CN108251519A - Pathogenic gene of pre-axial multi-finger disease and application thereof - Google Patents

Abstract

The invention provides a mutated LMBR1 gene and its application, a vector, a host cell and a kit containing the mutated LMBR1 gene. In addition, the present invention also provides diagnostic agents and kits for diagnosing preaxial polydactyly. The present invention provides new methods and tools for the diagnosis and treatment of PPD. In particular, the diagnostic method provided by the present invention and the primers and/or probes and/or antibodies used in the method can be used to quickly and effectively determine whether a subject suffers from PPD or is at risk of developing PPD.

Description

The causative gene of anterior axial polydactyly and its application

technical field

The present invention relates to the field of molecular genetics and the field of disease diagnosis and treatment; in particular, the present invention provides a mutated preaxial polydactyly-related disease-causing gene LMBR1 and its application, including a carrier and a host cell of the mutated LMBR1 gene and test kits. In addition, the present invention also provides diagnostic agents and kits for diagnosing preaxial polydactyly.

Background technique

Research reports from 1996 to 2000 show that the average incidence rate of polydactyly (toe) in my country is about 0.1%, and the incidence of polydactyly (toe) ranks second among all birth defects. Therefore, the study of its genetic cause and molecular mechanism is of great significance and value. By further clarifying its pathogenesis and locating disease targets, it can promote the development of clinical PPD-specific diagnosis and treatment technology, and deepen the understanding of PPD. Our understanding of the mechanisms of limb development.

Preaxial polydactyly (PPD) is a common congenital limb deformity with four subtypes. The pathogenic gene and mechanism of preaxial polydactyly type I (PPD-I) are unknown. Congenital polydactyly (polydactyly) is a common limb deformity, with an incidence rate of 7-14 per thousand [1]. Preaxial polydactyly (PPD) is the main subtype of congenital polydactyly (PPD), and the main clinical manifestation is the hyperplasia of more than normal number of fingers (toes) on the radial (tibial) side of the limbs.

According to the location of the affected fingers (toes), preaxial polydactyly can be divided into 4 subtypes (I-IV): type I (OMIM174400): polydactyly of the two knuckles of the thumb; type II (OMIM 174500): three fingers Polydactyly of thumb; type III (OMIM 174600): polydactyly of index finger; type IV (OMIM 174700): polydactyly and 3 syndactyly. In addition to occurring independently, polydactyly (toe) deformity can also appear as part of symptoms of some syndromes, such as crossed polydactyly (OMIM175690), triphalangeal thumb syndrome (triphalangeal thumb syndrome) -polysyndaetyly, TPT-PS, OMIM190605) etc.

Preaxial polydactyly (toe) is mostly an autosomal dominant (AD) disease, from the simple increase of a single phalanx of the three-knuckle thumb to the generation of a complete extra digit (toe), the phenotype of affected individuals has obvious difference. Through clinical and genetic studies on 12 families, Zguricas et al. believed that the key pathogenic gene region of PPD is located in the 1.9cM region between the microsatellite markers D7S55 and D7S2423; Heus et al. used the method of constructing the physical and transcriptional map of chromosome 7q36, The candidate region was further narrowed down to a region of about 450kb, where most of the PPD pathogenic genes reported so far are almost located. The widely reported polydactyly-related genes include SHH, HLXB9, HOXD13, GL13, LMBR1, etc., most of which are located on chromosome 7, especially in the 7q36 region. For PPD-II, the study found that all reported mutations were concentrated in the ZRS (zone of polarizing activity regulatory sequence) region located in the fifth intron of the 7q36LMBR1 (MIM*605522) gene. Studies have shown that in the ZRS region, there have been more than 20 1 point mutation, 10 repeat mutations, 1 triploid and a paired base insertion were reported to be associated with the pathogenesis of PPD-II. The mutation of GLI3 gene was proved to be related to PPD-IV, while SHH in the limb bud The expression of the front is actively inhibited by transcription factors such as GLI3, which constitutes a negative feedback regulatory mechanism. In addition, finger deformities such as PPD-II/III, TPT-PS, type IV (syndactyly type IV, SD4, MIM186200) and Werner Mesomelic syndrome (WMS, MIM 188770) have been reported to be associated with mutations in the ZRS region (mutation) or duplication (duplication) related. However, the pathogenic genes of PPD-I and PPD-III have not yet been confirmed. However, as a genetic hotspot of polydactyly, the ZRS region has attracted close attention from scholars at home and abroad.

Regarding the pathogenic mechanism of PPD caused by the abnormal expression of SHH regulated by ZRS mutation, studies have shown that the highly conserved ZRS region of about 800 bp in the LMBR1 gene is the cis-regulation of the SHH (Sonic hedgehog, MIM*600725) gene about 1 Mb downstream Element, this ZRS cis-regulatory element regulates the spatiotemporal expression of SHH and plays an important role in the normal development of fingers (toes). SHH protein is expressed and secreted at a site called ZPA (zone of polarizing activity) on the posterior edge of early limb buds, and is regulated by ZRS. ZRS mutations can cause ectopic expression of SHH protein, resulting in abnormal knuckles. It is proved that ZRS mutation leads to the occurrence of other subtypes of PPD by regulating the abnormal expression of downstream target gene SHH. However, the molecular mechanism of how ZRS mutation regulates the abnormal expression of SHH across the 1MB distance is still unclear.

