JP7570087B2 - Pathogenic variant-based prostate cancer testing methods - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act 25th Academic Meeting of the Japanese Society of Family Oncology Dates June 14-15, 2020 78th Academic Meeting of the Japanese Cancer Society September 26-28, 2020 JNCI J Natl Cancer Inst (2020) 112 (4): djz124 https://www. ncbi. nlm. nih. gov/pmc/articles/PMC7156928/ June 19, 2020

The present invention relates to a method for testing prostate cancer based on gene sequence analysis. More specifically, the present invention relates to a method for testing prostate cancer using genetic testing for pathogenic variants.

Prostate cancer is the second most common cancer in the world and the fourth most common cancer in Japanese men, and the number of patients is rapidly increasing. Prostate cancer is known to be highly hereditary, and the causes include (1) "single nucleotide polymorphisms (SNPs)" that have been revealed by genome-wide association studies (GWAS) so far and have a small effect on disease onset but are frequent, and (2) "pathological variants" that are very rare but greatly increase the risk of disease onset. As for the former, SNPs in the 15 region of the short arm of chromosome 5 or the IRX4 gene, SNPs in the GPRC6A/RFX6 gene, SNPs in the 22 region of the long arm of chromosome 13, SNPs in the C2orf43 gene, and SNPs in the FOXP4 gene have been reported (Patent Document 1). As for the latter, eight types of causative genes for prostate cancer have been reported, including BRCA1 and BRCA2, which are also known as causative genes for breast cancer (Non-Patent Document 1).

As with breast cancer, it is known that drugs such as PARP inhibitors are effective when prostate cancer patients have pathological variants in BRCA1 or BRCA2. Therefore, if genetic testing reveals that a prostate cancer patient has these pathological variants, it is expected that treatment with PARP inhibitors will be effective, just as it is with breast cancer. Furthermore, it is expected that other cancers contributed by the same genes will be detected early, and that genetic testing of close relatives can be used to prevent the onset of these cancers.

To carry out this type of "genomic medicine," large-scale data on the interpretation of genetic test results, the onset risk of disease in carriers of pathogenic variants, and clinical characteristics are essential. In 2018, the inventors' group built a database of pathogenic variants in hereditary breast cancer in Japanese people. However, unlike breast cancer, such data is lacking worldwide for prostate cancer, and currently there are no guidelines for treatment using genomic information.

Patent Publication No. 2012-157283

Lancet. 2016 Jan 2;387(10013):70-82.

The objective of the present invention is to clarify the relationship between causative genes, disease onset risk, and clinical information for prostate cancer using large-scale samples, thereby enabling efficient prediction of prostate cancer risk and genomic medicine.

In order to solve the above problem, the inventor analyzed the DNA of 7,636 prostate cancer patients (disease group) and 12,366 control group collected by Biobank Japan using a genome analysis method developed by the inventor (Hum Mol Genet. 2016;25(22):5027-5034.) for 8 genes known to cause prostate cancer, and identified 1,456 gene variants. The identified gene variants were then confirmed in the international database ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and each of the 1,456 gene variants was examined for whether or not they were pathogenic using the guidelines created by the American College of Clinical Genetics and Genomics (ACMG). As a result, 136 of the 1,456 identified gene variants (9.3%) were determined to be pathogenic variants, based on the results of ClinVar. The researchers then discovered that the frequency of these pathological variants is significantly higher in prostate cancer patients, particularly those under the age of 59, compared to healthy individuals, and found that prostate cancer risk can be efficiently tested based on the presence or absence of the identified pathogenic variants, thereby completing the present invention.

The gist of the present invention is as follows.
[1] A method for testing for prostate cancer, comprising using a sample derived from a subject and analyzing the presence of one or more of the 136 pathogenic variants listed in Table 1.
[2] The method according to [1], characterized in that the presence of one or more pathogenic variants selected from Nos. 3, 5, 7, 8, 10, 11, 13, 15, 17, 18, 22-27, 29, 30, 32, 34, 36, 39-41, 44-56, 59, 61, 62, 64-75, 77-86, 88-93, 95, 101-111, 113-116, 118, 122-124, 126, 128, 129, and 131-136 is analyzed among the 136 pathogenic variants in Table 1.
[3] The method according to [1] or [2], wherein the presence of a pathogenic variant in the BRCA2, HOXB13 or ATM gene is analyzed among the pathogenic variants.
[4] The method according to [3], wherein the presence of at least one of pathogenic variants No. 24, No. 61, No. 75, and No. 114 listed in Table 2 is analyzed.
[5] The method according to any one of [1] to [4], wherein at least 10 or more pathogenic variants among those listed in Table 1 are analyzed.
[6] The method according to any one of [1] to [5], wherein the prostate cancer is early-onset prostate cancer.
[7] A reagent for testing prostate cancer, comprising a reagent for analyzing the presence of one or more pathogenic variants among the 136 pathogenic variants listed in Table 1.

