Supporting Information Targeted sequencing of ...

Supporting Information Targeted sequencing of candidate genes Each sample was barcoded during enrichment and sequenced using Illumina HiSeQ 2000 system. Sequencing reads were mapped to the human genome using Bowtie (Langmead, et al 2009). Sequence variations were called using SAMtools (Li, et al 2009) and, annotated and functionally classified by ANNOVAR (Wang, et al 2010). A minimum coverage of 30 reads and a minimum of 5% allele burden were considered for this analysis. Germline polymorphisms previously reported in 1000 genome project and ESP5400 databases were excluded. Finally, variants located in non-coding regions, as well as synonymous variants were filtered out. RNA Sequencing Libraries were sequenced on the SOLiD 5500 Wildfire system according to the manufacturer's instructions (LifeTechnologies) with a read length of 75 bp bases. The sequencing reads were mapped to the human genome hg19 using the whole transcriptome analysis pipeline in LifeScope v2.5.1 (LifeTechnologies) and applying default settings. Sequenced reads were assembled into transcripts and the ones overlapping on the genome were grouped into loci by Cufflinks (Trapnell, et al 2010) version 2.1.1. Relative abundance (fragments per kilobase of exon per million of fragments mapped, FPKM) was calculated and differential expression and exon-usage were estimated by Cufflinks. Assembled transcripts were annotated by the NCBI RNA reference sequences collection (Pruitt, et al 2007) December 21, 2010 snapshot. Taqman Low Density Array (TLDA) Any failed measurements and Ct values larger than 35 were set as 36. Threshold cycle (Ct) data for all target genes and control gene GAPDH were used to calculate ∆Ct values [∆Ct = Ct (target gene) – Ct (GAPDH)]. Then, ∆∆Ct values were calculated by subtracting the

calibrator (control) from the ∆Ct values of each target. Finally, the normalized expression ratio (fold change) of the target gene was counted according to the formula 2-∆∆Ct. Genes differentially expressed between patients and healthy controls were determined by Student ttest with a level of significance PA p.G740E

2

RCMD-RS

F

77

89

105

No

46.XX

c.2098 A>G p.K700E

3

RCMD-RS

F

74

93

95

No

46.XX

c.1986 C>A p.H662Q

4

RCMD-RS

M

91

94

101

Yes

46.XY

c.1866 G>C p.E622D

5

RCMD-RS

M

75

108

112

No

46.XY

c.2098 A>G p.K700E

6

RARS

F

74

109

106

No

46.XX

c.1997 A>T p.K666M

7

RARS-T

M

66

92

94

Yes

46.XY

8

RCMD-RS

M

68

132

102

No

9

RCMD-RS

M

73

88

97

10

RARS

M

82

115

11

RARS

M

83

12

RARS

F

13

RARS

14

Sex Age

Hb MCV Transfusion (g/L) (fL)

Karyotype

SF3B1 mutation

Amino acid change

Patient n.

Other recurrent mutations and amino acid change

% % Erythroid RS BM cells 63.5

32

68

24

17.5

33

6

70

47

32

30

21

c.1866 G>C p.E622D

none ATRX (p.L1592H); TET2 (p.F125fs) IDH1 (p.A341S); TET2 (p.H1881Y) NA ABCA12 (p.M2189V); TET2 (p.L1322Q); JAK2 (p.V617F) NRD1 (p.P959S); TET2 (p.A827T) JAK2 (p.V617F)

44

16

46.XY

c.1876 A>G p.N626D

none

49

42

Yes

46.XY

c.2098 A>G p.K700E

none

27

26

106

No

46.XY

c.1997 A>G p.K666R

none

32

45

99

100

No

46.XY

c.1866 G>C p.E622D

CDKN2B (p.A19D)

20

52

74

101

95

No

46.XX

c.1997 A>G p.K666R

none

42.5

53

F

77

118

99

No

46.XX

c.2098 A>G p.K700E

47

33

RARS

M

49

122

100

No

46.XY

c.1988 C>T

53

70

20

RCMD-RS

M

85

112

100

No

46,XY

c.1997 A>G p.K666R

DNM3TA (p.V296M) CEBPA (p.P197Q); TET2 (p.P319fs) none

26

40

21

RCMD-RS

F

63

110

113

No

46,XX

c.2098 A>G p.K700E

IDH2 (R140Q)

