JP6164683B2 - Method for analyzing deletion site of spermatogenesis region on Y chromosome and Y chromosome - Google Patents

Description

  The present invention relates to a test for male infertility by a method for analyzing a deletion site of a Y chromosome and a spermatogenic region on the Y chromosome.

  10% of couples who want to have a baby have infertility, half of which are attributed to men. A test considered to be necessary for male infertility is a test for a deletion site in the spermatogenic region (AZF) of the Y-chromosome long arm (Azospermia Factor: AZF).

  The genomic region involved in spermatogenesis is present on the Y chromosome long arm. In 1976, Tiepolo et al. Named AZF the spermatogenesis candidate region at the distal part of the long arm of the Y chromosome (Non-patent Document 1). 1996 Vogt et al. Revealed that microdeletions were concentrated in three regions as a result of comparison of microdeletions on the Y-chromosome long arm and testicular tissue phenotype in patients with azoospermia. AZFb and AZFc, testicular tissue type of AZFa-deficient is Sertoli cell only syndrome (SCO), AZFb-deficient is maturationarrest, and AZFc-deficient is reported to show various phenotypes Patent Document 2). Currently, deletion classification based on AZFa, AZFb, and AZFc regions is used.

  When the Y chromosome genome was decoded in 2002, it was found that there was a Y chromosome-specific product (amplicon) on the distal side of the Y chromosome and a palindromic structure (palindrome structure). There are eight palindromic structures (P1 to P8) in the Y-chromosome-specific product region, and P1 to P3 are further divided into smaller subamplicons to form a palindromic complex (Non-patent Document 3).

  Chromosome recombination occurs between pairs with homologues. In general, not only recombination occurs between chromosomes, but also homologous recombination frequently occurs within the same chromosome. That is, recombination is also performed within the Y chromosome, which is a haploid chromosome. As a result, it became possible to explain that the microdeletion of the Y chromosome is caused by intrachromosomal homologous recombination (Non-patent Document 4).

(Deletion of AZFa)
In the AZFa region, there are two HERV (human endogenous retrovirus) 15yq1 and HERV15yq2 in tandem repeats, and completely homologous nucleotide sequences ID1 and ID2 exist in pairs in the same direction. It is considered that deletion occurred as a result of homologous recombination in ID1 and ID2. DFFRY and DDX3Y (DBY) are present in this region, and deletion of this region causes azoospermia.

(AZFb, c)
Conventionally, regions classified as AZFb and AZFc include palindromes P1 to P5 (FIGS. 1-2). The palindrome in AZFc forms an even smaller subpalindromic complex (FIGS. 2-3). Repeat sequences in this complex are deleted by homologous recombination and spermatogenesis is impaired.

{Deletion of AZFc (b2 / b4 deletion)}
The deletion of the AZFc region is considered to result from the deletion of BPY2, DAZ, and CDY2 as a result of homologous recombination due to the homology of subamplicons b2 and b4 (99.9% or more) in FIG.

(Gr / gr deletion)
Deletions due to recombination between palindrome (g1, g2), (r1, r3), (r2, r4) can be considered (FIG. 2). The genes DAZ1 / DAZ2 and DAZ3 / DAZ4 exist in r1, r2, r3, and r4, respectively. The former two and the latter two are a set. In this deletion, all DAZs are not deleted, so it is considered that spermatogenic ability exists. Thus, the gr / gr deletion is believed to have no deleted gene and cause no clinical impairment despite a large deletion of about 1.5 Mb. Incidentally, the gr / gr deletion in Japan is a DAZ1 / DAZ2 deletion and is considered to exist in almost 30% of all men (Jomon) and does not affect fertility (Non-patent Document 5). .

  Deletion regions that have been elucidated after the entire genome has been elucidated theoretically have a difference from the previously considered deletion regions. In addition, many of the markers conventionally used as markers for Y gene deletion are on the repeat sequence, and do not specifically reflect the position on the chromosome.

  6% of patients with azoospermia are AZF-deficient patients. Infertile couples due to azoospermia undergo sperm collection (TESE) and intracytoplasmic sperm injection (ICSI). However, depending on the deletion site of AZF deletion, TSE may not be indicated. That is, AZFa deletion, AZFb deletion, and AZFb + c deletion cannot obtain a therapeutic effect even by TSE. In order to avoid unnecessary physical burdens and medical expenses, analysis of the deletion site of AZF is indispensable, and it is necessary to accurately diagnose the deletion site in a timely manner.

