JP2020184992A - Methods for detecting hla class i allele-lacking leukocytes - Google Patents

Abstract

To provide quick and inexpensive methods for detecting HLA-LL without HLA typing to improve the medical care of bone marrow failure.SOLUTION: Disclosed is a method for detecting HLA class I allele-lacking leukocytes comprising a step of detecting loss of function mutation at the 19th nucleotide in the coding sequence of HLA-A gene and/or HLA-B gene using DNA derived from the blood of a subject of unknown HLA type.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting HLA class I allele-deficient blood cells and a kit for that purpose.

Bone marrow failure is a condition in which a decrease in bone marrow function results in a decrease in all blood cells. Idiopathic bone marrow failure caused by qualitative and quantitative abnormalities of hematopoietic stem cells is sometimes collectively referred to as "bone marrow failure syndrome". In recent years, the number of patients with bone marrow failure syndrome has been increasing with the aging of the population. The category of bone marrow failure includes aplastic anemia (AA), myelodysplastic syndrome (MDS), paroxysmal nocturnal hemoglobinuria (PNH), etc., but they overlap each other and have no boundaries. Since it is clear, it is important to perform treatment according to the pathological condition.

Aplastic anemia presents with various symptoms such as palpitation and shortness of breath associated with anemia, susceptibility to leukopenia, purpura due to thrombocytopenia, and bleeding tendency due to a decrease in hematopoietic stem cells. The onset of regenerative anemia includes direct damage to hematopoietic stem cells by cytotoxic T cells (CTL) and suppression of hematopoiesis by immunological mechanisms such as excess of T cell hormones that suppress hematopoietic stem cells. It is believed to be involved in many cases. On the other hand, in some cases, it develops due to abnormalities of hematopoietic stem cells themselves, but an effective method for differentiating these causes has not been established.

In the treatment of aplastic anemia and myelodysplastic syndrome, it is most important to determine whether the immune pathology is involved in their development. This is because when immunological conditions are involved in these diseases, immunosuppressive therapy has a high response rate and it is possible to avoid inadvertent use of another highly toxic treatment. The simplest way to determine the involvement of an immune pathology is to test blood cells for paroxysmal nocturnal hemoglobinuria (PNH) traits using flow cytometry. PNH-type blood cells are blood cells lacking the GPI-anchored membrane protein characteristic of PNH. Hematopoietic stem cells lacking the GPI-anchored membrane protein become normal stem cells in an environment where high concentrations of this cytokine are present in the bone marrow because they are less sensitive to "inflammatory cytokines that keep hematopoietic stem cells in a quiescent state." They are more likely to be activated in comparison and they produce GPI-anchored membrane protein-deficient blood cells. This is considered to be the reason why PNH type blood cells are easily detected in bone marrow failure due to immune pathology.
However, PNH-type blood cells cannot be accurately detected at all institutions, and even if PNH-type blood cells are negative, the immune pathology cannot be ruled out. Therefore, immunopathological markers other than PNH-type blood cells are required.

The most direct evidence that aplastic anemia is caused by the attack of hematopoietic stem cells by CTL is the presence of human leukocyte antigen (HLA) class I allergen-deficient blood cells in peripheral blood. The reason why CTL attacks the target cell is that the T cell receptor contained in CTL recognizes the self-antigen peptide presented by the HLA class I molecule of the target cell. When the "immune tolerance" of T lymphocytes to "self-antigens highly expressed by hematopoietic stem cells" is disrupted due to viral infection or exposure to environmental poisons, and as a result, CTLs against hematopoietic stem cells are induced. Conceivable. Hematopoietic stem cells with mutations in various genes are present in the bone marrow of healthy subjects, but they do not contribute to hematopoiesis because they are usually in a quiescent state. However, when a CTL attack on hematopoietic stem cells occurs, the stem cells that contribute to hematopoiesis decrease, while the sleeping stem cells are mobilized for hematopoiesis to supplement it. If any of these "raised" stem cells are hematopoietic stem cells that lack a specific HLA class I allele due to a genetic abnormality in HLA, they survive the attack of CTL because they are not recognized by T cell receptors. Produces HLA-deficient blood cells.
HLA-deficient leukocytes can be detected by flow cytometry (FCM) using a specific antibody against the HLA molecule.

Mechanisms for deleting this HLA class I allele include loss of the HLA gene haplotype due to heterozygosity loss (6pLOH) of the short arm of chromosome 6 in which the HLA gene group is present, and somatic mutation of the HLA class I allele. Can be mentioned. When one of the short arms (6p) of chromosome 6 encoding the HLA gene is deleted during cell division, the remaining chromosomes make up for the deletion to maintain the balance of the chromosomes. Form. The result is 6pLOH. Due to this phenomenon, leukocytes having only one HLA are detected in about 13% of all aplastic anemias (Non-Patent Document 1). This 6pLOH has been conventionally investigated by a method called SNP array analysis that can quantify the copy number of a specific gene region.
Another method for detecting the presence of 6pLOH is to quantify the imbalance between two allelic amounts by droplet digital PCR (ddPCR) (Non-Patent Document 2). In this method, the gene amount is 1: 1 when each allele is amplified by PCR under normal conditions, but in patients with 6pLOH, only one is amplified, so the amplified gene amount is not 1: 1. This is to detect the existence of 6pLOH.

On the other hand, somatic mutations in the HLA class I gene were clarified by examining the HLA class I gene region in detail using a next-generation sequencer (Non-Patent Documents 2 and 3).

As described above, clarifying the presence of HLA class I allele-deficient blood cells (hereinafter, may be abbreviated as "HLA-LL") diagnoses the pathophysiology of bone marrow failure and is appropriate for the pathology. It is very important for the treatment. However, the existing detection method has the following problems.
(1) In order to detect HLA-LL by ddPCR method or FCM, it is necessary to carry out expensive HLA typing in advance.
(2) Since HLA typing usually takes more than one week, it takes 1-3 months to clarify the presence or absence of HLA-LL, including the subsequent assay.
(3) The types of HLA antigens that can be detected with monoclonal antibodies are limited, and the detection of 6pLOH blood cells by the ddPCR method is not applicable to all cases.
(4) Detection of somatic mutations in HLA alleles by a next-generation sequencer is not practical because it is expensive and time-consuming.
(5) Since the detection sensitivity of HLA-LL is 1% or more in all of these methods, HLA-LL with a lower ratio cannot be detected.