According to the present invention, the risk of developing prostate cancer can be efficiently predicted based on the analysis of pathogenic variants. This makes it possible to realize genomic medicine according to the type of variant.

A Loriplot showing the location of the pathogenic variant for each gene and the number of people who carry it. The horizontal axis shows the position of the amino acid, and the vertical axis shows the number of people who carry the pathogenic variant. Four pathogenic variants shared by more than 10 people are shown in the figure. For example, "p.Arg 2318*" means that the 2318th Arg (arginine) in the BRCA2 gene is changed to a stop codon. The underlined pathogenic variants (p.Asp1540Asp and p.Gly132Glu) are those that were first revealed in the research of this invention. Note that a functional loss refers to a variant that stops the translation of an amino acid or causes a frameshift (fs: a shift in the reading frame of a codon due to the deletion or insertion of a base), resulting in the loss of the function of the amino acid, a nonsynonymous substitution refers to a variant that changes the amino acid sequence, and a synonymous substitution refers to a variant that does not change the amino acid sequence. Graph showing the proportion of patients with pathogenic variants by age at prostate cancer diagnosis. A) Proportion of patients with pathogenic variants. Two-tailed Cochran-Armitage test was used (P<0.001). B) Proportion of genes containing pathogenic variants.

The prostate cancer testing method of the present invention is characterized in that a sample derived from a subject is used and analyzed for the presence of one or more of the 136 types of pathogenic variants listed in Table 1. Of the pathogenic variants listed in Table 1, it is preferable to analyze 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more.
In addition, "analyzing for the presence of a pathogenic variant" refers to analyzing the nucleotide sequence of a region containing a pathogenic variant and analyzing whether or not the sequence contains a pathogenic variant.

The prostate cancer is not particularly limited as long as it is a malignant tumor occurring in the prostate, but it may be early-onset prostate cancer occurring in subjects aged 59 or younger.

A pathogenic variant is a mutation in a gene sequence, such as a substitution, deletion, insertion, or rearrangement, that causes a disease.

Nos. 1 to 7 in Table 1 are pathogenic variants present in the NBN gene.
The NBN gene is a gene that encodes a protein involved in the DNA double-strand break repair mechanism and the DNA damage checkpoint mechanism, and is known to be associated with prostate cancer (Non-Patent Document 1). For example, the pathogenic variant No. 5 is a C/A polymorphism at base 90992961 of the genome sequence of chromosome 8 registered in the Genome Research Consortium human genome build 37/UCSC human genome 19 (GRCh37/Hg19), and if this base is A of the mutant type (Alt), it can be said that the pathogenic variant is possessed.

Nos. 8 to 38 in Table 1 are pathogenic variants in the ATM gene.
The ATM gene is a cell cycle checkpoint kinase that encodes a protein involved in the regulation of tumor suppressor proteins such as p53 and BRCA1, and is known to be associated with prostate cancer (Non-Patent Document 1). For example, the pathogenic variant No. 1 is a C/T polymorphism at base 108098418 of the genome sequence of chromosome 11 registered in GRCh37/Hg19, and if this base is the mutant type (Alt) T, it can be said that the patient has a pathogenic variant.

No. 39 to No. 90 in Table 1 are pathogenic variants present in the BRCA2 gene.
The BRCA2 (breast cancer susceptibility gene II) gene is known as a tumor suppressor gene and is a causative gene for breast cancer, but is also known to be associated with prostate cancer (Non-Patent Document 1). For example, the pathogenic variant No. 39 is a T/C polymorphism at base 32893464 of the genome sequence of chromosome 13 registered in GRCh37/Hg19, and if this base is the mutant type (Alt) C, it can be said that the pathogenic variant is possessed.

