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!