Katagiri et al., Blood, 118: 6601-6609 (2011) Zaimoku et al., Blood, 129: 2908-2916 (2017) Babushok et al., Blood Adv, 1: 1900-1910 (2017)

Therefore, an object of the present invention is to provide a method for detecting HLA-LL quickly, with high sensitivity, and at low cost without performing HLA typing, thereby dramatically improving the medical care for bone marrow failure. ..

In order to achieve the above object, the present inventors detailed the HLA class I gene of patients with 6 pLOH using a next-generation sequencer in order to search for genetic abnormalities common to HLA-LL-positive patients. Analyzed. As a result, in 12 of the 22 analyzed cases, 1-3 types of loss-of-function mutations (median 1 type) were detected per case. Surprisingly, most of the loss-of-function mutations detected were concentrated at specific locations in the exon 1 region of the HLA-A or HLA-B gene, regardless of the type of HLA class I allele. This common mutation was previously found in the analysis of the HLA class I allele (HLA-B ^* 40 ^: 02), which is most frequently carried by patients with aplastic anemia (Non-Patent Documents 2 and 3), but is optional. It was not recognized at all that it was a common mutation among HLA class I alleles.

The present inventors consider that if this common mutation can be detected easily and with high sensitivity, the presence or absence of HLA-LL can be rapidly evaluated without performing HLA typing in advance, and ddPCR, which is a next-generation PCR method, is used. Focused on the law. Since the HLA gene is the most polymorphic gene among all genes, it was extremely difficult to prepare primers and probes that amplify only a specific region, but the present inventors conducted trial and error. As a result, we succeeded in designing a primer-probe set that detects this mutation with high sensitivity. When the ddPCR method was performed using DNA derived from peripheral blood as a template using this primer-probe set, the common mutation was detected in 101 of 353 patients with aplastic anemia (about 29%). In addition, it was clarified that when this method is combined with the conventional PNH type blood cell test, the immune pathology can be detected in about 4/5 cases. Notably, a common mutation was detected in 34 (31%) of the 108 PNH blood cell negative cases. Therefore, it was clarified that using this method, immune pathology can be detected even in cases where pathological diagnosis is difficult by PNH type blood cell test.

As a result of further research based on these findings, the present inventors have completed the present invention.

That is, the present invention is as follows.
[1] HLA class including the step of detecting a loss-of-function mutation in the 19th nucleotide of the coding sequence of the HLA-A and / or HLA-B gene in DNA derived from blood collected from a subject whose HLA type is unknown. Method for detecting I allele-deficient blood cells.
[2] The method according to [1], wherein the loss-of-function mutation is a nonsense mutation.
[3] The method according to [1] or [2], wherein a loss-of-function mutation is detected using a digital PCR method.
[4] The method according to any one of [1] to [3], wherein the subject is a patient with or suspected of having bone marrow failure syndrome.
[5] The method according to [4], wherein bone marrow failure is caused by a disease selected from the group consisting of aplastic anemia, myelodysplastic syndrome and paroxysmal nocturnal hemoglobinuria.
[6] The method according to any one of [1] to [5], which is used in combination with the detection of PNH type blood cells.
[7] A kit for detecting HLA class I allele-deficient blood cells in a subject whose HLA type is unknown.
(A) A primer set that can amplify a partial nucleotide sequence of an HLA-A and / or HLA-B gene, including the 19th nucleotide of the coding sequence.
(B1) Among the PCR products amplified by the primer set of (a) above, a probe capable of specifically hybridizing the 19th nucleotide to a normal one, and (b2) the primer set of (a) above. A kit comprising a probe capable of specifically hybridizing to an amplified PCR product in which the 19th nucleotide is mutated to cause a loss-of-function mutation.
[8] The kit according to [7], wherein the loss-of-function mutation is a nonsense mutation.
[9] (a1) The primer set capable of amplifying the partial nucleotide sequence of the HLA-A gene is a forward primer having a sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 1, and SEQ ID NOs: 2-5. Containing each of one or more nucleotide sequences selected from the nucleotide sequences represented by and one or more reverse primers consisting of substantially the same sequence, and / or (a2) a partial nucleotide sequence of the HLA-B gene. The primer set that can be amplified includes one or more forward primers having a sequence substantially the same as that of each of the one or more nucleotide sequences selected from the nucleotide sequences represented by SEQ ID NOs: 6 to 8, and SEQ ID NOs: 9 to 9. The kit according to [7] or [8], which comprises one or more reverse primers consisting of substantially the same sequence as each of one or more nucleotide sequences selected from the nucleotide sequences represented by 11.
[10] The probe of (b1) has substantially the same sequence as the nucleotide sequence represented by SEQ ID NO: 12, and the probe of (b2) has substantially the same sequence as the nucleotide sequence represented by SEQ ID NO: 13. The kit according to [8] or [9], which comprises the same sequence.

According to the present invention, HLA-LL can be detected easily and highly sensitively without performing HLA typing, so that immunization of bone marrow failure is extremely quick and inexpensive as compared with the conventional method requiring HLA typing. The pathological condition can be diagnosed.

