Nature Genetics: doi:10.1038/ng filepubertas praecox vera, (j) Brachytelephalangy, longitudinal ridges on finger nails, and keratosis pilaris on forearm. (k) Bulging of right humerus

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Supplementary Figure 1

Syndromic features of human SMARCD2 deficiency.

(a–d) Phenotype of patient AII.1. (a,b) Low-set, posteriorly rotated, simple ears, with prominent concha and broad incisura intertragica. (c) Hallux valgus (filled arrowhead) and plantar hyperkeratosis of left foot (empty arrowhead). (d) Sandal gap/increased interdigital space D1–D2 (asterisk) and dorsal feet hyperkeratosis D1 (filled arrowhead). (e–l) Phenotype of patient BII.1. (e,f) Thin hair, ear with dysplastic helix, neck with buffalo hump, and acanthosis nigricans. (g) Misaligned, dysplastic teeth, incomplete amelogenesis imperfecta (filled arrowhead), and accessory teeth in gingival plicae (empty arrowheads). (h,i) Features of metabolic syndrome and pubertas praecox vera, (j) Brachytelephalangy, longitudinal ridges on finger nails, and keratosis pilaris on forearm. (k) Bulging of right humerus diaphysis (empty arrowhead) and osteochondroma (filled arrowhead). (l) Increased interdigital space D1–D2 (asterisk), dorsal

feet hyperkeratosis D1, D2 left (filled arrowhead), brittle (5th) nail (empty arrowhead), and pes planus. Images have been partially cropped.

Nature Genetics: doi:10.1038/ng.3833

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Development of MDS/AML in SMARCD2 deficiency.


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(a–f) HE staining of bone marrow biopsies. (a,d) In 04/2008, HE-stained sections show altered bone marrow microarchitecture with reversion of the myeloid/erythroid ratio and dysplastic megakaryocytes. (b,e) In 10/2008, HE-stained sections show a dysplastic bone marrow with marked fibrosis (filled arrowheads). (c,f) In 04/2011, HE-stained sections show a hypercellular bone marrow with focal and peritrabecular infiltration by blast cells (asterisks). (g–i) Glycophorin C staining of bone marrow sections. (g) In 04/2008, irregular erythroid islands were observed. (h) In 10/2008, abnormal erythroid islands located along fibrotic strands were noted. (i) In 04/2011, erythroid islands are present in the immediate vicinity of blast cells (asterisk). (j–l) CD61 staining of bone marrow sections. (j) In 04/2008, small dysplastic megakaryocytes with hypolobated nuclei were observed. (k) In 10/2008, dysplastic micromegakaryocytes were noted. (l) In 04/2011, hypolobated dysplastic megakaryocytes in differing intensities, reminiscent of inadequate megakaryocyte

maturation and megakaryocyte dysplasia, were detected. Images have been partially cropped. Scale bars, approximately 20 m.


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Decreased expression of granulocyte-specific proteins in SMARCD2-deficient hematopoietic cells.

(a) Immunohistochemical detection of lactoferrin (LTF) in healthy donor (HD) bone marrow cells showing strong expression of LTF in


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myeloid cells. (b) Immunohistochemical detection of CD15 on healthy donor bone marrow, showing strong expression of CD15 by mature neutrophilic cells. (c) Immunohistochemical detection of myeloperoxidase showing normal amounts and distribution of myeloperoxidase (MPO)-expressing neutrophilic cells presenting at all stages of maturation. (d) Immunohistochemical detection of LTF in patient AII.1 showing lack of LTF-expressing cells. (e) Immunohistochemical detection of CD15 in patient AII.1 showing reduced intensity and decreased cell numbers of CD15-expressing cells. (f) Immunohistochemical detection of MPO in patient AII.1 bone

marrow cells showing a hypercellular bone marrow with presence of neutrophilic cells and blast cells expressing MPO with considerably lower intensity as compared to HD. (g) Immunohistochemical detection of LTF in patient BII.2 shows false positive background staining in a postmortem sample (note that LTF deficiency in cells from BII.1 has previously been documented by ELISA). (h)

Immunohistochemical detection of CD15 on patient BII.2 bone marrow cells shows a hypercellular bone marrow with almost complete absence of CD15-expressing cells. (i) Immunohistochemical detection of MPO in patient BII.2 bone marrow cells shows hypercellularity. MPO-positive neutrophilic cells are reduced in numbers and intensity in comparison to HD. (j) Immunohistochemical detection of LTF in patient CII.1 showing a hypercellular bone marrow and a lack of LTF-expressing cells. (k) Immunohistochemical

detection of CD15 in patient CII.1 showing a hypercellular bone marrow with markedly reduced intensity and decreased cell numbers of CD15-expressing cells in comparison to HD. (l) Immunohistochemical detection of MPO in patient CII.1 bone marrow cells shows a

hypercellular bone marrow with clusters of neutrophilic cells and blast cells. MPO expression is reduced. Images have been partially

cropped. Scale bars, approximately 20 m.


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Zebrafish models for Smarcd2 deficiency.

(a) Knockdown of smarcd2 by the splice-blocking morpholino oligonucleotides was examined by RT–PCR. Morpholino SB2 (MO SB2)

failed to disrupt smarcd2 splicing. Top, amplification of smarcd2 exon boundaries; bottom, -actin control; (+) and (–) indicate samples

prepared with and without reverse transcriptase, i.e., monitoring for genomic DNA contamination. Image cropped from one gel. The full-length gel image is shown in Supplementary Data 3b. (b) Fluorescence image of Danio rerio strain Tg(mpx:EGFP)

i114, control versus

smarcd2-morphant embryo. Reduced numbers of GFP-expressing neutrophils are observed in morphant versus control fish embryos. Acquired images: CTRL (n = 15 images), ATG MO (n = 15 images), SB1 MO (n = 15 images) and SB2 MO (n = 15 images). (c) Comprehensive analysis of neutrophil numbers in Tg(mpx:EGFP)

i114 at 72 h.p.f. after injection of morpholino oligonucleotides

(nonspecific control versus translation-start-site blocker (ATG) and splice-site blocker (SB1 and SB2) MOs against smarcd2. Numbers

represent fluorescence-labeled neutrophils per individual fish embryos. Data were pooled from two independent morpholino experiments: CTRL n = 15, ATG n = 15, SB1 n = 15, SB2 n = 15 fish. Center value, mean; error bars, s.d. P values were calculated by two-tailed unpaired t test. Replicates: 2. Please compare to Supplementary Tables 19 and 20. These results are an independent verification of the data in Figure 3b in another neutrophil reporter line. (d) Sanger chromatogram of the CRISPR/Cas9-edited frameshift

allele smarcd21/1

(1/1) versus the smarcd2 wild-type allele (wt) indicating the induced mutation c.66dup (Mut).


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Smarcd2 knockdown in zebrafish is specific for granulopoiesis.

