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10 All Content © Immucor, Inc. The Jolly Blood Banker and “Other” Blood Groups Jill R. Storry, PhD FIBMS Associate Professor, Lund University Technical Director, Immunohematology Clinical Immunology and Transfusion Medicine, Lund Blood systems covered in this talk:

• MNS • Kell • Lewis • Duffy • Kidd 36 blood group systems are carried on functional molecules

Host defence/innate immunity ABO P1PK Enzymes Lewis FORS GLOB H (Glyco)proteins of I structural or unknown function

LW Xg Transporters Duffy and channels Lutheran Indian Scianna Raph Complement JMH regulation Oka Adhesion molecules The MNS Blood Group System (ISBT 002)

• MNS antigens are carried on:  GPA  GPB  Hybrids of GPA & GPB • Specific to red blood cells • Highly polymorphic system – 49 antigens • Function of GPA and GPB not completely understood: Chaperone proteins? Protection by sialic acid? Antigens of the MNS blood group system

Prevalence n Antigens Variable 4 M N S s High 10 Ena ENKT ENEP ENEH ENAV U ‘N’ ENDA ENEV JENU Low 35 He Mia Mc Vw Mur Mg Vr Me Mta Sta Ria Cla Nya Hut Hil Mv Far sD Mit Dantu Hop Nob Or DANE TSEN MINY MUT SAT ERIK Osa HAG MARS MNTD SARA KIPP MNS antigens are carried on GPA and GPB

20 M/N

20 ’N’ Trypsin sites α-chymotrypsin at aa 50 & 58 site at aa 51 Papain site Papain site ~aa 54 at aa 78/80 48 S/s

RBC lipid bilayer

91 Numbering: 150 Counted from initiating N-linked sugar Methionine (19aa leader O-linked sugar sequence included) The GYPA gene family

Chromosome 4q31.21

GYPA GYPB GYPE

GYPA

1 2 3 4 5 6 7

GYPB

1 2 3 4 5 6

GYPE

1 2 3 4 5 6

High nucleotide identity Low nucleotide identity Molecular Basis of MNS Antigens

Mechanisms for generating diversity: • Single nucleotide polymorphisms (SNPs) • Deletions/insertions • Gene recombination events

All of the above are reported for the MNS system MNS SNPs

Single nucleotide polymorphisms are an effective method of creating diversity, e.g. S/s antigens

GYPB exon 4 GA GAA ATG GGA CAA CTT GTC CAT CGT TTC ACT GTA CCA G S E M G Q L V H R F T V P GA GAA ACG GGA CAA CTT GTC CAT CGT TTC ACT GTA CCA G s E T G Q L V H R F T V P SNPs Encode Low Incidence Antigens

20 M/N 23 Mg 24

36 MNTD 46 Nya 20 47 Vw/Hut 22 MV ‘N’ 24 50 Or 66 Vr 73 Osa 76 Ria 48 S/s 77 Mta SARA 80 78 ERIK 54 Mit 82 MARS 84 HAG 58 sD

Numbering: 91 GPB Counted from initiating GPA Methionine (19aa 150 sequence included Homology Between Genes Creates Diversity

GYPA

1 2 3 4 5 6 7

t GYPB

1 2 3 4 5 6

High nucleotide identity Low nucleotide identity

Recombination hotspot Creation of MNS hybrid genes

Unequal crossover

GP.Sch GYPB GYPA GYPB GYPA St(a+) Novel antigens are GP.Hil (MINY+, Hil+) created at GYPA GYPB GP.JL (MINY+, TSEN+) crossover points GP.TK (SAT+)

Gene conversion

New antigens are encoded by GYPA GYPB -A -B the novel hybrid sequence

DNA exchange by double GYPA GYPB strand break repair (DSBR) mechanism leaves one chromosome unaltered Different mechanisms can produce the same antigen

GP.Hil G GP.Mur P Generated by G P Generated by E unequal crossover P B E gene conversion A E T A Hil G Hil E G T G P G P B D B L

GP.Hil GPMur GP.Bun GP.HF MNS Antigens Produced by Hybrids

• Mia, Hut, Mur, MUT, Hil, TSEN, MINY, Hop, Nob, DANE, Sta, Dantu, SAT • Antibodies can cause HDN • The GP.Mur (Mi.III) phenotype is ~15% in Chinese populations • HDN due to ”anti-Mia” is a concern • Anti-Mur +/- MUT • One of the most common antibodies in Asian populations High Prevalence MNS antigens

