MOLECULAR VARIATION IN

THE SOLUTE CARRIER FAMlLY 4,

ANION EXCHANGER MEMBER 1 CENE;

CHARACTERUATION OF THRISE

LOW INCIDENCE ERYTHROID ANTIGENS

KIRK MCMANUS

A Thesis Submitted to the Faculty of Graduate Studies in Partial Fulfilment of the Requirements for the Degree of

MASTER OF SCIENCE

Department of Human Genetics University of Manitoba Winnipeg, Manitoba National Library Biblioth$ue nationale NB ~fc-da du Cana a Acquisitions and Acquisitions et Bibliographie Services seivices bibliographiques 395 W@lhgtonStreet 395, rue Wellington OttiwnON KlAON) OnrwaON KlAW cana& cmadn

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Molmilrr Variation In the Sdutc Cirrkr Frmlly 4, Mon Excbinger Mcmkr 1 Cent; Chamcterizrtion of TbLow Incidence Erythroid Antigena

Kirk McManur

A Therir/Pncticum aubmittd to the Ficulty of Cndute Stpdkc of The Udvenity

of Manitoba in partial tùl1Ulment of the requirementi of the dcgcc

of

Maiter of Science

Kirk McMrnui@1999

Permidon hrr kcn grinted to the Llbrary of The Udvenity of Mdtobr to knd or WU copia of tLh tbcrlr/pmetieum, to the Nrtloarl Libruy of Cam& to microfüm thia thair ud to kdor wU capiea of the film, rad to Diuertatbna Abstmcts Intcmtioul to prbiiah rn rbrtnct of thh tbddp~cticum.

The ruthor mmr otkr publication Wb,and atitlier tLh tûcdJptidiam BOT extendve extracta from tt MY k printed or otheMae rtptoduced without the ruthorta written peiahdon. Abstract

The low incidence erythroid antigens, BOW, NFLD, and Fr', are excellent Diego biood group system candidates based on a vanety of serological. biocliemical, and molecular data. Diego system antigens are canied on the erythroid membrane band 3, and are controlled by SLCM I (solute carrier farnily 4, anion exchanger member 1 gene). Band 3 contains two functionally distinct domains; an N-terminal cytoplasmic domain and a C- terminal transmembrane domain.

DNA fiom controls and individuals segregating for BOW, NFLD, and Fr", was amplified by PCR and screened for polymorphisms by single strand conformation polymorphism analysis (SSCP). Only exons corresponding to the transmembrane domain ofband 3 (exons 1 1-20 ofSLC4AI) were analyzed. DNAs displaying SSCPs were subjected to sequencing.

DNA fiom BOW+ individuals displayed a SSCP in exon 14 and cxliibited a single

C-tT mutation, corresponding to a Pro56 1Ser substitution in band 3. DNA from al1 tested

NFLD+ individuals displayed SSCPs in both exon 12 and 14. Subsequent DNA sequencing

identified A-+T and C-tG mutations in exons 12 and 14 that give rise to Glu429Asp and

Pro561Ala substitutions in band 3, respectively. Exon 13 SSCPs were evident in the DNA of Fr(a+) individuals tested. DNA sequencing revealed a G-+A mutation underlying a

Glu480Lys substitution.

We conclude that point mutations in SLC4Al representing amino acid substitutions

in the erythroid protein band 3 account for al1 three antigens. In light of these findings, the i i international Society of Blood Transfusion (ISBT) Working Party on Terminology for Red

Cell Surface Antigens has granted BOW, NFLD and Ff, Diego blood group system status and designated them DI 1 5, DI 16, and DI20 respectively. iii

Fint and forcmost 1 wouid like to thad my advisor and mentor Dr. Teresa Zelinski, for providing the opportunity to kgh my r~sea~hcarcer; 1 have tmly enjoyed my tirne in the Rh Laboratory. I cm't Say enough how much 1appreciate al1 the things you have done for me Tmy... both academicaliy and personally. 1 am grateful for al1 the things that you have taught me - th& you so very much for YOIU the and patience! This thesis stands as a testament to you as a ptnon and as a teachcr.

1 would also îike to extaid a luge thank you to Ms. Gai1 Coghlan for performing and teaching me the serological techniques involved during my the at the Rh Laboratory. 'Ihnnk you ûail for king more than just another wann body in the lab, thanks for king there when I needcd someone to talk to and sharing your life experiences with me!

1would like to acknowledge my cornmittee mcmbers, Dr. A. Dawson and Dr. RD Gietz, for their input and critical miew of tbis thesis.

A special thank you gas out to all the fourth year project studtnts who have conducted their re-h in the labomtory and dl the studcnts in the Deparhnent of Human Genetics.

1 would also like to acknowledge and thank Dr. Marion Lewis for her insight and commcnts on past prcsentations and papers.

1 wish to extend a huge thank you to my family for their continued love, support, patience and understanding duMg my timc as a Master's studcnt: Mom, Dad, Wendy, Mike, Ryan, Matthcw, Brian, Annmarie, and Tempe. A thank-you just isn't enough!!

Finaily, 1 would like to thank Jacki for her love and support. 1 am tndy gratefbl for evcrything you have done to heip me out! iv List of Figures

Figure 1 . Schematic npresentatioa of thc putative mode1 for band 3 ...... 5 Figure 2 . Localization of amino acid substitutions underlying band 3 antigens ...... 23 Figure 3 . Chromosome diagram depicting the localization of the various blood group genes ...... 30 Figure 4 . Pedigree of the Canadian kindred segregating for NFLD ...... 70 Figure 5 . Pedigrees of two Mennonite kindreds segregating for FZ (Panels A and 3) ...... 72 Figun 6. Molecular analysis of exon 14 of SLC4A 1 fiom BO W+ individuals (Panels A and B) ...... 87 Figure 7 . Molecular analysis of exon 12 and exon 14 of SLC4Ai fiom members of a family segregating for NFLD ...... 90 Figure 8. DNA sequence analysis of exon 12 and exon 14 of SLC4Al ...... 92 Figure 9. Molecular charactenzation of exon 1 3 of SLC4A 1 (Panels A and B) ...... 94 Figure 10 . Schematic representation of the transrnembrane domain of band 3 ...... 98 List of Tables

Table 1, Abnormal cellular phenotypes defhed by molecular analysis ofSLC4AI ...... 14 Table 2. Definition of RBC antigens carried on band 3 ...... 22 Table 3. Established blood group systems and their controlling genes ...... 29 Table 4. Definition of DNA primers used to ampli@ exons 1 1-20 of SLC4A1 ...... 81 Table 5. Definition of the three low incidence erythroid antigens investigated in this study ...... 86 Table 6. Lods for linkage between NFLD and SSCPs in exon 12 or exon 14 ofSLC4AI ...... 91 Table 7. Lods for linkage between FP and SSCPs in exon 13 of SLC4AI ...... 95 Table 8. Identification of restriction endonuclease cleavage sites within specific exons of SLC4AI comsponding to various Diego blood group system antigens ...... 102 Table 9. Exarnples of blood group products potentially involved in micro-organism invasion ...... 108 List of Ab breviations

A adenine ATP adenosine triphosphate AE2 anion exchanger 2 AE3 anion exchanger 3 band 3 HT band 3 hi& transport bp base pair C cytosine C 1- cm centimetre DI Diego blood group system (ISBT symbol) dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate DiDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonicacid DNA deoxyribonucle ic acid dNTP deox ynucleotide triphosphate dRTA distal renal tubule acidosis dTTP deoxythymidine triphosphate EDTA ethylenediamine tetraacetic acid g gram G guanine GPA glycophotin A H+ hydrogen ion; proton HCI hydrochloric acid HCOi bicarbonate HDN hemolytic disease of the newbom hr hour HS hereditary spherocytosis ISBT International Society of Blood Transfusion kb kilobase KC1 potassium chloride Da kiloDalton lod 1ogarith.m of the odds M molar; moles L" minute magnesium chloride millilitre mm millimetre mM millimolm NaOH hydrox ide NaEDTA disodium salt EDTA nanogram NH,Cl ammonium chloride NR nonrecombinant PCR polymerase chah reaction RWs) red blood (s) tpm revolutions per minute SDS SA0 Southeast Asian ovalocytosis SLC4A 1 4, anion exchanger member 1 gene SSCP single-strand conformation polymorphism T thymine TAE tris-acetate-EDTA buffer Ta9 Thermus aqiroticus TBE tris-borate-EDTA buffer TE tris-EDTA buffer TEMED N,N,N',N'-tetramethyleihylenediamine Tris tris (hydroxymethyl) aminomethane u units w ultravioiet 0 theta; recombination fraction pCi microcurie PL microlitre W Watts 33p radioisotope of phosphoriis

1.9.5 Complement Regulatory Glycoprotein Molecules ...... 45 1.9.5.1 Decay-Accelerating Factor PAF) and the Cromer Blood Group System ...... 46 1.9.5.2 Complement Receptor Type- 1 (CRI) and the Knops Blood Group System ...... 46 1.9.5.3 C4AJC4B and the Chido/Rogers Blood Group System ...... 48 1.9.6 Blood Group Enzymes or Putative Enzymes ...... 48 1.9.6.1 Acetylcholinesterase (AChE) and the Yt Blood Group System 48 1.9.6.2 Putative Metalloendopeptidase and the Kell Blood GroupSystem ...... 48 1.9.7 Xg glycoprotein and the Xg Blood Group Sy stem ...... 49 1.9.8 Carbohydrate Structures and the ABO, H, Lewis and P Blood Group Systems ...... 50 1.9.9 Do Glycoprotein and the Dombrock Blood Group System ...... 50 1.9.10 Blood Group Products of Unknown Function ...... 50 1.10 Diego Blood Group System and Band 3 Associated Antigens ...... 51 1.10.1 Diego; Dia and Dib;DII and DU ...... 52 1.10.2 Wright; Wf and WP;DI 3 and DI4 ...... 53 1.10.3 Waldner; WdD;DIS ...... 54 1.10.4 Redelberger; Rb8;DI6 ...... 55 1.10.5 Wanior; WARR; DI7 ...... 56 1.10.6 ELO;ELO; DI8 ...... 57 1.10.7 Wulfsberg; Wu; DI9 ...... 57 1.10.8 Bishop; BpD;DI10 ...... 58 1.10.9 Moen; Mou;DI11 ...... 59 1.10.10 Hughes; Hg'; DI12 ...... -59 1.10.11 VanVugt; Vg'; DI13 ...... 60 1.10.12 Swann; SUr; DI14 ...... 60 1.10.13 Bowycr;BOW;DIlS ...... 61 1.10.14 NE WFOUNDLAND; NFLD; DI 16 ...... 62 1.10.1 5 Nunhart; Jna; DI 17 ...... 63 1.10.16 hpic; KREP; DI18 ...... 63 1.10 .1 7 Traversu; Tr'. DI1 9 (Reserved) ...... $64 1.10.18 Froese; Fr'; DUO ...... $64 1.1O .1 9 Swann Class 1; SWI; DI2 1 ...... -65

2 OBJECTIVES ...... 66 2.1 BOW ...... 66 2.2 NFLD ...... 67 2.3 Ff ...... 67

3 MATERIALS ...... 68 3.1 Individuals and Families Stwiied ...... 69 4 METHODS ...... 73 4.1 Preparation of Red BldCe11 Suspensions ...... 73 4.2 Slanted Capillary Method of Blood Group Phenotyping ...... 73 4.3 Antibody Absorption and Chlorofonn Heat Elution ...... 74 4.4 indirect Antiglobulin Test ...... 75 4.5 Isolation of Genomic DNA ...... 76 4.6 Quantitation of Genomic DNA ...... 77 4.7 Preparation of 6% Polyacrylarnide/lO% Glycerol Non-denaturing Gels ...77 4.8 Preparation of 6% Polyacrylamide Denaturing Gels ...... 78 4.9 Preparation of 1% Agarose Gels ...... 79 4.10 Single Strand Conformation Polymorphism Analysis ...... 80 4.1 1 Linkage Analysis ...... 82 4.12 Exon-specific PCR-amplification and Product Isolation ...... 82 4.13 DNA Sequencing ...... 84

5 RESULTS ...... 86 5.1 BOW ...... 86 5.2 NFLD ...... 88 5.3 Ff ...... 93

6 CONCLUSION ...... 96

7 DISCUSSION ...... 97 7.1 Comments on Common Ancestry ...... 100 7.2 Alternative Methods for Determinhg MCPhenotype ...... 101 7.3 Second Putative Extracellular Loop of Band 3 ...... 104 7.4 Serological Relationship ...... 105 7.5 Molecular Analysis of Blood Oroup Polymorphisrns ...... 107 7.6 Funut Directions ...... 109

8 REFERENCES ...... 110

9 AppendbcA ...... 130 1 INTRODUCTION

1.1 Structure and Organization of the Humnn Solute Carrier Family 4, Anion

Exchanger Member 1 Gene

Solute carrier family 4, anion exchanger member 1 gene (SLCdAI),earlier known as

AEI, EPB3, and EMPB3, was first cloned in 1988 (Tanner et al., 1988). SLC4A 1 encodes erythroid band 3 and controls expression of Diego blood group systeni antigens. Originally localized to chromosome 17 (1 7q2 1-qter) by somatic ceIl hybridization

(Showe et al., 1987), Zelinski et al. (1993) refined the position of the locus to 17q2 1-q22 through linkage analysis. The entire genomic sequence of erythroid SLC4Al has been determined (Lux et al., 1989) and is available at the GenBank website

(http://www2.ncbi.nlm.nih.gov/genbank/queryryform.hl accession num ber L35930).

Human erythroid SLCJAI spans approximately 21 kb of genomic DNA and contains 20 exons and 19 introns. With the exception of exon 20 (2 153 bp), the exons range in size from

62 bp to 254 bp, while introns range in size fiom 86 bp to 5123 bp in length. Spread throughout the gene are nine Alu repetitive elements approximately 300 bp in length; three in intron 1, one in intron 13, two in intron 16, two in intron 1 7 and one in exon 20, al1 of which are followed by a poly-A rich sequence. Seven of the Alu repeats are in direct sequence alignment with the gene while two are inverted (Sahr et al., 1994).

Transcription initiation can begin hmeither of two tissue specific promoters and translation of these products produces two different band 3 isoforms, an erythroid form and a fonn. Erythioid SLC4Al transcription begins at a potential erythroid specific promoter mgion upstnam of exon 1. The promoter lacks TATA and CCAAT boxes but does 2 contain a cluster of potential transcription factor binding sites including SPI (GC boxes),

Ap 1 (activator protein 1), Ap2 (activator protein 2), GATA (erythroid factor 1), NF 1 (nuclear factor 1), GT boxes and several direct or inverted CACCC boxes (Schofield et al., 1994).

Transcription of kidney SLC4A I begins at a non-erythroid SLC4A 1 promoter located inside the erythroid gene. The kiâney specific promoter has not been well defined but does include transcription factor binding sequences (ie. GT boxes, CACCC boxes and Ap2). Located downstream fiom this cluster of transcription factor binding sites is a TATA box. Kidney exon 1 (K 1) is located in the third intron of the erythroid gene. The 3' end of K 1 has a splice site that in fully processed kidney transcripts has been shown to be spliced to the 5' end of exon 4 of the erythroid SLC4A 1 (Schofield et al., 1994).

A polyadenylation signal has been identified in the 3' non-coding region of exon 20

(AATAAA), 20 bp upstrearn of the RNA cleavage site. The total length of exon 20 is 2 153 bp, however 2075 bp is non-coding sequence. The non-coding region of exon 20 also contains several direct repeats and one Alu repetitive element. A composite SLCU I cDNA

(predicted to be 4959 bp in length) has been assembled fiom published cDNA sequences and the complete 3' non-coding region of exon 20 (Sahr et al., 1994).

1.1.1 SLC4Al in Other Organisms

SLC4AI has also been identified and characterized in a number of other hcluding mouse (Kopito and Lodish, 1985a; Kopito and Lodish, 1985 b), rat (Kudrycki and

Shull. 1989). rabbit (Vimr and Carter, 1976; Chow et al., 1992; Ligi et al., 1W8), cattle

(Nakashima and Makino, l98O), eel (Romano et al., 1995). trout (Hubner et al., 1 992), turtle

(Drenckhahn et al., 1987; Staknau et al., 1991) and chicken (Cox and Lazarides, 1988; 3

Romano et al., 1995). The gene organization, exon-intron splice sites, and DNA sequence of the human and mouse SLCQAI genes are highly conserved. Sahr et al. (1994), have show that the sequence immediately upstream of erythroid exon i contains recognition sequences for several transcription factors that arc highly conserved between mouse and man and are somewhat conserved in the rat. Except for exons 1, 3,4, 8. 1 1, and 14, ail of the human and mouse SLC4A I exons are the sarne size. in exons of different size, the difference is always a multiple of three and therefore does not alter the reading frame. The average exon coding sequence identity between human and mouse is approximately 80% at boih the nucleotide and amino acid levels. Hurnan and mouse intron sequences do not exliibit the same degree of homology (only up to 40% sequence similarity) as the exons, but show size similarities. The location of the kidney-specific intemal transcription initiation site has been show (Sahr et al., 1994) to be conserved in the rat and human SLCJAI genes. The mouse and rat kidneySLC4A I product is lacking the 79 most N-terminal aniino acid residues found in the erythroid isoform, while the human protein product lacks the 65 most N-terminal amino acids. This indicates that the SLC4AI gene has two separate tissue-specific promoters, an upstream promoter that hinctions in erythroid cells and an intemal prornoter that funetions in kidney cells (Sahr et al., 1994).

1.2 Human Membrane Protein, Band 3

Found in approximately 1,200,000 copies per RBC, band 3 is a prevalent RBC membrane protein (Tanner, 1993) that can exist as homodimers or homotetramers in situ, and controls expression of Diego blood group systern antigens. Band 3 migrates as a diffuse band of approximately 95 to 105 kDa on a SDS (sodium-dodecyl-sulfate) polyacrylamide 4

gel as a result of size variation in the attached N-glycan chain (see section 1.2.1). Band 3 is

91 1 amino acids in length and is comprised of two bctionally distinct and separable domains (Steck et al., 1976); a 40 kDa N-terminal domain and a 55 kDa C-terminal domain.

Mild proteolytic treatment of band 3 with a-chymotrypsin or low concentrations of trypsin

or papain separates each domain with minimal loss of function (Steck et al., 1976; Grinsteiii

et al., 1978; Low, 1986). The N-terminal cytoplasmic domain, consisting of amino acid

residues 1-403 (exons 1- 10 of SLClA 1), interacts with several different membrane skeleton

(see below), several glycolytic enzymes, and hernoglobin. The C-terminal

transmembrane domain (exons 1 1-20) is comptised of amino acid residiies 404-9 1 1. The C-

terminal domain is involved in respiration, facilitating the reciprocal exchange of HCO; and

CI' across the RBC membrane. Although no three-dimensional structure of band 3 is

available, a putative model has been proposed based largely on hydropathy analyses that

predict band 3 to traverse the membrane 14 times resulting in seven extracellular and six

intracellular loops (Fig. 1) (Tanner, 1993). Based on this model, approximately 90 amino

acid residues are extracellularly located, any one of which could underlie a potential

antigenic deteminant. The reader is reminded that although Figure 1 depicts band 3 in two-

dimensions, its dynamic three-dimensional structures may generate antigen epitopes.

A tmncated band 3 isofom has also been identified in type A intercalated cells of the

distal tubule fiom the kidncy (Wagner et al., 1987; Tanner, 1993). Translation begins at

Ma66 of the erythroid transcript and produces a band 3 molecule lacking the 65 N-terminal

arnino acids found in the erythroid form. The kiàney isoform functions in acidhase

homeostasis (Tanner, 1993; Kollert-Jons et al., 1993).

1.2.1 Carbobydrote Variation

The single N-lactosaminoglycan chah attached to band 3 at Asn 642 (Jay, 1986; Lux et al., 1989) exhibits size variation as a result of different numbers of repeating N- acetyllactosarnineunits. (Tsuji et al., 1980; Fukuda et al., l984a; Fukuda et al., 1984b). The lactosaminoglycan chahs are linear unbranched chahs that express the i antigen in fetal

RBCs (Fukuda et al., 1984a) while they are extensively branched in adult FU3Cs and carry the 1antigen (Fukuda et al., l984b). The carbohydrate blood group determinants of the AB0 system are also carried on band 3 (Daniels, 1999). Functional assays have concluded that nmoval of the N-glycan chain does not affect the band 3 anion exchange property or funciion (Tanner, 1993), nor does it alter the expression of Diego blood group systeiii antigens. However, in RBC precurson fiom individuals where glycosyl transferase deficiencies occur (ie. congenital dyserythropoietis anemia type II), anomalous band 3 clustering has been found suggesting that the normal carbohydrate chain is involved in band

3 assembly or membrane targeting dunng erythrocyte synthesis (Fukuda et al., 1984~).

1.3 Band 3 Function

As previously mentioned band 3 has two functionally distinct domains; the N- terminal domain and the C-terminal dornain. The N-terminal domain functioiis in RBC membrane stability, while the C-terminal domain functions in anion transport. In RBCs the anion exchange facilitates respiration, while in the kidneys, anion exchange aids in acid/base homeostasis. 1.3.1 Membrane Stability

RBCs are dynamic entities that must withstand shearing forces encountered in the capillaries of the cuculatory system. The inherent flexible properties of the RBC are due in part to membrane architecture. The RBC membrane is comprised of a plasma membrane tethered to an underlying protein membrane scaffold known as the membrane skeleton. The membrane skeleton is comprised of a- and P-spectrin tetramers cross-linked io short actin filaments atjunctional complexes, which include protein 4.1, myosin, tropomyosin, addiicin, p55 and protein 4.9 (Gilligan and Be~ett,1993). The membrane skeleton is attached to the plasma membrane at two sites. The major attachment site occurs via , a protein witli high affmity binding sites for the p-spectrin subiinit (Bennett and Stenbeck, 1979) and the cytoplasmic domain ofband 3 (Be~ettand Stenbeck, 1980). The ankyrin-band 3 interaction is believed to be strengthened through the association of an additional membrane skeleton protein, band 4.2 (Korsgren and Cohen, 1988; Rybicki et al., 1988). The second anachment site involves junctional complexes where protein 4.1 binds to the , glycophonn C (Reid et al., 1990).

