KR20200002164A - Novel Analysis Strategy for Human Leukocyte Antigen Typing using Sequence-Specific Oligonucleotide Probe Method - Google Patents

Abstract

The present invention relates to filtering and scoring (FnS) that is an analysis method which can quickly and accurately determine a final type of a human leukocyte antigen during human leukocyte antigen (HLA) type inspection by using a sequence-specific oligonucleotide probe. By using the method mentioned above, rapid and accurate HLA type determination can be possible regardless of race of a subject to be inspected.

Description

Novel Analysis Strategy for Human Leukocyte Antigen Typing using Sequence-Specific Oligonucleotide Probe Method in Human Leukocyte Antigen Assay by Sequence-Specific Oligonucleotide Probe Method

The present invention relates to an analytical method capable of quickly and accurately determining the final typing of human leukocyte antigen in human leukocyte antigen typing by using a sequence-specific oligonucleotide probe.

Human Leukocyte Antigen (HLA), a type of Major Histocompatibility Complex (MHC) in humans, produces an immune response that produces key factors that cause rejection in transplantation of hematopoietic stem cells (bone marrow) and organs. Regulatory genes. When foreign substances such as bone marrow cells or viruses of others enter the body, the human body takes action to remove them in order to protect itself, and plays an important role in determining whether the substance is the same as itself. It is HLA.

To increase the success rate of bone marrow or organ transplantation, the HLA types of patients and donors must be consistent. If the HLA is inconsistent, the transplanted hematopoietic stem cells are considered foreign to the patient, resulting in a rejection that is attacked, or conversely, a graft-versus-host disease in which the donor's T-lymphocytes treat the patient as foreign and attacks the patient. Symptoms such as skin disease, diarrhea and liver dysfunction can be caused and, in severe cases, can lead to death. In both cases, HLA typing is an essential test before organ and hematopoietic stem cell transplantation.

Current HLA test methods are classified into low resolution, middle / intermediate resolution, and high resolution according to the resolution of the test. Low-resolution serological methods require highly skilled skills and experience, and inconsistencies in comparison with DNA tests have resulted in DNA tests that are more easily performed in smaller laboratories. It is rapidly replacing.

The sequence specific oligonucleotide probe (SSOP) method used for the medium resolution analysis is a method of determining the result based on the hybridization pattern with the probe immobilized on the strip after PCR amplification. The SSOP method has the advantage of being slightly readable due to less ambiguity due to cross reactions and weak reactivity, which are commonly found in serological tests, and the ability to simultaneously process a large amount of samples. However, the most suitable allele pair is compared by comparing the 'probe probe pattern' of the candidate allele pairs with the 'measured probe pattern', which is a combination of approximately 100 SSO probes and the subject's HLA gene PCR product. Since it is selected, it takes a long time to make the final determination of HLA type. Therefore, it is necessary to use a fast and accurate data analysis method for final HLA classification in HLA classification by SSOP method.

1. Republic of Korea Patent No. 10-1782806

It is an object of the present invention to provide a method for final determination of the type of human leukocyte antigen in human leukocyte antigen typing using sequence-specific oligonucleotide probes.

In order to achieve the above object, an aspect of the present invention provides a "Filtering and Scoring (FnS)", which is a method for screening a human leukocyte antigen (HLA) type of individual comprising the following steps:

혼 hybridizing the HLA allele amplification product of the individual with the sequence-specific oligonucleotide probe to obtain a measurement probe pattern;

(B) grouping the HLA alleles into one allele group with alleles exhibiting the same predictive probe pattern;

Removing alleles that violate probes that have a definite negative response (eg, percent deviation less than -60%) in the grouped candidate allele group list;

조합 combines the remaining two HLA allele groups to form an allele pair, and removes allele pairs that violate probes with a positive positive response (e.g., more than + 60% percent deviation) from the list of candidate allele pairs Making; And

(B) comparing the predicted probe patterns of the individual allele pairs remaining in the candidate list with the measured probe patterns of the subjects to score a match, and finally selecting the allele pairs having the least mismatch for each HLA type of the subject.

