New mutant disease-causing gene of Alport syndrome, encoded protein and application thereof

A technology of gene coding and syndrome, applied in the field of mutated disease-causing genes, can solve the problems of children's kidneys not being observed and the difficulty of rapid diagnosis, etc.

Active Publication Date: 2014-12-17
石家庄华大医学检验实验室有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The typical pathological changes are the ultrastructural changes of the renal basement membrane (thinning, thickening, and rupture), but the pathological ch

Method used

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  • New mutant disease-causing gene of Alport syndrome, encoded protein and application thereof
  • New mutant disease-causing gene of Alport syndrome, encoded protein and application thereof
  • New mutant disease-causing gene of Alport syndrome, encoded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Sample acquisition

[0047] The inventor has collected two ATS families in China in recent years. Family 1 has 4 generations of 30 Han family members ( figure 1 ), showing autosomal dominant inheritance, family 2 has 4 generations of 10 Han family members ( figure 2 ), showing X-chromosomal dominant inheritance, all family members in family 1 and family 2 underwent routine urine and renal function tests. Both the proband of family 1 (III:12, case 1) and the proband of family 2 (III:1, case 2) were found to have total and segmental sclerosis and mesangial expansion by renal biopsy, and glomerular basement membrane was found by electron microscopy (GBMS) showed irregular thickening and splitting, based on clinical and biochemical indicators and imaging evidence, combined with the familial cases we collected, it suggested that ATS was more likely. 10 patient samples were selected in family 1 (II:1, II:3, II:5, II:7, III:7, III:9, III:10, III:12, III:14, and I...

Embodiment 2

[0049] Embodiment 2: sample DNA preparation

[0050] OMEGA Blood DNA Midi Kit whole blood DNA extraction kit was used to extract DNA from peripheral blood samples, and the extraction steps were as follows:

[0051] (1) Take 2ml whole blood sample, add 150ul OB Protease, 2.1ml Buffer BL and 20ul RNase A, vortex at maximum speed for 1 minute, and mix thoroughly.

[0052] (2) 65°C water bath for 15-20 minutes, and vortex 5 times during the water bath.

[0053] (3) Add 2.2ml of absolute ethanol, vortex at maximum speed for 30 seconds, and mix thoroughly.

[0054] (4) Transfer 3.5ml of the lysate into a 15ml centrifuge tube with a filter column, centrifuge at 4000 rpm for 5 minutes, take out the filter column, pour off the filtered liquid, and put it back into the filter column.

[0055] (5) Add the remaining lysate from step 3 into a 15ml centrifuge tube with a filter column, centrifuge at 4000 rpm for 5 minutes, take out the filter column, pour off the filter liquid, and put it...

Embodiment 3

[0063] Example 3: Exon capture and sequencing

[0064] The inventor used Agilent SureSelect Human All Exon Capture Kit (Agilent SureSelect Human All Exon Kit) in combination with Solexa high-throughput sequencing technology to sequence the exome sequence of the sample of Example 1 selected, as follows:

[0065] 1) Genomic DNA was randomly broken into fragments of about 150-200bp, and then adapters were connected to both ends of the fragments to prepare a hybrid library (see the Illumina / Solexa standard library construction instructions provided by http: / / www.illumina.com / ) .

[0066] 2) After the library is purified, it undergoes ligation-mediated PCR (ligation-mediated PCR (LM-PCR)) linear amplification and SeqCap EZ Oligo pool for hybridization enrichment, and then performs sequencing on the machine after linear amplification of LM-PCR . The sequencing platform is Illumina Hiseq2000, the read length is 90bp, and the average sequencing depth of each sample is at least 50×. ...

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Abstract

In the invention, two new mutations of two disease-causing genes relative to Alport syndrome are identified through an exon sequencing method. The inventor, by employing two ATS families as research objects, particularly performs exon sequencing and comparison respectively to diseased individuals and non-diseased individuals in the two ATS family and finds a frame shift deletion mutation (c.3213delA,p.Lys1071fs*5) on a COL4A4 gene of one family and a frame shift deletion mutation (c499delC,p.Pro167Glnfs*36) on a COL4A5 gene of the other family, wherein the two frame shift deletion locus are actual disease-causing locus of the two families. On the basis of above, the invention provides a mutant COL4A4 gene and a mutant COL4A5 gene, encoded protein and application thereof and includes carriers, host cells and kits of the mutant COL4A4 gene and/or the mutant COL4A5 gene. By means of the mutant COL4A4 gene and/or the mutant COL4A5 gene, molecular diagnosis and diseasing risk evaluation of the Alport syndrome can be carried out. The two mutant genes and the encoded proteins thereof also can be used as medicine targets for treating the Alport syndrome.

Description

technical field [0001] The invention relates to a human body mutated gene, in particular to a new mutation-causing gene of Alport syndrome. The invention also relates to the coding protein and application of the new mutation-causing gene of Alport syndrome, the carrier, host cell and kit containing the mutation-causing gene of Alport syndrome. Background technique [0002] Alport syndrome (Alport syndrome, ATS) is a genetic disease characterized by familial progressive hemorrhagic nephritis, eye disease and deafness, with an incidence of about 1 / 5000. In 1927, Alprot first reported the disease, and the affected family The patient showed progressive hematuria and deafness. The male patient died of uremia at an early age, and the female patient survived longer. The first symptom of ATS is generally renal symptoms, which manifests as periodic microscopic or gross hematuria in childhood, and the onset of male patients is earlier than that of female patients. Other symptoms inc...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/78C12Q1/68C07K16/18A61K39/395A61P13/12A61P27/02A61P27/16
Inventor 肖晶晶管李萍张建国邓昊徐洪波宋治郑文
Owner 石家庄华大医学检验实验室有限公司
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