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Skin keratinization-less hair-leukonychia syndrome new disease-causing gene and coding protein and application thereof

A syndrome and protein technology, applied in the mutated GJA1 gene and its encoded protein, to screen biological samples susceptible to the disease, the new pathogenic gene of keratinization-hypo-leukonychia syndrome-GJA1 gene field, It can solve problems such as unclear pathogenic genes

Active Publication Date: 2014-12-31
BGI GENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, the disease was classified as autosomal dominant palmoplantar keratosis-hypotrichosis syndrome (OMIM 104100), and the causative gene is still unclear

Method used

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  • Skin keratinization-less hair-leukonychia syndrome new disease-causing gene and coding protein and application thereof
  • Skin keratinization-less hair-leukonychia syndrome new disease-causing gene and coding protein and application thereof
  • Skin keratinization-less hair-leukonychia syndrome new disease-causing gene and coding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Sample acquisition

[0048] The inventor has collected a KHLS family in China in recent years. There are three generations in the family, including normal individuals and KHLS patients. In this family, 4 patient samples and 2 normal family samples were selected as research samples. A total of 6 samples were taken as research samples. 2ml of peripheral blood samples were collected from each sample, anticoagulated with EDTA, and stored at -80°C.

[0049] Randomly collect 100 normal individuals unrelated to the family 1 and family 2 as secondary verification samples, collect 2ml of peripheral blood samples from each, add EDTA anticoagulant, and store at -80°C.

Embodiment 2

[0050] Embodiment 2: sample DNA preparation

[0051] 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:

[0052] (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.

[0053] (2) 65 degrees Celsius water bath for 15-20 minutes, and vortex 5 times during the water bath.

[0054] (3) Add 2.2ml of absolute ethanol, vortex at maximum speed for 30 seconds, and mix thoroughly.

[0055] (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.

[0056] (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 liqu...

Embodiment 3

[0064] Example 3: Exon capture and sequencing

[0065] The inventor used the Agilent SureSelect Human All Exon Kit (Agilent SureSelect Human All Exon Kit) combined with Solexa high-throughput sequencing technology to sequence the exome sequence of the sample selected in Example 1, as follows:

[0066] 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 / ) .

[0067] 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 Hiseq 2000, the read length is 90bp, and the average sequencing depth of each sample is at least 50×.

[0068]...

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PUM

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Abstract

The present invention identifies skin keratinization-less hair-leukonychia syndrome new disease-causing gene-GJA1 gene. Specifically, skin keratinization-less hair-leukonychia syndrome diseased family is taken as a study object, by exon group sequencing and comparison of a diseased individual and a non-diseased individual in the family, the GJA1 gene is found to be the skin keratinization-less hair-leukonychia syndrome disease-causing gene, the following a plurality of pathogenic mutations of c.23Ggt; T (p.Gly8Val), c.412Ggt; C (p.Gly138Arg) and C.689-690delAT (p.Tyr230Cysfs * 7) are found in the GJA1 gene, and the pathogenic mutations cause GJA1 protein translation errors or early interrupts. On the basis, the invention provides mutant GJA1 gene and a coding protein thereof, a carrier containing the mutant GJA1 gene, host cells, and a system, a kit and a method for screening biological samples which are susceptible to the disease. Through use of the mutant GJA1 gene, molecular diagnosis and disease risk evaluation of the skin keratinization-less hair-leukonychia syndrome can be performed, and the mutant GJA1 gene and the coding protein can be used as drug targets for treatment of the skin keratinization-less hair-leukonychia syndrome.

Description

technical field [0001] The invention relates to a human body variation gene, in particular to a new pathogenic gene-GJA1 gene of skin keratosis-hypohair-leukonychia syndrome. The present invention also relates to the mutated GJA1 gene and its encoded protein, vectors comprising the mutated GJA1 gene, host cells, and systems, kits and methods for screening biological samples susceptible to the disease. Background technique [0002] Keratoderma-Hypotrichosis-Leukonychia Totalis Syndrome (KHLS) is an extremely rare autosomal dominant skin disease, including the work of the inventor, and only 3 cases have been reported so far (including 2 familial cases). Cutaneous keratosis-hypotrichosis-leukonychia syndrome mainly manifests as thin hair and white nails shortly after birth, and gradually follicular keratotic papules appear on the trunk, extremities, and perianal area, which may be accompanied by well-defined erythema. Follicular keratotic papules can coalesce at the site of f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/47C07K16/18C12Q1/68C12M1/00C12M1/34A61K39/395A61P17/00
Inventor 戴兰兰管李萍张建国杨勇林志淼赵嘉惠汪慧君
Owner BGI GENOMICS CO LTD
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