Band 3 is also believed to play a critical dein the maintenance of RBC membrane stability. Membrane shedding leading to severe spherocytosis and hemolysis has been observed in band 3 deficient RBCs. Membrane stability is thought to be achieved througli band 3-lipid interactions and loss of these interactions may be a key pathogenic event in hereditary spherocytosis (Peters et al., 1996). Previously, it was believed that spectrin and

PnLyrin bind to the RBC membrane and form the membrane skeleton only af'ter band 3 synthesis. Gcnc knockout studies of SLCQAI 4- mice conducted by Peten et al. (1996) cal1 8 this theory into question. They observed normal membrane skeleton formation in the total absence of band 3 and hypothesized that additional spectrin attachent sites may exist iii the overlying plasma membrane. It is their contention that band 3 plays a role in membrane stability rather than membrane skeleton formation.

Two hypotheses exist regarding the mechanism of plasma membrane loss in hereditary spherocytosis -a corpuscular hemolyiic anemia. The first argues that the is stabilized through interactions with spectrin at the membrane skeleton. It is proposed that spectrin deficient regions would tend to shed off membrane vesicles more readily resulting in spherocytosis. The second contends that band 3 serves to stabilize the plasma membrane through lipid interactions. Here it is proposed that in band 3 deficient

RBCs, the lipid anchoring interactions would be absent, resulting in the loss of unsupported lipids again leading to spherocytosis (Peters et al., 1996). Observations made by Peters et al. (1996) of band Ideficient RBCs fiom mice, tend to support the second hypothesis.

Further support for this mode1 was provided through studies of band 3-deficient cattle. Inaba er ai. (1996) conducted experiments on cattle with SLC4Al nonsense mutations that resiilt in the non-expression of band 3. The band 3-deficient cattle exhibit moderate uncompensated anemia with hereditary spherocytosis, retarded growth and defective

Cl'MCO; exchange causing mild acidosis. Deficiencies of many of the membrane skeleton proteins, namely spectrin, ankyrin, actin, and protein 4.2 have also been repoited. These results suggested that band 3 contributed to red ce11 membrane stability, CO2transport, and acid-base homeostasis, but was not essential to viability. 132 Respiration

Band 3 plays a critical role in respiration (CO2removal) by facilitating the reciprocal exchange of Cl- and HCO,' across the RBC membrane. Since CO, has very low solubility in H,O, it must be hydrated to form HCO,' prior to removal by the blood. nie conversion of CO2to carbonic acid (H,CO,) is catalyzed by carbonic anhydrase, a major cytoplasmic enzyme of the RBC (Tanner, 1993). At neutral pH, H,CO, readily dissociates into H' and

HCO,'. As intracellular HCO,' concentrations increase. band 3 facilitates its removal into the plasma in exchange for Cl- until a HCO,' concentration equilibrium is achieved. The resulting protons (H') are buffered by binding to which causes a shift in the 0: dissociation curve thereby releasing more O?in CO?rich environments (ie. tissue capillaries). in O, rich environments (ie. lung capillaries), a system reversal occurs and hemoglobin deprotonation nsults in increased O2binding affinity allowing for an influx of HCO,' and efflux of CIm(Tamer,1993; Vince and Reithmeier, 1998).

1.3.3 Acid/Base Homeostasis

Kidney band 3 is found only in the type A intercalated cells of the distal tubule where it contributes to urinary acidification by expelling HC03'across the basolateral membrane.

Briefly, the kidney s maintain acidhase homeostasis by reciaiming al1 filtered HCO ,' and excreting additional acid in sutficient quantity to match that produced by systemic metabolism. Although the proximal tubule cm reabsorb approximately 80-90% of filtered

HCO;, the final stage of acid excretion in the distal nephron is mediated largely by the type

A intercalated cells of the cortical and outer medullary collecting ducts (Jay, 1986;

Tanphaichitr et al., 1998). 1.3.4 Band 3 Associations

The N-terminal domain of band 3 associates with a number of glycolytic enzymes and hemoglobin. This domain is predominantly hydrophilic in nature with only 4 small hydrophobie regions. The first 33 amino acid residues of band 3 are extremely negative in charge, with 18 of the residues king either glutamic acid (Glu) or aspartic acid (Asp); no basic amino acid residues are located in this portion of the protein (Kaul et al., 1983).

Tyrosine (Tyr) of band 3 occurs primarily at Tyr8 (Dekowski et oz., 1983) although several minor sites have also ken identified (Yannoukakos et al., 199 1 a). Tyrosine phosphorylation occurs via and casein kinase and has been shown to inhibit glycolytic enzyme and hemoglobin binding affinity as a result of the added negative charge

(Low, 1986).

Several hi& affmity glycolytic enzyme [glyceraldehyde 3-phosphate dehydrogenase

(G3PDH), aldolnse and phosphofnictokinase (Murthy et al., 198 1 ;Tsai et al., 1982; Jenkins et al., 1985; Hams and Winzor, 1990)], binding sites in the extreme N-terminal domain of band 3 have been identified. Alterations in pH or ionic strength as well as cornpetitive inhibition have al1 been demonstrated to abolish afiity for these molecules (low, 1986).

Cystallographic examinations of the 33 extreme N-terminal amino acids have suggested that it is sterically possible for G3PDH and aldolase to bind simultaneously to band 3 (Walder et d.,1984). It is suggested that aldolase and share the same binding site on band 3 as they can displace each other but not G3PDH (Low, 1986). Stoichiometry experiments have show that a 1: 1 ratio for the band 3:aldolase complex nsults in 90% inhibition of aidolase activity while a 1: 1 ratio for the band 3 :G3PDH complex leads to total Il inactivation of G3PDH. Binding of phosphofhctokinase induces the dissociation of tetrameric phosphofiuctokinase to dimeric phosphofructokinase resulting in total inactivation. Low et al. (1986) suggested that associations of band 3 with glycolytic enzymes rnay be a way to store these enzymes for the 120 day life-span of a RBC.

Hemoglobin (Hb)has also ken shown to associate with the N-terminus of band 3.

Synthetic peptides for the first 1 1 arnino acid residues of band 3 were shown to bind Hb, most preferably de-oxy (de-oxygenated) Hb (Walder et al., 1984). Crystallographicanalyses of interactions between de-oxyHb and band 3 synthetic peptides suggest that five to seven amino acid residues of these peptides fit into the central cavity of Hb (Walder et of., 1984).

Binding is believed to be stabilized through ionic interactions that can be readily dissolvable at physiological pH or ionic strength. Binding is significantly reduccd by Hb oxygenation or by cross-linking of the Hb P-chains. Walder et al. (1984) suggested that cornpetition between G3PDH and Hb for band 3 binding may provide a potential means of linking RBC glycolysis to oxygen transport; if de-oxyHb afinity for band 3 is sufficirntly high enough to displace G3PDH, the overall rate of glycolysis rnay increase thereby altering the concentrations of glycolytic intermediates resulting in changes in the relative rates of reaction of the next steps in the reaction pathway.

Until recently, no interactions with the extreme C-terminal cytoplasmic tail of band

3 had been identified. The C-tmninal tail is comprised of 33 amino acids, 12 of which are acidic. in 1998, Vince et al. (1998) provided evidence that carbonic anhydrase II (CAII) bhds to this region. CAII is one of two isoforms found in erythrocytcs. CAII is present in roughiy 1,000,000 copies pet RBC, an amount similar to the number of band 3 molecules 12 per RBC. Immunoprecipitation (Vince et cri., 1998) with an antibody directed against the C- terminal tail of band 3 precipitated both band 3 and CAII from solubilized RBC membranes suggesting that CAII binds to the region closest to the lipid bilayer in a 1: 1 stoichiometry.

1.4 Related Anion Excbangers

The human (AE) is comprised of at least three members; their controlling genes SLC4A1, SLC4A2 and SLCIA3 encode band 3, AE2 and AE3, respectively (Gehrig et al., 1992; Yannoukakos et al., 1994). The chromosomal locations of al1 three genes have been identified; l7q2 1-22 (SLC4A 1) (Zelinski et al., 1993), 7q3 5-36

(SLCIA2)(Palumbo et al., 1986). and 2q36 (SLCIA3)(Yannoukakos et of., 1994). Although band 3 is restricted to cells of erythroid and kidney origin, AE2 and AE3 are found in a wide variety of cell types.

Protein sequence analysis of the C-terminal transmembrane domain of al1 three proteins reveal that they are highly homologous. AE2 and AE3 each contain approxiiiiately

1,200 amino acids, nearly 300 N-terminal residues more than erythroid band 3 (Tanner,

1993). Al1 three protein products are membrane molecules capable of anion transport. Botli

AE2 and AE3 exhibit Cl*/HCO,'exchange activity when expressed in mammalian ce11 lines, while AE2 also mediates CI' infiux when expressed in Xenopus oocytes (Lee et cil., 199 1).

Lee et al. (1991) demonstrated that the anion exchange activities of AE2 and AE3 are enhanced at high pH, an observation consistent with their possible involvement as pH regdaton (Zhang et al., 1996). While the isolated membrane domains display anion transport activity (Kopito et al., 1989; Lindsey et al., 1 990), the function(s) of the extended

N-teminal domain has yet to k elucidated. 1.5 Band 3 Variants

As many as 60 different erythroid and kidney band 3 variants have been identified and are summarized in Table 1. Identification of most of these variants was prompted by an observable phenotype, such as hereditary spherocytosis, Southeast Asian ovalocytosis, distal renal tubule acidosis, or congenital acanthocytosis. Characterization of these variants was accomplished largely through the molecular analysis of the controlling gene, SLC4A 1. Below are descriptions of clinical phenotypes for which band 3 variants have been identified through the characterization of SLC4A I mutations.

1.5.1 Hereditary Spherocytosis

Hereditary spherocytosis (HS) is the most prevalent fonn of corpuscular hemolytic anemia with a reported fiequency of 1 in 5,000 in Europeans (Morton et d., 1962). HS occurs when spheroidal, osmotically fragile RBCs resulting fiom the progressive loss of surface area, are prematurely destroyed in the spleen (Perrona et al,. 1999). The principal biochemical abnormality of HS (50% of reported cases) is a spectrin-deficicncy (Reinhart et al., 1994; Jarolirn et al., 1995; Hassoun et al., 1996; Hassoun et al., 1997). However deficiencies in band 4.2 (Takaoka et al., 1994), ankyrin (Hayette et al., 1998) and band 3 have also been reported. Band 3-deficiencies are proposed to result from the variants' inability to assume their proper structure or initiate membrane insertion. It is estimated that only 10-20% of HS cases result fiom band 3 deficiencies (Jarolim et al., 1995). Table 1. Atmormal cellular phenotypes defdby molecular analysis of SLC4AI

Variant Phenotypet SLC4Al Band 3 Reference DeSignation MutPtion$ Variation pW3w9 HS 10 bp duplication Frameshift - premature stop Jarolim et al., 1994a mu HS CGG+CAG Arg760Glu Jarolim et al., 1995 mm HS CGG-+TGG Arg870Trp Jarolim et al., 1995 -IV HS TGG+TGA T1p8 1 Stop Jamlim et al., 1994b; Jarolim et al., 1996 mv HS GGG-GAG Gly455Glu Jarolim et al., 1994b; Jarolirn et al., 1996 R.gue VI HS G+A intron 12 abnormal exon splice Jarolim et al., 1994b; Jarolim et al., 19% PWW vu HS del C (codon 616) premature stop Jaroiim et al., 1994b; Jarolim et al., 1996 mvm HS CTG+CCG Leu707Pro Jarolim et al., 1994b; Jarolim et al., 1996 WradccKralove HS CGG-+TGG Arg760Trp Jarolim et al., 1995 Hradec Hove II HS CAG+AAG splice site alteration Jarolim et al., 1994b; Jarolim et al., 1996 Jablonec HS CGC-+TGC Arg808Cys Jarolim et al., 1995 LY~ HS CGA+TGA Arg 15OStop Alloisio et al., 1996 Gtoas 4 G+A in promoter region Alloisio et al., 1996 Coimbra HS GTG+ATG Val488Met Alloisio et a1.,1993; Alloisio et al., 1997 Monàego * GAG+AAG Glu40Lys Alloisio et al., 1997 CCT-+TCT Pro 147Se Noirterre CAG-TAG Glu330Stop Gallagher et al., 1994 Chur GGC-GAC Gly77 1Asp Maillet et al., 1995 Milano 69 bp duplication Bianchi et al., 1997 Foggia del C (codon 31 1) fiameshift Miraglia del Guidice et al., 1997 Napoli ins T (codon 447) fiameshifi Miraglia del Guidice et al., 1997 Napoli ïï ATC-+AAC Ile783Asn Miraglia del Guidice et al., 1997 Worcester ins G (codon 1 70- 172) premature stop Jarolim et al., 1994b; Jarolim et al., 1996 Princeton ins C (codon 2730275) premature stop Jarolim et al., 1 994 b; Jar01im et al., 1 996 Truînov TAC-TAA Tyr428Stop Jarolim et al., 1994b; Jarolim et al., 1996 wa I)FnmV1 EXXX Table 1. Abnonnal cellular phenotypes defined by molecular analysis of SLC4AI

Variant Phenotypet SLC4Al Band 3 DeSignation Mutation% Variation

~3)-namd dRTA 13 bp duplication premature stop Karet et al.,1998 Memphis 1 M AAG-+GAG Lys56Glu Yannoukakos et al., 199 1 b; Jarolim et al., 1992b Memphis II M AAG+GAG LysS6Glu with DT (Leu854) Bruce et al., 1994 un-aamd CA CCG-+CTG Pro868Leu Kay et al., 1988; Bruce et al., 1993 t HS = hefeditary sphrrocytosis; dRTA = distal renal tubule acidosis; SA0 = Southeast Asian ovalocytosis; M = Mobility variation on a SDS-poiy8crylamide gel; CA = conge~talacanthocytosis; + = acts in tram to aggravate alleles causing HS t bp = base pair; del = deletion; ins = insertion; nt = nucleotide 17

HS is a heterogeneous syndrome with respect to clinical severity (ie. asymptomatic to severe hemolysis) (Reinhart et al., 1994), mode of inheritance (ie. autosomal dominant or recessive), RBC morphology and the underlying molecular defects (Palek and Jarolim,

1993). To date, approxirnately 40 mutations in SLC4AI have been identified as contributing to HS and represent the largest group of SLC4AI mutations identi fied by phenotype. The nature of the mutations include insertions, duplications, deletions, and point mutations nsulting in amino acid substitutions, piemature protein temination, protein elongation or loss of amino acids in band 3 (Table 1). In addition two mutations, Genas and Mondego, are believed to act in iram to aggravate the HS band 3 alleles Lyon and Coimbra, and result in a more severe form of HS (Alloisio et al., 1996; Alloisio et al., 1997).

1.5.2 Southeast Asian Ovalocytosis

Southeast Asian ovalocytosis (SAO) is a form of hereditary elliptocytosis charactenzed visually by the presence of oval RBCs, many of which contain one or two transverse ridges or longitudinal slits. SA0 is prevalent where malaria is endemic (ie.

Malasia, Papua New Guinea, Philippines and Indonesia) and is found at Frequencies approaching 30% in certain populations (Palek and Jarolim, 1993). Studies of ovalocytic

RBCs have identified many structural and hctional abnonnalities. However, affected individuals are often asymptomatic and have no clinically detectable hemol ysis (Liu et al.,

1990). Ovalocytic RBCs are rigid (Mohandas et al., 1984; Saul et al., 1984), a trait believed to have deleterious effects on survival. Ovalocytic RBCs also exhibit decreased osmotic fiagility (Honig et (il., 19711, increased thermal stability (Kidson et al., 198 1) and altered/decrcased expression of cythroid antigens (Booth et al., 1977). In addition, 18 ovalocytic RBCs are tesistant to in vitro invasion of the malaria 1 parasites Plosnrodiirm falciparum and PIasmodium knowlesi, suggesting that they may confer a selective advantage through parasite resistance (Kidson et al., 1981 ; Hadley et al., 1983). In vivo studies of ovalocytic RBCs have found reduced numbers of intracellular parasites in regions wliere malaria is endemic (Liu et al., 1990).

In 1990, Liu et al. (1990) demonstrated that band 3 was functionally and structurally abnonnal in individuals with SAO. Molecular analysis of SLC4Al from individuals with

SA0 identified a 27 bp deletion (codons 400-408) resulting in a deletion of nine amino acid residues hmband 3 (Table 1). These amino acids are localized to the boundary region of the cytoplasmic and membrane domains of band 3. Afinity analyses identified a stronger afiity between SA0 band 3 and ankyrin in intact RBCs (Liu et ol., 1990). Increases in tyrosine phosphorylation (Jones et al., 1990; Husain-Chishti et al., 1991) and markedly restricted lateral and rotational mobility of band 3 in the biological membrane (Liu et ni.,

1990; Tilley et al., 1991; Mohandas et al., 1992) have also been noted. Mohandas et 01.

(1992) suggested that the cytoplasmic domain conformation of the SA0 band 3 variant is altered resulting in its entanglement in the skeletal protein network. They hypothesized that this prevents the normal unwinding and stretching of the spectrin tetramers required for membrane extension thereby explaining the increased rigidity of the ovalocytic RBC membrane.

1.5.3 Distal Renal Tubule Acidosis

Recently, mutations in SLC4AI and the nsultant band 3 variant products have been hplicated as a key pathogenic event in the manifestation of distal renal tubule acidosis 19

(dRTA) in the acid-secreting type A intercalated cells (IC) of the kidney (Bruce et of., 1997;

Tanphaichitr et al., 1998). Mutations in SLC4Al resulting in dRTA fall into one of two categories; those exhibiting autosomal dominant inheritance and those exhibiting autosomal recessive Uiheritance.

In 1998 Jarolirn et al. (1998a) described an autosornal dominant form of dRTA in individuals from three unrelated families. Family members with dRTA have short stature, renal calcification and in some members repeated urinary tract infections. DNA seqiience analysis of SLC4AI fiom dRTA individuals identified a single heterozygous missense mutation underlying an Arg589His (R589H)substitution in band 3 (Table 1). Recombinant kidney band 3 (R589H) expressed in Xenopus oocytes had a 20 to 50% reductioii in CI*

NCOi exchange compared to the wild type (Jarolim et al., 1998a).

Bruce et al. (1997) identified members fiom four unrelated families exhibiting decreased anion transport leading to dRTA. Molecular characterization ofSLC4A 1 identified three different mutations resulting in three band 3 variants; Arg589His, Arg589Cys, and

Sm61 3Phe (Table 1). Family analysis determined each variant was inherited in an autosomal dominant fashion. The variant SK4Al DNA was expressed in Xenopus oocytes and anion transport activity was measured. Normally, Xenopus oocytes do not express endogenous band 3 but can be induced to express pure band 3 variants allowing for measurements of anion transport (ie. CI' uptake). Expression of the ArgS89His or the Arg589Cys band 3 variants resdted in decreased (up to 60%) Cl' uptake, while expression the Ser613Phe variant exhibitcd a very slight reduction in Cl- uptake as cornpared to wild type band 3.

Intenstingly, CO-expressionwith A (GPA) enhanced Cl- uptake for both variant and wild type band 3 suggesting GPA may be important for anion transport.

Recently, Tanphaichitr et al. (1 998) described an autosomal recessive form of dRTA

(Gly701Asp). Anion transport was analyzed in the Gly701Asp band 3 variant in Xenoplis oocytes. Transport activity levels for the kidney Gly7O 1Asp variant band 3 were 1.7% and

2.4% (at 20 OC and 37 OC respectively) of wild type function, while erythroid Gly70 1Asp transport activity levels were 11.7% and 7.2% of wild type. As with the previously mentioned example, CO-expressionofvariant band 3 with GPA completely resciied transport activity to that of wild type. Tanphaichitr et al. (1 998) hypothesized that since GPA and band 3 are CO-expressedin type A IC, this rescue may result in the milder form of dRTA. hterestingly, they also noted that total and surface associated band 3 variant levels were increased compared to those of the wild type. Despite the definition of CO-segregating mutations in SLC4AI that alter band 3 sequence, the mechanisms by which band 3 variants cause dRTA remain unclear.

1.S.4 Congenital Acanthocytosis

in 1988, Kay et al. (1 988) reported a single family with congenital acanthocytosis, an imgular RBC morphology which includes spine-like projections. Investigations of RBCs fkom family members identified a decrease in the number of high affinity ankyrin binding sites and an increase in anion transport. Protein analysis identified a band 3 variant that migrated slower on SDSRAGE than control band 3 and was later proven, through DNA sequence analysis, to be distinct fiom band 3 Memphis. This new variant termed band 3 HT

(high transport), exhibited significant increases in anion transport over controls (Bruce et al.

1993). DNA scquencing of SLCQAI hmaffected family membea identified a single C+T mutation in the 3' end of SLC4AI resulting in a Pro868Leu substitution in band 3. Bruce et al. (1 993) suggested that this substitution may prevent proper band 3 tetramerization thereby altering ankyrin binding affinity and facilitating the formation of the acanthocytic spine-like projections. They also suggested that the altered conformation of band 3 may prevent the binding of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), an anion transport

inhibitor used in anion transport assays, accounting for the observed increase in anion transport (Bruce et al. 1993).