Contents of the invention

In order to overcome the deficiencies of the above-mentioned prior art, the present invention locates the pathogenic gene and mutation site of PPD-I family through genetics-related research methods (whole genome type detection technology, linkage analysis and candidate region sanger sequencing) .

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the terms and laboratory procedures related to molecular genetics, nucleic acid chemistry and molecular biology used herein are all terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, when a specific sequence is used to describe a gene or nucleic acid, it includes not only the gene or nucleic acid represented by the specific sequence, but also the gene or nucleic acid represented by the complementary sequence of the specific sequence. In this application, although for the sake of convenience, in most cases only the sequence of one chain is given for a gene or nucleic acid, those skilled in the art can clearly know the sequence of its complementary chain. Therefore, the present application actually also discloses the sequence of said complementary strand.

For example, when referring to the LMBR1 gene, it includes not only the sense strand sequence, but also the complementary sequence of said sense strand. As another example, when referring to SEQ ID NO:1, it includes not only the sequence shown in SEQ ID NO:1, but also the complementary sequence of SEQ ID NO:1.

Nucleic acid sequences in this application include both DNA and RNA forms. Unless the context indicates otherwise, the nucleic acid sequences of the present invention include not only DNA forms, but also RNA forms. For example, when referring to SEQ ID NO: 1, it includes not only the DNA form, but also the RNA form (eg, the mRNA sequence).

As used herein, the term "mutation", when used to describe a gene or DNA, refers to the addition, deletion and/or substitution of one or more (eg, several) bases in a gene sequence or DNA sequence.

As used herein, the term "heterozygous mutation" refers to a mutation that is present in only one gene of a pair of alleles. As used herein, the term "homozygous mutation" refers to a mutation that occurs simultaneously in both genes of a pair of alleles.

As used herein, the term "c.446" refers to the 446th base of the ZRS sequence, wherein "c." means ZRS DNA, and the number "446" means the 446th base. Other similar terms used herein have similar meanings.

As used herein, the term "c.446T→A" refers to the mutation of the 446th base of the ZRS DNA sequence from T to A. Other similar terms used herein have similar meanings.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. Such vectors can be introduced into host cells by transformation, transduction or transfection, so that the genetic material elements carried by them can be expressed in the host cells. Vectors are well known to those skilled in the art, including but not limited to: plasmids; bacteriophages; cosmids and the like.

The vector may contain expression control sequences operably linked to the gene of interest. As used herein, the term "operably linked" means that the linked molecules are linked in such a way that the intended function is achieved. For example, the operative linking of the expression control sequence and the gene coding sequence can realize the control effect of the expression control sequence on the expression of the gene coding sequence. As used herein, the term "expression control sequence" is a control sequence required to achieve gene expression, which is well known in the art. Expression control sequences generally must include a promoter, often also include a transcription termination sequence, and may also include other sequences, such as enhancer sequences. Gene expression refers to transcription for siRNA, miRNA, etc., and can also include post-transcriptional processing; for protein-coding sequences, it generally refers to transcription and translation, resulting in protein.

In addition, a selectable marker may also be included in the vector. Such selectable markers are well known to those skilled in the art, such as, but not limited to, antibiotic resistance genes, such as penicillin resistance genes, erythromycin resistance genes, and the like.

As used herein, "PCR primer" refers to a polynucleotide fragment, typically an oligonucleotide, e.g. containing at least 5 bases, e.g. 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more base polynucleotide fragments.

Those skilled in the art know that the primer does not have to be completely complementary to the target gene to be amplified or its complementary chain, as long as it can specifically amplify the target gene. As used herein, the term "specifically amplified" refers to a primer capable of amplifying a gene of interest through a PCR reaction without amplifying other genes. For example, specifically amplifying the LMBR1 gene means that the primers only amplify the LMBR1 gene in the PCR reaction, but do not amplify other genes. The design of such primers is well known to those skilled in the art.

Usually, the primers are substantially identical to the target gene to be amplified or its complementary strand, so that the target gene can be specifically amplified. For example, the primers have at least 60% sequence identity, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91% sequence identity with the gene of interest to be amplified or its complementary strand. %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

As used herein, the term "hybridization" refers to the annealing of two single-stranded nucleic acid molecules with complementary sequences under certain conditions (suitable temperature and ionic strength, etc.) to form a double-stranded nucleic acid according to the principle of complementary base pairing. process. Nucleic acid hybridization can be between DNA-DNA, but also between DNA-RNA or

Between RNA-RNA, as long as there is a complementary sequence between them, base pairing can be performed. Generally, the two parties to the hybridization are a test nucleic acid molecule and a known nucleic acid molecule. Known nucleic acid molecules in hybridization systems are called probes. Nucleic acid hybridization includes solid-liquid phase hybridization and liquid phase hybridization. Liquid-phase hybridization is a hybridization reaction carried out in a solution, which refers to the annealing of a nucleic acid molecule to be tested and a known nucleic acid molecule (probe) in a solution to form a hybrid complex.

As used herein, the term "specifically detects LMBR1 gene mutation" means that the probe is capable of distinguishing LMBR1 gene with mutation from LMBR1 gene without mutation. In general, the stringency of the hybridization conditions can be controlled so that the probe can distinguish between genes containing mutations and genes not containing mutations. For example, under highly stringent conditions, a probe that is exactly complementary to the LMBR1 gene can hybridize to a LMBR1 gene that does not contain a mutation, but not to a LMBR1 gene that contains even a single point mutation, thereby distinguishing between the two. Likewise, a probe that is precisely complementary to a mutated LMBR1 gene can also be designed such that it hybridizes under highly stringent conditions to the mutated LMBR1 gene but not to a LMBR1 gene that does not contain the mutation.