EnaFS 20 47 ENEH/Vw/Hut ‘N’ 24 Ena

68&71 ENKT 48 EnaFR 81 ENEV 82 ENAV/MARS U 84 ENEP/HAG 59

GPA 91 GPB

150 ENDA: encoded by GYPA absent from JENU: encoded by GYPB, absent from GP.Dane hybrid GP.Mur hybrid Null Phenotypes in the MNS System

• En(a−): absence or alteration of GPA • S−s− : absence or alteration of GPB • MkMk phenotype: absence of GPA and GPB Clinical significance of MNS antibodies

Anti- Ig class HTR HFDN Comments M IgM/IgG Rarely Rarely Worth monitoring in pregnancy N IgM>IgG No No S IgG Yes Yes s IgG Rarely Rarely U IgG Yes Yes Ena IgG Rarely Rarely Can be an autoantibody ’N’ IgG Yes Yes ’Lows’ IgM>IgG No Yes Mabye naturally occurring

Many low prevalence MNS antigens have been detected as a consequence of HDFN in women with negative antibody screens. Disease can range from mild to severe. The Kell Blood Group System (ISBT 006)

• 1st antibody – anti-K, identified in 1946 in a report of HDFN • 36 antigens • Kell is expressed on: • RBCs, erythropoietic tissues, testes • Lower expression in brain, lymphoid organs, muscle (heart and skeletal) Kell blood group system antigens 001 002 003 004 005 006 K k Kpa Kpb Ku Jsa 007 008 009 010 011 012 Jsb ------Ula K11 K12 013 014 015 016 017 018 K13 K14 --- K16 K17 K18 019 020 021 022 023 024 K19 Km Kpc K22 K23 K24 025 026 027 028 029 030 VLAN TOU RAZ VONG KALT KITM 031 032 033 034 035 036 KYO KUCI KANT KASH KELP KETI 037 038 039 KHUL KYOR KEAL Low High Geographic variation in expression of low prevalence antigens

Antigen Ethnic group Occurrence Others

K Whites 9% <2% Arabs 25%

Jsa Blacks (USA) 20% <0.01% Ula Finns 2.6% <0.01% Kpc Japanese 0.32% <0.01% The KEL gene is located on chromosome 7

Chromosome 7q33 - KEL

19 exons 21,5 kb

introns exons

..ccccctctctctcctttaaag Exons contain coding nucleotides CTT GGA GGC TGG CGC ATC TCT GGTIntrons AAA containTGG ACT non TCC-coding TTA nucleotides: AAC TTT AAC CGA ACG CTG AGA. HighCTT percentageCTG ATG AGT of theCAG gene TAT is GGCnoncoding CAT TTC but itCCT TTC TTC AGAcontains GCC TACimportant CTA GGA regulatory CCT CAT sequences CCT GCC TCT CCA CAC ACA CCA GTC ATC CAG gtgagggatg...... The Kell glycoprotein

• Type 2 membrane protein

732aa; Mr 93 kDa 15 cysteine residues: predicts a heavily folded protein Glycosylated 5 (4) N-linked branched oligosaccharides • One of M13 family of neutral zinc endopeptidases • Specifically cleaves big endothelin-3 to ET-3 ET-3 is a powerful vasoconstrictor • Biological role on RBCs is still unknown Kell antigens are created by missense SNPs

K23–/K23+ 1145A>G TOU+/TOU– 1217G>A K13+/K13– 986T>C KUCI+/KUCI– 1271C>T K22+/K22– 965C>T KANT+/KANT– 1283G>T KETI+/KETI– 1391C>T KTIM+/KYIM– 913G>A K19+/K19– 1475G>A 11 12 K11/K17 905T/C 10 13 Ul(a–)/Ul(a+) 1481A>T KHUL/KEAL 877C>T KYOR/KYO 875G/A 9 14 Kpb/Kpc 842G/A Kpa/Kpb 841T/C 8 15 K12+/K12– 1523A>G KELP+/KELP– 780G>T KASH+/KASH– 758A>G 7 16 RAZ+/RAZ– 745G>A VLAN–/VLAN+ 743G>A 17 Jsa/Jsb 1790C/T VONG–/VONG+ 742C>T 6 KELP+/KELP– 2024G>A K/k 578T/C 5 18 K14/K24 538G/C KALT+/KALT– 1868G>A 4 19 K18+/K18– 388C>T; 389G>A 3 Reid, Lomas-Francis & Olsson The blood group antigen factsbook, 3rd ed. 2 Slide modified from ES Wester et al. Transfusion 2005,45:545 1 KEL Kell glycoprotein interacts with XK

