CN107619862B

CN107619862B - Sperm production disorder pathogenic gene NFATC1 for mild anemia men and detection method thereof

Info

Publication number: CN107619862B
Application number: CN201711112876.5A
Authority: CN
Inventors: 黄志玲
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-11-13
Filing date: 2017-11-13
Publication date: 2020-10-13
Anticipated expiration: 2037-11-13
Also published as: CN107619862A

Abstract

The invention provides a mutant form of a pathogenic gene NFATC1 of spermatogenic disorder in men with mild anemia, and the sequence of the mutant form is shown as SEQ ID NO. 1. The invention also provides a detection method of the disease-causing gene NFATC1 for male spermatogenic dysfunction in mild anemia, which is based on the amplification of the NFATC1 disease-causing gene fragment and Sanger sequencing thereof and mainly relates to an amplification primer pair shown by SEQ ID NO. 2-SEQ ID NO. 3 sequences. The NFATC1 pathogenic gene form provided by the invention is not reported, and can provide a basis and lay a foundation for analysis of the pathogenic mechanism of spermatogenic disorder of mild anemia men, detection of pathogenic genes, targeted prevention and treatment and the like; the constructed NFATC1 pathogenic gene detection method has the characteristics of accuracy, reliability, simplicity, convenience and quickness.

Description

Sperm production disorder pathogenic gene NFATC1 for mild anemia men and detection method thereof

Technical Field

The invention relates to a gene and a detection method thereof, in particular to a spermatogenesis deficiency pathogenic gene NFATC1 of men with mild anemia and a detection method thereof.

Background

About 5% of men of childbearing age have infertility problems, 40% of which are sperm quality abnormalities, mainly manifested by spermatogenic disorders such as oligospermia, azoospermia and poor sperm motility. Male fertility depends on functional sperms, and the processes of generation and development of the sperms in the testis are controlled by a plurality of related genes, and the abnormal occurrence of the genes can influence the formation of the sperms to cause male sterility. Mutations, insertions/deletions and genetic polymorphisms of spermatogenesis-associated genes are currently considered to be important genetic causes of male spermatogenesis deficiency, however, most of them have not been resolved. Therefore, it is necessary to explore the characteristics of male spermatogenic disorder pathogenic genes, which is the basis for disclosing the male sterility genetic mechanism, pathogenic gene detection and targeted prevention and treatment. In recent years, with the development of next-generation sequencing technologies and the reduction of sequencing costs, research on disease-causing genes at the genome level can be realized. However, since diseases often have complexity, the secondary sequencing results must be combined with basic signs and clinical information to better analyze the characteristics of pathogenic genes from specific disease subpopulations.

From the sequencing results of the whole exome of a sample of 25 patients with spermatogenic dysfunction, the applicant found that 4 patients showed the c.19839C > G mutation of NFATC1 gene, and the 4 patients all had mild anemia. Through further verification, the applicant of the invention firstly discloses that the gene NFATC1 is a gene related to spermatogenic disorder of mild anemia men and provides a pathogenic mutation form of the gene, and a mutation detection method based on PCR amplification and Sanger sequencing is constructed on the basis.

Disclosure of Invention

The invention provides a pathogenic gene corresponding to a spermatogenic disorder subgroup of men with mild anemia, and a method for detecting the pathogenic gene is constructed on the basis.

The technical scheme adopted by the invention for solving the technical problems is as follows:

one of the purposes of the invention is to provide a male spermatogenic disorder pathogenic gene NFATC1 for mild anemia, wherein the sequence of the fragment in which the mutation is located is shown as SEQ ID NO. 1, the corresponding mutation is c.19839C > G, and the mutation occurs in Exon 2; the mutant gene form is verified to be a spermatogenesis dysfunction pathogenic gene of men with mild anemia. It is noteworthy that SEQ ID NO 1 exhibits only the fragment sequence in which the mutation is located, the rest of the sequence of the gene is detailed in the wild-type reference gene sequence of accession number NG _029226.1 on GeneBank, and c.19839C > G indicates the 19839 site C mutation to G of the NFATC1 gene.

The disclosure of the pathogenic gene of the spermatogenic disorder of the mild anemia male can be used for preparing a diagnosis chip and a kit for the spermatogenic disorder of the mild anemia male, constructing a detection method for the spermatogenic disorder of the mild anemia male and preparing a medicine for treating the spermatogenic disorder of the mild anemia male.

