Probe set for multi-sample HLA gene typing using the capture method and typing method using the same

Abstract

This invention provides a highly practical probe set and a method for typing HLA genes using it, which enable efficient and highly accurate simultaneous typing of a large number of samples using the sequence capture method. [Solution] A probe set for HLA gene typing is provided, each consisting of oligonucleotides having a specific base sequence. Furthermore, an HLA gene typing method using the sequence capture method with the probe set is provided.

Description

Technical Field

[0001] The present invention relates to a novel probe set suitable for use in multi-sample HLA gene typing using a sequence capture method. More specifically, the present invention relates to a probe set capable of comprehensively and efficiently capturing genes in the polymorphic HLA region and enabling simultaneous typing of multiple samples, and a typing method using the same.

Background Art

[0002] Human Leukocyte Antigen (HLA), which is the Major Histocompatibility Complex (MHC) of humans, is deeply involved in the induction of immune responses by presenting peptides derived from foreign proteins such as pathogens and peptides derived from self-proteins to T cells. Six main types of antigens are known, including class I molecules (HLA-A, HLA-B, and HLA-C) expressed in almost all cells and class II molecules (HLA-DR, HLA-DQ, and HLA-DP) mainly expressed in immune system cells.

[0003] The gene region encoding HLA is located on the short arm p21.3 of human chromosome 6. From the telomere side towards the centromere side, it is arranged in the order of the class I region (HLA-A, HLA-C, HLA-B, etc.), the class III region, and the class II region (HLA-DRA, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, etc.). Many genes are encoded at a very high density, and associations with transplantation, blood transfusion, drug side effects, and various diseases have been reported. Note that there are no HLA genes in the class III region.

[0004] Even when HLA genes share identical base sequences in exon 2 or exon 3, which are known to have many polymorphisms, polymorphisms can still exist in other exons, resulting in a large number of HLA alleles. In recent years, it has also become clear that the 5'UT region, 3'UT region, and introns of HLA genes also contain many functional polymorphisms involved in transcriptional regulation.

[0005] Because HLA exhibits a high degree of polymorphism, it is known that there are an extremely large number of allele types. On the other hand, DNA typing to determine HLA alleles has been reported to be related not only to the matching of tissue compatibility between donors and recipients during transplantation, but also to protection and severity in lifestyle-related diseases, autoimmune diseases, cancer, viral infections, and drug side effects.

[0006] In recent years, the emergence of so-called "next-generation sequencers (NGS)" has made high-throughput DNA typing possible. However, the mainstream DNA typing method currently employed is one in which PCR products produced by polymerase chain reaction (PCR) are sequenced using NGS.

[0007] This NGS-based DNA typing method has several advantages: it avoids the so-called phase ambiguity that occurs in conventional PCR-SSO (Sequence Specific Oligonucleotide)-Luminex and PCR-SBT (Sequence Based Typing) methods, where the cis-trans positional relationships of multiple polymorphisms cannot be accurately determined; it can detect polymorphisms in intron and promoter regions, enabling the detection of null alleles whose gene structure is the same as other expressed HLA genes but whose expression is suppressed; and it can type a large number of samples at once.

[0008] However, PCR, which is essential for these conventional methods including NGS, has fundamental drawbacks such as requiring at least 5 ng of DNA for typing, limiting the number of samples that can be processed at once, and being unable to use fragmented DNA. Furthermore, PCR has difficult-to-solve problems such as misincorporation of bases by DNA polymerase during PCR amplification, production of chimeric molecules due to DNA elongation crossing over to other chromosomes (e.g., from maternal to paternal chromosomes) or other highly homologous genes, and allele drop (a phenomenon in which specific alleles are not amplified due to polymorphisms in genomic regions corresponding to PCR primers). Patent Document 1 proposed a probe set suitable for comprehensively typing HLA genes using the sequence capture method without using PCR, which has these various problems. However, the probe set described in Patent Document 1 was designed with an emphasis on improving the typing accuracy of individual samples, and further optimization is needed to efficiently process multiple samples simultaneously.

