HLA typing by molecular methods have evolved from sequence-specific oligonucleotide probes to next-generation sequencing (NGS).
Introduction of NGS-based HLA typing significantly reduced the number of ambiguities observed through full HLA gene sequencing.
HLA enrichment methods will continue to improve the speed of HLA typing and enable additional content to be evaluated for hematopoietic cell transplant patients as well as solid organ transplant patients.
Application of NGS to the HLA region will further the study of HLA regulation and expression as it has an impact on transplant outcomes.
Long-read HLA sequencing will enhance understanding of HLA haplotypes and alter how laboratories perform high-resolution HLA typing.
The HLA region on chromosome 6p21 covers more than 224 annotated genes, covering a span of more than 3.6 megabases. Although that may seem large, it comprises less than 0.15% of the whole human genome
. This region has been one of the most studied regions of the human genome since its discovery and contains a plethora of genes that are crucial for immune cell function and regulation , . The major histocompatibility complex (MHC) class I molecules, encoded by HLA-A , HLA-B , and HLA-C , are used by nearly all cells in the human body to express endogenous proteins on their surface for immune cell surveillance . Through this mechanism the immune system can identify defective and pathogen infected cells . MHC class II molecules, encoded by HLA loci DPB1 , DM , DO , DQB1 , DQA1 , DRB1 , and DRA among others, are used by antigen-presenting leukocytes to exhibit pathogenic peptides for T-lymphocyte examination and recruitment . Additionally, the HLA region encodes the complement system proteins vital for the innate immune response, which is responsible for opsonization and neutralization of pathogens
HLA typing is essential for assessment and treatment of a variety of medical conditions, including hematologic, rheumatologic, autoimmune, and cardiologic, among other diseases. The prevalence of hematologic malignancy in the general population has been observed to be greater than 63 per 100,000 people
. Although there are a variety of treatment regimens for these malignancies, hematopoietic cell transplant (HCT) often is the treatment. Some patients can benefit from an autologous transplant; however, a large portion of patients need an HCT allotransplant. HLA typing is required to find an appropriate HLA match even if the HCT allotransplant donor is a sibling or other blood relative . In patients receiving a solid organ transplant, HLA typing has been observed to be most beneficial, particularly in cases where sensitized transplant patients have developed allele specific antibodies
. Lack of appropriate typing, particularly at the HLA-A, HLA-B , and HLA-DRB1 loci, puts these patients at risk of transplant rejection and possible chronic systemic disease.
Typing of HLA allele variants is associated with a diverse array of human diseases. There are a host of autoimmune diseases associated with HLA variants, including systemic lupus erythematosus, psoriasis, multiple sclerosis, and sarcoidosis, among others. For some of these autoimmune diseases, HLA-associated risk alleles have been identified as strong genetic predictors of disease development. Other diseases associated with HLA alleles include type II diabetes, schizophrenia, Parkinson disease, and coronary artery disease
. Furthermore, there are HLA alleles correlated with adverse drug reactions. Some examples include abacavir in patients with the HLA-B∗57:01 allele and carbamazepine and oxcarbazepine in patients with the HLA-B∗15:02, HLA-B15:11, or HLA-A∗31:01 allele . From the numerous examples available in the literature to date, typing of the HLA region has become a crucial part for diagnosing disease as well as a vital component of transplant medicine and treatment regimen implementation ,
HLA typing in the beginning
One of the first, and for decades considered the gold standard of, HLA typing is serologic typing
. This approach uses serologic and deductive methods to identify the patient HLA type. To perform this type of HLA typing, isolated recipient lymphocytes are placed in wells with dye, complement, and different sera with affinity for characterized HLA alleles. If the recipient lymphocytes display the HLA type that the sera are characterized for, then complement is able to bind to the cells and compromise the cell membrane. This permeabilization allows the cells to take up the dye. Cells in each well with the different sera are inspected under a microscope for dye. By identifying which combination of sera caused cell lysis, the HLA type for the recipient can be determined.
The advantage of this method in the past was the speed at which typing could be achieved. In several hours, a basic HLA typing could be assigned. The disadvantage to this method was that serologic typing has poor sensitivity for the detection of small amino acid differences in HLA proteins, which can elicit a significant immune response. Also, a laboratory could use only previously characterized sera for known HLA alleles. This becomes increasingly difficult as novel alleles are continuing to be identified and largely has been abandoned with the adoption of molecular typing techniques.