30

42

22

RARS

F

65

94

99

No

47,XX,+mar[20]

c.2098 A>G p.K700E

NA

20

57

23

RARS

M

76

121

108

No

TET2 (Q790X)

43

30

24

RCMD-RS

M

73

102

97

No

46.XY

wild type

-

SRSF2 (p.95_103del)

36

70

25

RCMD-RS

M

84

110

79

No

46.XY

wild type

-

none

48

20

p.T663I

44-45,XY, del(13)(q12?q13?) c.1986 C>A p.H662Q

  Twenty RARS, RCMD-RS, RARS-T PATIENTS were selected for this study. Median age was 74 years (49-91 years). Table displays haematologivcal parameters (Haemoglobin (Hb), Mean Corpuscolar Volume (MCV)), transfusion dependency and genetic profile of each patient. All samples were SF3B1 mutated and several also carried additional mutations detected by targeted screenings. Two samples (MDS#24 and MDS#25) were SF3B1 wild type. Transfusion dependency is prior sampling. None: no mutations was found. NA: not analyzed.

Supplementary Table 4. Gene annotation cluster of the 710 genes that failed to up-regulate in RARS

Supplementary Table 5. Gene annotation cluster of the 222 genes that failed to down-regualte in RARS Annotation Cluster 1

Enrichment Score: 3.182

Category

Term

SP_PIR_KEYWORDS INTERPRO SMART UP_SEQ_FEATURE

sh3 domain IPR001452:Src homology-3 domain SM00326:SH3 domain:SH3

Annotation Cluster 2

Enrichment Score: 2.266

Category

Term

GOTERM_MF_FAT GOTERM_MF_FAT GOTERM_BP_FAT GOTERM_BP_FAT

GO:0030695~GTPase regulator activity GO:0060589~nucleoside-triphosphatase regulator activity GO:0051056~regulation of small GTPase mediated signal transduction GO:0046578~regulation of Ras protein signal transduction IPR001331:Guanine-nucleotide dissociation stimulator, CDC24, conserved site GO:0035023~regulation of Rho protein signal transduction IPR000219:Dbl homology (DH) domain GO:0005085~guanyl-nucleotide exchange factor activity GO:0005089~Rho guanyl-nucleotide exchange factor activity SM00325:RhoGEF GO:0005083~small GTPase regulator activity GO:0005088~Ras guanyl-nucleotide exchange factor activity GO:0008047~enzyme activator activity domain:DH GO:0005096~GTPase activator activity guanine-nucleotide releasing factor GO:0007265~Ras protein signal transduction GO:0007266~Rho protein signal transduction IPR001849:Pleckstrin homology GTPase activation

INTERPRO GOTERM_BP_FAT INTERPRO GOTERM_MF_FAT GOTERM_MF_FAT SMART GOTERM_MF_FAT GOTERM_MF_FAT GOTERM_MF_FAT UP_SEQ_FEATURE GOTERM_MF_FAT SP_PIR_KEYWORDS GOTERM_BP_FAT GOTERM_BP_FAT INTERPRO SP_PIR_KEYWORDS

Count

%

P Value

Fold Enrichment

FDR

11 10 10 8

5.069 4.608 4.608 3.687

0.000 0.001 0.002 0.003

4.988 4.297 3.612 4.277

0.098 0.768 1.928 4.168

Count

%

P Value

Fold Enrichment

FDR

14 14 11 10

6.452 6.452 5.069 4.608

0.000 0.000 0.000 0.000

3.358 3.284 3.990 4.353

0.345 0.427 0.687 0.767

6 7 6 8 6 6 10 6 11 5 8 6 5 3 7 5

2.765 3.226 2.765 3.687 2.765 2.765 4.608 2.765 5.069 2.304 3.687 2.765 2.304 1.382 3.226 2.304

0.001 0.001 0.001 0.001 0.001 0.002 0.002 0.002 0.002 0.005 0.007 0.009 0.028 0.064 0.082 0.086

8.717 6.463 7.959 5.099 7.856 6.690 3.536 6.606 3.181 7.061 3.523 4.722 4.353 7.216 2.313 2.994

0.854 1.163 1.293 1.280 1.284 2.179 2.705 2.767 3.108 8.130 9.657 11.028 36.170 65.473 70.328 69.112