(a) Erythrocyte staining in zebrafish larvae using O-dianisidine staining. Zebrafish treated with smarcd2 SB morpholino (right) show staining equal to that of control-treated zebrafish (left). Gross comparison shows equal O-dianisidine staining in control and morphant fish. (b) Macrophage numbers in zebrafish treated with smarcd2 SB morpholino and control-treated zebrafish are visualized by mCherry-positive cells in the tail. The smarcd2 SB morpholino, inducing aberrant splicing and consequent smarcd2 deficiency, shows no effect on macrophage numbers. Center value, mean; error bars, s.e.m. P values were calculated by two-tailed unpaired t test. Acquired images: CTRL (n = 94 images) and SB1 MO (n = 48 images). (c) Thrombocyte numbers in zebrafish treated with smarcd2 SB

morpholino and control-treated zebrafish are visualized by EGFP-positive cells in the tail. The smarcd2 SB morpholino, inducing aberrant splicing and consequent smarcd2 deficiency, shows no effect on thrombocyte numbers. Center value, mean; error bars, s.e.m. P values were calculated by two-tailed unpaired t test. Acquired images: CTRL (n = 48 images) and SB1 MO (n = 63 images). Data are pooled from two independent experiments in b and c. Replicates: 2, See Supplementary Table 20 for raw data.


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Morphology of smarcd2-deficient neutrophils in zebrafish.

(a) FACS sorting strategy of zebrafish neutrophils. (b) Developmental stages of zebrafish granulopoiesis (May–Grünwald Giemsa staining). (c) May–Grünwald Giemsa staining of sorted neutrophils in control versus morpholino knockdown zebrafish. (d) No gross

histological differences were recognized nor was any overt difference revealed by a four-category differential count of the embryonic neutrophil population (n = 1 experiment, pooling of neutrophils from n = 150 embryos/group); see Supplementary Table 20 for raw

data.


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Generation of Smarcd2-knockout mice.

(a) Mouse Smarcd2 (ENSMUST00000021052)-mutant embryonic stem cells (ES cells with heterozygous deletion of Smarcd2; clone

AF4) were purchased from KOMP repository. Shown is Smarcd2tm1(KOMP)Vlcg

(deletion allele). The positions of the primer pairs for the wild-type allele (empty arrowheads) and the knockout allele (filled arrowheads) are shown in relation to the Smarcd2 gene and deletion allele, respectively. (b) PCR-based genotyping. F1 intercrosses of heterozygous mice resulted in Smarcd2

+/+, Smarcd2

+/–, and

Smarcd2–/–

embryos. PCR was performed according to the manufacturer (KOMP repository): wild-type allele, 264 bp (empty arrowheads) and knockout allele, 569 bp (filled arrowheads). Embryos 13, 15, and 17 are of the knockout genotype (Smarcd2

–/–), and

embryos 12, 14, and 16 are of the heterozygous genotype (Smarcd2+/–

). Images have been cropped; full-length images are shown in Supplementary Data 3d,e. (c–e) Mendelian inheritance pattern of mutant Smarcd2 alleles. (c) Knockout embryos are not viable after birth. The numbers of Smarcd2

+/+ (n = 24), Smarcd2

+/– (n = 52), and Smarcd2

–/– (n = 1, perinatal death) offspring born are displayed. (d)

The distributions of genotypes per litter (in percent) are displayed. In total, expected Mendelian ratios of the genotypes (percent of genotype per litter) are observed in utero. Data from 15 litters are shown; compare with Supplementary Table 20. Center value, mean; error bars, s.e.m. P values were calculated by two-tailed unpaired t test. (e) The number of embryos per genotype and gestational age are shown at 12.5, 13.5, 14.5, 15.5, and 16.5 d.p.c. In total, wild type n = 24, heterozygous n = 56, and knockout n = 27 embryos were observed. (f) Western blot analysis detecting members of the SWI/SNF complex in Smarcd2-knockout embryos.

Protein expression of SMARCD1 (BAF60a), SMARCD2 (BAF60b), SMARCD2 (BAF60c) (kindly provided by J. Lessard, Montreal), and SWI/SNF members BRG1, BAF170, BAF155, and BAF47 are shown. Images of membranes have been cropped; full images are available in Supplementary Data 4. Protein expression was determined in crude embryonic tissue lysates by western blotting. Embryos from two litters were analyzed: wild type n = 3, heterozygous n = 3, knockout n = 3. Replicates: 1. (g) Enumeration of total

white blood cells (CD45.2+) and the stem cell compartment (LSK cells and LSK CD150

+CD48

– cells) per embryo in wild-type,

heterozygous, and knockout mouse fetal liver hematopoiesis. Smarcd2–/–

embryos show reduced CD45.2+ blood cells but comparable

amounts of stem cells (LSK, LSK CD150+CD48

–). Data are from three litters: wild type n = 7; heterozygous n = 10; knockout n = 8.

Center value, mean; error bars, s.e.m. P values were calculated by two-tailed unpaired t test. Replicates: 1.


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Colony count and flow cytometric analysis of GM colonies.

(a) Comprehensive analysis of myeloid cell colonies. Data are from five experiments with eight litters: wild type n = 10, heterozygous n

= 13, knockout n = 12. Numbers of colonies have been normalized to the input of 500 LSK cells. Center value, mean; error bars, s.e.m. P values were calculated by two-tailed unpaired t test. The F test when performed to compare the variances of the wild-type, heterozygous, and knockout groups showed, that variances differed significantly in total colonies (wild type vs. heterozygous, ***P < 0.001; wild type vs. knockout, ***P < 0.001), GM colonies (wild type vs. heterozygous, ***P < 0.001; wild type vs. knockout, **P = 0.003), M colonies (wild type vs. knockout, *P = 0.011), and G colonies (wild type vs. heterozygous, *P = 0.036; wild type vs. knockout, ***P < 0.001). Unpaired t tests with Welch’s correction showed that differences in means are significant in total colonies (wild type vs. heterozygous, *P = 0.037; wild type vs. knockout, **P = 0.004), in GM colonies (wild type vs. heterozygous, *P = 0.019; wild type vs. knockout, *P = 0.013), in M colonies (wild type vs. knockout, *P = 0.022), and in G colonies (wild type vs. knockout, *P = 0.011). This is likely to be due to sampling of experiments, outliers, and low numbers of colonies in M/G knockout samples. (b) Representative FACS plots of myeloid GM colonies derived from a showing expression of myeloid surface markers. Numbers are representative of the


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percentage of CD11b+Gr1

+ and CD11b

+Ly6c

+ cells. CD11b

+Gr1

+ and CD11b

+Ly6c

+ cells are absent in Smarcd2

–/– colonies. Data shown

are from one experiment (wild type n = 1, heterozygous n = 2, knockout n = 2), subsequently confirmed by a second experiment with wild type n = 5, heterozygous n = 4, and knockout n = 6, for which data are not shown. Replicates: 2.


