阅读说明：本技术 Plekhg5基因甲基化在弱精症诊断剂中的应用及试剂盒 (Application of PLEKHG5 gene methylation in asthenospermia diagnostic agent and kit ) 是由 杜野 薛兴奎 古艳丽 陈晓玲 马晓瑞 于 2019-11-21 设计创作，主要内容包括：本发明涉及人PLEKHG5基因启动子区的甲基化水平在制备检测精子活力的检测剂中的应用,所述人PLEKHG5基因启动子区位于人1号染色体第6545514至6548214位对应的区域。本发明通过基于全基因组甲基化测序结果进行分析和研究并通过验证,发现人PLEKHG5基因启动子区是否存在部分甲基化位点被甲基化在弱精症患者与健康人之间存在着显著性差异,可作为分子标志来诊断受试者是否患有弱精症。当该区域的所有甲基化位点均未被甲基化时,受试者患有弱精症的可能性较大。该分子标志可为弱精症的诊断提供辅助评价信息,结合其他诊断指标一起鉴定受试者是否患有弱精症。(The invention relates to application of methylation level of a promoter region of a human PLEKHG5 gene in preparing a detection agent for detecting sperm motility, wherein the promoter region of the human PLEKHG5 gene is positioned in a region corresponding to 6545514 to 6548214 of a human chromosome 1. According to the invention, through analysis and research based on a whole genome methylation sequencing result and verification, the fact that whether partial methylation sites exist in the promoter region of the human PLEKHG5 gene and are methylated is found to have a significant difference between a patient with asthenospermia and a healthy person, and the promoter region can be used as a molecular marker to diagnose whether a subject suffers from asthenospermia. When all of the methylation sites in this region are unmethylated, the subject is more likely to have asthenospermia. The molecular marker can provide auxiliary evaluation information for diagnosis of asthenospermia, and can be combined with other diagnosis indexes to identify whether a subject suffers from asthenospermia.)

1. The application of the methylation level of the promoter region of the human PLEKHG5 gene in preparing a diagnosis agent for asthenospermia.

2. The use of claim 1, wherein the promoter region of the human PLEKHG5 gene is located in the region corresponding to positions 6545514 to 6548214 of human chromosome 1.

3. The use of claim 2, wherein the promoter region of human PLEKHG5 is the region corresponding to the sequence shown in SEQ ID NO. 1.

4. A kit for diagnosing asthenospermia, which is characterized by comprising a primer for detecting the methylation level of a promoter region of a human PLEKHG5 gene.

5. The diagnostic kit according to claim 4, comprising a first primer pair consisting of two primers having sequences of SEQ ID NO 2 and 3, respectively, and a second primer pair consisting of two primers having sequences of SEQ ID NO 4 and 5, respectively.

6. The diagnostic kit of claim 5, further comprising a bisulfite solution.

7. The diagnostic kit of claim 6, wherein the bisulfite solution is a sodium bisulfite solution.

Technical Field

The invention relates to the field of human reproductive health, in particular to application of methylation level of a promoter region of a human PLEKHG5 gene in preparation of a diagnosis agent for asthenospermia and a diagnosis kit for asthenospermia.

Background

Over 40% of the infertile men are detected to have asthenospermia. The etiology of asthenospermia is multifactorial and, for example, may be closely related to endocrine disorders, environmental factors, life experiences, and even aging. Studies of knock-out and mutation in mice have shown that some gene mutations are associated with altered sperm motility, but that these genes have been shown to be independent of sperm motility in human studies. Although nearly 50% of cases of infertility are thought to be caused by genetic defects, in some cases of male infertility, genetic factors appear not to be major factors. There is increasing evidence that abnormal epigenetic changes also contribute to male infertility.

