THERE IS MORE TO HLA C THAN EXONS 2 AND 3: SEQUENCING OF EXONS 1,4 AND 5

THERE IS MORE TO HLA C THAN EXONS 2 AND 3: SEQUENCING OF EXONS 1,4 AND 5
Saskia A van der Vlies, Christina EM Voorter and Ella M van den Berg-Loonen. Tissue Typing Laboratory, University Hospital Maastricht, Maastricht, The Netherlands.

DNA typing techniques have proven to be accurate and efficient for the detection of HLA-C alleles. The introduction of these techniques has revealed a high degree of polymorphisms of HLA-C alleles at the nucleotide level. At present, 67 Cw alleles have been identified based on published sequences.
The allelic diversity of the HLA-C locus is predominantly present in the a1 and a2 domains of the heavy chain, encoded by exons 2 and 3. We have recently described a high resolution SBT approach for typing HLA-C by sequencing exons 2 and 3 without prior LR typing. Based on exons 2 and 3 sequencedata the alleles Cw*1701/02, *15051/2, *12021/2, *0701/06 and *1801/02 can not be distinguished. Additional sequence information of exons 1, 4 or 5 is needed. Therefore primers were developed for direct sequencing of these exons. Amplification primers for exon 1 were located in the 5'UT region and exon 2. For exons 4 and 5 primers were located in intron 3 and exon 6. The sequencing primers were located in 5'UT region, introns 3 and 4 respectively.
A panel of 16 individuals were sequenced for exons 1, 4 and 5 to verify correct amplification of the HLA-C alleles. They represented all HLA-C alleles detected in our laboratory. Furthermore, the sequence of exon 1 was determined in another 31 individuals, exon 4 in 20 and exon 5 in 68. No allelic drop-outs were observed in either of the three exons for all individuals typed. The panel was also studied for the known polymorphisms present in exons 1, 4 and 5. The sequences of exons 1 and 4 showed no discrepancies with the published sequence data. For exon 5 the sequences of Cw*0302, *0501 and *0701 did not correspond with the officially reported sequences. Sequencing of exons 5 of the original cells resulted in correction of the previously published sequences. Two new HLA-C alleles were identified with nucleotide substitutions outside exons 2 and 3.