Previous serologic studies found that Kna
was a high-incidence antigen in Caucasian and African American persons. Our earlier studies of native African persons indicated that Kna
was also a high-incidence antigen in this group.14
Thus, it was surprising to find that 36 percent of the samples from Bandiagara, Mali, typed as Kn(a−). Furthermore, 14 percent of the Bandiagara samples exhibited the Knops-null or Helgeson phenotype. These observations had not been previously made in African American persons or in other test sites in Mali where the incidence of the Helgeson phenotype was approximately 1 percent.2,7,14
Because we were unable to serologically type or Knb
, it is unknown whether these samples were heterozygotes. It has previously been reported that heterozygosity at the McC and Sl loci can result in false-negative phenotypes.9
Alternatively, the phenotype differences may relate to the ethnicity of the current study group. The other Mali sites have ethnic groups composed predominately of Bambaran and Melinke persons whereas the major ethnic groups in Bandiagara are Dogon and Peuhl persons.
To identify potential regions of the CR1 gene that might encode the Kna
polymorphism, inhibition studies were performed with various CR1 deletion constructs. Tamasauskas and colleagues15
reported that three examples of anti-Kna
were mostly inhibited with LHR-C whereas three others were inhibited by LHR-D. It was found that only the LHR-D+-containing constructs were able to completely neutralize all of our anti-Kna
including the original (DK) serum sample. DNA sequencing of this region identified a SNP at bp 4708 that changed the wild-type amino acid valine (No. 1561) to methionine. Interestingly, the corresponding amino acid in LHRs A, B, and C also carry valine. Because antibodies can recognize a series of six amino acids for binding, it is possible that adjoining amino acids are also important in the antigenic site. When analyzing the six amino acids on either side of 1561, we find that heterogeneity exists between the LHRs (). This may explain some of the differences in the inhibition studies between the previous reported work and the current study.
Alignment of amino acid sequences in homologous regions of human CR1 where the *Kna/Knb polymorphism is located. Bold letters indicate differences between the LHRs.
Because the Kna
mutation did not introduce a RFLP, primers were designed that would introduce a restriction enzyme site into the mutant DNA. The McC and Sl SNPs are also in the same exon; thus, the PCR allowed for the genotyping at all three loci. With the PCRRFLP method, the allele frequencies were 0.90 for Kna
and 0.10 for Knb
in the Bandiagara population. By direct allele count the projected phenotype frequency of Knb
in Bandiagara would be 18.3 percent, which is considerably higher than previously reported for any group. Other serologic studies have reported that the frequency of Knb
ranged from 1.2 percent among African American persons living in Philadelphia2
to 4.7 percent among 63 random Caucasian donors.16
Its role in CR1 function as well as rosetting and malaria are presently under investigation.
The PCR-RFLP was also used to genotype for the McC
loci in several areas of Mali as well as among African American persons. As shown in , the gene frequencies for Sl1
in African American persons are almost equal (0.48 vs. 0.52) whereas Sl2
is greatly increased in Africa, reaching its highest frequency among the Bambaran people (0.77). The Sl2
gene is almost completely absent in Caucasian and Asian persons.17
Adding our data to that previously published, over 1800 samples from Western Africa (Gambia, Senegal, Guinea, Sierra Leone, Ivory Coast, Ghana, and Mali) have been genotyped.9,17
The combined gene frequencies for McCa
range from 0.63 to 0.69 and for McCb
0.31 to 0.37 whereas the frequencies for Sl1
are 0.19 to 0.23 and 0.77 to 0.81 for Sl2
This is in contrast to studies of more than 1200 Kenyan persons who exhibited a lower frequency of McCb
(0.16) and Sl2
(0.67) (P. Zimmerman, personal communication). The data suggest that the McCb
genes may be under selective pressure in Africa.
Our data provide interesting insights into problems that often occur when trying to perform phenotyping of RBCs for the various Knops system antigens. It has previously been reported that false-negative results are obtained when the RBCs have low CR1 copy number either owing to an inherited or acquired deficiency or after prolonged storage of the blood sample.18
Accurate phenotypes can be obtained on most samples from Caucasian persons that have moderate to high CR1 levels and who are homozygous for Kna
, and Sl1
alleles (assuming all alleles are expressed).
The situation in African persons, however, is much more complex. In Bandiagara, 66 percent of the apparent McC(a−) and 81 percent of Sl1 samples were heterozygous by genotyping and, thus, were false-negative phenotypes. Similar results have been found in an earlier study of Malian donors from Bamako and Tienequebougou.9
Further complicating the phenotype status is the unequal expression of alleles.9
For example, if an individual is positive for an allele but that gene is only weakly expressed, false-negative serologic typings may even though the total CR1 copy numbers are in the normal range. Some family studies have also indicated that an amorph may exist in the Knops blood group as has been described for most other systems (J.M. Moulds, unpublished data). The molecular mechanism for this is currently under investigation. Once identified, genotyping for expression as well as the Knops blood group-related SNPs will make it possible to accurately type samples for research or transfusion purposes.