CN113308527A

CN113308527A - Gene composition, chip and kit for screening refractory hereditary bone diseases

Info

Publication number: CN113308527A
Application number: CN202110255299.5A
Authority: CN
Inventors: 章振林
Original assignee: Shanghai Sixth Peoples Hospital
Current assignee: Shanghai Sixth Peoples Hospital
Priority date: 2021-03-09
Filing date: 2021-03-09
Publication date: 2021-08-27

Abstract

The invention provides a gene composition, a chip and a kit for screening refractory hereditary bone diseases, wherein the gene composition comprises 322 hereditary bone disease related pathogenic genes. Aiming at the requirement of difficult diagnosis and treatment of difficult osteopathia in China, the group of genome compositions related to hereditary osteopathia and related products (gene chips and kits) provided by the invention are developed by combining genome sequencing and big data analysis on the basis of the existing knowledge of hereditary osteopathia and can be used for diagnosing various hereditary osteopathia, and the kit has the advantages that: the probe can be flexibly customized, the target gene and region can be captured specifically, and especially the pathogenic region not contained in WES can also be captured and sequenced; the method can supplement and optimize areas which cannot be captured by WES, achieves better capturing effect, and is high in coverage rate and high in variation detection precision; the cost performance is higher, the clinical detection is quicker, and the cost is lower.

Description

Gene composition, chip and kit for screening refractory hereditary bone diseases

Technical Field

The invention relates to the technical field of biology, in particular to a gene composition, a chip and a kit for screening difficult hereditary bone diseases.

Background

Hereditary osteopathy refers to a hereditary disease with abnormal skeletal development caused by genetic factor change, and the clinical phenotype includes growth retardation, short and small stature, long bone deformity, limited joint activity and the like, and the clinical and genetic heterogeneity is large. Although the incidence of diseases is low, the diseases are teratogenic and disabling, most of the diseases are born in young children and are inherited generation by generation or generation after generation, and huge mental and economic burdens are brought to patients and family members of the patients. Therefore, the definite diagnosis has great significance for the subsequent treatment and the prepotency of the patients.

At present, the diagnosis of hereditary bone diseases by clinicians is generally based on clinical history data, laboratory examination indexes, imaging examination and genetic detection of patients, but most of patients with hereditary bone diseases are not diagnosed and treated completely, and the following reasons specifically exist: 1. 461 hereditary bone diseases are discovered in total in 2019, the bone diseases are various and rare, most of the bone diseases are often only shown as skeletal deformity without hematuria biochemical abnormality, doctors have no systematic training, and the diseases cannot be diagnosed clearly due to insufficient cognition; 2. the phenotype of partial osteopathia is overlapped, most of the hereditary osteopathia can show short stature, but causes of short stature are different, and the hereditary chondrogenesis is imperfecta, spondyloepiphyseal dysplasia and pseudochondrogenesis imperfecta can show short stature; 3. the genetic heterogeneity of hereditary osteopathy is high, the same disease can have a plurality of pathogenic genes, for example, osteogenesis imperfecta can be caused by a plurality of gene mutations such as COL1A1, COL1A2, IFITM5, FKBP10, and the like, and the clinical diagnosis can only be carried out on the whole according to the medical history data of patients, and the individual treatment cannot be carried out by specific typing; different types of diseases caused by the same gene mutation, for example, COL2A1 gene mutation can cause spinal epiphyseal dysplasia, multiple epiphyseal dysplasia with vision and hearing disorders, Kniest bone dysplasia and the like, and great difficulty is added to diagnosis.

The pathogenic genes of hereditary bone diseases are more, doctors often cannot accurately locate the pathogenic genes, and the pathogenic genes or the pathogenic genes are determined by sequencing one by one candidate genes, so that the sequencing cost and time are increased invisibly. In the sequencing process, the sequencing data has the defects of overlarge N content, low sequencing quality, high base number proportion, low reads quality caused by sequence pollution and the like, influence on subsequent analysis, and poor coverage rate and low capture rate caused by high GC content.