As used herein, the term 'human leukocyte antigen (HLA)' is a glycoprotein molecule produced by the major histocompatibility complex (MHC) gene, which is present in all vertebrates and is a human MHC gene. HLA gene, the product of which is called HLA. HLA is expressed on the surface of all tissue cells in the human body and is also expressed on blood cells such as leukocytes and platelets. HLA is known to be involved in self and non-self cognition, immune responses to antigenic stimulation, regulation of cellular and humoral immunity and susceptibility to disease.

HLA genes are classified into classes I, II, and III according to the structure and function of antigens, of which class I includes genes such as HLA-A, B, and C, and class II contains HLA-DR, DQ, and DP genes. Included. Each loci of HLA has a large number of alleles, indicating the most severe polymorphism of human genes. Since the HLA genes are present in close proximity to each other on chromosome 6, they are inherited in a cluster when inherited from parent to child, and the combination of these alleles is called HLA haplotype.

According to one embodiment of the invention, the HLA allele amplification product in vi may be produced by polymerase chain reaction (PCR) known in the art. In addition, amplifying the HLA allele and hybridizing with the probe may use a commercially available HLA typing kit.

As used herein, the term "probe" refers to a nucleic acid sequence that exhibits specificity for human HLA gene sequence. According to one embodiment of the present invention, the probe may use Lifecodes HLA-SSO typing kits (Immucor, USA).

As used herein, the term 'probe hybridization pattern' refers to a pattern obtained by allocating positive / negative patterns according to hybridization of HLA alleles amplified in an individual's blood and individual probes, and measuring probes Also called patterns or complete reaction patterns for all probes (CRPoAPs).

As used herein, the term 'predictive probe pattern' is a pattern that combines the positive / negative status of known individual probes (approximately 100) for individual or two allele pairs, and provides information about the HLA kit manufacturer. .

According to one embodiment of the invention, the HLA type determination method is a probe (e.g., a probe with a percent deviation of -60% or less, or a percentage deviation in the lower third) Alleles that violate the belonging probe) can be removed from the candidate list.

According to one embodiment of the present invention, the HLA type determination method is a probe (eg, a probe having a percent deviation of + 60% or more, or a percentage deviation of the top half) in a positive positive response (absolute positive) at ⒞. And removing allelic pairs that violate the probes belonging to the candidate list.

As used herein, the term 'percent deviation' is a measure of probe response, which assigns positive numbers for positive and negative values for individual probes. The percent deviation is expressed as a percentage after subtracting the cutoff value from the corrected median fluorescent intensity (MFI) value divided by the cutoff value (Equation 2).

The calibrated MFI value of each probe can be calculated by dividing the original MFI value by subtracting the control blandk for probe MFI value from the consensus MFI value by subtracting the MFI value of the control blank for consensus. have.

Filtering and Scoring (FnS) method (HLA type determination algorithm) according to an embodiment of the present invention can determine the final human leukocyte antigen type of the individual quickly and accurately regardless of race, HLA type The FnS method can be applied to analysis software provided with commercial test kits for testing.

Figure 1 shows the results of obtaining a complete reaction pattern for all probes (CRPoAPs) using Lifecodes HLA-B typing kits in the HLA typing test of a subject.
2 shows an example of determining HLA-B type of an individual by using a filtering and scoring (FnS) method.
Figure 3 shows the results of comparing the time to analyze the HLA type using the EPM (exact pattern matching) and FnS (Filtering and Scoring) method.

Hereinafter, one or more embodiments will be described in more detail with reference to Examples. However, these examples are provided to illustrate one or more embodiments by way of example, but the scope of the present invention is not limited to these examples.

Experiment method

1. HLA Typing Target

507 HLA typing (human leukocyte antigen typing, histocompatibility testing; 119 HLA-A, 124 HLA-B, 130 HLA-DR and HLA-DQ) for 152 patients who needed a kidney transplant at Kyungpook National University Hospital. 134) test results were used. HLA typing results were anonymously assigned a new serial number.

