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Chapter 136 : Molecular Methods: HLA Alleles
Molecular biology techniques aimed at identification of HLA polymorphism at the gene level have largely replaced HLA typing assays based on identification of HLA proteins. The HLA genes encode at least six different HLA molecules, HLA-A, -B, and -C (class I molecules) and HLA-DR, -DQ, and -DP (class II molecules). The HLA molecules are encoded by the most polymorphic genetic loci known in humans. Each HLA allele is designated by the name of the gene followed by an asterisk and a four- to eight-digit number indicating the allele. Different HLA alleles defined by DNA typing can specify HLA proteins which are indistinguishable by methods based on protein identification, such as serology. DNA from HLA-characterized reference cells is usually derived from Epstein-Barr virus-transformed B lymphoblastoid cell lines. Molecular biology-based HLA typing methods utilize DNA as a starting material. PCR is used to generate a large number of copies of an HLA gene for rapid detection of HLA types. PCR uses sequence-specific primer pairs (a 5’ [sense] primer and a 3’ [antisense] primer) and Taq polymerase to specifically amplify selected segments of DNA. DNA is negatively charged due to its phosphate composition; thus, amplified DNA will move toward the positively charged pole in an agarose gel matrix. Amplification is used to obtain sufficient copies of a specific HLA gene for analysis by DNA sequencing.
DRB1 nucleotide sequences from a few DRB1 alleles. Dashes indicate identity with the sequence of DRB1*0101. The numbers indicate the codons. Only part of the sequence of the DRB1 gene is shown; the polymorphic exon 2 includes codons 5 through 95. Probes identifying polymorphic sequences distinguishing these alleles are boxed.
Example of two heterozygous allele combinations that cannot be distinguished from one another when both HLA-A alleles are characterized as a heterozygous mixture (i.e., ambiguous combinations). Some of the polymorphic nucleotides that distinguish the alleles and their cis and trans associations are diagrammed in the figure. The heterozygous combination A*02010101 and A*6601 (ambiguous combination 1) cannot be distinguished from the alternative, A*023501 and A*2603 (ambiguous combination 2), unless a single allele is isolated and characterized. Identification of the alleles present relies on the laboratory’s ability to link the first polymorphic sequence (located at codons 9-10, TTC ACA) to one of the two alternative second polymorphisms (CAC or CAG at codon 70). If CAC is in cis (i.e., found on the same DNA strand) with TTC ACA, then the first ambiguous combination is present.
Primer locations for sequence-based typing of the HLA-B gene. The locations at which amplification and sequencing primers anneal to the HLA-B gene are indicated by arrows. BIn3 includes both BIn3 and BIn3-AC primers. Ex, exon; Int, intron; F, forward; R, reverse.
Comparison of HLA typing methods
References a providing primer strategies (SSP) for determining HLA types
References a providing probe strategies (SSOPH) for determining HLA types
References a for alternate methods of DNA-based testing
References a on direct DNA sequencing (SBT)
Kits for the isolation of DNA
Examples of sequence-specific primer typing kits a
Examples of SSOPH typing kits a