Nos. 91 to 96 in Table 1 are pathogenic variants in the PALB2 gene.
The PALB2 gene is a gene that encodes a protein that binds to the BRCA2 protein and is involved in stable localization and accumulation in the nucleus, and is known to be associated with prostate cancer (Non-Patent Document 1). For example, the pathogenic variant No. 91 is a T/A polymorphism at base 23634387 of the genome sequence of chromosome 16 registered in GRCh37/Hg19, and if this base is A of the mutant type (Alt), it can be said that the pathogenic variant is possessed.

No. 97 to No. 110 in Table 1 are pathogenic variants present in the BRCA1 gene.
The BRCA1 (breast cancer susceptibility gene I) gene is known as a tumor suppressor gene and is a causative gene for breast cancer, but is also known to be associated with prostate cancer (Non-Patent Document 1). For example, the pathogenic variant No. 101 is a G/A polymorphism at base 41226411 of the genome sequence of chromosome 17 registered in GRCh37/Hg19, and when this base is A of the mutant type (Alt), it can be said that the pathogenic variant is possessed.

No. 111 to No. 115 in Table 1 are pathogenic variants in the HOXB13 gene.
The HOXB13 gene is a transcription factor that encodes a protein important for embryonic development and is known to be associated with prostate cancer (Non-Patent Document 1). For example, the pathogenic variant No. 111 is a G/A single nucleotide polymorphism at base 46804205 of the genome sequence of chromosome 17 registered in GRCh37/Hg19, and if this base is A of the mutant type (Alt), it can be said that the pathogenic variant is possessed.

Nos. 116 to 127 in Table 1 are pathogenic variants in the BRIP1 gene.
The BRIP1 gene is a gene that forms a complex with the BRCA1 protein and encodes a protein that has a DNA double-strand break repair mechanism, and is known to be associated with prostate cancer (Non-Patent Document 1). For example, the pathogenic variant No. 116 is a T insertion mutation at the position next to the 59760814th base in the genome sequence of chromosome 16 registered in GRCh37/Hg19, and it can be said that a pathogenic variant is possessed when this base is an insertion of mutant (Alt) T.

Nos. 128 to 136 in Table 1 are pathogenic variants in the CHEK2 gene.
The CHEK2 gene is a gene that encodes a protein that has the functions of cell cycle checkpoint control and cancer suppression, and is known to be associated with prostate cancer (Non-Patent Document 1). For example, the pathogenic variant No. 128 is a deletion mutation of G at base 29083910 of the genome sequence of chromosome 22 registered in GRCh37/Hg19, and it can be said that a pathogenic variant is possessed when this base is a deletion type of mutant type (Alt).

Among these, it is preferable to analyze for one or more pathogenic variants among Nos. 3, 5, 7, 8, 10, 11, 13, 15, 17, 18, 22-27, 29, 30, 32, 34, 36, 39-41, 44-56, 59, 61, 62, 64-75, 77-86, 88-93, 95, 101-111, 113-116, 118, 122-124, 126, 128, 129, and 131-136 in Table 1.
In addition, variants other than those mentioned above in Table 1 were detected at a high rate in healthy individuals in this analysis; however, by increasing the analysis population, it is thought that the rate would be higher in prostate cancer patients. Therefore, it is possible to analyze prostate cancer risk based on the presence of pathogenic variants other than those mentioned above.

Among the above eight genes, it is preferable to analyze pathogenic variants in one or more of the BRCA2, HOXB13, and ATM genes.

In particular, it is preferable to analyze one or more, two or more, three or more, or all four of the following four pathogenic variants found at high frequencies in prostate cancer patients in this analysis:

Variant No. 24 is a single nucleotide polymorphism located in the ATM gene on chromosome 11 and is listed in GenBank
This refers to a single nucleotide polymorphism of cytosine (C)/thymine (T) at the 108,164,048th base of Accession No. NC_000011.10, and when this base is T, it is a pathogenic variant, and the possibility or risk of developing prostate cancer is high. In addition, when the analysis is performed taking into account alleles, the possibility or risk of developing prostate cancer increases in the order of TT > CT > CC. Therefore, it is possible to prevent or treat prostate cancer in subjects with this base as T.