  In Patent Document 1, a polynucleotide obtained by amplifying a specific locus of the Y chromosome from a patient sample is subjected to agarose or polyacrylamide gel electrophoresis, and a corresponding polynucleotide obtained by amplification from a normal male sample. Compare with At the time of filing these patents, the palindromic structure was not elucidated, and the deletion site was not elucidated accurately, making accurate analysis difficult. In addition, the method described in the patent document requires a complicated electrophoresis operation and takes a long time.

Japanese translation of PCT publication No. 2002-510962

Tiepolo, L.M. , Hum. Genet. , 34: 119, 1976 Vogt, P.A. H. , Hum. Mol. Genet. , 5: 933, 1996 T. Koei et al., Hormone and Clinical 57 (1) 59-65 2009 Kuroda-kawaguchi et al., Nat Genet 2001; 29: 279-286. Sin HS et.al., Hum Reprod. 2010 Sep; 25 (9): 2396-403.

  It is an object of the present invention to provide an analysis method that enables simple, rapid, and accurate examination of male infertility by analyzing and specifying a deletion site of AZF.

In order to solve the above-mentioned problems, the present invention provides a method for newly analyzing a deletion pattern of AZF. That is, this invention consists of the following.
1. A method for analyzing a deletion site in a Y chromosome and / or a spermatogenesis region on a Y chromosome, wherein AZFc deletion is analyzed by sy2858 deletion analysis.
2. The analysis method according to item 1, further comprising a deletion analysis of one or more STS selected from sy1191, sy1307, and sy1206.
3. Y chromosome and / or Y chromosomal spermatogenesis region characterized by analyzing AZFb deletion by deletion analysis of one or more STS selected from sy1024, sy1967, sy1309, sy3199, sy1233, sy3010, sy2990 and sy1197 Method for analyzing the deletion site of.
4). The chromosomal formation on the Y chromosome and / or on the Y chromosome characterized by analyzing AZFb deletion, AZFc deletion and / or AZFb + c deletion by combining the analysis method of the preceding item 1 or the analysis method of the preceding item 2 Method for analyzing region deletion sites.
5. A method for analyzing a deletion site of a Y chromosome and / or a spermatogenesis region on a Y chromosome, wherein AZFa deletion is analyzed by deletion analysis of one or more STS selected from sy1324, sy1316, and sy1714.
6). Y chromosome and / or Y chromosome characterized by analyzing AZFa deletion, AZFb deletion, AZFb deletion and / or AZFb + c deletion by combining any two or more analysis methods of the preceding items 1-3, 5 A method for analyzing a deletion site in the upper spermatogenesis region.
7). 7. The analysis method according to item 6 above, further comprising deletion analysis of one or more STS selected from sy14, sy3118 and sy1251.
8). A method for analyzing a deletion site of a Y chromosome and / or a spermatogenesis region on a Y chromosome, wherein the deletion of one or more STS selected from the group consisting of the following (1) to (19) is analyzed.
(1) sY14
(2) sY3118
(3) sY1251
(4) sY1324
(5) sY1316
(6) sY1714
(7) sY1024
(8) sY1967
(9) sY1309
(10) sY3199
(11) sY1233
(12) sy3010
(13) sy2990
(14) sy1197
(15) sy1191
(16) sy1307
(17) sy1206
(18) sy2858
(19) sy3159
9. 9. The analysis method according to item 8 above, wherein deletion of all STSs selected from the group consisting of (1) to (19) is analyzed.
10. A method for analyzing a deletion site of a Y chromosome and / or a spermatogenesis region on a Y chromosome, wherein the deletion of one or more STSs in each of the following groups I to VI is analyzed.
Group I: (1) sY14, (2) sY3118, (3) sY1251
Group II: (4) sY1324, (5) sY1316, (6) sY1714
Group III: (7) sY1024, (8) sY1967, (9) sY1309, (10) sY3199, (11) sY1233, (12) sy3010, (13) sy2990, (14) sy1197
Group IV: (15) sy 1191, (16) sy 1307, (17) sy 1206
Group V: (18) sy2858
Group VI: (19) sy3159

  The analysis method of the present invention enabled efficient and comprehensive analysis of AZF deletion sites. Further, by analyzing not only the conventional AZF but also Y chromosome abnormality, Y chromosome microdeletion (subclassification) and Y chromosome polymorphism by one analysis, spermatogenic ability can be evaluated.