The invention also aims to provide a method for detecting the mutation of the pathogenic gene of the spermatogenic disorder in the mild anemia men, which mainly comprises the steps of amplifying the NFATC1 pathogenic gene fragment by using the primer pair shown in SEQ ID NO:2-SEQ ID NO:3, and carrying out Sanger sequencing on the amplified product to judge whether the c.19839C > G mutation occurs.

Wherein, the primer pair shown in SEQ ID NO. 2-SEQ ID NO. 3 amplifies the gene segment where the c.19839C > G mutation exists, the amplified segment corresponds to 19711-20087 of the wild-type reference gene sequence with the accession number NG-029226.1 on GeneBank, and the length of the amplified segment is 377 bp. The amplification primer pair SEQ ID NO. 2-SEQ ID NO. 3 of the pathogenic gene NFATC1 fragment can be applied to the preparation of diagnosis chips and kits for male spermatogenic disorders with mild anemia and the construction of male spermatogenic disorder detection methods.

The invention provides a c.19839C > G mutant gene form of male spermatogenic disorder in mild anemia caused by NFATC1 gene and a pathogenic gene detection method based on PCR amplification and Sanger sequencing, which can provide basis and lay the foundation for analysis of pathogenic mechanism, pathogenic gene detection, targeted prevention and treatment and the like.

Drawings

FIG. 1 is the Sanger sequencing map of the NFATC1 gene c.19839C > G mutation amplification fragment of patient P1 in example 1, with the mutation site localized, and the arrow pointing in the middle is the mutation site.

FIG. 2 is a genetic map of the family of patient with spermatogenic dysfunction No. P1 in example 1, and patient No. P1 is II1 in FIG. 2. In the two figures, squares represent males and circles represent females; filled represents spermatogenic impaired individuals, open represents normal individuals, "\" represents signs of mild anemia; the "+" in FIG. 2 indicates that the c.19839C > G mutation was detected in the amplified fragment of NFATC1 gene; "-" indicates that no c.19839C > G mutation was detected in the amplified fragment of NFATC1 gene; i and II represent the 1 st and 2 nd generation, respectively, and the numbers are the member numbers.

Detailed Description

The invention is further illustrated below with reference to specific examples.

Example 1

25 male patients with spermatogenic dysfunction diagnosed in clinical examinations were selected for genetic testing and analysis with informed consent. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures without specifying the specific conditions in the following examples are generally carried out under the conditions as described in the molecular cloning Experimental guidelines, J. SammBruk et al, third edition, scientific Press, or under the conditions recommended by the manufacturers. In addition, the embodiments relate to the diagnosis of patients with spermatogenic disorders such as oligospermia and asthenospermia and the determination of mild anemia and the like in accordance with the existing conventional clinical and physical criteria.

(1) Gene mutation assay for male patients with spermatogenic disorders

Taking 5ml of elbow venous blood of a male patient, extracting the genomic DNA of a sample of the patient to be detected by using a QIAamp blood genome extraction kit of Germany Qiagen, and carrying out whole exon capture sequencing on the large gene of the male patient after the sample is qualified. The whole Exon sequencing library is built by adopting a SureSelect Human All Exon Kit V1 Kit of Agilent company in the United states, a second-generation sequencer is Hiseq2500 of Illumina company in the United states, and the average sequencing depth is more than 500 x. And aiming at the original sequencing data, determining mutation site information by utilizing invalid filtering and polymorphism information of databases such as a dbSNP database, a thousand-person genome database, 8 HapMap exome databases and the like.

(2) Subgroup definition of male spermatogenic disorders of specific signs

A subgroup of 25 male patients with spermatogenic disorders with the same specific gene mutation was selected, while the number of patients in the subgroup was not less than 3 and common signs were found. As a result of control gene mutation, together with information on patients who were examined by inquiry and clinical routine examination, there were found subgroups of patients with mild anemia (4 patients, numbers P1, P2, P3 and P4 in total) and c.19839C containing NFATC1 gene>The specific results of the G mutation are shown in Table 1. P1-P4 the 4 patients are all adult males (average age 27 years), hemoglobin is between 90-120G/L (average hemoglobin is 102G/L, normal male standard is about 120G/L, normal female standard is about 110G/L), and the number of red blood cells is less than 3.4 × 10¹²(Male Normal Standard is about 4.0 × 10¹²Female normal standard is about 3.5 × 10¹²) The hematocrit values were all below 0.4 (about 0.42 for male normal and about 0.37 for female normal), and were all diagnosed as mild anemia. c.19839C of NFATC1 gene of these 4 patients>G mutation, and the sequence of the gene fragment in which the G mutation is located is shown as SEQ ID NO. 1. The remaining 21 patients with non-mild anemia had no c.19839C of NFATC1 gene>The detection of the G mutation can be regarded as a control.