UP_SEQ_FEATURE GOTERM_BP_FAT SMART INTERPRO

domain:PH GO:0008624~induction of apoptosis by extracellular signals SM00233:PH IPR011993:Pleckstrin homology-type

Annotation Cluster 3

Enrichment Score: 1.734

Category

Term

SP_PIR_KEYWORDS INTERPRO SMART KEGG_PATHWAY GOTERM_MF_FAT UP_SEQ_FEATURE INTERPRO GOTERM_MF_FAT SP_PIR_KEYWORDS INTERPRO SMART

SH2 domain IPR000980:SH2 motif SM00252:SH2 hsa05340:Primary immunodeficiency GO:0004715~non-membrane spanning protein tyrosine kinase activity domain:SH2 IPR008266:Tyrosine protein kinase, active site GO:0004713~protein tyrosine kinase activity tyrosine-protein kinase IPR001245:Tyrosine protein kinase SM00219:TyrKc

Annotation Cluster 4

Enrichment Score: 1.704

Category

Term

INTERPRO SP_PIR_KEYWORDS GOTERM_MF_FAT SP_PIR_KEYWORDS SP_PIR_KEYWORDS UP_SEQ_FEATURE INTERPRO GOTERM_MF_FAT UP_SEQ_FEATURE GOTERM_BP_FAT

IPR000719:Protein kinase, core phosphotransferase GO:0004672~protein kinase activity kinase serine/threonine-specific protein kinase domain:Protein kinase IPR017441:Protein kinase, ATP binding site GO:0004674~protein serine/threonine kinase activity binding site:ATP GO:0006468~protein amino acid phosphorylation

6 4 7 5

2.765 1.843 3.226 2.304

0.104 0.123 0.147 0.418

2.395 3.264 1.944 1.510

81.993 87.807 84.223 99.954

Coun t

%

P Value

Fold Enrichment

FDR

7 7 7 4 4 5 4 5 4 4 4

3.226 3.226 3.226 1.843 1.843 2.304 1.843 2.304 1.843 1.843 1.843

0.001 0.001 0.003 0.005 0.008 0.019 0.089 0.091 0.110 0.137 0.195

6.005 5.772 4.852 11.176 9.452 4.928 3.775 2.918 3.431 3.103 2.608

1.435 1.877 3.464 5.353 10.801 25.380 73.301 72.406 78.452 87.677 91.859

Coun t

%

PValue

Fold Enrichment

FDR

15 9 16 17 5 13 13 12 14 16

6.912 4.147 7.373 7.834 2.304 5.991 5.991 5.530 6.452 7.373

0.001 0.001 0.001 0.002 0.004 0.004 0.004 0.005 0.005 0.006

2.884 4.264 2.558 2.353 7.935 2.623 2.615 2.704 2.444 2.193

1.011 1.659 1.809 3.126 4.586 6.261 5.736 6.120 7.451 9.204

SP_PIR_KEYWORDS INTERPRO GOTERM_BP_FAT GOTERM_BP_FAT INTERPRO SMART SP_PIR_KEYWORDS UP_SEQ_FEATURE KEGG_PATHWAY UP_SEQ_FEATURE GOTERM_BP_FAT SP_PIR_KEYWORDS INTERPRO GOTERM_MF_FAT GOTERM_MF_FAT GOTERM_MF_FAT GOTERM_MF_FAT GOTERM_MF_FAT GOTERM_MF_FAT GOTERM_MF_FAT GOTERM_MF_FAT SP_PIR_KEYWORDS UP_SEQ_FEATURE GOTERM_MF_FAT INTERPRO SP_PIR_KEYWORDS SMART

serine/threonine-protein kinase IPR002290:Serine/threonine protein kinase GO:0006793~phosphorus metabolic process GO:0006796~phosphate metabolic process IPR017442:Serine/threonine protein kinase-related SM00220:S_TKc transferase active site:Proton acceptor hsa04270:Vascular smooth muscle contraction nucleotide phosphate-binding region:ATP GO:0016310~phosphorylation ATP IPR008271:Serine/threonine protein kinase, active site GO:0030554~adenyl nucleotide binding GO:0017076~purine nucleotide binding GO:0001883~purine nucleoside binding GO:0001882~nucleoside binding GO:0032555~purine ribonucleotide binding GO:0032553~ribonucleotide binding GO:0032559~adenyl ribonucleotide binding GO:0000166~nucleotide binding nucleotide-binding domain:AGC-kinase C-terminal GO:0005524~ATP binding IPR000961:AGC-kinase, C-terminal atp-binding SM00133:S_TK_X