2. Reverse SSOP HLA Typing

Medium resolution reverse SSOP HLA typing (reverse sequence-specific oligonucleotide probe HLA typing) was performed by the following method. Genomic DNA was isolated from peripheral blood samples using the Wizard Genomic DNA Purification Kit (Promega, USA). Then, HLA-A, -B, -DR and -DQB1 typing were performed using Lifecodes HLA-SSO typing kits (Immucor) according to the manufacturer's protocol. PCR reaction solution was prepared by mixing 15 μl of Lifecodes Master Mix (Immucor), 200 ng of genomic DNA and 2.5 U of Taq polymerase, and adjusting the final volume to 50 μl. PCR performed the denaturation reaction at 95 ° C. for 5 minutes, and the amplification step performed 8 cycles of 30 seconds at 95 ° C., 45 seconds at 60 ° C., and 45 seconds at 72 ° C., 30 seconds at 95 ° C., 45 seconds at 63 ° C. And 32 cycles of 45 seconds at 72 ° C. followed by an extension reaction at 72 ° C. for 15 minutes. Hybridization reaction was carried out by mixing 15 µl of the probe mix and 5 µl of the PCR result, 5 minutes at 97 ° C, 30 minutes at 47 ° C, and 10 minutes at 56 ° C. 170 μl of the streptavidin-phycoerythrin solution diluted 1: 200 with the hybrid reactant was mixed and analyzed within 30 minutes using a Luminex 200 system (Luminex Corp.).

Assay data, the complete reaction pattern (CRO) of the probe is constructed from positive / negative based on probes at the HLA-A, -B, -DR and -DQB1 loci, and complete reaction pattern for all probes (CRPoAPs). Is also called a probe hit pattern, a measurement probe pattern.

3. Data Analysis for HLA Typing

3-1. Initial shared steps

Common analysis steps for data analysis were performed under the same conditions using exact pattern matching (EPM) software from Lifecodes and filtering and scoring (FnS) software developed in the laboratory. Before running the software, hybridization patterns were identified by measuring the fluorescence of the microspheres in each well using Luminex 200 and its xPONENT software (Luminex Corp.), and a comma-separated values (csv) file was created. The csv file was imported into each data analysis software and used. The csv file contains median fluorescent intensity (MFI) values for each probe (microsphere). The obtained MFI values were used to assess the positive or negative reactivity of the probe. For quality control, the MFI value of the consensus SSO probe should exceed the threshold value defined in the package insert sheet. Lot-specific background control values were then subtracted from the raw MFI values of the samples and background-corrected data was generated. Next, the MFI value was corrected according to Equation 1 below to generate normalized data.

Percent deviation for a specific probe was calculated according to Equation 2 below.

Figure 1 shows the results of identifying CRPoAPs using Lifecodes HLA-B typing kits in the subject to determine the HLA type.

A of FIG. 1 shows an allele and probe hit result showing a portion of the expected CRPoAP of a homozygous allele pair. 1B is the Match It! The relationship between the CRPoAP calibration value and the probe name of the sample analyzed by DNA software is shown in FIG. 1B: green graph means negative probe, red graph means positive probe, grid graph means probe and black adjusted with positive probe Dots represent cutoff values for each probe.

3-2. EPM with Match It! Software Analysis

The CRPoAPs of the samples were compared with the expected CRPoAPs of allele pairs. The allele pair is a combination of all alleles or common well-documented alleles based on the IPDIMGT / HLA sequence database release 3.11.0. In order to find an exact match allele, probes suspected of showing false positive / negative reactions were changed before comparing the two patterns. For certain probes, the lower the absolute percentage deviation, the higher the probability of causing a false response.

3-3. Analysis with FnS Software

A dedicated software program based on the FnS method was written in Visual Basic version 6.0 (Microsoft Co.). The software uses Farpoint Spread version 3.0 (GrapeCity, Sendai, Japan) as a spreadsheet and grid component, and built a Korean HLA database in Access (Microsoft Co.).