Therefore, there is a need for an optimized product and method for detecting a gene associated with hereditary bone disease.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a gene composition, a chip and a kit for screening difficult hereditary osteopathia, and the kit is suitable for screening hereditary osteopathia of Chinese.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a genomic composition for screening refractory hereditary bone diseases, which comprises the following 322 genes:

ACAN, ACP5, ACVR1, ADAMTS10, ADAMTSL 10, AGA, AGPS, AKT 10, ALPL, ALX 10, AMER 10, ANKH, ANO 10, ANTXR 10, ARHGAP 10, ARSB, ATP6V0A 10, B3GALT 10, B4GALT 10, BHA LH72, BMP 10, BMPER, BMPR1 10, CA 10, CANT 10, CASR, CC2D 10, CCDC 10, CDC 10, CDH 10, CDKN1 10, CDT 10, CEP290, CHST 10, CHSY 72, CK3672, CACKAP 210, GRECD 10, CAFFC 10, CDC 10, CDCOGN 10, CDC 10, CAFFC 10, HES7, HGSNAT, HOXA11, HOXA13, HOXD13, HPGD, HSPG2, ICK, IDH1, IDH2, IDS, IDUA, IFITM5, IFT122, IFT140, IFT172, IFT43, IFT80, IHH, IKBKG, IL1 80, IMPAD 80, INPPL 80, KAT6 80, KIF 80, LBR, LEMD 80, LEPRE 80, LIFR, LMNA 80, LMNA 1 LMX 80, LONP 80, LPIN 80, LRP 80, LTBP 80, MAFB 80, MAN2B 80, MAN2C 80, MANBA, MATN 80, MENFN 80, MENFR 6863672, HO 80, HONP 72, PHNFP 80, NFP 80, NFR 80, NFP 80, NFR 80, NFP 36NRNPNFP 80, 36NRNPNFP 80, 36NRPSN 80, 36NRNPNFP 80, 363672, 80, 36363672, 80, 36NRNPNFP 36P 80, 36P 80, 363636P 36P 80, 36P 80, 36P 80, 36P 80, 363672, 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P 80, 36P, SKI, SLC17A, SLC26A, SLC29A, SLC34A, SLC35D, SLC39A, SLCO2A, SLCO5A, SMAD, SMARCAL, SMC1, SMC, SNRPB, SNX, SOST, SOX, SP, SULF, SUMF, TBCE, TBX, TBXAS, TCF, TCIRG, TCOF, TCTN, TGDS, TGFB, TGFBR, THPO, TMZEM 216, TMEM38, TMEM, TNFRSF11, TNFR, TP, TRAPPC, TREM, TRIP, TRPS, TRPV, TTC21, TWIST, TYROBP, WDR, WISP, WNT10, WNT, WNSF 5, WNT, YZT, XZT, XLT and XLT.

In a second aspect, the invention provides an application of the above genome composition in preparing a kit for screening refractory hereditary bone diseases.

In a third aspect, the present invention also provides a gene chip for screening refractory hereditary bone diseases, which comprises a probe for the above gene composition.

In a fourth aspect, the invention also provides a targeted capture sequencing kit comprising probes against the above genomic composition.

In a fifth aspect, the present invention also provides a kit for screening problematic hereditary bone diseases, which comprises the above gene chip or a probe for the above gene composition.

Further, the kit further comprises the following instructions:

a. establishing a DNA library comprising the gene composition;

quality detection and high-throughput sequencing of the DNA library to obtain FastQ data; and

c. the FastQ data described above was analyzed.

Further, the instruction a specifically includes the following steps:

(1) detecting the quality of the sample DNA;

(2) sorting, purifying and fragmenting DNA;

(3) carrying out end repairing reaction;

(4) adding single adenylic acid at the 3' end;

(5) connecting a sequencing joint;

(6) screening gene library fragments;

(7) PCR amplifying the gene library;

(8) hybridizing the target area chips;

(9) cleaning and purifying the hybridization library; and

(10) the hybridization library was amplified by PCR.