Annotation Cluster 5

Enrichment Score: 1.602

Category

Term

KEGG_PATHWAY GOTERM_BP_FAT

hsa04660:T cell receptor signaling pathway GO:0050671~positive regulation of lymphocyte proliferation

11 9 20 20 10 9 24 14 5 18 16 7 9 24 28 24 24 26 26 22 30 24 3 21 3 19 3

5.069 4.147 9.217 9.217 4.608 4.147 11.060 6.452 2.304 8.295 7.373 3.226 4.147 11.060 12.903 11.060 11.060 11.982 11.982 10.138 13.825 11.060 1.382 9.677 1.382 8.756 1.382

0.007 0.008 0.009 0.009 0.017 0.019 0.020 0.022 0.025 0.026 0.027 0.038 0.040 0.050 0.051 0.058 0.062 0.081 0.081 0.083 0.105 0.113 0.114 0.117 0.120 0.154 0.158

2.749 3.181 1.879 1.879 2.550 2.674 1.639 2.013 4.366 1.770 1.828 2.824 2.327 1.475 1.414 1.452 1.443 1.372 1.372 1.424 1.295 1.355 5.161 1.378 4.993 1.364 4.197

8.731 10.188 13.708 13.708 21.275 19.501 23.093 29.454 24.585 33.190 36.033 39.801 43.772 50.126 50.323 55.220 57.549 67.987 67.987 69.165 77.494 79.226 84.798 81.473 83.713 88.786 86.327

Coun t

%

P Value

Fold Enrichment

FDR

7 5

3.226 2.304

0.001 0.003

6.338 8.310

0.729 4.675

GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT KEGG_PATHWAY GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT GOTERM_BP_FAT  

GO:0032946~positive regulation of mononuclear cell proliferation GO:0070665~positive regulation of leukocyte proliferation GO:0051251~positive regulation of lymphocyte activation GO:0051249~regulation of lymphocyte activation GO:0002696~positive regulation of leukocyte activation GO:0050867~positive regulation of cell activation GO:0042102~positive regulation of T cell proliferation GO:0050863~regulation of T cell activation GO:0050870~positive regulation of T cell activation GO:0002694~regulation of leukocyte activation GO:0050865~regulation of cell activation GO:0050670~regulation of lymphocyte proliferation GO:0032944~regulation of mononuclear cell proliferation GO:0070663~regulation of leukocyte proliferation GO:0008284~positive regulation of cell proliferation GO:0042129~regulation of T cell proliferation GO:0002684~positive regulation of immune system process hsa04672:Intestinal immune network for IgA production GO:0042127~regulation of cell proliferation GO:0042110~T cell activation GO:0046649~lymphocyte activation GO:0001775~cell activation GO:0010647~positive regulation of cell communication GO:0045321~leukocyte activation GO:0009967~positive regulation of signal transduction GO:0006955~immune response

5 5 6 7 6 6 4 6 5 7 7 5 5 5 11 4 7 3 14 4 4 5 5 4 4 7

2.304 2.304 2.765 3.226 2.765 2.765 1.843 2.765 2.304 3.226 3.226 2.304 2.304 2.304 5.069 1.843 3.226 1.382 6.452 1.843 1.843 2.304 2.304 1.843 1.843 3.226

0.003 0.003 0.004 0.006 0.006 0.007 0.009 0.009 0.009 0.010 0.012 0.013 0.013 0.013 0.015 0.030 0.046 0.086 0.087 0.158 0.368 0.380 0.482 0.491 0.625 0.767

8.161 8.161 5.654 4.323 5.174 4.941 9.375 4.687 6.014 3.854 3.656 5.506 5.441 5.441 2.429 5.897 2.688 5.987 1.626 2.902 1.837 1.592 1.389 1.511 1.239 0.927

4.985 4.985 6.390 8.589 9.171 11.007 13.022 13.491 14.074 14.287 17.859 18.564 19.257 19.257 21.531 38.583 52.765 63.040 76.709 93.667 99.938 99.954 99.997 99.998 100.000 100.000