Probe design and hybridization techniques are well known in the field of molecular biology. Typically, the probes are labeled so that after the hybridization reaction is complete, the hybridized double strands can be separated and detected by using the label on the probe. Also, the primers can be labeled so that after PCR, by using the labels on the primers, the amplification products can be isolated and detected. Labels that can be used to label probes and primers are known in the art and include, but are not limited to, radioactive isotopes such as 125I, enzymes, substrates for enzymes, luminescent substances such as isoluminol and acridinium esters, fluorescent substances Such as fluorescein and rhodamine, biotin and colored substances such as latex particles and colloidal gold.

As used herein, the term "siRNA" means that siRNA can specifically silence or inhibit the expression of a specific gene through RNA interference.

In one aspect, the invention provides a mutated LMBR1 gene, which differs from the wild-type LMBR1 gene by one mutation, and the mutated LMBR1 gene causes the occurrence of preaxial polydactyly (PPD); the mutation is: c.446T→A in the ZRS region (SEQ ID NO.1) of the fifth intron of the LMBR1 gene.

In another aspect, the present invention provides a vector comprising the above-mentioned mutated LMBR1 gene.

Preferably, the vector is selected from cloning vectors and expression vectors.

Preferably, the vector further comprises an expression control sequence operably linked to the mutated LMBR1 gene. More preferably, the expression control sequence is selected from promoters, enhancers and terminators.

Preferably, the vector also comprises a selectable marker.

In another aspect, the present invention provides a host cell comprising the above-mentioned mutated LMBR1 gene or the above-mentioned vector.

In another aspect, the present invention provides the use of the above-mentioned mutated LMBR1 gene or the above-mentioned vector or the above-mentioned host cell for producing a PPD animal model, or for preparing a kit for producing a PPD animal Model.

In another aspect, the present invention provides a diagnostic agent for diagnosing PPD, said diagnostic agent comprising a probe capable of specifically detecting LMBR1 gene mutation, or an antibody or antigen-binding fragment thereof capable of specifically recognizing a mutated LMBR1 protein; Wherein, the LMBR1 gene mutation is c.446T→A in the ZRS region (SEQ ID NO.1) of the fifth intron.

Preferably, the probe or antibody or antigen-binding fragment thereof is labeled.

In another aspect, the present invention provides the use of primers capable of specifically amplifying the ZRS region of the fifth intron of LMBR1 gene in the preparation of a diagnostic agent for diagnosing PPD.

Preferably, the sequence of the primer is selected from SEQ ID NO:2 and 3.

Preferably, the primers are a pair of primers as shown in SEQ ID NO:2 and 3.

Preferably, said primers are labeled.

In another aspect, the present invention provides a kit comprising the above-mentioned diagnostic agent.

Preferably, the kit further comprises reagents for PCR, reagents for nucleic acid extraction, and/or secondary antibodies for detecting the antibodies or antigen-binding fragments thereof.

Preferably, the reagents for PCR include dNTPs and polymerase.

In another aspect, the present invention provides the use of the above-mentioned diagnostic agent in the preparation of a kit for detecting the mutation of LMBR1 gene and/or for diagnosing PPD.

In another aspect, the present invention provides the use of primers capable of specifically amplifying the ZRS region of the fifth intron of LMBR1 gene in preparing a kit for diagnosing PPD.

Preferably, the kit further comprises reagents for PCR or reagents for nucleic acid extraction.

Preferably, the reagents for PCR include dNTPs and polymerase.

Preferably, the sequence of the primer is selected from SEQ ID NO:2 and 3.

Preferably, the primers are a pair of primers as shown in SEQ ID NO:2 and 3.

Preferably, said primers are labeled.

In summary, the beneficial effects of the present invention are:

(1) The present invention provides new methods and tools for the diagnosis and treatment of PPD. In particular, the diagnostic method provided by the present invention and the primers and/or probes and/or antibodies used in the method can be used to quickly and effectively determine whether a subject suffers from PPD or is at risk of developing PPD.

(2) The present invention lays an important foundation for the study of the pathogenesis of PPD, and provides a new theoretical basis for the treatment of PPD patients. In addition, the identification of the causative gene of PPD is of great significance for future genetic counseling, prenatal diagnosis and gene therapy. In addition, the disease animal model obtained by using the PPD pathogenic gene is a powerful tool for studying the pathogenesis and treatment of PPD.