[0009] Recently, the "AlloSeq Tx17" (registered trademark) (manufactured by CareDx), a kit for HLA typing using the capture method, was launched. However, the amount of DNA required for typing is said to be 50 ng or more, and, similar to the probe set in Patent Document 1, the problem of simultaneous typing of multiple samples remains unresolved. In other words, there is a need for a new probe set suitable for high-throughput applications of the capture method. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] WO2017 / 135396 publication [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The present invention aims to overcome the technical challenges of conventional PCR-based typing methods and to provide a highly practical probe set and a typing method using the same that can efficiently and accurately type a large number of samples simultaneously using a sequence capture method. [Means for solving the problem]

[0012] To solve the aforementioned problems, the inventors conducted extensive research and, as a result, discovered that it is possible to simultaneously determine the sequences of multiple samples by using a probe set consisting of oligonucleotides having the base sequences shown in SEQ ID NOs: 1 to 448, thus completing the present invention.

[0013] In other words, the present invention provides a probe set for HLA gene typing, comprising oligonucleotides having the base sequences shown in SEQ ID NOs: 1 to 448. Furthermore, the present invention provides a method for typing HLA genes using the probe set. [Effects of the Invention]

[0014] The probe set of the present invention has a base sequence designed relative to a reference sequence uniquely designed by the inventors, and is designed to comprehensively type 11 genes included in the HLA region (HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5). Therefore, it becomes possible to simultaneously determine alleles from genes in multiple samples (determining allele names named in the database of THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM: IMGT® / HLA).

[0015] The sequencing capture method using the probe set of the present invention allows for the typing of even minute amounts of DNA, as small as 1 ng. Furthermore, the sequencing capture method of the present invention enables the typing of fragmented DNA samples (e.g., cfDNA (cell-free DNA) or FFPE (formalin-fixed paraffin-embedded) samples), which was difficult with conventional methods using PCR. Therefore, the method of the present invention does not require high-quality DNA samples such as blood, and can be used to type DNA from, for example, oral swab samples or swab-derived DNA, enabling non-invasive testing that does not burden the subject. Moreover, since the method of the present invention allows for the simultaneous processing of multiple samples, the cost per sample can be reduced (estimated to be about 1 / 5 compared to conventional methods using PCR and about 1 / 13 compared to commercially available capture method kits), making it suitable for large-scale research and clinical applications. [Brief explanation of the drawing]

[0016] [Figure 1] This figure shows a schematic of a gene typing method using the sequence capture method according to the present invention. [Modes for carrying out the invention]

[0017] Human genome reference sequences are being updated sequentially, and "GRCh38 / hg38" is widely known as a relatively new reference sequence. Conventional probes were generally designed based on known reference sequences. The inventors created a unique reference sequence based on known reference sequences in some exon regions that is not identical to any human genome sequence, but is similar to all human genome sequences, and designed probes using this unique reference sequence. Including these, 448 novel probes effective in specifically capturing each gene in the HLA region were designed.

[0018] Each probe included in the probe set of the present invention is fundamentally based on having the base sequence shown in SEQ ID NOs: 1 to 448, but may also have a base sequence in which one to several bases (or within 10%, preferably within 5%, more preferably within 3%, and even more preferably within 1%) of the bases constituting each probe are substituted, deleted, or inserted. However, this is limited to probes that exhibit the probe function intended in the present invention.

[0019] Furthermore, the present invention provides a method for typing HLA genes, including sequence capture using the above-described probe set. Figure 1 shows an overview of the typing method using the sequence capture method. Specifically, this method includes the following steps. (1) A process of obtaining DNA fragments by fragmenting the DNA contained in a sample obtained from a subject. (2) A step of mixing the DNA fragment obtained in step (1) with the probe set of the present invention and hybridizing it to obtain a DNA library. (3) A step to concentrate the DNA library hybridized with the probe. (4) A step of desorbing the probe from the concentrated DNA library and determining the sequence of the obtained DNA library using NGS.

[0020] (1) The process of fragmenting the DNA contained in the sample obtained from the subject is carried out using a standard method in the field. For example, DNA fragmentation using enzymatic cutting is preferred. The ends of the fragmented DNA are repaired (blunted) and adenine added as necessary. The amount of DNA contained in the sample should be 1 ng or more, preferably 5 ng or more, more preferably 10 ng or more. The upper limit of the DNA amount is not particularly limited, and for example, it can be less than 100 ng, less than 75 ng, less than 50 ng, less than 40 ng, less than 30 ng, or less than 15 ng, etc. When using fragmented DNA samples such as cfDNA or FFPE as the sample, the step of further fragmenting the DNA may be omitted. As the sample, body fluids such as blood, or samples derived from non-invasively collected swabs, etc. can be used.

