CN110714066A - DNA library for detecting and diagnosing corneal dystrophy disease-causing gene and application thereof - Google Patents
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Abstract
The invention relates to a DNA library for detecting and diagnosing corneal dystrophy disease-causing genes and application thereof, wherein the library comprises 24 corneal dystrophy related disease-causing genes, the 24 corneal dystrophy disease-causing genes are preferably selected, a probe pool is designed, a target region library aiming at the 24 corneal dystrophy disease-causing genes is established, the library is sequenced by using a high-throughput sequencing technology, and disease-causing mutation is searched for, so that genetic and molecular biological bases are provided for clinical diagnosis. The 24 gene detection regions can detect various common corneal dystrophies including epithelial and sub-epithelial corneal dystrophy, anterior elastic layer corneal dystrophy, stromal corneal dystrophy, posterior elastic layer and corneal endothelial dystrophy, and have important significance and clinical value for diagnosis and differential diagnosis of corneal dystrophy.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a DNA library for detecting and diagnosing corneal dystrophy pathogenic genes by a targeted high-throughput sequencing technology and application thereof.
Background
Corneal dystrophy is a general term for a group of progressive corneal transparency loss and vision-degrading keratopathy with genetic heterogeneity. The disease is mostly inherited with dominant chromosomes, the onset of the disease is more than 20 years old, the eyes are symmetrical, the disease course is slow, and the vision of a patient can be completely lost when the disease at the late stage affects the whole layer of cornea. At present, no specific treatment medicine exists for the disease, and people with serious vision influence can only treat the disease through corneal transplantation operation or laser treatment, but can still relapse after the operation.
The majority of pathological and morphological histological features of corneal dystrophies are the deposition of abnormal substances in different layers of the cornea of both eyes, and therefore the typing of corneal dystrophies has been determined based on the morphology of deposits observed under slit lamps, the affected corneal lamellae and histopathological features. With the progress of the deep knowledge and the molecular genetic characteristics, a plurality of genes related to corneal dystrophy are discovered in sequence. Morphological-based classification deficiencies have emerged, for example, corneal dystrophy due to mutations in the same gene has different phenotypes, and different genetic defects can lead to the same clinical manifestations. The 2008 international classification committee on corneal dystrophy promulgated a new classification of corneal dystrophy, supplemented with relevant genetic, clinical and pathological features based on previous anatomical classification. In addition, the determination of the pathogenic gene of corneal dystrophy provides a theoretical basis for understanding pathogenesis and exploring new treatment methods. Therefore, comprehensive, rapid and accurate screening of the pathogenic genes is an essential prerequisite for accurate diagnosis, prognosis judgment, family consultation plan formulation and personalized treatment of corneal dystrophy.
The clinical conventional diagnosis technology of corneal dystrophy is mainly slit lamp inspection. However, slit-lamp examination is poorly sensitive to early stage lesions and the clinical appearance of some patients is atypical, much less capable of diagnosing patients without yet showing clinical symptoms. Therefore, various corneal dystrophies cannot be accurately distinguished at present by only combining clinical symptoms and slit lamp examination. In particular, some clinical syndromes with corneal dystrophy as one of the phenotypes are easy to miss diagnosis and misdiagnose before the clinical manifestations of the eye do not appear. Therefore, genetic diagnosis has become the only solution to this problem. At present, the traditional Sanger sequencing method is mostly adopted in clinical laboratories to detect gene mutation, and if a plurality of genes related to corneal dystrophy are simultaneously detected, the workload is huge, the detection efficiency is low, more importantly, precious DNA samples are wasted, the detection cost of gene diagnosis is obviously increased, and the large-scale application of the gene diagnosis in clinical molecular diagnosis is severely restricted. Therefore, there is a need to find a new method for detecting corneal 5 dystrophie causative gene mutation, which improves the accuracy of diagnosis, reduces cost and labor intensity, and improves timeliness.
Disclosure of Invention
The invention aims to provide a DNA library for detecting and diagnosing a pathogenic gene of corneal dystrophy by a targeted high-throughput sequencing technology, which can improve the diagnosis accuracy and reduce the cost and labor intensity.
The second object of the present invention is to provide the use of said DNA library.