Description of drawings

Fig. 1 is the PDD-I family pedigree diagram (I-IV represents the algebra in the family); wherein, T\T and T\A are the results of sanger sequencing (for c.446);

Figure 2 shows the X-ray and physical imaging results of the hands of PPD-I family members (patients, suspected cases, and normal controls); among them, Figure 2A, B: II-11 (patient number), two typical distal Distal phalanx, left thumb with incomplete distal phalanx duplication; Figure 2C: IV-1 (patient) similar to II-11 phenotype; Figure 2D,E: III-8 (patient), bilateral thumb symmetry Incomplete duplication of the distal phalanx; Figure 2F: III-12, incomplete duplication of the distal phalanx of the right thumb, and normal left thumb; There is a small sesamoid bone between the knuckle and the middle phalanx; Figure 2H:III-5, normal control;

Figure 3 is the results of genome-wide Linkage analysis;

Figure 4 is the result of Sanger sequencing to identify the patient's ZRS region;

Fig. 5 EMSA experiment (A) and Super-shift (B) experiment detect the interaction of mutant DNA fragment and transcription factor/nucleoprotein (K: protein); Wherein, B-MT-DNA is biotin-labeled mutant DNA probe ;NE is nucleoprotein; Anti-HnRNPK is K protein antibody; Anti-HnRNPD is D protein antibody; Anti-HnRNPA/B is A/B protein antibody; Anti-HnRNPF is F protein antibody; IgG G is negative control antibody;

(C) EMSA experiments after overexpressing K protein and knocking down K protein in Caco-2 cells; where, B-MT-DNA is biotin-labeled mutant DNA probe; B-WT-DNA is biotin-labeled wild DNA probe; HnRNPK O is an overexpressed K protein; siHnRNPK s48 is an interfering RNA that can knock down the expression of K protein;

(D) is the result of chromatin immunoprecipitation-qPCR experiment (ChIP-qPCR experiment), the HnRNPK group was added K protein antibody, and the IgG group was a negative control antibody;

Figure 6 is a graph showing the results of the DNA pull-down experiment;

Figure A in Figure 7 is the dual luciferase reporter gene detection results;

Figure 7B shows the observation results of luciferase signal intensity changes in the three groups, in which K protein gene expression was knocked down by siHnRNPK s48;

Figure 7C shows the expression of K protein knocked down by transfection of siHnRNPK s48 in Caco-2 cells, and the changes of Shh mRNA were observed; among them, scrambled is the control group, and siHnRNPK s48 is the experimental group;

Figure 7D shows the results of transfection of siHnRNPK s48 in Caco-2 cells, and semi-quantitative observation of Shh protein expression by western blot;

Figure 8A and B are mice with point mutations in the ZRS region;

Figure 9 is a map of plasmid pEZX-GA03.

Detailed ways

The present invention aims at a rare PPD-I family of 31 people of four generations of the Han nationality, using the whole genome genotyping and linkage analysis and Sanger sequencing of candidate regions, and locates the new occurrence of the ZRS region (Sonic hedgehog, SHH remote regulator) in the LMBR1 gene Point mutation, located in the pathogenic hotspot of PPD—the heterozygous mutation of T>A in the ZRS region of the LMBR1 gene at 7q36 was co-segregated in this family, which is the pathogenic mutation of PPD-I, and none of the 50 normal controls carry a mutation in this gene. Our study suggests that this mutation is a de novo pathogenic mutation of PPD-I, but the mechanism of this mutation leading to PPD-I phenotype still needs to be further studied. Experiments proved that the point mutation caused a significant increase in the affinity between the ZRS region and the transcription factor K protein, and clarified the molecular mechanism of K protein mediating ZRS mutation, regulating the abnormal expression of SHH, and leading to the occurrence of PPD-I. It should be noted that the transcription factor K protein is nuclear heterogeneous ribonucleoprotein K, hnRNP K, with a molecular weight of 65Kd, located on chromosome 9, GeneID: 3190 on NCBI, and its main functional structure is three guide DNA-RNA With a linked KH domain and a unique KI domain, hnRNP K not only regulates gene expression at the transcriptional level through a CT element-dependent or CT-independent pathway, but also alters mRNA translation through its own phosphorylation Efficiency, as well as regulation of gene translation and transduction of intracellular signals, and plays an important role in the cell cycle.

Based on the above research results, we propose the scientific hypothesis that the new point mutation of ZRS in PPD-I family may activate the abnormal expression of SHH gene by changing the binding ability to transcription factors/proteins. In the previous pre-experiment: the inventors used Caco-2 cells endogenously expressing the SHH gene and related transcription factors to extract their nuclear proteins, and performed gel electrophoresis migration experiments with wild-type/mutant DNA probes synthesized in vitro ( Electrophoretic Mobility Shift Assay, EMSA), the results proved that both the wild-type and mutant probes detected the same specific DNA/protein binding bands, but the mutant probes significantly improved the binding ability to transcription factors/proteins. Further protein spectrum analysis of the binding band showed that there was a transcription factor K protein in the binding band. Super-shift experiments proved that the addition of K protein antibody significantly weakened the binding of the mutation probe to the protein, and the chromatin immunoprecipitation assay (ChIP) The experiment reversely verified that the K protein can bind to the DNA sequence of the ZRS region (there is no literature report on the interaction between the transcription factor K protein and the ZRS region), indicating that the K protein may mediate the new mutation of the ZRS leading to the occurrence of PPD-I, while Whether K protein is involved in mediating ZRS mutation to regulate the abnormal spatiotemporal expression of SHH leading to the occurrence of PPD-I still needs further in vitro and in vivo experiments.

This application takes the new pathogenic mutation of PPD-I family as the entry point, and on the basis of the newly discovered K protein, further through in vitro DNA-pull down, ChIP-sequence, dual luciferase reporter gene detection technology and small Mouse animal model to clarify the mechanism of how K protein mediates ZRS mutation to regulate the abnormal temporal and spatial expression of SHH leading to the occurrence of PPD-I, in order to provide a theoretical basis for the improvement of gene therapy and disease prevention and treatment.