For variant No. 24, the base sequence of the region containing the mutation is shown in SEQ ID NO: 1 (wild type: Ref.) and SEQ ID NO: 2 (mutated type: Alt.).
Ref. TTTTAAATTATATTTAGGTATTGGACTTGTTGAAATACTTAGTGATAGATA (SEQ ID NO: 1)
Alt. TTTTAAATTATATTTAGGTATTGGATTTGTTGAAATACTTAGTGATAGATA (SEQ ID NO: 2)

Variant No. 61 (rs80359520) is a deletion mutation located in the BRCA2 gene on chromosome 13, and refers to a mutation (del) in which the 32914068_32914071 base (AATT) of GenBank Accession No. NC_000013.10 is deleted. When this base is deleted, it is a pathogenic variant, and there is a high possibility or risk of developing prostate cancer. In addition, when the analysis takes into account alleles, the possibility or risk of developing prostate cancer increases in the order of (del/del) > (AATT/del) > (AATT/AATT). Therefore, it is possible to prevent or treat prostate cancer in subjects with this base del.

For rs80359520, the base sequence of the region containing the mutation is shown in SEQ ID NO: 3 (wild type: Ref.) and SEQ ID NO: 4 (mutated type: Alt.).
Ref. AAATCGTTTGTGTTTCACATGAAACAATTAAAAAAGTGAAAGACATATTTACAG (SEQ ID NO: 3)
Alt. AAATCGTTTGTGTTTCACATGAAAC AAAAAAGTGAAAGACATATTTACAG (SEQ ID NO: 4)

Variant No. 75 (rs80358920) is a single nucleotide polymorphism located in the BRCA2 gene on chromosome 13, and refers to a single nucleotide polymorphism of cytosine (C)/thymine (T) at base 32,920,978 of GenBank Accession No. NC_000013.10. When this base is T, it is a pathogenic variant, and there is a high possibility or risk of developing prostate cancer. In addition, when the analysis is performed taking into account alleles, the order of possibility or risk of developing prostate cancer is TT > TC > CC. Therefore, it is possible to prevent or treat prostate cancer in subjects who have this base as T.

For rs80358920, the base sequence of the region containing the mutation is shown in SEQ ID NO: 5 (wild type: Ref.) and SEQ ID NO: 6 (mutated type: Alt.).
Ref. TTGTTTCCTAGGCACAATAAAAGATCGAAGATTGTTTATGCATCATGTTTC (SEQ ID NO: 5)
Alt. TTGTTTCCTAGGCACAATAAAAGATTGAAGATTGTTTATGCATCATGTTTC (SEQ ID NO: 6)

Variant No. 114 is an SNP located in the HOXB13 gene on chromosome 17, and refers to a single nucleotide polymorphism of cytosine (C)/thymine (T) at base 46,805,561 of GenBank Accession No. NC_000017.10. When this base is T, it is a pathogenic variant, and there is a high possibility or risk of developing prostate cancer. In addition, when the analysis is performed taking into account alleles, the order of possibility or risk of developing prostate cancer is TT > CT > CC. Therefore, it is possible to prevent or treat prostate cancer in subjects where this base is T.

For variant No. 114, the base sequence of the region containing the mutation is shown in SEQ ID NO: 7 (wild type: Ref.) and SEQ ID NO: 8 (mutated type: Alt.).
Ref. CATAGGCTGGTAGGTTCCCGGATATCCCGGATAGAAGGCAAACTCAGTGGG (SEQ ID NO: 7)
Alt. CATAGGCTGGTAGGTTCCCGGATATTCCGGATAGAAGGCAAACTCAGTGGG (SEQ ID NO: 8)

Note that the positions of the pathogenic variants in Table 1 are those in the human genome sequence registered in the database. The above sequence may vary slightly at positions other than the pathogenic variants due to differences in race, etc. In such cases, the positions should be compared with the positions of the pathogenic variants in Table 1, and the relevant bases should be analyzed at the corresponding positions.

In addition, the variants analyzed in the present invention are not limited to those described above, and mutations in bases in linkage disequilibrium with the above bases may be analyzed. Here, "a base in linkage disequilibrium with the above base" refers to a base that satisfies the relationship of preferably ^r2 >0.8, more preferably ^r2 >0.9 with the above base. ^r2 is the linkage disequilibrium coefficient. Bases in linkage disequilibrium can be identified using, for example, the HapMap database (http://www.hapmap.org/index.html.ja) or the like.

