141-P: Absence of the HLA-G*0105N allele in Amerindian populations from the Brazilian Amazon regions: A possible role of natural selection

Abstract

HLA Sequencing Based Typing is an accurate and flexible approach for high-resolution typing. Genotype ambiguities are resolved by allele separation, enabling determination of the cis-trans configuration of polymorphisms.We routinely apply Group Specific Sequencing Primers (GSSP) to selectively sequence one of the two alleles. This approach only needs additional sequencing reactions on the same PCR template, and therefore is easy and quick to apply. SBTengine® facilitates easy analysis of the combined generic and GSSP sequence results.The GSSPs are designed following the criteria that the polymorphic nucleotide positions necessary for allele identification are located upstream the GSSP primers. The resulting sequence determines the cis-trans relations of the critical ambiguous polymorphisms. This approach significantly reduces possibilities for errors, hence the discriminatory polymorphisms are included in the selective primers. In the final allele assignment to resolve the genotype ambiguity the primer sequence and specificity is thus not required.For the three class I loci, release IMGT 2.17.0, the number of pairs of ambiguous genotypes are 889 for HLA-A, 3483 for HLA-B, and 440 for HLA-C. More than 95% can be resolved using GSSPs.Following our criteria the number of GSSPs needed to resolve all these ambiguities are for HLA-A: 20, HLA-B:36, and for HLA-C: 16. This low number of GSSPs can easily be managed in a routine typing laboratory. In combination with the Dynamic Ambiguity Resolving Tool (DART™) feature of SBTengine®, which identifies the GSSPs required for resolving the ambiguity, ambiguities of any kind easily are resolved in daily routine typing practice.