Knops Blood Group Antigens Research Articles

No Evidence that Knops Blood Group Polymorphisms Affect Complement Receptor 1 Clustering on Erythrocytes

Clustering of Complement Receptor 1 (CR1) in the erythrocyte membrane is important for immune-complex transfer and clearance. CR1 contains the Knops blood group antigens, including the antithetical pairs Swain-Langley 1 and 2 (Sl1 and Sl2) and McCoy a and b (McCa and McCb), whose functional effects are unknown. We tested the hypothesis that the Sl and McC polymorphisms might influence CR1 clustering on erythrocyte membranes. Blood samples from 125 healthy Kenyan children were analysed by immunofluorescence and confocal microscopy to determine CR1 cluster number and volume. In agreement with previous reports, CR1 cluster number and volume were positively associated with CR1 copy number (mean number of CR1 molecules per erythrocyte). Individuals with the McCb/McCb genotype had more clusters per cell than McCa/McCa individuals. However, this association was lost when the strong effect of CR1 copy number was included in the model. No association was observed between Sl genotype, sickle cell genotype, α+thalassaemia genotype, gender or age and CR1 cluster number or volume. Therefore, after correction for CR1 copy number, the Sl and McCoy polymorphisms did not influence erythrocyte CR1 clustering, and the effects of the Knops polymorphisms on CR1 function remains unknown.

Severity of malaria in relation to a complement receptor 1 polymorphism: a case–control study

Background:Studies on the relationship between complement receptor 1 (CR1) polymorphisms in exon 29 encoding the Knops blood group antigens (Swain–Langley (Sl) and McCoy (McC)) and outcome of clinical malaria have produced inconsistent results.Methods:Blood samples from Ghanaian children (n = 150) aged 1–12 years with complicated and uncomplicated malaria were genotyped for the Sl and McC blood group alleles by polymerase chain reaction and restriction fragment length polymorphism. Effect of Sl and McC genotypes on the clinical outcome of malaria was evaluated using logistic regression.Results:McCa/b genotype was significantly associated with more than two-fold increased susceptibility for severe malaria (OR = 2.31; 95% CI: 1.03–5.20, P = 0.043). However, McCb/b was associated with an 88% reduced risk of severe malaria (OR = 0.12; 95% CI: 0.02–0.64, P = 0.013). In contrast, there was no significant association between severe malaria and Sl1/1, Sl1/2, Sl2/and McCa/a genotypes. There was a trend towards decreased susceptibility to both cerebral malaria (CM) (OR = 0.13; 95% CI: 0.02–1.15, P = 0.07) and severe malarial anaemia (SA) (OR = 0.14; 95% CI: 0.02–1.19, P = 0.07) for McCb/b genotype when compared with the McCa/a genotype. There were no significant associations between Sl1/2 or Sl2/2 genotype and CM or SA when compared with Sl1/1 genotype.Conclusions:McCa/b was associated with increased susceptibility to severe malaria and McCb/b associated with reduced risk of severe malaria. Further studies with large sample size in other malaria endemic regions in Africa are warranted to confirm these findings.

Extensive Genomic Variability of Knops Blood Group Polymorphisms is Associated with Sickle Cell Disease in Africa

Sickle cell disease (SCD) is a multisystem disorder characterized by chronic hemolytic anemia, vaso-occlusive crises, and marked variability in disease severity. Patients require transfusions to manage disease complications, with complements, directed by complement regulatory genes (CR1) and its polymorphisms, implicated in the development of alloantibodies. We hypothesize that CR1 polymorphisms affect complement regulation and function, leading to adverse outcome in SCD. To this end, we determined the genomic diversity of complement regulatory genes by examining single nucleotide polymorphisms associated with Knops blood group antigens. Genomic DNA samples from 130 SCD cases and 356 control Africans, 331 SCD cases and 497 control African Americans, and 254 Caucasians were obtained and analyzed, utilizing a PCR—RFLP (polymerase chain reaction–restriction fragment length polymorphism) assay. Analyzing for ethnic diversity, we found significant differences in the genotypic and allelic frequencies of Sl1/Sl2 (rs17047661) and McCa/b (rs17047660) polymorphisms between Africans, African Americans, and Caucasians (P < 0.05). The homozygote mutant variants had significantly higher frequencies in Africans and African Americans but were insignificant in Caucasians (80.2% and 59.6% vs 5.9% for Sl1/2; and 36% and 24% vs 1.8% for McCa/b). With SCD, we did not detect any difference among cases and controls either in Africa or in the United States. However, we found significant difference in genotypic (P < 0.0001) and allelic frequencies (P < 0.0001) of Sl1/Sl2 (rs17047661) and McCa/b (rs17047660) polymorphisms between SCD groups from Africa and the United States. There was no difference in haplotype frequencies of these polymorphisms among or between groups. The higher frequency of CR1 homozygote mutant variants in Africa but not United States indicates a potential pathogenic role, possibly associated with complicated disease pathophysiology in the former and potentially protective in the latter. The difference between sickle cell groups suggests potential genetic drift or founder effect imposed on the disease in the United States, but not in Africa, and a possible confirmation of the ancestral susceptibility hypothesis. The lower haplotype frequencies among sickle cell and control populations in the United States may be due to the admixture and the dilution of African genetic ancestry in the African American population.