It is a figure which shows the result of having analyzed the presence or absence of mutation of the HLA class I gene in 22 HLA-LL positive patients using the next-generation sequencer. The pie chart on the left shows that in 12 of 22 cases, loss-of-function mutations were found in addition to 6 pLOH. The figure on the right schematically shows what kind of mutation occurs at which position of the HLA-A and HLA-B genes. (A) It is a figure which shows an example of the result of the detection method of this invention using the ddPCR method. (B) The state of determination of the cutoff value in the detection method of this invention is shown. The left shows the test result of the detection sensitivity using the dilution series, and the right shows the detection result in 24 healthy subjects (all cases are less than 0.07%). It is a figure which shows the actual inspection result (Example 1) of the detection method of this invention using the ddPCR method. The upper part shows HLA-LL positive cases, and the lower part shows HLA-LL negative cases. It is a figure which shows the breakdown of the presence / absence of PNH type blood cell, and the presence / absence of a common mutation of this invention in 303 patients of aplastic anemia (Example 1). It is a figure which shows the comparison of the test result of Example 1 (top) and Example 2 (bottom) in the same case. By changing the types of primers and PCR reagents, the amplification efficiency was improved, the reaction time of ddPCR could be shortened, and the separability was also significantly improved. It is a figure which shows the breakdown of the presence or absence of PNH type blood cell, and the presence or absence of a common mutation of this invention in 353 patients of aplastic anemia (Example 2).

[I] Detection Method of the Present Invention The present invention provides a method for detecting HLA class I allele-deficient blood cells (HLA-LL) in a subject whose HLA type is unknown (hereinafter, also referred to as "detection method of the present invention"). provide.
An allele refers to an individual gene located at the same locus on a pair of homologous chromosomes. "HLA class I allele-deficient blood cells (HLA-LL)" lack one or more HLA class I (eg, HLA-A, HLA-B, HLA-C) alleles by 6pLOH or are specific It means a leukocyte that does not express the HLA class I molecule encoded by the allele on the surface due to a loss-of-function mutation of the HLA class I allele.

The detection method of the present invention is a loss-of-function type in the 19th nucleotide (hereinafter, also referred to as “target nucleotide”) of the coding sequence of the HLA-A and / or HLA-B gene in DNA derived from blood collected from a subject. Includes the step of detecting mutations.

In the detection method of the present invention, the "subject" to be measured is a patient with bone marrow failure syndrome or a person suspected of having bone marrow failure syndrome. Bone marrow failure is a condition in which all blood cells are reduced due to a decrease in bone marrow function, and "bone marrow failure syndrome" is a general term for idiopathic bone marrow failure caused by qualitative and quantitative abnormalities of hematopoietic stem cells. The categories of bone marrow failure syndrome include aplastic anemia (AA), myelodysplastic syndrome (MDS), paroxysmal nocturnal hemoglobinuria (PNH), large granulolymphocyte hyperplasia (LGL), and pure red cell aplasia. PRCA), etc. are included. The main subject in the detection method of the present invention is a patient with or suspected of having aplastic anemia, myelodysplastic syndrome or paroxysmal nocturnal hemoglobinuria whose immune pathology may be involved in the development of bone marrow failure. Is a person.

The most characteristic feature of the detection method of the present invention is that the presence or absence of HLA-LL in a subject can be quickly detected without performing HLA typing. Therefore, the subject in the detection method of the present invention does not need to know the HLA type.

Examples of the subject-derived sample used in the detection method of the present invention include genomic DNA extracted from peripheral blood containing leukocytes. For example, genomic DNA can be isolated from blood collected from a subject by normal blood collection by a phenol extraction method or the like. At that time, for example, QI Amp
Commercially available genomic DNA extraction kits and devices such as the Circulating Nucleic Acid Kit may be used.

The loss-of-function mutation to be detected in the detection method of the present invention is a loss-of-function mutation in cytosine (c.19C), which is the 19th nucleotide of the coding sequence of the HLA-A and / or HLA-B gene. The nucleotide is located within exon 1 of the HLA-A and HLA-B genes and is the first nucleotide of the 7th codon (CGA). Examples of the loss-of-function mutation include nonsense mutations and frameshift mutations. The nonsense mutation detected in the present invention is a mutation (c.19C> T) that produces a stop codon (TGA) by substituting thymine at position 19 cytosine. On the other hand, a frameshift mutation includes a deletion or insertion of 1 or 2 nucleotides at the position of the 19th nucleotide, and is preferably a frameshift mutation due to a deletion or deletion of the 19th cytosine (c.19delC). More preferably, the loss-of-function mutation to be detected in the detection method of the present invention is a nonsense mutation due to the substitution of cytosine at position 19 with thymine (c.19C> T).

In general, the presence or absence of a loss-of-function mutation in a gene can be determined by any polymorphism analysis method known in the art. For example, a method using a PCR method can be mentioned. Methods using the PCR method include amplification of the target nucleic acid by the PCR method and detection of mutations in the amplification product by fluorescence or luminescence, PCR-RFLP (restriction fragment length polymorphism) method, and PCR-SSCP (single strand conformation polymorphism) method (Orita, M. et al., Proc. Natl. Acad. Sci., USA, 86, 2766-2770 (1989), etc.), PCR -SSO (specific sequence oligonucleotides) method, ASO (allele specific oligonucleotides) hybridization method (Saiki, Nature, 324, 163) that combines the PCR-SSO method and the dot hybridization method. -166 (1986), etc.), TaqMan (registered trademark, Roche Molecular Systems)-PCR method (Livak, KJ, Genet Anal, 14,143 (1999), Morris, T. et al., J. Clin. Microbiol., 34 , 2933 (1996)), real-time PCR method, digital PCR method (eg, droplet digital PCR (ddPCR) method, micropore distribution type digital PCR method, etc.) and the like.

Other methods include cycling probe method, Invader (registered trademark, Third Wave Technologies) method (Lyamichev V et al., Nat Biotechnol, 17,292 (1999)), and FRET (Fluorescence Resonance Energy Transfer) method (Heller, Academic Press Inc, pp. 245-256 (1985), Cardullo et al., Proc. Natl. Acad. Sci. USA, 85, 8790-8794 (1988), International Publication No. 99/28500, JP 2004- 121232, etc.), methods using DNA chips or microarrays (Wang DG et al., Science 280, 1077 (1998), etc.), Southern blot hybridization methods, dot hybridization methods (Southern, E., J. Mol) . Biol. 98,
503-517 (1975)) and so on.