Supplementary Table 6. Top 20 up and down-regualted genes between RARS and NBM at day 4 a. Up-regulated genes Locus

FPKM NBM

*VCAM1  

chr1:101185271-­‐101204771  

0.058  

5.245  

6.493  

0.004  

Integrin  binding  and  cell  adhesion  molecule  binding  

*CD5L  

chr1:157800703-­‐157811634  

0.070  

3.350  

5.578  

0.004  

Scavenger  receptor  activity,  possible  role  as  an  inhibitor  of   apoptosis  

DES  

chr2:220283098-­‐220291461  

0.480  

9.118  

4.248  

0.004  

Cytoskeletal  protein  binding  and  constituent  of  cytoskeleton  

PLEK2  

chr14:67853682-­‐67878868  

0.305  

5.392  

4.142  

0.004  

Phospholipid  binding  and  cytoskeletal  arrangement  

NR4A1  

chr12:52445190-­‐52453497  

0.700  

8.382  

3.582  

0.004  

Nuclear  transcription  factor,  its  translocation  to  mitochondria   induces  apoptosis  

CXCL12  

chr10:44865604-­‐44880545  

0.243  

2.862  

3.559  

0.004  

Chemokine  activity  and  receptor  binding  

CD1A  

chr1:158223926-­‐158228058  

0.132  

1.542  

3.551  

0.004  

Antigen-­‐presenting  protein,  lipid  and  glycolipid  antigens  binding  

NCRNA00200   chr10:1205707-­‐1214628  

0.345  

3.915  

3.506  

0.004  

SLC25A21  

chr14:37547735-­‐37548227  

0.499  

5.538  

3.472  

0.004  

*HBM  

chr16:215972-­‐216767  

0.734  

6.944  

3.242  

0.007  

Hemoglobin µ,    iron  ion  binding  and  oxygen  binding  

GZMB  

chr14:25100077-­‐25103527  

0.851  

7.946  

3.222  

0.004  

Recognize,  bind  and  lysis  of  specific  target  cells  

DLK1  

chr14:101193201-­‐101201467  

0.276  

2.575  

3.220  

0.004  

Neuroendocrine  differentiation    

NEO1  

chr15:73344824-­‐73597621  

0.268  

2.269  

3.080  

0.004  

Member  of  Ig  superfamily,    cell  growth  and  differentiation  and     cell-­‐cell  adhesion  

GALNT5  

chr2:158114037-­‐158171356  

1.161  

9.500  

3.033  

0.004  

Oligosaccharide  biosynthesis  

ANKRD57  

chr2:110371910-­‐110376564  

0.141  

1.117  

2.986  

0.004  

Unknown  function  

Gene

FPKM MDS log2(MDS/NBM)

FDR

Function

Long  intergenic  non-­‐protein  coding  RNA  200,  affiliated    with  the   lncRNA  class   Transport  of  oxodicarboxylates  across  the  inner  membranes  of   mitochondria  

SNORD114-­‐14   chr14:101438439-­‐101438513  

178.663  

1342.060  

2.909  

0.009  

Small  Nucleolar  RNA,  C/D  box  114-­‐14,  function  unknown  

AKR1C1  

chr10:5005453-­‐5020158  

0.299  

2.206  

2.881  

0.004  

Oxidoreductase  activity,  conversion  of  progesteron  into  its  active   form  

SLCO2B1  

chr11:74861909-­‐74917502  

0.329  

2.387  

2.861  

0.004  

Mediator  of    Na(+)-­‐independent  transport  of  organic  anions  

CD163  

chr12:7623409-­‐7656441  

0.350  

2.534  

2.858  

0.004  

MEG3  

chr14:101292444-­‐101327379  

2.303  

16.415  

2.833  

0.004  

Scavenger  receptor,  clearance  and  endocytosis  of   hemoglobin/haptoglobin  complexes   RNA  gene  affiliated  with  the  lncRNA  class,  inhibitor  of  tumor   proliferation  in  vitro  