Sperm are highly diverse cell populations that exhibit a high degree of diversity in both DNA and sperm quality parameters. Researchers have begun investigating the diversity within samples of sperm DNA methylation, including sperm DNA methylation diversity between different sperm cells and sperm DNA methylation diversity between sperm regions of different mass in the same ejaculate. The inventors of the present application discovered and reported during the course of their studies (Duye et al, Human Reproduction, Vol.31, No. 1pp.24-33,2016, Human target double detailed sequences DNA methylation with low sperm motility in astenoospermia) that methylation at certain regions was significantly different between sperm cells of patients with weak sperm and sperm cells of normal controls, presumably where methylation at certain regions may be associated with the onset of weak sperm.

However, the methylation regions reported at present as molecular markers of asthenospermia are very few and are difficult to be used for accurately diagnosing whether a subject suffers from asthenospermia, so that more methylation regions related to asthenospermia need to be identified, thereby providing more bases for subsequent diagnosis and treatment.

Disclosure of Invention

Based on the previous studies, we continued intensive studies to find and analyze differentially methylated regions associated with asthenospermia. Based on the gene, the invention provides the application of the promoter region of the human PLEKHG5 gene in preparing a diagnosis agent for asthenospermia.

In a specific embodiment, the promoter region of the human PLEKHG5 gene is located in the region corresponding to positions 6545514 to 6548214 of human chromosome 1.

In a specific embodiment, the promoter region of the human PLEKHG5 gene is a region corresponding to the sequence shown in SEQ ID NO. 1.

The invention also provides a kit for diagnosing asthenospermia, which comprises a primer for detecting the methylation level of the promoter region of the human PLEKHG5 gene.

In a preferred embodiment, the diagnostic kit comprises a first primer pair consisting of two primers having sequences of SEQ ID NO 2 and 3, respectively, and a second primer pair consisting of two primers having sequences of SEQ ID NO 4 and 5, respectively.

In a preferred embodiment, the test kit further comprises a bisulfite solution.

In a preferred embodiment, the bisulfite solution is a sodium bisulfite solution.

According to the invention, through analysis and research based on a whole genome methylation sequencing result and verification, the fact that whether partial methylation sites exist in the promoter region of the human PLEKHG5 gene and are methylated is found to have a significant difference between a patient with asthenospermia and a healthy person, and the promoter region can be used as a molecular marker to diagnose whether a subject suffers from asthenospermia. When all of the methylation sites in this region are unmethylated, the subject is more likely to have asthenospermia. The molecular marker can provide auxiliary evaluation information for diagnosis of asthenospermia, and can be combined with other diagnosis indexes to identify whether a subject suffers from asthenospermia.

Drawings

FIG. 1 is a statistical plot of the methylation levels of the promoter regions of the human PLEKHG5 gene for sperm (Na) in fraction a from 8 healthy volunteers versus sperm (Aa) in fraction a from 7 asthenospermic patients in a whole genome sequencing study;

FIG. 2 is a statistical chart of the methylation levels of the promoter regions of the human PLEKHG5 gene verifying sperm (Na) in fraction a of 50 healthy volunteers and sperm (Aa) in fraction a of 30 asthenospermia patients in the study.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

1. Sperm separation

7 asthenospermia volunteers (group A) and 8 healthy volunteers (group N) were recruited, sperm samples were obtained from the volunteers, and whole genome methylation level sequencing studies were performed. Sperm morphology was assessed using a computer-assisted Sperm analysis (CASA) using a Sperm Class Analyzer with a Diff-Quik Rapid staining method. Sperm cells having different viability were separated by the following method: centrifugation at 300 Xg for 20min produced non-continuous density gradient fractions (divided into 47.5, 57, 76 and 95% concentration layers), high-viability sperm cells were collected from the 95% concentration layer (fraction a), and low-viability sperm cells were collected from between the 57% and 76% concentration layers (fraction c). The collected sperms are washed twice by PBS, and genome DNA is extracted

2. Library construction and methylation sequencing

Genomic DNA was extracted from sperm cells using the kit for library construction. 1. mu.g of genomic DNA was fragmented into a fragment of approximately 200-300 bp. After purification, the DNA fragment was treated with T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase to form blunt-ended DNA fragments, which were 3' adenylated and then ligated with linkers to obtain a library. After quantification of the library, it was used for solution phase hybridization capture and bisulfite sequencing.

