Document 1. Supplemental methods, tables, and figures - Blood

Svensson/Hult et al.

3

Supplemental Method 1

Glycolipid preparation and thin layer chromatography-enzyme immune assay (TLC-EIA)

The lysed blood units were thawed and total neutral glycolipids were isolated with a

system of solvents and silica/ion exchange chromatography as previously reported.1,2

Glycolipids from A1/A2/B/O individuals were used as controls. Fs glycolipids from

canine epithelial cells, and para-Forssman (p-Fs) from human RBCs were used as

immunoassay references. p-Fs from human RBCs and Fs from chicken RBCs were

used as structural references. These antigens are similar in structure and may cross-

react with anti-A reagents. Excluding these compounds is critical to unambiguous

identification of Fs.

Total neutral glycolipids were extracted from RBC lysates resulting in 200 and 160

mg of glycolipids from Apae#1 and Apae#2, respectively. The total glycolipids from

each of Apae#1 and #2 were fractionated by silica chromatography column in a

system of chloroform (C) methanol (M) solvent mixes, starting with the least polar (50

mL fractions). The first solvent CM 9:1 was followed by CM 88:12; CM 85:15; CM

82:18; CM 75:25; CM 65:35; CM 60:40; CM 25:75; M; and finally CM 40:40 mix with

12 parts of water (CMW 40:40:12) v/v.

The TLC method used is based on that of Schnaar et al.3 and reported to be

sensitive for the characterization of weak A subgroups.2 Lane loading concentrations

depended on sample homogeneity with 20 µg of unfractionated total neutral

glycolipids, or 10 µg of semi-fractionated open-column fractions, or 0.1 to 1 µg of high

purity glycolipid fractions (e.g. Fs and p-Fs reference glycolipids) loaded per lane.

Glycolipids were eluted on the TLC plates in solvent mix of chloroform-methanol-

water 60:35:8 v/v, air-dried and then plasticized. In brief, the TLC plate was then

reacted with a primary antibody (Table S1) followed by a species-appropriate alkaline

phosphate conjugated anti-immunoglobulin; goat anti-rat IgM (SAB-211I; Nordic

Biosite, Sweden) or; goat anti-human polyvalent immunoglobulins (A3313, Sigma) or;

goat anti-mouse immunoglobulins (A0162, Sigma). Alternatively, biotin-labeled lectin

Helix pomatia (diluted at 1:3000) was followed by an alkaline phosphate conjugated

Streptavidin (S2890; Sigma). Reactions were visualized with NBT/BCIP chromogenic

substrate (B5655; Sigma).


4


Genetic testing

ABO genotyping. DNA was extracted from the buffy coat with a modified salting-out

procedure.4 Genomic typing of the ABO locus was performed with the established

and validated methods used at the Nordic Reference Laboratory for Genomic Blood

Group Typing in Lund, Sweden, including PCR-RFLP5 and PCR-ASP.6

Sequencing of GBGT1. Oligonucleotide primers used in this study were designed

using the human GBGT1 sequence ENST00000372040 in Ensembl and synthesized

by Invitrogen (Invitrogen, Carlsbad, CA). PCR was performed with primer pairs as

described in Table S2. Amplification was performed in a reaction volume of 20 µL

with 0.5 µmol/L of each primer, 2 nmol of each dNTP, and 100 ng of genomic DNA.

The thermostable enzyme used was 0.5U from the Expand High Fidelity PCR

System in the supplied buffer 2 with a final Mg2+ concentration of 1.5 mM according to

the manufacturer (Roche Molecular Systems, Pleasanton, CA). After an initial

denaturation step at 95°C for 2 min was followed by 35 cycles of denaturation (95°C

for 20 s), annealing (60°C for 35 s) and extension (72°C for 1 min for exons 1-6 and 2

min for exon 7) and a final extension at 72°C for 2 min. All amplification products

were separated by high-voltage electrophoresis on 3% agarose gels (Seakem, FMC

Bioproducts, Rockland, ME) stained with ethidium bromide (0.56 mg/L gel, Sigma

Chemicals, St. Louis, MO). Products were purified using the Qiaquick gel extraction

kit (Qiagen GmbH, Hilden, Germany), sequenced with the BigDye terminator kit v1.1

(Applied Biosystems, Foster City, CA) and analysed on a 3130 Avant/Genetic

analyser (Applied Biosystems). For cis/trans assignment PCR fragments were cloned

in a TOPO TA Cloning vector (Invitrogen) according to the manufacturer’s

instructions and subsequently sequenced with M13 primers included in the kit.