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Differentially expressed genes in mouse Smarcd2-deficient early progenitors affect myeloid pathways.

(a) Differentially expressed genes in Smarcd2+/+

versus Smarcd2–/–

mouse LSK cells cluster in 11 groups by Reactome FI spectral

clustering. Pathways cluster significantly in immune system and myeloid pathways. Smarcd2 is highlighted in red. Relevant pathways and interactions between pathways are indicated. The sources of pathway annotations are indicated in parentheses: C, CellMap; R, Reactome; K, KEGG; N, NCI PID; P, Panther; B, BioCarta. A complete list of pathways enriched per cluster is shown in

Supplementary Table 6a. (b) Venn diagram of differentially expressed genes in Smarcd2+/+


LSK cells and CEBP

target genes (Supplementary Table 6c). (c) Differentially expressed genes in Smarcd2+/+


mouse LSK cells overlap

with CEBP targets (13 genes) and interact via MYC, FOS, EP300, and the ubiquitin system. Genes in green diamonds have been

introduced as linker genes; differentially expressed genes are color-coded by continuous mapping of log(fold change) generated in LSK Smarcd2

+/+ versus Smarcd2

–/– RNA–seq experiments (blue, reduced expression in knockout; red, increased expression in knockout).

Analysis and display of the network were carried out using Cytoscape 3.3.0 and the Reactome Functional Interaction (FI) plugin (solid line with arrow for activating/catalyzing FI, solid line with block for inhibitory FI, solid line (alone) for FIs extracted from complexes or inputs, and dotted lines for predicted FIs). The genes for all intersections are listed in Supplementary Table 6c.


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Absence of GMP progenitors in Smarcd2–/–

fetal liver hematopoiesis.

(a–c) Representative example of wild-type (a) and knockout (b) fetal liver hematopoiesis (per embryo) and gating strategy (c). Cells

were first discriminated by FSC/SSC (data not shown) and then by SSC-H/CD45. CD45+

cells were gated on Lin–c-Kit

+ cells. CD45

+Lin

–

Sca-1+c-Kit

+ (LSK) cells were sorted, and CD45

+Lin

–CD117

+Sca-1

– (myeloid progenitor) cells were further separated into CD45

+Lin

–

Sca-1–c-Kit

+CD34

–CD16/32

lo MEPs, CD45

+Lin

–Sca-1

–c-Kit

+CD34

+CD16/32

int CMPs, and CD45

+Lin

–Sca-1

–c-Kit

+CD34

+CD16/32

hi

GMPs. In comparison to a, GMP cells are almost absent in b. (c) Numbers in black boxes refer to the percentage of wild-type cells, and

numbers in white boxes refer to the percentage of knockout cells.


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Promoter occupancy of target genes for the CEBP and BRG1 proteins in SMARCD2-deficient NB4 cells.

(a–c) CEBP and BRG1 bind to the promoter regions of MMP8 (a), CAMP (b), and SERPIN A1 (c). Although binding is not significantly

reduced in cells transduced with shRNA1 (P = ns) or shRNA2 (P = ns) in comparison to cells transduced with a nonspecific control shRNA (CTRL), a trend can be observed. The experiment was performed two times. Data points from two experiments with a total of five biological replicates (five independent cultures and ChIP immunoprecipitations) are merged. Center value, mean; error bars, s.d. P values were calculated by two-tailed unpaired t test. Replicates: 3.


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Immunoprecipitation of endogenous CEBP .

(a–d) Experiment 1. (a) Immunoblotting for SMARCD2 detects SMARCD2 in a CEBP immunoprecipitate in short and long exposures;

a distinct band is noted (red arrow) at the expected size of ~56 kDa, corresponding to input and coimmunoprecipitated SMARCD2

proteins. (b) Cropped lanes of a showing immunoprecipitation with isotype control and antibody to CEBP (a2). (c) CEBP is detected

in input and immunoprecipitate; the image was cropped (c3) for summary. (d) Cropped images highlighting bands from experiment 1 shown with standard kDa marker (PageRuler Prestained Protein Ladder, Thermo Fisher). (e–g) Experiment 2. (e) Immunoblotting for

SMARCD2 detects SMARCD2 in two independent CEBP immunoprecipitates from the same lysate (technical replicates). Distinct

bands are noted (red arrows) at the expected size of ~56 kDa, corresponding to input and coimmunoprecipitated SMARCD2 proteins

(e1). Immunoblotting for GAPDH shows the presence of GAPDH in input but not in immunoprecipitates (e3). (f) CEBP is detected in

input and immunoprecipitates (red arrows) (f2). (g) Cropped images highlighting bands from experiment 2 shown with standard kDa marker (PageRuler Prestained Protein Ladder, Thermo Fisher). (h–j) Experiment 3. (h) Immunoblotting for SMARCD2 detects

SMARCD2 in the CEBP immunoprecipitate. A distinct band is noted (red arrow) at the expected size of ~56 kDa, corresponding to

input and coimmunoprecipitated SMARCD2 (h1). Immunoblotting for GAPDH shows the presence of GAPDH in Input but not in

immunoprecipitates (red arrow) (h3). (i) CEBP is detected in input and immunoprecipitates (red arrows) (i2). (j) Cropped images

highlighting bands from experiment 3 shown with standard kDa marker (Precision Plus Protein Kaleidoscope Prestained Protein Standards, Bio-Rad). The protein standard ladders differ between experiment 1 and 2 and experiment 3.


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SMARCD2 knockdown in human NB4 AML cells compromises myeloid pathways.

(a) Differentially expressed genes in undifferentiated AML-NB4 cells (shRNA-mediated SMARCD2 knockdown versus control) cluster in 11 groups by Reactome FI spectral partition and show significant enrichment for myeloid pathways. SMARCD2 is highlighted in red. Relevant pathways and interactions between pathways are indicated. The sources of pathway annotations are indicated in parentheses: C, CellMap; R, Reactome; K, KEGG; N, NCI PID; P, Panther; B, BioCarta. A complete list of the pathways enriched per cluster is given in Supplementary Table 6e. (b) Differentially expressed genes in ATRA-differentiated AML-NB4 cells (shRNA-

mediated SMARCD2 knockdown versus control) cluster in 12 groups by Reactome FI spectral partition and show significant enrichment for myeloid pathways. SMARCD2 is highlighted in red. Relevant pathways and interactions between pathways are indicated. The sources of pathway annotations are indicated in parentheses: C, CellMap; R, Reactome; K, KEGG; N, NCI PID; P, Panther; B, BioCarta. A complete list of the pathways enriched per cluster is shown in Supplementary Table 6f. (c) Venn diagram of differentially

expressed genes (undifferentiated and ATRA differentiated) and CEBP target genes. Genes from all intersections are listed in

Supplementary Table 6d. (d) Differentially expressed genes in ATRA-differentiated AML-NB4 cells overlap with CEBP targets (21

genes) and interact via EP300, TP53, STAT1, and the ubiquitin system. Genes in green diamonds have been introduced as linker genes; differentially expressed genes are color-coded by continuous mapping of log(fold change) in SMARCD2-knockdown cells versus control cells (blue, reduced expression in SMARCD2-knockdown cells; red, increased expression in SMARCD2-knockdown cells). Analysis and display of the network were carried out with Cytoscape 3.3.0 and the Reactome Functional Interaction (FI) plugin: solid lines with arrows for activating/catalyzing FIs, solid lines with blocks for inhibitory FIs, solid lines (alone) for FIs extracted from


22

complexes or inputs, and dotted lines for predicted FIs. This data set is derived from NB4 cells exposed to 1 M ATRA for 2 d and,

thus, cannot directly be compared to the qPCR data shown in Figure 5a.