The probes with absolute positive and absolute negative responses were defined as probes with absolute percent deviations of 60% or greater in the measurement results for the samples: absolute positive probes> + 60% and absolute negative probes <-60%. Partial reaction pattern with only definite probes (PRPwoDP) was defined as a pattern consisting of the following probes: positive PRPwoDP consisting of absolute positive probes and negative PRPwoDP consisting of absolute negative probes.

The standard method of finding the best-fit allele for the sample CRPoAP of Korean subjects is as follows: o The second or third resolution level based on the presence or absence of an allele previously identified in an individual in Korean. filter by resolution level; 개별 filter individual alleles based on negative PRPwoDP: for probes belonging to the negative PRPwoDP, if the predictive response of one candidate allele is positive, then remove the allele from the candidate allele list; (B) combine the remaining alleles two by one to get a list of allele pairs; ⑷ allele pairs are filtered based on positive PRPwoDP: for probes belonging to the positive PRPwoDP, if the predictive response of one candidate allele pair is negative, remove the allele pair from the list of candidate allele pairs; O A mismatch score is obtained by comparing the raw CRCRAP obtained from the subject with the expected CRPoAP of the individual allele pair remaining in the candidate allele pair list.

Alternative methods are designed for ethnically diverse or mixed population groups (Table 1): The ethnic-based filtering step groups individual alleles according to the same probe hit pattern. It was replaced by a grouping step. The grouping step and the race-based filtering step were performed once before data analysis, when a new lot of information entered the software's database. Therefore, the time required to perform the two steps is not included in the data analysis time.

Mismatch scores were calculated separately for the sample CRPoAP and the predicted CRPoAP of the specific candidate allele pair. The mismatch score consists of the sum of the number of mismatch probes and the percent deviation (Σ% deviation), and the sum of the percent deviations is calculated according to the following equation:

This scoring was repeated for all remaining gene pairs and the allele pair with the smallest value among the mismatch scores was chosen as the most suitable allele pair. Final HLA typing was determined by prioritizing allele pairs containing all CWD alleles. In order to confirm the accuracy of HLA typing when the number of mismatched probes was 3 or more, the test was repeated using the same method and HLA-SBT.

2 shows an example of typing HLA-B type using an alternative method (filtering and scoring method). An allele indicated in yellow in FIG. 2 means a CWD allele.

Table 1 below compares the standard and alternative methods for typing HLA types.

step Explanation Standard way Alternative way Individual alleles Allele pair Individual alleles Allele pair One Early common steps 3,958 7,834,861 3,958 7,834,861 2 Grouping alleles having the same probe hit pattern NA NA 1,771 1,569,106 3 Filter based on the presence of alleles identified in Koreans 239 28,680 NA NA 4 Filtering based on negative PRPwoDP (absolute negative probe with percent deviation <-60%) 16 - 135 - 5 Combining the remaining alleles into allele pairs - 136 - 9,180 6 Filtering based on positive PRPwoDP (absolute positive probe with percent deviation> + 60%) - 10 - 92 7 Computing a mismatch score between the original CRPoAP of the subject sample and the expected CRPoAP of the residual candidate allele pair - One - One

3-4. Comparison of Results Analysis Time for Reverse SSOP HLA Typing

The time needed to analyze the data was compared between Match It!

For half of a total of 507 HLA typing tests, two experts performed data analysis simultaneously using different methods. For the other half, data analysis was performed using different methods. The correctness of the final HLA typing determination was determined according to the following criteria: All candidate CWD allele pairs with the smallest mismatch scores were determined by final HLA typing or typing of a given HLA locus.

3-5. Statistical analysis

Statistical analysis was performed using Excel 2013, and the two methods were compared by paired p- test. When the P value was 0.05 or less ( P <0.05), it was judged to have statistical significance, and the results were described as mean.

