AB0 Blood Group Discrepancies

Gregory Denomme, PhD, FCSMLS(D)BloodCenter of Wisconsin

Historical Perspective Ancestral (consensus) sequence is A1

Initial evolution uncertain– Cell-to-cell recognition (glycocalyx)?– Variations evolved to counteract the ability

of microorganisms to invade cells

Evolution due to microorganisms– Bubonic plague 1340s – Group O survivors– Regional – Group B among Asians

Objectives

To review the structure/function of glycosyltransferases

To identify the molecular basis for the common transferase activities

To summarized the nucleotide changes that lead to ABO discrepancies

ABO 101 (years that is) Discovered by Landsteiner 1901 Status determined by:

– expression of a sugar (glycosly) due to an enzyme (ase) and the presence of reciprocal antibody(ies)

– e.g. red cells: Group A, plasma: anti-B

Hematological disorders confound status

Expression does not always adhere to classical Mendelian inheritance

1990 nucleotide consensus sequence for ABO– better understand of spurious and conflicting

observations

The A and B antigens are structurally similar!

A antigen B antigen

Golgi

Location of Glycosyltransferases

Gene to Gene Product

Cytoplasmic tail

Stem region

Globular Head

Transmembrane domain

A

B

mRNA

GalNAc1α3Galβ-R|α2Fuc

Gal1α3Galβ-R|α2Fuc

9q34….

1 2 3 4 5 6 75’ UTR 3’ UTR

Exons

RNA

****

354 amino acids

Patenaude et al., 2002

• Divided into two domains separated by a cleft

General structure

•Active centre is located within the cleft

z N-terminal• Recognizes nucleotide donor

General structure

Patenaude et al., 2002

General structure

Patenaude et al., 2002

Donor (UDP)

General structure

Patenaude et al., 2002

C-terminal• Recognizes acceptor (H)

General structure

Patenaude et al., 2002

Acceptor (H)

General structure

Patenaude et al., 2002

Mn2+

Disordered loop (amino acids 179-194)

4 penultimate amino acids

Only 4 amino acids affect substrate specificity or the rate of reaction

Amino acid position 176 235 266 268Glycosyltransferase A Arg Gly Leu GlyGlycosyltransferase B Gly Ser Met AlaNucleotide position 526 703 796 803

261 467 526 703 796/803 1060802

Common ABO Alleles: exon 6 - 7

A1

B

O2

O1v

O1

∆G

A2

∆C

A2

B B AA

A1

Exon 3 Deletion B3 Phenotype – altered function

GTGAGTGC...Exon 3B Exon 4

Exon 3 GTGAATGC... Exon 4B3

mRNA

ABO∆3

Macroscopic MF aggl’n

Equal Crossover

Occurs in Meiosis during Metaphase II

Pair of chromosomes Non-sister chromatidexchange

Non-sister Chromatid Exchange261 467 526 703 796/803 1060

802

AX

B

O1v

O2

O1v

AX

Group A child from non-A parents!

Non-deletional O2 Alleles (O301)Blood grouping discrepancies between ABO genotype and phenotype caused by O alleles.Yazer MH, Hosseini-Maaf B, Olsson ML. Curr Opin Hematol. 2008 Nov;15(6):618-24

PURPOSE OF REVIEW: In the modern transfusion service, analysis of the ABO allele underlying a donor or recipient's A or B subtype phenotype is becoming a mainstream adjunct to the serological investigation.

RECENT FINDINGS: Although the most common group O alleles share a common crippling polymorphism, a growing number of alleles feature other polymorphisms that render their protein nonfunctional yet are similar enough to the consensus A allele that an errant phenotype would be predicted from the genotype… Some of these O alleles might actually encode a protein with weak and variable A antigen synthetic ability.

SUMMARY: ABO genotyping can be a powerful asset in the transfusion service, but a thorough knowledge of the confounding factors that can lead to genotype/phenotype discrepancies is required.

Forward type as Group O Back type as Group A (no anti-A)

261 467 526 703 796/803 1060802

Common ABO Alleles: exon 6 - 7

A1

B

O2

O1v

O1

∆G

A2

∆C

A2

B B AA

A1

Weak/altered transferase activity Missense mutations tend to cluster

– nts 539-548 (2) 641-721 (6) 829-873 (5) 965-1061 (7)

Polymorphisms in these regions suggest that the areas critical to functional substrate/ acceptor sites or are necessary for the appropriate 3D structure– Areas to explore in in vitro synthetic enzyme production

A few alleles (A1, A2, B, B(A), O2), expressed in synthetic systems, show cause and effect relationships between the amino acid alterations and the associated phenotypes

5’ enhancer region ABO gene has no obvious promoter region An active 5’ enhancer (minisatellite sequence)

has been identified requires CBF/NF-Y The enhancer region is located 43 kbp

upstream of the start codon and is 43 bp in length

Variable number of tandem repeats (VNTR)– One 43 bp motif associated with A1 and O2 alleles– 4 – 43 bp motifs associated with A2, B, O1 and O1v

Olsson and Chester 2001

A2B A1A1A1O1

Olsson and Chester 2001

5’ enhancer transcription The alleles with 4 repeats give rise to 100

times more mRNA than the single motif

Regardless, the A1 enzyme is more efficient than B or A2 despite only one enhancer

This observation suggests that mutations giving rise to B are detrimental to enzyme activity

Somehow the enhancer region compensates by the change in the donor sugar

Enzyme Dimerization The G871A (Asp291Asn) change

interferes with the α-helical structure; likely reason for decreased enzyme activity

Enzyme kinetics is better if it dimerizes with a functional enzyme

Observation seen with Ax phenotype with the B glycosyltransferase

One allele – two blood groups? Dual enzyme activity alleles identified 4 nucleotides define A and B activity

– the latter two are crucial for A or B specificity

cis-AB phenotype: G803C (B-associated nt)– represented by the AAAB designation

cis-AB phenotype: A796C (A-associated nt)– represented by the BBAB designation

A2

B B A A

526 703 796/803

cis-ABA A A B

526 703 796/803

cis-ABB B A B

526 703 796/803

B(A) activity Normal B glycosyltransferase transfers

some A sugar onto H substance– not detectable using antibody probes

A monoclonal anti-A was discovered that reacted with some phenotype B– Found an A703G (Ser235Gly)– represented by BABB

– aka weak cis-AB… make anti-A!

cis-ABB A B B

526 703 796/803

ABO Summary Transferase activity defined by 4 amino acids

Missense mutations have taught us much about the functional domains of transferases– Source of weak transferase activity

Missense mutations of the 4 positions lead to gain of function

The common alleles can be identified by their nucleotide changes at 4 positions– with one exception; Group B with Group O non-

deletional allele → the Ax phenotype!

THANK YOU!