Supplementary Figures

Supplementary Fig. 1. Representative transmission electron microscopy (EM) images of platelets from two unrelated healthy controls and seven cases from pedigrees A, B and E. Cases have normally sized platelets but some of their platelets have reduced numbers of alpha granules. Platelets depleted of alpha granules contain empty vacuoles (*) and some of them have membrane complexes (#). Scale bar of 1 μM for magnification x7,000.

Supplementary Fig. 2. A. Cumulative frequency distributions of the number of dense granules per platelet, quantified by whole mount EM analysis, for two cases from pedigree E (lines) and the mean+-1 standard deviation (SD) across 57 controls (shaded area). There was a slight but significant reduction in the number of dense granules per platelet in the cases (p = 4.01x10-6 (E II.6) and p=9.199x10-5 (E II.3), Kolmogorov-Smirnov test). B. Representative EM image for case E II.6. Scale bar of 1 μM for magnification x30–40,000.

Supplementary Fig. 3. A. Western blot analysis of HEK293 transfected cells with myc-tagged IKZF5 WT and mutants after extraction of different subcellular fractions as indicated (left panels). GAPDH and HIST3H3 serve as loading controls (right panel). HEK293 transfected with empty vector (Mock) express no IKZF5. Representative blots of triplicated expression experiments loading equal amounts of proteins. B. Western blot analysis of HEK293 transfected cells with myc-tagged IKZF5 WT and mutants after loading total cell extracts made from 75000 cells. The expression of the mutant ranged between 45-63% compared to WT IKZF5 (experiment 1). C. Western blot analysis of HEK293 transfected cells with myc-tagged IKZF5 WT and mutants after loading total cell extracts made from 75000 cells. The expression of the mutant ranged between 74-109% compared to WT IKZF5 (experiment 4).

Supplementary Fig. 4. Confocal immunofluorescence using an anti-IKZF5 antibody (green) and phalloidin (Actin; red) showing punctate, nuclear staining for WT IKZF5 while the mutants remain largely outside the nucleus after transfecting HEK293 cells. Scale bar of 10 μM.

Supplementary Fig. 5. Representative images of proplatelet forming megakaryocytes for each studied culture from six unrelated healthy controls and seven cases from pedigrees A, B and E. Cases have a reduction in proplatelet formation by megakaryocytes compared to controls. However, there was no discernible difference in the number or spatial distribution of VWF-positive alpha granules. Scale bar of 20 μM. Factin (red), VWF (green).

Supplementary Fig. 6. Bar plot of the number of RNA-seq read pairs aligned to the reference human transcriptome for samples from three cases and 14 controls. Within each bar, the number of aligned and unaligned read pairs are shown.

Supplementary Tables

IDPLT in PRP (x109/l)

Collagen1 μg/ml

Ristocetin1.5 mg/ml

AA1mM

U466191 μM

ADP3 μM

ADP5 μM

TRAP50μM

B II.1 62 normala normalb normal ND normalc normalc ND

B II.2 105 normala normalb normal ND normalc normalc ND

C II.3 120 normal normal normal normal normal normal normal

D III.2 160 reduced reduced reduced ND ND reduced ND

E II.3 195 reduced normal reduced ND ND reduced reduced

Supplementary Table 1. Platelet light transmission aggregometry (LTA) in response to various agonists. Agonist doses used in B II.1 and B II.2 were slightly different to those used in other cases: acollagen 2 ug/ml, bristocetin 1.25 mg/ml, cADP 2, 3 & 4μM. LTA for C II.3 was performed on two occasions and there were normal responses to all agonists on both occasions. PRP: Platelet Rich Plasma, AA: Arachidonic Acid, TRAP: Thrombin Receptor Activating Peptide. ND: not done.

IDNumber and mode of delivery

Platelet augmentation required Bleeding Transfusions

C III.3 1 vaginal delivery No No No

D II.2 1 vaginal delivery No No No

D II.3 1 vaginal delivery No No No

E II.8 3 vaginal deliveries No

Yes in the first delivery but not in the other two

Red blood cell transfusion due to immediate postpartum hemorrhage after the first delivery

E III.7 5 vaginal deliveries No No No

Supplementary Table 2. Pregnancies in carriers of IKZF5 variants including need for platelet augmentation during pregnancy or delivery, clinically significant bleeding and transfusion requirement.

Supplementary Table 3. Please refer to separate spreadsheet.