Experiment result

1. Data analysis time

As shown in Table 2 below, the average analysis time (minutes: seconds) for the FnS method is shown as 00:21 (00: 08-01: 47), which is 01:04 (00: 15-) of the standard analysis method EPM method. 23:45) was found to be shorter ( P <0.05).

analysis
time
(Minutes: seconds) Number of mismatch probes (sample count) 0 (334) 1 (98) 2 (49) 3 (11) 4 (10) 5 (3) 6 (1) 7 (1) Sum
(507) EPM 00:35
(00: 15-02: 31) 01:06
(00: 15-04: 23) 01:54
(00: 36-05: 06) 02:54
(00: 35-05: 29) 03:39
(00: 32-08: 39) 11:02
(01: 36-23: 45) 17:00 15:00 01:04
(00:15
-23: 45) FnS 00:19 (00: 08-01: 01) 00:23
(00: 11-01: 08) 00:27
(00: 09-01: 34) 00:36
(00: 11-01: 47) 00:41
(00: 25-01: 30) 00:35
(00: 17-00: 52) 00:38 00:43 00:21
(00:08
-01: 47)

In addition, as shown in Figure 3 using the EPM method it can be seen that the analysis time increases significantly as the number of mismatch probes increases. On the other hand, using the FnS method it was confirmed that the analysis time is relatively constant regardless of the number of mismatch probes.

2. Accuracy of HLA Typing

As shown in Table 3 below, the results of the EPM and FnS methods were inconsistent in four of the 507 HLA typing samples. Considering the sum of allele frequencies, number of mismatched probes, and percent deviations in Koreans, HLA typing with the FnS method was accurate in all four samples.

Specifically, when the EPM method was used, it was not possible to identify a plurality of CWD allele pairs with the smallest sum of percent deviations in two samples. The other two samples could not identify a single candidate CWD allele pair with the smallest number of mismatch probes or with the smallest sum of percent deviations.

HLA
Gene locus EPM Method FnS method HLA final typing method Specified allele pairs Mismatch
Score Specified allele pairs Mismatch
Score Sample 1 HLA-A A * 24: 02, 24:02 0, 0 A * 24: 02, 24:02 0, 0 FnS A * 24: 02, 24:20 0, 0 A * 24: 20, 24:20 0, 0 A * 24: 20, 24:20 0, 0 Sample 2 HLA-A A * 24: 02, 24:02 0, 0 A * 24: 02, 24:20 0, 0 FnS A * 24: 02, 24:20 0, 0 A * 24: 20, 24:20 0, 0 A * 24: 20, 24:20 0, 0 Sample 3 HLA-B B * 35: 01, 55:01 2, 105 B * 35: 01, 55:02 1, 11 FnS Sample 4 HLA-DQ DQB1 * 04: 01, 04:02 3, 73 DQB1 * 04: 01, 04:01 2, 55 FnS

Through the above experimental results, it was confirmed that the FnS method for typing HLA types could increase the accuracy of the analysis while reducing the analysis time. In addition, the FnS method can type HLA typing of individuals regardless of race.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (3)

Human leukocyte antigen (HLA) type determination method of an individual comprising the following steps:
혼 hybridizing the HLA allele amplification product of the individual with the sequence-specific oligonucleotide probe to obtain a measurement probe pattern;
(B) grouping alleles having the same predictive probe pattern among the HLA alleles into one allele group;
(B) removing from the candidate list allele groups that violate a probe that has a definite negative response (eg, percent deviation less than -60%) in the grouped candidate allele group list;
조합 combines the remaining two HLA allele groups to form an allele pair, and lists allele pairs in the candidate allele pair list that violate the probe with a positive positive response (eg, percent deviation greater than + 60%) Removing from; And
(B) comparing the predicted probe pattern of the individual allele pair remaining in the candidate allele pair list with the measurement probe pattern of the subject to score a match, and finally selecting allele pairs having the least mismatch for each HLA type of the subject.
The method of claim 1, wherein the method removes from the list of candidate alleles the alleles whose percent deviations are in violation of probes belonging to the lower third.
The method of claim 1, wherein the method removes from the list of candidate allele pairs alleles that violate probes in the upper half of ⒟.

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