[0021] Next, an adapter is ligated, and different index sequences (up to 384 types) are added to each subject (specimen) by PCR. At this time, it is preferable to measure the DNA fragment length and DNA concentration. The DNA libraries are mixed at an equimolar concentration to obtain a sample.

[0022] (2) Next, the DNA library obtained in (1) is mixed with the probe set of the present invention and hybridized. Before hybridization, it is necessary to add a label to each probe contained in the probe set that can bind to the label carried on the separation carrier such as beads in the subsequent concentration step. As the substance for labeling the probe, biotin is preferably used.

[0023] (3) Next, for example, the DNA library hybridized with the biotin-labeled probe is adsorbed to streptavidin-immobilized beads (magnetic beads). The DNA fragments that did not hybridize with the probe are removed by washing, and the target DNA library adsorbed to the magnetic beads is concentrated.

[0024] (4) Finally, the concentrated DNA library and the probe are detached using a conventional method. The DNA library from which the probe has been detached is, if necessary, PCR amplified using a primer specific to the adapter sequence, and then the nucleotide sequences of up to 384 specimens are determined using NGS. Thereafter, data analysis based on the nucleotide sequence information is performed.

[0025] The present invention will be explained below with specific examples. However, the following examples do not limit the scope of the present invention. Also, the numbers assigned to "Sample" in the table below are numbers that identify the individual (subject) from whom the DNA sample was collected. [Examples]

[0026] Example 1 448 probes (Sequence IDs: 1-448) of the present invention were synthesized. For 384 DNA samples containing 1 to 50 ng of DNA, the target DNA was enriched using the 448 probes obtained by sequence capture, the probes were removed, and then the DNA was sequenced using NGS.

[0027] The specific steps are as follows: A DNA library for HLA gene sequencing was prepared using a commercially available kit. Specifically, DNA was cut into fragments of approximately 300 bp using an enzyme, and the library was prepared through the processes of DNA fragment end repair, adenine addition, and adapter ligation. The adapters were prepared by PCR to add 384 different index sequences, each unique to each sample.

[0028] The ligated DNA libraries were analyzed for fragment length using a 4150 TapeStation system (Agilent Technologies Inc.), and DNA concentration was measured using an Invitorogen Qubit4 Fluorometer (Thermo Fisher Scientific). Based on the measurement data, 384 prepared DNA libraries were mixed at equimolar concentrations.

[0029] The probe of the present invention was labeled with biotin. Using reagents from xGen® Hybridization and Wash v2 Reagents (Integrated DNA Technologies Inc.), the probe was hybridized to the DNA library obtained above according to the same protocol.

[0030] The biotin-avidin reaction was used to enrich the probe-bound DNA library. Following standard procedures, the probe was detached from the DNA library, and then PCR was performed using the adapter sequence and specific primers. The DNA libraries from the 384 obtained samples were sequenced using NextSeq2000 (Illumina Inc.) (using P1 reagent). The results are shown in Tables 1-28 below.

[0031] As shown in each table, using the method described above, we were able to type the DNA of each sample beyond the first region (2-digit level) for identifying serological HLA types and the second region (4-digit level) for identifying alleles with amino acid substitutions within the same serological HLA type, up to the third region (6-digit level) for identifying alleles with base substitutions without amino acid mutations. In bone marrow transplantation, it is said that if the HLA types of the transplant recipient and the donor are a perfect match at the four-digit level, the success rate of the transplant will improve and the incidence of severe graft-versus-host disease (GVHD) will decrease. In light of this, the typing method of the present invention has been confirmed to be a method for obtaining clinically useful information.