1.6 RedCellAntigensLocalizedtoBand3

At present, amino acid substitutions underlying 2 1 Diego system antigens have been characterized and localized to band 3 (Table 2). Although the single DNA sample from a

Tr(a+) individual is not enough to conclude it controls Tt' expression, it has been included

in this section as an identified band 3 polymorphism. Figure 2 depicts the location of the

band 3 amino acid substitutions (black circles) as summarized in Table 2. One must be carefùl not to equate a single amino acid substitution with antigenicity, as antigen expression may require other factors such as altered side chain length, charge, three-dimensional conformation, or associations with other molecules (ie. Wrband glycophorin A) (Issiit et al.,

1975; Ring et al., 1994; Bruce et al., 1 995). Table 2. Definition of RBC antigens dedon band 3

Antigcn ISBT SLC4AI Exon Amino Acid Extracellular Reference Symbol Numba Mutation Substitutiont Loop

DiB DI 1 CCC-CTG Pro854Leu Bruce et al. 1994 Dib DI2 CCC Pro854 Bruce et al. 1 994 wr' DI3 GAG-AAG Glu658Lys Bruce et al. 1995 wf DI4 GAG Glu658 Bruce et al. 1995 wd' DIS GTG-ATG ValSS7Met Bruce et al. 1 9%; Jarolim et al. 1997a Rb. DI6 CCA-CTA Pro548Leu Jarolim et al. 1997a WARR DI7 ACT+ATT ThrS52lle Jarolim et al. 1997b EL0 DI8 CGG-+TGG Arg432Trp Zelinski et al. 1998a wu DI9 GGC+GCC Gly565Ala Zelinski et al. l998b BP' DI 10 AAC-AAA Asn569Lys Jarolim et a!. 1 W6b; larolim et al. 1998 b Mo8 DI1 1 CGT-CAT Arg656His Jarolim et al. l996b; larolim et al. 1 998b Hg. DI12 CGT+TGT Arg656Cys Jarolim et al. 1996b; Jarolim et al. I 998b VB. DI1 3 TAC-KAC Tyr555His Jaroiim et al. 1 996b; Jarolim et al. 1 998 Svrr DI 14 CGG-+CAG Arg646Gln Zelinski et al. 1997 BOW DI15 CCC+TCC Pro56 1Ser McManus et al. 1998; Poole et al. 1998; Jarolim et al. 1998b; McManus et ai. 1999 NFLD DI16 GAA-GAT Glu429Asp McManus et al. 1998; McManus et al. 1999 CCC+GCC Pro56 1Ala In8 DI17 CCC-+TCC Pro566Ser Poole and Hallewell, 1997 KREP DI18 CCC+GCC Pro566Ala Poole et al. 1998 Tr' DI 19* AAGjAAC Lys55 1Asn Jarolini et al. 1997a Ff DI20 GAG* AAG Glu480Lys McManus et al. (unpu blished observation) SWI DI2 1 CGG-TGG Arg646Trp Zelinski et al. (unpublished observation) t nurnber indicates band 3 amino acid position

+ only one DNA sample fiom a TP+ individual was available for analysis Figure 2. Localization of amino acid substitutions underlying band 3 antigens. Open circles represent intracellular and extracellular amino acids; grey circles represent proposed amino acids localized to the membrane. Black circles indicate the locations of ainino acids underlying band 3 akigeiis. 24

Characterization of Diego blood group system antigens through identification of amino acid substitutions has contributed to the current understanding of RBC antigens and band 3 topology. With the exception of Bpa,al1 substitutions underlying Diego blood group system antigens have extracellular locations. Presently, the amino acid substitutions for 2 1

RBC antigens have been reported in five extracellular loops of band 3 (loops 1,2,3,4 & 7).

Sixteen ofthe substitutions underlying RBC antigens are contained within extracellular loops

3 and 4 alone. Two different amino acid substitutions localized to extracellular loop I of

band 3 underlie two distinct RBC antigens, namely EL0 and NFLD, while loops 2 and 7 each harbor single arnino acid substitutions accounting for the Fr" and Dia/Dib

polymorphisms respectively. Extracellular loop 3 cames substitutions accounting for 10 different polymorphisms including Rba, WARR, Vg', Wda, NFLD,BO W, Wu, Jn" KREP

and TI? Two different amino acid substitutions at positions 561 and 566 underlie the

NFLDIBO W and Jn8/KREP blood group polymorphisms respective1y. Amino ac id

substitutions in the fourth extracellular loop account for the Sg,SWI, Mo', Hg", Wr" and

Wrb antigens with the Sw' or SWI substitution occumng at position 646, the Mo-r Hg"

substitution at position 656, and amino acid residue 658 accounting for the Wr" and Wrb antigens. Finally, a substitution in band 3 accounting for Bp", appears to be localized to the putative transmembrane portion of extracellular loop 3. Recall that the current band 3 mode1 depicted in Figun 2 was created based on rnodelling techniques and may not accurately represent the three-dimensional conformation of band 3 in vivo. Although the substitution underlying Bpa expression appears to reside in the membrane it is possible that it is extnccllularly located considering in vivo membrane fluidity and band 3 three-dimensional conformation .

Several Diego blood group system antigens also exhibit a-chymotrypsin sensitivity.

Two cleavage sites are located in the third extracellular loop of band 3 at tyrosines residues

553 and 555. Treatment of WARR+, Wd(a+), Tr(a+), Wu+, BOW+, Inta+), and KREP+

RBCs with a-chymotrypsin abolishes antigen recognition by their defining antibodies.

Enzymatic treatment of Rb(a+), ELO+, and WLD+ RBCs has yielded variable results, while a-chymotrypsin sensitivity of Vg(a+) and Bp(a+) RBCs has not been documented.

The remaining antigens are reported to be unaffected by a-chymotrypsin treatrnent (Reid and

Lomas-Francis, 1997; Coghlan, unpublished observations).

1.7 Human Blood Croups

The birth of blood group science began in 1875 when Landois cornpared the

agglutination reaction observed between the mixing of RBCs fiom an of one species

with the serum of an animal fiom a different species, to that of the phenornenon observed following the mixing of bacteria with appropriate immune sera (Race and Sanger 1975). He

suggested that the RBC agglutination was a direct result of the antigens contained on their surface teacting with antibodies in the sem. The first example of agglutination of human

RBCs by serum belonging to the same species had to wait an additional 25 years. in 1900,

Landsteiner made the startling observation that the sera of some of his colleagues agglutinateci the RBCs of some of the others (Race and Sanger, 1975). This discovery rcvolutionized the field of immunology and bore the fields of serology and blood group science. 26

The importance of blood group incompatibility as a potential cause of transfusion reaction and hemolytic disease of the newbom (HDN) has been known for decades. As a result, more chan 250 unique human erythrocyte antigens have been described to date, with contributions to the fields of genetics and molecular biology being made along the way.

For instance, FY (DufQ blood group system gene) was the first gene assigned to a specific chromosome through linkage with an inherited variation in heterochromatin (Donahue et of .,

1968), linkage between LU:SE (Lutheran blood group system gene : secretor gene) provided the first example of autosomal linkage in humans (Mohr, 195 1) and provided the first proof of crossingsver between homologous chromosomes (Sanger and Race, 1958). Linkage between RMELI (Rh gene : elliptocytosis gene) in some families and not in othen provided the first evidence that an inherited disease could have two genetic backgrounds (Goodall et al., 1953), and studies of AB0 provided proof of Mendelian inheritance of a normal characteristic (Epstein and Otenberg, 1908). Recent technological advances have allowed blood group scientists to characterize these clinically important antigens and document their interaction with the antibodies defming them.

1.8 International Society of Blood Transfusion Nomenclature

Currently, the International Society of Blood Transfusion (IS BT) Working Party on

Red Ce11 Terminology has four classifications for erythroid antigens; established blood group systems, collections, high incidence antigens and low incidence antigens. Blood group systems are dehed as genetically distinct groups of antigens controlled by a single gene or by closely linked homologous genes (Lewis et cil., 1990). The establishment of a new blood gioup system requins the identification of either, an antigen whose production is govemed 27 by a 'polymorphic' gene that is distinct fiom the genes involved in the established systems, or, an antigen whose production is govemed by a usually 'non-polymorphic' gene that is distinct fiom the genes involved in the established systems and that has a chromosoinal assignment (Lewis et al., 1990). Blood group collections are a series of antigens related as a result of serological, biochemical or genetic data. High incidence antigens (ISBT's 90 1 series) are antigens that have not been placed into a collection or system and are found witli a fiequency greater than 90% in a random Caucasian population. Low incidence antigens

(ISBT's 700 series) are antigens not belonging to a blood group collection or system and are found at fiequencies lower than 1% (sometimes substantially lower than 1%) in a randoin

Caucasian population (Daniels et al., 1995). To be included in a sy stem a new antigen must display either: i) an antithetical relationship between the new antigen and one already assigned to a system or ii) evidence fiom linkage analysis of family data, that the controlling allele is probably a newly recognized form of the pertinent gene, with siipporting serological or biochemical data (Lewis et al., 1990).

1.9 Blood Croup Systems

During the century that followed the original discovery of the AB0 blood groups more than 250 additional blood group antigens have been identifieci (Daniels et al ., 1999).

Early work involved serologic investigations of blood groups and determination of reaction patterns and mode of inheritance. Later, with advances in biochemistry, the biosynthesis of antigen structures were investigated. For example in the 1960s and 70s the carbohydrate biosynthetic pathways underlying AB0 blood antigen expression were elucidated (Heam et al., 1968; Race and Watkins, 1969; Watkhs, 197 1). Continucd technological advances 28 facilitated protein sequencing, and in the late 1970s the amino acid sequence of GPA (MN antigens) was determined (Blumenfeld and Adamany. 1978). More recently , molecular techniques have been employed to characterize many of the genes controlling blood group expression, and most recently have been applied in the characterization of the polymorphisms giving rise to many of the 22 1 blood group antigens of the established blood gioup systems (Daniels et al., 1999).

Currently there are 25 established blood group systems each containing at least one antigen (Daniels et al., 1999). Each blood group system is controlled by a single gene or by closely linked homologous genes. Chromosomal locations for al1 of the controlling genes have ken identified and are listed in Table 3 and depicted in Figure 3 (Reid et ni., 1998).

Blood group loci are spread throughout the genome residing on 12 different autosomes and the X chromosome. Eight chromosomes contain only one blood group gene locus, while two chromosomes, 1 and 19 harbor six and five loci respectively. Seventeen of the loci have been localized to chromosomal regions, whereas eight have been localized to a single liighly resolved band of a G-banded metaphase chromosome spread (Fig. 3).

Although gene products for al1 but one (RAPH)of the blood groups have beeii identified, the function of many of these has yet to bc elucidated (Reid et al., 1998). Several of these proteins are thought to be transporters, receptors and adhesion molecules, glycoproteins or hruisferases @aniels, 1999). Bnef descriptions of blood group system products and theù possible hctions, either known or Uiferred, follow. 29 Table 3. Established blood group systems and their controlling genes

ISBT Blood Gene Chromosomal Gene Product Goup Systemt Location

AB0 A = a-3-N-acetyl-D- galactosaminy 1-tram ferase B = a-3-D-galactosyl-transferase MNS (002) [43] GYPA GPA GYPB GPB GYPE GPE Pi Galactosy1-transferase RHD Acylated RhD protein RHCE Acylated RhCE protein Lutheran (005) [18] LU LU glycoprotein & B-CAM Kell(O06) [23] KEL Kell glycoprotein Lewis (007) [6] FüT3 a-3/4-L-fucosy ltransferase DuffY (008) 161 FY Fy glycoprotein Kidd (009) [3] SLC14AI Urea transproter (HUT1 I ) Diego (010) [2 11 SLC4A I Anion Exchanger 1 (Band 3) Yt (O1 1) [2] ACHE Acety lcholinesterase xs (012) 111 XG Xg glycoprotein Scianna (0 1 3) [3] SC Sc glycoprotein Dombrock (0 1 4) [SI DO DO glycoprotein Colton (01 5) [3] AQP 1 Channel-forming integral protein (AQP- 1) Landsteiner- LW LW glycoprotein Wiener (0 16) [3] Chido/ C4A C4A Rogers (O 17) [9] C4B C4B Hh (018) [Il Fm a-2-L-fucosy l-transferase (019) 111 XK Kx glycoprotein Gerbich (020) [7] GYPC GPC & GPD Cromer (02 1) [1 O] DAF CD55 (DAF) Knops (022) [SI CRI CD35 (CRI) Indian (023) [2] CD44 CD44 OK (024) [1] OK CD147 RAPH (025) [l] MER2 Not Defmed t numkr in round parentheses 0 denotes the ISBT bldgroup system number designation; numbers in square pemithcses idcatc the cunent nurnber of antigens contained within each systcm Figure 3. Chromosome diagram depicting the localization of the various blood group genes. individual chromosomes are numbered. The long and short msof each chromosome are separated by a horizontal line (centromere). Bldgroup genes (itdicized) encased in brackets indicate the boundaries of theu assignment. 1.9.1 Membrane Transport Molecules

RBC membrane transport proteins facilitate the movement of biologically important molecules such as anions, urea and water in and out of the cell. This group of membrane proteins have cytoplasmic termini, and an even number of transmembrane spa~inga-helical regions. Three RBC membrane transporters are known to have blood group activity, band

3 (Diego), AQP-1 (Colton) and HUTl1 (Kidd). Two additional proteins of undetermined function (ie. Rh glycoproteins and the Kx glycoprotein) are included in this section due :O their structural similarities with other transport proteins.

1.9.1.1 Band 3 and the Diego Blood Croup System

Currently the Diego blood group system is comprised of 21 blood group antigens including two pairs of antithetical antigens, Dia/Diband WP/Wrb, and 17 low incidence antigens. The underlying amino acid substitutions have al1 mapped to erythroid band 3 which appears to be associated with GPA in the membrane; immunoprecipitation studies using a monoclonal antibody directed against the C-terminal domain of band 3 CO- precipitated band 3 and GPA (Wainwright et al., 1989; Leddy et al., 1994). In addition the presence of GPA has been proven essential for Wib expression. Brire et of. (1995) demonstrated that in the absence of GPA, individuals with the Wp allele are serologically typed as Wr(a-b-). Further analysis suggested that amino acid residues 55 to 68 of GPA interact with the eighth tnuismembrme spanning domain of band 3. Since band 3 and the

Diego blood group system are major constituents of this thesis they are described in detail elscwhcrc. 1.9.1.2 Aquaporin-1 (AQP-1) and the Colion Blood Group System

The Colton blood group system consists of three blood group determinants; the polymorphic Co8and cab antigens and the highly prevalent Co3 antigen present on al1 RBCs, except those of the extremely rare Colton-nul1 phenotype, Co(a-b-) (Reid and Lomas-

Francis, 1997).

AQP-1 is a member of the farnily of channel-forming integral glycoproteins. AQP- 1 is encoded by AQPI which has been localized to chromosome 7pl4 (Moon et al., 1993).

AQP-1 is thought to span the membrane six times with both C- and N-termini having cytoplasmic locations. A single N-glycan is attached to an Asn residue located in the first extracellular loop (Preston and Agre, 199 1). AQP- 1 is present in approximately 1 20,000 to

160,000 copies per RBC and is believed to exist as a homotrimer in vivo (Denker er al.,

1988). The amino acid substitution contmlling Coaand Cobexpression is located in the first extracellular loop (Smith et al., 1994). AQP- 1 has also been identified in the kidney, choroid plexus and various other epithelial and endothelial tissues forming a water-selective charnel in the plasma membrane that enhances the transport of osmotically driven water. AQP- 1 may play a role in the proximal tubule and descending loop of Henle by reabsorbing water fiom the glomedat filtrate and may facilitate rehydration after shrinkage in the hypertonie environment of the muil medulla. AQP- 1 expression assays in Xenopus oocytes suggest it may aîso k involved in CO2 transport in the peripheral blood as increases in CO2 permeability were obsewed in the prescnce of carbonic anhydrase (Nakhoul et al., 1998). 33

Analysis ofAQPl fiom three healthy unrelated Co(a-b-) individuals identified three different homozygous mutations. Two individuals had no detectable levels of membrane

AQP-1; one had an AQPI deletion encompassing almost the entirety of exon 1, while the other had a single bp insertion that altered the reading fiame. The third Co(a-b-) propositus had barely detectable levels of RBC membrane AQP- î and DNA sequencing identiiied a single missense mutation underlying a Pro38Leu substitution. Jhterestingly, an immiinoblot assay of the renal tubules did not detect any AQP-1 calling into question the significance of

AQP-1 in renal function and suggesting a level of functional redundancy (Preston et al.,

1994).

1.9.1.3 Human UnaTransport Protein Il (HUT11)and the Kidd Blood Croup System

The Kidd (K)blood group system consists of three determinants; the polyinorphic

Jka and Jkb antigens resulting fiom a single amino acid substitution (Asp28OAsn) and the high fiequency Jk3 antigen present on al1 RBCs except those of the Kidd-nul1 phenotype,

Jk(a-b-). The Kidd glycoprotein (HUTI1) is a comprised of 39 1 amino acids. HUTI 1 traverses the membrane 10 times with both temini having cytoplasmic locations. A single N-glycan chain is attached to HUTl1 in the third extracellular loop (Reid and Lomas-Francis, 1997). in addition to RBC localization, HüTl 1 has been identified in endothelial cells of the vasa recta and in the vascular supply of the renal medulla, but not in the renal tubules (Daniels, 1999).

in RBCs, HUTl1 serves two main functions; it transports urea in and out of the RBC pnventing shrinkage that might otherwise occur in the renai medulla (a environment rich in ma), or subsequent swelling as they Ieave; and it prevents removal of urea from the renal 34 medulla thus preserving urea concentration in the kidney (Macey and Yousef, 1988).

The Jk(a-b-) phenotype is extremely rare in Caucasian populations but has a fiequency of 1J4OO in Polynesians (Henry and Woodfield, 1995). Jk(a-b-) individuals have impaired urea transport and urea concentrating defects. Analyses of Jk(a-b-) RBCs demonstrate a 1000 times slower urea transport rate than normal cells (Frohlich et al., 199 1).

In general, the Jk(a-b-) phenotype is not associated with any clinical syndrome which may be due to the presence of a homologous urea transporter, HUT2 (Olives et al., 1996).

1.9.1.4 Rh Glycoproteins and the Rh Blood Group System

The Rh blood group system is the most complex of al1 the blood group systems with at least 45 defined antigens (Daniel et al., 1999). The Rh system is encoded by two highly homologous closely linked genes, RHD and RHCE. RHD and RHCE encode the RhD (D antigen) and RhCE (CcEe antigens) glycoproteins respectively (Reid et al., 1998). RhD and

RhCE traverse the membrane 12 times with both termini being cytoplasmically located. The

Rh glycoproteins are closely associated with Rh50 an Rh-associated glycoprotein (RhAG).

Rh glycoproteins and RhAG exhibit 36% homology and adopt similar membrane conformations. These three protehs may form a membrane complex together with the LW

glycoprotein, Fy glycoprotein, CD47 and GPB @miels, 1999).

The rare Nd,phenotype occurs when no Rh antigens are expressed, and it is usuaily

associated with some degrec of hemolytic anemia, characterized by stomatocytosis and

spherocytosis (Daniels, 1999). RBCs from Rh,,,,, individuals exhibit functional and rnorphological abnomalities that can include abnomal membrane phospholipid organization

(Kuypers et al., 1984). Additionai abnomalities include increased cation permeability, 35 reduced cation and water content and decreased membrane cholesterol content (Daniels,

1999).

Although the precise function of the Rh glycoproteins has yet to be elucidated, stnictural similarities to other known membrane transporters have been observed (Cherif-

Zahar et al., 1990). Structural analyses suggest that they may be involved in solute transport or maintenance of membrane phospholipid symmetry by functioning as flippases. Although

RBC membranes fiom mu,,individuals have abnormal phospholipid organization, phosphatidylserine flippase activity is normal (Schroit et al., 1990).

1.9.1.5 Kx Glycoprotein and the Kx Blood Croup System

The Kx glycoprotein is 444 amino acids in length and potentially traverses the RBC membrane 10 times. Its predicted topographie arrangement shares structural similarities with memben of a family of proteins that CO-transporta neurotransmitter together with Na' and

Cr . The primary structure is most consistent with that of Na'-dependent glutamate transporters. The Kx glycoprotein is unglycosylated and associates with the Kell glycoprotein via disulfide bonds (Daniels, 1999).

Absence of the Kx glycoprotein results in McLeod syndrome which is characterized by depressed Kell-antigen expression, acanthocytic RBCs, and muscular and neurologie defects (Daniels et al., 1996). McLeod syndrome manifests clinically in muscle wasting, diminished deep tendon reflex, choreiforni movements and cardiomyopathy. Ln many cases, elevated ltvels of senun creatine kinase are also found. The association between deficiencies in Kx glycoprotein and neuroacenthocytosishas yet to be determined but the relationship to mammalian neurotrensmitter tnnsporters rnay provide a clue. 1.9.2 Glycophorin Molecules

The glycophorin farnily is comprised of two pairs of glycoproteins, GPA/GPB and

GPCIGPD. Al1 four glycoproteins have a highly glycosylated N-terminai domain, a single membrane spanning domain, and a C-terminal cytoplasmic domain. Although they have undetemined function al1 contain high amounts of sialic acid that might prevent RBC aggregation. Glycopho~scomprise a major portion of the carbohydrate matrix encompassing the RBC, called the giycocalyx, and are believed to protect FU3Cs from microbial attack and mechanical damage (Daniels, 1999).

1.9.2.1 Glycophorin A (CPA)/Glycophorin B (GPB) and the MNS Blood Group System

Antigens ofthe MNS blood group system result from polymorphisms found in GPA and GPB. At least 43 system antigens have been identified (Daniels et al. 1999). Most of the molecular determinants underlying these antigens arise through genetic rearrangements between the two closely linked controlling genes, GYPA and GYPB (Reid et ai., 1998).

These rearrangements include unequal cross-over. gene conversion, and splice site mutations that result in the formation of hybrid proteins. GPA is a major integral membrane protein found at nearly the same fkequency as band 3 - 1,00,000 copies per RBC. GPA is N- glycosylated and found either as a homodimer or a heterodimer with GPB. GPA andfor GPB nul1 phenotypes have ken identified but are quite rare. Most arc believed to anse by gene deletion events (Daniels, 1999).

Recently, evidence has been provided suggesting that GPA associates with band 3 in the RBC membrane. Co-expression studies of GPA and band 3 in Xenopus oocytes demonstrated increased Cf transport and higher levels of membrane band 3 than oocytes 37 expressing band 3 alone. Investigaton concluded (Groves and Tanner, 1992)that GPA may

fonn intracellular complexes with band 3 facilitating its translocation to the RBC membrane.