b. Down-regulated genes *ULK4P3  

chr15:32698800-­‐32727250  

1.769  

0.219  

-­‐3.015  

0.028  

ULK4  pseudogen  3,  unknown  function  

C10orf82  

chr10:118422741-­‐118429509  

1.691  

0.225  

-­‐2.907  

0.004  

Chromosome  10  Open  Reading  Frame  82,  unknown  function  

FCRL1  

chr1:157764022-­‐157789940  

2.889  

0.477  

-­‐2.599  

0.004  

B-­‐cells  activation  and  differentiation  

EPX  

chr17:56270087-­‐56282678  

408.417  

69.545  

-­‐2.554  

0.004  

Peroxidase  activity  and  heme  binding  

KRT81  

chr12:52679696-­‐52685299  

4.766  

0.920  

-­‐2.374  

0.004  

CD96  

chr3:111260925-­‐111371206  

5.725  

1.151  

-­‐2.315  

0.004  

RNASE2  

chr14:21423629-­‐21424594  

246.053  

51.195  

-­‐2.265  

0.004  

Ribonuclease  activity  and  nucleic  acid  binding  

*RNF220  

chr1:45019114-­‐45019165  

4573.930  

1017.600  

-­‐2.168  

0.049  

Ubiquitination  and  proteasomal  degradation  of  SIN3B  

CIRH1A  

chr16:69203324-­‐69204431  

1.160  

0.274  

-­‐2.082  

0.034  

Positive  regulator  of  HIVEP1  virus  type  I  enhancer  binding   protein  1  

LUM  

chr12:91497187-­‐91505542  

4.815  

1.235  

-­‐1.963  

0.004  

Extracellular  matrix  structural  constituent  and  collagen  binding.  

HRH4  

chr18:22040592-­‐22060044  

2.689  

0.711  

-­‐1.919  

0.009  

Histamine  receptor  activity  predominantly  expressed  in   haematopoietic  cells  

CD79A  

chr19:42381189-­‐42385439  

2.239  

0.598  

-­‐1.904  

0.018  

B  lymphocyte  antigen  receptor      

Keratin  81,  structural  molecule  activity,  formation  of  hairs  and   nails   Adhesive  interaction  of    T  and  NK  cells  during  late  immune   response  

DNTT  

chr10:98064084-­‐98098321  

4.629  

1.268  

-­‐1.868  

0.004  

DNA  nucleotidylexotransferase  activity  and  DNA-­‐directed  DNA   polymerase  activity  

FAM18B2  

chr17:15423233-­‐15424159  

1.358  

0.374  

-­‐1.860  

0.046  

Affiliated  with  the  lncRNA  class,  unknown  function  

C16orf74  

chr16:85740013-­‐85740841  

1.482  

0.414  

-­‐1.840  

0.037  

Unknown  function  

MAP3K9  

chr14:71191316-­‐71275921  

1.343  

0.386  

-­‐1.800  

0.019  

LOC339524  

chr1:87595447-­‐87634886  

4.301  

1.256  

-­‐1.776  

0.018  

QPRT  

chr16:29698063-­‐29699828  

3.792  

1.109  

-­‐1.774  

0.004  

Key  enzyme  in  catabolism  of  quinolinate  

CDK15  

chr2:202671101-­‐202760128  

2.972  

0.883  

-­‐1.751  

0.012  

Serine/threonine-­‐protein  kinase  involved  in  the  control  of  the   cell  cycle  

PRG2  

chr11:57154151-­‐57158444  

1211.500  

362.554  

-­‐1.741  

0.004  

Cytotoxin  involved  in  antiparasitic  defense  mechanisms    

Serine/threonine  kinase  of  the  MAP  kinase  signal  transduction   pathway   RNA  gene,  and  is  affiliated  with  the  lncRNA  class,  Biological   oxidations  

  The up-regulation of CXCL12 was validated by TLDA. (*) Genes commonly deregulated both day 0 and day 4.

Supplementary Table 7. Mis-spliced genes in RARS versus NBM at day 0 of the erythoid culture Gene symbol TFCP2 TFCP2 TFCP2 MBIP MBIP MBIP MBIP MBIP MBIP MBIP EIF2B4 EIF2B4 TGIF2 TGIF2 TGIF2 TGIF2 CYP2S1 CYP2S1 CYP2S1 PROCR PROCR DUT DUT MIAT MIAT MIAT MIAT ZNF518B ZNF518B ZNF518B ZNF518B ZNF518B C21orf59 C21orf59 C21orf59 C21orf59 TMEM14C TMEM14C AMZ2 AMZ2 AMZ2 AMZ2 AMZ2 AMZ2 NUDT7 NUDT7 NUDT7 FLJ27354 FLJ27354 FLJ27354 FLJ27354 TMEM91 TMEM91 TMEM91 TMEM91 TMEM91 TMEM91 LOC402778 LOC402778 LOC402778 ACBD3 ACBD3 ACBD3 ACBD3 ACBD3 ACBD3 ACBD3 SNX3 SNX3