In order to detect a small amount of mutated DNA, a method using the PCR method is preferable from the viewpoint of simplicity and sensitivity. In addition, unlike conventional real-time PCR, digital PCR, which is a next-generation PCR that can perform absolute quantification without worrying about amplification efficiency, has high accuracy, high sensitivity, and excellent processing performance, especially ddPCR, is particularly popular. preferable.

When the PCR method is used, the detection method of the present invention can be carried out, for example, as follows.
(1) A step of contacting a DNA sample with a probe set that specifically hybridizes with a target nucleotide (2) A step of amplifying a region containing the target nucleotide by PCR (3) A step of including the PCR product amplified in the step (2). Step of measuring fluorescence intensity of solution (4) Step of detecting loss-of-function mutation in target nucleotide based on the measurement result of (3) above

(1) Step of contacting the probe set with the DNA sample (a) Probe set The probe set that specifically hybridizes with the target nucleotide used in the step (1) includes the HLA-A and / or HLA-B gene. Oligonucleotides consisting of a contiguous nucleotide sequence of about 5 to about 30 bases, preferably about 7 to about 20 bases, that hybridize to a partial nucleotide sequence containing the target nucleotide are used. The probe set consists of two types of probes whose target nucleotide portion is a normal type (cytosine) and a mutant type (thymine when the loss-of-function mutation is a nonsense mutation).

In a preferred embodiment of the invention, the probe set is an oligonucleotide of substantially the same sequence as the nucleotide sequence represented by SEQ ID NO: 12 (CCCCGAACC) for the HLA-A and HLA-B genes. (Normal type) and an oligonucleotide (variant type) having a sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 13. Here, the oligonucleotide having substantially the same sequence as the nucleotide sequence represented by SEQ ID NO: 13 (CCCTGAACC) is a probe for detecting a nonsense mutation in the target nucleotide of the HLA-A and HLA-B genes. The sequence represented by SEQ ID NO: 12 corresponds to the 16th to 24th nucleotide sequences of the coding sequences of both the HLA-A and HLA-B genes, and in most alleles, the sequences are completely matched between the two genes. , Both HLA-A and HLA-B can be measured with one probe set.

Here, "substantially identical sequence" means not only an completely identical sequence but also "hybridize to a partial nucleotide sequence containing a target nucleotide of the HLA-A and / or HLA-B gene under the conditions of PCR reaction". A sequence in which 1 or 2 nucleotides, preferably 1 nucleotide, excluding the nucleotide corresponding to the target nucleotide is substituted, deleted, inserted or added in the nucleotide sequence represented by each of the above SEQ ID NOs, provided that it is possible. It also means to include. Preferably, the probe set used in the present invention is an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12 (normal type) and an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 13 (variant type). ..

A labeling substance can be attached to each probe. Examples of the labeling substance include fluorescent dyes and the like. Various such fluorescent dyes are commercially available, for example, 6-FAM (fluorescein), HEX, TE, Quasar 670, Quasar 570, Quasar 705, Pulsar 650, TET, HEX, VIC, JOE, CAL Fluor Orenge. , CAL Fluor Gold, CAL Fluor Red, Texas Red, Cy, Cy5
And so on. It is preferable that the fluorescent dye bound to the normal probe and the fluorescent dye bound to the mutant probe are different. By adopting this configuration, an HLA fragment having a normal target nucleotide (c.19C) has a normal probe, and an HLA having a target nucleotide (c.19C> T in the case of a nonsense mutation) that causes a loss-of-function mutation. By hybridizing each mutant probe to the fragment, two types of fluorescence derived from the fluorescent dye of each probe are detected.

It is preferable that each probe is further bound with a quencher capable of quenching the fluorescence from the fluorescent substance. The quencher is not particularly limited as long as it can quench the fluorescence from the fluorescent dye, and may be a fluorescent dye or a non-fluorescent dye, but a non-fluorescent dye is preferable from the viewpoint of detection accuracy. There can be. Specific quenchers include, for example, 6-carboxytetramethylrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), Eclipse Dark Quencher, Iowa black FQ (IBFQ), minor groove binder (MGB), non- Fluorescent quencher (NFQ) and the like.

Examples of the building blocks of the probe include ribonucleotides and deoxyribonucleotides. The nucleotide residue contains sugars, bases and phosphoric acid as components. Ribonucleotides have a ribose residue as a sugar and bases adenine (A), guanine (G), cytosine (C) and uracil (U) (which can also be replaced with thymine (T)). Deoxyribonucleotide residues have deoxyribose residues as sugars and bases such as adenine (dA), guanine (dG), cytosine (dC) and thymine (dT) (which can also be replaced with uracil (dU)). Have.

These nucleotides may be modified (modified nucleotide residues may be referred to as "modified nucleotide residues") or unmodified (unmodified nucleotide residues are referred to as "unmodified nucleotides". Residues). The inclusion of modified nucleotide residues can improve nuclease resistance and thus stability. When having a modified nucleotide residue, the position and proportion of the modified nucleotide residue in the probe are not particularly limited. In addition, it may contain a linker region composed of an unmodified nucleotide residue or a molecule different from the modified nucleotide residue.

The modified nucleotide residue may be modified, for example, by any of the components of the unmodified nucleotide residue. In the present invention, "modification" includes, for example, substitution, addition and / or deletion of the component, substitution, addition and / or deletion of an atom and / or functional group in the component. Examples of the modified nucleotide residue include naturally occurring nucleotide residues, artificially modified nucleotide residues, and the like. As the naturally occurring modified nucleotide residue, for example, Limbach et al. (Limbach et al., 1994, Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183 to 2196) can be referred to. In addition, examples of the modified nucleotide residue include residues that are substitutes for nucleotides.