RNA, cDNA preparation and transcript analysis. Buffy coats from the two Apae

individuals were frozen in Trizol® (Invitrogen) immediately upon receipt. Five in-house

control samples were frozen in parallel. Bone marrow from an apparently healthy

donor was obtained following informed consent. CD34-positive cells from primary

bone marrow were enriched and cultured towards erythroid development as

previously described7. RNA was extracted from the thawed Trizol aliquots according

to the manufacturer’s instructions. cDNA was synthesized using the High Capacity


5

RNA-to-cDNA kit (Applied Biosystems) and the GeneAmp® PCR System 2700

(Applied Biosystems).

Real-time quantitative PCR was performed on 3 µL of cDNA with TaqMan probes

and a sequence detection system (Model 7500, Applied Biosystems), according to

the manufacturer's instructions. Exon boundary 2 to 3 was detected with a TaqMan

gene expression assay (Hs 01063930_m1, Applied Biosystems). All samples were

run in triplicate. Transcript target quantities were normalized to Beta Actin (Assay

Hs99999903_m1, Applied Biosystems). For analysis the sample with the lowest cycle

threshold (CT) value was used as calibrator. We considered as positive the results

from any sample with at least two detected (CT < 40) values within the triplicate.

Detailed information on PCR-ASP settings. Five pmol of each primer (1; i6:838F -

ex:7:887A-R, 2;ex:7:363A-F - ex7:1139R) and 0.5 pmol each of the internal positive

control primers (JK-F3-L and JK-R3-L) was used in each reaction. For primer details

see Table S2. The primers were mixed with 100 ng of genomic DNA, 2 nmol of each

dNTP, 0.5 U of DNA polymerase (AmpliTaq Gold, Perkin Elmer/Roche Molecular

Systems, Branchburg, NJ), glycerol, and cresol red at final concentrations of 5 and

0.01 percent, respectively, in the buffer supplied. The final reaction volume was 10

µL. Thermocycling was performed with a PCR system (GeneAmp 2700, Perkin Elmer

Cetus, Norwalk, CT). For PCR-ASP, the following cycling conditions were used: Initial

denaturation at 96°C for 7 minutes was followed by 32 cycles at 94°C for 30 seconds,

65°C (1) or 63°C (2) for 30 seconds, 72°C for 1 minute and 30 seconds, and a final

extension 72°C for 2 minutes. PCR products were separated electrophoretically on 2

to 3% agarose gels (Seakem, FMC Bioproducts, Rockland, ME) stained with

ethidium bromide (0.56 mg/L gel, Sigma Chemical Co., St. Louis, MO) after high-

voltage electrophoresis at 150 to 160 V and visualized on a UV light board (Multi

Image Light cabinet, software Alpha View version 3.0.3.0, Alpha Innotech, Cell

Biosciences, Santa Clara, CA).


6


Flow cytometry

RBCs and transfected cell line MEG-01 were tested by flow cytometry. The cells

were washed three times in phosphate-buffered saline (PBS), and 10 µL packed and

washed RBCs were suspended in 400 µL PBS. A 96-well plate (NUNCTM Apogent,

Denmark) was used for sample preparation: approximately 500,000 RBCs were

added to each well containing a total volume of 50 µL. For anti-Forssman testing

RBCs were fixed for 10 min by the addition of 100 µL of 0.1% glutaraldehyde to

reduce agglutination of antigen-positive cells. Incubation was performed at room

temperature under constant mixing. The plate was then centrifuged at 300 x g for 1

min and the supernatant discarded.