11 12 10 13 9 14 15 8 15 16 7 H Exons 1-19 Kell E 6 L 17 L H 5 18 4 COOH 19

3 XK 2

1 NH2 KEL N-glycans Cys347=Cys72 Preferential processing of big ET-3 by wild-type s-Kell

• Null phenotype of the Kell system  All Kell antigens are absent  Expression of Kx is elevated  Amount of XK protein is reduced • No apparent physiological defect • Reported in all populations • May produce anti-Ku (KEL5) • Many molecular backgrounds  Missense mutations  Nonsense mutations  Altered splicing due to intron mutations Preferential processing of big ET-3 by RBCs of common Kell phenotype:

comparison with K0 phenotype

Lee S et al. Blood 1999;94:1440-1450

©1999 by American Society of Hematology Reference allele KEL*02 encodes KEL2, KEL4, KEL5, KEL7, KEL11, KEL12, KEL14, KEL18, KEL19, KEL22, KEL26, KEL27, KEL29, KEL30, KEL32, KEL33, KEL34, KEL35, KEL36, KEL37, KEL38 Phenotype Allele Name Nucleotide change† Intron/ Amino acid change Exon

K0 KEL*01N.01 c.1678C>G 15 p.Pro560Ala

K0 KEL*01N.02 c.244T>C 4 p.Cys82Arg

K0 KEL*02N.01 c.223+1g>c Intron 3 Alternative splicing: p.Arg75fs

K0 KEL*02N.02 c.382C>T 4 p.Arg128Ter c.1790C (17)

K0 KEL*02N.03 C246T>A 4 p.Cys82Ter

K0 KEL*02N.04 c.1042C>T 9 p.Gln348Ter http://www.isbtweb.org/nc/workingK0 KEL*02N.05 c.2027G>A-parties/red-cell18 -immunogeneticsp.Ser676Asn-and-blood- K0 KEL*02N.06 c.223+1g>a Intron 3 Alternative splicing: group-terminology/ p.Arg75fs

K0 KEL*02N.07 c.574C>T 6 p.Arg192Ter

K0 KEL*02N.08 c.526−2a>g Intron 5 Alternative splicing Mod phenotypes Classification of a mod phenotype may depend on the reagents used. Kmod; KEL:1weak KEL*01M.01 c.578C>G 6 p.Thr193Arg Kmod KEL*02M.01 c.1088G>A 10 p.Ser363Asn Kmod KEL*02M.02 c.2030A>G 18 p.Tyr677Cys Kmod KEL:−13 KEL*02M.03 c.986T>C 9 p.Leu329Pro Kmod KEL*02M.04 c.2107G>A 19 p.Gly703Arg Kmod KEL*02M.05 c.1719C>T 16 p.Gly573Gly Kmod KEL*0M2.06 c.306C>A, c.1298C>T 4, 11 p.Asp102Glu, p.Pro433Leu Alloantibodies to Kell Antigens

• Usually IgG1 • Clinically important antibodies:  Cause of HTR  Mild to severe HDFN • Anti-K is the most common alloantibody specificity outside ABO & Rh systems • Anti-K often found in sera containing antibodies to high incidence Kell antigens Anti-K and HDFN • Kell antigens are well developed on fetal RBCs • Clinical presentation is very different than HDFN due to other non-Kell antibodies • Prediction of disease severity based on antibody titre or amniotic bilirubin is unreliable • Antibodies inhibit erythropoeisis • Post-delivery:  Hyperbilirubinaemia is lower than expected  Reduced reticulocytosis  Reduced erythroblastosis  Very anaemic babies Why is the HDFN so severe?

Onset of RBC proteins in cultured cord blood

Time in culture (days)

Southcott MJG, et al. The expression of human blood group antigens during erythropoiesis in a cell culture system. Blood 1999;93:4425-35

”… may have an important role in the early stages of hematopoiesis or cell lineage determination.” The Lewis blood group system (ISBT 007) • 6 antigens recognized by ISBT: Lea, Leb, LebH, ALeb, BLeb, Leab • Adsorbed onto RBCs from the plasma • ”Histo-blood” group antigens:  Glycoproteins in saliva and blody fluids  Lymphocytes, platelets  Epithelia of various tissues • Synthesized by α(1,3/4) fucosyltransferase encoded by FUT3 Lewis antigens and Secretor Status

• FUT2 encodes α2FucT2, which synthesizes H antigen on soluble glycoproteins

Blood group RBC Antigens Genes Secretor?