Through literature query and database comparison, the c.19839C > G mutation of the NFATC1 gene of the male spermatogenic disorder with mild anemia is found to be the first report of the invention. The NFATC1 gene is an important member of the NFAT family, and was first recognized as a regulatory factor for T cell activation, and has an important function in regulating immune cells. Subsequent researches find that the factor can regulate and control various genes such as COX-2, Apob and the like closely related to hypertension, atherosclerosis and dyslipidemia. However, no research report about the gene related to spermatogenic dysfunction of men with mild anemia is available.

TABLE 14 Gene test and verification results for patients with male spermatogenic disorder due to mild anemia

Patient numbering	Gene	Mutational events	Sanger sequencing results	Family separation	Sequence of
						P1	NFATC1	c.19839C>G	FIG. 1, in agreement	Is that	SEQ ID NO.1
P2	NFATC1	c.19839C>G	See fig. 1 for consistency	Is that	SEQ ID NO.1
						P3	NFATC1	c.19839C>G	See fig. 1 for consistency	Is that	SEQ ID NO.1
P4	NFATC1	c.19839C>G	See fig. 1 for consistency	Is that	SEQ ID NO.1

Aiming at the c.19839C > G mutation, a PCR primer pair is designed to amplify the gene segment where the PCR primer pair is located. The gene fragment in which c.19839C > G (19711-20087 for the wild-type reference gene sequence corresponding to accession number NG _029226.1 in GeneBank) was amplified using the primer set shown in SEQ ID NO:2-SEQ ID NO:3, the amplification system is detailed in Table 2, and the length of the amplified fragment is 377 bp.

TABLE 2 amplification System

Numbering	Composition of	Adding amount of
			1	10X PCR buffer(Mg²⁺)	2.5ul
2	dNTP Mix(10mM)	2ul
			3	Upstream primer (10uM)	1ul
4	Downstream primer (10uM)	1ul
			5	rTaq	0.2ul
6	Template DNA(30ng/ul)	1ul
			7	Sterilization ddH₂O	17.3ul

The amplification conditions were: pre-denaturation at 94 ℃ for 3 min; 30 cycles: denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 30 s; final extension at 72 ℃ for 5 min.

P1-P4 the products of NFATC1 gene amplification in 4 patients were sent to Shanghai Bioengineering Co., Ltd for Sanger sequencing. Sanger sequencing results were all found to be consistent with the second generation sequencing results (FIG. 1, Table 1).

(3) Family analysis and verification of pathogenic gene mutation

Since the c.19839C > G mutation of the NFATC1 gene is not reported, blood samples of family-related members of the 4 patients with mild anemia male spermatogenic dysfunction are collected from P1-P4, and the NFATC1 gene fragment is subjected to PCR amplification and Sanger sequencing. Taking patient P1 as an example, the patient has the c.19839C > G mutation of the NFATC1 gene. As shown in FIG. 2, patient P1 is II1, and the patient is 30 years old and has been pregnant and diagnosed in marriage 4 years; semen routine analysis shows that the density of sperms in 3 successive semen examinations is less than 1500 ten thousand per milliliter, and the parents are healthy and have 28 years sisters. The menstrual blood of 4 persons in the family is routinely tested, and the patients P1 and the mothers are found to be mild anemia, and the parents are good sisters. Collecting blood samples of parents and sisters of a patient to extract genomic DNA, amplifying the NFATC1 gene fragment by using primer pairs shown in SEQ ID NO:2-SEQ ID NO:3 according to the conditions in the step (2), and Sanger sequencing shows that the mother of the patient also has c.19839C > G mutation, and the father and the sisters do not have the site mutation. The results show that all the patients with mild anemia contain the c.19839C > G mutation of NFATC1 gene, and the men carrying the c.19839C > G mutation simultaneously have spermatogenic disorder, while the women do not produce sperm and therefore do not have the symptom.

The other 3 cases in Table 1, namely P2-P4 patients also have the c.19839C > G mutation of NFATC1 gene (see Table 1 in detail), and the analysis shows that the pedigrees of the patients are also the sign of mild anemia, all the patients contain the c.19839C > G mutation of the NFATC1 gene, and the males carrying the c.19839C > G mutation have spermatogenic disorder at the same time, namely the phenomenon of coseparation of the pedigree with genotype-phenotype appears, thereby proving that the NFATC1 gene is the related gene of the spermatogenic disorder of the mild anemia males, the pathogenic mutation is c.19839C > G, and the sequence of the fragment is shown as SEQ ID NO: 1.

