HLA Ambiguous Alleles

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In our CareDx TruSight Assign HLA reports, ambiguous alleles are identified with a double dash (- -). For example, “- -:01:01” indicates an ambiguity in the first field, “01:- -:01” indicates an ambiguity in the second field, and “01:01:- -” indicates an ambiguity in the third field.  Ambiguous alleles are an artifact of the current limitations of sequence-based HLA typing.  Here is a brief description of one type of ambiguous allele.

Heterozygous basepair combinations.

Fig. 1

Fig. 1 shows the nucleotide sequences from two subjects for the HLA-B gene, exon 3, loci 551-570 (1).  The top two sequences are derived from Subject #1 and the bottom two sequences are derived from Subject #2.  For each subject, allele #1 is shown first (black) and allele #2 is shown second (purple).  The HLA type for Subject #1 is: B*07:02:01/B*07:24. The HLA type for Subject #2 is: B*35:03/B*35:33.

Both subjects display heterozygous alleles at loci 559 (G/C; C/G) and 560 (A/T; T/A).  The allele pairs at locus 559 are identical between subjects, and likewise the allele pairs at locus 560 are also identical between subjects.  Thus, the basepair combinations at each locus yield ambiguous results; we cannot determine which allele pair should be assigned to which subject.

Note that although the chromosome pairs are color-coded in this example, they are not uniquely identifiable in actual sequence runs.

Excluded polymorphisms.

In some cases, polymorphisms that distinguish alleles may not be interrogated by the primer probeset for a particular sample.  Currently, HLA library kits are designed to sequence a subset of the entire MHC (Major Histocompatibility Complex) genomic region.

 

LociTarget Sequence
HLA-A4.1 kb (entire gene)
HLA-B2.8 kb (exon 1 - intron 6)
HLA-C4.2 kb (entire gene)
HLA-DRB1/3/4/54.6 - 5.1 kb (exon 2 - intron 4)
HLA-DQB16.9 kb (entire gene)
HLA-DPB19.7 kb (exon 2-3' UTR)
HLA-DQA17.3 kb (entire gene)
HLA-DPA110.3 kb (entire gene)

Table 1

Table 1 shows the HLA genes, exons, introns and UTR’s covered by the CareDx TruSight HLA kit.  As is the case with other HLA kits, this kit does not completely sequence the entire MHC region.  Specific exons or sections of exons that contain polymorphisms may not be covered by this kit.  These excluded polymorphisms can result in ambiguous allele calls.

 

References

  1. S.D. Adams, K.C. Barracchini, D. Chen, F. Robbins, L. Wang, P. Larsen, R. Luhm, D.F. Stroncek, Ambiguous allele combinations in HLA Class I and Class II sequence-based typing: when precise nucleotide sequencing leads to imprecise allele identification. J. Transl. Med. 2, 30-35 (2004)