  As described above, the analysis method of the present invention enables the simultaneous analysis of the presence or absence of AZF deletion sites and a plurality of deletion sites, enables simple, rapid and accurate testing, and provides the correct test results to many patients. Can be provided. Thereby, unnecessary operations such as TSE can be avoided, and appropriate treatment can be provided to the patient.

It is a figure showing the genome structure of Y chromosome, and the position of AZF. It is a figure which shows the deletion site | part based on the palindrome complex containing a sub palindrome, and a palindrome. Arrangement of STS in AZFb, c region. It is a Y chromosome map including the position of STS (Source: http://breakpointmapper.wi.mit.edu/mapper.html). Analysis result of microdeletion of Y chromosome (main classification) (“1” in the figure means no deletion, “0” means that there is a deletion) Analysis result of Y chromosome abnormality Analysis result of Y chromosome microdeletion (subclassification) Y chromosome polymorphism analysis results

(Method for analyzing AZF deletion site of the present invention)
The present invention is characterized by analyzing STS (sequence tag site: a region of DNA on a chromosome used as a guideline for a molecular gene mapping approach) deletion described in Table 1 below. The present invention relates to a method for analyzing a deletion site of AZF. The analysis method of the present invention will be described in detail below.

Sy757 (number 0) in Table 1 above is an STS on the X chromosome and can be used as a positive control in the analysis method of the present invention.
The deletion pattern of sy1291 (No. 20) in Table 1 is different depending on races. Therefore, it adds to STS of the detection object of the analysis method of this invention as needed (refer: nonpatent literature 5).

{Sperm formation region (AZF)}
The spermatogenic region (AZF) in the present invention is a genomic region involved in spermatogenesis that exists on the long arm of the Y chromosome, and is classified into AZFa, AZFb, and AZFc based on the position and phenotype on the genome (see: 1-3).

(Deletion site of AZF)
The AZF deletion site in the present invention is a partial deletion region on AZF. Y chromosome microdeletion of AZF on Y chromosome (main classification), Y chromosome abnormality affects spermatogenesis, for example, AZFa deletion, AZFb deletion and AZFb + c deletion are not compatible with TSE.

{Y chromosome microdeletion (main classification)}
In the analysis of AZF Y-chromosome microdeletion (main classification), the presence or absence of AZFa deletion, AZFb (P5 / proximal P1) deletion, AZFb + c deletion (P5 / distal P1) and AZFc deletion (b2 / b4) Analyze (Ref: Figure 5).
Doctors, researchers, and their assistants can determine the presence or absence of spermatogenic potential and the level of spermatogenic potential based on the analysis results of the main classification of AZF.

(Y chromosome abnormality)
In the analysis of Y-chromosome abnormality, Y-chromosome deletion, Y-chromosome long-arm deletion, Y-chromosome long-arm partial deletion I (distant from AZFa), Y-chromosome long-arm partial deletion II (distant from AZFb), Y-chromosome long arm Analysis of the presence of deletion III (distant from AZFb), Y-chromosome long arm deletion IV (distant from AZFb), Y-chromosome long arm partial deletion V (distant from AZFb) and Y-chromosome long arm partial deletion VI (distant from AZFb) (Ref: FIG. 6).
The Y chromosome abnormality corresponds to the deletion of the main classification. For example, the deletion beyond AZFb is the same as the AZFb + c deletion because the deletion includes AZFb containing AZFb and the heterochromatin of the Y-chromosome long arm.
Thus, by combining the analysis result of the Y chromosome abnormality with the analysis result of the main classification of AZF, an exhaustive and detailed deletion site can be detected easily and with high accuracy.

