The Nature of Polymorphism of the HLA Class I Non-Coding Regions and Their Contribution to the Diversification of HLA

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Abstract
The sequence database of HLA class I genes is mainly derived from mRNA analysis. Little is known about the non-coding sequences of the different class I alleles. In this study we have determined the sequence of the 1st through 3rd introns of the majority of HLA-A and -B alleles. The few published sequences emerged to contain substantial errors. The introns turned out to be highly polymorphic with a variability of 14.6% in the 1st intron decreasing to 6.2% in the 3rd intron. Against all expectations, this variability is not characterised by random point mutations but by a highly systematic diversity reflecting the ancestral relationship of the HLA alleles. The variability is arrested on the level of the serological diversity. The striking conservation within each ancestral lineage suggests that point mutations have been negatively selected. This finding could be explained by the evolutionary pressure on base order, promoting the potential to extrude single-strand stem-loops from supercoiled duplex DNA, which is believed to be important for combination. Moreover, the GC content was found to be as high as 78% in the 1st and 2nd introns and 55% in the 3rd intron. These CpG islands are directly involved in the exchange of short stretches of DNA in unequal crossing-over events. Additionally, conversion between different class I sequences is facilitated by regions of strong homology, stabilizing the pairing of variable regions. All these observations indicate the potential of a substantial contribution of introns to the recombinational activity of class I genes. The exclusive clustering of CpG islands in the 1st and 2nd introns restricts the gene conversion events to the regions of the 2nd and 3rd exons and therefore protects the conservation of the 5 flanking region and the 3 part of the gene. Since there are less diversification forces acting on introns they may be more conserved in a trans-species manner than exons. Therefore, they could provide the answer for the controversy regarding intra- or trans-species evolution.