[0032] [Table 1]

[0033] [Table 2]

[0034] [Table 3]

[0035] Table 4

[0036] Table 5

[0037] Table 6

[0038] Table 7

[0039] Table 8

[0040] Table 9

[0041] Table 10

[0042] Table 11

[0043] Table 12

[0044] Table 13

[0045] Table 14

[0046] Table 15

[0047] Table 16

[0048] Table 17

[0049] Table 18

[0050] Table 19

[0051] Table 20

[0052] Table 21

[0053] Table 22

[0054] Table 23

[0055] Table 24

[0056] [Table 25]

[0057] [Table 26]

[0058] [Table 27]

[0059] [Table 28]

[0060] Example 2 (The present invention) Typing was performed using the capture method for the 24 DNA samples listed in Tables 29-34 below (TYGD001-TYGD024: each containing 1-50 ng of DNA), in accordance with the method described in Example 1. Equipment used: NextSeq2000 (manufactured by Illumina)

[0061] Comparative Example 1 (Conventional Method) Typing using PCR (conventional method) was performed on the aforementioned 24 types of DNA samples (TYGD001 to TYGD024; each containing 50 ng of DNA). Reagents used: AllTypeNGS11-LociAmplification Kit (Manufactured by OneLambda) Equipment used: NextSeq2000 (manufactured by Illumina) Methods and conditions: The procedure was carried out according to the manufacturer's protocol.

[0062] Tables 29-34 show the DNA typing results for each sample obtained using the conventional PCR method (Comparative Example 1) and the method of the present invention (Example 2). "TRUE" in the tables indicates that the typing results for Comparative Example 1 and Example 2 were identical. As shown in each table, the DNA typing results obtained using the conventional method (Comparative Example 1) and the method of the present invention (Example 2) were completely identical for all measured samples. In other words, it was confirmed that the method of the present invention enables accurate DNA typing, similar to conventional methods using PCR. In addition, the cost required for the method of the present invention is lower than that required for conventional methods, and the cost per sample using the method of the present invention was approximately 1 / 5 of that of conventional methods.

[0063] [Table 29]

[0064] [Table 30]

[0065] [Table 31]

[0066] [Table 32]

[0067] [Table 33]

[0068] [Table 34]

[0069] Example 3 (The present invention) For the eight DNA samples listed in Tables 35-36 below (TYGD003, 004, 007, 008, 013, 014, 019, and 020; each containing 1 to 50 ng of DNA), typing was performed using the capture method in accordance with the method described in Example 1. Equipment used: NextSeq2000 (manufactured by Illumina)

[0070] Comparative Example 2 (Commercial Kit Method) Typing using the capture method (commercially available kit) was performed on the eight DNA samples mentioned above (TYGD003, 004, 007, 008, 013, 014, 019, and 020; each containing 50 ng of DNA). Reagents used: AlloSeq Tx17 (CareDx) for 24 samples Equipment used: NextSeq2000 (manufactured by Illumina) Methods and conditions: The procedure was carried out according to the manufacturer's protocol (using 50 ng of DNA).

[0071] Tables 35-36 show the DNA typing results for each sample obtained using a commercially available kit method with a capture method (Comparative Example 2) and the method of the present invention (Example 3). "TRUE" in the table indicates that the typing results for Comparative Example 2 and Example 3 were identical. As shown in each table, the DNA typing results obtained using the commercially available kit method (Comparative Example 2) and the method of the present invention (Example 3) were completely identical for all measured samples. In other words, it was confirmed that the method of the present invention enables accurate DNA typing similar to that of capture methods using commercially available kits. In addition, the cost required for the method of the present invention is lower than that required for conventional methods, and the cost per sample using the method of the present invention was approximately 1 / 13 of that of the commercially available kit method.

[0072] [Table 35]

[0073] [Table 36]

Claims

1. A probe set for HLA gene typing, consisting of oligonucleotides, each having a nucleotide sequence shown in Sequence ID No. 1 to 448.

2. The probe set according to claim 1, wherein biotin is added.

3. (1) A process of obtaining DNA fragments by fragmenting the DNA contained in the sample obtained from the subject, (2) A step of mixing the DNA fragment obtained in the above step with the probe set described in claim 1 or 2 and hybridizing it to obtain a DNA library. (3) A step of concentrating the DNA library hybridized with the probe, (4) A step of removing the probe from the concentrated DNA library and determining the sequence of the obtained DNA library using NGS. A method for typing HLA genes, including [specific gene names].

4. The typing method according to claim 3, wherein the probe set is the probe set according to claim 2, and the concentration step is carried out by adding streptavidin-immobilized beads.

5. The typing method according to claim 3 or 4, further comprising the step of PCR amplification of the DNA fragment before sequencing the DNA fragment.

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