Different volumes of primary antibody were added to each well (anti-Forssman 25 µL,

plasma 20 µL, eluate 10 µL) and PBS were added to a total volume of 50 µL. The plate

was incubated for 10 min at room temperature under constant mixing and then

incubated for an additional 50 min at 4oC. The cells were washed twice with 150 µL

PBS. Another 50 µL PBS and 5 µL of secondary antibody were added, incubated for 10

min in darkness at room temperature under constant mixing and washed twice with 150

µL PBS. Finally, the RBCs were resuspended in 300 µL PBS and transferred to 5 mL

BD FalconTM plastic tubes (BD Biosciences, MA).

Flow cytometric analysis was performed on a FACScan/FACSCalibur flow cytometer

(Becton Dickinson) using FACSflowTM (BD Biosciences, Erembodegem-Aalst,

Belgium) sheath fluid. From a sample volume of 300 µL, 10,000-50,000 events were

collected at a flow rate of 60 µL/min. Log fluorescence data were gated on a linear

forward scatter versus linear side scatter dot plot. The analysis gate was set to

include the viable part of the cell population, excluding deformed and aggregated

cells i.e. cells that were high on side scatter/ forward scatter. When testing MEG-01

7AAD was added to exclude nonviable cells. Appropriate controls were included in

each run. For RBCs FL1 detection was included in all dot plots to avoid the risk that

autofluorescence (e.g. developing in RBCs transported under suboptimal conditions)

would be interpreted as a weak positive signal. FL1 and FL2 PMTs were adjusted to

ensure that the negative control RBCs fell within the set FL1 and FL2 markers.


7


Blood group serology

Native and papainised RBCs from Apae donors were tested (including control RBCs of

blood group A and O) with a variety of ABO reagents, anti-A (n=16) and anti-A,B

(n=8). Lectin studies were performed as part of the original serological study to

exclude known forms of polyagglutination (see reference 13 in the main manuscript),

and were not repeated here. Testing was performed by both gel column agglutination

and in tubes. Gel card; 50 µL of ABO reagent and 25 µL of a 3% RBC suspension

were incubated in neutral gel cards (50520, DiaMed/Bio-Rad, Cressier, Switzerland)

and spun in a gel card centrifuge for 10 min. Tubes; One drop of ABO reagent and

one drop of a 3% RBC suspension were incubated for 20 min at room temperature

and then centrifuged for one minute. Grading was done according to standard blood

bank practice.

The plasma from Apae donors was previously (reference 13 in the main manuscript)

shown to exhibit the expected antibodies found in group O plasma. To identify donors

with a strong anti-Forssman in their plasma, crossmatch was performed against

RBCs from the two Apae donors. Native and papainised RBCs were incubated with

plasma from random blood donors. RBCs in a 0.8% suspension incubated with

plasma in ID-card (50531) LISS/Coombs’ gelcards (DiaMed/Bio-Rad) incubated at

37ºC for 20 min and then centrifuged in a gel card centrifuge for 10 min. In addition,

RBCs in a 3% suspension were incubated in glass tubes at 4ºC (native cells) or room

temperature (papainised RBCs) for 1 h and then centrifuged. Tube tests were graded

both macro- and microscopically according to standard blood bank practice. Eluates

were prepared according to Judd8 using Apae RBCs and plasma that gave a strong

reaction in serological testing with native Apae RBCs. Appropriate controls were tested

in parallel as described8. Eluates were tested against human (phenotypes A1, A2, B,

O and Apae) and animal (from sheep and dog) RBCs.

Hemolysin testing (adapted from Mollison: Blood Transfusion in Clinical Medicine,

10th edition, 1997) was performed by mixing one drop of 10% RBC saline

suspension with 2 drops of freshly drawn serum. The samples were incubated for 30

minutes at 37º and then centrifuged for 3 minutes at 1000 x g and the supernatants

H. Clausen Denmark 26 Anti-AB Polyclonal Human DiaMed Cressier,

Switzerland Hemagglutination

27 Anti-AB Polyclonal Human Immucor Norcross, USA Hemagglutination 28 Anti-AB Polyclonal Human Biotest Dreieich, Germany Hemagglutination 29 Anti-AB Monoclonal IgM Mouse F125 Dominion Dartmouth, Canada Hemagglutination 30 Anti-AB Monoclonal IgM Mouse BIRMA-1 ES-4