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Regions of altered chromatin accessibility cluster around transcription start sites.

(a) Regions of open chromatin cluster around transcription start sites (TSSs) in DMSO/mock-treated NB4 cells. Peaks were binned into

500-bp windows with respect to their distance to the TSS. Each bar represents the fraction of peaks falling into one bin against all peaks of one group. The peaks are grouped as all peaks (black), upregulated peaks (red), and downregulated peaks (blue). The depicted peak distribution is based on undifferentiated (DMSO vehicle control) NB4 cells. (b) As in a but based on ATRA-differentiated

NB4 cells.


SUPPLEMENTARY NOTE 1"

This note describes methods and analyses for which there was not sufficient room in the 2"

Online Methods section. 3"

4"

Consanguinity estimates 5"

To estimate the (unknown) consanguinity of the affected individuals, we implemented a 6"

variant of the Froh method72, using only the runs of homozygosity (roh) of lengths > 1Mbpb. 7"

This is more cautious than the threshold of 500kbp recommended in the original publication. 8"

That analysis gave estimated inbreeding coefficients for the index patient of each pedigree as 9"

follows. Pedigree A: 0.090; Pedigree B: 0.014; Pedigree C: 0.087. The (estimated) inbreeding 10"

coefficient of the patients is equivalent to the kinship coefficient of the carrier parents. Thus, 11"

the parents of the affected individuals in families A and C are more closely related than first 12"

cousins, who would have a kinship coefficient of 0.0625. The parents of the affected in 13"

individuals in family B are almost as closely related as second cousins who would have a 14"

kinship coefficient of 0.015625.""15"

"16"

Statistics - homozygosity mapping 17"

Asymptotic LOD scores, assuming the frequency of the disease-associated marker haplotype 18"

decreases in the limit towards 0, were computed by hand using the principle that each meiosis 19"

after the first contributes log102 to the score. We used FASTLINK v. 4.1P73-75 to check the 20"

asymptotic scores and to compute scores with more realistic marker allele frequencies. For 21"

LOD score computations, we assumed full penetrance and a rare disease-associated allele. 22"

The scores shown in Results were computed for the two families separately and summed. 23"

24"

25"

26"


Sanger sequencing of SMARCD2 (adding information to Online Methods) 27"

DNA was extracted from adherent cells or suspension cells with QIAamp DNA Blood Mini 28"

Kit (Qiagen #51106) and used for further application. RNA was extracted with Qiagen 29"

RNeasy Micro Kit (#74004), RNase inhibitor ribolock (Thermo) was added, RNA was stored 30"

at -80°C or used for cDNA transcription using High-Capacity cDNA Reverse Transcription 31"

Kit (Life Technologies, # 4368813). 32"

33"

Sanger sequencing was performed on both gDNA and cDNA. The exonic regions of gDNA 34"

were amplified by PCR reaction. Per reaction, 2.5 µl HiFi buffer, 2.5µl dNTP 2 mmol/l, 0.125 35"

µl HiFi polymerase, DMSO 1.25 µl or betaine 5 µl, 1.5 µl primer forward/ reverse 10 pmol/l, 36"

≥20 ng DNA, up to 25 µl nuclease free water. The PCR reaction conditions were 95 °C 37"

melting for 5-10’, followed by 35-40 cycles of loops consisting of 90 °C melting for 30 sec, 38"

56 °C annealing for 30sec, 72 °C elongation for 30 sec (primer list for exons upon request) or 39"

90 sec for amplification of full length cDNA exon 1 to exon 13 of ENST00000448276; 40"

NM_001098426.1 (Supplementary Table 3). The full length SMARCD2 transcript 41"

amplification was performed with the aim to enrich all potential transcript variants conserved 42"

in the N and C termini of SMARCD2-001 ENST00000448276; NM_001098426.1. It includes 43"

mutated transcripts of SMARCD2-001 (ENST00000448276; NM_001098426.1) altered by 44"

exon skipping, intron retention or insertion/ duplications. Bands were visualized on 1% 45"

agarose gel. PCR products were cleand up using ExoSAP-IT (Affymetrix, AF 78202) or with 46"

QIAquick Gel Extraction (Qiagen #28706). Sanger sequencing was performed in house on a 47"

ABI 3130xl Genetic Analyzer or outsourced to Eurofins Company, Munich, Germany. 48"

Results were analyzed using the Seqman (DNASTAR) or ApE (M. Wayne Davis, Utah53) 49"

software packages. 50"

51"

52"


Cell lines 53"

The standard cell line NB4, (ACC 207) was purchased from the German Collection of 54"

Microorganisms and Cell Cultures (DSMZ). NB4 cells can be partially differentiated into 55"

neutrophil-like cells by all-trans retinoic acid (ATRA). Patient cell lines were cultivated from 56"

skin biopsies (fibroblasts) or peripheral blood after infection with Epstein-Barr virus (EBV) 57"

(the term we use for the transformed cells lines is EBV-transformed lymphoblastoid cell 58"

lines). Adherent cell lines 293T (kindly provided by Dr."C"Baum,"MHH"Hannover) and 59"

fibroblasts from healthy donors and patients AII.1 and BII.1 were cultured in DMEM, 60"

supplemented with 10% FCS, 50U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-Glu. 61"

Suspension cell lines NB4 and EBV-LCL were cultured in RPMI, supplemented with 10% 62"

FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-Glu, 10 mM HEPES buffer. 63"

Mycoplasma"testing"is"done"on"a"regular"scheme"following"SOP"in"the"lab."All"64"

experiments"were"done"on"mycoplasma"free"cells. 65"

66"

Plasmids and molecular cloning 67"

SMARCD2 was amplified from a human healthy donor sample or from patients’ cDNA 68"

(Supplementary Table 3). Analysis on agarose gels revealed differences in fragment size 69"

and/or number of fragments in patients versus healthy donor. Fragments were gel extracted 70"

and cloned into an in-house, CMV driven plasmid modified from the pCHGFPW plasmid76 71"

using 5’ prime XhoI primer with Kozak and FLAG tag and 3’ SpeI primer (Supplementary 72"