Furthermore, the method adopted by the sample DNA quality detection comprises agarose gel electrophoresis and Nanodrop 2000/Qubit.

Further, the instruction c specifically includes the following steps:

(1) obtaining a preliminary comparison result by using BWA software; and

(2) the Picard software is used for counting the primary alignment results, and the statistics include the number and the proportion of the sequences in the alignment, the proportion of redundant sequences (Duplicate Reads) caused by PCR amplification in the sample preparation and capture experiment processes, the proportion of sequences Q20 and Q30, the average coverage depth and the coverage proportion of the target area from 1X to 100X.

Further, the kit further comprises the following instructions:

d. and (3) correcting the primary comparison result: the method adopts a GATK standard flow to correct the repeated sequence caused by PCR amplification, error comparison generated by InDel and base quality, accurately identifies SNV and InDel, and greatly reduces false positive and false negative generated in the sequencing and comparison processes;

e. SNV filtering is carried out on the high-throughput sequencing data, and then pathogenicity analysis is carried out to evaluate by combining clinical symptoms, so that a pathogenic gene is determined.

Further, the above pathogenicity analysis uses software or databases including SIFT, POLYPHen V2, MutationTaster, and Cadd Raw.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

aiming at the requirement of difficult diagnosis and treatment of difficult osteopathia in China, the group of genome compositions related to hereditary osteopathia and related products (gene chips and kits) provided by the invention are developed by combining genome sequencing and big data analysis on the basis of the existing knowledge of hereditary osteopathia and can be used for diagnosing various hereditary osteopathia, and the kit has the advantages that: the probe can be flexibly customized, the target gene and region can be captured specifically, and especially the pathogenic region not contained in WES can also be captured and sequenced; the method can supplement and optimize areas which cannot be captured by WES, achieves better capturing effect, and is high in coverage rate and high in variation detection precision; the cost performance is higher, the clinical detection is quicker, and the cost is lower.

Drawings

FIG. 1 is a graph of base quality distribution of raw data for 15 patients according to an embodiment of the present invention.

Detailed Description

The invention provides a gene composition, a chip and a kit for screening refractory hereditary bone diseases according to the characteristics of hereditary bone diseases of Chinese people, wherein the gene composition comprises the following 322 genes:

The specific position and coverage information of the 322 genetic bone disease related pathogenic genes are shown in the following table 1:

TABLE 1322 detailed location and overlay information of hereditary osteopathy-related pathogenic genes

In addition, the gene chip and the kit comprise probes for the 322 genes, and more than 400 known hereditary bone diseases can be detected.

The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.

In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.

Example 1

The embodiment provides a method for screening refractory hereditary bone diseases, which is based on the principle that a target capture sequencing kit is customized according to the 322 genes, and comprises the following steps:

DNA library establishment:

1) sample DNA Quality detection (Evaluation of DNA Quality): high quality genomic DNA is a prerequisite for conducting genome sequencing experiments. The quality detection of the genome DNA is carried out by the following two methods: agarose gel electrophoresis for genomic DNA integrity: the electrophoretic bands are required to be clearly visible without obvious tailing; the method comprises the following steps of (1) detecting the concentration and the quality of genome DNA by using a Nanodrop 2000/Qubit: the required concentration is more than or equal to 50 ng/mu L, the total amount is more than or equal to 2 mu g, and OD260/280 is 1.8-2.0.

2) Sorting, purifying and fragmenting DNA (DNA Purification and cleavage): taking genome DNA for ultrasonic fragmentation. After fragmentation, the DNA length is mostly between 100-500 bp.

