Full-length HLA-DRB1 coding sequences generated by a hemizygous RNA-SBT approach.

Currently 1582 HLA-DRB1 alleles have been identified in the IMGT/HLA database (v3.18). Among those alleles, more than 90% have incomplete allele sequences, which complicates the analysis of the functional relevance of polymorphism beyond exon 2. The polymorphic index of each individual exon of the currently known allele sequences, shows that polymorphism is present in all exons, albeit not equally abundant. Full-length HLA-DRB1 RNA sequencing identifies polymorphism of the complete coding region. Here we describe a hemizygous full-length RNA sequence-based typing (SBT) approach based on group-specific HLA-DRB1 amplification and subsequent sequencing. RNA full-length sequences can easily be accessed because of the short amplicon length (801 bp). The RNA-SBT approach was successfully validated on a panel of DRB1 alleles having fully known coding sequences according to the IMGT/HLA database, and cover all serological equivalents. Subsequently, the approach was applied on a panel of 54 alleles with incomplete allele sequences, resulting in full-length coding sequences and the identification of one new and one corrected allele. This study shows the universal applicability of the RNA-based sequencing approach to identify full-length coding sequences and to define the polymorphic content of HLA-DRB1 alleles.

Allele and haplotype frequencies of HLA-DPA1 and -DPB1 in the population of Guadeloupe.

Genetic polymorphism of human leukocyte antigen (HLA)-DPA1 and -DPB1 loci was studied in 154 unrelated individuals from Guadeloupe, an archipelago of five islands located in the Carribean Sea. Thirty different DPB1 and eight different DPA1 alleles were observed with a heterozygosity index of 0.87 and 0.78, respectively. This high degree of heterozygosity corresponds with those found in African populations. The DPB1* 01:01:01 allele was most frequent (0.260), followed by 02:01:02 (0.143) and 04:01:01 (0.127). The DPA1 alleles 01:03 (0.380), 02:01 (0.302), 02:02 (0.175) and 03:01 (0.123) were identified in >35 individuals each, whereas 01:04, 01:05 and 04:01 were present only once. Haplotype estimations revealed the presence of 39 different haplotypes, with DPB1*01:01:01-DPA1*02:02 and DPB1*02:01:02-DPA1*01:03 as the most frequent (0.143 and 0.140, respectively). A striking difference was observed in DPB1/DPA1 associations between DPB1*04:02 and *105:01, that have identical exon 2 sequences. DPB1*04:02 was exclusively associated with DPA1*01:03, whereas DPB1*105:01 was present with DPA1*03:01, *03:02 or *04:01. This implies that the DP molecules are actually different, and this difference is relevant to consider in studies on the function of HLA-DP molecules in transplantation. Overall, HLA-DPA1 and DPB1 allele frequencies and haplotypes of the population of Guadeloupe were most similar to African populations, with characteristic alleles and haplotypes that bespeaks the admixture with other ethnicities.

Allelic polymorphism in introns 1 and 2 of the HLA-DQA1 gene.

Human leukocyte antigen (HLA) class II antigens are highly polymorphic membrane glycoproteins, encoded by the A and B genes of DR, DQ, and DP. The polymorphism is mainly located in exon 2, with the exception of DQA1. Of the 27 DQA1 alleles presently known, 18 cannot be identified on the basis of exon 2 alone, but need additional information from the other exons. DQA1 has been reported to be the most ancient class II gene. For evolutionary comparison and to assess the degree of polymorphism outside the exons, the sequences of introns 1 and 2 were determined from 30 different cell lines, encompassing 15 different DQA1 alleles. The sequences revealed major nucleotide differences between the different lineages, whereas within each lineage few differences were present. Phylogenetic analysis of intron and exon sequences confirmed this lineage specificity. Altogether, the present data indicate that the HLA-DQA1 lineages represent ancient entities. The observed variation of the introns in alleles with identical exon sequences implicates conservative selection of the exons within a given lineage. Intron sequences may provide the means to set up an accurate typing system.