Table 3). Due to individual truncations at the 3´- terminus of mutated proteins, cloning was 73"

restricted to 5´-FLAG tags for mutated proteins. SMARCD2-specific pGIPZ. shRNA 74"

constructs and pGIPZ non-silencing control were purchased from Thermo Scientific (shRNA 75"

1-3: clone ID V3LHS_300463; V3LHS_300461; V3LHS_400374 and non-silencing control # 76"

RHS4531). The shRNA sequences were cloned via Mlu1 and Xho1 restriction sites into 77"

pGIPZ.SF.GFP.2 plasmid (kindly provided by Axel Schambach, MHH Hannover). Viral 78"


particles were produced in 293T cells with gag-pol, VSVG and rev helper plasmids. NB4 cell 79"

lines were stably transduced, GFP sorted and expanded. Knockdown efficiency was 80"

determined by expression of protein (Western blot) and mRNA/cDNA (qPCR method, see 81"

section: Expression of neutrophil specific granule genes in NB4 cells). 82"

83"

Immunoprecipitation experiments and Western blotting 84"

Transfection of wild-type and mutant SMARCD2 Flag-tagged proteins or HA-tagged CEBPE 85"

with calcium phosphate into semi-confluent 293T with 10 µg plasmid/10 cm dish was 86"

performed. Cells were harvested on day 3, pelleted and lysed in freshly prepared RIPA Buffer 87"

(450 mM NaCl, 25 mM TrisHCl pH7.5, 1 mM EDTA, 1% NP40, 5% Glycerol, 25 mM Na-88"

Pyrophosphate, 50 mM Na-Fluoride, EDTA-free protease inhibitor (Roche)). For each 89"

immunoprecipitation, one confluent 10 cm dish with 293T cells has been used. Lysates were 90"

cleared by centrifugation (21,000 g for 15 min at 4 °C). IP with FLAG affinity gel (Sigma) or 91"

HA affinity gel (Thermos Scientific) or Anti-CEBPE (sc-158x, SCBT) mAb was performed 92"

overnight at 4 °C on a rotating laboratory wheel. Samples were washed five times in RIPA 93"

buffer. Elution was performed with FLAG peptide 37.5 µg (Sigma F3290-4MG) or boiling in 94"

2x Laemmli sample buffer. Lysates were analyzed by Western blot. Immunoprecipitation of 95"

endogenous CEBPE was performed in NB4 cells, treated with ATRA 1 µM for 3 days. Cells 96"

were lysed in RIPA Buffer (250 mM NaCl, 25 mM, TrisHCl pH7.5, 1 mM EDTA, 1% NP40, 97"

5% Glycerol, 25 mM Na-Pyrophosphate, 50 mM Na-Fluoride, EDTA-free protease inhibitor 98"

(Roche)) and lysates were mildly sonicated to disrupt DNA. First, lysates were incubated with 99"

Anti-CEBPE (C-22X, sc-158x)77 or isotype control for 4hrs at 4°C, followed by incubation 100"

with protein-A beads for 1.5hrs at 4 °C. Next, beads were washed and bound proteins were 101"

eluted in 2x Laemmli sample and analyzed by Western blot. 102"

103"


Similarly, SMARCD2 expression in fibroblasts and EBV-LCLs of healthy donor and patients 104"

was analyzed. Cells were lysed with freshly prepared RIPA buffer. Lysates were cleared by 105"

centrifugation (21,000 g, 10 min, 4 °C). Protein quantification was performed with Bradford 106"

reagent (Bio-Rad Laboratories) using ELISA plate readers (Synergy H1 Hybrid Reader, 107"

BioTek; infinite M200, Tecan). Equal amounts of protein - achieved by Bradford 108"

(SMARCD2 expression in healthy donor and patient cells) or counting of input cells 109"

(Immunoprecipitation) were separated by sodium dodecyl sulphate polyacrylamide (SDS-110"

PAGE) gel electrophoresis and blotted onto polyvinyl difluoride (PVDF) membranes. The 111"

membranes were blocked in PBS containing 0.1 % Tween-20 (PBS-T) supplemented with 5% 112"

BSA or 5% non-fat dry milk for 2 hrs, followed by incubation with primary antibodies 113"

overnight at 4 °C or for 1 h at room temperature in (PBS-T) with 5 % BSA or 5% non-fat dry 114"

milk. 115"

116"

Primary antibodies used included anti-Flag, mouse, clone M2, F1804-200ug (Sigma) or anti 117"

FLAG-HRP, A8592-.2MG (Sigma), SMARCD2, mouse monoclonal antibody clone F-34, 118"

(sc-101162, SCBT), rabbit anti- SMARCD1, SMARCD2, SMARCD3 (generated, profiled 119"

and kindly provided by Julie Lessard, Montreal, Canada), GAPDH mouse monoclonal 120"

antibody clone 6C5 (sc-32233, SCBT), anti-HA, rabbit (ab9110, Abcam), anti BRG1, rabbit, 121"

clone EPNCIR111A (ab110641, Abcam), anti SMARCC2/BAF170, rabbit ( #8829S, CST), 122"

anti SMARCC1/ BAF 155, rabbit, clone D7F8S (#11956S, CST), anti SNF5/BAF47, rabbit, 123"

clone D9C2 (#8745S, CST),. After washing the PVDF membranes in PBS-T, secondary 124"

horseradish peroxidase conjugated antibodies anti-mouse (BD Pharmingen), anti-rabbit (CST) 125"

or conformation specific anti-rabbit IgG (L27A9, CST) were added for 1h at room 126"

temperature. After development with chemiluminescent substrate (Pierce ECL Western 127"

blotting substrate), digital images were acquired on a Chemidoc XRS Imaging System (Bio-128"

Rad Laboratories). Blots were stripped between detection of different antibody probes using 129"


Restore Western Blot Stripping Buffer (Thermo Scientific). Data analysis was performed 130"

using Image LabTM software (Bio-Rad Laboratories). 131"

132"

Statistics - Flow cytometry 133"

All"viable"embryos"were"processed."For immunological phenotyping, a minimum sample 134"

size of n=5 per genotype was chosen to allow detection of 50% difference between genotypes 135"

(assumed s.d. 20% in each population, Type 1 Error 0.01 and Type 2 Error 0.1). However, the 136"

number of genotyped samples was limited by litters. Therefore, experiments were repeated. F 137"

test was performed to compare variances of each group Wt, Ht, and Ko. Variances differed 138"

significantly in Figure 5b,c: CD11b+Gr1+ Wt vs Ko (F test p < 0.0001 (***)), and 139"

CD11b+Ly6c+ Wt vs Ko (F test p < 0.0001 (***)). This is possibly due to the fact that values 140"

in Ko samples are particularly low (near to zero). Here, we assume the difference in variance 141"

between Wt and Ko to be a limitation of measuring the abundance of a near-to-absent 142"

population. To analyze erythroid progenitor stages Figure 5g,h two-way ANOVA testing was 143"

used to compare multiple groups of data i.e. Wt vs Ht and Wt vs Ko in stage S0-S5. In 144"

addition, F test was performed to compare variances of Wt vs Ko in stage S1 and S3. This 145"

showed that variances differ significantly. Therefore, unpaired t tests with Welsh´s correction 146"

for these stages were performed in addition, which demonstrate significant differences of the 147"

means for Wt vs Ko in stage S1 (p < 0.001 (***)) and stage S3 (Wt vs Ko p < 0.001 (***). 148"