ES-15 Immucor Norcross, USA Hemagglutination

31 Anti-AB Monoclonal IgM Mouse BD63/85 Biotest Dreieich, Germany Hemagglutination 32 Anti-AB Monoclonal IgM Mouse GAMA110/112 Immucor Norcross, USA Hemagglutination 33 Anti-AB Monoclonal IgM Mouse ES-4/ES-15 Sanquin Amsterdam,

Netherlands Hemagglutination

34 Anti-AB 2-39 Monoclonal IgG3 Mouse HW5 ETS Bretagne, France

TLC-EIA

35 Anti-AB 2-40 Monoclonal IgM Mouse CL178 ETS Bretagne, France

TLC-EIA

36 Anti-AB 2-41 Monoclonal IgM Mouse BH517 ETS Bretagne, France

TLC-EIA

37 Anti-AB 2-38 Monoclonal IgM Human HIRO-40 Japanese Red Cross Centre

Osaka, Japan

TLC-EIA

38 Anti-Galactosyl-A

Monoclonal IgM Mouse HH8 HH9

Kind gift from H. Clausen

Copenhagen, Denmark

TLC-EIA

39 Anti-ALeb Monoclonal IgM Mouse HH3 Kind gift from H. Clausen

Copenhagen, Denmark

TLC-EIA

40 Anti-Lea Monoclonal IgM Mouse LM 112-161/78 FR 2.3

Biotest Dreieich, Germany TLC-EIA

41 Anti-Leb Monoclonal IgM Mouse LM 129-181/96 FR 2.10

Biotest Dreieich, Germany TLC-EIA

42 Anti-X2 Monoclonal IgM Mouse TH2 Kind gift from

H. Clausen Copenhagen, Denmark

TLC-EIA

Lectins 43 Helix pomatia Sigma Saint Louis,

Missouri, USA Hemagglutination

44 Helix pomatia, biotin-labelled

Sigma Saint Louis, Missouri, USA

TLC-EIA

(Table S1 -

p.2)

Secondary, AP-conjugated

Used with primary antibody # and lectin

45 Anti-rat Monoclonal IgM Goat SAB-211I Nordic Biosite Täby, Sweden TLC_EIA 1 46 Anti-mouse Polyclonal IgG,A,M Goat Sigma Saint Louis,

Missouri, USA TLC-EIA 3-4; 18-25;

34-36; 38-42 47 Anti-human Polyclonal IgG,A,M Goat Sigma Saint Louis,

Missouri, USA TLC-EIA 37

48 Streptavidin Sigma Saint Louis, Missouri, USA

TLC-EIA 44

Secondary,

FITC-conjugated

49 Anti-human IgM

Polyclonal F(ab’)2 Rabbit Dako Glostrup, Denmark Flow cytometry Plasma/eluate

Secondary,

PE-conjugated

50 Anti-mouse kappa

Monoclonal IgG1 Rat X36 Becton Dickinson

San José, USA Flow cytometry 3, 4

51 Anti-rat IgM Polyclonal F(ab’)2 Goat Beckman Coulter

Paris, France Flow cytometry 1

(Table S1 - p.3)

Supplemental Table S2. Oligonucleotide primers and primer combinations used in this study.

a. exon amplification b. allele specific amplification c. sequencing d. PCR-ASP e. control primers for PCR-ASP f. mutagenesis and/or expression

Primer name Nucleotide sequence 5’-3’ Location Amplified region

Fragment length (bp)

Use

5’-80F TCC TGC TCC CAG GCC GCA 5’ Exon 1 357 a, c i1:51R GCC CCT TGC GTG GCA GGG Intron 1 i1:1142F AGA GAA GAG GTT GCA GTG AAT Intron 1 Exon 2 583 a, c i2:272R CCT ATG AGG AGA TCA AGG TTT Intron 2 i2:735F GAG ACC TGC ACT CCC AAT G Intron 2 Exon 3 239 a, c i3:59R TGA ACT TAA ATA CTC TCC TGT C Intron 3 i3:5326F CCC GCA GGG GCC TCT GA Intron 3 Exon 4-5