Upon data processing analysis, we found that the global DNA methylation levels of both fraction a and fraction c of group a and group N were almost equal with no significant difference. However, in some regions, there is methylation variability between groups A and N, or between different fractions of the same group.

3. Identification of differentially methylated regions

By analyzing the differentially methylated regions described above, we found that there was a difference in the methylation level in the region between 6545514-6548214 of chromosome 1 (reference Genome: GRCh37/hg19, UCSC Genome Browser), which is the promoter region of PLEKHG5 gene, covering positions-2711 to-11 of the gene (SEQ ID NO: 1). Our study showed that the methylation levels in this region were significantly different between fraction a of group N and fraction a of group a. As shown in table 1 and fig. 1, all methylation sites in this region were unmethylated in group a fraction a sperm cells, while 5 sperm cells in 8 group N fraction a sperm cells were methylated to varying degrees in this region with a very significant P value of 0(< 0.01). Therefore, we speculate that the methylation level of sperm in fraction a at this site may be a molecular marker for diagnosing whether a subject has asthenospermia.

TABLE 1 methylation levels of the promoter region of the PLEKHG5 gene in 8 healthy people of group N

4. Clinical validation

We additionally recruited 50 healthy volunteers and 30 asthenospermic volunteers, whose semen samples were collected for clinical validation.

Sperm cells having different viability were separated by the following method: centrifugation at 300 Xg for 20min produced non-continuous density gradient fractions (divided into 47.5, 57, 76 and 95% concentration layers), high-viability sperm cells were collected from the 95% concentration layer (fraction a), and low-viability sperm cells were collected from between the 57% and 76% concentration layers (fraction c). The collected sperm was washed twice with PBS, and genomic DNA was extracted.

Primers were designed and site specific bisulfite sequencing (LBS) was used to detect methylation rates in the region between 6545514-6548214 of chromosome 1. The method comprises the following steps:

bisulfite treatment was performed using the EZ DNA Methylation-Direct Kit (Zymo Research). Carrying out denaturation on the genome DNA at 99 ℃ for 8min, and then immediately adding a bisulfite solution to incubate at 64 ℃ for 3.5h for conversion; the converted genomic DNA was recovered by a recovery column and used as a template for PCR.

Site-specific PCR was performed using HotStarTaq Master Mix (Qiagen). According to the region to be detected, two pairs of primers (table 2) are respectively designed for amplification and sequencing, the length of an amplified fragment of each pair of primers is about 2kb, an ABI3730 sequencer is used for completing the amplification product after the amplification product is recovered, and the methylation state is detected by comparing C-T conversion.

TABLE 2 PCR primers

The results are shown in fig. 2, 38 of the 50 healthy human fraction a sperm cells had different degrees of methylation in this region, accounting for 76% of all healthy human sample fraction a samples, and 30 asthenospermia sperm cells had all methylation sites in this region that were not methylated, with a very significant P value of 0(< 0.01). Thus, when all of the methylation sites in this region of the subject are unmethylated, there is a greater probability that the subject will have asthenospermia. It can be seen that the presence or absence of methylation in the promoter region (-2711 to-11) of the PLEKHG5 gene can be used as an auxiliary criterion for diagnosing whether a subject has asthenospermia.

It should be noted that GRCh37/hg19 is used herein as a reference genome to locate the relevant region, and other reference genomes in the art, and the indication of the relevant region may be different from that in the present disclosure, but it is within the scope of the present invention as long as the final alignment by the blast-like alignment tool corresponds to the region indicated in the present study. Similarly, although SEQ ID NO 1 is shown as an example, it will be clear to those skilled in the art that the sequences in the genome are not completely conserved and that variations are possible, and that the sequence detected need not be identical to SEQ ID NO 1, and only a final alignment by blast et al to SEQ ID NO 1 is required to be considered as falling within the scope of the present invention.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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