The purpose of the invention is realized by the following technical scheme: a DNA library for diagnosing corneal dystrophy based on high throughput sequencing technology, the library comprising 24 corneal dystrophy-associated disease genes, wherein said 24 corneal dystrophy-associated disease genes are shown in the following table:
。
the DNA library provided by the invention covers various common types of corneal dystrophy, including epithelial and epithelial corneal dystrophy, anterior elastic layer corneal dystrophy, stromal corneal dystrophy, posterior elastic layer and corneal endothelial dystrophy, and the like, and comprises 24 corneal dystrophy pathogenic related genes. These 24 gene selections are based on the internationally reported and recognized clinical pathogenic gene database (OMIM database, HGMD database, ClinVar database). The pathogenic mutations on the 24 genes can cause diseases alone or in combination to cause more serious and complex clinical phenotypes, and the invention can comprehensively sequence the pathogenic mutations at one time for the first time.
According to the invention, a probe pool which can cover the exon of the gene and the adjacent +/-20 bp intron region is designed according to 24 corneal dystrophy pathogenic genes, a target region library containing the 24 corneal dystrophy pathogenic genes is established by utilizing probe target capture, the library is sequenced by utilizing a high-throughput sequencing technology, pathogenic mutation is searched, the genetic cause of corneal dystrophy is determined, and the theoretical basis of genetics and molecular biology is provided for clinical diagnosis.
The application of the DNA library in preparing a kit for diagnosing corneal dystrophy.
The application comprises the following steps:
1) clinical data and clinical biological samples of patients with corneal dystrophy are collected, preferably, the clinical biological samples refer to various samples derived from human bodies, including but not limited to peripheral blood, body fluid, tissue and organ samples from subjects, such as saliva, hair or oral mucosa of the subjects.
2) The genomic DNA of the sample is extracted, preferably, the extraction method comprises a DNA extraction kit or various manual extraction methods.
3) Quantifying the extracted genomic DNA and constructing a library, preferably, the quantification methods include, but are not limited to, fluorescence quantification methods and electrophoresis; wherein, the library construction comprises the following steps:
a) fragmenting genomic DNA, preferably by methods including but not limited to ultrasonication, transposase cleavage, and restriction endonuclease cleavage;
b) carrying out end repair on the fragmented genomic DNA and simultaneously carrying out 3' end addition of A;
c) ligating the product of adding A at the 3' end to a linker for amplification of the ligation-effective product, preferably from a high throughput sequencing library kit;
d) performing PCR amplification on the ligation product by using a universal primer, and adding a complete joint, wherein the universal primer is preferably from a high-throughput sequencing and library building kit;
e) targeting and capturing a target region by using a probe aiming at the 24 corneal dystrophy related genes, wherein the capture method preferably comprises but is not limited to liquid phase probe capture and solid phase chip hybridization capture;
f) washing off the uncaptured library, and only reserving the target area library;
j) obtaining a target region capture library.
4) The library is subjected to a quantitative procedure, preferably, the quantitative method includes, but is not limited to, a fluorescent quantitative method and electrophoresis.
5) Performing high throughput sequencing of the library using a sequencing device, preferably, the sequencing device includes, but is not limited to, a Novaseq series, a Hiseq series, a Nexeseq series, a BGIseq series second generation nucleic acid sequencer;
6) and comparing the obtained sequencing data by bioinformatics, and obtaining the related information of the pathogenic site by mutation interpretation.
A diagnostic kit comprising said DNA library.
Compared with the prior art, the invention has the advantages that:
1. corneal dystrophy is a genetic blinding disease seriously harming visual health, and is a primary, progressive, binocular and blinding corneal lesion. The determination of the pathogenic gene of corneal dystrophy can not only understand the function of the disease-related protein, but also provide more useful clinical information for patients with corneal dystrophy, for example, the screening and analysis of the pathogenic gene can help to make accurate diagnosis of clinical suspected cases and atypical phenotypes. The invention aims to explore the genetic cause of corneal dystrophy, thereby helping to understand pathogenesis, assisting clinical diagnosis, prognosis judgment and prenatal diagnosis and laying a foundation for gene therapy in the future.
2. The gene diagnosis is helpful for clinical genetic counseling, prenatal diagnosis and the like. Because corneal dystrophy has great harm to the visual health of individuals, and clinical heterogeneity and expression difference exist in diseases, gene detection can provide more genetic information for families of patients. Through clinical genetic consultation, family members can know the possibility of suffering from diseases; parents or the patients can guide the birth through gene detection, and the health of the next fetus in the future is ensured. Corneal dystrophy is mostly frequently-stained dominant inheritance, but also has an autosomal inheritance pattern, and accurate health and reproduction guidance can be provided only by performing gene detection. Finding carriers of asymptomatic young virulence genes in families also helps to develop home medical and rehabilitation programs earlier.
