Abstract HGVS meeting Toronto, October 26, 2004 Phenotypes vs Genotypes in the World of Blood Group Antigens Olga O. Blumenfeld1 and Santosh K. Patnaik2 Deparments of Biochemistry1 and Cell Biology2. Albert Einstein College of Medicine. New York, NY 10461 Blood group antigens are proteins, glycans or glycolipids, of a variety of functions, whose common feature is that all are expressed on the surface of red cells and are polymorphic in the population. The hallmark of each antigen is an epitope or a linear or spatially arranged sequence of amino acids, or a carbohydrate sequence which, due to its variant nature, can be recognized as non-self by the immune system. The science (art) of serology is based on this recognition, and its goal is to decipher and assign blood group phenotypes using antibodies to the polymorphic epitopes as tools. A blood group system is a set of variant antigens encoded by alleles of a single locus, each expressing a related form of a common blood group phenotype. The Blood Group Antigen Gene Mutation Database* documents 39 gene loci encoding 29 blood group systems and comprising a total of 688 alleles that result in surface expression of at least 400 different blood group phenotypes. Here we examine the correlation between the genotype, the structure of the allele and the blood group phenotype. This is a rare example in which a direct correlation between a DNA alteration and a single physiologic function (antibody response) can be established, with modifier genes or environmental factors playing a minimal role. In the database, DNA alterations were documented in donors who were selected for study on the basis of a variant blood group phenotype. Thus an alteration of the epitopic and/or coding regions was expected. As generally observed for many documented human sequence variations, among total DNA alterations, single nucleotide mutations predominated (sense ~8%; missense ~ 50%, nonsense ~6%). Nonsense mutations and small deletions, insertions, or splice site alterations, often accompanied by additional upstream or downstream mutations, gave rise to a number of related alleles whose products were truncated or defective and resulted, directly or indirectly (inactive glycosyltransferases), in the absence of epitopes from the cell surface. Thus, for several blood group systems such different alleles resulted in the null phenotype (for example, the O phenotype of the ABO system). Gene rearrangements based on different mechanisms (gene conversions, unequal recombinations) could also give rise to the same epitopic sequence and the same phenotype that specified different hybrid alleles (the MN system). In contrast, in most instances, missense mutations linked to the epitope, gave rise to variant phenotypes each characteristic of a specific mutation and the amino acid change (Rh, Diego, Kell, others). This was observed whether the protein exhibited a single or multiple epitopes and the effect of the mutation could be direct or indirect (affecting level of cell surface expression). The majority of DNA alterations had no apparent effect on the function of the erythrocyte. The subtle relationships among a single amino acid replacement affecting the epitopic region, the immune response and the ability to detect it, become apparent from a survey of different blood group systems documented in the database. *http://www.bioc.aecom.yu.edu/bgmut/index.htm

Olga O. Blumenfeld & Santosh K. PatnaikDepartments of Biochemistry (OOB) & Cell Biology (SKP)

Albert Einstein College of Medicine, New York

www.bioc.aecom.yu.edu/bgmut/index.htm

Phenotypes in the World of Blood Group Antigens

documented in theDatabase of DNA Variation in Genes Encoding Blood Group Antigens

Blood Group Antigens

proteins, glycans or glycolipids

variety of functions

expressed at the surface of red cells

polymorphic in the population

Y YY

Blood Group System

A set of variant antigens

resulting from alleles of a single locus,

each defining a common serological phenotype.

Summary: 29 blood group systems, 40 genes, 707 allelesAlso detailed: non-human counterparts for H/h, MN, Rh

System Locus Funcion Alleles

ABO ABO enzyme 115Chido- Rodgers C4A, factor 7+

C4BColton AQP1 channel 7Cromer DAF receptor 13Diego SLC4A1 exchanger 78Dombrock DO unknown 9Duffy FY receptor 7Gerbich (Ge) GYPC structure 9GIL AQP3 channel 2H/h FUT1, enzymes 57

FUT2I GCN2 enzyme 8

(IGnT)Indian (IN) CD44 adhesion 2JMH SEMA7A signaling 0Kell (with Kx) KEL, enzyme 67

XKKidd SLC14A1 transport 8Knops CR1 receptor 24+

System Locus Function Alleles

Landsteiner- ICAM4 adhesion 3Weiner (LW)Lewis FUT3, enzymes 36

FUT6,FUT7

Lutheran LU adhesion 16MNS GYPA, unknown 43

GYPB,GYPE

OK BSG adhesion 5P-related A4GALT, enzymes 27

B3GALT3RAPH-MER2 CD151 3Rh RHCE, transport 126

RHD, RHCGRHAG, RHBG

Scianna ERMAP adhesion 4Xg XG, adhesion 0

CD99 (MIC2)YT ACHE enzyme 4

Summary of DNA alterations

Locus Sense Missense Nonsense Splicing Insertion Deletion Rerrangements Gross Recurrentidenticalalterationsat same sites