Intron 4 348 a, c i5:72R CTG ATG TTA GCA CCT CCA CT Intron 5 i5:519F GGG TCA CAC AGT GTG AGA G Intron 5 Exon 6 322 a, c i6:85R CAG CAG GGT CCT GTG TGC Intron 6 i6:841F GGT AAG TGG ACC CCA GCC T Intron 6 Exon 7 1503 a, c 3’+78R CCA AGA TCA CGC CAC TGA AC 3’ i6:838F GCT GGT AAG TGG ACC CCA G Intron 6 PCR-ASP 887A 644 a, d ex:7:887A-R GTT CAG GTG GCT TTC CTC CT Exon 7 b, d ex:7:363A-F CGG GTG ATC ATC ACT AGG TAA Exon 7 PCR-ASP 363A 816 b, d ex7:1139R GGT TGA ATT CGC CTG ACT GA Exon 7 a, c, d ex7:618R TTC CGA AAC ACC ATG TCC ACA Exon 7 c ex7:946F CGT CCA AGG TGC TGT CCC Exon 7 c ex7:1468F CAT ATT GCC CTT AAC GTA GCA Exon 7 c ex:7:363C-F CGG GTG ATC ATC ACT AGG TAC Exon 7 b ex:7:887G-R GTT CAG GTG GCT TTC CTC CC Exon 7 b JK-F3-L GCA TGC TGC CAT AGG ATC ATT GC e JK-R3-L GAG CCA GGA GGT GGG TTT GCC e GBGT1-expr-F GCC GAA TTC ACC ATG CAT CGC CGG AGA CTG G Exon 1 ORF 1068 f GBGT1-expr-R GGC GGA TCC CCC TCA GCT CCT CAG GCA GCT G Exon 7 f mutGBGT1 ex7:887A-F CTG CCT GGC AGG AGG AAA G Exon 7 f mutGBGT1 ex7:887A-R CTT TCC TCC TGC CAG GCA G Exon 7 f B3GALNT1-expr-F GCC GAA TTC GCC TGG ATG GCC TCG GCT CTC Exon 5 ORF 1023 f B3GALNT1-expr-R GCC GGA TCC AAG TTA ATA ATG GCA TGT GGT GTT C Exon 5 f

Supplemental Table S3. Glycolipid structures identified by MS and NMR in fractions Apae#1 and Apae#2. Below each sugar residue the ppm value for the anomeric resonance is given at 30°C for samples dissolved in DMSO:D2O (98:2). Also given are two reference structures (p-Fs and A-6-2) discussed in the text. ___________________________________________________________________________________________ Trivial name VI V IV III II I Reference ___________________________________________________________________________________________ GbO4 GalNAcβ3 Galα4 Galβ4 Glcβ1 Cer 1 4.509 4.796 4.244 4.165 nLc4 Galβ4 GlcNAcβ3 Galβ4 Glcβ1 Cer 2 4.199 4.647 4.249 4.160 P1 Galα4 Galβ4 GlcNAcβ3 Galβ4 Glcβ1 Cer 3 4.782 4.198 4.651 4.252 4.16 H-5-2 Fucα2 Galβ4 GlcNAcβ3 Galβ4 Glcβ1 Cer 4 5.04 4.33 4.63 4.26 4.16 x2 GalNAcβ3 Galβ4 GlcNAcβ3 Galβ4 Glcβ1 Cer 5 4.59 4.28 4.65 4.26 4.17 GbO5 Galβ3 GalNAcβ3 Galα4 Galβ4 Glcβ1 Cer 6 4.20 4.61 4.80 4.26 4.17 Forssman (Fs) GalNAcα3 GalNAcβ3 Galα4 Galβ4 Glcβ1 Cer 1 4.692 4.511 4.781 4.252 4.195 para-Forssman (p-Fs) GalNAcβ3 GalNAcβ3 Galα4 Galβ4 Glcβ1 Cer This work 4.527 4.459 4.794 4.255 4.16 A-6-2 GalNAcα3 (Fucα2) Galβ4 GlcNAcβ3 Galβ4 Glcβ1 Cer 7 4.943 5.157 4.396 4.600 4.259 4.183 __________________________________________________________________________________________