3. The DNA library of the application refers to a large number of documents on the basis that the inventor carries out high-throughput gene detection service for many years and accumulates a large number of clinical cases of corneal dystrophy in China, and 24 genes are selected from a large number of corneal dystrophy pathogenic genes by adopting a disease-gene correlation screening method provided by ClinGen and other power-welfare databases. The selected gene gives consideration to comprehensiveness, accuracy and scientificity. The invention preferably selects 24 corneal dystrophy pathogenic genes, designs a probe pool, establishes a target region library aiming at the 24 corneal dystrophy pathogenic genes, and utilizes a high-throughput sequencing technology to sequence the library, search pathogenic mutation and provide genetic and molecular biological basis for clinical diagnosis. The 24 gene detection regions can detect various common corneal dystrophies including epithelial and sub-epithelial corneal dystrophy, anterior elastic layer corneal dystrophy, stromal corneal dystrophy, posterior elastic layer and corneal endothelial dystrophy, and have important significance and clinical value for diagnosis and differential diagnosis of corneal dystrophy.
4. The invention adopts a high-throughput sequencing technology to sequence the target region capture library, and can simultaneously detect all exons and adjacent regions thereof of 24 related pathogenic genes involved in the invention in one sequencing reaction. Compared with the traditional sequencing technology, the method has the advantages of obviously improved detection efficiency and obviously reduced cost, has great advantages in the aspects of gene mining and pathogenic gene screening of corneal dystrophy, and is an efficient, reliable and economic corneal dystrophy gene detection technology.
5. In conclusion, the DNA library and the application thereof have the characteristics of accuracy, flexibility, rapidness and low cost; through clinical evaluation, the invention has good auxiliary diagnosis value on the corneal dystrophy.
Drawings
FIG. 1 is a diagram showing quality control information for creating a target region-targeting DNA library for 24 corneal dystrophy genes in proband samples in example 1.
FIG. 2 is data coverage information for sequencing the target region of the 24 corneal dystrophy genes of proband samples in example 1.
Fig. 3 is data amount information for sequencing a target region of 24 corneal dystrophy genes in the proband sample in example 1.
Fig. 4 is a family map of a family with decreased vision in example 1, in which arrows indicate probands, solid icons indicate patients and those with abnormal vision test, and open icons indicate healthy individuals.
Detailed Description
The invention provides a DNA library for detecting and diagnosing corneal dystrophy disease-causing genes and application thereof, which are further described in the following with reference to specific embodiments.
Unless otherwise indicated, the techniques used in the examples are conventional and well known to those skilled in the art, and may be performed according to the fourth edition of the molecular cloning, laboratory Manual, or related information, and the reagents and products used are also commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources, trade names, and components of the reagents used are indicated at the time of first appearance, and the same reagents used thereafter are the same as those indicated at the first appearance, unless otherwise specified.
Example 1:
in this embodiment, a Hiseq sequencing platform of Illumina corporation is used to detect genomic DNA of peripheral blood of a human subject, and the specific implementation steps are as follows:
1. sample source
A family of vision deterioration from Fujian province in China, wherein the predecessor is a 52-year-old male, and the vision deterioration is gradually caused by the repeated appearance of corneal epithelial erosion symptoms such as redness, ophthalmalgia, photophobia and lacrimation about 20 years ago. And (3) slit lamp inspection: irregular elliptical clouding of the edges was visible under the central epithelium and superficial stroma of the cornea in both eyes. The 25-year-old daughter of the probate has no obvious clinical symptoms, but the slit lamp examination result shows that the upper subcutaneous part of the cornea of both eyes is scattered in a punctate turbid state, and the eye examination of the 23-year-old son and the probate wife of the probate is not abnormal. The family of the patient is shown in fig. 4, with the arrows pointing to probands, the solid icons representing patients and the open icons representing healthy individuals. Informed consent was obtained from all participants and 10ml of venous blood (EDTA anticoagulated) was collected from the elbow vein of family members of this example for testing.
2. Extraction of specimen genomic DNA:
genomic DNA extraction kit (HiPureblood) from magenta was used according to the instructions provided by the trade company&Tissue DNA Kit) genomic DNA was extracted from peripheral blood samples, the purity of the DNA was measured using Nanodrop one, and OD of the genomic DNA obtained260nm/OD280nmAll are located between 1.7 and 2.0, and the concentration of the DNA is measured by using Nanodrop one, and the concentration of the obtained genomic DNA is 50 to 100 ng/. mu.L, and the total amount is 5 to 10. mu.g.