As described above, prostate cancer can be tested for based on the presence of a pathogenic variant. For example, the presence of a pathogenic variant in one or more genes selected from the pathogenic variants in Table 1, preferably the BRCA2, HOXB13, and ATM genes, can be analyzed, and when a subject has one or more, two or more, three or more, four or more, or five or more pathogenic variants, it can be determined that the subject has a high risk of developing or progressing prostate cancer. In other words, the present invention can provide data for diagnosing prostate cancer.

Preferably, the pathogenic variants in Table 1 are analyzed in combination.
In this case, a risk score can be calculated taking into account the number of alleles of each variant and the odds ratio, and the risk of prostate cancer can be determined based on the obtained risk score.
In addition, the pathogenic variants listed in Table 1 may be analyzed in combination with other variants such as SNPs.

Prostate cancer can be tested for by determining the base types in the variants and associating the presence of one or more pathogenic variants or a risk score based on the presence of pathogenic variants with prostate cancer.
For any variant, either strand of the double-stranded DNA may be analyzed. For example, either the sense strand or the antisense strand of each sequence may be analyzed.

In the present invention, the subject of analysis is a human, for example, a human suspected of being at risk of prostate cancer. There is no particular limitation on the race of the subject, and examples of the subject include Asians, including Japanese.
The sample used for the analysis of the presence of a pathogenic variant (hereinafter simply referred to as variant analysis) is not particularly limited as long as it is a sample derived from a subject and contains chromosomal DNA. Examples of samples used for variant analysis include body fluids such as blood, urine, and cerebrospinal fluid, cells from the cervix and oral mucosa, and body hair such as hair. Although these samples can be used directly for variant analysis, it is preferable to isolate chromosomal DNA from these samples by a conventional method and use it for analysis.

Analysis of variants can be performed by standard gene sequence analysis methods, including, but not limited to, sequence analysis, PCR, hybridization, and the Invader method.

Sequence analysis can be performed by standard methods. Specifically, a sequence reaction is performed using a primer set several tens of bases 5' from the site where the pathogenic variant exists (hereinafter simply referred to as the variant site), and the type of base at the corresponding site can be determined from the analysis results. Note that it is preferable to amplify a fragment containing the variant site by PCR or the like beforehand, prior to the sequence reaction.

Variants can also be analyzed by examining whether amplification has occurred by PCR. For example, primers are prepared that have a sequence corresponding to the region containing the base at the variant site and whose 3' ends correspond to each base. PCR is performed using each primer, and the type of base can be determined based on the presence or absence of an amplified product. Amplification can also be examined by the LAMP method (Patent No. 3313358), NASBA method (Nucleic Acid Sequence-Based Amplification; Patent No. 2843586), ICAN method (JP Patent Publication 2002-233379), SmartAmp method (Patent No. 3897805), etc. Alternatively, a single-strand amplification method may be used.

Also, a DNA fragment containing a variant site can be amplified, and the type of base can be determined based on the difference in electrophoretic mobility of the amplified product. One such method is the PCR-SSCP (single-strand conformation polymorphism) method (Genomics. 1992 Jan 1;12(1): 139-146.). Specifically, first, DNA containing the target variant is amplified, and the amplified DNA is dissociated into single-stranded DNA. The dissociated single-stranded DNA is then separated on a non-denaturing gel, and the type of base can be determined based on the difference in mobility of the separated single-stranded DNA on the gel.

Furthermore, if the base at the variant site is included in a restriction enzyme recognition sequence, it can also be analyzed by the presence or absence of cleavage by the restriction enzyme (RFLP method). In this case, a DNA sample is first cleaved with a restriction enzyme. Next, the DNA fragments are separated, and the type of base can be determined based on the size of the detected DNA fragment.

It is also possible to analyze the type of base by examining the presence or absence of hybridization. That is, by preparing a probe corresponding to each base and examining which probe a base hybridizes to, it is possible to determine which base the variant is, i.e., whether it is a pathogenic variant or not.

In this way, by analyzing which base the variant is, the presence of a pathogenic variant can be determined, thereby providing data for testing for prostate cancer.
The results determined by the method of the present invention are provided to a doctor or the like as necessary. The doctor or the like who has been provided with the results can diagnose the risk of onset or progression after performing necessary tests such as blood tests, X-ray tests, and CT scans. If the doctor or the like diagnoses that the risk of onset or progression of prostate cancer is high, treatment such as surgery or anticancer drug treatment can be performed. In addition, if the doctor or the like diagnoses that the risk is low, these treatments, which are a heavy burden on the patient, can be avoided.