Unless otherwise indicated, the experiments and methods described in the examples (eg, molecular biology and nucleic acid chemistry experiments) were performed essentially according to conventional methods well known in the art and described in various references. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), all incorporated by reference Incorporated into this article. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products. Those skilled in the art understand that the examples describe the present invention by way of example and are not intended to limit the scope of the claimed invention.

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Collection, phenotype identification and classification of embodiment 1PPD families

The inventors of the present application organized and carried out case collection of PPD pedigrees within Guangdong Province, and collected a rare four-generation PPD-I family with 31 individuals including 8 patients. The diagnosis of PPD is based on family history, physical examination, and X-ray imaging examination of skeletal deformities in the patient's hands and feet. Classified according to the methods established by Temtamy S and WinterRM.

Result analysis: As shown in Figure 1, the PPD-I type family of 31 persons, including 8 patients with obvious PPD phenotype and 9 patients with indeterminate disease phenotype, was autosomal dominant. X-ray imaging examination showed that the patients had different degrees of PPD-I phenotype, and obvious phalange deformities were visible to the naked eye; some members had small sesamoid bones between the distal phalanx and the middle phalanx of the thumb, but no obvious phalanx deformity was visible to the naked eye. Because there is a certain proportion of sesamoid bone phenomenon in the normal population, this part of the members is tentatively designated as suspected patients

The localization of embodiment 2 pathogenic genes

Using QIAamp DNAblood Mini kits (Qiagen, Germany) to extract all members of the PPD-I family and 50 normal control peripheral blood genomic DNA samples.

a. Genome-wide genotyping (LMBR1 gene) typing

Select 11 family members, including the DNA samples of 7 patients (II3, II7, II11, III4, III8, III11, IV1) and 4 normal people (II4, II8, II12, III5) in Figure 1 for denaturation and fragmentation Hybridize with the IlluminaHumanOmni2.5-Quad Beadchip chip after chemicalization and amplification, and capture the DNA after adding the labeled nucleic acid. The HumanOmni2.5-Quad Beadchip covers putative functional variants curated from exome and whole genome sequences of more than 12,000 individuals, combining 2.5 million SNP tags from HapMap and the 1000 Genomes Project ( >2.5% MAF), and >240,000 exonic markers. DNA samples were denatured, neutralized and incubated overnight at 37°C. The amplified DNA is a suspension fragment, which is hybridized with the Beadchip overnight at 48°C, while unhybridized and non-specifically hybridized DNA samples are eluted. Labeled nucleotides are added to extend the captured DNA fragments. The iScan system was used for imaging, and the GenomeStudio (v.2011.1) software system was used for data analysis. 2.5million SNPs (single-nucleotide polymorphisms) were obtained for each sample.

b. Linkage and copy number variation (CNV) analysis

From 2.5million SNPs, 7,037 SNPs across autosomes were selected for scatter linkage analysis. First, a genome-wide affected-only model-free linkage analysis was performed on the PPD-I family. Based on the monogenic autosomal dominant inheritance mode, the linkage regions obtained above were further analyzed based on Model of multi-point linkage analysis (multipoint model-based linkage analysis). The copy number variation (CNV) in all regions was analyzed by cnvPartitionv.1.2.1 software, and the results are shown in Figure 3.

c. Sanger sequencing

Sanger sequencing was performed on the linked region using Bigdye Terminator Cycle Sequence Kit 3.1 (ABI Applied Biosystems) to identify the pathogenic gene and mutation site of PPD-I.

Result analysis: As shown in Figure 3, it is the result of genome-wide Linkage analysis, in which 5 regions were identified, including 1p13(~45cM), 4q35(~10cM), 7q36(~15cM), 10p15(~10cM) and 21q21( ~15cM), LOD>1.8. The Chr7 mutation site is located in the ZRS region, located in the 7q36 region of chromosome 7.

As shown in Figure 4, the ZRS region of the patient was identified by Sanger sequencing, where WT represents the wild type; MT represents the mutant type; the sequencing results show that the patient has a T/A point mutation, while the normal person is T/T, and 7 patients have T /A point mutation, 4 normal persons were T/T. The primers required for PCR amplification of the ZRS region of the fifth intron of the LMBR1 gene are shown in Table 1 below.

Table 1 LMBR1 Gene No. 5 Exon ZRS Region PCR Amplification Primer Sequence

Forward 5'-CTGATTTTGTAAGGAACTCTGG-3' (SEQ ID NO.2) Reverse 5'-GCGTATGGGAACTCAGAAA-3' (SEQ ID NO.3)

Result analysis: The results of genome-wide genotyping analysis of 7 patients and 4 normal subjects are shown in Figure 3. From 2.5 million SNPs, 7,037 SNPs across autosomes were selected for scatter-point linkage analysis, and 5 linkages were identified Regions 1p13, 4q35, 7q36, 10p15 and 21q21 (LOD>1.8), since the ZRS is located in the 7q36 region linked to it, we first performed Sanger sequencing on the ZRS of other members of the family, and the PCR amplification primer sequences are shown in Table 1. The results A T/A point mutation was identified in the ZRS region of all patients and suspected patients (III-9), while T/T was identified in normal and other suspicious patients (Figure 4), which was present in patients and normal controls total separation. In the above regions, the cnvPartition v.1.2.1 software analysis did not find the co-segregation phenomenon of CNV abnormal cases and controls, excluding the possibility of copy number variation leading to PPD-I type.

