Pheno- and Genotype in Blood Group Variants - Two Sides of the Same Coin 260,00 CHF. Swisstransfusion 2018 Bern. 23 rd of August 2018,

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1 Pheno- and Genotype in Blood Group Variants - Two Sides of the Same Coin Swisstransfusion 2018 Bern 23 rd of August 2018, ,00 CHF Erwin A. Scharberg, Baden-Baden Christoph Gassner, Zürich

2 some ABO some SNP typing 260,00 CHF some specialities

3 some ABO some SNP typing 260,00 CHF some specialities

4 Genetic background of the ABO System Chromosome 9 ABO-Gen ~15 kb genom. DNA Exon Exon 6 Exon 7 Allele A 1 (wt) G C C G C G G G C A 2 - T del B - - T A A - C A - O 1 del O A Phenotype A 1 A 2 B O O 4 positions in the DNA sequence code the specificity and activity of the glycosyl transferase and determine the A 1, A 2, B und O blood groups

5 Anti-A1: + Anti-A2: neg Anti-B: ++++ Anti-O/H: neg Genotype: Ax01/O01

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7 Bx03/O02

8 Genetic variants in the ABO system 216 Gene variants with known phenotypes (August 2018) A alleles (84): Phenotype *A1 2 alleles A 1 *A2 18 alleles A 2 *A3 7 alleles A 3 *Ael 10 alleles A el *Aw 47 alleles A weak B alleles (55): Phenotype *B1 3 alleles B *B3 8 alleles B 3 *Bel 11 alleles B el *Bw 33 alleles B weak O alleles (77): Phenotype *O1 (261delG) 58 alleles O *O2 (non-261delg) 19 alleles O; weak or missing isoagglutinins ERYTHROGENE:

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10 Ax03/O03 Ax01/O01

11 Donors with aberrant ABO phenotypes 206,262 donors 412 samples with aberrant ABO phenotypes (0.20%) 45 samples with weak antigens Ax01 (646T>A): n=15 Aw06 (502C>G): n=5 Aw13 (2T>C): n=3 Ael01 (804insG): n=2 Ax03, Aw04, Aw09, B(A)03: n=4 (one each) novel variant (Bw21): n=1 367 samples with negative antigens and weak or absent isoagglutinins non-deletional O alleles (O03, Aw08, O51): n=255 other alleles (Ax03, Ax01): n=6 novel variants (Bw20, Aw15): n=3 molecular basis unknown: 103 molecular basis unknown: n=15

12 Geno-/Phenotypes in the ABO System Serological border line Phenotype 0 A el A x /A weak A 3 A 2 A 1 Allele O01 O03 Ael01 Aw01 A301 A201 A101 O02 Aw08 Ael02 Ax01 A302 A202 A Ax Die genotype-phenotype-correlation in the ABO variants is a quantitative effect. The antigen does not change - the antigen density is different. This quantitative effect is not clearly determinable by molecular methods.

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14 Loss of the A antigen (patient with myelodysplastic syndrome)

15 ABO-Genotyping (SSP-PCR): O1/O1!!!

16 ABO-Genotyping (SSP-PCR): O1/O1!!! Repeated ABO-Genotyping (SSP-PCR): O1/???

17 ABO-Genotyping (SSP-PCR): O1/O1!!! Repeated ABO-Genotyping (SSP-PCR): O1/??? ABO-Genotyping (buccal swab) A101/O101

18 an observation (Immunohematology, FACS Zürich) Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

19 first thoughts Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

20 first thoughts Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

21 first thoughts Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

22 first thoughts «Twin Chimeras» two or more zygotes «Dispermic Chimeras» single zygote «Chimeras of Unestablished Type» Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

23 however, gel cards delivered much more cases, and in 2007 for RHD Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

24 however, gel cards delivered much more cases, and in 2007 for RHD Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

25 however, gel cards delivered much more cases, and in 2007 for RHD apparently some «chimerism» is caused by «somatic mutations» Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