<2> Prostate cancer testing reagent of the present invention The present invention also provides a testing reagent such as a primer or a probe for testing prostate cancer. Such a probe includes a probe that contains a base sequence covering the above-mentioned variant site and can determine the type of base at the variant site based on the presence or absence of hybridization. Specifically, the probe includes a probe having a length of 15 or more bases that contains a base at the variant site of any of the base sequences shown in SEQ ID NOs: 1 to 8 (pathogenic variant sequences are SEQ ID NOs: 2, 4, 6, and 8) or a complementary sequence thereof. The length of the probe is preferably 15 to 35 bases, more preferably 20 to 35 bases.

The primer may be a primer that can be used in PCR to amplify the variant site, or a primer that can be used to sequence the variant site. Specifically, the primer may be a primer that can amplify or sequence a region containing the variant site of any of the base sequences shown in SEQ ID NOs: 1 to 8 (pathogenic variant sequences are SEQ ID NOs: 2, 4, 6, and 8). The length of such a primer is preferably 15 to 50 bases, more preferably 15 to 35 bases, and even more preferably 20 to 35 bases.

Examples of primers for sequencing the variant site include a primer having a sequence in the 5' region of the base at the variant site, preferably 30 to 100 bases upstream, and a primer having a sequence complementary to the 3' region of the base at the variant site, preferably 30 to 100 bases downstream. Examples of primers used to determine the type of variant based on the presence or absence of PCR amplification include a primer having a base sequence including the variant site and containing the variant base at the 3' end, and a primer having a complementary sequence to the base sequence including the variant site and containing the complementary base of the variant base at the 3' end.

In addition to these primers and probes, the testing reagent of the present invention may also contain polymerase and buffer for PCR, hybridization reagents, etc.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following aspects.

Methods
Study samples: All study samples were obtained from Biobank Japan database, which collects DNA and clinical information from patients with various diseases, including prostate cancer, from all over Japan between 2003 and 2018. In this study, a hospital registry study was conducted among 7,744 prostate cancer patients and 12,520 male controls. All participants provided informed consent. This study was approved by the Ethics Committee of the Institute of Medical Science, University of Tokyo and the RIKEN Center for Integrative Medical Sciences.

Sequence and bioinformatics analysis Eight genes reported to be related to prostate cancer in Non-Patent Document 1 (ATM, BRCA1, BRCA2, BRCA1-interacting protein C-terminal helicase 1 [BRIP1], CHEK2, HOXB13, NBN, and PALB2) were selected.
The complete coding regions and 2-bp flanking intron sequences of all eight genes (37,982 bp) were analyzed by a multiplex PCR-based targeted sequencing method (Hum Mol Genet. 2016;25(22):5027-5034.) Ultimately, 1456 gene variants were identified in 7,636 cases and 12,366 control individuals, with 99.98% of the target regions covered by at least 20 sequence reads.

Variant annotation Clinical significance was determined using the American College of Medical Genetics and Genomics and the College of Molecular Pathology (ACMG/AMP) guidelines (Genet Med. 2015;17(5):405-424, Nat Rev Genet. 2017;18(10):599-612.) and the disease association criteria registered in ClinVar (Nucleic Acids Res. 2016;44(D1):D862-D868.). Pathogenic variants were identified based on the ACMG/AMP guidelines and/or the ClinVar disease association classification.

Statistical Analysis Case-control association analyses were performed using Fisher's exact test under the dominant model. To investigate the association of pathogenic variants with demographic and clinical characteristics, t-tests for continuous variables and Fisher's exact test or Cochran-Armitage test for discrete variables were used. All statistical tests were two-sided, and P less than 0.05 was considered statistically significant, except when the Bonferroni correction was applied to the association analysis between each of the eight genes and PCa (P<0.006=0.05/8). All analyses were performed using the R statistical package (version 3.1.3).

<Results>
DNA from 7,636 prostate cancer patients (disease group) and 12,366 control groups collected by Biobank Japan was analyzed for eight genes known to cause prostate cancer, and 1,456 gene variants were identified. When the identified gene variants were checked in the international database ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), only 670 (46.0%) were registered. Of these, 58 were pathogenic variants, 216 were non-pathogenic gene variants, and the remaining 396 were indeterminate. Using the guidelines created by the American College of Clinical Genetics and Genomics (ACMG), we then examined each of the 1,456 gene variants to determine whether they were pathogenic or not. Finally, based on the results of ClinVar, 136 (9.3%) of the 1,456 gene variants identified in this study were determined to be pathogenic variants (Table 1).

