Capture probe group, amplification primer group and detection method of related genes of renal cystic diseases

Abstract

The invention relates to the technical field of molecular diagnosis of hereditary nephropathy, and particularly discloses a capture probe group, an amplification primer group and a detection method of related genes of renal cystic diseases. The probe group is used for capturing 42 gene regions closely related to renal cyst, such as ALG8, PKD1, PKD2, GANAB, TSC2 and the like, each probe is 120 bp and is flatly designed along a sense strand, and the density of the probes in a low GC region is increased; the primer group comprises four pairs of long-fragment primers for amplifying first to 34 exon segments of the PKD1 gene, has high specificity, and can effectively distinguish PKD1 from a pseudo gene. An amplification product and original genome DNA are mixed according to a specific ratio to build a library and are subjected to high-throughput sequencing, so that the sequencing coverage and mutation detection rate of the PKD1 are remarkably improved. In 59 clinical samples, the positive detection rate reaches 93.2%, and the method is suitable for screening, diagnosis and genetic typing of renal cystic diseases.

Description

Technical Field

[0001] This invention relates to the technical field of molecular diagnosis of hereditary kidney diseases, and in particular to capture probe sets, amplification primer sets and detection methods for genes related to renal cystic diseases. Background Technology

[0002] Renal cystic diseases are a group of clinical syndromes characterized by the formation of single or multiple fluid- or semi-solid cysts in the renal parenchyma. Based on their pathogenesis, they are mainly divided into two categories: non-hereditary and hereditary. Non-hereditary renal cysts, such as simple renal cysts, have a high incidence in adults and generally have a good clinical prognosis. Hereditary renal cysts, on the other hand, include various types that severely affect renal function, such as autosomal dominant polycystic kidney disease (ADPKD), polycystic kidney dysplasia, medullary cystic kidney, and renal cysts associated with tuberous sclerosis.

[0003] Currently, the clinical diagnosis of different types of renal cystic diseases relies heavily on imaging examinations and family history analysis. However, due to the striking similarity between certain congenital malformations (such as duplicated kidneys) and cysts on imaging, and the frequent presence of extrarenal manifestations such as hepatic cysts in hereditary diseases, clinical classification and accurate diagnosis remain significantly challenging. Furthermore, some key pathogenic genes, such as PKD1, possess highly homologous pseudogene regions in the genome, making it difficult for conventional PCR and sequencing technologies to accurately distinguish between true and false genes, leading to detection failures or false negatives.

[0004] In recent years, the development of next-generation sequencing (NGS) technology has provided new methods for gene testing of hereditary kidney diseases. However, if capture and library construction are performed directly, the detection results may be affected by PKD1 pseudogene interference, resulting in insufficient accuracy. Therefore, there is an urgent need to establish a stable, efficient, and highly targeted detection method that combines specific long-fragment PCR amplification with high-throughput sequencing to improve the molecular diagnostic capabilities for renal cystic diseases. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a capture probe set, amplification primer set, and detection method for genes related to renal cystic diseases.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0007] A capture probe set for genes associated with renal cystic disease, the probe set comprising nucleic acid probes for capturing 42 genes associated with cystic kidney disease, the probe set comprising the following features:

[0008] (1) Each probe is 120 bp in length;

[0009] (2) The probe is continuously laid out along the positive strand region of the target gene, covering the corresponding gene exons and part of the intron regions;

[0010] (3) After the probe sequence is compared with the human whole genome sequence, its homology in the non-target region is less than 80%;

[0011] (4) In regions where the GC content is less than 45%, the coverage density of the probe is more than 1.5 times that of regions with high GC content.

[0012] Furthermore, the probe set includes any nucleic acid sequence from SEQ ID NO.1 to SEQ ID NO.365.