149"

Flow cytometry sorting 150"

Murine fetal liver Lin-Sca1+ckit+ (LSK) early progenitor cells, were isolated to perform CFU 151"

assays and RNA Sequencing (RNA-Seq). Individual embryos were genotyped, fetal liver 152"

tissue was suspended in HBSS with 3% FCS, and LSK cells were defined as follows: 153"

CD45.2+, lineage-, Mac1low/+, ckit+ and Sca1+ cells. Cells were then stained with anti-CD45.2-154"

FITC (BD Pharmingen), biotinylated lineage antibodies (anti-B220, -CD3, Gr-1, and -Ter-155"


119; all BD), anti-Mac1/CD11b-eFluor 450 (eBioscience), anti-CD117/c-kit-Alexa 156"

Fluor®780 (eBioscience) or PE, anti-Sca-1-PeCy7 (eBioscience), anti CD16/32-APC 157"

(eBioscience) and Anti-CD34-V450 (eBioscience). Biotinylated monoclonal antibodies were 158"

labeled by incubation with Streptavidin-PerCP/PerCP-Cy5.5 (eBioscience). LSK cells were 159"

sorted into Iscove's Modified Dulbecco's Medium (IMDM) with 3% FCS for CFU assays or 160"

directly into 1% TritonX supplemented with RNAse Inhibitor (Promega) for RNA-Seq. Cell 161"

sorting was performed using a FACS Aria III cell sorter and FACS Diva software. 162"

163"

Colony forming unit assays 164"

See Supplementary Note. Flow-sorted fetal liver LSK cells were washed and resuspended in 165"

50 µl IMDM without FCS. Between 500-1500 LSK cells were plated per 35mm Petri dishes 166"

containing 1.3 ml MethoCult® (M3231 or M3434, Stem Cell Technologies). M3434 (rm SCF, 167"

rm IL-3, rh IL-6, rh EPO) was used to examine erythro-myeloid maturation. M3231 (with 168"

addition of murine G-CSF [50 ng/ml], murine GM-CSF [50 ng/ml] or murine M-CSF [50 169"

ng/ml] (cytokines from Peprotech)) was used to assess myeloid maturation to specific 170"

cytokines. CFU colonies were assessed daily from day 3 onwards. Colony forming units (> 20 171"

cells) and lineage differentiation potential were assessed using an inverted microscope 172"

(Axiovert-II, Zeiss) at day 7 - 12. CFUs were photographed (data not shown), counted and 173"

analyzed by FACS. Cytological assessment was performed by May-Grünwald-Giemsa stain 174"

after cytospin centrifugation (Shandon Cytofunnel Thermo). 175"

176"

Statistics – CFU counts 177"

CFU counts were normalized to LSK cell input and analyzed by Prism software (GraphPad); 178"

statistical center value: Mean, standard error: SD or SEM, p-values, and two-tailed unpaired t-179"

tests were used. F tests were performed to compare variances of each group Wt, Ht, and Ko. 180"

In Figure 5 e, variances differed significantly in GM-CSF Wt vs Ht and Wt vs Ko (F test p 181"


=0,018 (*) and p= 0,005 (**)) and G-CSF Wt vs Ht and Wt vs Ko (F test p= 0,017 (*) and p= 182"

0,002 (**)). Unpaired t tests with Welsh´s correction shows, that the difference in means of 183"

G-CSF CFU Wt vs Ko is significant (p=0,024 (*)).If variances differed significantly between 184"

groups, F test p-values were given and an unpaired t test with Welch´s correction was 185"

performed in addition. Results are indicated in legends. 186"

Zebrafish morpholino knockdown experiments (adding information to Online Methods) 187"

Microinjection of morpholino oligonucleotides was performed as follows: Antisense 188"

morpholino oligonucleotides (Gene Tools, LLC (Eugene, OR)) (Supplementary Table 3) were 189"

resuspended as stock in milli-Q water at 1 mM and microinjected at highest non-toxic 190"

concentration for each morpholino (700 µM for all MO-smarcd2). 10-20 min post fertilization 191"

embryos carrying the reporter gene of interest were collected in egg water and placed on a 4% 192"

agarose gel block aligned to grooves on the gel surface. Microinjection of 1-cell embryos was 193"

performed using a standard microinjection apparatus and large-bore needle, positioned at the 194"

border of cell and yolk sac. 195"

196"

Knockdowns of smarcd2 by the splice-blocking morpholino oligonucleotides were examined 197"

by RT-PCR as follows: Whole embryo RNA was extracted using TRIzol® Reagent (Life 198"

Technologies) and cDNA synthesis was performed using SuperScript™ III Reverse 199"

Transcriptase (Invitrogen). Phusion High Fidelity DNA Polymerase (Thermo Scientific) was 200"

used for cDNA amplification. 50 µl PCR reaction was consisting of 1 µl Phusion DNA 201"

Polymerase, 10 µl 5X Phusion HF Buffer, 1 µl dNTP (10 mM), 1 µl forward primer (10 µM), 202"

1 µl reverse primer (10 µM), 2 µl RT reaction product (cDNA) and 34 µl of nuclease free 203"

water. Biorad T100 thermal cycler with following program was used for amplification: 90 204"

seconds at 95 °C as initial denaturation, followed by 30 cycles of 30 sec at 95 °C for 205"

denaturation, 30 sec at 56 °C for annealing, 30 sec at 72 °C for extension, and final extension 206"

at 72 °C for 5 min. Primer sequences are in Supplementary Table 3. Total numbers of 207"


fluorescent neutrophils, macrophages and thrombocytes in digital images of control and 208"

morphant embryos were manually counted at 72 hrs post fertilization (hpf) using an Olympus 209"

MVX10 microscope fitted with an Olympus DP72 camera. 210"

211"

Phenotype analysis of zebrafish 212"

EGFP-positive neutrophils in digital images of control and F2 Tg(mpx:EGFP) embryos of 213"

various smarcd2 allelotypes were manually counted at 72 hrs post fertilization (hpf) in the tail 214"

distal to the tip of the yolk extension, which includes the leukocyte-rich caudal hematopoietic 215"

tissue (CHT) using Olympus MVX10 microscope fitted with an Olympus DP72 camera. 216"

Three dpf whole embryos were stained by o-dianisidine to label erythroid globin-containing 217"

globin (erythroid cells) as previously described78. 218"

The mCherry-positive macrophages and EGFP-positive thrombocytes were manually counted 219"

at 72 hpf respectively in digital images of Tg(mpeg1:Gal4FF)gl25/(UAS-220"

