CN112458104A - Mutant N4BP2 gene related to non-syndromic cleft lip and palate and application thereof - Google Patents
Mutant N4BP2 gene related to non-syndromic cleft lip and palate and application thereof Download PDFInfo
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Abstract
The invention discloses a mutated N4BP2 Gene related to non-syndrome cleft lip and palate and application thereof, wherein the mutated N4BP2 Gene contains a non-frameshift deletion mutation site 4:40133481-40133489 delaGATATTT in the 13 th exon and a missense mutation site 4:40123905T > G and a missense mutation site 4:40122958C > T in the 9 th exon respectively compared with an unmutated N4BP2 Gene with the accession number of Gene bank as NM-018177.6, and the mutated N4BP2 Gene can be used for preparing a diagnostic kit for detecting the non-syndrome cleft lip and palate diseases. The discovery of the mutation site can guide the family offspring to the good prenatal and postnatal care, and can be used as a carrier screening and prenatal diagnosis screening site of the genetic disease of non-syndrome cleft lip and palate for guiding the good prenatal and postnatal care of the population.
Description
Technical Field
The invention belongs to the field of medical gene diagnosis, and relates to a pathogenic gene N4BP2 mutation site of hereditary disease non-syndrome cleft lip and palate and application thereof in gene diagnosis.
Background
Cleft lip and palate is among the most common structural birth defects of congenital facial cleft jaw, and the worldwide neonatal incidence is about 1/700. Congenital cleft lip and palate infant has severe eating problems, dysphasia, middle ear infection and tooth defects.
Cleft lip and palate is high in prevalence rate (1.42/1000) in people in China. Cleft lip and palate can be classified into complex cleft lip and palate and non-complex cleft lip and palate according to whether other deformities are accompanied. Among them, non-syndromic cleft lip and palate are more common, including single cleft lip, cleft palate or cleft lip and cleft palate, accounting for more than 70% of the deformity of the whole cleft face. The facial development is originated from migration and proliferation of neural crest cells, and cleft lips are caused by fusion failure of maxilla and inner nasal process during the facial development; the developmental stage of palatine bone is derived from the differentiated, transformed, proliferative, apoptotic migration of mesenchymal and epithelial cells during the embryonic stage, cleft palate is due to the failure of midline fusion of the two palatal plates.
Non-syndromic cleft lip and palate belongs to polygenic hereditary diseases, so far, more than 100 genes are related to non-syndromic cleft lip and palate through domestic and foreign researches, but the potential pathogenic mechanism of specific genetic variation of new candidate genes still needs to be determined. The genetic risk gene research of cleft lip and palate suggests that a plurality of genes such as IRF6, BCL-3, MTHFR, PTCH1, BMP4, ZNF533, DLX and the like are susceptibility genes for maxillofacial development.
The N4BP2 gene is one of the candidate genes discovered in the early stage of the research, and the functional research thereof is very limited at present. N4BP2 was first reported in 2002 and was named NEDD4 binding protein 2 in combination with the N-terminus (52-422aa) of NEDD4 protein. NEDD4 is a protein highly expressed in neural precursor cells and embryonic chondrocytes in the middle of pregnancy in the process of embryonic development, and is involved in the regulation and control of important embryonic development genes. NEDD4 knockout mice display involvement in the TGF β -MAPK signaling pathway due to neural crest cell deficiency leading to embryo-derived bone development and severe maxillofacial malformations. N4BP2 was found to be evenly distributed in the cytoplasm, similar to NEDD 4. N4BP2 is degraded by proteasomes (proteosomes) mediated polyubiquitination by NEDD4 protein. The N-terminal of the N4BP2 protein structure is provided with a polynucleotide kinase (PNK) structure domain, can bind and hydrolyze ATP, and can play a role as a 5' -polynucleotide kinase. N4BP2 protein specifically binds to protooncoprotein BCL-3 through the PNK domain, and thus N4BP2 is also called BCL-3binding protein (B3 BP). Multiple sites of the BCL-3 gene are associated with the occurrence of