Modifications of the nucleotide residues include, for example, modification of a sugar-phosphate skeleton (the skeleton also includes a base) (hereinafter, sugar phosphate skeleton). In the sugar phosphate skeleton, when the sugar is ribose, for example, a ribose residue can be modified. The ribose residue can, for example, modify the 2'-carbon, and specifically, for example, the hydroxyl group bonded to the 2'-carbon can be modified with a methyl group, or the hydroxyl group can be replaced with a halogen such as hydrogen or fluoro. .. Further, by substituting the hydroxyl group of the 2'-carbon with hydrogen, the ribose residue can be replaced with deoxyribose. The ribose residue can be replaced with, for example, a stereoisomer, and may be replaced with, for example, an arabinose residue. Hereinafter, the nucleic acid in which the hydroxyl group bonded to the 2'-carbon of the sugar is modified with a methyl group as described above may be referred to as a 2'-O-methyl modified nucleic acid. Further, in the present invention, "nucleic acid" includes nucleic acid monomers such as nucleotides.

The sugar phosphate skeleton may be replaced, for example, with a non-ribose phosphate skeleton having non-ribose residues (including non-deoxyribose residues) and / or non-phosphate. Is also included in the modification of the sugar phosphate skeleton. Examples of the non-ribose phosphate skeleton include uncharged substances of the sugar phosphate skeleton. Substitutes for the nucleotides substituted with the non-ribos phosphate skeleton include, for example, morpholino, cyclobutyl, pyrrolidine and the like. Other examples of the alternative include artificial nucleic acids. Specific examples include PNA (peptide nucleic acid) and bridged Nucleic Acid (BNA). Examples of BNA include Locked Nucleic Acid (LNA) and 2'-O, 4'-C-Ethylene Bridged Nucleic Acid (ENA: 2'-O, 4'-C-Ethylenebridged Nucleic Acid). However, it is preferably LNA.

In the step (1), the method of bringing the probe set into contact with the DNA sample is not particularly limited, but the probe set and the DNA can be added to the same solution, for example.

In addition, the above solution usually contains a PCR primer set and a DNA polymerase.

(B) PCR primer set The PCR primer set consists of a forward primer and a reverse primer of about 15 to about 30 bases capable of specifically amplifying a partial nucleotide sequence containing a target nucleotide of the HLA-A gene and the HLA-B gene. The HLA gene shows the highest degree of polymorphism among the functional genes, so that the region containing the target nucleotide can be amplified for any HLA type. Based on the sequence information of HLA-A and HLA-B genes of multiple individuals, it is possible to select the region with the lowest polymorphism in the region sandwiching the target nucleotide and design multiple types of primers as needed. desirable.

As the nucleotide constituting the primer set, the same nucleotide as the probe set can be used, or the same modification as the nucleotide constituting the probe may be applied.

In a preferred embodiment of the present invention, the primer set includes a forward primer having a sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2 for the HLA-A gene. A primer set containing each of one or more nucleotide sequences selected from the nucleotide sequences represented by ~ 5 and one or more reverse primers consisting of substantially the same sequence can be mentioned. Further, for the HLA-B gene, one or more forward primers having substantially the same sequence as each of the one or more nucleotide sequences selected from the nucleotide sequences represented by SEQ ID NOs: 6 to 8 and one or more forward primers. A primer set comprising each of one or more nucleotide sequences selected from the nucleotide sequences represented by SEQ ID NOs: 9 to 11 and one or more reverse primers having substantially the same sequence can be mentioned.

Here, "substantially identical sequence" means that not only the completely identical sequence but also "each target sequence of HLA-A and / or HLA-B gene can be hybridized under the condition of PCR reaction". As a condition, it means that in the nucleotide sequence represented by each of the above SEQ ID NOs, 1 or 2 nucleotides, preferably 1 nucleotide is substituted, deleted, inserted or added. Preferably, the primer set used in the present invention is represented by SEQ ID NOs: 2 to 5 and a forward primer consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and / or 14 for the HLA-A gene. A primer set comprising one or more reverse primers consisting of each of one or more nucleotide sequences selected from the nucleotide sequences. Further, for the HLA-B gene, one or more forward primers consisting of each of one or more nucleotide sequences selected from the nucleotide sequences represented by SEQ ID NOs: 6 to 8 and SEQ ID NOs: 9 to 11 are shown. A primer set comprising one or more reverse primers consisting of each of one or more nucleotide sequences selected from the nucleotide sequences to be obtained.

(C) DNA polymerase The DNA polymerase is not particularly limited as long as it is a DNA polymerase usually used in the PCR method.

(2) Step of Amplifying the Region Containing the Target Nucleotide by PCR In the step (2), the method of amplifying the region containing the target nucleotide by PCR is not particularly limited, but it is preferably amplified by the digital PCR method. Digital PCR is performed, for example, by the following procedure. A reaction solution containing a probe set, a DNA sample, a PCR primer set and a DNA polymerase is set in a digital PCR device. Here, the mixing ratio of each reaction solution component can be appropriately selected and optimized within a range known per se. For example, the mixing ratio shown in Examples described later may be used, but it can be appropriately changed depending on the primer set, probe set, and the like to be used. However, in patients with bone marrow failure syndrome, a sufficient amount of DNA may not be obtained even with whole blood, so the amount of DNA sample is preferably 50 to 200 ng, preferably 80 to 150 ng per reaction solution.
The digital PCR apparatus distributes the reaction solution to a large number of reaction wells, thousands to tens of thousands, provided on the chip. The reaction well is, for example, a minute well having an opening size of several tens of μm or a nanoliter-sized droplet. When the reaction mix is added to the chip, some reaction wells on the chip preferably contain about one copy of the HLA-A or HLA-B allele, and the other reaction wells do not contain the allele. The reaction mix is distributed to the reaction wells. When PCR is performed in a reaction mix distributed to multiple reaction wells, a region of the allele is amplified in the reaction well where the desired HLA allele is present, due to the nuclease activity of DNA polymerase. The probe bound to the target region is cleaved, the fluorescent dye dissociates, and fluorescence is emitted.