Human genetic polymorphisms in the Knops blood group are not associated with a protective advantage against Plasmodium falciparum malaria in Southern Ghana

BackgroundThe complex interactions between the human host and the Plasmodium falciparum parasite and the factors influencing severity of disease are still not fully understood. Human single nucleotide polymorphisms SNPs associated with Knops blood group system; carried by complement receptor 1 may be associated with the pathology of P. falciparum malaria, and susceptibility to disease.MethodsThe objective of this study was to determine the genotype and haplotype frequencies of the SNPs defining the Knops blood group antigens; Kna/b, McCoya/b, Swain-Langley1/2 and KCAM+/- in Ghanaian patients with malaria and determine possible associations between these polymorphisms and the severity of the disease. Study participants were patients (n = 267) admitted to the emergency room at the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana during the malaria season from June to August in 1995, 1996 and 1997, classified as uncomplicated malaria (n = 89), severe anaemia (n = 57) and cerebral malaria (n = 121) and controls who did not have a detectable Plasmodium infection or were symptomless carriers of the parasite (n = 275). The frequencies were determined using a post-PCR ligation detection reaction-fluorescent microsphere assay, developed to detect the SNPs defining the antigens. Chi-square/Fisher’s exact test and logistic regression models were used to analyse the data.ResultsAs expected, high frequencies of the alleles Kna, McCb, Sl2 and KCAM- were found in the Ghanaian population. Apart from small significant differences between the groups at the Sl locus, no significant allelic or genotypic differences were found between the controls and the disease groups or between the disease groups. The polymorphisms define eight different haplotypes H1(2.4%), H2(9.4%), H3(59.8%), H4(0%), H5(25.2%), H6(0.33%), H7(2.8%) and H8(0%). Investigating these haplotypes, no significant differences between any of the groups were found.ConclusionThe results confirm earlier findings of high frequencies of certain CR1 alleles in Africa; and shed more light on earlier conflicting findings; the alleles McCb, Sl2, Knb and KCAM- or combined haplotypes do not seem to confer any protective advantage against malaria infection or resulting disease severity. Based on these findings, in a very well-characterized population, malaria does not seem to be the selective force on these alleles.

Lack of Evidence from Studies of Soluble Protein Fragments that Knops Blood Group Polymorphisms in Complement Receptor-Type 1 Are Driven by Malaria

Complement receptor-type 1 (CR1, CD35) is the immune-adherence receptor, a complement regulator, and an erythroid receptor for Plasmodium falciparum during merozoite invasion and subsequent rosette formation involving parasitized and non-infected erythrocytes. The non-uniform geographical distribution of Knops blood group CR1 alleles Sl1/2 and McCa/b may result from selective pressures exerted by differential exposure to infectious hazards. Here, four variant short recombinant versions of CR1 were produced and analyzed, focusing on complement control protein modules (CCPs) 15–25 of its ectodomain. These eleven modules encompass a region (CCPs 15–17) key to rosetting, opsonin recognition and complement regulation, as well as the Knops blood group polymorphisms in CCPs 24–25. All four CR1 15–25 variants were monomeric and had similar axial ratios. Modules 21 and 22, despite their double-length inter-modular linker, did not lie side-by-side so as to stabilize a bent-back architecture that would facilitate cooperation between key functional modules and Knops blood group antigens. Indeed, the four CR1 15–25 variants had virtually indistinguishable affinities for immobilized complement fragments C3b (K D = 0.8–1.1 µM) and C4b (K D = 5.0–5.3 µM). They were all equally good co-factors for factor I-catalysed cleavage of C3b and C4b, and they bound equally within a narrow affinity range, to immobilized C1q. No differences between the variants were observed in assays for inhibition of erythrocyte invasion by P. falciparum or for rosette disruption. Neither differences in complement-regulatory functionality, nor interactions with P. falciparum proteins tested here, appear to have driven the non-uniform geographic distribution of these alleles.

