Duffy

The Duffy blood group system (ISBT 008) is clinically significant due to its role in transfusion reactions and HDFN, but it holds unique biological interest because its primary antigens, Fy^a and Fy^b, reside on a protein that acts as a receptor for chemokines and, crucially, is used by the Plasmodium vivax malaria parasite to invade red blood cells. The antigens are defined by amino acid variations on the Duffy Antigen Receptor for Chemokines (DARC) protein, encoded by the DARC gene. The common Fy(a-b-) phenotype, prevalent in individuals of African descent, provides strong resistance to P. vivax malaria and has a distinct molecular basis compared to other blood group null phenotypes

Genetics: The DARC Gene

• The Duffy antigens are encoded by the DARC gene (also historically known as FY), located on chromosome 1
• The main alleles determining the Fy^a and Fy^b antigens are FY*A and FY*B

Biochemistry: The DARC Protein

• Structure: The DARC gene codes for the Duffy Antigen Receptor for Chemokines (DARC) protein. This is a glycosylated, multi-pass transmembrane protein (passing through the RBC membrane 7 times), typical of many cell surface receptors
• Function: DARC has two well-established functions:
  1. Chemokine Receptor It binds a wide variety of pro-inflammatory chemokines (small signaling proteins involved in inflammation and immune responses), particularly those of the CC and CXC families. It’s considered a “silent” or “scavenging” receptor, potentially regulating inflammation by binding excess chemokines without initiating traditional signaling cascades. It’s expressed not only on RBCs but also on endothelial cells lining blood vessels (especially post-capillary venules), Purkinje cells in the cerebellum, and other tissues
  2. Malaria Receptor It serves as the essential receptor for the merozoite stage of the malaria parasite Plasmodium vivax (and the related monkey malaria parasite P. knowlesi) to enter red blood cells

The Major Duffy Antigens: Fy^a and Fy^b

These are the two main, antithetical antigens of the system:

• Biochemical Difference: The difference between Fy^a and Fy^b arises from a single nucleotide polymorphism (SNP) in exon 2 of the DARC gene. This SNP results in a single amino acid substitution at position 42 on the first extracellular loop of the DARC protein:
  • Fy^a (FY1): Has Glycine (Gly) at position 42
  • Fy^b (FY2): Has Aspartic Acid (Asp) at position 42
• Inheritance: An individual inherits one DARC allele from each parent, leading to three common genotypes:
  • FY*A/FY*A → Phenotype: Fy(a+b-)
  • FY*B/FY*B → Phenotype: Fy(a-b+)
  • FY*A/FY*B → Phenotype: Fy(a+b+)
• Prevalence: Antigen frequencies vary significantly among ethnic populations:
  • Caucasians: ~66% Fy(a+), ~83% Fy(b+)
  • Black individuals: ~10% Fy(a+), ~23% Fy(b+) (High prevalence of Fy(a-b-))
  • Asians: >99% Fy(a+), ~18% Fy(b+)

The Null Phenotype: Fy(a-b-)

This phenotype, where red cells lack both Fy^a and Fy^b, is particularly important and has a fascinating molecular basis, especially in African populations:

• Common Mechanism (African Ancestry)
  • Genetics: Most Fy(a-b-) individuals of African descent possess the FY*B allele but have an additional SNP in the promoter region of the DARC gene, specifically at position -33 (T>C). This mutation occurs within a binding site for the GATA-1 erythroid transcription factor
  • Biochemistry/Expression: The GATA-1 factor is essential for gene expression in red blood cell precursors. The -33C mutation disrupts GATA-1 binding, preventing transcription of the DARC gene only in erythroid cells. Therefore, DARC protein (carrying the Fy^b antigen) is absent from their red blood cells
  • Crucially: DARC expression is NOT affected in other tissues (like endothelial cells) because gene expression there is controlled by different transcription factors. These individuals are often designated as having the FY*B^ES allele (Erythroid Silent)
  • Malaria Resistance: This RBC-specific absence of DARC confers strong resistance to invasion by Plasmodium vivax
• Rare Mechanisms (Other Populations)
  • In other populations (e.g., Caucasians), the rare Fy(a-b-) phenotype usually results from inheriting two non-functional DARC alleles with mutations within the coding sequence itself (nonsense or frameshift mutations). These individuals lack DARC expression on all tissues, not just RBCs

Other Duffy Antigens

• Fy3: A high-prevalence antigen present on cells expressing Fy^a or Fy^b. It is likely located on a different part of the DARC protein than the Fy^a/Fy^b site. Fy3 is absent on RBCs from individuals with the rare Fy(a-b-) phenotype caused by mutations in the coding region. However, Fy3 antigen is present (though perhaps weakly) on the RBCs of individuals with the common GATA-box Fy(a-b-) phenotype, as some residual protein might be expressed. Anti-Fy3 is made only by the rare “true null” individuals
• Fy5: Another antigen whose expression seems to depend on an interaction between the DARC protein and the Rh protein complex. Fy5 is weakened or absent on Rh_null cells
• Fy6: An epitope on the DARC protein that appears to be the actual binding site for P. vivax merozoites. It is conformation-dependent and sensitive to enzyme treatment

Effect of Reagents on Duffy Antigens

This is a defining characteristic used in antibody identification:

• Proteolytic Enzymes (Ficin, Papain, Bromelin): Duffy antigens (Fy^a, Fy^b, Fy6) are DESTROYED by common enzyme treatments. The enzymes cleave the DARC protein, removing the antigenic sites
• Sulfhydryl Reagents (DTT, 2-ME): Duffy antigens are RESISTANT to treatment with DTT or 2-ME

Duffy Antibodies (Anti-Fy^a, Anti-Fy^b, Anti-Fy3)

• Type: Usually IgG (often IgG1), reacting best at the AHG (Coombs) phase
• Stimulation: Typically immune stimulated (require exposure through transfusion or pregnancy). Anti-Fy^a is encountered more commonly than anti-Fy^b, likely reflecting antigen prevalence and perhaps slightly higher immunogenicity
• Dosage: Often show dosage (react more strongly with homozygous cells)
• Complement Activation: Generally do not activate complement efficiently
• Clinical Significance: Clinically Significant!
  • Hemolytic Transfusion Reactions (HTRs): Can cause both acute and delayed HTRs, typically extravascular
  • Hemolytic Disease of the Fetus and Newborn (HDFN): A well-recognized cause of HDFN, which can range from mild to severe
• Anti-Fy3: Rare antibody made by individuals with the “true” Fy(a-b-) null phenotype (coding region mutations). Reacts with almost all cells except those lacking DARC protein. Clinically significant

Key Terms

• DARC Gene: Gene on chromosome 1 encoding the Duffy protein
• DARC Protein: Duffy Antigen Receptor for Chemokines; the protein carrying Duffy antigens
• Chemokine: Small signaling proteins involved in inflammation and immune cell trafficking
• Fy^a/Fy^b: Antithetical antigens differing by Gly/Asp at position 42
• Fy(a-b-): Null phenotype lacking Fy^a and Fy^b on RBCs
• GATA-1: Erythroid transcription factor essential for gene expression in RBC precursors
• Promoter Mutation: Genetic change in the regulatory region upstream of a gene (e.g., the -33 T>C mutation in DARC causing the common Fy(a-b-) phenotype)
• Plasmodium vivax: Malaria parasite that uses DARC to invade red blood cells
• Enzyme Treatment: Use of proteolytic enzymes (ficin, papain) which destroy Duffy antigens