In the cross-research field of modern medicine and immunology, the iterative innovation of Human Leukocyte Antigen (HLA) typing technology continues to promote the development of precision medicine. As an important bridge between gene polymorphism and immune response regulation mechanism, HLA typing plays a core supporting role in organ transplantation donor-recipient matching and autoimmune disease pathogenesis research.
Compared with the traditional serological typing method based on antibody-specific recognition, Molecular HLA Typing technology has realized the paradigm shift from antigen phenotype analysis to accurate genotype identification through in-depth analysis of the nucleotide sequence of HLA loci. This technical breakthrough has significantly improved the resolution of HLA typing and established a new research framework in the fields of rare allele discovery, transplant rejection prediction, and prognosis evaluation.
This paper comprehensively introduces the molecular HLA typing technology from PCR-based method to NGS, analyzes and compares the mainstream technologies and the technology selection based on demand.
What is Molecular HLA Typing
Molecular HLA Typing, that is, HLA typing at the molecular level, refers to the method of directly detecting the nucleotide sequence of the HLA gene by molecular biology technology, to determine the HLA allele type of individuals. As the core component of the major histocompatibility complex (MHC), HLA is highly polymorphic, especially HLA-A, HLA-B, and HLA-DRB1, and the number of alleles has reached thousands. The core of Molecular HLA Typing is to analyze the polymorphism of the HLA gene from the DNA level, which provides a molecular basis for precision medicine and immune-related research.
Structure of HLA Class I and Class II molecules (Deshpande., 2017)
Difference from Traditional Serological Methods
Traditional HLA typing mainly relies on serological methods, which classify HLA antigens on the cell surface by antibodies. However, serological methods have obvious limitations.
- Low resolution: Serological methods can only identify the antigen specificity level, but can not distinguish alleles with highly similar amino acid sequences, which is difficult to meet the needs of clinical high-resolution typing.
- Depending on the quality of antiserum: The preparation and preservation of antiserum are strict, and there are differences between batches, which may lead to inaccurate typing results.
- Unable to detect new alleles: For the new HLA alleles that have not yet been discovered, serological methods cannot identify them because of the lack of corresponding antibodies.
In contrast, Molecular HLA Typing has obvious advantages:
- Based on DNA detection: The HLA gene sequence is directly analyzed, which avoids the influence of abnormal expression of cell surface antigen on typing results.
- High-resolution typing: It can be accurate to the allele level, even distinguish alleles with only a single nucleotide difference, and provide more accurate matching basis for clinical applications such as organ transplantation.
- Discovery of new alleles: Through molecular biology techniques, especially NGS, new HLA alleles can be discovered and identified, and the HLA database will be continuously enriched.
- The high degree of standardization: The operation flow and data analysis of molecular typing technology can realize standardization, reduce the interference of human factors, and improve the repeatability and comparability of results.
Allelic ambiguities of next generation sequencing (NGS) vs sequence-specific oligonucleotide probe (SSOP) (Smith et al., 2019)
Molecular HLA Typing Technology Comparison
In the evolution of HLA typing technology, the change from serological method to molecular biological method marks significant progress in this field. Molecular typing technology has built a diversified technical system with distinct levels and complementary functions. Each technical method is based on the unique principles of molecular biology and shows its outstanding performance in the application of HLA genotyping.
PCR-SSP
Based on the PCR amplification of allele-specific primers, PCR-SSP designed specific primers whose 3' end is strictly complementary to the target sequence for HLA gene polymorphism sites. When there are corresponding alleles in the template DNA, the primers are accurately combined with the template and start amplification, resulting in specific bands that can be detected by electrophoresis. Its technical core lies in constructing a combinatorial library containing hundreds of pairs of specific primers and realizing simultaneous detection of multiple alleles by multiplex PCR.
A. Technical characteristics
a) Simple and rapid operation: Typing can be completed within hours without complicated probe hybridization or sequencing steps.
b) High resolution: It can reach the level of medium-resolution typing and is suitable for most clinical routine matching.
c) The requirements for experimental conditions are low: Ordinary PCR instruments can complete it, which is suitable for basic laboratories.
A. Limitations
a) Primer design is complex: It is necessary to design specific primers for a large number of HLA alleles, and the primer library is huge and the cost is high.
b) Unknown alleles cannot be detected: Only known alleles can be detected, and there is nothing to do with newly discovered alleles.
c) Heterozygote resolution is limited: for complex heterozygote samples, primer competition inhibition may occur, resulting in inaccurate typing results.
Number of ambiguities for HLA-A typing for exons 1 to 5 (HDkit/XRkit) (Bouthemy et al., 2018)
PCR-SSO
PCR-SSO technology first amplifies the target region of the HLA gene by PCR and then hybridizes the amplified product with a sequence-specific oligonucleotide (SSO) probe immobilized on a membrane or chip. According to the existence and intensity of the hybridization signal, the HLA allele type is judged.