E1b:Eco.NfsBmCherry)c264 49 and Tg(itga2b:eGFP)la2 50 embryos injected with either 221"

smarcd2 SB1 or standard control morpholinos. Cell numbers were quantified tail area distal 222"

to the tip of the yolk extension. 223"

EGFP-positive neutrophils from Tg(mpx:EGFP)i114 19 embryos were isolated for morphology 224"

analysis in Monash Flowcore facility using an Influx cell sorter (BD), data were analyzed 225"

using FlowJo software (version 7.6.1)., and cells were stained by May Grünwald-Giemsa78. 226"

227"

Statistics - Zebrafish phenotype 228"

Descriptive and analytical statistics were prepared in Prism 5.0c (GraphPad Software Inc). p-229"

values are from two-tailed unpaired t-tests, statistical center value: Mean, error bar: standard 230"

deviation. 231"

232"

233"


Statistics- Zebrafish morpholino experiments 234"

To determine sample size in morpholino experiments, several different scoring methods were 235"

used on 3dpf embryos: neutrophils in CHT, neutrophils in whole animals. In practice, the 236"

power / sensitivity of test was limited by the maximum practical number of embryos for each 237"

test. This was typically approximately n = 20 / group. To put this in context: in smarcd2+/+, 238"

the neutrophils/CHT score was typically 200+/-30 (SDmax). Assuming a similar SD in the 239"

test group, and a hypothesized 1-sided difference: a sample size of n=13 provides a power (1-240"

β)=0.8 and type 1 error α = 5% for detection of a 15% reduction in neutrophil numbers in the 241"

test group. Test and control MOs were microinjected by a standard protocol into 1-2 cell 242"

embryos randomly selected from pools of embryos collected from matings of groups of male 243"

and female adult fish. Loss of microinjected embryos from death and developmental defects 244"

was comparable to the control embryos and typically <10%. All"viable"embryos"were"245"

processed."Embryos were randomly allocated to control and test groups by their random 246"

selection during the process of microinjection. The MO experiments were scored unblinded. F 247"

tests were performed to compare variances of each group CTRL, ATG, SB1, SB2; variances 248"

do not differ significantly. 249"

250"

Statistic - Zebrafish CRISPR/Cas9 experiments 251"

The sample size was determined by a large number of different CRISPR alleles. All available 252"

-/- embryos of the selected single mutant allele were included in the analysis, after molecular 253"

confirmation of genotyping. The inclusion of embryos was denominated by their accurate 254"

genotyping; genotyping was attempted on all possible contributory embryos. In this case, 255"

randomization was not appropriate, as the hypothesis was that a phenotype segregated with a 256"

particular genotype. The phenotyping (counting neutrophils) was performed prior to 257"

genotyping and then neutrophil counts in the homozygous mutant embryos were included in 258"


analysis. The outcome was therefore assessed fully blinded. An F test was performed to 259"

compare variances of smarcd2+/+ and smarcd2-/- groups; variances do not differ significantly. 260"

261"

Chromatin Immunoprecipitation protocol 262"

Chromatin Immunoprecipitation was performed as previously described60-63. Cells were 263"

harvested, crosslinked with 1.5 mM ethylene glycol bis-succinimidyl succinate (EGS) for 30 264"

min at room temperature, followed by 1% formaldehyde for 10 min at room temperature. 265"

Chromatin was washed three times in PBS and 10%FCS and was sheared in Buffer B (50 mM 266"

Tris-HCl, pH 8, 10 mM EDTA, 1% SDS, protease inhibitor) on a Covaris sonicator. Lysate 267"

was spun down and incubated with antibodies overnight at 4 °C and consecutively Dynabeads 268"

ProteinG for 2 hrs at 4 °C. Antibodies used for ChIP experiments included IgG control rabbit 269"

(ab37415, Abcam), SWI/SNF-ATPse BRG1, (07-478, Millipore), CEBPE (sc-158X, C-22 270"

SCBT)77. 271"

272"

After that, beads were washed four times in buffer A (10 mM TrisHCl (pH 7.5), 1 mM 273"

EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Sodium-deoxcycholate, 140 mM 274"

Sodium chlorid, protease inhibitor) and one time in buffer C (10 mM TrisHCl, pH8.0, 10 mM 275"

EDTA) and eluted at 65°C for 20min and shaking. Eluate was reverse cross-linked for 20min 276"

at 95°C, followed by RNase and proteinase K digestion. DNA of input and 277"

immunoprecipitated samples were extracted by the Isoamyl-phenol-chloroform method and 278"

DNA was precipitated in 100% ethanol for at least 1hr at -80 °C. After that, DNA was 279"

concentrated by centrifugation in Eppendorf bench top centrifuge at 4 °C for 30 min at 20,000 280"

g, washed in 70% ethanol followed by centrifugation at 4 °C for 6 min. Pellets were vacuum 281"

dried and DNA was resuspended in nuclease free water and analyzed by qPCR. Primers used 282"

for LTF, MMP8, CAMP, and AAT are in the Supplementary Table 3. 283"

284"


Statistics - ChIP 285"

Analysis was performed using the percent input method79,80. In brief, we adjusted inputs of 286"

10% to 100% by calculating Inputadj =CT Input-Log10 10 and calculated percent of input as 287"

100*2^(Inputadj - Ct (IP)). 288"

289"

Statistics - Human RNA-Seq and ATAC-Seq data analysis (adding information to 290"

Online Methods) 291"

The R/Bioconductor67 package GenomicAlignments was utilized to generate count data for 292"

the RNA-Seq data based on the gene level. The same gene model as in the alignment step 293"

was used. Only reads mapping uniquely to one feature were counted by setting the 294"

summarizeOverlaps function option to mode=‘Union’. Furthermore all other options were set 295"

to FALSE (ignore.strand=FALSE, inter.feature=FALSE, fragments=FALSE). To reduce 296"

noise from genes with low expression levels, genes were kept only if the 95 % quantile of the 297"

coverage across all samples was below 10 reads as suggested by DESeq259 package. DESeq2 298"

was utilized to carry out a differential expression analysis between shRNA treated cells (clone 299"

1 V3LHS 300463 and clone 2 V3LHS 400374) and the control cells (RHS4531) of the 300"

remaining 13,244 genes. Furthermore, a differential expression analysis was performed 301"

between ATRA treated cells and DMSO treated cells as control. A significance cutoff of p < 302"

0.005 was applied to the results before further downstream analysis. The results of 303"

differentially expressed genes are listed in Supplementary Table 15 and 16.. 304"

"305"

