). Isolated neutrophils were incubated with antibodies, washed with Dulbecco's PBS twice and measured by means of flow cytometry. Ten thousand cells were counted and analyzed by the FACSDiva software. Fc blocking and isotype controls were applied to exclude unspecific bindings. CD11b (mouse monoclonal IgG1, PE conjugated), CD116/GM-CSF-R alpha (mouse monoclonal IgG1, PE conjugated), CD131w/GM-CSF-R beta (mouse monoclonal IgG1, purified) and secondary antibody for CD131w (rat anti-mouse IgG1, PE-conjugated).