Thus, the obtained sequencing results fully demonstrate that the heterozygous mutation identified in the present invention causes PPD disease. That is, the c.446T→A mutation in the ZRS region (SEQ ID NO.1) of the fifth intron of the LMBR1 gene is related to the occurrence of PPD disease.

Example 3 Molecular mechanism of ZRS mutation regulating abnormal expression of downstream SHH gene

1) ZRS mutation changes its ability to bind to transcription factors/specific proteins in the nucleus

EMSA experiment: Using human-derived Caco-2 cells (expressing SHH protein, and the transcription factor that regulates SHH expression in the nucleus) as a cell model, construct biotin-labeled wild-type and mutant ZRS probes in vitro, and collect Caco-2 cell nuclear protein , Gel electrophoretical mobility shift assay (Electrophoretical mobility shift assay, EMSA) to detect differences in the binding of nucleoproteins to wild-type and mutant ZRS probes.

The specific binding band cut gel in EMSA was identified by protein spectrum, and the transcription factor K protein was obtained. The K protein antibody was added to the EMSA binding system for Super-shift experiments and ChIP experiments to verify the interaction between K protein and ZRS.

1. ChIP experiment:

K protein and chromatin are cross-linked to form a complex, which is broken into small chromatin fragments within a certain length by ultrasonic waves, and then the complex is precipitated by a K protein-specific antibody to specifically enrich the K protein-bound DNA Fragments, through the purification and qPCR detection of the target fragments, it is proved in reverse that the K protein can bind to the DNA sequence of the ZRS region; wherein, the PCR primers for the ZRS region are the same as the primer sequences provided in Table 1. The reaction system is 2ul of 1:1 mixture of front and rear primers, 5ul of MIX, and 3ul of DNA, and the amplification reaction is the amplification reaction of ordinary qPCR.

Result analysis: As shown in Figure 5, both the mutant probe (SEQ ID NO.7) and the wild-type probe (SEQ ID NO.8) lanes showed band S binding to the specific protein, but the mutant probe was obviously Increased its ability to bind to nucleoproteins (Fig. 5A). The protein in this lane was analyzed by mass spectrometry, and it was found that the specifically bound protein may be HnRNPK protein, HnRNPD, HnRNPA/B, HnRNPF, and K protein antibody, D protein antibody, A/B protein antibody, The F protein antibody was used for Super-shift experiments, and the results shown in Figure (5B) found that the band S band in the lane where the K protein antibody was added was significantly weakened (the absence of supershift bands may be due to the fact that the molecular weight of the DNA-protein complex was too large and did not enter the gel. The results have been repeatedly verified), indicating that the protein combined with ZRS-DNA may be K protein.

In Figure C, the K protein is overexpressed through the plasmid, and the Band S band is widened; the expression of the K protein is reduced by knocking down the K protein mRNA by siHnRNPK s48, and the Band S band is weakened, which proves that there is a K protein in the Band S band. In Figure D, the chromatin immunoprecipitation-qPCR experiment uses the K protein antibody to separate the K protein-bound DNA sequence through chromatin immunoprecipitation, and then performs qPCR (real-time fluorescent quantitative PCR). The experimental materials are lymphocytes separated from the blood cells of patients (MT group) and normal people (WT group). It was found that the qPCR signal of the MT group, that is, the mutant group, was significantly higher than that of the WT group, and it was reversely verified that the K protein can bind to the DNA sequence in the ZRS region. Wherein, the sequence of siHnRNPK s48 is shown in Table 2 below.

Table 2 Sense strand and antisense strand of siHnRNPK s48

2. The steps of ChIP-qPCR experiment : K protein and chromatin are cross-linked to form a complex, which is broken into small chromatin fragments within a certain length by ultrasonic waves, and then the complex is precipitated by a K protein-specific antibody. Continually enrich the DNA fragments bound to K protein. Through the purification and qPCR detection of the target fragments, the ZRS (%input) is 0.024, and the GAPDH (%input) is 0.012 (the relative expression of the target gene region is 2 times that of the negative control gene) .

The results are shown in FIG. 5D , thus conversely proving that the K protein can bind to the DNA sequence in the ZRS region. It is further indicated that the protein combined with the DNA sequence in the ZRS region is K protein.

2) K protein is involved in the regulation of SHH expression

DNA pull down MS-Shh promoter experimental steps: construction of SHH promoter probe in vitro, collection of Caco-2 nucleoprotein, binding reaction, binding products for protein mass spectrometry analysis, identification of transcription factors/proteins that bind to SHH promoter, and searching for possible interactions with K protein interacting protein (or K protein itself). The Shh promoter probe sequence is shown in SEQ ID NO.6.

DNA pull down MS-ZRS experimental steps: construct probes in vitro with 29 nucleotide sequences before and after the ZRS point mutation, and other steps are the same as above.

Among them, the ZRS point mutation probe: (SEQ ID NO.7) (A in bold underline is the detected mutation site); ZRS point wild-type probe: CCCAGTGGCTAATTTGTATCAGGCCTCCA (SEQ ID NO.8).

Result analysis: As shown in Figure 6, the DNA pull down MS experiment proved that protein K can bind to the promoters of ZRS and Shh. This experiment proved that protein K can indeed bind to the promoters of ZRS and Shh, but failed to prove the binding ability Caused changes in gene expression, which were next demonstrated using a dual-luciferase reporter gene assay.