[0013] Furthermore, the probe set is used to capture the nucleic acid regions of the following 42 genes: ALG8, ALG9, ANKS6, B9D2, BBS4, CEP164, COL4A1, DNAJB11, DZIP1L, GANAB, GLIS2, HNF1B, INVS, KAT6B, LRP5, MAPKBP1, MUC1, NEK8, NOTCH2, NPHP1, NPHP3, NPHP4, OFD1, PAX2, PKD1, PKD2, PKHD1, PRKCSH, RPGRIP1L, SDCCAG8, SEC63, TMEM216, TMEM231, TMEM237, TMEM67, TSC1, TSC2, UMOD, VHL, WDR19, XPNPEP3, and ZNF423.

[0014] A method for detecting genes related to renal cystic disease using the above-described capture probe set includes the following steps:

[0015] (1) Using a set of capture probes for renal cystic disease-related genes to perform hybridization capture on the nucleic acid samples to be tested;

[0016] (2) Construct a library from the captured target sequence and perform high-throughput paired-end sequencing;

[0017] (3) The sequencing results are compared with the reference genome to identify gene mutations associated with cystic nephropathy.

[0018] Furthermore, the high-throughput paired-end sequencing was performed using the Illumina platform, and the mutation identification was interpreted according to the HGVS nomenclature standard and the ACMG pathogenicity classification guidelines.

[0019] A primer set for long-fragment PCR amplification of exons 1 to 34 of the PKD1 gene, comprising the following 4 pairs of primers:

[0020] PKD1-E1F: SEQ ID NO.366;

[0021] PKD1-E1R: SEQ ID NO.367;

[0022] PKD1-E2-12F: SEQ ID NO.368;

[0023] PKD1-E2-12R: SEQ ID NO.369;

[0024] PKD1-E13-21F: SEQ ID NO.370;

[0025] PKD1-E13-21R: SEQ ID NO.371;

[0026] PKD1-E22-34F: SEQ ID NO.372;

[0027] PKD1-E22-34R: SEQ ID NO.373;

[0028] The exons 1 through 34 are annotated based on transcript number NM_001009944.2.

[0029] Furthermore, the first amplified fragment is 2278 bases long, corresponding to the sequence segment containing exon 1;

[0030] The second amplified fragment is 8640 bases long and corresponds to the continuous sequence segment containing exons 2 to 12.

[0031] The third amplified fragment is 8043 bases long and corresponds to the continuous sequence segment containing exons 13 to 21.

[0032] The fourth amplified fragment is 7800 bases long and corresponds to the continuous sequence segment containing exons 22 to 34.

[0033] A method for constructing sequencing samples using the above-mentioned amplification products, wherein the amplification products are mixed with the original genomic DNA in the following proportions for library construction:

[0034] Original genomic DNA, 3G in length, was added in an amount of 400ng;

[0035] The product obtained from the amplified region containing exon 1 was 2278 bases in length, and the amount added was 0.0034 ng.

[0036] The product of the continuous region containing exons 2 to 12 obtained by amplification has a length of 8640 bases and the amount added is 0.013 ng;

[0037] The product of the continuous region containing exons 13 to 21 obtained by amplification has a length of 8043 bases and the amount added is 0.012 ng;

[0038] The product obtained from the amplification of the continuous region containing exons 22 to 34 has a length of 7800 bases and was added in an amount of 0.0117 ng.

[0039] Application of the above-mentioned probe set or detection method in genetic screening, early diagnosis and genetic typing of cystic nephropathy.

[0040] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following aspects:

[0041] 1. This invention designs four pairs of specific long-fragment PCR primers to amplify the exon 1 to 34 regions of the PKD1 gene, which can effectively eliminate the interference of six pseudogenes (PKD1P1 to PKD1P6) that are highly homologous to PKD1, ensuring that the amplification products are derived from the true PKD1 gene, and overcoming the problem of false positives or false negatives that are easy to occur in the existing NGS direct library construction.