26 in a first step, we want to distinguish «chimerism» from «somatic mutations» Is there three-, or tetraploidy at polyallelic genetic loci? Test by e.g. HLA-typing for HLA-A. (Check tissues) Affects several blood group systems. Is there a deviation from a 1:1 ratio, measured at any heterozygous SNP of the affected blood group in whole blood? Test by e.g. digital PCR (ABO). Affects one blood group system. Check cell-lines specific for hematopoiesis. Are there alleles known causing chimerism? NO Consequently, typing for ABO-alleles seems useless. Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

27 in a first step, we want to distinguish «chimerism» from «somatic mutations» Is there three-, or tetraploidy at polyallelic genetic loci? Test by e.g. HLA-typing for HLA-A. (Check tissues) Affects several blood group systems. Is there a deviation from a 1:1 ratio, measured at any heterozygous SNP of the affected blood group in whole blood? Test by e.g. digital PCR (ABO). Affects one blood group system. Check cell-lines specific for hematopoiesis. Are there alleles known causing chimerism? NO Consequently, typing for ABO-alleles seems useless. Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

28 in a first step, we want to distinguish «chimerism» from «somatic mutations» Is there three-, or tetraploidy at polyallelic genetic loci? Test by e.g. HLA-typing for HLA-A. (Check tissues) Affects several blood group systems. Is there a deviation from a 1:1 ratio, measured at any heterozygous SNP of the affected blood group in whole blood? Test by e.g. digital PCR (ABO). Affects one blood group system. Check cell-lines specific for hematopoiesis. Are there alleles known causing chimerism? NO Consequently, typing for ABO-alleles seems useless. Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

29 in a first step, we want to distinguish «chimerism» from «somatic mutations» Is there three-, or tetraploidy at polyallelic genetic loci? Test by e.g. HLA-typing for HLA-A. (Check tissues) Affects several blood group systems. Is there a deviation from a 1:1 ratio, measured at any heterozygous SNP of the affected blood group in whole blood? Test by e.g. digital PCR (ABO). Affects one blood group system. Check cell-lines specific for hematopoiesis. Are there alleles known causing chimerism? NO Consequently, typing for ABO-alleles seems useless. Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

30 some ABO some SNP typing 260,00 CHF some specialities

31 SNP -> aminoacid change -> change of N-glycosylation recognition site Asn-Arg-Thr in k (KEL2, KEL*02) to Asn-Arg-Met in K (KEL1, KEL*01) N O T T T PHE 2HN O N O A A C ASN 2HN H N N O C G A ARG +2HN HO N N O A C THR G 193 N O C T G LEU

32 SNP -> aminoacid change -> change of N-glycosylation recognition site Asn-Arg-Thr in k (KEL2, KEL*02) to Asn-Arg-Met in K (KEL1, KEL*01) N O N O T T T PHE 2HN O N O 2HN O N O A A C ASN 2HN H N N O 2HN H N N O C G A ARG +2HN HO N N O N O +2HN A C THR G 193 S N N O N O A T MET 193 G C T G LEU

33 SNP genotyping for blood groups is highly accurate & may be improved, considering newly observed genetic polymorphism Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

34 SNP genotyping for blood groups is highly accurate & may be improved, considering newly observed genetic polymorphism Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

35 Low Frequency Antigen genotyping Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

36 Low Frequency Antigen genotyping Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

37 Low Frequency Antigen genotyping +

38 Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

39 Total number of red cell antigens recognized by the ISBT 360

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42 o o o + o + o o o o o +

43 IAT o o o + o + o o o o o +

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46 Blood group alleles ( 42) tested for the inhouse-panel: Do(a/b); Yt(a/b); Di(a/b), LW(a/b) Js(a/b); K11/K17 Lu:8,14; Au(a/b); Sc:1,2 Kn(a/b); McC(a/b); Sl:1,2; Yk(a+/-); KCAM+/- In(a/b); Cx+/-; Vel+/- Ch1-6, Rg1,2 Additional Serological Testing: Ch(a), Rg, Cs(a), Yk(a), Kn(a)

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49 Patient: Cw+ (D+ C+ c- E- e+), Genotyping: Cw+/Cw+ Anti-MAR-like confirmed using 5 Cw+/Cw+ test cells Additional antibody: anti-s