non-syndromic cleft lip and palate. BCL-3 is involved in cell proliferation, a cell adhesion function, whose down-regulation may lead to disruption of ectodermal development, and plays an important role in facial formation. BCL-3 is also involved in cell division and migration regulation and is screened as a susceptibility gene for cleft lip and palate. N4BP2 can also be combined with histone acetyltransferase p300/CBP protein through PNK structural domain. p300 histone acetyltransferase activity is critical for Wnt dependent mesenchymal cell proliferation and migration, and loss of p300 function in humans and mice leads to craniofacial defects. The Smr structural domain is highly conserved in prokaryotic and eukaryotic cells and has the function of a nicked endonuclease to participate in gene recombination. N4BP2 is the first found Smr domain-carrying protein in human cells, and Smr domain DNA is located at the C-terminal 1688-1770aa position of N4BP2 protein, and the most conserved loop region is involved in the binding of DNA to single-stranded or double-stranded chain. Since N4BP2 has Smr structure domain and possesses the function of nick endonuclease, the extension loop from single nucleotide to unpaired base can recognize double-stranded DNA mismatch, and can open the supercoiled DNA into nicked open loop. N4BP2 is involved in important DNA mismatch repair (MMR) and gene recombination (including mitosis and meiosis) processes, especially the coupling of transcription with repair or recombination. In 2017, in 1995 researches on low-frequency variation genes of patients with non-syndrome cleft lip and palate and a control, it was found that the low-frequency coding variation of the N4BP2 gene is related to the increased risk of non-syndrome cleft lip and palate. The results suggest that N4BP2 may participate in the development of cleft lip and palate birth defects through various ways.
Disclosure of Invention
The invention aims to provide a mutated N4BP2 gene related to non-syndrome cleft lip and palate and application thereof, and the invention has important significance for clarifying pathogenesis of non-syndrome cleft lip and palate, enriching pathogenic gene mutation spectrum of non-syndrome cleft lip and palate and establishing a genetic diagnosis method for the genetic disease.
The purpose of the invention is realized by the following technical scheme:
compared with an unmutated N4BP2 Gene with the Gene bank accession number of NM-018177.6, the mutated NEDD4 binding protein 2 (N4 BP2) Gene related to the non-syndrome cleft lip and palate, the mutated N4BP2 Gene respectively contains a non-frameshift deletion mutation site 4:40133481, 40133489delAAGATATTT and a missense mutation site 4:40123905T > G and a missense mutation site 4:40122958C > T in the 13 th exon, and the mutated N4BP2 Gene can be used for preparing a diagnostic kit for detecting the non-syndrome cleft lip and palate disease.
Compared with the unmutated N4BP2 with Gene bank accession number NP-060647.2, the mutated N4BP2 contains amino acid mutation sites of p.Lys1531_ Phe1533del, p.Thr1076Met and p.Phe1392Val, and the mutated N4BP2 can be used for preparing a diagnostic kit for detecting the non-syndrome cleft lip and palate disease.
A PCR primer composition for detecting N4BP2 gene mutation related to non-syndrome cleft lip and palate is composed of an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.2, and the PCR primer composition can be used for preparing a diagnostic kit for detecting non-syndrome cleft lip and palate diseases. The PCR primer composition is only suitable for detecting the 4:40133481, 40133489 delaGATATTT mutation sites of the 13 th exon of the N4BP2 gene.
Compared with the prior art, the invention has the following advantages:
1. the invention provides three new non-syndrome cleft lip and palate pathogenic gene mutation sites, enriches the pathogenic gene mutation spectrum, provides new data for clarifying the pathogenesis of non-syndrome cleft lip and palate and the design of a gene diagnostic chip aiming at the disease, and can be used for carrier screening and prenatal gene diagnosis.
2. The discovery of the mutation site can guide the family offspring to the good prenatal and postnatal care, and can be used as a carrier screening and prenatal diagnosis screening site of the genetic disease of non-syndrome cleft lip and palate for guiding the good prenatal and postnatal care of the population.