3) Dual luciferase reporter gene detection: K protein (or protein interacting with K protein) is used to mediate ZRS mutation to regulate SHH gene expression, and detect the expression difference between luciferase groups.

experiment method:

(1) The map of the pEZX-GA03 plasmid is shown in Figure 9, which is used as a control empty vector. The following plasmids were constructed on the basis of the pEZX-GA03 plasmid:

Plasmid 1: Replace mini cmv with minimal promoter SHH, named S-NEG-GA03 (i.e. CS-NEG-GA03-02);

Plasmid 2: replace minicmv with ZRS wild type (SEQ ID NO.10)+minimal promoter SHH, named CS-ZRS01-GA03;

Plasmid 3: replace minicmv with ZRS mutant (SEQ ID NO.9)+minimal promoter SHH, named CS-ZRS02-GA03;

Plasmid 4: ZRS wild type (SEQ ID NO.10)+mini cmv, named CS-ZRS01-GA03-1;

Plasmid 5: ZRS mutant (SEQ ID NO.9)+mini cmv, named CS-ZRS02-GA03-2;

Plasmid 6: the empty vector removed by mini cmv, named CS-pEZX-GA03 (i.e. CS-NEG-GA03-01);

Among them, the sequence of minimal promoter SHH is shown in SEQ ID NO.6; the sequence of 150 bases near the ZRS mutation point is as follows:

(SEQ ID NO.9), wherein A in italics is a mutant base. The wild-type sequence corresponding to SEQ ID NO.9 is shown in SEQ ID NO.10.

(2) Transfecting the plasmid with the luciferase reporter gene into Caco-2 cells, wherein the plasmids involved include:

The Shhprom group plasmid is the promoter of the Shh gene added on the plasmid with the luciferase reporter gene (i.e. plasmid 1);

The WT-Shhprom group plasmid added wild-type ZRS (150 base sequences before and after the mutation site) in front of the Shh gene promoter sequence (i.e., plasmid 2);

The MT-Shhprom group plasmid is the 150-base sequence before and after the ZRS mutation site added in front of the Shh gene promoter sequence (ie, plasmid 3).

Result analysis: as shown in FIG. 7 , the results in FIG. 7A were obtained by constructing the above-mentioned plasmids 1, 2, and 3 and transfecting them into Caco-2 cells. Because the Shh promoter is constructed in the plasmid, once the promoter starts to express, the subsequent luciferase will also be expressed, and the luciferase does not need to be modified to have biological activity. By adding a specific luciferase substrate , luciferase will emit biofluorescence during the reaction with the substrate, and then the bioluminescence released during the reaction can be measured by a fluorometer; Figure A proves that the WT-Shhprom group is compared with the Shhprom group, and the MT-Shhprom group is compared with the Shhprom group The ZRS region enhances the expression of Shh, comparing the WT-Shhprom group and the MT-Shhprom group, it can be seen that the mutant ZRS enhances the expression of Shh is stronger;

In Figure B, siHnRNPK s48 was used to knock down the expression of K protein, and the luciferase-induced fluorescence signals of the three groups were observed to decrease, which reversely proved that the K protein was the cause of the difference in the fluorescence signals of the three groups in Figure A;

In Figure C, Caco-2 cells were transfected with siHnRNPK s48 to knock down the expression of K protein, and the reduction of Shh mRNA synthesis was observed, which proved that intracellular K protein can promote the expression of Shh;

Figure D uses Western blot at the protein level to prove that the decreased expression of K protein can lead to the decreased expression of Shh.

4) Using CRISP/Cas9 technology to construct mouse animal models with point mutations in the ZRS region

experiment method:

Construct a CRISPR/Cas9 vector, and a gRNA (guide RNA) targeting the ZRS region of the LMBR1 gene (gRNA is located near the new mutation in ZRS), and microinject it into mouse fertilized eggs together, and verify it by conventional sequencing, Western-blot and PCR ZRS mutant form, confirm that the model is successfully constructed, and obtain a ZRS mutant mouse model, as shown in Figures 8A and 8B.

Analysis of the results: As shown in Figure 8, the phenotype of the ZAS heterozygous mutant mice is polydactyly on both lower limbs (Fig. 8A, B), indicating that the point mutation in the ZRS region can lead to the polydactyly phenotype.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> The First Affiliated Hospital of Sun Yat-sen University