(3) Step of Measuring Fluorescence Intensity of Solution Containing PCR Product The fluorescence intensity in the step (3) can be measured, for example, by using a fluorescence reader known per se. When the PCR is performed by digital PCR, the fluorescence intensity can be measured by, for example, measuring the number of reaction wells in which fluorescence is detected by a reader of a digital PCR device. When a plurality of probes having different fluorescent dyes are used, the plurality of fluorescence intensities can be measured at once by detecting the fluorescence derived from each fluorescence.

(4) Step of detecting loss-of-function mutation in target nucleotide In the step (4), based on the measurement result of the step (3), the loss-of-function mutation in the target nucleotide of the HLA-A and / or HLA-B gene Can be detected. That is, from the above measurement results, the number of copies of normal DNA and the number of copies of mutant DNA are obtained for each gene, and the number of copies of mutant DNA is divided by the sum of the number of copies of normal DNA and the number of copies of mutant DNA. , The allergen frequency of mutation can be calculated.

If the allele frequency of the resulting mutation is higher than the cutoff value, the subject can be determined to be HLA-LL positive. Here, for the cutoff value, for example, a PCR reaction is carried out in the same manner as above using DNA derived from blood collected from a certain number or more of healthy subjects, and the allergen frequency (false positive rate) of mutation is calculated. It can be appropriately determined by a value sufficiently higher than the obtained median or mean value (for example, + 3SD) and higher than the detection limit of the PCR reaction. The cutoff value depends on the amount of DNA used for the measurement, but when the amount of DNA is 100 ng per reaction solution as in the examples described later, the false positive rate in healthy subjects is 0 to 0.042% (median 0.009%). Therefore, the cutoff value was set to 0.07% together with the result of the sensitivity test.

When HLA-LL positive is determined by the detection method of the present invention, it can be determined that the subject is likely to develop bone marrow failure due to damage to hematopoietic stem cells by an immunological mechanism. Therefore, it can be predicted that immunosuppressive therapy, such as administration of anti-thymocyte immunoglobulin (ATG) and / or cyclosporine (CsA), will be effective for the subject.
From the above viewpoint, the present invention also implements the detection method of the present invention, and as a result, administers immunosuppressive therapy, for example, ATG and / or CsA, to a subject determined to be HLA-LL positive. Provided are methods for diagnosing and treating immune pathologies in bone marrow failure, including the above.

The detection method of the present invention can be used in combination with the PNH type blood cell test. As described above, PNH-type blood cells cannot be accurately detected at all facilities, and even if PNH-type blood cells are negative, the immune pathology cannot always be ruled out. Therefore, it is particularly desirable to carry out the detection method of the present invention for a subject who is negative in the PNH type blood cell test. When the detection method of the present invention is determined to be HLA-LL positive, it is considered that bone marrow failure is caused by damage to hematopoietic stem cells by an immunological mechanism even if PNH type blood cells are negative. Therefore, immunosuppressive therapy can be predicted to be effective in these cases. The PNH type blood cell test can be performed by, for example, the method described in Sugimori et al., Blood, 107: 1308-1314 (2006), Japanese Patent Application Laid-Open No. 2012-122954, but is not limited thereto.

[II] Kit of the present invention The present invention also provides a kit for detecting HLA class I allele-deficient blood cells in a subject whose HLA type is unknown (hereinafter, also referred to as “kit of the present invention”). The kit is suitable for carrying out the above-mentioned detection method of the present invention, and includes the following primer set (a) and the following probe sets (b1) and (b2) as a configuration.
(A) Primer set capable of amplifying a partial nucleotide sequence of the gene, which contains the 19th nucleotide of the coding sequence of the HLA-A and / or HLA-B gene (b1) Amplified by the primer set of (a) above. A probe in which the 19th nucleotide of the PCR product can specifically hybridize to a normal one (b2) Of the PCR products amplified by the primer set of (a) above, the 19th nucleotide loses its function. A probe that can specifically hybridize to a mutated one that causes a type mutation

In a preferred embodiment, the loss-of-function mutation at the 19th nucleotide of the coding sequence of the HLA-A and / or HLA-B gene is a nonsense or frameshift mutation, more preferably a nonsense mutation.

It is desirable that the primer set of (a) is capable of amplifying the region containing the 19th nucleotide of the HLA-A and / or HLA-B gene from DNA derived from a subject having an arbitrary HLA type. .. Since the HLA gene exhibits the highest degree of polymorphism among functional genes, in order to be able to amplify the region containing the target nucleotide for any HLA type, the primer sequence should be placed in the region with the lowest possible polymorphism. It is desirable to set.

In one preferred embodiment, as the primer set of (a) above,
(A1) The primer set capable of amplifying the partial nucleotide sequence of the HLA-A gene is represented by a forward primer having a sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NOs: 2-5. Each of one or more nucleotide sequences selected from the above nucleotide sequences contains one or more reverse primers consisting of substantially the same sequence, and / or (a2) a partial nucleotide sequence of the HLA-B gene can be amplified. The primer set is represented by SEQ ID NOs: 9 to 11 and one or more forward primers having a sequence substantially the same as that of each of the one or more nucleotide sequences selected from the nucleotide sequences represented by SEQ ID NOs: 6 to 8. Examples thereof include one containing one or more reverse primers consisting of substantially the same sequence as each of one or more nucleotide sequences selected from the nucleotide sequences to be prepared.
Here, "substantially the same sequence" is synonymous with the case of "(b) PCR primer set" in the detection method of the present invention.

More preferably, the primer set of (a) is
(A1) The primer set capable of amplifying the partial nucleotide sequence of the HLA-A gene is a forward primer consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and / or 14, and the nucleotide sequence represented by SEQ ID NOs: 2-5. A primer set comprising one or more reverse primers consisting of each of one or more nucleotide sequences selected from and / or capable of amplifying a partial nucleotide sequence of the (a2) HLA-B gene is available in SEQ ID NOs: 6-8. It consists of one or more forward primers consisting of each of one or more nucleotide sequences selected from the represented nucleotide sequences, and one or more nucleotide sequences selected from the nucleotide sequences represented by SEQ ID NOs: 9 to 11. It contains one or more reverse primers.