References to Supplemental Table S3. 1. Dabrowski J, Hanfland P, Egge H. Structural analysis of glycosphingolipids by high-resolution 1H nuclear magnetic resonance spectroscopy. Biochemistry. 1980;19(24):5652-5658. 2. Clausen H, Levery SB, Kannagi R, Hakomori S. Novel blood group H glycolipid antigens exclusively expressed in blood group A and AB erythrocytes (Type 3 chain H). J Biol Chem. 1986;261(3):1380-1387. 3. Diswall M, Ångström J, Karlsson H et al. Structural characterization of α1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies. Xenotransplantation. 2010;17(1):48-60. 4. Holgersson J, Jovall PÅ, Samuelsson BE, Breimer ME. Structural characterization of non-acid glycosphingolipids in kidneys of single blood group O and A pigs. J Biochem. 1990;108(5):766-777. 5. Thorn JJ, Levery SB, Salyan MEK et al. Structural characterization of x2 glycosphingolipid, its extended form, and its sialosyl derivatives: Accumulation associated with the rare blood group p phenotype. Biochemistry 1992;31(28):6509 6517. 6. Kannagi R, Levery SB, Ishigami F et al. New globoseries glycosphingolipids in human teartocarcinoma reactive with the monoclonal antibody directed to a developmentally regulated antigen, stage-specific embryonic antigen 3. J Biol Chem. 1983;258(14):8934-8942. 7. Clausen, H, Levery SB, McKibbin JM, Hakomori S. Blood group A determinants with mono- and difucosyl type 1 chain in human erythrocyte membranes. Biochemistry 1985;24(14):3578-3586.

Supplemental Table S4. Prevalence of SNPs relevant for this study. Values are given as the percentage of tested alleles in random donors.

SNP-ID

Data set

58C>T

(rs2073924)

363C>A

(rs35898523)

887G>A

(-)

Tested subjects

(n)

This study

28

3.5

0

9/113/256*

1000genomes† 31 4.9 0 1000§

NHLBI-ESP‡ 22 6.4 0.008 6503§

* Only nine random donors were tested for 58C>T (by sequencing) whilst 113 and 256 donors were screened by PCR-ASP for 363C>A and 887G>A, respectively. None of screening-positive donors were homozygous for 363C>A. † The thousand genomes project, Phase 1 integrated variant call set (www.1000genomes.org) ‡ National Heart Lung and Blood Institutes Exome Sequencing Project (www.evs.qs.washington.edu) § All subjects tested in these cohorts have been included independent of ethnic/geographic background.


SUPPLEMENTAL FIGURE LEGENDS

Figure S1. Extended thin layer chromatography analysis.

Monoclonal anti-A type 2, 2-24 (clone NaM200-16C5) activity against total glycolipids

and concentrated glycolipid column fractions.

Figure S2. Sequencing and PCR analysis of the GBGT1 gene.

DNA sequencing chromatograms highlighting two regions of particular interest in

GBGT1, including consensus and variant human samples at and around nucleotide

position 887 (left) and 363 (right). The upper middle panel shows the GBGT1

sequence from a gorilla. The same result was obtained for the other non-human

primates tested (not shown: baboon, gibbon, chimpanzee, macaque and orangutan).

In the lower middle panel, gel electropherograms of products obtained following

PCR-ASP assays to detect the two new mutations (887G>A and 363C>A) detected

in exon 7 of the GBGT1 gene are shown. For 887G>A, the lanes are labelled as

follows: Apae#1; pos = Apae#2 as positive control, i.e. a donor with 887G>A; neg =

negative control, i.e. DNA from a random blood donor lacking 887G>A; H2O = water

control; M = molecular size marker ΦX174 DNA/HaeIII. For 363C>A the lanes are

labelled as follows: Apae#1; pos = random blood donor with 363C>A as positive

control; neg = random blood donor lacking 363C>A as negative control; H2O = water

control; M = molecular size marker ΦX174 DNA/HaeIII.