{Y chromosome microdeletion (subclassification)}
In the analysis of Y chromosome microdeletion (subclassification), Ym-1 (P5 + P4) deletion, Ym-2 (P5 + P4) deletion, Ym-3 AZFb partial deletion, Ym -4 AZFb partial deletion, Ym-5 (P3 deletion) deletion, Ym-6 (P1 + P2 + P3 deletion) deletion, Ym-7 (P1 + P2 + P3 deletion) deletion, Ym-8 (b1 / b3 deletion) Deletion, Ym-8 (P3 deletion) deletion, Ym-9 (P3 deletion) deletion, Ym-10 (b2 / b3 deletion) deletion, Ym-11 (gr / gr deletion) deletion And Ym-12 (P1 deletion) is analyzed for the presence or absence of deletion (see: FIG. 7).
By combining the analysis results of the Y chromosome microdeletion (subclassification) with the analysis results of the main classification of AZF and the Y chromosome abnormality analysis results, comprehensive and detailed deletion sites can be easily and more accurately detected. be able to. In addition, the above subclassification, clinical phenotype has not been established yet.

(Y chromosome polymorphism analysis)
Y chromosome polymorphism analysis (see: FIG. 8) detects Y chromosome polymorphism due to a single nucleotide deletion that is included in any of the subject STSs and does not affect the clinical phenotype, and Y chromosome microdeletion By combining with the analysis result of (main classification) and the Y chromosome abnormality analysis result, an exhaustive and detailed analysis result can be reflected.

(Analysis method)
In the method for analyzing a spermatogenic region on the Y chromosome according to the present invention, the STS of the AZF region present on the genomic DNA in the sample (see: Table 1 above) is amplified using amplification primers, and amplified. Get the product. Further, the amplification product is detected using a probe capable of detecting the deletion site of STS. Each probe can hybridize with the corresponding STS on AZF, and can detect the corresponding STS on the genome in the sample.
In the analysis method of the present invention, for example, amplification is performed from patient genomic DNA using a plurality of primers, but if there is a deletion, it is not amplified, so that it is not hybridized and the deletion site is not detected.

In the method for analyzing the spermatogenic region on the Y chromosome of the present invention, in order to detect the most detected AZFc deletion (b2 / b4) from the result of the microdeletion of the Y chromosome (main classification) in Example 1 below. The deletion of sy2858 is analyzed.
In addition, one or more STS deletions selected from sy1191, sy1307 and sy1206 are analyzed to detect AZFb + c deletion (P5 / distal P1) and / or AZFc deletion (b2 / b4).
Next, one or more STS deletions selected from sy1024, sy1967, sy1309, sy3199, sy1233, sy3010, sy2990 and sy1197 are detected to detect AZFb deletion and / or AZFb + c deletion (P5 / distal P1). analyse.
Next, one or more STS deletions selected from sy1324, sy1316 and sy1714 are analyzed to detect AZFa deletion.
Next, one or more STS deletions selected from sy14, sy3118, sy1251 are analyzed to detect Y chromosome deletion.

  In a preferred embodiment of the method for analyzing a spermatogenic region on the Y chromosome of the present invention, at least one or more, preferably 10 or more, and more preferably all STS deletions in Table 1 are analyzed.

In addition, in another preferred embodiment of the method for analyzing a spermatogenic region on the Y chromosome of the present invention, one or more STS deletions in each of the following groups I to VI are analyzed.
Group I: (1) sY14, (2) sY3118, (3) sY1251
Group II: (4) sY1324, (5) sY1316, (6) sY1714
Group III: (7) sY1024, (8) sY1967, (9) sY1309, (10) sY3199, (11) sY1233, (12) sy3010, (13) sy2990, (14) sy1197
Group IV: (15) sy 1191, (16) sy 1307, (17) sy 1206
Group V: (18) sy2858
Group VI: (19) sy3159

(Analysis method)
The analysis method of the present invention preferably includes the following steps, but is not particularly limited.
(1) Step of extracting genomic DNA from a sample (2) Amplifying the corresponding STS present on the DNA using the extracted genomic DNA as a template using the STS amplification primers shown in Table 1 above And (3) a step of hybridizing the amplification product and a probe (4) a step of detecting hybridization of the amplification product and the probe

(sample)
The sample in the present invention is a sample obtained from an organism containing genomic DNA, preferably from a mammal, and includes blood, body fluid, tissue extract and the like. Human blood is preferable.

(DNA extraction)
Methods for extracting DNA from a sample are well known in the art, and for example, extraction with phenol / chloroform can be used. Alternatively, a commercially available DNA extraction reagent may be used.