Drawings
FIG. 1 is a 4-generation non-syndromic cleft lip and palate family, in which black arrows indicate probands; black symbols represent the affected state; slash indicates that the product has passed;
FIG. 2 is a family of non-syndromic cleft lip and palate generations 2;
FIG. 3 is a sequencing diagram of the PCR products of the affected individuals IV 2 in the family 1;
FIG. 4 is a Western blot result chart of N4BP2 overexpression plasmid and mutant plasmid transfected HEK293T cell;
FIG. 5 is a graph of the immunohistochemistry results of wild-type C57/B6 mouse E12.5 and E13.5 facial tissue sections;
FIG. 6 is a mouse phenotype graph, A-D are body weight measurement graphs, E is a comparison graph of mouse dorsal aspect, and F is an X-ray result graph.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.
The invention collects information of non-syndrome cleft lip and palate family and sporadic case, draws family spectrum for family patients, and confirms the pathogenic gene mutation site by extracting genomic DNA of sporadic patients and family members, exon sequencing technology, PCR amplification of candidate pathogenic gene mutation site and Sanger sequencing. Then, the site is detected for other patients and normal persons in the family, and whether the mutation site is the pathogenic cause of non-syndromic cleft lip and palate in the family is confirmed. The method comprises the following specific steps:
1. collecting the family and sporadic cases of non-syndromic cleft lip and palate patients
The method is cooperated with a clinical hospital, and according to the principle of informed consent, the method is used for collecting peripheral blood samples and collating basic information and clinical data of patients with non-syndrome cleft lip and palate, family individuals and sporadic patients. And (4) drawing a family map and judging the genetic mode of the non-syndromic cleft lip and palate in the family.
2. Gene diagnosis DNA analysis non-syndrome cleft lip and palate pathogenic gene mutation
Candidate pathogenic gene mutation sites are obtained by peripheral blood genome DNA extraction and exon sequencing technologies of non-syndromic cleft lip and palate patients and other family members. Designing primers for the candidate pathogenic gene mutation sites, carrying out PCR amplification and Sanger sequencing on the patients and other family members, and carrying out comparison analysis to confirm the pathogenic gene mutation sites.
3. In vitro functional validation
An overexpression plasmid (Shanghai Jikai gene chemistry technology, Co., Ltd.) of N4BP2 is constructed, a corresponding mutation plasmid is constructed through a point mutation kit, a HEK293T cell line is transfected in vitro, and whether the expression and the function of the N4BP2 protein can be influenced by the corresponding mutation site is detected.
4. In vivo functional verification
The phenotypic differences of the knockout mice and the wild mice were compared by constructing N4bp2 knockout mice (cantonese seiko biotechnology limited).
Based on the above research, the invention detects that the 13 th exon of the N4BP2 gene of the patient (proband) and the mother of the patient has a non-frameshift deletion mutation in the 4 th generation non-syndromic cleft lip and palate family 1, namely, the 9 th base AAGATATTT deletion at the 4:40133481 and 40133489 sites on the 4 th chromosome causes the deletion of the encoded amino acids of N4BP2, namely lysine (Lys), isoleucine (Ile) and phenylalanine (Phe) (GRCh37. p13; NM-018177.6; NP-060647.2). The mother of the patient is a mutant individual, and the father of the patient is a normal person and does not have the mutation site.
The invention detects that the 9 th exon of the N4BP2 gene of a patient and the mother thereof has a missense mutation in another 2-generation non-syndromic cleft lip and palate pedigree 2, namely, the mutation from T to G at the 4:40123905 site on the 4 chromosome causes the encoded amino acid to be changed from phenylalanine (Phe) to valine (Val) (GRCh37. p13; NM-018177.6; NP-060647.2). The mother of the patient is a mutant individual, and the father of the patient is a normal person and does not have the mutation site.
In two non-syndromic cleft lip and palate sporadic cases, the invention detects that the 13 th exon of the N4BP2 gene of a patient has a missense mutation, namely, the 4:40122958 site on the 4 chromosome is mutated from C to T, so that the coded amino acid is changed from threonine (Thr) to methionine (Met) (GRCh37. p13; NM-018177.6; NP-060647.2).