Example 2

In addition, 10 cases of patients with mild anemia and male spermatogenic dysfunction, 20 cases of patients with non-anemia and 10 cases of patients with mild anemia and non-spermatogenic dysfunction were collected, and their blood samples were subjected to genome extraction. For each sample, the NFATC1 disease gene fragment was amplified and the amplified product was subjected to sequencing analysis, according to the system and method in example 1. As a result, only 10 patients with male spermatogenic dysfunction with mild anemia were found to have the c.19839C > G mutation of the NFATC1 gene, and the related data are shown in Table 3.

Table 310 sequencing results of NFATC1 gene fragment of male patients with mild anemia and dysspermia

Sample numbering	Mutational events	Sequence of
			A1	c.19839C>G	SEQ ID NO:1
A2	c.19839C>G	SEQ ID NO:1
			A3	c.19839C>G	SEQ ID NO:1
A4	c.19839C>G	SEQ ID NO:1
			A5	c.19839C>G	SEQ ID NO:1
A6	c.19839C>G	SEQ ID NO:1
			A7	c.19839C>G	SEQ ID NO:1
A8	c.19839C>G	SEQ ID NO:1
			A9	c.19839C>G	SEQ ID NO:1
A10	c.19839C>G	SEQ ID NO:1

The c.19839C > G mutation of the NFATC1 gene appears in 10 patients with mild anemia and spermatogenic disorder in the table 3, while the c.19839C > G mutation of the NFATC1 gene does not exist in 20 patients with non-anemia and 10 men with mild anemia and non-spermatogenic disorder, so that the c.19839C > G mutation form of the NFATC1 gene provided by the invention is proved to be the pathogenic gene of the mild anemia and male spermatogenic disorder from the positive and negative aspects, and the detection method provided by the invention has good reliability and accuracy.

Based on the c.19839C > G pathogenic mutation form of the NFATC1 gene newly identified by the invention, the applicant of the invention purifies the amplicon in which the mutation is located, clones the amplicon into a commercial plasmid and other vectors, then introduces the amplicon into host bacteria such as escherichia coli, and the like, and the bacterial liquid is stored in a freezing tube with the final concentration of 20% (V/V) glycerol and is stored in a refrigerator at minus 80 ℃ for a long time. On the other hand, because of the disclosure of the pathogenic gene sequence, the pathogenic gene sequence can be prepared from a wild type gene by adopting a site-directed mutagenesis mode or obtained by adopting a sequence synthesis mode.

While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the invention is susceptible to various modifications and alternative forms. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Dingzhiling

<120> male mild anemia spermatogenesis dysfunction disease-causing gene NFATC1 and detection method thereof

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>377

<212>DNA

<213> Intelligent (Homo sapiens)

<400>1

ccacaacctt cagacctcca caccgggcat catcccgccg gcggatcacc cctcggggta 60

cggagcagct ttggacggtg ggcccgcggg ctacttcctc tcctccggcc acaccaggcc 120

tgatgggggc cctgccctgg agagtcctcg catcgagata acctcgtgct tgggcctgta 180

ccacaacaat aaccagtttt tccacgatgt ggaggtggaa gacgtcctcc ctagctccaa 240

acggtccccc tccacggcca cgctgagtct gcccagcctg gaggcctaca gagacccctc 300

gtgcctgagc ccggccagca gcctgtcctc ccggagctgc aactcagagg cctcctccta 360

cgagtccaac tactcgt 377

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ccacaacctt cagacctcca 20

<210>3

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

acgagtagtt ggactcgtag g 21

Claims

1. Use of a pathogenic gene in the preparation of a diagnostic kit for spermatogenic disorders in men with mild anemia, characterized in that said pathogenic gene is the NFATC1 gene which has undergone a g.19839C > G mutation.

2. The use according to claim 1, characterized in that the kit comprises primer pairs represented by the sequences SEQ ID NO 2-SEQ ID NO 3.

3. Use according to claim 2, characterized in that the kit amplifies the NFATC1 mutant gene fragment carrying the g.19839c > G mutation under the conditions: pre-denaturation at 94 ℃ for 3 min; 30 cycles: denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 30 s; final extension at 72 ℃ for 5 min.