3. Establishing a genome amplification library:
according to the instructions provided by the trade company, the Kit of KAPA company (KAPA Hyperplus library preparation Kit) is used for carrying out enzyme digestion and fragmentation, end repair, 3' end A addition, linker ligation and PCR amplification on genomic DNA, and finally, the obtained library is quantified and quality checked, and the specific implementation steps are as follows:
1) and (3) carrying out genome DNA fragmentation reaction, wherein the reaction system is as follows:
| name of reagent | Dosage (mu L) |
| Genomic DNA (100ng) | 1 |
| NF H2O | 16.5 |
| KAPA Frag Buffer | 2.5 |
| KAPA Frag Enzyme | 5 |
| |
25 |
Reaction conditions are as follows: the reaction was carried out at 37 ℃ for 12 min.
2) End repairing and 3' end adding A reaction, wherein the reaction system is as follows:
| name of reagent | Dosage (mu L) |
| Fragmented DNA in step 1) | 25 |
| KAPA End Repair&A-Tailing Buffer | 3.5 |
| KAPA End Repair&A-Tailing Enzyme Mix | 1.5 |
| Total volume | 30 |
Reaction conditions are as follows: reacting at 20 ℃ for 1 min; reacting at 65 ℃ for 30 min; keeping the temperature constant at 20 ℃.
3) Linker linking reaction, the reaction system is as follows:
reaction conditions are as follows: the reaction was carried out at 20 ℃ for 20 min.
4) The first PCR amplification reaction comprises the following reaction systems:
| name of reagent | Dosage (mu L) |
| DNA obtained in step 3) | 16 |
| KAPA HiFi HotStart Ready Mix(2×) | 20 |
| T5*Primer(10μM) | 1.5 |
| T8*Primer(10μM) | 1.5 |
| Total volume | 39 |
Reaction conditions are as follows: 45s at 98 ℃; (98 ℃ for 15s,60 ℃ for 30s,72 ℃ for 30 s). times.6 cycles; 1min at 72 ℃; storing at 12 deg.C.
5) DNA quantification and quality control:
measuring the concentration of the PCR amplification product by using Nanodrop one, preparing 2% agarose gel, mixing the obtained PCR amplification product with a loading buffer solution, and observing the position, brightness and uniformity of a band by electrophoresis, wherein the size of the band is between 200 and 800bp, and the band is qualified.
4. Constructing a target region targeting library based on probe hybridization capture:
according to the instruction operation provided by the trade company, the library construction kit of IGT company is adopted for the library construction, the probe is designed according to the candidate 24 cornea malnutrition pathogenic gene sequence, and the synthesis and biotin labeling are carried out, and the specific implementation steps are as follows:
1) and (3) probe hybridization:
mixing 5 mu g of PCR product obtained in the step 3 with 5 mu L of Cot-1human DNA, and carrying out vortex oscillation; adding 2.5 × Ampure XP beads according to the total volume, uniformly mixing and centrifuging, and standing for 5min at room temperature; the magnetic beads are centrifugally washed for 2 times by 80% ethanol, and are eluted by a hybridization solution for hybridization reaction, wherein the system of the hybridization solution is as follows:
hybridization conditions: reacting at 95 ℃ for 10 min; 65 ℃ overnight.
2) The probes hybridized with the sample target sequences were captured on magnetic beads by binding biotin to Streptavidin using 80. mu.L of Streptavidin-labeled magnetic beads (M-270Streptavidin beads). The beads were resuspended in 20. mu.L NF Water using 200. mu.L of 1 × Bead WashBuffer washed three times in sequence at 65 ℃ and room temperature, 3min each.
3) And carrying out a second PCR amplification reaction on the captured target sequence, wherein the reaction system is as follows:
| name of reagent | Dosage (mu L) |
| KAPA HiFi Hot start Readymix(2×) | 25 |
| X Gen Library Amplification primer-Ts Mix | 5 |
| DNA with magnetic beads obtained in step 2) | 20 |
| |
50 |
Reaction conditions are as follows: 45s at 98 ℃; (98 ℃ for 15s,60 ℃ for 30s,72 ℃ for 30s)7 cycles; 1min at 72 ℃; storing at 4 ℃.
4) And (3) PCR product quantification and quality inspection:
measuring the concentration and the strip distribution condition of the PCR amplification product by using Nanodrop one; preparing 2% agarose gel, mixing the obtained PCR amplification product with the sample loading buffer solution, and observing the position, brightness and uniformity of the band by electrophoresis, wherein the size of the band is qualified between 200 and 800 bp.