GPA is not believed to increase the amount of cellular band 3 but rather enhance the rate of

membrane band 3 accumulation. Interestingly , gene knock-out experiments involving

SLC4AI (the gene controlling band 3 expression) in mice resulted in a total absence of

membrane GPA despite the presence of GPA mRNA (Hassoun et al., 1998). This strongly

suggests that band 3 may function as a GPA chaperone directing it to the membrane. In

human cells however, expression of GPA can occur in the absence of band 3. Further

investigations detennined that GPA expression occurs pnor to band 3 expression during ex

vivo erythropoiesis (Southcott et al., 1997). It has also been proposed that GPA may play

a role in signal transduction (Daniels, 1999). If however a transmembrane signal can be

induced by ligand binding to an extracellular domain, the signal most likely passes to the

membrane skeleton via band 3 as native GPA does not contact the membrane skeleton.

1.9.2.2 Clycophorin C (GPC)/Glycophorin D (GPD) and the Gerbich Blood Group

System

Diffenntial translation initiation of the same mRNA results in two isoforms, GPC

and GPD. GPD is 21 amino acid residues shorter than GPC and results froin translation

initiation of Met-22 of GPC mRNA. The single Kg1ycan chah is attached to an Asn residue

located in the first 21 amino acid residues of GPC, the portion lacking in GPD. RBCs

lacking one or mon of the three high fkquency antigens (Ge2, Ge3, and Ge4) result from

missense mutations or exon deletions, while the expression of the four low fiequency

antigens (Wb, Ls: An8, and W') rcsult hm missense mutations or duplication or triplication of exon 3 (Daniels, 1999).

The function ofthe extracellular domain is unclear although the cytoplasmic domains of GPC and GPD are attached to the membrane skeleton hou& interactions with protein

4.1 and phosphoprotein p5S. Protein 4.1 is a putative protein link to the underlying spectrin/actin network while p55 is thought to be a stabilizing protein (Marfatia er al., 1997;

Workman and Low, 1998).

The rare Gerbich-nul1 phenotype (Leach phenotype) has been identified and results fiom the total absence of GPC and GPD. RBCs fiom these individuals exhibit altered RBC membrane morphology. Approximately 20 to 60% of these RBCs are classified as elliptocytic resulting in decreased membrane stability (Daniels et al., 1986). Concurrent decreases of up to 25% for protein 4.1 and of up to 98% for p55 were also observed (Cliishti et al., 1996).

1.9.3 Recepton and Adhesion Moleculta

Based on protein structural information (prirnary, secondary, tertiary or quaternary), many RBC surface proteins appear to be recepton or adhesion molecules. The function of most of these proteins in RBCs is unknown but can be inferred from observations of known fùnctions of the same proteins expressed in different tissues. Ce11 surface receptors bind specific ligands, such as hormones, activating effectors that produce intracellular signals.

Ce11 adhesion molecules facilitate ce11 to ce11 adhesion or adhesion events to the extraceilular matrix. It is not apparent why RBCs requk ce11 adhesion molecules although it has been suggested that the RBC proteins may be vestigal in nature serving some function during erythropoicsis. Them arc at least two RBC receptors that exhibit blood group activity; the 39

DufQ glycoprotein (Du@ system) and CD44 (Indian system). in addition, there are at least three supplementary rnembers that belong to the immunoglobulin-superfamily which will be discussed later; the Lutheran glycoprotein (Lutheran systern), ICAM-4

(LandsteinerIWiener system) and CD 147 (Okasystem).

1.9.3.1 DulTy Glycoprotein and the Duffy Blood Croup System

in people of Asian and European ancestry, the Duw (FY) blood group system consists of two determinants, Fy' and ~y~ giving rise to three phenotypes; Fy(a+b-),

Fy(a+b+), and Fy(a-b+). in Black Afiicans a third allele Fy is also present which does not produce Fy' or Fyb. in parts of West Afnca the fiequency of Fy approaches 100%. The Fy" blood group results fiom a Gly44Asp substitution in the FY glycoprotein. The Fy allele is identical to F# except for the presence of a second mutation in the erythroid specific transcription binding (GATA) consensus factor. in RBCs this mutation prevents FY glycoprotein formation but allows for expression of Fy in non-erythroid cells (Reid and

Lomas-Francis, 1997; Daniels, 1999).

The FY glycoprotein or DARC (Dufi Antigen Receptor for Chemokines) is 338 arnino acids in length. (Reid and Lomas-Francis, 1997). The protein is believed to traverse the membrane seven times with the N-terminus being extracel ularly located and the C- terminus being cytoplasmically located. The 65 amino acid N-terminus contains two potential N-glycosylation sites. The exact function(s) of FY glycoprotein in RBCs is unclear, however the structure of the FY glycoprotein is similar to the large superfamily of

O-proteins (Dohlmm et al., 1991). Members of this superfamily bind a variety of ligands that mediate the actions of extracellular sipals including light, odorants, neurotransmitters 40 and peptide hormones (eg. chemolcines). The FY glycoprotein bas been identi fied in RBCs, in endothelial cells lkgpost-capillary venules throughout the body, in epithelial cells of the renal collecting ducts and pulmonary alveoli, and in the Purkinje neurons of the cerebellum (Hadley et al., 1994; Chaudhuri et al., 1997; Horuk et al., 1997). Based on conservation of FY across species, the FY glycoprotein is believed to play an important role through interactions or communications with other cell-type dependent membrane components to elicit tissue specific responses upon ligand binding.

FY glycoprotein binding assays demonstrated its afinity for a variety of pro- inflamrnatory cytokines that in turn activate leukocyte recruitment. Examples of cytokines bound to the FY glycoprotein include interleukin-8 (IL-81, melanoma growth stimulatoiy activity (MGSA) of the C-X-C class and monocyte chernotactic protein- l (MCP- 1 )

(Darbonne et al., 1991; Neote et al., 1993; Homk et al., 1993). The chemokine binding pocket is proposed to include the fust and fourth extracellular domains that are thought to interact via disulfide bonds (Tournamille et al., 1997). The FY glycoprotein does not appear to be coupled to GTP binding proteins thereby downplaying its possible role in signal transduction. Unforhmately the function ofthe FY glycoprotein is unknown, however it may play a role as a clearance nceptor for inflamrnatory mediators or act as a scavenger for unwanted chemokines. The limited function of the FY glycoprotein in the RBC is exemplified through the fairly prevalent Fy-nul1 phenotype observed in many healthy individuals fiom West Afnca (Daniels, 1999). Finally, the Duw glycoprotein is essential to Plasmodium vivm invasion as it acts as a receptor (Hadley and Peiper, 1997). P. vivm is widcly distributcd throughout Africa and is unable to infect Fy(a-b-) individuals suggesting that the nul1 phenotype arose through selective advantage.

1.9.3.2 CD44 and the lndian Blood Croup System

CD44 is a ubiquitous glycoprotein with multiple isofonns. CD44 carries the low fiequency Ine and hi& fiequency Inb antigens (Spring et al., 1988). The multiple isofonns result fiom alternative splicing of at least half of the 20 exons present in the controlling gene,

CD44. The standard RBC and leukocyte isoform CD44H, contains a C-terminal cytoplasmic tail, a transmembnne domain, and an N-terminal extracellular domain. The globular structure of the N-terminal domain is a result of disulfide bonds. The N-terminus contains a region homologous to cartilage link protein (Daniels, 1999).

Many functions have been attributed to CD44 and include, participation in B and T ce11 activation, leukocyte adhesion to the extracellular matrix, endothelial cells and stroma1 cells, modelling of the extracellular matrix during wound healing and embryonic development, and lymphocyte-endothelial ce11 interactions involved in the localization of lymphocytes to the sites of infiammation (Borland et al., 1998). Most of the above mentioned îùnctions involve specific interactions between CD44 and hyaluronan, an extracellular matrix component (glycoamino glycan) of high molecular weight. CD44 has also been show to bind additional extracellular matrix components such as collagen, fibronectin and lamiriin. CD44 may function in erythropoiesis as it appears to be involved in the adhesion of burst fodgunits-erythroid (BFU-E) to the bone rnarrow (Daniels,

1999). 1.9.4 Imrnunoglobulin-Superfamily Glycoproteins

The Immunoglobin-Super family (IgSF) molecules are abundant on RBCs, leukocytes and a variety of other cell types. Members of the IgSF contain repeating extracellular domains that share sequence homology with the immunoglobin variable (V) and constant (Cl or C2) domains. The IgSF domains contain approximately 100 amino acid residues that interact to form hvo P-sheets stabilized by disulfide bonds. Many members are bctional receptors or adhesion molecules that may be involved in signal transduction

(Williams and Barclay, 1988; Daniels, 1999). There are at least three RBC IgSF glycoproteins exhibiting blood group activity; the Lutheran glycoprotein (Lutheran system),

ICAM-4 (LandsteinerIWiener system) and CD 147 (OkQysstem).

1.9.4.1 Lutheran Glycoprotein and the Lutheran Blood Croup System

The Lutheran blood group system consists of at least 18 antigens encoded in part by four allelic pairs (Daniels et al., 1999). A rare nul1 phenotype (Lu(a-b-)) has been observed and characterized. Lu(a-b-) arises from three different genetic backgrounds: a) homozygosity for a nonsense (inactivating) mutation in L 11, b) heterozygosity for an unlinked dominant inhibitor gene, ht(Lu) and c) hemi ygosity for an X-linked inhibitor gene

(Daniels, 1999). in RBCs, two isoforms of the Lu glycoprotein have been observed, an 85 kDa fom and a 78 kDa fonn that is identical to B-CAM.Both isoforms contain two V and three C2 IgSF domains, arranged as V-V-C2-C2-C2,and a ttansmembrane spaming domain

(Parsons et al., 1995). The 85 kDa isoform also contains a cytoplasmic tail that contains a

Src 3 homology domain suggesting a potential receptor and signal transducer function. The extracellular portions of both isofoms contain consensus integrin binding motifs. The Lu 43 glycoprotein is structurally similar to ce11 adhesion molecules, the human melanoma MUC 18 melanoma antigen and rdchicken neural adhesion molecules; the 78 kDa isoform is identical to B-CAM (Parsons et al., 1995).

The Lu glycoprotein is widely distributed throughout the body and has been identified in RBCs, the basement membrane of superficial epithelia and around mucous glands (Parsons et al., 1995). The late appearance of the Lu glycoprotein on erythroid progenitors suggests it may play a role in erythropoiesis (Southcott et al., 1997; El Nemer et al., 1998). Bot.isofonns have the same binding capacity for laminin, a major basement membrane component. Binding assays have demonstrated thst laminin binding corresponds with the level of Lu glycoprotein expression. Lu(a-b-) RBCs (ie. no Lu glycoprotein) do not bind laminin despite the expression of CD44,a putative laminin binding protein. RBCs frein patients with sickle ce11 disease expressing high levels of Lu glycoprotein exhibit increased quantities of laminin binding comsponding to the increase in Lu glycoprotein (Daniels,

1999). Although the exact function of RBC Lu glycoprotein is unknown, the information summarized above may provide potential answers.

1.9.4.2 Intemllular Adhesion Molecule 4 (ICAM-4) and the Landsteiner-Wiener Blood

Group System

The LW (Landsteiner-Wiener) blood group system consists of three antigens; the high incidence antigens LW and LWab,and the infiequent antigen LWb. The LW blood group system is serologically associated with the Rh blood group system (Daniels, 1999) as

RhD+ RBCs more shongly express LW antigens than RhD negative RBCS,and mu,,RBCs arc also LW-null. The LW-nul1 phcnotypc, LW(a-b-ab-), is extremely rare and nsults fiom 44 a premature termination codon arising fiom a 10 bp deletion in the first exon of LW

(Hermand et al., 1996). The LW gene contains three exons and has been localized to

19~13.3(Hermand et al.. 1995). The gene encodes ICAM-4 which contains three major domains, a C-terminal cytoplasmic tail, a transmembrane domain, and an N-terminal extracellular domain comprised of two C2 IgSF domains. The first C2 domain shares 30% sequence identity with ICAM-1 (CD54),ICAM-2 (CD102) and ICAM-3 (CD50)(Bailly et al., 1994).

ICAM-1, -2 and -3 are al1 members of the IgSF and al1 bind CD 1 1a/CD 18 integrins present on lymphocytes, granulocytes, monocytes and macrophages (Daniels, 1999). Also present on leukocytes, ICAMs are important regulaton of T-ce11 activation. They enhance interactions between T cells and antigen presenting cells. Binding interaction analyses demonstrate that many leukocyte cell lines (eg. monocytes, B cells, T cells and NK cells) can be bound to immobilized ICAM-4 and that this binding can be blocked by antibodies directed against CD18 or in the case of T cells, CD1 la. Ce11 lines derived from patients lacking CD1 laICDl8 did not bind LW glycoproteins. Bailey et al. (1994) concluded that the LW glycoprotein is a ligand for CD1 1fCDI 8 inteqins based on their observation that immobilized CD1 lafCDl8 and CD1 1bICD 18 integrins bind RBCs lacking ICAM- 1, -2, and

-3, rendering the name ICAM-4. Although the function ofthe LW glycoprotein in RBCs has yet to k resolved, it is curious why a RBC would have an adhesion molecule for integrins that is also localized to ledcocytes and the vascular endothelium. 1.9.4.3 CD147 and the OK Blood Croup System

nie OK blood group system contains a single blood group antigen, OP. Oka is found at extremely high frequency and only eight unrelated Japanese Ok(a-) individuals have been identified (Daniels, 1999). The absence of Oka results fiom a Gld2Lys substitution in

CD147 encoded by OK. CD147 is comprised of one V and one C2 domain organized in an unusual C2-V arrangement (Spring et al., 1997). CD147 has been identified on RBCs, leukocytes and in human leukemic ceIl Iines. Although CD147 function is unknown, the highly conserved cytoplasmic and trammembrane domains suggest it may be a men1 ber of a signal transduction cornplex. bterestingly, expression of CD 147 on leukocytes is strongly upregulated upon ce11 activation suggesting it may also be a leukocyte activation-associated glycoprotein (Kasinrerk et al., 1992). Sequence analysis of the lgSF domains is most consistent with ce11 adhesion molecules or growth factor recepton. Finally, CD147 on tumour cells induces the production of collagenase and other extracellular matrix metalloproteinases (MMP) and may be involved in tumour invasion and metastasis. In healthy tissue CD147 rnay function in embryonic development or wound healing by causing dermal fibroblasts to increase their MMP production, thus facilitating tissue remodelling.

1.9.5 Complement Regulatoy Glycoprotein Molecules

nien are at least two RBC glycoproteins that protect against destruction by autologous complement and have blmd group activity; decay-accelerating factor (Cromer system) and complement receptor type 1 (Knops). Two additional proteins, C4A and C4B, carrying ChidolRogers bldgroup antigens regulate the complement cascade. 1.9.5.1 Decay-Accelerating Factor PAF) and the Cromer Blood Group System

The Cromer blood group system contains at least 10 blood group antigens, seven of high fkequency and three of low fiequency (Reid and Lomas-Francis, 1997). The Cromer- nul1 phenotype (Inab) arises fiom different single base pair mutations resulting in the insertion of a premature stop codon (Lublin et al., 1994; Wang et al., 1998).

The DAF glycoprotein is a member of the RCA (regulator of complement activation) farnily of proteins that regulate the complement cascade. DAF contains between two and 30 disulfide bonded repeating domains called complement control protein domains or short consensus repeats (SCRs). Each SCR contains 56-79 amino acid residues including four cysteine residues. The two disulfide bonds present in SCRs give rise to its loop structure.

DAF contains four SCRs but does not traverse the membrane. It is tethered to the membrane by a glycosylphosphatidylinositol (GPI)anchor (Daniels, 1999).

DAF has been identified on all blood cells, vascular endothelial cells, is widely distributed on epithelia, cells of the gastrointestinal and genitourinary tracts, and is also fond in the cells of the central nervous system. A soluble form of DAF has also been identified in plasma and other body fluids. DAF hctions by inhibiting the amplification stage of complement activation - DAF pnvents the association of specific complement factors that when associated, form the C3 convertases of the classical and alternative pathway S.

1.9.5.2 Complement Receptor Type-1 (CRI) and the Knops Blood Group System

CR1 is a large integral RBC membrane protein that belongs to the RCA superfamily.

The Knops blood group system contains five antigens, four of relatively high fnquency and 47 one of lower fipquency. The Knops antigens Vary greatly in strength rcsulting in variable

CR1 expression in difierent individuals. The number of CR 1 molecules per RBC also varies significantly from person to person, ranghg fiom 20 to more than 800 (Moulds et nl.,

1992a). No tme Knops-nul1 phenotype has been observed although in the Helgeson phenotype the Knops system antigens cannot be detected by conventional serological methods due to the very low CR1 copy number. The molecular basis of Knops systeni antigens has yet to be detemined.

CR1 has four isoforms of varying molecular weights, each of which have transmembrane domains and short cytoplasmic C-terminal tails. The extracellular domain of the cornmon A isofonn contains 30 SCRs of which the 28 most N-terminal SCRs can be divided into four larger regions called long homologous repeats (LHRs) based on sequence homology (Aheam and Fearson, 1989). CR1 is also expressed on peripheral blood cells, follicular dendritic cells and kiâney glomerular podocytes.

The major fûnction of RBC CRL is to bind and process C3b/Clb coated immune complexes and transport them to the spleen and liver for removal (Aheam and Fearson,

1989). CR1 also demonstrates decay accelerating activity for C3 convertase complexes of the classical and alternative pathways of the complement cascade. CR1 is a cofactor for factor4 mediated cleavage of C3b and C4b. Phagocytosis by monocytes and neutrophils of

C3b and C4b coated particles is believed to be enhanced by CRI. CR1 may also play a role in the pcevention of complement mediated lysis of RBCs (Aheam and Fearson, 1989).

Intercstingly, Knops system antibodies do not significantly reduce the swival of transfused incompatible RBCs despite relatively large quantities of IgG bound to CR1 (Daniels. 1999). 1.9.5.3 C4NC4B and the ChidolRogers Blood Group System

nie C4A and C4B glycoproteins are encoded by Clci and C1B which control

Chido/Rogers blood group expression. C4A and C4B regulate the complement cascade;

C4B forms part of the C3 convertase enzyme that activates the next step in the complement cascade by cleaving C3. In general, C4A harbors the Rogers antigens while C4B harbors the

Chido antigens, however occasional gene cross-overs produce exceptions (Giles, 1987).

1.9.6 Blood Group Enzymes or Putative Enzymes

At least two blood group system products fa11 into this category, one of known function and one with inferred fùnction based on structural information; the Yt and Kell blood group system products, namely acetylcholinesterase (AChE) and a putative metalloendopeptidase respectively.

1.9.6.1 Acetylcholinesterase (AChE) and the Yt Blood Group System

AChE is attached to RBCs by a GPI-anchor. AChE hydrolyses the neurotransmiiter acetylcholine upon its release fiom the pre-synaptic terminal thereby producing a shon signal. AChE on RBCs is enzymatically active (Spring et al., 1992) although its function in RBCs is unclear. The Yt8 and Ytb antigens represent a single arnino acid substitution in

AChE (Bartels et al., 1993).

1.9.6.2 Putative Metalloendoptptidase and the Kell Blood Group System

The Kell blood group system consists of 23 antigens most of which arise from single amino acid substitutions in the Kell glycoprotein (Daniels et al., 1999). The Keli-nul1 phenotypc has many differcnt molecular backgrounds, al1 of which prevent the translocation of mature processeci Kell glycoprotein to the RBC membrane. The Kell glycoprotein is an 49 integral membrane protein containhg an Kterminal cytoplasmic tail, a single tnuismembrane domain, and a long C-terminal extracellular domain. The extracellular domain contains 665 amino acid residues, six potential N-glycan attachment sites and 1 5 cysteine residues (Lee, 1997). Cys72 is attached to Cys347 of the Kx glycoprotein via a disulfide bond. Recall that the McLeod syndrome is associated with Kx-deficient RBCs and nduced expression of Kell antigens.

The Kell protein contains a pentarneric zinc-binding motif present in members of the zinc-dependent endopeptidase family. It shares sequence homology with neutrai endopeptidase 24.1 1 (NEP or CDlO) and endothelium-converting enzyme (ECE) (Turner and Tanzawa, 1997). NEP inactivates a wide variety of peptide hormones including encephalins. ECE catalyses the biosynthesis of endothelin, a vasoconstrictor enzyme. A substrate for the Kell glycoprotein has not been identified. The Kell protein may be involved in early stages of erythropoiesis as it appears prior to GPA or band 3. Suggestions of its involvement in modulating peptide growth factors on the ce11 surface do not seem implausible (Vaughan et al., 1998).

1.9.7 Xg Glycoprotein and the Xg Blood Croup System

The Xg blood group system consists of a single antigen (Xga),that is fully developed at birth. The XG gene has ken localized to the pseudoautosomal region of the X chromosome, is not subject to X inactivation and encodes the Xg glycoprotein. (Daniels,

1995). DNA sequena analysis ofXG predicts a 180 amino acid protein with an extracellular

N-teminiil domain, a trammembrane domain, and a cytoplasrnic C-terminal domain.

Containeci within the appmxirnately 140 extemal amino acid residues are 16 potential O- 50

glycosylation sites (Daniels, 1995). Little is known of the Xg glycoprotein's function.

1.9.8 Carbohydrate Structures and the ABO, H, Lewis and P Blood Group Systems

The ABO, H and Lewis oligosaccharides are ubiquitous structures found in many tissues and are often referred to as the histo-blood groups. The AB0 and H antigenic

determinants are carried on various glycolipids and glycoproteins of the RBC membrane

@miels, 1995; Daniels, 1999). The majority of AB0 and H specificity is determined by the

N-glycan of band 3 and a RBC membrane . The Lewis antigens are not

produced in or by the RBC but rather they are acquired from the plasma. Not much is known

about the P blood group system or the gene controllhg its expression (Pl). Pl is known to

code for a galactosyltransferase (Reid and Lomas-Francis, 1997; Reid et al., 1998). The

single antigen in the P blood group system is carried on a carbohydrate determinant linked

to a glycosphingolipid through ceramide. nie functions of al1 three blood group structures

are unknown, apart fiom their contributions to the glycocalyx. These may include resistance

to micro-organism invasion, or protection from mechanical stresses encountered in the

capillaries.