A. Technical characteristics
a) High resolution: High-resolution typing can be achieved, especially for HLA-II gene typing.
b) High flux: Multiple loci and alleles can be detected simultaneously by chip technology.
c) The results can be quantified: The intensity of the hybridization signal can reflect the abundance of the target sequence, which helps analyze heterozygote samples.
B. Limitations
a) The operation steps are complicated: PCR amplification, hybridization, membrane washing, and signal detection are required, which takes a long time.
b) Sensitive to experimental conditions: slight changes in hybridization temperature and salt concentration may affect the hybridization results.
c) It is difficult to maintain the probe library: with the increasing number of HLA alleles, the probe library needs to be updated constantly, and the cost is high.
Number of null allele ambiguities for HLA-A, HLA-B, and HLA-DRB1 typing (Bouthemy et al., 2018)
Sanger Sequencing
Sanger sequencing is a DNA sequencing technology based on the dideoxy chain termination method, which determines the allele type by reading the nucleotide sequence of the HLA gene. For HLA typing, Sanger sequencing is usually used after targeted amplification, that is, the target exon of the HLA gene is amplified by PCR first, and then the amplified products are sequenced in two directions.
A. Technical characteristics
a) Gold standard method: Sanger sequencing was once the gold standard for HLA typing, with high accuracy and reliability.
b) Direct reading sequence: The nucleotide sequence of the HLA gene can be obtained directly, which provides the most direct evidence for allele identification.
c) Suitable for the identification of new alleles: New HLA alleles can be found and identified.
B. Limitations
a) Low flux: One sequencing can only analyze one fragment of a sample, which can't meet the requirements of Qualcomm quantitative typing.
b) High cost: Sequencing reaction and reagent cost is high, especially for the detection of multiple sites and exons.
c) Complex data analysis: For heterozygous sub-samples, it is time-consuming and laborious to manually splice and analyze the sequence.
Time and effort for next generation sequencing (NGS) and sequence-specific oligonucleotide probe (SSOP) workflows (Smith et al., 2019)
NGS
NGS technology, also known as the new generation sequencing technology, can simultaneously sequence a large number of HLA gene fragments through large-scale parallel sequencing. For HLA typing, targeted capture or multiplex PCR is usually used to amplify all exons or full-length sequences of HLA genes, and then high-throughput sequencing is used to compare the sequencing reads to the HLA reference database through bioinformatics analysis, so as to determine the allele type.
A. Technical characteristics
a) Qualcomm amount: One sequencing can complete multiple HLA loci typing of multiple samples, which significantly improves the detection efficiency.
b) High resolution: the full-length sequence analysis of HLA genes can be realized, reaching the level of high-resolution typing and even haplotype analysis.
c) Comprehensive coverage: it can detect all exons of the HLA gene, including highly polymorphic regions, and avoid missing important polymorphic sites.
d) Discovery of new alleles: Through unbiased sequencing, new HLA alleles can be efficiently discovered and identified, and the update of the HLA database can be promoted.
B. Limitations
a) High requirements for equipment and technology: professional NGS equipment and bioinformatics analysis platform are needed, and high requirements for laboratory conditions and personnel technology are required.
b) Complex data analysis: massive sequencing data need complex bioinformatics processes to analyze, including sequence alignment, allele allocation, etc., which requires high computing resources and algorithms.
c) Higher cost: Although the cost of single sequencing is decreasing with the development of technology, the cost of equipment investment and data analysis in the early stage is still high.
Comparison of common HLA typing methods (Shahrabi et al., 2019)
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Advantages of NGS in Molecular HLA typing
In the field of HLA high-resolution typing, NGS technology reshapes the detection paradigm with its unique advantages. It realizes the full coverage of HLA genes through Qualcomm parallel sequencing, can accurately analyze complex heterozygotes, significantly improve the detection rate of rare alleles, provide accurate haplotype data for transplantation matching and disease association research, and promote HLA typing to leap from medium resolution to accurate allele identification.
Whole Gene Coverage
Traditional HLA typing techniques, such as PCR-SSP and PCR-SSO, usually only detect some highly polymorphic regions of HLA genes and may miss polymorphic sites in other regions. NGS technology can cover all exons or full-length sequences of HLA genes through targeted capture or multiplex PCR amplification. The advantages of this whole gene coverage are:
- Avoid omitting important polymorphic sites: The polymorphism of the HLA gene not only exists in the classical antigen-binding groove region but also may be distributed in other regions. The polymorphism of these regions may affect the expression and stability of HLA molecules or the interaction with other molecules, thus affecting the immune response. Full gene coverage can ensure that all polymorphic sites that may affect HLA function are detected.
- Accurate analysis of complex alleles: Some HLA alleles may have mutation combinations of multiple loci, and only detecting some regions may lead to the wrong identification of alleles. Whole gene coverage can provide complete sequence information and ensure accurate analysis of complex alleles.
- Haplotype analysis is supported: HLA haplotypes can be inferred through whole gene sequencing and bioinformatics analysis, which is of great significance for organ transplantation matching and disease association research.