5. Generating information analysis and variant interpretation of sequencing data:
NGS sequencing results were aligned to the human reference genome UCSC NCBI37/hg19 using Novocraft Novoalign to obtain a unique aligned sequence aligned to the genome. The VarScan mpileup2snp and VarScan mpileup2indel detection was used to determine the variation of the 24 corneal dystrophia-related disease-causing gene regions. Common variations in dbSNP and ExAC databases were removed using Remove RunCommon Variants and Remove Global Common Variants software. The variants were then annotated using Interactive Biosoftware Alamut Batch. The database used for annotation includes: dbSNP, ExAC, 1000g, ClinVar, OMIM, etc., and utilizes the software FATHMM, FATHMMMKL, METALR, METASVM, MUTATIONASSESSOR, MUTATIONTASTERAGGGD, AGVGD, LRT, PROVEAN, SIFT to predict the variant function. According to the ACMG genetic variation classification standard and guideline, mutation sites which are meaningful for diagnosing the corneal dystrophy are obtained by analysis.
6. Sequencing results interpretation and analysis
Through the DNA library for detecting and diagnosing the corneal dystrophy causing gene provided by the invention, 24 corneal dystrophy related genes of 4 testees are sequenced at one time. Taking prover as an example, through quality control analysis, the size of the obtained DNA library is mainly distributed in 200-800bp, the average length is 346bp, and the concentration and the fragment size meet the sequencing requirement, thereby prompting that the quality of the library construction is qualified (as shown in FIG. 1). The DNA library comprises coding regions of the 24 genes with the length of 60171bp, non-coding regions with the length of 5455bp, and the total length of 65626 bp. Wherein the coding region has a sequencing coverage of 50X over 98% and 20X over 99% (see FIG. 2). In Total, fragment read data (Total Reads) was obtained 201691 with a match rate (Aligned) of 88.82% (see FIG. 3). The sequencing result meets the data requirement of variation interpretation. Analysis of the sequencing data revealed corneal dystrophy-associated genetic variation associated with the subject's clinical phenotype, as shown in the following table:
by the embodiment, the heterozygous variation of c.535c > T (p.r 179) of TGFBI gene carried by peripheral blood of the examinee and the examinee daughter is found, and finally, the patient is determined to be a patient with corneal dystrophy, and a basis is provided for clinical diagnosis. The asymptomatic predecessor daughter also carries pathogenic gene variation, is a hereditary predecessor, can generate corneal dystrophy in the future, can be used for performing targeted vision protection, and can be used for performing third-generation test-tube babies or prenatal diagnosis if a birth plan exists, so as to ensure healthy offspring. Asymptomatic proband son carries no disease-causing genetic variation and the risk of corneal dystrophy is significantly reduced (see fig. 4).
Reference throughout this specification to the description of "one embodiment," "this embodiment," "an embodiment," or the like, means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention and is not specifically referred to.
It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.
Claims (8)
1. A DNA library for diagnosing corneal dystrophy based on high throughput sequencing technology, comprising: the library comprises 24 pathogenic genes related to the corneal dystrophy, wherein the 24 pathogenic genes related to the corneal dystrophy are shown in the following table:
2. use of the DNA library of claim 1 for the preparation of a kit for the diagnosis of corneal dystrophy.
3. Use according to claim 2, characterized in that: the application comprises the following steps:
1) extracting genomic DNA of a sample of a subject;
2) quantifying the extracted genomic DNA and constructing a library according to the following steps:
a) fragmenting the genomic DNA;
b) adding a base A to the 3' end of the fragmented genomic DNA while performing end repair;
c) connecting a product with a base A added at the 3' end with a joint;
d) performing PCR amplification on the ligation product, and adding a complete linker;
e) targeting and capturing a target region by using a probe aiming at the 24 corneal dystrophy related genes;
f) washing off the uncaptured library, and only reserving the target area library;
j) obtaining a target region capture library;
3) performing quantitative operation on the library;
4) high-throughput sequencing;
5) and (5) analyzing the data to obtain the related information of the pathogenic site.
4. Use according to claim 3, characterized in that: the sample in step 1) is from peripheral blood, body fluid and tissue organ samples of a subject.
5. Use according to claim 3, characterized in that: the quantitative method in the step 2) comprises a fluorescence quantitative method and electrophoresis.
6. Use according to claim 3, characterized in that: the fragmentation method in the step a) comprises ultrasonic crushing, transposase enzyme digestion and restriction enzyme digestion.
7. Use according to claim 3, characterized in that: the quantitative method in the step 3) comprises a fluorescence quantitative method and electrophoresis.
8. A diagnostic kit comprising the DNA library of claim 1.
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| CN114606302A (en) * | 2022-04-08 | 2022-06-10 | 复旦大学附属中山医院 | Method for extracting oral mucosa nucleic acid to perform whole genome high-throughput sequencing |
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