1.9.9 Do Glycoprotein and the Dombrock Blood Croup System

The Dombrock @O) blood group systern consists of five antigens localized to the

Do glycoprotein (Banks et al., 1995). nie Do glycoprotein is a GPI-linked glycoprotein of

approximately 48 to 60 kDa (Telen et al., 1990). Very little is known of its function.

1.9.10 Blood Group Products of Unimown Function

Two molecules with blood group activity fa11 into this category, the Sc glycoprotein

containhg Sciama (SC) blood pupsystem antigens and the yet to be defmed product of 5 1 the RAPH blood group system carrying the RAPH blood group antigen. The genes controlling the expression ofeach have been identified and their chromosomal locations have been detemined (Noades et al., 1979; Daniels persona1 communication, 1998).

1.10 Diego Blood Group System and Band 3 Associated Antigens

Diego blood group system (DI) expression is controlled by SLCJA 1. Currently, the

Diego systern is comprised of 21 antigens (ISBT numben DI1-2l), some of which are known to stimulate the production of clinically significant matemal antibodies (eg. anti-Di3, anti-Dib, anti-ELO, and anti-Wf') causing HDN.

Al1 characterized amino acid substitutions underlying the 2 1 Diego system antigens are located in the transmembrane domain of band 3 and more specifically in extracellular loops 1,2,3,4 and 7. The amino acid substitution underlying Bp' is the sole exception being located on the most extenor portion of the sixth transmembrane-spa~inghelix.

Since 1993, the Diego blood group system has seen a large expansion. Originally comprised of only two antithetical antigens, Di' and Dib, the system now contains 21 characterized antigens. The arnino acid substitutions underlying each of the 2 1 Diego blood group system antigens are listed in Table 2. The amino acid residues underlying the 2 1

Diego system antigens covet al1 4 families of amino acids; five are basic (WP, Bp" Moa,

Sur, and Fr'), one is acidic (NFLD), five are polar (WP,Se BOW, Jnm, and Trl), and 1 1 are non-polar (Di', Dib, Wd', Rb8,WARR, ELO, Wu, Hg', NFLD, KREP,and SWI). As there arc approximately 90 extracellular amino acid residues in band 3, many additional antigens cumntly fond in the ISBT's high and low incidence antigen series are potential Diego system candidates. 52

Although Figure 2 depicts the amino acid substitutions that underlie specific Diego blood group antigens, one must be carefbl not to equate antigenicity with the respective substitution. Rather, antigen expression seemingly involves additional factors such as three- dimensional conformation, amino acid charge, side chah length and in certain instances associations with other proteins (ie. the Wright antigens and GPA). Listed below are serological descriptions of each Diego blood group system antigen.

1.10.1 Diego; Di' and Dib;DI1 and Dl2

Dia was first mentioned in 1954 by Levine et al. and was the subject of a full length report (Layrisse et al., 1955) detailing the pattern of reactivity of an antibody produced in a Venezuelan woman that caused HDN. Subsequent investigations of the antibody found it to react with the RBCs of her husband and two of his three siblings (Levine et al., 1956).

Further investigations of the Venezuelan family, including members from four successive generations, suggested that the blood characteristic was inherited in an autosomal dominant fashion. Lewis et al. (L957), studying a Canadian-Japanese kindred segregating for Din confïnned autosomal dominant transmission of the trait.

Although Di8 was originally described in what was believed at the time to be a

Caucasian family, the family was thought to contain racial admixture based on the observation of physical features consistent with those of native Indians. Due to this astute observation, studies of Di8 distribution wen conducted on other genetic populations such as the Caribe and Arawaco Indians (Levhe et al., 1956; Junqueira et al., 1956), northem

Natives (Lewis et al., 1956a), Inuit (Lewis et al., 1956b), Chinese and Japanese (Layrisse and hncis, 1956; Lewis et al., 1956a), Afiican Americans, Western Afiicans, and 53

Caucasians. It was concluded that Dia was of very low frequency and was a characteristic generally identified in, but not exclusive to, people of Mongolian descent (ie. Chinese,

Japanese and native Arnericans).

In 1967, Thompson et al. (1967) described the antibodies defining the antithetical antigen Dib in two individuals. in the first case, the production of anti-Dib occurred as a result oftransfûsing a Di(a+) individual with Di@+) RBCs. The second appeared in a Di(a+) woman who had no reports of a previous blood transfusion but had nine children and was most likely exposed to Di(b+) RBCs through a fetal bleed. Dib is a very cornmon antigen that has been found in al1 populations tested to date. The Diego antigens were excluded froni the ABO, MNS, P, Rh, Kell, Dum, Kidd and Xg blood group systems and became the tenth established blood group system (ISBT number 0 1 0).

Dia/Dibexpression is controlled by a single Pro854Leu substitution in the seventh extracellular loop of band 3 (Bnice et al., 1994).

1.10.2 Wright; WP and Wrb; DI 3 and DI4

In 1953, Holman identified an antibody that defined Wr" (Holman, 1953). This antibody was responsible for HDN. The antigen was named for the family in which it was found. Examination of RBCs fiom family members identified Wr' in al1 three children.

Investigation of members fiorn the father's family also identified two Wr(a+) individuals and thus demonstrated its inheritance. Further tests did not detect any serologic relationship to other known bldgcoup antigens and concluded that WP was independent of ABO, Rh,

MNS and Kidd. Additional cases of severe HDN have since been reported (Wiener and

Brancato, 1953) as have cases of severe transfusion reaction (TR) (van Loghem et al., 1955; Metaxas and Metaxas-Buhler, 1963).

Eighteen yean later, in the serum of a Wr(a+) individual, antibodies to what was believed to be the highly prevalent antithetical antigen, WP (Adams et al. 1971) were identified. The serum fiom the propositus reacted with al1 cells tested except for her own leading researchen to believe that she was homozygous for Wr". Titration tests have shown that her RBCs reacted consistently to a higher titer than did the cells of unrelated or related

Wr(a+) individuals.

A single Glu658Lys substitution in the fourth extracellular loop of band 3 gives rise to Wi' (Bruce et al., 1995).

1.10.3 Waldner; Wd'; Dl5

Mile testing Red Cross blood donors for Fr(a+) propositi, Lewis and Kaita (1 98 1) fortuitously identified Fr(a-) RBCs fiom three individuals that reacted with a serum containing multiple antibodies. Approximately 4,000 additional unrelated samples were tested without fmding a similarly reactive sample. Further investigations of the three propositi (Manitoba Hutterites) and their families identified an additional 10 reactive samples. Hutterites are the descendants of 2 15 people who migrated to South Dakota from

Gennany between 1874 and 1877 and later moved to Canada. This previously unrecognized antigen was detennined to have an autosomal dominant mode of inheritance and was desipated Waldner as al1 original propositi had that sumame. In the original paper, Wda was excluded fiom the ABO, Chido, Colton, Dombrock, Du@, Kidd, MNS, P, and Rh blood group systems and the use of Wbas a potential Hutterite marker for genetic studies was suggested (Lewis and Ksita, 1981). 55 Wd8 has also been identified on the RBCs fiom members of a family from Holland

(Lewis et al., 1985) and on the RBCs firom two individuals of a family from South Africa belonging to the Hei//om (Moores et al., 1990). The Hei//om are descendants of the Khoisan race which is compri~dof the Khoi and San people that live in the region of the Cape of

Good Hope. This area was htcolonized in the Vhcentury by people of Dutch and

Geman descent and it is well known that the colonists often had Khoi wives. Moores et al.

(1990) postulated that Wd' was introduced into the Khoi population through these nieans.

Wd' expression results fiom a Va1557Met substitution in the third extracellular loop of band 3 (Bruce et al., 1996; Jarolim et al., 1997a).

1.10.4 Redelberger; Rb8; DI6

Rb' is a low fkquency antigen that was first identified in three blood donors. Family analysis suggested that Rb' was inherited in an autosomal dominant fashion (Contreras et al.,

1978). Ln hopes of Mercharacterizhg this antigen, samples were sent to several reference laboratories for testing. Al1 concluded that this was a newly defined antigen and it was given the designation Rb'. A second Rb(a+) blood sample 0 was identified after screening approximately 10,000 blood sarnples at the North London Blood Transfusion Centre. These

RBCs were observed to have the sarne pattern of reactivity with a series of anti-sera as the

RBCs fiom the original propositus, Mr. Redelberger. Absorption studies conducted with

RBCs fiom Mr. Redelberger or NM removed the activity for their own and the other's cells.

A third propositus was found when she required a transfusion and an unexpected reaction with one of several sera was observtd. Additional studies conducted on the RBCs of the third propositus rcvealed identical teaction patterns as with the RBCs of Mr. Redelberger. 56

Rba segregates independently from the ABO, MNS, P, Rh, Kell, Du@ and Kidd blood groiip systems. Although the fiequency of Rba is extremely low, the fiequency of anti-Rbais much greater and it is ofien found in serum containing antibodies to other low incidence antigens, such as Bpa(Contreras et al., 1978).

Molecular analysis of DNA fiom Rb(a+) individuals determined that a Pro548Leu substitution in the third extracellular loop of band 3 gives rise to the Rb' antigen (Jarolim er al., 1997a).

1.10.5 Warrior; WARR; DI7

Investigation of a mild case of HDN in an infant of a Native Amex-ican family led to the recognition of a new low incidence RBC antigen tenned WARR (Coghlan et al., 1995).

A systematic search of 8,275 individuals (1,083 with some Native American ancestory) for additional WARR propositi found only one additional WARR+ individual - the sister of the original propositus. Upon subscquent investigation, RBCs fiom the wife of a WARR+ family member were also found to be WARR+. A common WARR+ ancestor between the two families has not ken identified but is likely to have existed due to the rarity of this antigen. Genetic linkage data fiom two kinàreds excluded WARR nom the MNS, Lutheran,

Lewis, Duffy, Kidd, Xg, Chidd'odgers, Kx and Gerbich blood group systems, wliile serologic and molecular evidence suggested it was not likely part of the Kell or Yt blood group systems. In addition, it was obsewed that 26 of the authon' collection of sera containing multiple antibodies to low incidence antigens reacted with the propositus' RBCs by direct agglutination of untreated cells or by the indirect antiglobulin method. Serologic testing of RBCs firom one of the chilâren hmthe WARR+ x WARR+ mating gave 57 consistently stronger reactions with several examples of anti-WARR than did the cells from her parents or siblings (al1 WARR+) and was therefore interpreted to be a WARR homozygote (Coghlan et al., 1995).

The WARR antigen results fiom a Thr552Ile substitution in the third extracellular loop of band 3 (Jarolirn et al., 1997b).

1.10.6 ELO; ELO; DIS

Otiginally mentioned in 1956 (Milgrom et al. ,1956) in a list of 'private' antigens,

EL0 was not given an ISBT designation (700.5 1) until 1991 (Daniels er al., 1993). Genetic and serologic studies conducted by Coghlan et al. (1993) on a three generation family provided evidence for ELO's exclusion fiom the MNS, Rh, Duffy, Kidd, Xg, Kx, Gerbich,

Lutheran, Sciama, Dombrock and Colton blood group systems and probable exclusion from the Kell and Chido/Rodgers systems. EL0 has been found on the RBCs of individuals with a variety of genetic backgrounds (Belgian, Iranian, Greek, and Anglo-saxon) but is of extremely low fiequency.

An Arg432Trp substitution in the first extracellular loop of band 3 underlies the EL0 polymorphism (Zelinski et al., 1998a).

1.10.7 Wulfsbcrg; Wu; DI9

Investigation of an umxpected incompatible crossmatch led to the realization that

Mrs. Wulfskrg's RBCs had a unique low incidence antigen. Screening of 8,406 patient sera identifiedthm additional examples of antibodies to Mrs. Wulfsberg's RBCs. Unfortunately due to a small family size no inherîtancc information or exclusion information could be

BSCCrfainaî. In 1976 the first full length paper (Komstad et al., 1976) demonstrated that Wu 58 was inherited in an autosomal dominant fashion, segregated independently of sex and the

ABO, MNS, Rh, Lutheran, Du@, and Kidd blood group systems and was most likely independent of Kell. Four years later, four additional unrelated Wu+ samples were found in a total of 16,476 South Australian blood donors allowing for its exclusion fiom the P blood group system (Young et al., 1980). In recognition of al1 this information, the ISBT designated Wu as 700.13. Recently, three Wu homozygotes have been identified in a family segregating for Wu (Moulds et al., 1989). Antibodies defining Wu are commonly found in sen containing antibodies to various other low incidence antigens.

In the past, there has been some confusion surrounding the Wu antigen. in 1973 the identification of a "new" blood group antigen 'Hov', was reported (Szaloky et al., 1973).

In this report, Hov was found not to react with anti-Wu and thus the ISBT gave it the 700.38 designation. in 1992, Moulds et al. (1992b)detemined Wu and Hov to be identical through complementary and duplicative serologic experiments in four separate laboratories.

Consequently, the ISBT declared 700.38 (Hov) obsolete (Lewis et al., 1990).

Molecular analysis ofSLC4A 1 on DNA fiom Wu+ family members ident ified a point mutation giving rise to a Gly56SAla substitution in the third extracellular loop of band 3

(Zelinski et al., 1998b).

1.10.8 Bisbop; Bp8; DI10

Originally discovercd by Cleghorn in 1964, Bpawas listed in a table of low incidence antigens by Race and Sanger (1975). The first cornplete report carne in 1970 (Liotta et al.,

1970) when Bp8 was descrikd as an extremely rare antigen found only in two unrelated individuais. At the tirne, fmily analysis and scgngation information had excluded Bpafiom 59 al1 established blood group systems and detennined BpQo be inherited in an autosomal dominant fashion. Further, examples of anti-Bpa are oflen found with antibodies to other naturally occhglow incidence antigens and is fiequently found in conjunction with anti- Wi.

An Asn569Lys substitution in the a region proximal to the third extracellular loop underlies the Bp' antigen (Jarolim et al., 1996b; Jarolim et ai., 1998b).

1.10.9 Moen; Mo'; DI11

First described in an abstract to the 1972 American Association of BIood Banks and intern~tionalSociety of Hematology Joint Meeting in Washington, Moa has yet to be the subject of a full length report (Jarolim et al., 1998b). The presence of the antibody defining

MoBwas initially identified in a serum containing antiJna and has since been identified in five additional sera containing multiple antibodies. A second example of Mo' was identified among 5,000 surveyed Oslo blood donors (Jarolim et al., l998b).

The Mo8antigen results fiom a single Arg656His substitution located in the fourth extracellular loop of band 3 (Jarolim et al., 1996b; Jarolim et al., 1998b).

1.10.10 Hughes; Hf; DI12

Identification of the low incidence antigen Hg' in diree unrelated individuals and subsequent family analysis led Rowe and Hammond to conclude that Hga is inherited in an autosomal dominant fashion segngating independently of Rh, MNS and is not sex Iinked

(Rowe and Hammond, 1983). Examination of 18 multi-reactive sera containing anti-Hgadid not identify a single pure example of anti-Hg. Rather, al1 18 sera were found to contain various adbodies to other low hquency antigens and oAen contained antibodies to other Diego system antigens (ie. anti-Bp', anti-Wr", anti-Mo8).

A single Arg656Cys substitution in the fourth extracellular loop of band 3 gives rise to Hg' (Jarolim et al., 1996b; Jarolim et al., 1998b).

1.10.11 Van Vugt ;Vg; DI13

The extremely rare blood group antigen Van Vugt (Vga)was originally found after testing 17,209 Australian blood donors (Young, 1981). Family analysis identified five additional Vg(a+) individuals and demonstrated that the antigen was inherited as an autosomal dominant character that segregated independently from the MNS blood group system. As is the case for most of the Diego system antigens, antibodies defining Vga are found at higher frequencies than the antigen (1 1 out of 1,669 random samples), and are ofien found in sera containing antibodies to other low incidence antigens.

Molecular analysis of DNA fkom Vg(a+) individuals identi fied a Tyr5 5 5 His substitution in the third extracellular loop of band 3 that underlies the Vg polyrnorphism

(Jarolim et al., 1996b;Jarolim et al., lW8b).

1.10.12 Swann; Sw8; DI14

SHT was identified during routine compatibility tests with the serurn of a patient and the RBCs îrom a blood donor (MLSwann) in 1959 (Cleghom, 1959). Further testing of the semnvealed the presence of other antibodies to low incidence antigens (eg. anti-Wr').

Absoiption tests identified the presence of a new antibody within the serum that reacted to a new antigen present on the RBC9sof Mr. Swann. This new antigen was named SwP or

700.4. Tests on an additional 30,000 mdom blood donors identified four additional Sw(a+) individuals, two of domwm related. Family analysis revealed that Sw' was inherited as 6 1 a Mendelian dominant character. Continued investigations of families segregating for SM? have excluded it fiom al1 blood group systems except Diego, Colton, Landsteiner- Weiner, and H (Cleghom, 1960; Lewis et al., 1988). Anti-Swa is also found in sera containing antibodies to other low incidence antigens, narnely anti-Wt' and -Bya.

As differences in reaction patterns of RBCs previously typed as Sw(a+) with anti-Sw" were observed, four reference laboratories undertook comparative studies to determine the basis of the variability (Contreras et al., 1987). Some of the Sw(a+) RBCs exhibited identical reaction patterns to those of the original Sw(a+) propositus, while others gave some but not al1 the expected reactions. Serological investigations determined that the historically temied Sw(a+) phenotype was heterogeneous with two distinct categones. The fiat category which had the identical reaction pattern as the original propositus was termed Swam class

1 or SWI and was given its own ISBT designation 700:4,41, as antibodies that reacted solely

to it had ken identified. The second category termed Sw' or ïOO:4,-41, was not given its own ISBT desipation (ie. 700:4,42) as antibodies that react exclusively with it and not SWI

(Contreras et al., 1987) had not been (and still have not been) identified. Since then, the

locus of the gene goveming Sw' expression has been localized to chromosome 17q (Zelinski

et cil., 1996).

Sw' expression in controlled by an Arg646Gh substitution localized to the fourth

extracellular loop of band 3 (Zelinski et al., 1997).

1.10.13 Bowyer; BOW; DI15

Fht rccognized in 1975, BOW wasn't reported untii 1988 by Chaves et al. (1 988).

It was idcatifieci denthe semof a patient king matched to RBCs from a donor (Bowyer) 62 prior to a transfusion, gave a strong positive agglutination. Family analysis indicated that

BOW was inherited in an autosomal dominant fashion and segregated independently from the ABO, Rh, MNS, P and Kell blood group systems. A second BOW+ donor was identified, unfortunately no additional family information was available. BOW is known to be a-chymotrypsin sensitive (Reid and Lomas-Francis, 1997) and is serologically related to

Wu (Kaita et al ., l992), a previously charactenzed Diego system antigen, and NFLD a low incidence antigen now identified as belonging in the Diego system.

The BOW antigen arises fiom a single Pro56 I Ser substitution occumng in the third extracellular loop of band 3 (McManus et al., 1998; Poole et al., 1998; Jarolim et al., 1998b;

McManus et al., 1999).

1.10.14 NEWFOUNDLAND; NFLD; DI16

In 1984, Lewis et al. (1984) identified a new low incidence antigen NFLD in a family originally under investigation for a chromosome 3 inversion. Analysis of this large

Canadian kincûed demonstrated that NFLD was inherited as an autosomal dominant character and was independent of ABO, MNS, Rh, Kell, Du@, Kidd, Yt, Dombrock, and the sex-linked blood group systems. The NFLD antigen has since ken identified in two imrrlated individuals fiom Japan (Okubo et al., 1988). As previously mentioned, a serological relationship between NFLD, BOW and Wu has been established (Kaita et al.,

1992).

To date, NFLD is the only Diego system antigen controlled by hvo concurrent substitutions, Glu429Asp and ProS61Ala, localized to the fust and third loops of band 3 rcspectivtly (McManus et al., 1998; McManus et of ., 1999). 63

1.10.15 Nunhart; Jn'; DI17

The low incidence RBC antigen Ina was fortuitously identified in a blood donor (Mr

J. N.) fiom Prague. Jn8was not the subject of a full length report until 1967 (Komstad et al.,

1967). A positive agglutination reaction of Mr J. N.'s RBCs with a serum containing anti-

Wf was observed although the reaction was much weaker than that with Wr(a+) RBCs.

Further testing of Mr. J. Ne'sRBCs with additional examples of sera containing anti-Wr" and anti-Bpa produced clearly negative agglutination results. Two other examples of sera containing antiJn8 were identified and gave agglutination reactions with Mr. J. N.'s RBCs although these sera also contained antibodies that defme other low incidence antigens.

Analysis of a farnily segregating for Jna detennined it to be inherited as an autosomal dominant character independent of the MNS, P, Rh, Duw, Kidd and X-linked blood group systems (Komstad et al., 1967).

Molecular analysis has concluded that a Pro566Ser substitution in the third extracellular loop of band 3 gives rise to the Jna antigen (Pooie and Hallewell, 1997).

1.10.16 Krcpic; KREP; DI18

KREP was originally identified as a altemate fonn of Jna despite one multispecific serum reacting only to RBCs fiom the original KREP propositus and not to Jn(a+) RBCs

(Poole and Hallewell, 1997). Molecular analysis of SLCQAI from Jn(a+) and KREP+ individuals identified two distinct point mutations, C-rT and C+G respectively, occurring at the same nucleotide position. Clarification and distinction of KREP fiom Jn' did not occur until a ycar latcr whm Poole et al. (1998) observed the same point mutation in the DNA from the hthcr of the original propositus. 64

A single Pro566Ala substitution in the third extracellular loop of band 3 accounts for the KREP antigen (Poole et al., 1998).

