HLA-Cw allele analysis by PCR-restriction fragment length polymorphism: study of known and additional alleles.

Abstract

We describe a technique for HLA-Cw genotyping by digestion of PCR-amplified genes with restriction endonucleases. Locus-specific primers selectively amplified HLA-Cw sequences from exon 2 in a single PCR that avoided coamplification of other classical and nonclassical class I genes. Amplified DNAs were digested with selected enzymes. Sixty-three homozygous cell lines from International Histocompatibility Workshop X and 113 unrelated individual cells were genotyped for HLA-Cw and compared with serology. The present protocol can distinguish 23 alleles corresponding to the known HLA-Cw sequences. Genotyping of serologically undetectable alleles (HLA-Cw Blank) and of heterozygous cells was made possible by using this method. Six additional HLA-Cw alleles were identified by unusual restriction patterns and confirmed by sequencing; this observation suggests the presence of another family of allele-sharing clusters in the HLA-B locus. This PCR-restriction endonuclease method provides a simple and convenient approach for HLA-Cw DNA typing, allowing the definition of serologically undetectable alleles, and will contribute to the evaluation of the biological role of the HLA-C locus.

A high‐resolution genotyping method for HLA‐C alleles and possible shared HLA‐C‐B haplotypes between Japanese and Caucasians

“…Comparison of the data obtained in the present study with the DNA typing results for the Tenth IHW cell lines for HLA-C alleles (14,15) revealed that most HLA-C alleles except for Cw*0701, are shared by Japanese and Caucasians. On the other hand, comparison of the allele frequencies in the present Japanese population with those in a French population (6) showed that the predominant HLA-C alleles are different between these two populations. Cw*0102, 1403,0304,1202,0801 and Cw*0702 are the most frequent HLA-C alleles in the Japanese, while Cw*0702, Cw*0401, Cw*0501, and Cw*1601 are the most frequent in the French (Figure 2).…”

“…Cw*O401 is mainly associated with HLA-B62 in Japanese, and with B35 in Caucasians. Though both Cw*1402 and Cw*1502 were detected and found to be associated with HLA-B51 in Japanese and French, the predominant allele is Cw*1402 in Japanese and Cw*1502 in French (6).…”

“…As for Cw*08 alleles, Cw*0801 is predominant and Cw*0803 is also common in Japanese. On the other hand, Cw*0802 is predominant in French (6). Three alleles, Cw*0701, Cw*0802 and Cw* 1601, which may be common in Caucasians (15) have not been detected in Japanese, whereas a common allele in Japanese, Cw*1403, has not been detected in Caucasians.…”

“…The frequencies of HLA-C alleles in a French population have been reported (6). As for Japanese, Ando et al (7) reported performing HLA-C genotyping by a low-resolution PCR-SSP method.…”

HLA‐DRB4 genotyping by PCR‐RFLP: diversity in the associations between HLA–DRB4 and DRB1 alleles

“…Previously we developed the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for HLA class I1 typing based on the digestion of PCR-amplified DNAs with allele specific enzymes which have a single recognition site in some alleles but none in other alleles in the aniplified regions (16). This PCR-RFLP method is easy and sensitive because genotypes can be simply defined by checking whether the amplified DNAs are digested or not, facilitating simple reading of the generated RFLP band patterns (17)(18)(19)(20)(21)(22). In the present study, we have first developed a DRB4 allele typing technique by PCR-RFLP and examined genomic polymorphism of the HLA-DRB4 gene DRB4*0101 : l l C AGG AAT CAG AAA GGA CAC TCT GGA ClT CAG CCA ACA GCA CTC l T G AGC TGA ~~~4 * 0 1 0 3…”