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Elucidating Genetic and Biochemical Aspects of the P1 and Sda Carbohydrate Histo-Blood Group Antigens

Stenfelt, Linn LU orcid (2020) In Lund University, Faculty of Medicine Doctoral Dissertation Series
Abstract
Human histo-blood groups are inherited polymorphic variants that occur in the molecular structures on the human
red blood cell (RBC) surface. Introducing foreign RBCs into a recipient lacking an antigen may activate the humoral
defence leading to a hemolytic transfusion reaction. Antigenic differences can also cause hemolytic disease of the
fetus and newborn (HDFN). Blood group antigens are implicated as receptors in pathogen invasion and their
expression are often altered in cancerous tissues. Blood group antigens are carried by protein or carbohydrate
structures. Carbohydrate antigens are synthesized stepwise by glycosyltransferases and are carried on
glycosphingolipids or glycoproteins anchored into the RBC... (More)
Human histo-blood groups are inherited polymorphic variants that occur in the molecular structures on the human
red blood cell (RBC) surface. Introducing foreign RBCs into a recipient lacking an antigen may activate the humoral
defence leading to a hemolytic transfusion reaction. Antigenic differences can also cause hemolytic disease of the
fetus and newborn (HDFN). Blood group antigens are implicated as receptors in pathogen invasion and their
expression are often altered in cancerous tissues. Blood group antigens are carried by protein or carbohydrate
structures. Carbohydrate antigens are synthesized stepwise by glycosyltransferases and are carried on
glycosphingolipids or glycoproteins anchored into the RBC membrane. The aim of this work was to elucidate the
molecular genetic mechanisms behind the P1 and Sda antigens, as well as to study their glycan structures. The P1
antigen belongs to the P1PK blood group system. Silencing of A4GALT causes the null phenotype (Pk–, P1–) of this
system. However, the consequence of the genetic differences between the P1 (Pk+, P1+) and P2 (Pk+, P1–)
phenotypes, i.e. the molecular mechanism underlying how P1 antigen is expressed, has remained unknown.
Additionally, there have been divided views regarding the molecular carriers of the P1 antigen, Galα1-4Galβ1-
4GlcNAc-R. The Sda antigen GalNAcβ1-4(NeuAcα2-3)Gal-R was associated with the B4GALNT2 gene already in
2003. However, the genetic basis of the Sd(a–) phenotype was never revealed.
Through EMSA experiments the Runt-related transcription factor 1 (RUNX1) was identified to bind P1 alleles
specifically, dependent on rs5751348 in A4GALT. Knock-down of RUNX1 decreased the A4GALT mRNA levels,
establishing its effect as a P1/P2-discriminating factor. Based on these findings a genotyping assay was implemented
at the Nordic Reference Laboratory for Genomic Blood Group Typing in Lund, Sweden. P1 was also established to
be carried on glycoproteins in N-glycan conjugates, in addition to glycosphingolipids.
Sequencing of B4GALNT2 in nine Sd(a–) individuals identified the missense mutation rs7224888 as highly
associated with the phenotype. Additionally, the splice-site polymorphism rs72835417, and the rare missense
variants rs148441237 and rs61743617 were encountered in the Sd(a–) cohort. In silico studies identified a close
correlation between expression of B4GALNT2 and the cancer-associated lncRNA RP11-708H21.4 locus, located
directly downstream of the gene. Finally, the Sd(a–) associated SNP rs7224888 was shown to abolish Sda synthase
activity in over-expression experiments. The epitope was evaluated with DBA lectin binding, fluorescence
microscopy, enzyme immunoblots and mass spectrometry. The latter confirmed that the glycotransferase utilizes
substrates on both on N- and O-glycan elongation.
Understanding the molecular mechanism underlying the P1 antigen as well as defining the genetic background of
the Sd(a–) phenotype has enabled genotyping approaches for clinical practice. Additionally, the confirmation of
B4GALNT2 expressing the Sda synthase, has allowed the International Society of Blood Transfusion (ISBT) to move
the Sda antigen from the series of high-frequency antigens to its own, new blood group system designated SID, no.
038. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • professor Spitalnik, Steven L., Department of Pathology & Cell Biology, Columbia University, New York City, NY, USA
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Blood Group, P1 antigen, Sda antigen, A4GALT, B3GALNT2, glycosyltransferase, red blood cell, transfusion medicine
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
issue
2020:90
pages
76 pages
publisher
Lund University, Faculty of Medicine
defense location
Belfragesalen, BMC D15, Klinikgatan 32 i Lund
defense date
2020-09-18 13:15:00
ISSN
1652-8220
ISBN
978-91-7619-952-7
language
English
LU publication?
yes
id
cafbd22f-4ade-4200-bf34-7c1e4d7692c2
date added to LUP
2020-08-31 10:25:52
date last changed
2020-09-01 08:09:17
@phdthesis{cafbd22f-4ade-4200-bf34-7c1e4d7692c2,
  abstract     = {{Human histo-blood groups are inherited polymorphic variants that occur in the molecular structures on the human<br/>red blood cell (RBC) surface. Introducing foreign RBCs into a recipient lacking an antigen may activate the humoral<br/>defence leading to a hemolytic transfusion reaction. Antigenic differences can also cause hemolytic disease of the<br/>fetus and newborn (HDFN). Blood group antigens are implicated as receptors in pathogen invasion and their<br/>expression are often altered in cancerous tissues. Blood group antigens are carried by protein or carbohydrate<br/>structures. Carbohydrate antigens are synthesized stepwise by glycosyltransferases and are carried on<br/>glycosphingolipids or glycoproteins anchored into the RBC membrane. The aim of this work was to elucidate the<br/>molecular genetic mechanisms behind the P1 and Sda antigens, as well as to study their glycan structures. The P1<br/>antigen belongs to the P1PK blood group system. Silencing of <i>A4GALT</i> causes the null phenotype (Pk–, P1–) of this<br/>system. However, the consequence of the genetic differences between the P1 (Pk+, P1+) and P2 (Pk+, P1–)<br/>phenotypes, i.e. the molecular mechanism underlying how P1 antigen is expressed, has remained unknown.<br/>Additionally, there have been divided views regarding the molecular carriers of the P1 antigen, Galα1-4Galβ1-<br/>4GlcNAc-R. The Sda antigen GalNAcβ1-4(NeuAcα2-3)Gal-R was associated with the <i>B4GALNT2 </i>gene already in<br/>2003. However, the genetic basis of the Sd(a–) phenotype was never revealed.<br/>Through EMSA experiments the Runt-related transcription factor 1 (RUNX1) was identified to bind P1 alleles<br/>specifically, dependent on rs5751348 in <i>A4GALT</i>. Knock-down of RUNX1 decreased the A4GALT mRNA levels,<br/>establishing its effect as a P1/P2-discriminating factor. Based on these findings a genotyping assay was implemented<br/>at the Nordic Reference Laboratory for Genomic Blood Group Typing in Lund, Sweden. P1 was also established to<br/>be carried on glycoproteins in N-glycan conjugates, in addition to glycosphingolipids.<br/>Sequencing of <i>B4GALNT2</i> in nine Sd(a–) individuals identified the missense mutation rs7224888 as highly<br/>associated with the phenotype. Additionally, the splice-site polymorphism rs72835417, and the rare missense<br/>variants rs148441237 and rs61743617 were encountered in the Sd(a–) cohort. In silico studies identified a close<br/>correlation between expression of <i>B4GALNT2 </i>and the cancer-associated lncRNA RP11-708H21.4 locus, located<br/>directly downstream of the gene. Finally, the Sd(a–) associated SNP rs7224888 was shown to abolish Sda synthase<br/>activity in over-expression experiments. The epitope was evaluated with DBA lectin binding, fluorescence<br/>microscopy, enzyme immunoblots and mass spectrometry. The latter confirmed that the glycotransferase utilizes<br/>substrates on both on N- and O-glycan elongation.<br/>Understanding the molecular mechanism underlying the P1 antigen as well as defining the genetic background of<br/>the Sd(a–) phenotype has enabled genotyping approaches for clinical practice. Additionally, the confirmation of<br/><i>B4GALNT2</i> expressing the Sd<sup>a</sup> synthase, has allowed the International Society of Blood Transfusion (ISBT) to move<br/>the Sda antigen from the series of high-frequency antigens to its own, new blood group system designated SID, no.<br/>038.}},
  author       = {{Stenfelt, Linn}},
  isbn         = {{978-91-7619-952-7}},
  issn         = {{1652-8220}},
  keywords     = {{Blood Group; P1 antigen; Sda antigen; A4GALT; B3GALNT2; glycosyltransferase; red blood cell; transfusion medicine}},
  language     = {{eng}},
  number       = {{2020:90}},
  publisher    = {{Lund University, Faculty of Medicine}},
  school       = {{Lund University}},
  series       = {{Lund University, Faculty of Medicine Doctoral Dissertation Series}},
  title        = {{Elucidating Genetic and Biochemical Aspects of the P1 and Sd<sup>a </sup>Carbohydrate Histo-Blood Group Antigens}},
  url          = {{https://lup.lub.lu.se/search/files/83267163/Linn_Stenfelt_web.pdf}},
  year         = {{2020}},
}