Le(a+b−) Lea FUT3 No Le(a−b+) Leb FUT2, FUT3 Yes Le(a−b−) Neither (FUT2) Yes or No Structure of Lewis Antigens on RBCs

Lea and Leb are not synthesized on RBCs, but adsorbed from plasma

β1-3 β1-4 β1-1 Lea β1-3 R Type 1 chain α1-4

β1-3 β1-3 β1-4 β1-1 Leb R Fucose α1-2 α1-4 GlcNAc Galactose Glucose R = Core structure Function of Lewis Antigens

• Receptors for various pathogens • Leb identified as the receptor for Helicobacter pylori • Sialyl-Lea is a tumour marker – diagnostic marker in carcinomas such as colorectal, pancreatic and gastric cancers • The related sialyl-Lex is not found on RBCs but expressed on endothelium – Ligand for selectins Helicobacter pylori and Gastritis

H. pylori BabA adhesin recognizes Leb on gastric epithelium resulting in binding

Mahdavi et al. Science 297:573-578, 2002 Slide courtesy of S. Spitalnik Helicobacter pylori and Gastritis

Inflammation increases SLex expression on gastric epithelium, which is recognized by H. pylori SabA adhesin

Mahdavi et al. Science 297:573-578, 2002 Slide courtesy of S. Spitalnik Helicobacter pylori and Gastritis

Neutrophils, which express high levels of SLex, infiltrate epithelium. Phase variation in H. pylori leads to decreased SabA expression and evasion of phagocytosis

Mahdavi et al. Science 297:573-578, 2002 Slide courtesy of S. Spitalnik Antibodies to Lewis antigens

Blood group Anti- Lea Anti- Leb

Le(a+b−) No Very rarely Le(a−b+) Very rarely No Le(a−b−) Occasionally Occasionally • Mostly IgM • Oftern naturally occurring • More often found in plasma from pregnant women • Readily neutralised by saliva • Anti-Leb is not clinically significant • Most anti-Lea are not clinically significant The Duffy blood group system (ISBT 008) • Consists of 5 antigens: Polymorphic: Fya, Fyb (Fya is a high prevalence antigen in South East Asia) High prevalence: Fy3, Fy5, Fy6 (Fy6 is defined by a monoclonal antibody only) • Carried by Atypical Chemokine receptor 1 (ACKR1) on RBCs • Also found on endothelial cells i capillaries, epithelia of the kidneys, lungs and in brain Atypical Chemokine receptor 1 (ACKR1)

• Major isoform of glycoprotein is 336 aa • Fya/Fyb at position 42 (Gly42Asp) • Susceptible to papain/ficin treatment of RBCs

Fy6 Fy3 Fya/Fyb

RBC membrane

N-glycans Genetic basis of Duffy phenotypes

−67 125 265 298

Exon 1 Exon 2

t/c A/G C/T G/A Fya/Fyb ”Fyx”

• Polymorphism at −67 (t>c) interrupts a GATA-1 binding site and the gene is not transcribed • No protein on the RBC • Fy(a−b−) phenotype  Found in >90% West Africans, ~60% of African Americans (FY*B)  Also found in Papua New Guinea (FY*A) Function of ACKR1 • Binds different chemokines – RBC chemokine ”sink” • Associated with hematopoeisis: neutropenia in healthy Fy(a−b−) individuals of African ancestry has been associated with absence of ACKR1

Nature Immunology 2017 ACKR1 is a receptor for Plasmodium spp. • Major receptor for P. vivax and P. knowlesi • Individuals with Fy(a−b−) phenotype are protected from infection • BUT… evidence that ACKR1 protein helps PF4 from platelets kill P. falciparum

Platelet Factor 4 and Duffy Antigen Required for Platelet Killing of Plasmodium falciparum McMorran et al. Science 2012;338:1348-51. Fig. 4

McMorran et al. Science 2012;338:1348-51. Antibodies to Duffy blood group antigens • Readily formed following transfusion • IgG antibodies • Anti-Fya and anti-Fyb can cause both transfusion reactions and HDFN • Anti-Fy3 is generally weak and not clinically important but…

 Anti-Fy3 produced by true Fynull individuals (mutations in FY gene) can be clinically important • Anti-Fy5 is very rare • Anti-Fy6 only described as a monoclonal antibody reactive with a papain-sensitive epitope on ACKR1 The Kidd blood group system (ISBT 009)

Antigens Gene SLC14A1 Jka, Jkb and Jk3 (HUT11A), UT-B Chromosome 18, 10 exons 7 8 6 9 Properties of SLC14A1 5 Jka/Jkb 10 389 amino acids Multipass membrane protein 4 11 Function 3 Urea transporter 2 Found on RBCs, kidney, colon 1 SLC14A1 Null phenotype Antibodies Jk(a−b−) Delayed HTR Reduced urine concentration HDFN Anti-Jk3 Olives, Lucien, Sidoux-Walter et al Institute National de la Transfusion Sanguine, Paris JK alleles

3 st Single Nucleotide Polymorphisms (SNPs)

588A 838G JK*01 Jk(a+)

-46 a/g 588G 838A JK*02 Jk(b+)

Olives et al., J. Biol. Chem. 1994.