Figures S3 and S4. Pedigrees with phenotype and genotype information for the two unrelated Apae families studied.

Traditional pedigree symbols are used to depict the family members and their

relationships. The investigated propositi (Apae#1 in Fig.S3 and Apae#2 in Fig.S4) are

indicated by arrows. Phenotypes (non-italics) and genotypes (italics) are given below

the symbols. Family members were typed by PCR-ASP for both the 887G>A

(activating) and 363C>A (truncating) mutations in GBGT1. In the individuals

heterozygous for both mutations their location in trans was verified by sequencing of

allele-specific fragments. None of the samples were homozygous for these

mutations. Black and white symbols represent family members with and without the


Apae phenotype, respectively. The grey square indicates an individual positive for the

887G>A substitution (based on a DNA sample from saliva) whilst no blood sample

was obtained and the phenotype has therefore not been verified.

Figure S5. Semi-quantification of GBGT1-mRNA levels.

Analysis of GBGT1-mRNA by real-time PCR. Forssman (GBGT1) transcript levels

were normalized to an appropriate house-keeping gene, beta-Actin. Diagram (a) and (b) represent two different experiments where all samples were tested in triplicate.

The sample displaying the lowest CT value, i.e. the highest level of transcripts, was

set to 100%. The displayed values were calculated as 2-[delta] [delta]CT and

expressed as % of the highest value. CT is the threshold value for the number of

cycles required for the signal to reach a set level. The error bars represent the

standard error of the mean (S.E.M.) for the four samples with common phenotypes.

(a) Erythroid bone marrow culture on day 9 vs. peripheral blood from four different

donors with common phenotypes; (b) Results from peripheral blood from the same

four donors as in (a) compared to Apae#1 and Apae#2.

Anti-A type 2 (2-24)

Figure S1.

A1

Apae

#2

#2 C

M88:1

2+

85:1

5

#2 C

M82:1

2

#2 C

M75:2

5

#2 C

M65:3

5

#2 C

M60:4

0

#2 C

M25:7

5

Fs

p-F

s

Apae

#1

#1 C

M88:1

2+

85:1

5

#1 C

M82:1

2

#1 C

M75:2

5

#1 C

M65:3

5

#1 C

M60:4

0

#1 C

M25:7

5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Human consensus 887G

Human Apae#1 887G>A

(Arg296Gln)

Gorilla consensus 887G

Human consensus 363C

Human Apae#1 363C>A

(Tyr121Stop)Apae#1 pos neg H2O M

PCR-ASP 887G>A

Apae#1 pos neg H2O M

PCR-ASP 363C>A

Figure S2.

Apae#1O1O1GBGT1887G/A363C/A

A

AA1O1GBGT1887G/G363 C/A

ApaeO1O1GBGT1887G/A363C/A

O

OO1O1GBGT1887G/G363C/A

nt

AA1O1GBGT1887G/G363C/A

nt

A AA1O1GBGT1887G/G363C/A

I

II

IIIA

A1O1GBGT1887G/G363C/A

Figure S3:Apae#1 Pedigree

n.t.O1O1GBGT1887G/A363C/C

Apae#2O1O1vGBGT1887G/A363C/C

ApaeO1O1vGBGT1887G/A363C/C

O

†

OO1O2GBGT1887G/G363C/C

OO1O1vGBGT1887G/G363C/C

OO1vO1vGBGT1887G/G363C/C

I

IIO

O1O1vGBGT1887G/G363C/C

Apae

III

nt

Figure S4:Apae#2 Pedigree

0102030405060708090

100

BM n=1 Blood n=4

% o

f hig

hest

exp

ress

ion

0102030405060708090

100

Blood n=4 Apae#1 Apae#2

% o

f hig

hest

exp

ress

ion

a

b

Figure S5.

GBGT1-mRNA from peripheral blood in common vs. Apae phenotypes

GBGT1-mRNA from bone marrow vs. peripheral blood

Apae#1 Apae#2

Documents

Document 1. Supplemental methods, tables, and figures - Blood