[0042] 2. This invention establishes a strategy for mixing long fragment amplified PKD1 products with original genomic DNA in a specific ratio. Without affecting the detection efficiency of other genes, it effectively increases the proportion of PKD1 in the sequencing library and improves sequencing coverage depth and variant detection rate.

[0043] 3. This invention designs a capture probe set covering 42 genes closely related to renal cysts, including ALG8, PKD1, PKD2, GANAB, and TSC2. It adopts a 120bp flat-lay specific design, and the probe density is increased in regions with low GC content, which effectively improves the whole genome capture efficiency and reduces sequencing costs.

[0044] 4. In clinical sample application, the method of this invention detected definite pathogenic or suspected pathogenic mutations in 55 out of 59 patients with nephropathy phenotypes, with a positive detection rate of 93.2%. Among them, a large number of newly discovered PKD1 variants were found, which further verified the practical value of this invention in clinical early screening and genetic typing. Attached Figure Description

[0045] Figure 1 This is an agarose gel electrophoresis image of the PCR amplification product of the PKD1 gene.

[0046] Figure 2 This is a graph showing the percentage of sequencing results after mixing the PCR amplification product and the original genome sequence of the PKD1 gene. Detailed Implementation

[0047] The present invention will now be described in detail with reference to the embodiments.

[0048] Example 1

[0049] This embodiment discloses a capture probe set for genes related to renal cystic kidney disease, including nucleic acid probes for capturing 42 genes related to cystic kidney disease. The probe set includes the following features:

[0050] (1) Each probe is 120 bp in length;

[0051] (2) The probe is continuously laid out along the positive strand region of the target gene, covering the corresponding gene exons and part of the intron regions;

[0052] (3) After the probe sequence is compared with the human whole genome sequence, its homology in the non-target region is less than 80%;

[0053] (4) In regions where the GC content is less than 45%, the coverage density of the probe is more than 1.5 times that of regions with high GC content.

[0054] The genes captured by the capture probe set and their chromosomal coordinates are shown in the table below:

[0055]

[0056]

[0057]

[0058] The probe set includes any of the nucleic acid sequences in SEQ ID NO.1 to SEQ ID NO.365.

[0059] This embodiment also discloses a method for detecting genes related to renal cystic diseases using the above-mentioned capture probe set, characterized by comprising the following steps:

[0060] (1) Using a set of capture probes for renal cystic disease-related genes to perform hybridization capture on the nucleic acid samples to be tested;

[0061] (2) Construct a library from the captured target sequence and perform high-throughput paired-end sequencing;

[0062] (3) The sequencing results are compared with the reference genome to identify gene mutations associated with cystic nephropathy.

[0063] High-throughput paired-end sequencing was performed using the Illumina platform, and mutation identification was interpreted according to the HGVS nomenclature standard and the ACMG pathogenicity classification guidelines.

[0064] This embodiment also discloses a primer set for long-fragment PCR amplification of exons 1 to 34 of the PKD1 gene, including the following 4 pairs of primers:

[0065]

[0066] Exons 1 through 34 are annotated based on transcript number NM_001009944.2.

[0067] The PKD1 gene PCR amplification kit and amplification experimental system are shown below:

[0068] PKD1-E1F / PKD1-E1R Amplification Kit:

[0069] Consumables Input volume 2×Phanta Max Master Mix 25.0ul Forward primer F (10 μM) 1.0ul Reverse primer R (10 μM) 1.0ul DNA template (30 ng / ul) 3.0ul Deionized water 5.0ul Betaine (1.5M) 5.0ul

[0070] PKD1-E1F / PKD1-E1R amplification experimental system:

[0071]

[0072] PKD1-E2-12F / PKD1-E2-12R, PKD1-E13-21F / PKD1-E13-21R, PKD1-E22-34F / PKD1-E22-34 Amplification Kits:

[0073] Reaction components reaction volume 2X Phanta Max Master Mix 25.0ul Forward primer F (10 μM) 2.0ul Reverse primer R (10 μM) 2.0ul DNA template (30 ng / ul) 3.0ul Deionized water 5.0ul Betaine (1.5M) 5.0ul