1.10.17 Traversu; TF;DI19 (Resewed)

Very little information is available on the low incidence antigen Tt' (700.8). Personal communications in the 1960s between Cleghorn and Race and Sanger described the

identification of two Tr(a+) propositi among approximately 38,000 English blood donors

(Race and Sanger, 1975). Family analysis excluded TI" from the ABO, MNS,P, Rh, Du@,

Kidd and the X-chromosome blood group systems. The serum containing anti-Tr" aiso

contained antibodies to other known low incidence antigens including anti-Wr' (Jarolim et

al., 1997a).

Although DNA from only one Tr(a+) sample was available for sequencing, a single

nucleotide mutation in SLC4Al giving rise to a Lys551Asn substitution in the third

extracellular loop of band 3 was observed (Jarolim et al., 1997a). Based on this information

the ISBT reserved a Diego system number (DI19) for Tr'. To date Tt' is the only antigen of

the approximately 270 known antigens to have two designations; one in the 700 low

incidence series (700.8) and one in the Diego blood group system (DI19).

1.10.18 Froese; Fr'; DI20

in 1978, Lewis et al. (1 978) described the new low incidence blood group antigen Fr'.

Segregation information detemined Fr' to be inherited in an autosomal dominant fashion,

distinguishable from the ABO, MNS, P, Rh, Kell, Luthetan, Du*, Kidd, Dombrock and

Scianiir, blood group systcrns. Two years latet, testing of approximately 2,000 individuals

for the pnsence of Ff identified two additional cases and allowed for exclusion fiom the 65

Colton blood group system (Kaita et al., 1980). Antibodies de fining Fr' have been identified in sera containing antibodies to other low incidence antigens such as Rbhnd Wr". Fr' has been identified in several Mennonite families and information gleaned from families segregating for Ff was used in linkage analysis between FR and two chromosome 17q markers (Zelinski et al., 1996). A peak lod score of 5.72 with no evidence of recombination

was obtained, thereby establishing linkage.

Molecular analysis of SLC4AI identified a single point mutation giving rise to a

Glu480Lys substitution that accounts for Fr' expression (McManus, unpublished

observation).

1.10.19 Swann Class I; SWI; DI21

As previously mentioned the low incidence antigen SWI(7OOA 1 ) represents the first

subclass comprishg the SHr' epitope identified on the RBCs of the original propositus, Mr.

Swann (Contreras et al., 1987). A serological relationship between SWI and Sw' was

evidenced through absorptions with anti-SM (anti-700.4). Two distinct antibodies react to

SWI, one that reacts exclusively to SWI and one (anti-700.4) ihat also reacts with, Sw'.

Antibodies to SWI have been observed in sera containing multiple antibodies to other low

incidence antigens, including anti-Wf. Family analysis of segregation patterns determined

that the SWI antigen was inherited in an autosomal dominant fashion, excluding it from al1

but the Diego, Colton, Landsteiner-Weiner and H blood group systems (Cleghom, 1960;

Lewis et al., 1988).

SWI arises fiom an Arg646Trp substitution in the fourth extracellular loop of band

3 (&linski, unpublished observation). 2 OBJECTIVES

The primary objective of my Master's program was to characterize SLC4,d 1 at the molecular level. It involved characterizhg nucleotide differences which rnay or may not underlie specific red ce11 antigens. In addition to documenting variation within SLC4A1, contributions to the understanding of erythroid band 3 topology and the Diego blood group systems were made.

Based on the proposed mode1 of erythroid band 3, approximately 90 amino acids occupy extracellular locations. Differences in some of these may account for specific erythroid antigens. To investigate this possibility 1 undertook the molecular analysis of

SLC4Al in an attempt to defuie three low incidence erythroid antigens, BOW,NFLD and

Fr', based on the following information.

2.1 BOW

The low incidence antigen, BOW,was first described in 1988 (Chaves et al., 1 988) and was excluded fiom 5 of 25 established blood group systems (Lewis el al., 1990).

Enzyme treatment of BOW+ RBCs with a-chymotrypsin abolishes recognition by defining antibodies. As there are two a-chymotrypsin cleavage sites in the third putative extracellu lar loop of band 3 (Tyr553 and TyrSS5) it is plausible that an arnino acid substitution underlying

BOW expression rnay be localized to this loop. In addition, a serological relationship (Kaita et al., 1992) between BOW, NFLD and Wu(DI9), a now characterized Diego system antigen

(Zeliaski et al., 1998b). had ken demonstrated. 2.2 NFLD

Fortuitously identified in OUI laboratory (Lewis et ai., 1988), NFLD was found to segregate in a Canadian family of French ancestry originally under investigation for a chromosome 3 inversion. Family analysis excluded NFLD fiom 10 of the 25 established blood group systems, but not Diego (Daniels et al., 1995). As previously mentioned, a serological relationship with BOW and DI9 (Kaita et al., 1992) had been demonstrated.

2.3 FP

First nported in oulabontory (Lewis et al., 1978), Fr' was found to segregate in a

Mennonite family fiom Manitoba. Ff has been excluded fiom 17 of the established blood group systems, and the gene controllhg Ff expression has been localized to chromosome

17q (Zelinski et al., 1W6), the same region as SLC4A 1. 3 MATERIALS

The Rh Laboratory is an International Reference Centre for the resolution of blood group incompatibilities. This activity ensured that al1 of the prerequisite resources (eg. RBCs carrying the rare antigens of interest and sera containing antibodies defining them) required to coaduct my studies were available. in addition, genealogical and serological records maintained in the Laboratory provided critical information on relationships between family members and complete blood group phenotypes. Al1 serological investigations were performed by, or under the guidance of, an expert serologist, Gail Coghlan.

For reference purposes, Appendix A contains a list of al1 solutions used during these investigations. Chernicals wen of molecular biology grade and were purchased from Gibco,

Fisher, Bio-Rad, and Mallinckrodt.

Members of the Canadian kindnd segregating for NFLD were contacted by letter outlining the premise of the proposed study. Individuals interested in participating visited their family physician and shipped their blood sample (in supplied containers) to the Rh

Laboratory. The Japanese blood sample was provided by Dr. Y. Okubo (Osaka Red Cross

Centre, Japan). Followiag a telephone conversation that outlined the proposed study, members of the two Me~onitefamilies segregating for Fr" were invited to participate.

Intensted individuals demonstrated their willingness to participate by contacthg the Rh

Laboratory and ananging for blood samples to be taken at their home by our serologist, Gail

Coghlan. nie BOW+ blood samples were gifts fiom GamrnaBiologicals(Houston, TX) and the National Blood Service (London and the South East, United Kingdom). Control DNA used in these studics was prcviously isolated fiom blood samples obtained witb permission 69 from random individuals. At the time of donation, each individual was asked of their willingness to participate in fiiture genetic research projects involving normal blood markers.

If agreeable, their DNA was isolated and stored at 4 "C in the laboratory.

3.1 Individuals and Families Studied

A blood sample was obtained fiom the original BOW+ propositus (Chaves et al.,

1988). DNA was extracted fiom isolated white blood cells and the RBCs were serologically tested for the presence of the BOW antigen. A second DNA sample fiom an unrelated

BOW+ individual was received fiom GammaBiologicals, Houston, Texas.

The NFLD antigen was identified in family members of a large Canadian kindred

(Lewis et al., 1984). Previously stored DNA from several members from this kindred was available. Arrangements were made to have fieshly drawn blood samples shipped to our laboratory fiom several of the original family members and fiom several other family members not previously investigated. DNA was extracted fiom isolated white blood cells and RBCs were serologically tested. A pedigree of the NFLD kindred studied is depicted in Figure 4. Not al1 individuals depicted in Figure 4 were serologically and molecularly tested. in addition, a blood sample fiom an unrelated Japanese NFLD+ individual (Okubo et al., 1988) was obtained fiom Dr. Okubo. This sample was treated and studied in the same mmet as the blood samples fiom farnily members.

7 1

DNA tkom members of two Manitoba Mennonite kindreds segregating for Fr" was available for analysis. RBCs fiom individual members were previously tested for the presence or absence of the Ff antigen. The family pedigrees are depicted in Figure 5 (Panels

A and B). One individual in generation 1 of Panel B, denoted by (*) ,was deceased and a blood sample was unavailable for serologic or molecular analysis. Presumably he was Fr(a+) based on two factors; his sister is Fr(a+) and four of his children are Fr(a+). A blood sample fiom his wife was available and determined to be Fr(a-). Figure 5. Pedigrees of two Mennonite kindreds segregating for Ff'(Panels A and B). Fr(a+) individuals are represented by half-filled symbols and Fr(a-) individuals represented by open symbols. Roman numerals (1, II, III) indicate generation. Genealogical information obtained fiom family members did not identify a common ancestor between the two kindreds. (*) denotes an individual from whom a bldsample was not available for serological or inolecular analysis. 4 METHODS

4.1 Preparation of Suspensions

Blood sarnples were obtained by venepuncture and drawn into heparin collection tubes. Each sample was centrifugeci for 5 min at 2,200 rpm (IEC, Centra-HN). Serum was removed using a glass pipette and stored at -20 OC. The white blood cells were removed by glass pipette and placed in a fiesh tube. The remaining RBCs were stored by adding a volume, equivalent to the removed plasma, of Alserver's solution. RBCs were prepared for serological testhg by placing one or two drops of the sample in a small test-tube (10mm x

7Srnrn) and adding 0.9% saline (using at least 1O volumes of saline to 1 volume of red cells).

The sarnples were mixed gently, centrifuged briefly (maximum 1 min) at 2,000 rpni (IEC

Clinical Centrifuge) to lightly pack the RBCs and the saline was removed (Chown and

Lewis, 1951). This RBC washing procedure was repeated three additional times. AAer the fial wash, RBCs were suspended in saline to approximately 20% - 30% (volume/volume).

4.2 Slanted Capillary Method of Blood Croup Phenotyping

Glass capillary tubes (9cm long and a bore size of approximately 0.4rnrn) were placed in the appropriate antisenun (ie. anti-NnD). The antiserum was allowed to enter the capillary tube, to a height of approximately 2cm. The exterior of the tube was wiped. A

20%-30% saline suspension of four times-washed RBCs was allowed to enter the capillary tube to a cornbined height of 4cm at the same end as the serum. The capillary tube was held in a vertical position ehinating bubble formation at the serum-RBC interface and wiped at the exterior ofthe tube to prevent cross contamination. The capillary tube was inverted and placed on a 45degne angîed illuminated agglutination stand so that the suspended RBCs 74 were above the semand fell into and through the serum. Agglutination was monitored afier 5- 10 min (Chown and Lewis, 195 1). RBCs carrying the antigen of interest fom complexes with the defming antibodies producing visible clumps in the capillary tube.

RBCs not canying the antigen of interest will not fom complexes but rather, form long nmwcontinuous nbbons or needles of cells throughout the capillary.

4.3 Antibody Absorption and Chloroform Heat Elution

One volume of washed, packed RBCs was placed in a disposable glass culture tube

(1Omm x 75mm)and two volumes of anti-sera were added. The tube was mixed gently and stored at 4°C for a minimum of 2 hr to a maximum of 18 hr. Following incubation, the tube was centrifuged at 2,200 rpm (IEC Centra-HN) for 5 min. The supematant was removed and placed in a second disposable glass culture tube containing packed RBCs identical to the

RBCs used in the fvst absorption. The remaining packed RBCs with bound antibodies were collected and stored for antibody elution (see below). The RBCsIsupernatant were mixed and incubated at 4°C for 2 hr. The tube was centrifuged for 5 min, and the supernatant collected and placed into a third tube containing identical packed RBCs. The sarnple was mixed, incubated at 4OC for 2 hr and centrifuged for 5 min. nie supematant was then tested for the presence/absence of the antibody of interest by the slanted capillary method of blood group phenotyping (4.2) using positive and negative RBC controls. If the antibody was still pmcnt the absorption steps were repeated until the antiôoây was no longer present. If absent, the sera could then be used in additional blood group phenotypings.

RBCs with bound antibodies fiom the first absorption step (see above) were washed four tirnes with 0.9% saline as outlincd in the prcparation of red blood ce11 suspensions (4.1 ). 75 niesupematant fiom the fourth wash was collected and stored for later testing. One volume of washed, packed RBCs with bound antibodies (from above) was placed in a lOmm x

75mm glass tube and an equal volume of 6% albumin or inert AB serum was added. The tube was mixed gently and one volume (equal to the packed RBCs) of chloroform was added. Again the tube was mixed gently. A cork stopper was inserted loosely into the top of the tube and the tube was placed in a 56 OCwater bath for 5 min. The tube was vigorously shaken while in the water bath. The tube was placed in a centrifuge (EC Clinical

Centrifuge) and spun at 900 to 1,000 x gravity for 5 min. The supematant eluate was transferred to a clean glass tube and tested in parallel with the supematant saline from the fourth wash (Branch et al., 1982).

4.4 Indirect Aatiglobulin Test

A fine-tipped glass pipette was prepared by stretching a glass vacuum tube (1Omm x 70mm) under a flame. Using this pipette, three volumes of antisera were added to a labelled Coombs tube (50mm x 5mm). One volume of saline washed, packed RBCs were added to the Coombs tube containhg the antisera. The mixtures were incubated at room temperature for 2 - 4 hr in a sealed container. Subsequent to incubation, the RBCs were washed four times with 0.9% saline. AAer each saline wash the supematant was removed with the fine-tipped pipette. Approximately 4 cm of anti-hwnan immunoglobulin reagent was drawn into glass capillary tubes. An aliquot (approximately 2 cm) of the previously incubated RBCs was added. RBCs were allowed to enter the capillary to approximately

20mm. The tubes wen inverted and placed on the viewing box. Agglutination was monitorcd hm0-20 min. Tubes in which agglutinationreactions did not occur after 20 min were recorded as negative.

4.5 Isolation of Genomic DNA

White blood cells isolated fiom 4.1 were transferred to l O mL polypropy lene tubes.

The remaining RBCs were lysed by the addition of 5 volumes of red ceIl lysis mixture at

37OC for 10 min. Samples were mixed and centnfbged (IEC Centra-HN) for 10 min at 2,200 rpm. The supematant was removed and a second treatment with the red cell lysis mixture was performed. The supematant was removed and the white ce11 pellets were washed twice with 5 mL of 0.9% saline. After centrifugation, the white ce11 pellets were re-suspended in

2 rnL high TE. White ce11 lysis mixture (approximately 2 mL) was tlien added under pressure (ie. 3 mL syringe and an 18 gauge needle) to each white ceIl suspension.

Approxirnately 4 mL (1 volume) of Tris-buffered (pH 8.0) phenol was added to the white ce11 lysates. The samples were then mixed for 10 min and centrifuged at 2,200 rpm for 7 min to achieve phase separation. The aqueous phase was removed and placed in a fresh polypropylene tube. Again, 1 volume of Tris-buffered phenol was added, the samples were mixed (10 min) and centrifuged for 7 min. The aqueous phase was removed and 4 inL of a 24: 1 chlorofom:isoamyl alcohol solution was added. Gentle mixing ( 10 min), followed by brief centrifugation (6 min) at 2,200 rpm separated the phases. The aqueous phase was removed and placed in a fksh polypropylene tube. 400 pL of 4M ammonium acetate was added irnmedietcly followed by the addition of 4.5 mL of ice-cold isopropanol. Samples werc inverted gently until DNA shands became visible. The DNA strands were transferred to a 1.5 mL microcentrifuge tube and centrifigeci at 14,000 rpm (IEC Micro-MB) for 1 min. nie supernatant was rcmoved and the DNA pellet was washed three times; twice with 70% 77 ethanol and once with 95% ethanol. ABer the fmal wash, the DNA pellets were dried under vacuum at room temperature for 1.5 hr. Subsequent to drying, volumes (400-700pL)of low

TE were added to each pellet depending on its size.

4.6 Quantitation of Genomic DNA

Approximations of DNA concentrations were performed visually by comparison of newly extracted genomic DNA samples with previously extracted genomic DNA samples whose concentrations had been detemiined spectrophotometrically. Briefly, 5 pL of each genomic DNA sample was diluted in 20 pL low TE mixed, and centrifuged briefly in a microcentrifuge (IEC Micro-MB). A 5 pL aliquot of this dilution was added to 5 pL of

Orange G, mixed and centrifuged briefly. Two genomic DNA standards were selected and prepared in the same manner as describcd above; one 500ng/pL sample and one 1000ng/pL sample. The 10 pL aliquots were nsolved on a 30 rnL 1% agarose gel (1 2 well comb)

(Fisher Biotech) and resolved at lSOW for 1.5 hr. Gels were then placed on an W- transillurninator (Spectroline TR-365)and sample htensities were compared to standards and recorded.

4.7 Preparatioo of 6% Polyacrylamide/lOOh Glycerol Non-denaturing Gels

Single strand conformation polymorphism (SSCP)gels were prepared by combining

10 mL 30% acrylamide (29:1). 5 mL 1Ox TBE buffet, 5 mL glycerol and 30 mL ddH,O in a 50 mL graduatcd cylindcr. Following a bief but vigorous mixing, the gel solution was dispensed into a 125 mL side arm flask and de-aerated under vacuum for 15 min. Following de-aeration, 156 pi, 20% ammonium persulfate (Mallinckrodt) was added immediately followed by the addition of 33 pL TEMED (Bio-Rad). The gel solution was mixed gently 78 and pound between two S2 Sequencing System (Gibco BRL, Life Technologies) glass plates separated by 0.4mm spacers. Two shark tooth combs were inserted and clamped and the gel was allowed to polymerize for 1 hr. Gels were stored ovemight at 4°C. Pnor to use, the combs were removed, the large wells were rinsed with 1 x TBE buffer, and the cornbs were n-inserted in the opposite orientation to create 2 x 24 wells. Gels were run on a S2

Sequencing System apparatus at either room temperature at 7W for 1 8 hr or at 4 OCat 42 W for 6 hr.

4.8 Preparation of 6% Polyacrylamide Denaturing Gels

Gels were prepared by dissolving 2 1 .Og urea in 10 mL 30% acry lamide ( 19: 1), 5 mL

10x TBE buffer, and 10 mL dW,O in a 125 mL flask at 60°C with occasional mixing. Once dissolved, the solution was placed in a 50 mL graduated cylinder and brought up to a total volume of 50 mL with ddH,O. The gel solution was placed in a 125 mL side am flask allowed to cool to room temperature and de-aerated under vacuum for 1 5 min. Finally, 33 3 pL 10% ammonium peaufate was added followed immediately by the addition of 22 pL

TEMED (Bio-Rad). The solution was hand mixed gently and poured between the two glass plates of the S2 Seqwncing System (Gibco BRL, Life Technologies) separated by 0.4mm spacers. Two shark tooth combs were inserted, clamped and the gel was allowed to polyrnerize for 1 hr. The gel was stored ovemight at 4°C. Prior to sequencing, the gel was removed fiom the cold and allowed to wann to room temperature for 1hr. The shark tooth combs wcre removed, the large well rinscd with lx TBE buffer, and the combs re-inseried in the opposite orientation to form 2 x 24 wells. Gels were pre-warmed for 1 hr at 67W. 4.9 Preparatioa of 1./0 Agarose Gels

Two different gel sizes were used depending on the experiment; small format gels were prepared for quantitation of genomic DNA and SLCJA I exon amplification procedures and mini-gels were prepared for DNA extraction with the Qiagen gel extraction kit.

Small format gels were prepared by placing 0.4g agarose (Fisher Biotech), in a 125 mL flask and adding 40 mL of lx TAE buffer. The solution was stirred and heated on a magnetic stimr with hotplate (Corning) until the agarose was fùlly dissolved. The flask was transfemd to s magnetic stirrer without heat and allowed to cool to approximately 65 OC.

Prior to pouring 4 pL ethidiurn bromide (1Omg/ml) was added to the gel solution. A volume of 30 mL was measured and poured into a small format gel casting tray sealed at both ends with masking tape containing a 12- or 16-well comb. Once solidified, the comb and the tape were removed and the gel was placed in an electrophoretic tank (1 2cm n 40cm) containing

250 mL lx TAE buffer.

Mini-gels were prepared by adding 1Sg agarose (IBI) to 1 SOmL l xTAE buffer in a 250 mL flask. The mixture was stirred and heated on a stirrerhotplate (Coming)until the agarose was Mly dissolved. Once dissolved the flask was transfemd to stirring pad without heat and allowed to cool to approximately 65 OC. Pnor to pouring 15 pL ethidium bromide was added. The gel solution was poured into a 14cm x 20cm casting tray (Kodak, BioMax) sealed at both en& with masking tape containing a rnodified 16-well comb. The comb was modified by taping 3 wells together to fom one larger well. Each modified comb contained only 4 larget wells. The gel was allowed to solidify kfore removing the comb and tape and was placed in îhe elcctrophoretic tank (Kodak, BioMax). 4.10 Single Strand Conformation Polymorphism Analysis

Mutational analysis of exons 1 1-20 of SLC4A 1, the region corresponding to the transmembrane domain of band 3, was conducted using the SSCP analysis protocol outlined by Orita et al. with slight modifications (Orita et al., 1989a; Orita et al., 1989b). To each 0.5 mL microcentrifuge tube containing 200-400ng genomic DNA, 25 PL of the SSCP PCR reaction mixture was added: [a-'2P]d~TP(2.5 ~Cilreaction),0.2pM of the appropriate exon- specific sense and antisense primer (Table 4), 1mM dCTP, 1mM dTP, 1mM dGTP, 0.5 mM dATP in lOmM Tris HCI (pH 8.3) containing SOmM KCl, 1SrnM MgCl,, 0.01% gelatin,

15% glycerol and 1U of Taq DNA polymerase (Gibco BRL, Life Technologies). Samples were overlain with a drop of mineral oil and amplified under the appropriate thermal cycler conditions for each individual exon using a 96 well thermal cycler (Hybaid, Omni Gene

Thermal Cycler). Briefly, each amplification undenvent 1 cycle at 95 OCfor 3 min, 35 cycles

of 95 OCfor 1 min, X°C (where X is the program temperature for each primer pair [Table 41)

for 1 .S min and 72OC for 2 min, followed by one cycle at 72°C for 10 min. Although the

melting temperature for each primer is listed in Table 4, the listed program temperature for

each primer pah was used and represents the optimized annealing temperature yielding the

best nsults. Slight modifications of the above protocol were used for exon 16: i) the Expand

High Fidelity PCR System (Boehringer Mannheim) was employed for amplification and ii)

27 cycles of DNA denaturing, primer annealing and elongation were used in place of 35.