Gene name transcription factor CP2 transcription factor CP2 transcription factor CP2 MAP3K12 binding inhibitory protein 1 MAP3K12 binding inhibitory protein 1 MAP3K12 binding inhibitory protein 1 MAP3K12 binding inhibitory protein 1 MAP3K12 binding inhibitory protein 1 MAP3K12 binding inhibitory protein 1 MAP3K12 binding inhibitory protein 1 eukaryotic translation initiation factor 2B, subunit 4 delta eukaryotic translation initiation factor 2B, subunit 4 delta TGFB-induced factor homeobox 2 TGFB-induced factor homeobox 2 TGFB-induced factor homeobox 2 TGFB-induced factor homeobox 2 cytochrome P450, family 2, subfamily S, polypeptide 1 cytochrome P450, family 2, subfamily S, polypeptide 1 cytochrome P450, family 2, subfamily S, polypeptide 1 protein C receptor, endothelial (EPCR) protein C receptor, endothelial (EPCR) deoxyuridine triphosphatase deoxyuridine triphosphatase myocardial infarction associated transcript (non-protein coding) myocardial infarction associated transcript (non-protein coding) myocardial infarction associated transcript (non-protein coding) myocardial infarction associated transcript (non-protein coding) zinc finger protein 518B zinc finger protein 518B zinc finger protein 518B zinc finger protein 518B zinc finger protein 518B chromosome 21 open reading frame 59 chromosome 21 open reading frame 59 chromosome 21 open reading frame 59 chromosome 21 open reading frame 59 transmembrane protein 14C transmembrane protein 14C archaelysin family metallopeptidase 2 archaelysin family metallopeptidase 2 archaelysin family metallopeptidase 2 archaelysin family metallopeptidase 2 archaelysin family metallopeptidase 2 archaelysin family metallopeptidase 2 nudix (nucleoside diphosphate linked moiety X)-type motif 7 nudix (nucleoside diphosphate linked moiety X)-type motif 7 nudix (nucleoside diphosphate linked moiety X)-type motif 7 hypothetical gene supported by AK130864 hypothetical gene supported by AK130864 hypothetical gene supported by AK130864 hypothetical gene supported by AK130864 transmembrane protein 91 transmembrane protein 91 transmembrane protein 91 transmembrane protein 91 transmembrane protein 91 transmembrane protein 91 IFITM10 interferon induced transmembrane protein 10 IFITM10 interferon induced transmembrane protein 10 IFITM10 interferon induced transmembrane protein 10 acyl-Coenzyme A binding domain containing 3 acyl-Coenzyme A binding domain containing 3 acyl-Coenzyme A binding domain containing 3 acyl-Coenzyme A binding domain containing 3 acyl-Coenzyme A binding domain containing 3 acyl-Coenzyme A binding domain containing 3 acyl-Coenzyme A binding domain containing 3 sorting nexin 3 sorting nexin 3

Transcript ID TCONS_00112738 TCONS_00112739 TCONS_00112740 TCONS_00148099 TCONS_00148100 TCONS_00148101 TCONS_00148112 TCONS_00148113 TCONS_00148114 TCONS_00148120 TCONS_00263359 TCONS_00263360 TCONS_00287582 TCONS_00287583 TCONS_00287584 TCONS_00287585 TCONS_00227180 TCONS_00227181 TCONS_00227182 TCONS_00287346 TCONS_00287347 TCONS_00157905 TCONS_00157906 TCONS_00303849 TCONS_00303850 TCONS_00303851 TCONS_00303852 TCONS_00363607 TCONS_00363608 TCONS_00363609 TCONS_00363610 TCONS_00363611 TCONS_00300747 TCONS_00300748 TCONS_00300749 TCONS_00300750 TCONS_00409015 TCONS_00409016 TCONS_00196570 TCONS_00196571 TCONS_00196572 TCONS_00196573 TCONS_00196574 TCONS_00196575 TCONS_00179605 TCONS_00179606 TCONS_00179607 TCONS_00033035 TCONS_00033036 TCONS_00033037 TCONS_00033038 TCONS_00227198 TCONS_00227210 TCONS_00227211 TCONS_00227212 TCONS_00227213 TCONS_00227214 TCONS_00082892 TCONS_00082893 TCONS_00082894 TCONS_00042991 TCONS_00042992 TCONS_00042993 TCONS_00042994 TCONS_00042995 TCONS_00043000 TCONS_00043022 TCONS_00435172 TCONS_00435173