(Amplification of STS on extracted genomic DNA)
Using the extracted genomic DNA as a template, for example, STS described in Table 1 present on the DNA is amplified by multiplex PCR. Preferably, a primer corresponding to Sy757 is also used as a control. A known base sequence can be used as the base sequence of the primer. By using the extracted genomic DNA as a template, even if amplification primers exist in the PCR reaction system, the corresponding STS amplification product is not generated if the extracted genome has a deletion.

  The primer can be obtained by chemical synthesis by a method known per se (for example, using a general-purpose DNA synthesizer). The length of the primer is 10 to 40 mer, preferably 15 to 30 mer. A primer set is prepared by synthesizing a forward primer and a reverse primer.

(Multiplex PCR)
Using the extracted genomic DNA as a template and the selected primer set, STS is amplified using multiplex PCR. For example, Ex Taq ^{(registered trademark)} (Takara Bio) can be used as the polymerase. Methods for selecting appropriate PCR reaction conditions based on the Tm of the primer are well known in the art.

(Adjustment of label amplification product)
In order to facilitate the detection of PCR amplification products, PCR can be performed using labeled nucleotides. As such a labeling substance, a radioisotope, a fluorescent substance, a chemiluminescent substance, digoxigenin (DIG), biotin and the like well known in the art can be used.

(Hybridization and detection)
The labeled amplification product and the probe are hybridized, and then the generated hybrid is detected. In the analysis method of the present invention, if hybridization between the amplification product and the probe is not detected, it indicates that the corresponding AZF site is deleted.
Preferably, the probe is immobilized on the array, and the amplified product and the probe are hybridized by bringing the labeled product into contact with the array, and then the generated hybrid is detected. If no hybridization of the probe on the array is detected, it indicates that the corresponding AZF site is missing.
The probe chemically synthesizes a DNA oligomer probe by a method known per se (for example, using a general-purpose DNA synthesizer).

  Hybridization and detection can be performed by methods well known in the art. For example, after hybridization, the array (membrane) is blocked, and the hybrid of the probe and the amplification product is visualized in blue using alkaline phosphatase-labeled anti-DIG antibody and NBT / BCIP. Then, the color development pattern and color development intensity of the array are observed with the naked eye, and the presence or absence and the deletion site in AZF are analyzed and evaluated.

The analysis method of the present invention preferably uses Luminex Corporation Luminex ^(TM) system. The system can simultaneously detect multiple AZF STS deletions with high sensitivity.

  The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto.

Analyzing the STS of the spermatogenic region on the Y-chromosome derived from males who have been complaining of male infertility, Y chromosome microdeletion (main classification), Y chromosome abnormality, Y chromosome microdeletion (subclassification), and Y chromosome polymorphism The mold was analyzed. Specifically, the deletion site in AZF was analyzed by PCR-Luminex method using PCR and Luminex ^{(registered trademark)} system (Luminex Corp.) using DNA derived from male blood as a chief complaint for male infertility. It was. Details are as follows.

(DNA extraction from blood)
DNA was extracted and collected from blood collected from 2013 males who had a complaint of male infertility using a commercially available kit.

(Preparation of amplification products by multiplex PCR)
PCR reaction solution was 1-5 μL of DNA sample (50-200 ng), 0.2 μM each primer, 200 μM dNTPs, 1 × PCR buffer (20 mM Tris-HCl (pH 8.3), 30 mM KCl, 2.5 mM) MgCl2) and 0.625 U Taq DNA Polymerase (Roche) were prepared as a total volume of 25 μL. Primers corresponding to each STS detection (primer for STS amplification) were designed based on the public database MSY Breakpoint Mapper (http://breakpointmapper.wi.mit.edu/mapper.html) and biotin at the 5 ′ end. The label was used. The PCR program was “final extension at 95 ° C. for 2 minutes, followed by 45 cycles of 95 ° C. for 30 seconds, 61 ° C. for 30 seconds, 72 ° C. for 60 seconds, and 72 ° C. for 5 minutes”. A GeneAmp9700 thermal cycler (Applied Biosystems) was used as the instrument.

