Advanced Clinical Molecular Diagnostics: DNA Based Tissue Typing

Introduction to the Major Histocompatibility Complex (MHC) and HLA

• The Major Histocompatibility Complex (MHC) locus is located on chromosome 6.
• The MHC locus represents a specific region of the human genome that includes Human Leukocyte Antigen (HLA) genes as well as other non-HLA genes.
• Human Leukocyte Antigens (HLA) are the specific gene products of the MHC region.
• Functionally, HLAs are membrane-bound proteins responsible for the vital recognition of "non-self" cells and tissues.
• HLA gene sequences are highly polymorphic, differing from one individual to another at the level of single base pairs.
• Each unique sequence variant is categorized as a different allele.

Molecular Structure of HLA Class I and Class II Molecules

• HLA Class I Molecule Structure:     * Consists of a heavy chain (the $\alpha$ chain) and a light chain ($\beta_2$-microglobulin).     * The $\alpha$ chain contains three extracellular domains: $\alpha_1$, $\alpha_2$, and $\alpha_3$.     * The structure is anchored in the cell membrane via the $\alpha$ chain which traverses the cytosol.     * The $\beta_2$-microglobulin is a non-covalently associated polypeptide.
• HLA Class II Molecule Structure:     * Consists of two non-identical membrane-spanning polypeptide chains: an $\alpha$ chain and a $\beta$ chain.     * The $\alpha$ chain includes two domains: $\alpha_1$ and $\alpha_2$.     * The $\beta$ chain includes two domains: $\beta_1$ and $\beta_2$.     * Both chains pass through the cell membrane into the cytosol.

Functional Classification of the MHC Region

• Class 1 MHC:     * Gene Products: $HLA-A$, $HLA-B$, and $HLA-C$.     * Tissue Location: Found on the surface of all nucleated cells.     * Primary Function: Responsible for the identification and destruction of abnormal or infected cells (such as viral-infected or tumor cells) by cytotoxic T cells.
• Class 2 MHC:     * Gene Products: $HLA-D$.     * Tissue Location: Found on B lymphocytes, monocytes, macrophages, dendritic cells, activated T cells, endothelial cells, and Langerhans cells.     * Primary Function: Identification of foreign antigens by helper T cells to coordinate an immune response.
• Class 3 MHC:     * Gene Products: Complement factors $C2$, $C4$, and factor $B$.     * Tissue Location: Plasma proteins.     * Primary Function: Defense against extracellular pathogens through the complement system.
• Cytokine Genes:     * Gene Products: Tumor Necrosis Factor-alpha ($TNF-\alpha$) and Tumor Necrosis Factor-beta ($TNF-\beta$).     * Tissue Location: Plasma proteins.     * Primary Function: Signaling molecules involved in cell growth and differentiation.

HLA Allele Nomenclature

• A standard nomenclature system has been established by the World Health Organization (WHO) Nomenclature Committee to manage the increasing number of identified HLA alleles.
• The nomenclature is used to describe specific DNA sequences required for DNA-level typing.
• Breakdown of a typical HLA allele name (e.g., HLA-DRB1):     * HLA: Indicates the gene region.     * DR: Indicates the subregion.     * B: Indicates the specific $\alpha$- or $\beta$-chain polypeptide (in this case, the $\beta$ chain).     * 1: Indicates the specific gene locus.

Inheritance and Clinical Significance of HLA

• Haplotype Inheritance: HLA alleles are inherited together in blocks known as haplotypes.
• Individual Variation: Every individual (with the exception of identical twins) possesses a unique set of HLA alleles.
• Transplantation (Allografts): In clinical transplantation, the donor organ and the recipient are genetically different, a state known as an allograft.
• Matching and Engraftment: The success of engraftment is highly dependent on the compatibility (matching) of the HLA alleles between the donor and the recipient.
• Resolution: This refers to the level of detail or specificity with which an HLA allele is determined (Low, Intermediate, or High resolution).

Serological (Protein-Level) HLA Typing

• Complement-Dependent Cytotoxicity (CDC) Test:     * Lymphocytes are typed by crossmatching them against Panel Reactive Antibodies (PRA).     * Procedure: Lymphocytes + Specific Antibodies + Complement.     * Positive Reaction: If the antibody recognizes the antigen on the lymphocyte, the complement is activated, resulting in a dead cell (indicated by dye uptake in the lab).     * Negative Reaction: If the antibody does not recognize the antigen, the cell remains alive.
• Bead Arrays and Flow Cytometry:     * Recipient antihuman antibodies are assessed by crossmatching them to known lymphocyte antigens that have been conjugated to microparticles (beads).     * Results are analyzed using flow cytometry to determine the presence of specific antibodies.

DNA-Based HLA Typing Methods

• Specimen Requirements: DNA typing typically utilizes whole-blood patient specimens collected in anticoagulants. Cell lines with known HLA types serve as reference samples.
• Restriction Fragment Length Polymorphism (RFLP):     * Analyzed via Southern blot to identify HLA class II alleles.     * Early DNA typing focused primarily on the most polymorphic loci: $HLA-B$ and $HLA-DRB$.
• Sequence-Specific Oligonucleotide Probe Hybridization (SSOP):     * Uses immobilized probes to identify patient alleles through hybridization.     * Bead-array SSOP utilizes probes immobilized on fluorescent beads for high-throughput testing.
• Sequence-Specific PCR (SSP-PCR):     * Uses allele-specific primers.     * PCR primers designed to recognize different alleles are typically supplied in a pre-loaded 96-well plate format.
• Sequence-Based Typing (SBT):     * The highest resolution method.     * Polymorphic regions are amplified by PCR and then subjected to direct DNA sequencing.
• Next-Generation Sequencing (NGS):     * Focuses on MHC regions utilizing long-range PCR.     * Used to resolve sequence ambiguities that other methods might miss.

Resolution Levels and Methodological Challenges

• Typing Discrepancies:     * DNA sequence changes do not always result in a change to the epitope (protein structure), meaning DNA and serology may not always correlate perfectly.     * Serology lacks the specificity to recognize every allele detectable by DNA analysis.     * Serological antibodies may be cross-reactive, binding to multiple different alleles.     * Ongoing discovery of new alleles means that retyping results today may differ from typing performed years ago.
• Resolution Level Hierarchies:     * Low-Resolution: CDC (Serology), PCR-SSP, PCR-SSOP, PCR-RFLP.     * Intermediate-Resolution: PCR-SSP, PCR-SSOP, SSP-PCR + PCR-RFLP, SSOP-PCR + SSP-PCR.     * High-Resolution: PCR-SSP, PCR-SSOP, SBT.
• Combining Methods for Higher Resolution:     * Technicians often combine tests to resolve ambiguities, such as using SSP-PCR followed by PCR-RFLP, or SSOP followed by SSP-PCR.     * SBT results can be further clarified by serological data if needed.