FPKM NBM 5.769 0.498 0.075 1.089 1.466 0.047 1.239 0.227 0.048 1.556 6.944 0.047 0.001 0.699 7.686 3.136 0.873 0.005 0.378 1.081 0.000 1.014 20.058 0.045 0.525 1.011 0.072 0.553 0.414 0.245 0.006 2.306 7.448 2.031 3.732 3.950 5.218 18.005 0.152 0.000 0.120 1.633 0.000 1.765 1.311 2.133 1.073 0.563 0.098 1.254 0.005 8.470 0.850 3.875 0.000 0.146 4.786 0.066 0.453 0.725 0.059 0.004 0.966 0.948 0.959 1.086 1.644 21.008 8.391

FPKM RARS log2 (RARS/NBM) 2.334 0.142 8.235 2.469 0.013 2.772 2.204 0.059 0.470 2.342 1.459 9.152 2.514 0.601 8.227 2.120 0.731 1.089 0.086 0.657 0.657 12.303 18.221 0.803 1.372 0.243 0.153 0.606 0.969 0.020 2.784 2.250 11.109 3.013 0.005 10.118 61.997 12.151 0.006 0.000 0.342 4.665 2.701 0.668 0.000 1.258 3.561 0.849 0.085 0.416 1.453 7.509 0.425 0.005 3.432 1.986 4.737 1.825 0.000 0.000 0.093 0.947 0.001 2.056 4.467 1.319 1.824 68.781 0.186

-1.305 -1.809 6.770 1.180 -6.804 5.894 0.831 -1.953 3.288 0.590 -2.251 7.602 11.926 -0.219 0.098 -0.565 -0.255 7.820 -2.133 -0.719 12.459 3.601 -0.139 4.169 1.386 -2.056 1.078 0.131 1.228 -3.610 8.744 -0.036 0.577 0.569 -9.548 1.357 3.571 -0.567 -4.714 0.000 1.504 1.514 14.547 -1.401 -14.227 -0.762 1.731 0.594 -0.190 -1.593 8.134 -0.174 -1.000 -9.709 0.000 3.763 -0.015 4.779 -14.567 -13.180 0.662 7.929 -9.938 1.117 2.220 0.281 0.150 1.711 -5.496

FDR

0.024

0.024

0.024

0.016

0.016 0.016 0.040

0.016

0.016

0.016

0.016

0.040

0.040

0.016

0.040

0.016

0.016

0.040

 

Relative abundance (fragments per kilobase of exon per million of fragments mapped FPKM), differential expression, exon-usage and FDR were determined by Cufflinks. Fold change is expressed as log2 of the ratio between RARS and NBM. At day 4 of erythroid culture only 2 genes, ANKHD1 and TMEM14C, showed altered splicing. TMEM14C was differentially spliced at both time points.  

    Supplementary Table 8. Primers designed to validate splice isoforms by qPCR

  Gene TMEM14C TFCP2

 

Isoform

Forward (5'-->3')

Reverse (5'-->3')

TCONS_00409015 CGTGAGACCGCTTGTTTTCT

ATGATCCCACCAGAAGCAAC

TCONS_00409016 TTCGTGAGACCGGTGCAG

ATGATCCCACCAGAAGCAAC

Exon 6

CACCTTTGGGCTTGAAAACT

AGAGGACATCTGTGTTTATTCAGG

Supplementary Table 9. Differentially expressed genes between RARS and NBM by TLDA

  Day 0 Gene P-value ABCB7 ALAS2 APOE AXL BAT2L2 CCL18 CD5L CHRNA6 CXCL12 EIF2C2 EPX ETNK1 HBB HIATL1 HPRT1 HS3ST2 ITFG1 KIAA0406 LYVE1 MFN2 MSL1 MSR1 NSMCE4A RNASE1 RTF1 SEPP1 TIMD4 TM2D1 TRNAU1AP

0.0026 p