(Hybridization reaction between PCR amplification product and probe on fluorescently labeled beads)
The amplified PCR product was subjected to a hybridization reaction with a sequence-specific probe of each STS that was solid-phase bonded to fluorescently labeled beads. The probe was a 15-30mer oligonucleotide containing the base sequence contained in the amplification region of each primer set. Each probe, based on the Luminex ^(TM) technology, was coupled via a carboxyl group coated microbeads stained with various concentrations combined two colors of fluorescent dyes. The microbeads to which the probe was bound and the PCR product were mixed and used for the hybridization reaction in the same tube. The hybridization reaction solution was 5 μL PCR product, 1 × hybridization buffer (75 mM Tris-HCl (pH 8.0), 0.6 mM EDTA (pH 8.0), 0.15% surfactant, 4.5 M tetramethyl chloride). Ammonium) 20 μL, 5 μL Prepared as a total volume of 30 μL containing probe-bound beads. The hybridization reaction was performed by incubating at 95 ° C for 2 minutes and then at 52 ° C for 30 minutes. Subsequently, 50 μL of PBS-Tween (1 × PBS (pH 7.5), 0.01% Tween-20) was added, centrifuged at 2,000 g × 1 min, the supernatant was discarded, and another 50 μL of PBS− The washing operation of adding Tween, centrifuging at 2,000 g × 1 min and discarding the supernatant was performed twice.

(Streptavidin labeled phycoerythrin reaction and measurement)
After the hybridization reaction, streptavidin-labeled phycoerythrin (SA-PE) was added to the reaction solution and incubated at 52 ° C. for 15 minutes for secondary labeling. A GeneAmp9700 thermal cycler (Applied Biosystems) was used as the instrument. The obtained reaction solution was prepared based on the Luminex ^{(registered trademark)} system (Luminex corp.) And the microbead array technology applying the principle of flow cytometry to the SA-PE of the beads to which the probe corresponding to each STS marker was bound. The origin fluorescence value was detected. For the DNA bound to the microbeads, a minimum of 50 beads were measured per measurement, and the quantified median fluorescence intensity (MFI) of PE was used.
An individual cut-off value was set for the median value of each probe-bound bead, and if it was equal to or greater than that value, it was determined that there was no STS deletion, and if it was less than that, it was determined that there was an STS deletion. The individual cut-off values were set with reference to numerical values obtained from healthy male subjects and healthy female samples or negative controls (distilled water).

(Analysis result of STS deletion in spermatogenic region on Y chromosome)
Fig. 5 shows the analysis result of the Y chromosome microdeletion (main classification), Fig. 6 shows the analysis result of the Y chromosome abnormality, Fig. 7 shows the analysis result of the Y chromosome microdeletion (subclass), and Fig. 7 shows the analysis result of the Y chromosome polymorphism. As shown in FIG.
As is clear from the results of FIGS. 5 to 8, it was possible to classify the STS deletion in the spermatogenic region on the Y chromosome of 879 males who had received a chief complaint of all male infertility (37 classifications). In addition, AZF deletion did not exist in 1134 men who visited the hospital with chief complaints of infertility.
As described above, the analysis method of the present invention enables the simultaneous analysis of the presence or absence of STS in the spermatogenesis region on the Y chromosome and a plurality of deletion sites, and enables simple, rapid, and accurate inspection, We were able to provide correct test results to men who complained of male infertility.

  The analysis method of the present invention enables simple, rapid and accurate analysis, provides correct test results to many people who are treating infertility, and provides patients with appropriate treatment.

Claims (3)

A method for analyzing a deletion site of a Y chromosome and / or a spermatogenesis region on a Y chromosome, wherein the deletion of all STSs selected from the group consisting of the following (1) to (19) is analyzed.
(1) sY14
(2) sY3118
(3) sY1251
(4) sY1324
(5) sY1316
(6) sY1714
(7) sY1024
(8) sY1967
(9) sY1309
(10) sY3199
(11) sY1233
(12) sY3010
(13) sY2990
(14) sY1197
(15) sY1191
(16) sY1307
(17) sY1206
(18) sY2858
(19) sY3159
The analysis method according to claim 1, wherein each STS is amplified by multiplex PCR in the analysis method.
The analysis method according to claim 1 or 2, wherein each STS is detected by Luminex ^{(registered trademark)} in the analysis method.