Following amplification the reactions were teminated through the addition of a stop solution

(95% forniamide, 4.8% 0.5M EDTA pH 8.0,O. 1% bromophenol blue, and 0.1 % cyanol). Tabit 4. Definition of DNA phers used to amplify exons 1 1-20 of SLC4A I

Primer Sequence (5'-3')

TCC TCC AGC TAC TCC CTC TGA CAG AGG GTC AGA GGC AAG AGT CCT GGA AAT GAT CTC CTG ACC T ACC ATG CAT CAG GCA GGT GGT CCT TGC CCC AAG ACC cm TGA C'IT ATA CAC AAC CTC CCG TGT G CCA AGC CAA AGC TGG GAG AGA GGG CTG GGA TAG GGC AGT GT GGG GAG TGA CTG GGC ACT GA ATG AGG ACC TGG GGG GTA TCA GCC C'IT GGC AIT C'IT ACC TGC T GCC AGG GAA AGG TCT CTG cc GGA GAA CCC TGC TïA CCC CTC GAG GAT GGT GAA GAC GCG ACC AAG CTG GGC TGA GAG TGT GCG TCT ACC ACC CCA GGC TGG Gd GGG GTG TGA TAG GCA CTG ACC CCC TCG CAT GCT CCC AGC TC CTC 'iTC TGG CCC CAG GGT CT TGC TCC AGG AGG ATC CTG AGT

> t Melting temperatures were cafculated using the following formula; Tm= 69.3 + (0.41 x %GC) - 650 Primer length 82

The products were divided in two and one set was denatured by boiling for 5 min, followed by immediate placement on ice. The products were separated using a S2

Sequencing System (Gibco BRL,Life Technologies) on a 6% polyacrylamide/lO% glycerol

non-denaturing gel for 18 hr at 7W (room temperature), or for 6 hr at 42W (4 OC). In the case of exon 13, products were separated on an 8% polyacrylamide/lO% glycerol non-denaturing gel at 7W for 22 hr at rwm temperature. Gels were then transfened to 3MM (Whatman) filter paper, àried under vacuum (Savant SGD2000 Slab Gel Dryer) at 72°C for 1 hr and exposed to X-ray film (Biomax-MR, Kodak) for 1 to 3 days. Films were developed in an

AFPm 14 XL automatic film processor.

4.1 1 Linkage Analysis

Materna1 and patemal lod scores were determined and totalled based on segregation counts at assumed recombination fractions (O) = 0.00,0.05,0.10,0.20,0.30 and 0.40 (Race and Sanger 1975). Peak lods and peak recornbination fractions were determined by scrutiny.

4.12 Eson-specific PCR-amplification and Product Isolation

Exons displaying SSCPs wen PCR-amplificd in triplicate fiom genomic DNA with the same exon-specific intronic primea described in Table 4. 25 PL of the PCR reaction mixture (1X PCR buffer, 0.2pM of the appropriate sense and antisense primer, 1mM dCTP,

1mM dTTP, I mM dGTP, 0.5mM dATP in 1OmM Tris HCl (pH 8.3) containing 5OmM KCI,

1SmM MgCl,, 0.01 % gelatin, 15% glycerol and 1U of Taq DNA polymerase (Gibco BRL,

Life Technologies) was added to each 200400ng genomic DNA in a 0.5 mi, m ic rocentri fige tube. A &op of mineral oil was placed on top of the reactions and they were placed in a

üinmai cycler (Hybaid, Omni Gcnc Thermal Cycler) and run under the appropnate exon- specific amplification conditions as outlined previously.

The amplified triplicate products were pooled and run on a 150 mL 1% agarose gel

(IBI Agarose, inter Science) containing 15pL ethidium bromide at lOOW for 2 hr in an electrophoresis tank (Kodak, BioMax) at room temperature to isolate the produci. The

agarose gel was placed on a W trans-illuminator (Spectroline, TR-365) and the product

bands were cut fiom the gel and placed in a 1.5 mi, microcentrifuge tube and extracted as described in the QIAquick Gel Extraction Kit manual (Qiagen hc).

Briefly, each gel piece containing the arnplified exon product was weighed, placed

in 1.5 mL microcentrifuge tubes and three volumes of QC buffer was added. The tubes were

placed in a 60°C incubator for 10 min with occasional mixing or until the gel pieces were

fully dissolved. One volume of isopropanol was added and the solution was mixed.

Approxirnately 750 pL of this solution was added to an QIAquick Spin Column and placed

in a 2 mL collection tube. The tubes were placed in a microcentrifuge (IEC Micro-MB) and centrifbged at 14,000 rpm for 1 min. The 80w Uirough was discarded and 500 pL of QC

buffer was added to each isolation tube. The tubes were centrifbged for an additional minute.

Again the flow through was discarded and 750 pL PE buffer was added to each isolation

tube. Prior to centrifugation for an additional minute, the tubes were allowed to incubate for

2 min. Once again the flow through was discarded and the tubes were re-centrifûged for an additional minute to removc traces of PE buffer. The isolation tubes were placed in clean

1.5 rnL microcenûifuge tubes and 50 pL elution buffer was added to the centre of each isolation tube. The samples werc incubated for 2 to 5 min pior to fmal centrifugation for

1 rnin. Approxhatcly 45 HL of amplified DNA was recovered and placed in a clean labelled 0.5 mL microcentrifuge tube.

4.13 DNA Seqoeocing

QLAquick isolated products (as described in section 4.12) were sequenced using the dsDNA Cycle Sequencing System kit (GIBCO BRL, Life Technologies), with the appropriate sense primers end-labelleci with [y-"PIATP according to the manufacturer's ncornmendations. Briefly, the protocol for sequencing DNA from three individuals was as follows: 4.5 pL dW,O, 4.5pL Kinase buffer, 4.5 pL T4 Kinase and 50pCi ['-"Pl ATP was added to 10 IL of the appropriate sense primer (diluted 1 :100). AAer mixing and a brief centrifugation, the mixture was incubated at 37°C for 10 min (Haake Dl water bath), followed by a 5 min incubation at 55 OC(Fisher Isotemp incubator). To three sets of tubes

labelled A, C, G, and T, 2.0 pL termination mix (A, C, G, and T) were added. The prereaction mixture was prepared by adding 6U (2.0pL)Taq DNA polymerase to 5 8 pL ddH20 and 17 pL Tuq Sequencing buffer. The prereaction mixture was mixed and centrifbged briefly. A 25 pL aliquot of the prereaction mixture was added to three tubes containing 10 PL template DNA (three individuals) previously amplified and extracted as outlined above. To this tube containing a total volume of 35 pL (prereaction mixture and template DNA), 9 pL of the end-labelled primer was added. This mixture was centrifûged

briefly prier to aliquoting 9pL to each tube set labelled A, C, G, and T. Reactions were

placed in a thermal cycler (Techne Genius) and amplified under the appropriate conditions.

Reactions were tenninated by the addition of 4 pL Stop solution provided in the kit. Products

werc separattd by electrophonsis on a 6% polyacrylamide denaturing gel (containing urea) on a S2 Squcacing System (Gibco BRL, Life Technologies), for 1.25-3 hr at 67W. Gels 85 wen transfemd to 3MM (Whatman) filter paper, dried under vacuum (Savant SGD2000

Slab Gel Dryer) at 76°C for 1.25 hr and exposed to X-ray film (Biomax-MR, Kodak) for 1-2 days. Films were developed in an AFPm 14 XL automatic film processor. Autoradiographs wcre scanned at 600 dpi with a Hewlen Packard 5200 Scanner. Image backgrounds were deleted using Adobe Photoshop v5.0. S RESUH"T'

5.1 BOW

SSCP analysis of exons 11 to 20 ofSLC4AI identified a product mobility shift in exon 14 of DNA fiom both unrelated BOW+ individuals (Fig. 6A; lanes 7 and 8), but not in the DNA of controls (lanes 1-6). No exon 14 product rnobility shifis were observed in the

DNA fiom 35 unrelated control individuals. DNA sequencing of exon 14 from both BOW+ individuals identified a single C-tT mutation giving rise to a Pro561 Ser substitution in band

3 (Fig. 6B). A similar point mutation was not observed in DNA 60m more than 20 unrelated controls. The results are summarized in Table 5,

Table 5. Defmition of the three low incidence erythroid antigens investigated in this siudy

Antigen Point SLC4A 1 Amino Acid Band 3 Symbol Mutation Exon Substitution Loop

NFLD GAA+GAT 12 Glu429Asp 1 CCC+GCC 14 Pro56 1Ala 3 BOW CCC+TCC 14 Pro56 1Ser 3 Fi GAG+AAG 13 Glu48OLys 2 14 Lysine 562 Lysine 562 ACGT ACGT 9 0

Prolinel Proline 561 Serine 561

G Control BOW+ G Valine 560 T T Valine 560 G G

Figure 6. Molecular analysis of exon 14 of SLC4A 1 fiom BOW+ individuals. Panel A; SSCP analysis of exon 14. Genomic DNA was amplified as described in Methods and separated on a 6% polyacrylamide gel for 18 hours at rcmm temperature. SSCPs are evident in the DNA from two unrelated BOW+ individiials, lanes 7 and 8. Lanes 1 to 6 are the products of unrelated controls. Panel B; DNA sequence analysis of exon 14. DNA sequencing fiom a control individual (lefi) and fiom an individual heterozygous for the BOW polymorphism (right). Samples were separated on a 6% denaturing polyacrylamide gel for 1 10 min at 67W. The black arrow indicates the location of the C +Tmutation resulting in a Pro561Ser substitution in band 3. 88

5.2 NFLD

SSCP analysis identified concurrent product mobility shifts in exons 12 and 14 of

SLC4AI fiom the DNA of 16 NFLD+ family memben and fiom the DNA of the unrelated

NFLW individual iiom Japan. Product mobility shifts were not obsented in the DNA from

10 NFLD negative family members or in the DNA fiom 35 unrelated controls. Figure 7A depicts SSCP analysis of exon 12 fiom family members segregating for NFLD; those individuals with DNA product mobility shiAs (lanes 1, 5, 6, 7, 8, 9, and 10) are NFLD+ while the DNA product fiom NFLD negative individuals do not exhibit mobility shifts (lanes

2, 3, 4, and 11). A SSCP is evident in the amplified DNA from the Japanese NFLD+ individual (lane 14), while lanes 12 and 13 are water blanks. Figure 7B depicts SSCP analysis of exon 14 from family members segregating for NFLD. NFLD+ individuals exhibit product mobility shifts (lanes 1,5,6,7,8,9, and IO) while NFLD negative members do not (lanes 2,3,4, and 1 1). A SSCP was also detected in DNA from the unrelated NFLD+ individual hmlapan (lane 12).

Availability of DNA from 17 children (four families) fiom the large kindred segngating for NFLD allowed for linkage analysis between NFLD and SSCPs in exon 12 or 14 of SLC4A I. The results are summarized in Table 6. A peak lod of 3.3 1 at 8 = 0.00 was deteminecl for patcmal meioses. Analysis of the two matemally informative families with five chiidren resulted in peak lods of 1.5 1 at 6 = 0.00. No evidence of recombination betwcen eacb pair was obsewed in either patemal (individual family counts of z = 9:O and

3NR [Non Recombinants]) or matemal (individual fmily counts of 3NR and 2NR) meioses.

Totd peak lods of 4.82 at 6 = 0.00 rcpnscnt odds of neady 100,000: 1 in favor of linkage and 89 thereby establish linkage between NFLD and SSCPs observed in either exon 12 or exon 14 of SLC4A 1.

DNA sequencing of exon 12 of SLC4A I fiom six NFLD+ individuals, including the unrelated NFLD+ individual fkom Japan, identified a single A-T mutation resulting in a

Glu429Asp substitution in band 3. A similar mutation was not identified in the DNA from eight NFLD negative fmily members and from 23 unrelated controls. Sequencing of exon

14 from 12 NFLD+ individuals, including the NFLD+ individual from Japan, identified a

C-rG mutation not observed in the DNA fiom eight NFLD negative family members or fiom

23 unrelated controls. The C+G mutation results in a Pro561Ala substitution in band 3.

DNA sequencing results are depicted in Figure 8 and surnmarized in Table 5. Figure 7. Molecular analysis of exon 1 2 and exon 1 4 of SLClA 1 from members of a family segregating for NFLD. Genomic DNA was amplifid as outlined in Methods and separated on a 6% polyacrylamide gel for 18 hours at room temperature. Half shaded symbols represent NFLD+ individuals, open synibols are individuals negative for NFLD. Panel A; exon 1 2 SSCPs are evident in lanes 1,5,6, 7,8,9, 10 and 14. Lane 14 is the amplified DNA product from the unrelated NFLD+ Japanese individual, while lanes 12 and 13 are water blanks. Panel B; Products in lanes 1, 5,6, 7, 8.9. 1 O and 12 exhibit exoo 14 SSCPs. Lane 12 is the amplified DNA product from the uiirelated NFLD+ Japa~ieseindividual. a O Table 6. Lods for iinkage between NFLD and SSCPs in exon 12 or exon 14 of SLC4AIt

Segrcgation Nmber of Number of Assumed Reçombination Fraction Information* Families Meioses 0.00 0.05 0.10 0.20 0.30 0.40 zA 6

5 = peiik lods; 6 = peak recombination fraction * P = patemal; M = matemal; T = total A G Lysine 430 A ACGT ACGT A Lysine 430 A 0 9 A 0 9 9 0 A 9 9 4 Am O O A Glutamic Acidl Glutamic Acid 429 A 0 9 Aspartic Acid 429 G 9 9 G 0 0 A 0 0 A Glycine 428 G Control NFLD+ G Glycine 428 G G

A Exon 14 A Lysine562 A Lysine 562 A CGT ACGT A A

G 9 0 U Valine 560 T T Valine 560 G Control NFLD+ G

Figure 8. DNA sequence analysis of exon 12 and exon 14 of SLC4A 1. Panel A; DNA sequence analysis of exon 12 fiom a control individual (lefi) and from an individual heterozygous for NFLD (right). The black arrow indicates the position of the A-+T mutation underlying the Glu429Asp substitution in band 3. Panel B; DNA sequence analysis of exon 14 fiom a control individual (lefi) and from an individual heterozygous for NFLD (right). The C-*G mutation is identified by the black arrow and results in a Pro561 Afa substitution in band 3. Saiiiples were separated on a 6% denaturing polyacrylamide gel at 67W for 1 10 (exoii 12) aiid 120 niin (exoii 14) respectively. a t 4 93

5.3 Fr'

SSCP analysis conducted on DNA fiom members of two Mennonite families segregating for Fr' identified product mobility shifis in exon 13 of SLC4A 1 from 2 1 Fr(a+) individuals. No product mobility shifts were observed in the DNA fiom 18 Fr(a-) family members or in the DNA from 31 unrelated controls. Figure 9A depicts SSCP analysis of exon 13 fiom family memben segregating for Fr'; those individuals with DNA product mobility shifts (lanes 1, 4, 7, and 9) are Fr(a+), while the DNA products fiom Fr(a-) individuals do not exhibit mobility shifts (lanes 2, 3, 5,6, and 8).

As a result of access to DNA fiom members of two kindreds segregating for Fr', linkage analysis between F9 and SSCPs in exon 13 of SLC4AI was conducted. Five families with 16 children wen analyzed for SSCPs in exon 13. A peak lod of 1.5 1 at 6 =

0.00 was determined for patemal meioses while analysis of the two matemally informative families with nine children resulted in peak lods of 2.1 1 at 8 = 0.00. No evidence of recombination between F4 and SSCPs in exon 13 ofSLC4AI was observed in either patemal

(individual farnily counts of z = 2:0, z = 2:O and 3NR) or matemal (z = 2:O and z = 7:O) meioses. Total peak lods of 3.61 at 6 = 0.00 exceeds the threshold required to establish linkage between FP and the exon 13 SSCP. Table 7 sumrnarizes the lod determinations.

DNA scquencing of exon 13 identified a G-rA mutation in SLC4A I fiom 13 Fr(a+) family members that was not observed in 10 Fr(a-) family members or in 16 unrelated controls (Fig. 9B). This mutation underlies a Glu480Lys substitution in band 3 as summarized in Table 5. ACGT ACGT G G 0 = 0 Glutamic Acidl Glutamic Add 480 A 9 - 0 A Lysine 480 9 4 G/A G 9 9 -99 0 9 C C Cysteine 479 G Control Fr (a+) G Cysteine 479 T T Figure 9. Molecular characterization of exon 1 3 of SLC4A 1. Panel A; SSCP analysis of exon 13 fiom family memben segregating for FP. Genomic DNA was amplified as described previoulsy and products were separated on an 8% polyacrylamide gel for 22 Iiours at room temperature. Open synibols represent Fr(a-) individuals while half shaded symbols are Fr(a+) individuals. SSCPs are evident in lanes 1.4, 7, and 9. Panel B; DNA sequence analysis of exon 13. DNA sequence analysis of exon 13 from a control (lefi) and a Iieterozygous Fr(a+) individual (right). Samples were separated on a 6% denaturing polyacrylamide gel for 75 min at 67W. The black arrow indicates the location of the G-FA mutation underlying the Glu480Lys substitution in band 3. C Table 7. Lods for linLagt Ween Fr" and SSCPs in exon 13 of SLC4A 1t

Sqmgaiion Nurnberof Nwnber of Assumed Recombination Fraction Infômation* Families Meioses 0.00 0.05 0.10 0.20 0.30 0.40 ii 8

-- 2 = perk loci~;6 = pteL ncombination fiaction

+ P = patemal; M = maternal; T = total 6 CONCLUSION

We conclude that amino acid substitutions in erythroid protein band 3 arising from point mutations in SLC4A I underlie the BOW blood group polymorphism and control NFLD and Fr' antigen expression. Single point mutations in exon 13 (G-+A) and 14 (C+T) of

SLC4A1, reprcsenting Glu480Lys and Pro561 Ser substitutions in band 3 account for the FP and BOW blood group polymorphisms respectively, while two concurrent mutations in exon

12 (A-tT) and 14 (C-+G) correspondhg to Glu429Asp and Pro561Ala control NFLD expression. Ln light of these findings, BOW, NFLD, and FiL have been granted Diego system statu by the ISBT Working Party on Terminology for Red Ce11 Surface Antigens, and assigned the system numbers D115, DM, and DIZO, respectively. 7 DISCUSSION

The expansion of the Diego blood group system to include 19 additional antigens

(Daniels et al., 1995; Daniels et al., 1999) has largely ken made possible through molecular analysis of the controlling gene, SLC4AI. Most of the newly designated Diego system antigens were former members of the low incidence series. According to the ISBT Working

Party on Red Ce11 Terminology guidelines, inclusion of a low incidence antigen in an established blood group system nquires experirnental evidence suggesting that the antigen is a newly recognized ailele of the controlling gene and supporting serological or biochemical data (Lewis et al., 1990). The present snidy fulfills these criteria in relation to the low incidence antigens BOW, NFLD and Fr'.

Characterization of a CCC-*TCCmutation in exon 14 of SLC4Al that gives rise to a Pro561Ser substitution in the third extracellular loop of band 3 defuies the BOW blood group polymorphism (Fig. 10). Our results are concordant with those of Jarolim et of.

(1 Wb), and because of these independent fïndings, the ISBT Working Party has designated

BOW as DI1 5 and rendered its previous designation (700.46) obsolete (Daniels et al., 1999).

With respect to NFLD, SSCP analysis of SLC4A I suggested that variation in both exon 12 and 14 might relate to NFLD expression. Due to the availability of a kindred segregating for NFLD we had the opportunity to investigate the linkage nlationship between

NFLD (detennined fiom RBC phenotype) and each SSCP of S'C4AI. Peak lods of 4.82 at 8

=0.00 forNFtD: exon 12 SSCPofSLC4AI andof4.82 at 8 = 0.00 forNFLD:exon 14 SSCP of SLC4AI represent odds of 104000:l in favour of linkage for each pair. Since NFLD status wu, based on RBC phcnotype, we conclude that NFLD expression is controlled by Figure 10. Schematic representation of the transmembrane domain of band 3. Amino acids are represented by circles; open circles are proposed extracellular and intracellular ainino acids and lightly shaded circles are those localid to the putative membrane spanning domain. Black circles indicate the locations of amino acid substitutions underlying previously characterizcd Diego system antigens while striped circles indicate the locations of the amino acid substitutions underlying NFLD, BOW and Ff expression. The single N-glycan chah is linked to band 3 in the fourth extr;icellular loop at Asn 642. 99

SLCQAI and is dependent on the presence of two mutations (GAA-rGAT in exon 12 and

CCC-+GCC in exon 14) that result in Glu429Asp and Pro561Ala substitutions in the first and third extracellular loops of erythroid band 3 respectively (Fig. 10). This conclusion, drawn from the analysis of DNA from NFLD+ family members is supported by Our analysis of DNA fiom the NFLD+ individual fiom Japan and the fact that we have not observed one or the other, or both mutations in DNA fiom eight NFLD negative family members or 23 unnlated control samples. in light of these fmdings, NFLD has been assigned to the Diego blood group system (DI16). and its 700.37 designation made obsolete (Daniels et al., 1999).