3) End Repair reaction (End Repair): adding a terminal completion system into the purified DNA fragment, wherein the single-stranded protrusion at the 3 'end is digested by the Exonuclease (Exonuclease) activity of T4 DNA Polymerase (T4 DNA Polymerase), and the 5' end protrusion is completed by the Polymerase (Polymerase) activity; meanwhile, phosphate groups necessary for subsequent ligation reaction are added to the 5 'end of Phosphokinase (PNK), and the mixture is purified by Agencourt AMpure XP magnetic beads to finally obtain a blunt-end DNA short fragment library containing the phosphate groups at the 5' end.

4) Reaction with "A" at the 3 'end (Adenlylate 3' Ends): to the above system was added an "A" buffer reaction system at the 3' end. The single adenosine A is added at the end of the double-stranded DNA3 'with the end modified, so that the blunt end self-connection between DNA fragments is prevented, and the single adenosine A can also be matched with the single T protrusion at the 5' end of the sequencing adaptor in the next step in a complementary manner, so that the accurate connection is realized, and the self-series connection between the library fragments is effectively reduced.

5) Ligation sequencing linker (Adapter Ligation): adding a connecting buffer solution and a double-stranded sequencing adaptor into the reaction system, and connecting the illumina sequencing adaptor to both ends of the library DNA by using T4 DNA ligase.

6) Library fragment screening (Size Selection): for the adaptor-added library, fragments of different sizes were separated by agarose gel electrophoresis, and fragments of appropriate length were recovered by gel cutting and used for the next PCR amplification. The purified library eliminates excessive sequencing joint and joint self-connection products in the system, avoids ineffective amplification in the PCR process and eliminates the influence on-machine sequencing.

7) PCR Amplification of DNA library (PCR Amplification): the original library was amplified using high fidelity polymerase to ensure sufficient library throughput. In addition, since only the DNA fragments with adapters attached to both ends can be amplified, this step can effectively enrich this DNA. The number of PCR amplification cycles is controlled to be between 10 and 12, and the deviation caused by overlarge number of amplification cycles is reduced on the premise of ensuring enough products; finally, the concentration of each library was accurately determined using Qubit.

8) Target area chip Hybridization (Library Hybridization): the library was hybridized to probes using the Agilent surelectxt custom Kit for region of interest capture.

9) Hybrid Library washing and Purification (Library Wash and Purification): by means of coupled biotin groups on the probe, using

MyOne^TMStreptavidin T1 magnetic beads enriched probes. After the hybridization step in step 8), the library DNA to which the probe specifically binds is enriched. After further washing to remove DNA non-specifically bound to the magnetic beads, the DNA belonging to the region of interest in the library is enriched.

10) PCR Amplification of DNA library (PCR Amplification): the original library was amplified using high fidelity polymerase to ensure sufficient library throughput. The number of PCR amplification cycles is controlled between 10 and 12. And on the premise of ensuring that the product is enough, the bias introduced due to the overlarge number of amplification cycles is reduced. And purifying the amplified library by magnetic beads to obtain the sequencing library which can be operated on a computer.

11) Quality testing of the Library (Library Quality Assessment): the library concentration and the library fragment length distribution were measured using the Qubit and Agilent 2100Bioanalyzer, respectively. The required concentration was >5 ng/. mu.l and the fragment lengths were centered between 300 and 400 bp.

12) Sequencing on a machine (Sequencing): the library was finally subjected to high throughput sequencing on the Illumina Hiseq platform in a2 × 150bp paired-end sequencing mode to obtain FastQ data.

2. And (3) data analysis: BWA software (http:// bio-bw. source form. net /) can accurately and rapidly align short sequences to a reference genome, and the software is continuously updated to indicate that a document is complete, so that the method is currently the alignment method of sequencing data recommended by the Broad center. And (3) comparing the raw data of each sample with a reference genome by using a mem algorithm of BWA to obtain a primary comparison result in a BAM format. The alignment results of each sample are counted by Picard software (https:// branched. githu. io/Picard /), including the number and ratio of sequences aligned, the ratio of redundant sequences (Duplicate Reads) caused by PCR amplification during sample preparation and capture experiments, the ratio of Q20 and Q30 sequences, average coverage depth, the coverage ratio of the target region from 1X to 100X, and the like. Generally, when the base coverage depth of a site is 10X or more, the SNV (single nucleotide variation) detected at the site is considered to be relatively reliable.