Particularly preferably, the primer set of (a) is
(A1) The primer set capable of amplifying the partial nucleotide sequence of the HLA-A gene includes a forward primer consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 14 (preferably SEQ ID NO: 14) and SEQ ID NOs: 2-5. A primer set containing four reverse primers consisting of each of the nucleotide sequences represented by and / or capable of amplifying a partial nucleotide sequence of the (a2) HLA-B gene is represented by SEQ ID NOs: 6-8. It contains three types of forward primers consisting of each of the nucleotide sequences and three types of reverse primers consisting of each of the nucleotide sequences represented by SEQ ID NOs: 9 to 11.
In (a1), when a forward primer consisting of the nucleotide sequence represented by SEQ ID NO: 14 is used, the amplification efficiency can be further improved and the separation ability can be improved, although it depends on the type of other PCR reagents.

Further, when the loss-of-function mutation at the 19th nucleotide of the coding sequence of the HLA-A and / or HLA-B gene is a nonsense mutation, it is preferable.
The probe of (b1) has a sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 12.
The probe (b2) has a sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 13.
Here, "from substantially the same sequence" is synonymous with the case of "(a) probe set" in the detection method of the present invention.

More preferably, the probe set is
The probe of (b1) is composed of the nucleotide sequence represented by SEQ ID NO: 12.
The probe of (b2) is composed of the nucleotide sequence represented by SEQ ID NO: 13.

In addition to the primer set of (a) and the probe set of the normal probe of (b1) and the mutant probe of (b2), the kit of the present invention comprises PCR in the detection method of the present invention. Includes reagents used in the reaction, such as DNA extraction reagents, DNA polymerase, dNTPs, reaction buffers, nucleic acids containing target regions that are positive controls for PCR, and containers, instruments, instructions, and the like. Can be done. In addition, the above-mentioned primer set or probe set may be included in the kit as a probe set and / or a primer set in which each primer or each probe is stored in a coexisting state so as not to adversely affect the reaction. it can.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Reference Example 1 Identification of genetic abnormalities common to HLA class I allele-deficient blood cell (HLA-LL) -positive patients
The HLA class I gene was analyzed using a next-generation sequencer (model: Miseq; illumina) in 22 HLA class I allele-deficient blood cell-positive patients. As a result, 1-3 types of loss-of-function mutations (median 1 type) were detected in 12 of 22 cases.

More interestingly, most of the loss-of-function mutations detected are concentrated on the 19th nucleotide (c.19C) of the coding sequence in exon 1 of the HLA-A and HLA-B genes, regardless of the HLA class I allele. It became clear that this was the case (Fig. 1). Therefore, it was suggested that this common mutation could be a useful marker for assessing the presence or absence of HLA-LL without typing HLA.

Example 1 Detection of HLA-LL using a common mutation of HLA class I gene as an index by ddPCR method Therefore, 303 patients with aplastic anemia and healthy subjects were used by ddPCR method to detect this common mutation with high sensitivity. The allelic frequency of the common mutation (nonsense mutation (c.19C> T)) identified in Reference Example 1 was measured for DNA samples extracted from whole blood collected from 24 subjects.
To cover all HLA class I gene polymorphisms, one FW primer (SEQ ID NO: 1) and four RV primers (SEQ ID NOs: 2-5) were designed for HLA-A. For HLA-B, 3 types of FW primers (SEQ ID NOs: 6 to 8) and 3 types of RV primers (SEQ ID NOs: 9 to 11) were designed. The FW and RV primers were mixed so that the final concentration was 500 nM. One probe was designed for each of the mutant sequence (SEQ ID NO: 13) and the normal sequence (SEQ ID NO: 12), and in order to increase the sensitivity, a probe in which LNA (Locked Nucleic Acids) was incorporated into a normal Taqman probe was designed. .. Each probe was adjusted to a final concentration of 250 nM. The normal probe was labeled with HEX and the mutant probe was labeled with FAM. Iowa black FQ (IDT) was used as a quencher on the 3'end side of the probe sequence.

Each reagent was mixed at the ratio shown in Table 3, the reaction solution was set up, and the reaction solution was set in the QX200 Droplet Generator (Bio-Rad) to prepare a Droplet.

After preparing the Droplet, a PCR reaction was carried out using a thermal cycler under the conditions shown in Table 4, and after the PCR was completed, the measurement was performed with a QX200 Droplet reader (Bio-Rad) and analyzed with Quantasoft software (Bio-Rad). An example of the result is shown in FIG. 2 (A). The mutation allele frequency was calculated by dividing the copy number of the mutant DNA of Mt by the copy number of the normal DNA of Wt plus the copy number of the mutant DNA.

Since patients with aplastic anemia may not be able to obtain a sufficient amount of DNA even with whole blood, the amount of DNA used in this protocol was set to a small amount of 100 ng. Since the median allele frequency of the common mutation in 24 healthy subjects was 0.009%, the cutoff value was set to 0.07% together with the result of the sensitivity determined by the dilution series (Fig. 2 (B)). ..

FIG. 3 shows an example of actual measurement results. The above cases were positive, and the allelic frequency of common mutations was calculated to be 1.28%. The case below was a negative case and was judged negative because the allele frequency was 0.01%, which was less than the cutoff value. When common mutations were detected in 303 cases using this ddPCR method, 99 of 303 cases (32.7%) were positive, and the median mutant clone size was 0.60% (range, 0.074-23.1%). ..

By combining the PNH type blood cell test and the common nonsense mutation test using ddPCR, as shown in FIG. 4, it has become possible to detect the immune pathology in about 4/5 cases. A particularly important finding is that this assay using ddPCR detected a common nonsense mutation in 30 of 92 cases (32.6%) of "PNH type blood cell negative", which was previously difficult to diagnose. is there.