[0074] Experimental systems for PKD1-E2-12F / PKD1-E2-12R, PKD1-E13-21F / PKD1-E13-21R, and PKD1-E22-34F / PKD1-E22-34:

[0075]

[0076]

[0077] This embodiment also discloses a method for constructing sequencing samples using the above-described amplification products, wherein the amplification products are mixed with the original genomic DNA in the following proportions for library construction:

[0078] DNA length Amount added (ng) gDNA 3G 400 Long-PCR amplification product 1 2278bp 0.0034 Long-PCR amplification product 2 8640bp 0.013 Long-PCR amplification product 3 8043bp 0.012 Long-PCR amplification product 4 7800bp 0.0117

[0079] This embodiment also discloses the application of the above-mentioned probe set or detection method in genetic screening, early diagnosis and genetic typing of cystic nephropathy.

[0080] Testing and verification

[0081] 1. Agarose gel electrophoresis was performed on the PCR amplification products of the PKD1 gene, and the results are as follows: Figure 1 As shown.

[0082] 2. The sequencing ratio of the mixed PCR amplification product and the original genome sequence of the PKD1 gene was as follows: Figure 2 As shown.

[0083] 3. Sixty patients with renal cyst phenotypes underwent 42-gene testing for renal cystic disease. Following the Human Genome Variation Society (HGVS) nomenclature principles and ACMG guidelines, the mutation sites screened in this study were named, their pathogenicity was rated, and Sanger sequencing was performed for verification. The statistical results are shown in the table below. Analysis revealed 55 mutation sites in 10 target genes in 59 patients with renal disease, including PKD1, PKD2, GANAB, PRKCSH, PKHD1, TSC1, TSC2, NPHP3, NPHP4, and PKHD1 genes, with a detection rate of 93.2% (55 / 59). Of these 55 mutations, 29 (29 / 55, 52.7%) were novel mutations, and 26 (26 / 55, 47.3%) were known mutations. 39 mutations were found in the PKD1 gene (21 novel mutations and 18 known mutations), 5 in the PKD2 gene (3 novel mutations and 2 known mutations), 1 novel mutation in the GANAB gene, 2 in the HNF1B gene (1 novel mutation and 1 known mutation), 1 novel mutation in the TSC1 gene, 1 novel mutation in the TSC2 gene, 1 novel mutation in the PRKCSH gene, 1 known mutation in the NPHP3 gene, 1 known mutation in the NPHP4 gene, and 3 in the PKHD1 gene (1 novel mutation and 2 known mutations). Among patients clinically diagnosed with ADPKD, the mutation rates for PKD1, PKD2, and GANAB were 79.6% (39 / 49), 10.2% (5 / 49), and 2.0% (1 / 49), respectively. Specific results are shown in the table below.

[0084]

[0085]

[0086]

[0087] The above detection and verification results show that the four pairs of PKD1 gene Long-PCR amplification primers provided by the present invention can effectively overcome the interference of six pseudogene homologous sequences and can effectively amplify exon 1 to exon 34 of the PKD1 gene.

[0088] This invention provides a mixing ratio of PKD1 gene Long-PCR amplification product and original genomic sequence, which can effectively increase the total amount of PKD1 gene sequence DNA without affecting the detection efficiency of other genes.

[0089] The above-mentioned capture probe set provided by the present invention can effectively capture genes related to cystic nephropathy, thereby reducing the detection cost of related genes and improving detection efficiency.

[0090] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A set of capture probes for genes related to renal cystic disease, characterized in that, The probe set includes nucleic acid probes for capturing 42 genes associated with cystic kidney disease, and the probe set includes the following characteristics: (1) Each probe is 120 bp in length; (2) The probe is continuously laid out along the positive strand region of the target gene, covering the corresponding gene exons and part of the intron regions; (3) After the probe sequence is compared with the human whole genome sequence, its homology in the non-target region is less than 80%; (4) In regions where the GC content is less than 45%, the coverage density of the probe is more than 1.5 times that of regions with high GC content.

