Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia by Phil J. Ancliff, Michael P. Blundell, Giles O. Cory, Yolanda Calle, Austen Worth, Helena Kempski, Siobhan Burns, Gareth E. Jones, Jo Sinclair, Christine Kinnon, Ian M. Hann, Rosemary E. Gale, David C. Linch, and Adrian J. Thrasher Blood Volume 108(7):2182-2189 October 1, 2006 ©2006 by American Society of Hematology

Identification of 2 WAS mutations. Identification of 2 WAS mutations. (A) Sequence analysis of P1 demonstrating 36201T>C leading to Ile294Thr. (B) Sequence analysis of P2 demonstrating 36143T>C leading to Ser272Pro. (C) Confirmation of the presence of the T>C mutation in P1 by PCR and DdeI restriction endonuclease digestion. Lane 1: undigested PCR product, lane 2: P1, lane 3: patient's sister (clinically healthy), lane 4: mother, lanes 5 and 6: healthy controls, and lane 7: water. (D) Confirmation of the T>C mutation in P2 by PCR and BpmI restriction endonuclease digestion. Lane 1: P2, lane 2: mother, lane 3: father, lane 4: maternal aunt, lane 5: maternal grandmother, lane 6: maternal uncle, and lane 7: DDW. Phil J. Ancliff et al. Blood 2006;108:2182-2189 ©2006 by American Society of Hematology

Presence of the Ile294Thr or Ser272Pro WASp mutation profoundly impaired myelopoiesis and led to increased levels of hematopoietic cell apoptosis. Presence of the Ile294Thr or Ser272Pro WASp mutation profoundly impaired myelopoiesis and led to increased levels of hematopoietic cell apoptosis. (A) CD34+ cell expansion after 14 days in liquid culture. (B) Annexin V–positive cells following anti-CD95 administration to cultured PB lymphocytes. Error bar indicates 1 SD from 33 healthy donors. (C) Spontaneous apoptosis in bone marrow progenitor cells. Phil J. Ancliff et al. Blood 2006;108:2182-2189 ©2006 by American Society of Hematology

Macrophages from P1 had abnormal podosome clustering and increased levels of F-actin. Macrophages from P1 had abnormal podosome clustering and increased levels of F-actin. (A) Normal macrophages were polarized with podosomes evenly distributed at the leading edge. (B-D) Macrophages from P1 exhibited a loss of polarization and uneven podosome distribution; typically podosomes were found in multiple clusters or ring structures. (E and F) Staining with antivinculin antibody revealed a ring of vinculin around an actin core for both normal and patient cells, respectively. Insets show a magnified view of an individual podosome. (G) Quantification of podosome clusters per cell. (H) Quantification of F-actin in normal and patient cells. Phil J. Ancliff et al. Blood 2006;108:2182-2189 ©2006 by American Society of Hematology

Live WASp-Ile294Thr macrophages exhibited abnormal shape and motility. Live WASp-Ile294Thr macrophages exhibited abnormal shape and motility. (A-C) Comparison of area, elongation factor, and dispersion factor for normal, WASp-deficient (–), and WASp-Ile294Thr macrophages. (D,E,F) Representative composite images of normal, WASp-deficient, and WASp-Ile294Thr macrophages, respectively. Dashed lines represent the outline of the cell body. (G) Quantification of podosome turnover. *P < .05; **P < .01; ***P < .001. Error bars indicate 1 SE. Phil J. Ancliff et al. Blood 2006;108:2182-2189 ©2006 by American Society of Hematology

Ile294Thr and Ser272Pro mutations enhanced the ability of WASp to stimulate actin polymerization in vitro. Ile294Thr and Ser272Pro mutations enhanced the ability of WASp to stimulate actin polymerization in vitro. Sepharose beads coated with WT or mutant GST-WASp constructs or GST-vector alone were incubated with 3 different dilutions of U937 cell lysates for 1 hour at room temperature. (A) SDS-PAGE of bound proteins stained with anti-GST and antiactin antibodies. (B) Actin polymerization activity for each mutant relative to WT. Phil J. Ancliff et al. Blood 2006;108:2182-2189 ©2006 by American Society of Hematology