For$the$ATACNSeq#analysis#the#reads#were#first#pulled#together#from#all#samples.#On#all#306"

reads&MACS281 was used with default parameters to call peaks. The resulting peaks were 307"

used as genomic features to generate count data for each sample with the GenomicAlignment 308"

package81. The count data were again subjected to filtering steps to reduce the noise as 309"

suggested by DESeq2. The remaining peaks where then annotated with the nearest gene and 310"


the distance to the transcribed region of it. We removed peaks, which were more than 2kb 311"

away from the transcribed region. To call significantly differently occupied genomic regions 312"

the Bioconductor package EdgeR82 was utilized. We compared the SMARCD2 knock-down 313"

against the control for the undifferentiated and for the ATRA differentiated cells. A 314"

significance cut-off of P value < 0.005 was applied to the results before further downstream 315"

analysis. The EdgeR results of the significantly differentially occupied peaks are listed in 316"

Supplementary Table 15 and 16 with their raw counts and their normalized counts. 317"

318"

Heatmaps were plotted based on the log2-fold-changes. The dendrogram was obtained by 319"

gene-wise and sample-wise hierarchical clustering with complete linkage. Fold changes and 320"

p-values per gene are given in Supplementary Table 15 (DMSO/undifferentiated) and 321"

Supplementary Table 16 (ATRA/differentiated). 322"

323"

324"

325"

326"

327"

328"

329"


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Author information:

The authors declare no competing financial interests. Correspondence and requests for

materials should be addressed to CK ([email protected])

"


Supplementary Table 1

Synopsis of clinical findings in pedigrees A, B and C

AII.1 BII.140 BII.2 CII.1

Country of origin Pakistan Pakistan Pakistan Lebanon

Froh –Consanguinity72 0.090 more closely related than 1st cousins (0.0625)

0.014 similar to 2nd cousin (~0.015625)

0.014 similar to 2nd cousin (~0.015625)

0.087 more closely related than 1st cousins (~0.0625)

Clinical findings delayed separation of umbilical cord, pneumonia, recurrent septicemia, intractable diarrhea, failure to thrive, otitis media, mastoiditis with bitemporal bone destruction, dacryocystitis,

delayed separation of umbilical cord, pneumonia, recurrent septicemia, intractable diarrhea, failure to thrive, pyogenic abscesses, septicemia

delayed separation of umbilical cord, pneumonia, recurrent septicemia, intractable diarrhea, failure to thrive, omphalitis, septic shock

delayed separation of umbilical cord, pneumonia, recurrent septicemia, intractable diarrhea, failure to thrive, otitis media, necrotizing blepharitis 1st week of life, visceral leishmaniasis, hemorrhagic necrotising fasciitis, septic shock

Pathogens (confirmed by culture)

S. aureus, P. aeruginosa (blood) Rhodococcus equi (blood) Enterococcus faecium (blood /lung) P. aeruginosa (blepharitis), Leishmania (BM aspirate, microscopy)

Hematological findings specific granule deficiency maturation arrest in BM blast excess in BM aplastic anemia, (hemoglobin pretransfusion 6.5g/dl) intermittent neutropenia (ANC <100/µl)

specific granule deficiency maturation arrest in BM blast excess in BM

specific granule deficiency maturation arrest in BM blast excess in BM post mortem BM showed accumulation of myeloid progenitors

specific granule deficiency maturation arrest in BM blast excess in BM intermittent severe neutropenia (ANC<100/µl), peripheral blasts (1-2%), thrombocytopenia (54.000/µl), anemia (7.9-15.9 g/dl) BM reticulin fibrosis, myelofibrosis, extramedullar hematopoiesis (liver)

Skeletal findings increased interdigital space D1-2 osteopenia brachydactyly D4

increased interdigital space D1-2 bulking of radius osteochondroma pes planus

normal N/D

Dermatological findings hyperextensible skin brittle, dysplastic, short nails increased callus at plantar metatarsal pads and dorsal digital phalanges (foot) hirsutism

brittle, dysplastic nails, increased callus on dorsal digital phalanges (foot), acanthosis nigricans, keratosis pilaris, hirsutism

normal N/D

Endocrine findings none premature thelarche, after chemotherapy: ovarian failure glucose interolerance managed on metformin

normal N/D

Neuropsychological development

moderate developmental delay, learning difficulties, regular schooling

global delay of 2y at the age of 9y, dyspraxia normal normal

Dental findings irregularly shaped teeth (partly conical) misalignment incomplete amelogenesis

irregular shaped teeth (partly conical) misalignment incomplete amelogenesis

N/A N/D

Outcome successful HSCT, aged 2 years successful HSCT, aged 1 year 1 month death, aged 1.5 month death, aged 5 years 4 month

N/A: not applicable, N/D: no documented, BM: bone marrow (smear) HSCT: hematopoietic stem cell transplantation



Effect of specific SMARCD2 mutations on reported transcripts (derived from ensemble.org)

Gray: reference transcript, chosen by degree of evidence (CCDS); N/A: not available; WT sequence, i.e. transcript not affected by mutation; § using nomenclature by IVS the effect of splice site mutation on various transcripts can be readily appreciated; # probably WT, duplication beyond stop codon/3´UTR.

Transcript Biotype CCDS Pedigree A Pedigree B Pedigree C

SMARCD2-001 ENST00000448276!!

NM_001098426! protein coding CCDS45756 c.1181+1G>A

IVS9+1_G>A§ c.414_438dup c.401+2T>C IVS2+2_T>C§

SMARCD2-002 ENST00000225742!!

No RefSeq protein coding N/A IVS9+1_G>A c.189_213dup IVS2+2_T>C

SMARCD2-003 ENST00000323347


SMARCD2-005 ENST00000577990

No RefSeq protein coding N/A WT WT WT

SMARCD2-006 ENST00000584400

No RefSeq nonsense mediated decay N/A WT c.229_*10dup # WT

SMARCD2-007 ENST00000580054

No RefSeq nonsense mediated decay N/A WT c.198_222dup IVS1+2_T>C

SMARCD2-010 ENST00000584483

No RefSeq retained intron N/A IVS4+1_G>A WT WT

SMARCD2-012 ENST00000450364

No RefSeq protein coding N/A IVS4+1_G>A WT WT

SMARCD2-201 ENST00000613943




Available material and diagnostics in patients

N/A: not available, PB: peripheral blood, BM: bone marrow, HSCT: hematopoietic stem cell transplantation.

All specimen were taken from non-transplanted (non-HSCT) patients, unless the opposite is stated.

AII.1 BII.1 BII.2 CII.1 Clinical history, routine hematological work-up

yes yes yes yes

viable SMARCD2-/- cells fibroblasts, fibroblast N/A EBV-transformed B cell line

Cytology 2 PB smear images 2 PB and 1 BM smear images N/A N/A

Flow cytometry Before and after HSCT (data not shown) N/A N/A N/A

Granule protein expression N/A reduced or absent40 unpublished N/A

Bone marrow histology 1 block N/A 1 block (post mortem) 4 blocks


Documents

Nature Genetics: doi:10.1038/ng filepubertas praecox vera, (j) Brachytelephalangy, longitudinal ridges on finger nails, and keratosis pilaris on forearm. (k) Bulging of right humerus