Example 2 Detection of HLA-LL using a common mutation of HLA class I gene as an index by ddPCR method (2)
DNA samples extracted from whole blood collected from 353 patients with aplastic anemia and 24 healthy subjects, including 303 patients examined in Example 1, were carried out except that some primers, reagents, and PCR conditions were changed. The allele frequency of the common mutation (nonsense mutation (c.19C> T)) was measured in the same manner as in Example 1.
For HLA-A, 1 type of FW primer (SEQ ID NO: 14) and 4 types of RV primer (SEQ ID NO: 2-5) were designed. For HLA-B, 3 types of FW primers (SEQ ID NOs: 6 to 8) and 3 types of RV primers (SEQ ID NOs: 9 to 11) were designed. The FW and RV primers were mixed so that the final concentration was 500 nM. One probe was designed for each of the mutant sequence (SEQ ID NO: 13) and the normal sequence (SEQ ID NO: 12), and in order to increase the sensitivity, a probe in which LNA (Locked Nucleic Acids) was incorporated into a normal Taqman probe was designed. .. Each probe was adjusted to a final concentration of 250 nM. The normal probe was labeled with HEX and the mutant probe was labeled with FAM. Iowa black FQ (IDT) was used as a quencher on the 3'end side of the probe sequence.

Each reagent was mixed at the ratio shown in Table 6, the reaction solution was set up, and the reaction solution was set in the QX200 Droplet Generator (Bio-Rad) to prepare a Droplet.

After preparing the Droplet, a PCR reaction was carried out using a thermal cycler under the conditions shown in Table 7, and after the PCR was completed, the measurement was performed with a QX200 Droplet reader (Bio-Rad) and analyzed with Quantasoft software (Bio-Rad).

The mutation allele frequency was calculated by dividing the copy number of the mutant DNA of Mt by the copy number of the normal DNA of Wt and the copy number of the mutant DNA, as in Example 1. The cutoff value was also set to 0.07% as in Example 1.

FIG. 5 shows a comparison of the measurement results of Example 1 and Example 2 for the same case. The upper part is the measurement result of Example 1, and the lower part is the measurement result of Example 2. By changing the forward primer and the PCR reagent, the amplification efficiency is improved as compared with the case of Example 1, the reaction time of ddPCR can be shortened by 1 hour or more, and the resolution (particularly Wt dots) is also remarkable. Improved. When common mutations were detected in 353 patients with aplastic anemia using this ddPCR method, 101 of the 353 patients (28.6%) were positive, and the median mutation clone size was 0.42% (range, 0.071-23.1). %)Met.

The result of combining the PNH type blood cell test and the common nonsense mutation test using ddPCR is shown in FIG. Similar to Example 1, the immune pathology could be detected in about 4/5 cases. In addition, a common nonsense mutation was detected in 34 of 108 cases (31.5%) who were "PNH type blood cell negative".

In the present invention, an assay system capable of finding a mutation at a specific site of the HLA-A and / or HLA-B gene common to HLA-LL-positive patients regardless of the HLA class I allele and detecting the common mutation with high sensitivity. Was established. According to this method, since HLA typing is not required, the presence or absence of HLA-LL can be clarified within one day at a low cost of about 2000 yen per case. As a result, it is possible to quickly and easily distinguish whether or not an immune condition is involved in bone marrow failure syndrome such as aplastic anemia and myelodysplastic syndrome, which is extremely useful in the treatment of bone marrow failure syndrome. is there.

Claims (10)

HLA class I allele deficiency, including the step of detecting a loss-of-function mutation in the 19th nucleotide of the coding sequence of the HLA-A and / or HLA-B gene in blood-derived DNA collected from subjects of unknown HLA type. How to detect blood loss cells. The method of claim 1, wherein the loss-of-function mutation is a nonsense mutation. The method according to claim 1 or 2, wherein a loss-of-function mutation is detected using a digital PCR method. The method according to any one of claims 1 to 3, wherein the subject is a patient with or suspected of having bone marrow failure syndrome. The method of claim 4, wherein bone marrow failure is caused by a disease selected from the group consisting of aplastic anemia, myelodysplastic syndrome and paroxysmal nocturnal hemoglobinuria. The method according to any one of claims 1 to 5, which is used in combination with the detection of PNH type blood cells. A kit for detecting HLA class I allele-deficient blood cells in subjects of unknown HLA type.
(A) A primer set that can amplify a partial nucleotide sequence of an HLA-A and / or HLA-B gene, including the 19th nucleotide of the coding sequence.
(B1) Among the PCR products amplified by the primer set of (a) above, a probe capable of specifically hybridizing the 19th nucleotide to a normal one, and (b2) the primer set of (a) above. A kit comprising a probe capable of specifically hybridizing to an amplified PCR product in which the 19th nucleotide is mutated to cause a loss-of-function mutation. The kit according to claim 7, wherein the loss-of-function mutation is a nonsense mutation. (A1) The primer set capable of amplifying the partial nucleotide sequence of the HLA-A gene is represented by a forward primer having a sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NOs: 2-5. Each of one or more nucleotide sequences selected from the above nucleotide sequences contains one or more reverse primers consisting of substantially the same sequence, and / or (a2) a partial nucleotide sequence of the HLA-B gene can be amplified. The primer set is represented by SEQ ID NOs: 9 to 11 and one or more forward primers having a sequence substantially the same as that of each of the one or more nucleotide sequences selected from the nucleotide sequences represented by SEQ ID NOs: 6 to 8. The kit according to claim 7 or 8, which comprises one or more reverse primers consisting of substantially the same sequence as each of one or more nucleotide sequences selected from the nucleotide sequences to be obtained. The probe of (b1) has substantially the same sequence as the nucleotide sequence represented by SEQ ID NO: 12, and the probe of (b2) has substantially the same sequence as the nucleotide sequence represented by SEQ ID NO: 13. The kit according to claim 8 or 9, which comprises an array.