**2.4. Clinical relevance of the HLA system**

A key limitation of this old system was that it only allowed for up to 99 alleles which differ in any of the pairs of positions. The HLA-A\*02 and B\*15 allele groups were the first to run into this problem when more than 99 alleles were detected. At that time, the WHO Nomenclature Committee for the HLA system decided to adopt the rollover sequences A\*92 and B\*95 respectively for A\*02 and B\*15. When A\*0299 was identified, the next A\*02 allele described was named A\*9201. Similarly when B\*1599 was identified the next B\*15 allele described was named B\*9501. Recently however, a number of other HLA types started to fast approach 99 alleles. These include A\*03, B\*40, B\*44 and DRB1\*11. Adopting rollover sequences for all of these was impractical. A rollover system of sorts had already been adopted for HLA-DPB1. When HLA-DPB1\*9901 was identified, the next HLA-DPB1 allele was named 'within the

In 2010, a new nomenclature system was adopted (Fig 2) [8, 9]. This introduced colons ':' as separators between pairs of digits. HLA-A\*02010102L therefore became HLA-A\*02:01:01:02L. The pairs of digits separated by colons are known as Fields. The first and second digits of the old nomenclature form the 1st Field of the new nomenclature. The third and fourth digits of the old nomenclature form the 2nd Field of the new nomenclature. To help reduce confusion in adopting the new nomenclature, the leading '0' in alleles 1-9 of each allele group was kept.

The introduction of the colons means that each Field is no longer restricted to 99 digits but can be expanded limitlessly. Once HLA-A\*03:99 was identified, the next A3 allele could be named

With the introduction of colons and therefore the removal of the artificial restriction of 99 digits, there is no more need for rollover sequences. HLA-A\*92 and B\*95 were renamed A\*02 and B\*15 respectively and their associated alleles remapped. A\*9201 became A\*02:101. A\*9202 became A\*02:102 etc. HLA-B\*9501 became B\*15:101. HLA-B\*9502 became B\*15:102 etc. HLA-

existing sequences' as HLA-DPB1\*0102.

374 Current Issues and Future Direction in Kidney Transplantation

**Figure 2.** New HLA nomenclature

HLA-A\*03:100.

The most important function of MHC molecule is in the induction and regulation of immune responses. T-lymphocytes recognize foreign antigen in combination with HLA molecules.

In an immune response, foreign antigen is processed by and presented on the surface of a cell (e.g. macrophage). The presentation is made by way of a HLA molecule. The HLA molecule has a section, called its antigen (or peptide) binding cleft, in which it has these antigens inserted. T-lymphocytes interact with the foreign antigen/HLA complex and are activated. Upon activation, the T cells multiply and by the release of cytokines, are able to set up an immune response that will recognize and destroy cells with this same foreign antigen/HLA complex, when next encountered. The exact mode of action of HLA Class I and HLA Class II antigens is different in this process. HLA Class I molecules, by virtue of their presence on all nucleated cells, present antigens that are peptides produced by invading viruses. These are specifically presented to cytotoxic T cells (CD8) which will then act directly to kill the virally infected cell. HLA Class II molecules have an intracellular chaperone network which prevents endogenous peptide from being inserted into its antigen binding cleft. They instead bind antigens (peptides) which are derived from outside of the cell (and have been engulfed). Such peptides would be from a bacterial infection. The HLA Class II molecule presents this "exogenous" peptide to helper T cells (CD4) which then set up a generalized immune response to this bacterial invasion. Thus it is apparent that MHC products are an integral part of immunological health and therefore it is no surprise to see a wide variety of areas of clinical and genetic implications.

#### **2.5. HLA and renal transplants**

HLA typing was applied to kidney transplantation very soon after the first HLA determinants were characterized [10-12]. The importance of reducing mismatched antigens in donor kidneys was immediately apparent with superior survival of grafts from HLA identical siblings compared to one haplotype matches or unrelated donors. It is apparent that the effect of HLA matching is significant, even with the highly efficient immunosuppression used today. The important things include need for ABO compatibility and the need for a negative T-lympho‐ cyte crossmatch (using cytotoxicity). Complement binding anti-HLA Class I antibodies present at the time of transplant will cause "hyperacute rejection" of the graft (i.e. when the T cell crossmatch is positive).

**3.3. Molecular genetic techniques**

same enzyme [15].

*3.3.2. Polymerase chain reaction*

*3.3.1. RFLP (Restriction Fragment Length Polymorphism)*

exercise to a technique that is essentially automatable.

a starting point for reconstruction of double stranded DNA at that site.

There are a number of PCR based methods in use. For example:

each with primers specific for different HLA antigens.

**3.4. Sequence Specific Oligonucleotide (SSO) Typing**

produced by cutting with a particular enzyme.

Restriction Fragment Length Polymorphism (RFLP) methods rely on the ability of certain enzymes to recognize exact DNA nucleotide sequences and to cut the DNA at each of these points [14]. Thus, the frequency of a particular sequence will determine the lengths of DNA

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The DNA for one HLA (Class II) antigen, e.g. DR15, will have these particular enzyme cutting sites (or "restriction sites") at different positions compared to another antigen, e.g. DR17. Consequently, the lengths of DNA observed when DR15 is cut by a particular enzyme, are characteristic of DR15 and different to the sizes of the fragments seen when DR17 is cut by the

The Polymerase Chain Reaction (PCR) is a revolutionary system for investigating the DNA nucleotide sequence of a particular region of interest in any individual [16]. Very small amounts of DNA can be used as a starting point, such that it is theoretically possible to tissue type using a single hair root. Sequencing DNA has been transformed from a long and laborious

The first step in this technique is to obtain DNA from the nuclei of an individual. The double stranded DNA is then denatured by heat into single stranded DNA. Oligonucleotide primer sequences are then chosen to flank a region of interest. The oligo- nucleotide primer is a short segment of complementary DNA, which will associate with the single stranded DNA to act as

If the oligonucleotide is chosen to be close to a region of special interest like a hypervariable region of HLA-DRB then the part of the DNA, and only that part, will become double stranded DNA, when DNA polymerase and deoxyribonucleotide triphosphates are added. From one copy of DNA it is thus possible to make two. Those two copies can then, in turn, be denatured, reassociate with primers and produce four copies. This cycle can then be repeated until there is a sufficient copy of the selected portion of DNA to isolate on a gel and then sequence or type.

**•** *Sequence Specific Priming (SSP) -* In this test, the oligonucleotide primers used to start the PCR have sequences complimentary to known sequences which are characteristic to certain HLA specificities. The primers, which are specific to HLA-DR15, for example, will not be able to instigate the PCR for HLA-DR17. Typing is done by using a set of different PCR's,

By this method, the DNA for a whole region (e.g. the HLA DR gene region) is amplified in the PCR. The amplified DNA is then tested by adding labeled (e.g. Radioactive) oligonucleotide