3. And (3) correcting the data comparison result: the primary alignment result obtained by correcting BWA software by adopting a GATK standard process is mainly corrected aiming at a repetitive sequence caused by PCR amplification, error alignment generated by InDel and base quality. Thus, the processed comparison result can more accurately identify SNV and InDel, and the false positive a and the false negative b generated in the sequencing and comparison processes are greatly reduced. a: the sources of false positives include (1) high sequencing error rate, which is likely to cause erroneous judgment when the sequencing depth is low; (2) the randomness of sequencing causes the detected times of two alleles to deviate from 1:1, and causes errors in genotype interpretation; (3) false positive sites are easily caused when repeated sequences, STR and the like exist; (4) the presence of homologous sequences results in partial reads being mis-spliced. b: sources of false negatives include (1) imbalances in sequencing, especially at lower sequencing depths, where partial regions fail to be covered; (2) influence of GC content.

4. Screening the sequencing data: SNV were filtered according to the following criteria: 1) population database frequency filtering. 1000genome < ═ 0.01, ExAC03_ EAS < ═ 0.01, gnomAD _ exosome _ EAS < ═ 0.01, geneskyexodb _ Freq < > 0.05 (sky hao 300 exon control database). 2) And (4) filtering functional mutation. The mutations in the exon or splicing region are retained, and nonsynonymous SNV type mutations such as missense mutation and frameshift mutation are retained. 3) The reported pathogenic mutations remain, if the mutation is DM (Disease-using mutations) at HGMD _ site _ class (pathogenicity classification of HGMD database), the mutation remains, when 1), 2 is not considered). And (3) carrying out pathogenicity analysis on the filtered data, carrying out pathogenicity prediction on the data by using SIFT, POLYPHen V2, MutationTaster, Cadd Raw and the like, determining the pathogenicity of the data, carrying out Sanger sequencing verification on the proband and family members of the proband and evaluating by combining clinical symptoms to determine a pathogenic gene and determine the carrying condition of a mutant gene of the family members.

Example 2

In this embodiment, for 15 suspected patients with hereditary bone diseases, the screening of candidate gene mutations related to problematic hereditary bone diseases is implemented by the following steps:

step one, customizing a target capture sequencing kit aiming at the 322 genetic bone disease related pathogenic genes, wherein the kit comprises a probe of the 322 genes, and the gene sequencing kit can detect more than 400 known genetic bone diseases;

step two, establishing a high-quality library for sequencing analysis, and specifically comprising the following steps: (1) taking genome DNA for ultrasonic fragmentation, adding exonuclease of T4 DNA polymerase and phosphokinase to repair the tail end, adding single adenylic acid at the tail end of DNA3' to prevent the tail end of the DNA fragments from self-connecting, and connecting an illumina sequencing joint to the two ends of the library DNA by using T4 DNA ligase. Fragments of different sizes were separated by electrophoresis, and excess sequencing adapters and adapter self-ligated products were removed. (2) The original library was amplified with high fidelity enzyme, hybridized with probes using the Agilent sureselectXT custom Kit, and the region of interest captured. By using

MyOne^TMStreptavidin T1 magnetic bead enrichment probe, after hybridization, non-specifically bound DNA fragments are discarded, and the original library is amplified again by high fidelity enzyme, so that the total amount of the library is ensured.

Step three, data analysis: BWA software (http:// bio-bw. source form. net /) can accurately and rapidly align short sequences to a reference genome, and the software is continuously updated to indicate that a document is complete, so that the method is currently the alignment method of sequencing data recommended by the Broad center. And (3) comparing the original data of each sample with the reference genome by using a mem algorithm of BWA to obtain a primary comparison result of the BAM format file. The alignment results for each sample, including the number and ratio of sequences aligned, the ratio of redundant sequences (Duplicate Reads) resulting from PCR amplification during sample preparation and capture experiments, the ratio of Q20 and Q30 sequences, the average coverage depth, the ratio of coverage of the target region 1X-100X, etc., were counted by Picard software (https:// branched amplification. Generally, when the base coverage depth of a site is 10X or more, the SNV detected at the site is considered to be relatively reliable.