Next, we created a Lori plot to confirm the amino acid position and frequency of pathogenic variants for each gene.The results showed that most (78.8%) pathogenic variants were found in only one patient out of 7,636 patients, making them extremely rare.
On the other hand, pathogenic variants that were frequently shared by 10 or more individuals were found in one ATM gene, two BRCA2 genes, and one HOXB13 gene (Figure 1). Of these, the two variants found in ATM and HOXB13 were new pathogenic variants that had not been registered in ClinVar before.

We also investigated the proportion of these 136 pathogenic variants in the disease and control groups. As a result, it was found that 2.9% of the disease group and 0.8% of the control group had pathogenic variants across the eight genes, and that having a pathogenic variant increases the likelihood of developing prostate cancer by approximately 3.7 times (Table 3). In addition, it was found that three genes, BRCA2, HOXB13, and ATM, contribute to the development of prostate cancer (Table 3).

Next, we investigated the clinical characteristics of pathogenic variant carriers, and found that pathogenic variant carriers were diagnosed with prostate cancer two years younger than non-carriers, that more of them had a history of smoking or drinking, that a higher proportion of them had family members with breast cancer, pancreatic cancer, lung cancer, or liver cancer, and that their clinical symptoms were worse in terms of TNM classification, Gleason score, and PSA (prostate-specific antigen) (Table 4).

When we investigated the proportion of genetic variant carriers by age at prostate cancer diagnosis, we found that a high proportion of those under 60 years old (7.9%) was found, and the proportion decreased with age, but remained almost constant at around 2-3% for those over 65 years old (Figure 2). Hereditary diseases caused by a single pathogenic variant are usually thought to develop early, but as with breast cancer, there is a certain contribution even in older age, and it was found that genetic testing is meaningful.

Claims (7)

A method for testing for the possibility and/or risk of developing prostate cancer, comprising using a sample derived from a subject and analyzing for the presence of one or more pathogenic variants selected from Nos. 8 to 43, 45 to 74, 77 to 90, and 111 to 115 in the BRCA2, HOXB13, and ATM genes among the 136 pathogenic variants listed in Table 1, the method comprising:
A polymorphism in a base corresponding to the base (Ref) in Table 1 of the base sequence containing the pathogenic variant is analyzed, and based on the analysis results,
When the base corresponding to the base (Ref) in Table 1 of the nucleotide sequence containing the pathogenic variant is the base (Alt) in Table 1,
A method for determining whether there is an increased possibility and/or risk of developing prostate cancer.

The method according to claim 1, characterized in that the presence of one or more pathogenic variants selected from No. 8, 10, 11, 13, 15, 17, 18, 22-27, 29, 30, 32, 34, 36, 39-41, 45-56, 59, 61, 62, 64-74, 77-86, 88-90, 111, and 113-115 out of the 136 pathogenic variants in Table 1 is analyzed. 3. The method of claim 2, wherein the presence of at least one pathogenic variant selected from Nos. 24, 61, and 114 is analyzed.

The method according to any one of claims 1 to 3, wherein at least 10 or more pathogenic variants among those listed in Table 1 are analyzed. The method according to any one of claims 1 to 4, wherein the prostate cancer is early-onset prostate cancer. A reagent for testing a risk of developing prostate cancer, comprising a probe for analyzing the presence of one or more pathogenic variants selected from Nos. 8 to 43, 45 to 74, 77 to 90, and 111 to 115, which are pathogenic variants in the BRCA2, HOXB13, and ATM genes among the 136 pathogenic variants listed in Table 1,
The probe is a probe for detecting a base in a base sequence containing the pathogenic variant, the base being one of the bases shown in Table 1 (Alt
) a complementary sequence of a sequence of 15 or more bases including the bases.

A reagent for testing a risk of developing prostate cancer, comprising primers for analyzing the presence of one or more pathogenic variants selected from Nos. 8 to 43, 45 to 74, 77 to 90, and 111 to 115, which are pathogenic variants in the BRCA2, HOXB13, and ATM genes among the 136 pathogenic variants listed in Table 1,
The testing reagent, wherein the primer is a primer capable of amplifying a region containing a base corresponding to the base (Alt) shown in Table 1 in a base sequence containing the pathogenic variant.

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