The underlying molecular polymorphism controllhg Fr' expression has also been determined. We had the oppominity to investigate the linkage relationship between Fr" and

SSCPs in exon 13 of SLCQAI due to the availability of DNA samples fiom many rnembers oftwo distinct Manitoba Mennonite kindreds segregating for Fr'. A peak lod score of 3-61 ai 4 = 0.00 between Fr" and SSCPs in exon 13 was calculated and represents odds greater than 1,000: 1 required to establish linkage. DNA fiom al1 Fr(a+) individuals tested exhibited exon 13 SSCPs, while no exon 13 SSCPs were observed in the DNA fiom 49 Fr(a-) individuals, including 18 Fr(a-) family members. DNA sequencing of exon 13 fiom 13

Fr@+) farnily rnembers identified a GAG-+AAG mutation not observed in the DNA of 26

Fr(a-) individuals, including 10 Fr(a-) fmily mernôers. This point mutation underlies a

Glu480Lys substitution in band 3 and is the fint characterkd substitution localized to the second extracellular loop (Fig. 10). In light of these findings the ISBT Working Party has assigned Ff to the Diego blood pupsystem (DUO)making the 700.26 designation obsolete

@Pniels personal commuaication, 1999). 7.1 Comments on Common Ancestry

The identification of two distinct point mutations in DNA fiom NFLD+ individuals of a kindred segregating for NFLD and fiom a NFLD+ individual fiom Japan represents the first timt a Diego system antigen is reporteci to result fiom two molecular detemihants. This rarity mises the possibility that the two mutations observed in both populations could have a common ancestor. Historically, it has been well documented that there has been intemediary contact between Newfoundland and the Orient since the late 15" century when

Portuguese explorers sailed the hi& seas. However, it is possible that these mutations arose independently in the two distinct populations. This explanation seems less likely in view of the rarity of de novo mutations defining any low incidence antigen, as evidenced by serologically determined phenotypic Grquencies. in addition, it should be mentioned that the unrelated BOW+ individuals are both fiom the United Kingdom and it is possible that they have a common ancestor. Finally, although no cornmon ancestor could be identified, it is highly probable that the two Manitoba Mennonite kindreds segregating for Fr' are related.

Investigations of relatcdness are potentially possible through haplotype analysis.

Polymorphic DNA marken selected fiom the region surrounding the SLC4A 1 locus (1 7q2 1-

22) could be used to determine rclatedness. Related individuals would be expected to share sirnilar haplotypes in markers surrounding the SLC4A I locus. However, haplotype anal y sis may not provide a definitive enswer. Cmssing over betwecn homologous chromosomes in subsequent gentrations since the cornmon ancestor may rcsult in different haplotypes. 7.2 Alternative Methods for Determiaing RBC Pbenotypc

In many instances, the rarity of, or lack of access to, antibodies defming rare antigens makes it impossible to test serologically for these rare antigens. Because of the molecular definition of SLCQAI, it is now possible to use PCR-based tests in conjunction with restriction endonucleases to identify the presence of a mutant allele. Restriction endonucleases are employed to cleave PCR amplified DNA at specific sequences producing

DNA hgments of hown sue. These enzymes could also be used to determine the zygosity of the individual tested (see below). Point mutations giving rise to a specific Diego system antigen may either create or abolish a restriction site. For example, the point mutation underlying BOW abolishes a Bad restriction site. Treatment of amplified exon 14 DNA fiom a control individual with Ban1 would produce two product bands (1 14 bp and 159 bp), while treatment of exon 14 amplified DNA fiom a BOW+ individual would produce three product bands (1 14 bp, 159 bp and 273 bp), with the third band (273 bp) corresponding to the mutated allele. Similarly, the exon 14 point mutation giving rise to NFLD creates a Mscl restriction site and would cleave the 273 bp non mutated allele to 103 bp and 170 bp.

However, the exon 12 point mutation giving rise to NFLD neither creates nor destroys a restriction site and thenfore would not be usefil in determining allele status. The point mutation controlling Fr' expression abolishes a Bsalrestriction site. Treatment of amplified exon 13 DNA hmFr(a+) individuals would produce three bands (56 bp, 236 bp and 292 bp). Many of the point mutations underlying Diego system antigens either create or abolish restriction endonuclcase sites and could k used to confimi the presence of the mutated

&le. Table 8 summuizes thtsc results and pmvides fiagrnent sizes of the cleaved product for each antigen.

Table 8. Identification of restriction endonuclease cleavage sites within specific exons of SLC4AI correspondhg to various Diego blood group system antigens

Ant igen Exon Restriction Site? Restriction Fragment Symbol Endonuclease Sizes

Di' Abolished Hpall

Dib ------O Wi Neither

w? HIIIIIILII Wd' Created Msl I Rb8 Neither WARR Abolished Bbsl EL0 Abolished HpaIl Created BstNI Wu A bolished A pal BP' Neither Mo* Neither Hg. Abolished Cac81 vg. Created DralII Sw' A bolished Hpall SmaI BOW Abolished BanI NFLD Neither Created Mc1 Jn' Abolished Apal KREP Created HueLi Abolished Apal SWI Abolished HpaII Ff Abolished Bsal tAbolishcd, abolished a restriction endonuclease site; Created, created a restriction endonuclease site; Neither, neithei abolished nor created a restriction endonuclease site. 1O3

Amplification of DNA and treatrnent of the product with the appropriate restriction endonuclease can also determine zygosity. In cases where the underlying point mutation results in the creation of a restriction endonuclease site, three amplified DNA product bands would be evidcnt after restriction endonuclease treatment of DNA fiom a hetero zygous individual; one band ofhigher molecular weight (more bp) corresponding to the non-mutated allele, and two bands of lower molecular weight (fewer bp) corresponding to the mutated allele. Treatment of amplified DNA &orn a homo y gote would result in the presence of two bands of lower molecular weight. For example, distinguishing Wbhetero- and homozygotes is possible by this method. When visualized on an agarose gel, treatment of amplified DNA fiom Wd' heterozygotes with Ml1 would result in three bands (90, 183, and 273 bp), while treatment of amplified DNA fiom Wd' homozygotes would result in two bands of lower molecular weight (90 and 183 bp). To date, no homozygous Wda individual has been identified. However, based on the fiequency of Wda in the Huttente population it is plausible that within the next few generations a mating between Wd' heterozygotes could produce a Wd8 homozygote. In instances where the underlying point mutation abolishes a restriction site, cleavage of DNA fiom a heterozygous individual would still result in three bands; the higher molecular weight band comsponding to the mutated allele, and the two lower molecular weight bands cornsponding to the non-mutated allele. Treatment of amplified DNA fiom a homozygote would rcsult in a single band of higher molecular weight. For exarnple, individuals heterozygous and homozy gous for Wu have been ident i fied and the underlying point mutation abolishes an ApaI restriction site. Visualization of Apd trcatcd amplified DNA from a Wu+ hetcroysote would rcveal three product bands (1 13, 1O4

1 14, and 227 bp), while Apd treatment of amplified DNA fiom a Wu+ homozygote would only reveal the single band of higher molecular weight (227 bp).

7.3 Second Putative Extracellular Loop of Band 3

The amino acid substitution giving rise to Fr' expression is the first blood group antigen epitope mapped to the second extracellular loop of band 3. Wang et al. (1997) provided evidence suggesting bat the second extracellular loop is vital to band 3's anion transport hction and that certain perturbations within this loop could be detrimental to its function. Complementation studies in Xenopur oocytes of CO-expressedband 3 fragments demonstrate that the second extracellular loop is crucial to anion transport Function.

Not surprisingly only one other substitution in this loop has ever been identified.

Alloisio et al. (1997) characterizcd a band 3 variant (Coimbra) in which a Va1488Met substitution gave rise to HS. No information about Coimbra's antigenicity is known.

If nsidue 480 were critical to anion transport, a Glu480Lys substitution in the second extracellular loop would have two major effects. First, a charge reversal would occur by

inserting a basic amino acid (side chah containing an amino group) in the place of an acidic amino acid (side chain containing a carboxyl group). Second, the extracellular loop would adopt a different conformation based on the difference in side chain size and the interaction each would have on the spatial1y smunding residues. Because of this it seems unli ke 1y that residue 480 is involvcd in anion transport and may cal1 into question the suggestion that extracellular loop 2 is critical to bction. To examine the effect of a Glu480Lys substitution on anion transport bction, we have contacted Dr. Peter Jarolim who has agreed to perfonn anion flux assays on RBCs hmFr(a+) individuals. However, to absolutely dismiss the IO5 importance of this loop in anion transport fiction would require Merexperimentation involvhg the smounding residues and site-directed mutagenesis.

7.4 Serological Reiationsbip

The results of our study allow for speculation on the previously documented serological relationship between NFLD,BOW and Wu. Kaita et al, (1 992) summarized the pattern of reactivity of 101 multispecific reagents in tests with NFLD+, BOW+, and Wu+

RBCs. Thuty-seven of the reagents recognized none of the three determinants and require no Merexplmation. Twenty-two of the reagents reacted with NFLD+ and BOW+ RBCs, but not with Wu+ RBCs. We assume that these 22 contain antibodies defining band 3 epitopes containhg amino acid residue 561. since both NFLD and BOW are altered in this position. Three of the multispecific reagents reacted with RBCs carrying NFLD, BOW or

Wu, suggesting they contain antibodies that recognize epitope(s) created within the region of amino acid residues 561 to 565. Thirty of the reagmts reacted only with BOW+ RBCs, three only with Wu+ RBCs and six only with WLD+ RBCs. These 39 nagents apparently recognize the distinct band 3 epitopes that define BOW (Pro561Ser), Wu (Gly565Ala) and

NFLD (Glu429Asp and Pro561Ala). For the latter, we suspect that the defining epitope is crcated through an association/interactionbetween the fust and third extracellular loops of

ôand 3. Finai pmfawaits SK4A1 site-directed mutagenesis, and band 3 expression studies.

Contreras et al. (1987) briefly described a unique and perplening serologic telationship betwem Fr@+) and Swann+ RBCs. Absorption of two multispecific sera with

Swann positive RBCs (either SM? or SWI) abolishes activity against three examples ofFr(a+)

RBCs. Howevcr, absorption of tbc same two sera with Fr(a+) RBCs does not abolish 1O6 activity against either type of Swann positive RBCs. It is evident why if one Swann type (ie.

Sw') removes reactivity with Ff+ RBCs, that the other (ie. SWI) does as well, as the molccular determinants underlying Sw" and SWI expression have been characterized and are known to result in an amino acid substitution at residue 646 of band 3 (Zelinski et al., 1997;

Zelinski, unpublished observations). If one assumes there are antibodies that recognize a band 3 epitope that includes the Swann site (646) and the Fr' site (480). it is not clear why absorptions of the sarne two sera with Fr(a+) RBCs does not remove the activity to RBCs of either Swann type. One possible explanation is the reactive antibody portion (Fab)

recognizes the Swann epitope more strongly than the Fr' epitope (ie. stronger affmity). The

stronger affinity inay result fiom the position of the substituted amino acid residue within

the Fab binding pocket. The Swann substituted residue may be located in the central portion

of the Fab pocket resulting in a stronger affinity while the Fr' substituted residue may be

located in the periphery of the Fab pocket resulting in a weaker affmity. Absorption of the

sera with Swann positive RBCs wodd remove al1 of this type of antibody and subsequent

testing would result in loss of reactivity with Fr(a+) RBCs. On the other hand, absorption

of these sera with Fr(a+) RBCs rnay not absorb al1 subtypes of this antibody because of a

lack of tight a=ty between antibody and antigen. Consequently reactivity to Swann

positive RBCs would not k lost. Only hughexamination of the defining antibody,

perhaps through epitope mapping, and a ktter understanding of the three-dimensional

structure of band 3 will the nactivity pattern of these two sera be fully undentood. 7.5 Molecular Analysis of Blood Group Polymorphisms

Although the hction of many of the blood group gene products is known, almost nothing is known about the purpose of blood group polymorphisms. We are able to identify

RBC polymorphisms by utilizing alloantibodies produced through blood incompatibilities

(transhion or prepancy related). It bas been theorized that for any polymorphism to have achieved a fkquency of mater than 1% in a large population it must currently have, or have had a selective advantage @miels, 1999). With the recent observations that certain micro- organisrns can attach to blood gmup products (glycoproteins or glycolipids) facilitating invasion, it seems possible that many of the polymorphisms arose from selective advantage.

Table 9 lists blood group products (glycoproteins and glycolipids) that may act as pathogen receptors and promote invasion of the host. It is evident why these pathogens or those of the past may have played a critical role in the genesis and evolution of blood group polymorphisms. Mutations in blood gioup genes nsulting in altered susceptibility of the host to invasion by a microsrganism but do not adverscly affect the function of the molecule would be beneficial to the host. Many cases of host invasion by a micro-organism do not involve RBC,but pdpsdo involve blood group gene products expnssed in other tissues.

Many of the blood gmup products am present in several diffennt types of cells. Perhaps it is these non-erythroid cells which are subject to invasion by micro-organisms and the polymorphisms obscrved and detectcd serologicaily (in RBC) result fiom a selective advantage. For example, dthougb P. vivox is found throughout A frica, Fy (a-b-) individuah are known to be mistant to invasion. Table 9. Examples ofblood group products potentially involved in microsrganism invasion

Micro-organism Receptor (Blood group system)

Plasmodium falciparum GPA (MNS), CRI (Knops), Band 3 (Diego) Pfusmodium knowlesi Band 3 (Diego) Plasmodr'um vivax DuQ Glycoprotein (Du@) Hel icobacter pylor i Leb(Lewis) Escherichia coli Pl (P), DAF (Cromer) Mjmbacterium leprae CR1 (Knops) Influenza virus; Sendai virus GPA (MNS)

Apart hmnsistance to invasion by micro-organisms there is no obvious advantage to blood group diversity. More than 250 blood group antigens have been characterized and many have frequencies less than 1%. Perhaps some of these low fiequency antigens arose through point mutations occurring randomly in the genome over long periods of time.

Presumably those mutations which negatively alter function would be lost.

Intenstingly null phenotypes have bmi obsewed for several of the blood group products, includhg Colton-null, Kidd-null, Rh,,,,,,, Gerbich-null, Duw-null, Lutheran-null, and Kx-null. These null phenotypes often result fiom mutations in the controlling gene that prevent a fully functioning proàuct fiom king ptduced or fiom migrating to its appropriate cellular location (ie. plasma membrane). Some individuals with nul1 phenotypes are nported to k healthy, suggcsting s level of functionai ndundancy in cells. That is, if one moiecule is absent and its function is lost, a different molecule will perfonn a similar function although usually at a rcduced rate. Many null phenotypes were believed to be lethal based on hctiodloss. Recently, a sevenly hydropic, anemic infant was delivered by emergency 1O9 caesarean section, who was homozygous for a SLC4AI mutation. Examination of the infant's RBCs revealed a total absence of band 3 and band 4.2. At eight months, the infant was apparentiy thriving through regular blood transfusions and daily supplements of sodium bicarbonate. This single human example calls into question the dogma that total absence of band 3 (specifically Cl'MCO,' exchange) is incompatible with life. Rather, it demonstrates that absence of band 3 can be compatible with life, albeit with extreme medical intervention.

7.6 Future Directions

With the characterization of the underlying point mutations giving rise to many of the blood group antigens cornes the ability to test for these antigens at the DNA level. This allows a11 laboratories to conduct analyses previously possible only at select laboratories by expert serologists. In fact, rare reagents (ie. sera containing the defining antibodies), are no longer required if the antigen has been previously characterized. Primer sets for al1 characterized antigens could be prepared and a series of amplification reactions could easily be performed by PCR. Point mutations either abolishing or creating a restriction endonuclease site could easily be used in the identification of certain rare antigens as well as provide information on ygosity. For those mutations that do not alter restriction endonuclease sites, DNA sequencing could be performed to test for the underlying point mutation. In the fiinut, it may be possible to design scquence specific flourescent primers for al1 antigens and ampli@ only a few samples to get complete blood typing based on color.

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1M Tris-HCl --- Name Amount Final Concentration Tris-HC1 15.8g 1M ddH,O up to L OOmL total volume Total Volume 1 OOmL titrate to pH 8.3 with 4M NaOH

4M NaOH Name Amount Final Concentration NaOH 40.0g 4M ddH& up to 1.OL total volume Total Volume 1.OL

Name Amount Final Concentration KC1 7.5g 1M dW20 up to 100.0mL total volume Total Volume 1ûû.OmL

Name Asnount Final Concentration Mgcl2 20.3g 1M ddH,O up to 100.0mL total volume Totai Volume 1Oû.OmL

2% Gelatin Name Amount Final Concentration Gelatin 02g 2%

Total: Volume 10.0mL 10x PCR Buffer Name Amount Final Concentration 1M Tris-HCI pH 8.3 100.OpL 1OOmM 1M KCl 5OO.OpL SOOmM 1M MgCl, 1 S.OpL 1 SmM 2% Gelatin 50.0pL O. 1% ddH,O 335.0pL Total Volume 1 .OmL

mwMixnirew- Name Amount Final Concentration ddHH,O 133.OpL 10xPCRBuffer 28.OpL lx SSCP nucleotides 28.0pL 50% Glycerol 80.0pL Sense Primer 4.0pL Anti-sense Primer 4.OpL Tuq DNA Polymerase 3.OpL Total Volume 280.0uL

SSCP PCR Reaction Mixturr (10 reactions) Name Amount Final Concentration dM2O 120.0pL I ûx PCR Buffer 25.OpL SSCP Nucleotides 20.0pL lx 50% Glycerol 75 .OpL Smsc Primer 5.OpL Anti-sense Primer 5.0@ Tuq DNA Polymerase (5U/pL) 2.0pL [a-3?]dATP 50.OpCi Total Volume 280.5pL 9- Name hount Final Concentration Tris 157.5g Boric Acid 27.8% N%EDTA 9.3g ddH,O up to 1.OL total volume Total Volume 1.OL

1 x TBE Buffer Name Amount Final Concentration

Total Volume 1 ,OL

-300/0 at 29:1) Name Amount Final Concentration Acry lamide 43.51 Bis-acrylarnide 1 .Sg ddH,O up to 150.0rnL total volume Total Volume 1SO.0rnL 5 Namc Amount Final Concentration Glycerol 5 ,OmL SSCP Acrylamide 1 O.OmL (30% at 29: 1) 1 0x TBE Buffer S. OmL ddH20 30.0d Total Volume 50.0mL

20% Ammonium Persulfiitc Name hount Final Concentration Ammonium Persuffite 0.2g 20% ddH,O 1.OniI.l Total Volume 1.OmL sscp. Name Amount Final Concentration Formamide 9.8mL 0.5M EDTA pH 8.0 O.2mL Bromophenol Blue 0.0 1 g Xylcne Cyan01 0.01 g Total Volume 1O.OrnL

Sequen-OY~ at 1 9: 1) Name Amount Final Concentration Acry lamide 42.750 Bis-acry lamide 2.25g ddH20 up to 150.0mL total volume Total Volume 150,OmL dGel Narnc Amount Final Concentration Urea 21g Sequencing Acrylamide 10.0mL (30% at 19: 1) 10x TBE Baer 5.0mL ddH,O up to 50mL total volume Total Volume 50,OmL

10% Ammonium Persulfate Name Amount Final Concentration Ammonium Persulfate O. 1 g

Total Volume 1 .OmL

Name Amount Final Concentration Tris 193.68 Acetic Acid 45.7mL NaDTA 29.8g Total Volume 4.0L -titrate to pH 8.1 with glacid acctic acid lx TAE Buffer Namt Amount Final Concentration 10x TAE BufFer 100.0rnL ddH-0 900,OmL Total Volume 1 .OL

Name Amount Final Concentration Tris 121.14g ddH,O up to 1 .OL total volume Total Volume 1 .OL -titrate to pH 8.0 with glacial acetic acid

0.5M EDTA DH8.0 Narne Amount Final Concentration EDTA 186.21 NaOH Zog dM2O up to 1 .OL total volume Total Volume 1 .OL -titrate to pH 8.0 with ION NaOH

Low TE Name Amount Final Concentration 1M Tris pH 8.0 1 .OmL 0SM EDTA pH 8.0 O.2mL ddH20 98.8mL Total Volume 1OO.OmL

High TE Name Amount Final Concentration 1M Tris pH 8.0 tO.OmL 1ûûm.M 0.5M EDTA pH 8.0 8.0mL 40mM da20 82.W Total Volumc 100.0mL Name Amount Final Concentration 1M Tris pH 8.0 5O.OmL 100% p-mercapto- 1 .OmL ethanol ddH20 449.0m.L Total Volume 500,OmL A. - - g-- Name Amount Final Concentration WC1 8.30~ ddH,O up to 1 .OL total volume Total Volume 1 .OL

Name Amount Final Concentration Tris 2.06g ddH20 up to 100.0mL total volume

Total Volume 100.Om.L - . .- -. -titrate to pH 7.65 with glacial acetic acid

Name Amount Final Concentration 0.155M W,CI 900.0mL 0.1 70M Tris pH 7.65 100.0mL Total Volume 1 .OL

Namc Amount Final Concentration NaCl 5.84g dd&O up to 100.0mL Total Volume 100.OniL 20% - Name Amount Final Concentration SDS 200.0g ddH20 up to 1.OL total volume Total Volume 1 .OL

_whiteSoiution __ Name Amount Final Concentration 1 M Tris pH 8.0 10.OmL 1 OOmM OSM EDTA pH 8.0 8.0mL 40mM SM NaCl 20.OmL 1M 20% SDS 1.OmL 0.2% ddH20 6 1 .OmL Total Volume 100.0rnL - -

4M Ammonium Acetate- Name Amount Final Concentration Ammonium Acetate 144.20 ddH20 up to 50O.Om.L Total Volume 500,OmL -

24: 1 Chloroform:Iso-amvl alcohol Name Amount Final Concentration CMoro form 240,OmL Iso-amvl alcohol 1 0.0mL Total Volume 2SO.OmL

70% Ethanol Name Amount Final Concentration 95% Ethanol 737.0rnL 75% ddH20 263 .O& Total Volume 1 .OL - Name Amount Final Concentration Agame 03g 1.0%1x TAE Buffer 30.0rnL Ethidium Bromide 3.0uL Total Volume 30.0pL -

Name Amount Final Concentration 0.5M EDTA 10.0m.L Ficol 1O.OmL ûrange G Crystals 0. log ddHH,O 80.0mL Total Volume 1OO.Om.L