TABLE 215 statistical table of sequencing data quality for patients suspected of having hereditary bone diseases

Whether the sequencing results are balanced or not is evaluated according to the base mass distribution (shown in figure 1) of the raw data of each sample, and samples with larger differences are rejected.

Step four, a mature data analysis system: the preliminary alignment results obtained using GATK standard protocol to calibrate BWA software were corrected primarily for repeat sequences resulting from PCR amplification, incorrect alignments by InDel, and base quality, with SNV/InDel numbers for 15 patient samples as shown in table 3 below.

TABLE 315 SNV/InDel number of patient samples

And step five, comparing and analyzing all SNV/InDel sites with known information such as a newly released group database, a function database, a disease database and the like, and evaluating the variation frequency, the function characteristics, the conservation, the pathogenicity and the like of the SNV/InDel sites. Therefore, the SNV/InDel locus with the most biological significance can be quickly found from a large amount of variation information, the analysis range is quickly reduced to hundreds or even tens, and the research efficiency is greatly improved on the premise of ensuring the research accuracy. 8-20 candidate disease-causing genes were initially selected for each sample (as shown in Table 4 below).

TABLE 415 candidate disease genes after preliminary screening of patient chip capture data

And step six, screening the candidate genes from the aspects of genetic mode, family cosegregation, clinical phenotype and the like to finally obtain more accurate diagnosis results (as shown in the following table 5).

Table 515 patients screened specific information of pathogenic gene

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. A gene composition for screening refractory hereditary bone diseases, which is characterized by comprising the following 322 genes:

2. Use of the genomic composition according to claim 1 for the preparation of a kit for screening for conditions associated with refractory hereditary bone diseases.

3. A gene chip for screening refractory hereditary bone diseases, which comprises a probe against the gene composition of claim 1.

4. A targeted capture sequencing kit comprising probes directed against the genetic composition of claim 1.

5. A kit for screening for problematic hereditary bone disease, comprising a probe for the gene composition of claim 1 or the gene chip of claim 3.

6. The kit of claim 5, further comprising instructions to:

a. creating a DNA library comprising said genetic composition;

c. the FastQ data was analyzed.

7. The kit according to claim 5, wherein the instructions a comprise in particular the steps of:

(1) detecting the quality of the sample DNA;

(2) sorting, purifying and fragmenting DNA;

(3) carrying out end repairing reaction;

(4) adding single adenylic acid at the 3' end;

(5) connecting a sequencing joint;

(6) screening gene library fragments;

(7) PCR amplifying the gene library;

(8) hybridizing the target area chips;

(9) cleaning and purifying the hybridization library; and

(10) the hybridization library was amplified by PCR.

8. The kit according to claim 5, characterized in that said instructions c comprise in particular the steps of:

(1) obtaining a preliminary comparison result by using BWA software; and

(2) and counting the primary alignment result by using Picard software, wherein the counted values comprise the number and the proportion of the sequences in alignment, the proportion of redundant sequences caused by PCR amplification in the sample preparation and capture experiment processes, the proportion of sequences Q20 and Q30, the average coverage depth and the coverage proportion of a target area from 1X to 100X.

9. The kit of claim 8, further comprising instructions to:

d. correcting the preliminary comparison result: the method adopts a GATK standard flow to correct the repeated sequence caused by PCR amplification, error comparison generated by InDel and base quality, accurately identifies SNV and InDel, and greatly reduces false positive and false negative generated in the sequencing and comparison processes;

10. The kit of claim 9, wherein the pathogenicity analysis employs software or databases comprising SIFT, polyphren V2, mutationmaster, and Cadd Raw.