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Low-frequency SNV (single nucleotide variant) marker related to assisted diagnosis of NOA (non-obstructive azoospermia) with unknown clinical reasons and application thereof

An auxiliary diagnosis and marker technology, applied in the fields of genetic engineering and reproductive medicine

Active Publication Date: 2015-01-07
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no report on the application of low-frequency and high-penetrance genetic markers in the diagnosis of NOA of unknown clinical cause. If the low-frequency SNV of NOA susceptibility of unknown clinical cause can be screened as biomarkers, and corresponding diagnostic reagents can be developed It will be a powerful impetus to the diagnosis status of NOA of unknown clinical cause in my country, and it will also open up a new way for its drug screening, drug efficacy evaluation and targeted therapy.

Method used

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  • Low-frequency SNV (single nucleotide variant) marker related to assisted diagnosis of NOA (non-obstructive azoospermia) with unknown clinical reasons and application thereof
  • Low-frequency SNV (single nucleotide variant) marker related to assisted diagnosis of NOA (non-obstructive azoospermia) with unknown clinical reasons and application thereof
  • Low-frequency SNV (single nucleotide variant) marker related to assisted diagnosis of NOA (non-obstructive azoospermia) with unknown clinical reasons and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The collection of embodiment 1 sample and the arrangement of sample data

[0070] The inventor collected a large number of blood samples of NOA patients from the Reproductive Medicine Center of Nanjing Medical University from April 2005 to January 2013. After sorting out the sample data, the inventor selected 5560 samples that met the following criteria. Experimental samples for microarray scanning and Sequenom MassARRAY genotyping of a single genetic marker:

[0071] 1. Repeated semen quality test / testicular biopsy, diagnosed as azoospermia;

[0072] 2. The semen of one ejaculation was centrifuged without sperm, and the cause of obstruction was ruled out;

[0073] 3. Normal sexual function; patients with known etiologies such as specific history of cryptorchidism, history of blood trauma, orchitis, vasectomy obstruction, vasectomy, polychromosomal abnormalities, and Y chromosome azoospermia factor microdeletion were excluded;

[0074] 4. Healthy male controls matched...

Embodiment 2

[0076] Example 2 Whole Exon Region Scanning of Low-Frequency SNVs in Peripheral Blood DNA

[0077] Among the above-mentioned eligible 960 patients with NOA of unknown clinical cause and 1800 healthy male controls, the two groups were age-matched. These two groups of people were passed through Illumina Human Exome BeadChip (Illumina Inc., San Diego, CA) chip detection obtained relevant results. The specific steps are:

[0078] 1. Add hemolysis reagent (i.e. lysate, 40 parts) to the peripheral blood stored in the 2ml cryopreservation tube. Dilute the TrisHcl solution to 2000ml, the same below), turn it upside down and mix it completely.

[0079] 2. Removal of red blood cells: Fill the 5ml centrifuge tube to 4ml with hemolysis reagent, mix by inverting, centrifuge at 4000rpm for 10 minutes, and discard the supernatant. Add 4ml of hemolysis reagent to the precipitate, invert and wash again, centrifuge at 4000rpm for 10 minutes, and discard the supernatant.

[0080] 3. Extrac...

Embodiment 3

[0090] Example 3 Sequenom MassARRAY Genotyping

[0091] The low-frequency and high-penetrance genetic markers found to be associated with the onset of NOA with clinically unknown causes were detected in another 1400 cases of NOA with clinically unknown causes and 1400 healthy male controls. The specific steps are as follows:

[0092] 1. Add the hemolysis reagent to the peripheral blood stored in the 2ml cryopreservation tube, mix it upside down and transfer it completely.

[0093] 2. Removal of red blood cells: Fill the 5ml centrifuge tube to 4ml with hemolysis reagent, mix by inverting, centrifuge at 4000rpm for 10 minutes, and discard the supernatant. Add 4ml of hemolysis reagent to the precipitate, invert and wash again, centrifuge at 4000rpm for 10 minutes, and discard the supernatant.

[0094] 3. Extract DNA: Add 1ml extract solution to the precipitate (each 300ml contains 122.5ml 0.2M sodium chloride, 14.4ml 0.5M ethylenediaminetetraacetic acid, 15ml 10% sodium dodecyl ...

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Abstract

The invention belongs to the fields of gene engineering and reproductive medicine, and discloses a low-frequency SNV (single nucleotide variant) marker related to assisted diagnosis of NOA (non-obstructive azoospermia) with unknown clinical reasons and application thereof. The marker is a combination of 32 low-frequency SNVs, including rs2294228, rs2180233, rs41360247, rs895497, rs6806528, rs3130785, rs117524265, rs9369640, rs34902660, rs2298090, rs13194053, rs11965377, rs3893259, rs9261588, rs1264429, rs2523480, rs2524276, rs2255221 and the like. The marker can be used for preparing an assisted diagnosis kit for non-obstructive azoospermia with unknown clinical reasons.

Description

field of invention [0001] The invention belongs to the fields of genetic engineering and reproductive medicine, and relates to a low-frequency SNV marker related to auxiliary diagnosis of NOA with unknown clinical cause and application thereof. Background technique [0002] Fertility is a complex process that cannot be completed by a single individual, but depends on both men and women, and can only be successfully completed when various conditions are suitable for both parties. Statistics from WHO show that infertility has become one of the most common diseases in the field of human reproductive health. About one out of every seven couples has various degrees of fertility obstacles, and nearly half of them are caused by male problems. Caused, thus known as male infertility. Male infertility has caused heavy mental and economic burdens to individuals and families, and has become one of the unstable factors of families and society. [0003] Among all the causes of male infe...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 胡志斌沈洪兵沙家豪林苑倪碧娴
Owner NANJING MEDICAL UNIV
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