Distribution of the number of reads per sample and per locus (Liu et al., 2020)
Rare Allele Detection
The high polymorphism of the HLA gene leads to the existence of a large number of rare alleles, which are less frequent in the general population but may be more common in a specific population or family. Traditional typing techniques are often difficult to detect rare alleles due to the limitation of primers or probe libraries, while NGS technology has obvious advantages in rare allele detection:
- Unbiased sequencing: NGS technology can detect HLA genes in samples without knowing the allele sequence in advance through high-throughput sequencing, so as to find and identify rare alleles.
- High sensitivity: NGS technology has a high sequencing depth, can detect low-frequency alleles, and can accurately identify the existence of rare alleles even in heterozygote samples.
- Promote the update of the HLA database: The discovery and identification of rare alleles will help enrich the HLA database and provide a more comprehensive reference standard for HLA typing around the world, especially for the study of rare diseases and transplantation matching of minority people.
Inter-lot comparison of the percentages of total reads mapped to each locus (Liu et al., 2020)
How to Select Proper Technology
With the development of HLA typing technology, it is very important to choose the appropriate sequencing method to obtain accurate and reliable HLA typing results. Different research purposes, resolution requirements, sample types, flux and cost considerations will all affect the selection of HLA sequencing methods.
Matching of HLA Typing Methods and Sample Types
The selection of HLA sequencing methods should be based on the sample type. The difference of DNA quality, integrity and source of different samples directly affects the applicability and accuracy of sequencing technology. From fresh blood to FFPE samples, from high-quality DNA to micro-degraded samples, accurate matching technology is needed to ensure the reliability and effectiveness of HLA typing.
A. Fresh blood
Fresh blood is one of the most commonly used sample types in HLA typing, which is rich in white blood cells and can extract high-quality DNA, which is suitable for many sequencing methods. For fresh blood samples, NGS technology is the first choice if high resolution typing results are needed. The quality of DNA extracted from fresh blood is high, which can meet the requirements of NGS technology for DNA quality and quantity. Through NGS, high-resolution typing with full gene coverage can be realized. If the sample size is small or the time is urgent, Sanger sequencing can also be used for HLA typing of fresh blood samples, especially for high-resolution analysis of a single sample.
B. Low quality DNA
For low-quality DNA samples, such as DNA extracted from trace blood, dried blood spots or other sources, its concentration and integrity may be poor. At this time, PCR-SSP technology may be more suitable, because it requires relatively little amount of DNA, and by optimizing primer design and PCR conditions, the amplification efficiency of low-quality DNA can be improved. If NGS technology must be used, it is necessary to pretreat low-quality DNA, such as whole genome amplification, but this may introduce bias and need to be considered in data analysis.
Accuracy of the three tools for HLA typing at the second field or the third field resolution for different depths and read lengths (Liu et al., 2021)
Disease Correlation Research
In the study of rheumatoid arthritis (RA), traditional PCR-SSP can only detect the specificity of HLA-DR4 antigen, while NGS can distinguish HLA-DRB1*04:01/04:04 subtypes. The researchers used NGS to sequence the whole HLA gene cluster, and found that HLA-DQB103:02 was independently related to the severity of the disease (OR=1.32, 95% CI 1.17-1.49) except the classic Shared Epitope. The haplotype linkage of HLA-DRB1-DQA1-DQB1 was successfully analyzed by using 10X Genomics long-read sequencing technology, which could not be achieved by short-read technology.
Positive prediction agreement of the seven algorithms for HLA typing at different resolutions and genes (Liu et al., 2021)
Conclusion
The development of Molecular HLA Typing technology has experienced the iteration from traditional serological methods to molecular biological techniques. At present, the mainstream molecular typing technologies include PCR-SSP, PCR-SSO, Sanger sequencing and NGS. Each technology has its unique advantages and limitations. In practical application, the appropriate technology should be selected according to specific requirements (such as resolution requirements, sample size, time and cost, etc.).
With the continuous development of technology and the reduction of cost, NGS technology will be more widely used in HLA typing. At the same time, combined with the development of bioinformatics and artificial intelligence, the accuracy and efficiency of HLA typing will be further improved, providing more solid technical support for the research and clinical application of precision medicine and immune-related diseases.
References:
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- Bouthemy C, Ralazamahaleo M, et al. "Improvement in HLA-typing by new sequence-specific oligonucleotides kits for HLA-A, -B, and -DRB1 loci." HLA. 2018 92(5): 279-287 https://doi.org/10.1111/tan.13382
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- Liu C, Duffy BF, Weimer ET, et al. "Performance of a multiplexed amplicon-based next-generation sequencing assay for HLA typing." PLoS One. 2020 15(4): e0232050 https://doi.org/10.1371/journal.pone.0232050
- Liu P, Yao M, Gong Y, et al. "Benchmarking the Human Leukocyte Antigen Typing Performance of Three Assays and Seven Next-Generation Sequencing-Based Algorithms." Front Immunol. 2021 12: 652258 https://doi.org/10.3389/fimmu.2021.652258
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