Recurrentdifferentalterationsat samesites

TotalDNAchanges1

ABO 17 29 2 - 1 2 22 - 19 - 53 (20)A4GALT 3 4 1 - 3 4 - - 4 - 15 (n/a)CR1 1 16 - - - - - - - - 17 (n/a)KEL 2 26 6 3 1 - - - - 3 38 (14)XK - 2 6 5 1 6 - 8 - - 28 (n/a)FUT1 1 16 4 - 1 3 - - - 2 25 (25)FUT2 3 6 3 - - 2 - - 5 - 14 (15)FUT3 - 11 - - - - - - 6 - 11 (10)

FUT6 7 9 1 - 1 - - - 13 - 18 (5)GYPA - 14 - 1 - - 243 2 - - 41 (1)GYPB 2 7 - - - - 243 2 - - 35 (n/a)RHCE 2 13 1 1 - 2 183 - - - 37 (12)RHD 2 45 3 - 1 2 24 - - - 77 (8)RHAG - 5 - 6 3 - - - - - 14 (11)SLC4A1 - 48 4 3 7 12 - - - - 74 (78)BGMUTdb total

312 19 19 33 92 12 497

HGMDtotal

19368 3207 2093 5498 340 1827 32333

1. For comparison, number of corresponding entries in HGMD is shown in parentheses;n/a: not available in HGMD. Not included: regulatory mutations, small indels, repeatvariations, gross insertions & duplications. Total documented for 1338 genes2. Minimal number; approximate numbers as breakpoints for the proposed intragenicrearrangements and gene conversions are unknown3. Rough estimate of all numbers because of a variety of numerous gene rearrangements

Phenotype vs genotype

A number of alleles give rise to the same blood group phenotype

Silencing mutationsnonsense, deletions, insertions, splicing, regulatory regions rearrangements, gross deletions (null phenotype in nearly all systems) Kell, Rh, Diego, ABO, others

Gene rearrangements GYPA, Rh ex. Sta - 7 genotypes; RH neg - 17 genotypes

A single allele gives rise to a unique blood group phenotype

Missense mutationsKell, Diego, RH, Duffy, ABO and others

Gene rearrangementsGYPA, Miltenberger series; Rh, weak D(Du), DAU & others

Same Blood Group Phenotype, Multiple Genotypes

Kell null

1 phenotype

11 genotypes

7 nonsense3 splicing1 deletion

Same Blood Group Phenotype, Multiple Genotypes

O gene-null

Same deletion in many alleles (261delG in 38 of 43 O null alleles)

19 variants

Popov et al. JBC.1997,272,18325

Band 3 Glycoprotein

del

del

19 blood group phenotypes54 other phenotypes (spherocytosis, etc.)

anion exchanger

anion ex.

Binds torbc cytoskeleton

Diego

19 of 19 alleles

Multiple BloodGroup Phenotypes,Multiple Genotypes

Sites and distribution of alterations vs location of epitopes:Kell (KEL)

Each polymorphic site can be assigned to a different Kell antigen

24 missense mutations at positions in extracellular domains

7 nonsense; 3 splicing ;1 del

Sets of polymorphic residues

Each expressed in different individuals at different frequencies.

Common phenotype

K-1, 2*; -3, -21, 4*; -6, 7*; 11*, -17; 14*, -24; 10, 5, 12, 13, 16, 18, 20, 22, -23

Known antithetical partners

K2/K1; K4/K3/K21; K7/K6; K11/K17

The Kell Glycoprotein

Single Blood Group Phenotype, Single Genotype

Kell 22 of 24 alleles

note

note

note

Same Blood Group Phenotype, Multiple Genotypes:Examples in the KEL blood group system

Single Blood Group Phenotype, Single Genotype

Rh

Examples of genotypes vs phenotypes due to DNA rearrangements in GYPA family

Gene Sequence Phenotype

GYPA ex3 EET ex4 GERVQL wild type

GYPB ex2 QTN ex4 GETGQL wild type s

GYPB ex2 QTN ex4 GEMGQL wild type S

hyb.A-Bs Recombination ex3 EET ex4 GETGQL HIL

hyb.A-BS Recombination ex3 EET ex4 GEMGQL SJLhyb.BAB Gene conversion ex3 EET ex4 GETGQL HIL

hyb.B-A Recombination ex2 QTN ex4 GERVQL Sta

Johe,Vengelen-Tyler,Leger,Blumenfeld Blood 1991,78:2456

Examples were provided showing that, on the red cell surface, single amino acid or carbohydrate alterations,

resulting from missense mutations or other DNA changes are recognized, as one might expect, as foreign by the immune system and, remarkably, can be detected by

serological approaches .

Acknowledgements

Contributors to the database

Departments of Biochemistry and Cell Biology, Albert Einstein College of Medicine

Thank you!