50 Antibodies identified due to the information derived from genotyping of the test cells ( ) 136 of 9,063 patients = 1.5% / 249 of 20,986 samples = 1.4%

51 some ABO some SNP typing 260,00 CHF 260,00 CHF some specialities

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55 Patient: M. D., 14 year old, female monoclonal reagents untreated cells monoclonal reagents untreated cells

56 monoclonal reagents untreated cells monoclonal reagents untreated cells polyclonal reagents untreated cells

57 monoclonal Anti-D reagents IgM monoclonal Anti-D reagents IgG monoclonal Anti-D reagents IgG

58 Father of the patient monoclonal reagents untreated cells monoclonal reagents untreated cells

59 Mother of the Patient monoclonal reagents untreated cells monoclonal reagents untreated cells

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61 Genotype: D+, C+, c+, E-, e+

62 monoclonal reagents E.H.: R(1)R2, (C)cD.E(e) JAHK positive (Rh53) monoclonal reagents

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64 Localization of the Ser122Leu and Ser122Pro Substitution on the RHCE protein 122 Ser (JAHK-) Leu (JAHK+) Pro (JAHK-)

65 monoclonal reagents monoclonal reagents r ( )G r, (C)cdde(e) JAHK positive (Rh53)

66 monoclonal anti-g titer in r r, Ccddee RBCs monoclonal anti-g titer in R o r, ccd.ee RBCs monoclonal anti-g titer in r ( )G r (C)cddee RBCs

67 HM10 P3X61 P3X212 P3X212 11F1 23B10 Anti-D mab IgM HM16 P3X35 P3X241 P3X249 P3X290 Anti-D mab IgG r ( )G r, (C)cdde(e) JAHK positive (Rh53) LHM LHM LHM LHM LHM LDM-1 76/55 76/64 70/45 59/19 169/80 Anti-D mab IgG

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69 Lutheran-null phenotyp (InLu type) bei EKLF/KLF1-Mutationen

70 gene regulation by transcription factors Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

71 GATA-transcription factors are Zinc finger proteins, efficiently binding DNA. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA2 and GATA3 but only poorly by GATA1. Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

72 GATA-transcription factors are Zinc finger proteins, efficiently binding DNA. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA2 and GATA3 but only poorly by GATA1. Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

73 GATA-transcription factors are Zinc finger proteins, efficiently binding DNA. 3 -A T A G-5 3 -A T A G-5 Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA2 and GATA3 but only poorly by GATA1. Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

74 Basal Cell Adhesion Molecule (BCAM) Lutheran Blood Group, CD 239, 19q13.2 exons bp introns bp Lu a / Lu b IgSF1 LU8 / LU14 IgSF2 SNPs rs T/C 0,840/0,160 rs G/T 0,800/0,200 Lu a His 77, LU1 A252 Lu b Arg 77, LU2 G252 1 / Lu(a+b-), cau Ex 3, IgSF1 rs A/G 0,700/0,300 rs3669 T/C 0,780/0,220 rs7026 C/T 0,690/0,310 LU8 Met 204, LU8 T611 LU14 Met 294, LU14 A611 1 / LU(8-14+), afr Ex 6, IgSF2 Au a Thr 539, LU18 A1615 Au b Ala 539, LU19 G / Au(a-b+), cau Ex 12, IgSF5 ( Auberger ) Au a / Au b IgSF5 Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

75 Blood Sep 1;112(5): Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

76 Blood Sep 1;112(5): Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

77 Two recent observations by the Lund group. Both involving transcription factors (GATA1, RUNX1) Christoph Gassner BLOOD TRANSFUSION SERVICE ZURICH, SRC+

78 There is a learning phase in Genetics, which is still ongoing. During this phase, Genetics is nothing without Phenotypes. All current blood group phenotypes may currently be explained by regular Mendelian inheritance. 260,00 CHF 260,00 CHF (Sometimes) transcription factors play a role in phenotypes. There are only very few exemptions, where e.g. epigenetics, or mirna, or other regulators play an important role.