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Method and kit for diagnosing male infertility caused by piwil1 gene mutation

A male infertility, kit technology, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc.

Active Publication Date: 2021-01-05
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although several SNPs in Hiwi were reported by Gu and colleagues in 2010, there are no related functional studies

Method used

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  • Method and kit for diagnosing male infertility caused by piwil1 gene mutation
  • Method and kit for diagnosing male infertility caused by piwil1 gene mutation
  • Method and kit for diagnosing male infertility caused by piwil1 gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1. Identification of Hiwi D-box mutations in azoospermia patients

[0131] Human Piwil1 gene is also called Hiwi gene. The sequence of wild-type Hiwi mRNA is shown in SEQ ID NO:10; the amino acid sequence is shown in SEQ ID NO:11.

[0132] The present inventor has carried out a large amount of analysis work to human genome sequence, has determined the D-box element coding of azoospermia patient Hiwi gene to straddle the 6th and the 7th exon sequence (about 1kb in full-length 45kb left and right sides) ). The present inventor screened 300 cases of normal people and 413 cases of non-obstructive azoospermia patients, and found that three of the azoospermia patients had mutations in the D-box element, located at the 217th, 220th or 220th position of the HIWI amino acid sequence. 224 bits, such as figure 1 A~C.

[0133] These mutations are located within the D-box sequence element, that is, within the 217-224th amino acid (including 217 and 224) of human PIWIL1 (...

Embodiment 2

[0137] Example 2, Construction and phenotypic identification of conditional gene knock-in D-box mutation mouse model

[0138] Based on the mutation status of patient No. 1, the inventors used mice to construct a mouse model (MIWI D-box mutant mouse) with conditional gene knock-in D-box mutation. The construction strategy was as follows: figure 2 a.

[0139] After analyzing the structure of the MIWI gene, it was found that the point mutation R218A (corresponding to the 217th R in the Hiwi amino acid sequence) was located on exon 7 (ENSMUSE00000313528), and the point mutation L221A (corresponding to the 220th L in the Hiwi amino acid sequence) was located on exon 8 (ENSMUSE00000313519).

[0140] The targeting vector of MIWI conditional point mutation mainly includes the following functional elements: the 5' and 3' loxP sites are inserted into intron5 and intron8 respectively; the exon 6-22 cDNA sequence of Piwil1 gene and 3×stop are located at the 5' and 3' between loxP; posi...

Embodiment 3

[0149] Embodiment 3, Miwi + / DB -Cre mouse sperm protamine replacement histone process is impaired

[0150] The homologue of the human Piwil1 gene in mice is the Miwi gene.

[0151] The inventor is further to Miwi + / DB -Cre mice were analyzed, and it was found that the mature sperm of this genotype mouse had histone retention, and it was speculated that the protamine replacement process of histone was hindered.

[0152] Such as Figure 4 A, Visible by immunofluorescence detection, Miwi + / DB - Histone H2A, H2B, H3, H4 in mature sperm of Cre mice compared with Miwi + / DB Mature spermatozoa in mice all accumulated in large quantities.

[0153] Such as Figure 4 B, Western blotting detection of histone and protamine expression in mature sperm can be seen, Miwi + / DB - Histone H2A, H2B, H3, H4 in mature sperm of Cre mice compared with Miwi + / DB There is a large accumulation of mature sperm in mice, Miwi + / DB -Protamine PRM1 and PRM2 in mature sperm of Cre mice compared with M...

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Abstract

The invention relates to a method and a kit for diagnosing male infertility caused by Piwil1 gene mutation. The invention discloses a gene related to male infertility such as azoospermia, namely the Piwil1 gene. Moreover, the fact that mutation at multiple positions of the Piwil1 gene can cause the occurrence of male infertility is identified. Thus, the Piwil1 gene can be used as a target gene fordiagnosing male infertility, and also can be applied to the development for the treatment medicines effective for the diseases. According to the technical scheme, explanation is given for the mechanism that PIwIl1 participates in the histone substitution by protamine for the first time.

Description

technical field [0001] The invention belongs to the field of gene mutation diagnosis, and more specifically, the invention relates to a method and kit for diagnosing male infertility caused by Piwil1 gene mutation, and a polypeptide capable of treating male infertility. Background technique [0002] Genomic DNA is mostly bound by histones in the form of octamers in eukaryotic cells, but histones will be replaced by protamine in the mature sperm of many animals. Protamine is a class of small, highly alkaline protein molecules that combine with DNA to form a more compact structure, thereby compressing and storing genomic DNA in the sperm head, which is essential for the formation of functional sperm . The replacement of histones by protamine is a complex process that occurs during the post-meiotic metamorphosis, when male germ cells pass through metamorphosis from spherical spermatids to form mature spermatozoa. Although it has been more than a hundred years since the discov...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/47C12Q1/6883G01N33/68A61K38/17A61P15/08
Inventor 刘默芳苟兰涛康俊炎戴鹏王鑫李锋
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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