Therefore, the invention provides the application of three mutant N4BP2 genes and three mutant N4BP2 genes related to non-syndrome cleft lip and palate as well as a mutant N4BP2 gene and a mutant N4BP2 gene in preparing a diagnostic kit for detecting non-syndrome cleft lip and palate diseases. Compared with the non-mutated N4BP2 Gene with the Gene bank accession number NM-018177.6, the 13 th exon of the mutated N4BP2 Gene contains a non-frame-shifting deletion mutation site 4:40133481, 40133489delAAGATATTT and two missense mutation sites 4:40123905T > G and 4:40122958C > T of the 9 th exon, wherein the mutation site is related to non-syndromic cleft lip and palate. Compared with the non-mutated N4BP2 with Gene bank accession number NP-060647.2, the mutated N4BP2 contains amino acid mutation sites of p.Lys1531-Phe1533del, p.Thr1076Met and p.Phe 1392Val.
Furthermore, the invention also provides a PCR primer composition for detecting the N4BP2 gene mutation related to the non-syndrome cleft lip and palate and application thereof in preparing a diagnostic kit for detecting the non-syndrome cleft lip and palate diseases. The PCR primer composition consists of an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.
Example (b):
1. collecting the family and sporadic cases of non-syndromic cleft lip and palate patients
According to the principle of informed consent, a northern area 4-generation non-syndromic cleft lip and palate family and 33 sporadic patients were collected, wherein the first patient in the family is IV 2, and the female is mainly characterized by left-side cleft lip. The patient mother also presented with a left cleft lip, normal paternal phenotype, not a close married. Pedigrees were drawn (FIG. 1). In 33 patients, 17 of the patients had cleft lips and 16 had cleft palate. Samples including peripheral blood samples from 2 patients (IV 2, III 17) and 1 normal individual (III 16) in the family and 33 patients in the distribution, and relevant basic information and clinical data were collected and numbered.
2. Gene diagnosis DNA analysis non-syndrome cleft lip and palate pathogenic gene mutation
Extracting the peripheral blood genome DNA of the IV 2 patient in the family 1, sending the extracted peripheral blood genome DNA to Beijing Nuo He genesis science and technology GmbH for whole exon sequencing to obtain a candidate pathogenic gene N4BP2 13 exon mutation site 4:40133481-40133489(GRCh37.p7), wherein the nucleotide sequence of the mutated N4BP2 13 exon is shown as SEQ ID NO. 3. Designing a primer for the candidate pathogenic gene mutation site, wherein the primer comprises the following components: 5'-GTGCAAGAGTACTCGTATTAC-3' (SEQ ID NO.1) and 5'-CTTGGAATAATACCCACAG-3' (SEQ ID NO. 2). Carrying out PCR amplification on the patient and other family individuals under the reaction condition of 95 ℃ for 3 min; 30 cycles of 95 ℃, 30s, 60 ℃, 30s, 72 ℃, 30 s; 72 ℃ for 5 min; the size of the product is 416 bp. Then, the PCR product was sent to Beijing Ongjinke Biological Technology Ltd for Sanger sequencing, and alignment analysis revealed that the N4BP2 gene mutation site was found in the patients (IV 2, III 17), the patient and their mother had a deletion of the 13 th exon corresponding base position of N4BP2 and the father had no mutation at the III16 corresponding base position AAGATATTT, as shown in FIG. 3. From the results, 9 bases AAGATATTT deletion exists at the 13 th exon 4:40133481 and 40133489 site of the N4BP2 gene of the affected individual IV 3; 9 bases AAGATATTT are deleted at the 13 th exon 4:40133481 and 40133489 sites of the N4BP2 gene of the affected individual mother III 17; the affected individual father III16 has the N4BP2 gene with the 13 th exon 4:40133481 and 40133489 site AAGATATTT and has no deletion (incidentally: family 2 affected individuals, parents and sporadic patients have no designed primer for PCR). The mutation site results in the N4BP2 gene coding change c.4591-4599 del; Lys1531-Phe1533del, encoding a deletion of lysine 1531 to phenylalanine 1533 of the protein (NM-018177.6; NP-060647.2).
Two sporadic patients CL656ZJJ and CP634LMT respectively have cleft lip and cleft palate II degrees, and complete exon sequencing is carried out to obtain a candidate virulence gene N4BP2 exon 9 mutation site 4:40122958C > T (GRCh37.p13), and the nucleotide sequence of the mutated N4BP2 exon 9 is shown as SEQ ID NO. 5. The mutation site results in an alteration of c.3227c > T encoded by the N4BP2 gene; Thr1076Met, threonine at position 1076 of the encoded protein to methionine (NM-018177.6; NP-060647.2).
