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Pathogenic gene mutation of hypothyroidism and diagnostic reagent based on same

A technology of thyroid function, diagnostic kit, applied in the field of medical diagnosis

Active Publication Date: 2017-11-24
JINAN CENTER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the application of SNP in the diagnosis of hypothyroidism. If the SNP of hypothyroidism can be screened out as a biomarker and a corresponding diagnostic kit can be developed, it will definitely promote the early diagnosis of hypothyroidism in my country. and open up new ways for drug screening, drug efficacy evaluation and targeted therapy

Method used

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  • Pathogenic gene mutation of hypothyroidism and diagnostic reagent based on same
  • Pathogenic gene mutation of hypothyroidism and diagnostic reagent based on same
  • Pathogenic gene mutation of hypothyroidism and diagnostic reagent based on same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 sample acquisition

[0042] The inventor discovered a family with four generations of hypothyroidism (referred to as hypothyroidism), and the clinical information of some members of the family with overt hypothyroidism is shown in Table 1. There are patients with hypothyroidism in the four generations of the family, and it is speculated that the disease is a dominant inheritance; in addition, there are females and males in the patients, so it is speculated that the disease is autosomal inheritance, which has nothing to do with gender. figure 1 Pedigrees for overt hypothyroidism are shown, where ● or ■ indicate affected individuals.

[0043] Diagnostic criteria:

[0044] You can refer to the 2007 edition of "Guidelines for Diagnosis and Treatment of Thyroid Diseases in China":

[0045] (1) Clinical manifestations: early stage patients with mild disease may have no specific symptoms. Typical patients experience chills, fatigue, swelling in hands and feet, ...

Embodiment 2

[0053] Example 2 Exon sequencing

[0054] 1. Instruments and equipment are shown in Table 2.

[0055] Table 2 Instruments and equipment

[0056]

[0057]

[0058] 2. Reagent consumables

[0059] Agilent SureSelect Human All Exon Kit (Agilent), Agilent DNA 1000Kit (Agilent), 1.5ml centrifuge tube (Eppendorf), 0.2ml centrifuge tube (Eppendorf), tip (Axygen), 96-well plate (Axygen), BigDye v3.1 (Thermo), absolute ethanol, POP7 gel (Thermo), capillary electrophoresis Buffer (Thermo), Sequence Standard (3500) (Thermo), blood genomic DNA extraction kit (TIANGEN), D2000 DNA Marker (TIANGEN), agarose (SunShineBio ), EB (Amresco).

[0060] 3. Reagent formula

[0061] 3.1 TBE electrophoresis solution

[0062] 5×TBE electrophoresis solution was prepared according to Table 3.

[0063] Table 3 Formula of 5×TBE electrophoresis solution

[0064] Reagent

volume

Tris

54g

boric acid

7.5g

EDTA (pH 8.0, 0.5mol / L)

20mL

wxya 2 o

...

Embodiment 3

[0106] Example 3 Sanger sequencing verification

[0107] Because exome sequencing has a certain degree of false positives, we further used Sanger sequencing to detect CHRNA1:NM_000079:exon6:c.G605A:p.R202Q and TRPM8:NM_024080:exon12:c.G1442C:p. The G481A site was verified. The CHRNA1:NM_000079:exon6:c.G605A:p.R202Q site and TRPM8:NM_024080:exon12:c.G1442C were performed on the 5 patients, 8 normal persons in the family line and 100 normal persons outside the family line in Example 1 respectively :p.G481A site genotype detection.

[0108] The specific method steps are as follows:

[0109] (1) DNA extraction

[0110] Genomic DNA in peripheral blood leukocytes was extracted according to the method in Example 1.

[0111] (2) Primer design

[0112] Primers were designed with reference to the human genome sequence database hg19 / build36.3.

[0113] The primer sequences for the CHRNA1:NM_000079:exon6:c.G605A:p.R202Q site are as follows:

[0114] 5'-AAACCTCACTTCCTTTTCTCAGGA-3' (...

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PUM

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Abstract

The invention discloses pathogenic gene mutation of hypothyroidism. According to the invention, exome sequencing technology firstly reveals that mutation at the site CHRNA1: NM_000079: exon 6: c. G605A: p. R202Q and site TRPM8: NM_024080: exon 12: c. G1442C: p. G481A may lead to hypothyroidism. Research results of the invention can be used for early screening of carriers of the pathogenic gene mutation of hypothyroidism, providing guidance for prenatal and postnatal care, and providing molecular diagnosis bases for patients with hypothyroidism.

Description

technical field [0001] The invention belongs to the field of medical diagnosis, and relates to a hypothyroid disease-causing gene mutation and a diagnostic reagent based on the gene mutation. Background technique [0002] High-throughput sequencing technology (High-throughput sequencing) refers to a sequencing technology that can perform sequence determination on hundreds of thousands to millions of DNA molecules in parallel at a time, and the read length of each sequence determination is generally short. High-throughput sequencing technology is a revolutionary change to traditional sequencing. With the rapid development of Roche's 454 sequencer in 2005 and the Solexa sequencer in 2006, high-throughput sequencing technology rose in China around 2009. There are many types of high-throughput sequencing, such as genome re-sequencing, whole exon sequencing, and transcriptome re-sequencing (also known as RNA-Seq). [0003] Whole exon sequencing (whole exon sequencing) refers to ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 姜强
Owner JINAN CENTER HOSPITAL
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