<120> The causative gene of preaxial polydactyly and its application

<130> 2018

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 788

<212>DNA

<213> Homo sapiens

<400> 1

aactttaatg cctatgtttg atttgaagtc atagcataaa aggtaacata agcaacatcc 60

tgaccaatta tccaaaccat ccagacatcc ctgaatggcc agagcgtagc acacggtctg 120

taggattaag aggttaactc ctataacttc aaacaaagtg cctgataata aaagcaaaaa 180

gtacaaaatt ttaggtaact tcctttctta attaattgga ctgaccaggt ggaagcgaag 240

agttctgtgc tggtgcttgg aatgtctata aagctgagca acatgacagc acaatagagg 300

aggaacaaag atttttttaa tatgtttcta tcctgtgtca cagtttgaaa ttgtcctggt 360

ttatgtccct tttggcaaac ttacataaaa gtgacctgta ctgtatttta tgaccagatg 420

actttttccc cccagtggct aatttgtatc aggcctccat cttaaagaga cacagagtga 480

gtaggaagtc cagcctctgt ctccacgagc tttcattgca ttctttcatt atttttgctc 540

gttttttgcc actgatgatc cataaattgt tggaaatgag tgattaagga agtgctgctt 600

agtgttagtg gcacatgcgc atatttggcc tggttctggt gggtgagagg aaatcacaga 660

caaaagggaa gcccctgctg ggaaccctgc aaggaaattt aacttgggtc atgttttgat 720

cttagtgttt attacagaaa atgaagccat atctcactaa ctattgttac gtgttaattt 780

gattttcc 788

<210> 2

<211> 22

<212>DNA

<213> Artificial sequence

<400> 2

ctgattttgt aaggaactct gg 22

<210> 3

<211> 19

<212>DNA

<213> Artificial sequence

<400> 3

gcgtatggga actcagaaa 19

<210> 4

<211> 21

<212> RNA

<213> Artificial sequence

<400> 4

cuagugucuu guuaguugau u 21

<210> 5

<211> 21

<212> RNA

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ucaacuaaca agacacuagu u 21

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<212>DNA

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actctgtaac tgcccttgag cacgcctcca tcccatttgc agagctctac aaaaaaaaaa 60

atgtattaat tgaagcacaa taaaagagca gtatttttac atttttctaa ggcacttaaa 120

gcatttcaaa taccagtgct ttaagttatg gtcactcata attatcctcg gtccacagag 180

gctgagtctt caggaggggc tcagtcctct ttcttcaaaa tgcagggaga gccagcctgc 240

actataaagt ccctggggaa gaaaaattaa ataacagctg ggcagctgtg cccctcaaat 300

gtgtgagcga ctctgtgctt gatgactgaa gcttgcgggg gtttaaactg tcctttaaaa 360

acttaagttg gttttaactg aagcacctac cattccaggg aaagtcaggg ggaaacgcaa 420

ggatgtacac aaagccggga gtaactgctg ttaccacaag acatgcgctc ctccctcccg 480

cccaccttta tcttaggaaa gatttttttt tttttaactc gaatccttca ggtgaaaata 540

aagcaacagc agcaacagaa aaaaaaaacg tagtcttctt cagggttaac atcagaagac 600

aagcttgtgc ggctttggcc aatcagatgc gcccctggca gctataatat agtgctaaag 660

gcagcctctc tcacaagctc tccaggcttg ctaccatta aaatcagact ctttttgtct 720

tttgattgct gtctcgcgac ccaactccga tgtgttccgt tatcagcggc cggcagcctg 780

ccattccagc ccctgtctgg gtggggagcg gtggagagtc ccccgcagcc gcggcgggca 840

aggttatata ggaagagaaa gagcgaggca gccagcgagg gagagagcga gcgggcgagc 900

cggagcgagg aagggaaagc gcaagagaga g 931

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<212>DNA

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cccagtggct aattagtatc aggcctcca 29

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<212>DNA

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<400> 8

cccagtggct aatttgtatc aggcctcca 29

<210> 9

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<212>DNA

<213> Homo sapiens

<400> 9

ttggcaaact tacataaaag tgaccttgta ctgtatttta tgaccagatg actttttccc 60

cccagtggct aattagtatc aggcctccat cttaaagaga cacagagtga gtaggaagtc 120

cagcctctgt ctccacgagc tttcattgca 150

<210> 10

<211> 150

<212>DNA

<213> Homo sapiens

<400> 10

ttggcaaact tacataaaag tgaccttgta ctgtatttta tgaccagatg actttttccc 60

cccagtggct aatttgtatc aggcctccat cttaaagaga cacagagtga gtaggaagtc 120

cagcctctgt ctccacgagc tttcattgca 150

Claims (10)

1. the LMBR1 genes of mutation, which is characterized in that its difference with wild type LMBR1 genes is 1 mutation, and institute State the LMBR1 genes of mutation causes to refer to the generation of sick (PPD) before axis more；The mutation is：The 5th introne of LMBR1 genes C.446T → A in ZRS areas (SEQ ID NO.1).

2. a kind of carrier, which is characterized in that the carrier includes the LMBR1 genes of the mutation of claim 1.

3. a kind of host cell, which is characterized in that the host cell includes the LMBR1 genes or power of the mutation of claim 1 Profit requires 2 carrier.

4. the purposes of the host cell of the LMBR1 genes of the mutation of claim 1 or the carrier or claim 3 of claim 2, It is used to generate PPD animal models or is used to prepare kit, and the kit is used to generate PPD animal models.

5. for diagnosing the diagnosticum of PPD, which is characterized in that the diagnosticum include can specific detection LMBR1 genes dash forward The probe of change, wherein, the LMBR1 gene mutations be the 5th introne ZRS areas (SEQ ID NO.1) in c.446T → A.

6. it is capable of purposes of the primer in the ZRS areas of the 5th introne of specific amplification LMBR1 genes in diagnosticum is prepared, The diagnosticum is used to diagnose PPD.

7. purposes according to claim 6, which is characterized in that the primer is such as SEQ ID NO:Primer shown in 2 and 3 It is right.

8. a kind of kit, which is characterized in that the kit includes the diagnosticum of claim 5.

9. the diagnosticum of claim 5 is preparing for detecting the mutation of LMBR1 genes and/or the kit for diagnosing PPD In purposes.

10. the primer for being capable of the ZRS areas of the 5th introne of specific amplification LMBR1 genes is preparing the examination for diagnosing PPD Purposes in agent box.