Knops blood group polymorphism and susceptibility to Mycobacterium tuberculosis infection

Complement receptor 1 (CR1) protein carries the Knops blood group antigens and is the receptor for the major ligand involved in Mycobacterium tuberculosis (Mtb) adhesion to macrophages. Erythrocyte CR1 binds immune complexes (ICs) formed during Mtb invasion, facilitating their clearance by the host immune system. The occurrence of specific Knops blood group genotypes among African populations was investigated to evaluate their impact on resistance or susceptibility to Mtb infection. The distribution of the Knops blood group genotypes (McC and Sl) was compared between tuberculosis (TB) patients with confirmed diagnosis of Mtb in isolates and negative controls. Conditional logistic regression was used to access the association between genotypes distribution and susceptibility to Mtb infection. At the McC locus, individuals heterozygous (McC(a) /McC(b) ) were more resistant to Mtb infection (odds ratio [OR], 0.42; 95% confidence interval [CI], 0.22-0.81; p = 0.007). Although less significant, a similar effect was conferred by Sl1/Sl2 genotype (OR, 0.05; 95% CI, 0.28-0.9; p = 0.02). This protective effect was maintained among individuals presenting the McC(b) /Sl2 haplotype (OR, 0.25; 95% CI, 0.08-0.74; p = 0.008). Acquisition of McC(b) and Sl2 alleles among African population is correlated with resistance to Mtb infection, adding this bacterium to the list of mechanisms underlying the selection of the Knops blood group polymorphism among these populations.

Molecular analysis of the York antigen of the Knops blood group system

Antigens of the Knops blood group system are present on complement component (3b/4b) receptor 1 (CR1/CD35), which is a transmembrane glycoprotein encoded by the CR1 gene. Eight of the nine known antigens of this system are linked to polymorphisms in Exon 29. The molecular background of one antigen, York (Yk(a)), has not yet been described. We aimed to identify a polymorphism associated with the absence of Yk(a) to enable molecular typing. Yk(a)-negative individuals were identified by serologic typing. Their CR1 gene was partially sequenced and compared to that of Yk(a)-positive individuals. Loss of Yk(a) antigen was investigated by expressing the SCR22/23 domain of both wild-type and mutated CR1 as a GPI-linked protein on HEK293 cells. We observed that absence of the Yk(a) antigen is caused by a mutation in Exon 26 of the CR1 gene. This 4223C>T mutation results in a 1408T>M change at the protein level. Ten of 117 donors (8.5%) were homozygous TT, confirming the Caucasian frequency of 8% Yk(a)-negative individuals. Serologically, these TT donors showed a Yk(a)-negative phenotype, while CC/CT individuals were Yk(a)-positive. While the Yk(a) antigen was present on HEK293 cells expressing wild-type constructs, cells expressing the 4223C>T variant were Yk(a) negative. We identified a 4223C>T sequence variation in the CR1 gene causing absence of the Yk(a) antigen of the Knops blood group system. With this finding, all polymorphisms of the known Knops blood group antigens have been revealed, enabling molecular testing to contribute to red blood cell alloantibody identification procedures.

Association between Knops blood group polymorphisms and susceptibility to malaria in an endemic area of the Brazilian Amazon.

Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the genotype and allele and haplotype frequencies of single-nucleotide polymorphisms (SNPs) of Knops blood group antigens and examine their association with susceptibility to malaria in an endemic area of Brazil. One hundred and twenty-six individuals from the Brazilian Amazon were studied. The CR1-genomic fragment was amplified by PCR and six SNPs and haplotypes were identified after DNA sequence analysis. Allele and haplotype frequencies revealed that the Knb allele and H8 haplotype were possibly associated with susceptibility to Plasmodium falciparum. The odds ratios were reasonably high, suggesting a potentially important association between two Knops blood antigens (Knb and KAM+) that confer susceptibility to P. falciparum in individuals from the Brazilian Amazon.

Analysis of Knops blood group antigens on CR1 (CD35) by the MAIEA test and by immunoblotting.

Kna, McCa, Sla and Yka are red cell antigens of relatively high frequency, located on complement receptor 1 (CR1, CD35). Antibodies to these Knops system antigens are not uncommon. They are not haemolytic and do not reduce the survival of transfused incompatible red cells, but they are a nuisance in transfusion laboratories as they can cause an incompatible crossmatch and must be identified before they can be dismissed as clinically insignificant. Human red cell alloantibodies can be shown to be Knops system antibodies by the monoclonal-antibody-specific immobilization of erythrocyte antigens (MAIEA) test, using murine monoclonal anti-CR1. In addition to confirming that Kna, McCa, Sla and Yka are located on CR1, the MAIEA test was used to confirm that Csa is not on CR1. Red cells of the Helgeson phenotype, the null phenotype of the Knops system by conventional serological methods, have levels of Kna, McCa, Sla and Yka intermediate between those of alpha-chymotrypsin-treated cells (which lack Knops system antigens) and those of positive control cells. Level of expression of Knops system antigens is very variable and intensity of staining of immunoblots probed with monoclonal anti-CR1 correlated with strength of Knops system antigens, as determined by the MAIEA test. In individuals heterozygous for alleles producing different allotypes, separate bands representing each allotype on an immunoblot showed identical intensity of staining, suggesting that the quantity of CR1 on red cells is controlled, at least in part, by a locus independent of CR1. An analysis of CR1 on red cells of individuals who have made Knops system antibodies suggested that the Knops system antigens and the antibodies that detect them are complex and heterogeneous.