Asp280Asn Jka Jkb JK alleles that encode weak antigen expression

Reference allele JK*01 encodes JK1, JK3 Phenotype Allele name Nucleotide change Exon Predicted amino acid change

JK:1 or Jk(a+) JK*01 or c.838A>G 8 p.Asn280Asp JK*A JK:2 or Jk(b+) JK*02 or JK*B Weak phenotypes Jk(a+W) JK*01W.01 c.130G>A 3 p.Glu44Lys Jk(a+W) JK*01W.02 c.511T>C 6 p.Trp171Arg Jk(a+W) JK*01W.03 c.28G>A 3 p.Val10Met Jk(a+W) JK*01W.04 c.226G>A 4 p.Val76Ile Jk(a+W) JK*01W.05 c.742G>A 7 p.Ala248Thr Jk(b+W) JK*02W.01 c.548C>T 6 p.Ala183Val Jk(b+W) JK*02W.02 c.718T>A 7 p.Trp240Arg The common Jka+w phenotype is due to an intracellular amino acid change

200

190 210

Pro196

180

220

Asp280Asn 170 230 Jka/Jkb 90 140

240 160 340 330 290 80 150 100 130 270 300 310

70 120 350 110 320

360

370

40 Glu44Lys 380

30 20 10 Function of Jk (SLC14A1)

• Major urea transporter on RBCs and kidneys • Absence of SLC14A1 has no clinical symptoms but individuals have reduced urine concentrating ability • Since SLC14A1 is an important functional protein in kidneys, Jka/Jkb are minor histocompatibility antigens in transplantation The Jk(a−b−) phenotype

• Rare phenotype but up to 1% in Polynesians • RBCs are resistant to hemolysis by 2M Urea

Phenotype Urea transport Hemolysis Jka/Jkb Passive/Active 1 min

Jknull Passive 30 min

Read after 2 min

Jk(a+b-) Jk(a-b-) Jk(a-b-) JK alleles that silence antigen expression Null phenotypes JK*02

JK:–3 or Jk(a–b–) JK*02N.01 c.342-1G>A Intron 4 p.Arg114_Thr156del; Alternative splicing JK:–3 or Jk(a–b–) JK*02N.02 c.342-1G>C Intron 4 p.Arg114_Thr156del; Alternative splicing JK:–3 or Jk(a–b–) JK*02N.03 c.222C>A 4 p.Asn74Lys

JK:–3 or Jk(a–b–) JK*02N.04 c.663+1G>T Intron 6 p.Leu223fs*?; Alternative splicing

JK:–3 or Jk(a–b–) JK*02N.05 c.723delA 7 p.Gly243Alafs*20 Identical to JK*01N07? (c.838A>G is located past termination) JK:–3 or Jk(a–b–) JK*02N.06 c.871T>C 8 p.Ser291Pro

JK:–3 or Jk(a–b–) JK*02N.07 c.896G>A 8 p.Gly299Glu

JK:–3 or Jk(a–b–) JK*02N.08 c.956C>T 9 p.Thr319Met

JK:–3 or Jk(a–b–) JK*02N.09 c.191G>A 4 p.Arg64Gln

JK:–3 or Jk(a–b–) JK*02N.10 c.194G>A 4 p.Gly65Asp

JK:–3 or Jk(a–b–) JK*02N.11 c.499A>G; c.512G>A 6 p.Met167Val; p.Trp171Ter

JK:–3 or Jk(a–b–) JK*02N.12 c.437T>C; c.499A>G 5; 6 p.Leu146Pro; p.Met167Val

JK:–3 or Jk(a–b–) JK*02N.13 c.499A>G; c.536C>G 6 p.Met167Val; p.Pro179Arg Antibodies to Kidd blood group antigens • Readily formed following transfusion • IgG antibodies • Anti-Jka, -Jkb and anti-Jk3 can cause severe hemolytic immediate/delayed transfusion reactions • HDFN is mild to moderate • Anti-Jka and anti-Jkb may disappear rapidly but are readily boosted by transfusion of antigen- positive blood For more information on blood group alleles….

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