2. The capture probe set for renal cystic disease-related genes according to claim 1, characterized in that, The probe set includes any nucleic acid sequence from SEQ ID NO.1 to SEQ ID NO.

365.

3. The capture probe set for renal cystic disease-related genes according to claim 1 or 2, characterized in that, The probe set is used to capture the nucleic acid regions of the following 42 genes: ALG8, ALG9, ANKS6, B9D2, BBS4, CEP164, COL4A1, DNAJB11, DZIP1L, GANAB, GLIS2, HNF1B, INVS, KAT6B, LRP5, MAPKBP1, MUC1, NEK8, NOTCH2, NPHP1, NPHP3, NPHP4, OFD1, PAX2, PKD1, PKD2, PKHD1, PRKCSH, RPGRIP1L, SDCCAG8, SEC63, TMEM216, TMEM231, TMEM237, TMEM67, TSC1, TSC2, UMOD, VHL, WDR19, XPNPEP3, and ZNF423.

4. A method for detecting genes related to renal cystic diseases using the capture probe set according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Using a set of capture probes for renal cystic disease-related genes to perform hybridization capture on the nucleic acid samples to be tested; (2) Construct a library from the captured target sequence and perform high-throughput paired-end sequencing; (3) The sequencing results are compared with the reference genome to identify gene mutations associated with cystic nephropathy.

5. The method for detecting genes related to renal cystic diseases according to claim 4, characterized in that, The high-throughput paired-end sequencing was performed using the Illumina platform, and the mutation identification was interpreted according to the HGVS nomenclature standard and the ACMG pathogenicity classification guidelines.

6. A primer set for PCR amplification of a long fragment of exons 1 to 34 of the PKD1 gene, characterized in that, Includes the following 4 pairs of primers: PKD1-E1F: SEQ ID NO.366; PKD1-E1R: SEQ ID NO.367; PKD1-E2-12F: SEQ ID NO.368; PKD1-E2-12R: SEQ ID NO.369; PKD1-E13-21F: SEQ ID NO.370; PKD1-E13-21R: SEQ ID NO.371; PKD1-E22-34F: SEQ ID NO.372; PKD1-E22-34R: SEQ ID NO.373; The exons 1 through 34 are annotated based on transcript number NM_001009944.

2.

7. The primer set for long-fragment PCR amplification of exons 1 to 34 of the PKD1 gene according to claim 6, characterized in that: The first amplified fragment is 2278 bases long and corresponds to the sequence segment containing exon 1. The second amplified fragment is 8640 bases long and corresponds to the continuous sequence segment containing exons 2 to 12. The third amplified fragment is 8043 bases long and corresponds to the continuous sequence segment containing exons 13 to 21. The fourth amplified fragment is 7800 bases long and corresponds to the continuous sequence segment containing exons 22 to 34.

8. A method for constructing a sequencing sample using the amplification product according to any one of claims 6 or 7, characterized in that, The amplification products were mixed with the original genomic DNA in the following proportions for library construction: Original genomic DNA, 3G in length, was added in an amount of 400ng; The product obtained from the amplified region containing exon 1 was 2278 bases in length, and the amount added was 0.0034 ng. The product of the continuous region containing exons 2 to 12 obtained by amplification has a length of 8640 bases and the amount added is 0.013 ng; The product of the continuous region containing exons 13 to 21 obtained by amplification has a length of 8043 bases and the amount added is 0.012 ng; The product obtained from the amplification of the continuous region containing exons 22 to 34 has a length of 7800 bases and was added in an amount of 0.0117 ng.

9. The application of a probe set or detection method as described in any one of claims 1 to 8 in genetic screening, early diagnosis and genetic typing of cystic nephropathy.

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