4. Genetic recurrence risk analysis
According to the results of genetic analysis and gene detection, genetic counseling and prenatal gene detection should be performed on the female with child bearing age III17 in family 1, the female with child bearing age IV 2 in family 2, the female with child bearing age 703 in family 2, and the female with child bearing age 701 in marriage to birth to prevent the child from being born. Namely, the carrier is ensured to select the fetus with the normal gene locus when in birth, so that the pathogenic gene can be prevented from being continuously transmitted in families.
The discovery of the pathogenic gene mutation site can carry out gene detection on the offspring individuals in the family to judge whether the offspring individuals are carriers or not and the incidence risks of regeneration nurses, and can carry out prenatal diagnosis and the guidance of good birth and good care. Has important significance for enriching the pathogenic gene mutation spectrum of non-syndrome cleft lip and palate and establishing a gene diagnosis method.
5. In vitro functional analysis of pathogenic sites
In this example, an overexpression plasmid pCMV-N4BP2 of N4BP2 was constructed, and a three-site mutation plasmid was constructed on the basis of the overexpression plasmid using a point mutation kit. The plasmids are respectively transfected into HEK293T cells by using a transfection reagent lipo3000, total cell protein is extracted after transfection for 48 hours, and the expression level of N4BP2 protein is detected by Western blot, and the result shows that the expression of the N4BP2 protein is reduced by three mutation sites, as shown in figure 4. Of these, 4:40122958C > T had the most severe effect on protein expression.
6. In vivo functional analysis of pathogenic genes
First, the expression of wild type C57/B6 mouse embryos E12.5 and E13.5 and N4bp2 protein in the development period of mouse cleft and palate embryos was detected by immunohistochemistry, and the result showed that N4bp2 protein was highly expressed in palatine stroma of mice E12.5 and E13.5, as shown in FIG. 5.
Then, a knockout mouse of N4bp2 was constructed, and it was found that the N4bp2 mouse has a certain embryo lethality, as shown in Table 1. Differences in phenotype after birth were observed in mice, and it was found that the pure and knockout mice were small in size, slow in development, and the X-ray results showed low bone density, as shown in fig. 6.
TABLE 1
| Genotpe | N4bp2+/+ | N4bp2+/- | N4bp2-/- |
| Number of |
18 | 24 | 5 |
| Frequency | 38.3％ | 51.1％ | 10.6％ |
| Mendel’s |
25％ | 50％ | 25％ |
Sequence listing
<110> Harbin university of medicine
<120> mutant N4BP2 gene related to non-syndromic cleft lip and palate and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4911
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atntngtgca agagtactcg tattaccttg gaataatacc cacagaaaag ggagaccctt 60
atgtttgaaa aagattgtgc cactaaacta aaggagaagc agctctttcc agccattaac 120
caaaattttc tggtggacat tttcaaggac cacaacccaa gagacgatga agatattatc 180
tctgaaaaag aagaaaatat tttatcttta tctttgaagc atctagagtt cactgaagag 240
aagaatcttg atgtaaccaa agaaacaatg ttacctgaga atgttgcata tctctctaat 300
gcagatttaa acaaaagaag aaaagaaata agtgatatga atcctagcat tcaaagtgct 360
ttaattctgg aaactccaca catgtatttt tctgactctg aaagcaaact acaggcaaca 420
gacaaaagtg aaaacgagca aatagaaatg gtggctgtaa aagggtatag taaaactgac 480
acagatagtt ctatggagag agtatcacct agtacttgct gtagtgaaaa taatcaagaa 540
gactgtgatc ttgcaaatag tggaccactt caaaatgaaa aatcctcacc tggtgaaata 600
gtggaagaaa gagcaacagt aacgaaaaaa gcctttggga aacaaaaaag caaatcgact 660
ttggaaaagt tcccaagaca tgagctatca aattttgttg gtgactggcc agttgataag 720
actattggtc agaggacaaa aaggaacaga aaaactgaaa aaacttcatc cgtacaaagc 780
gacaaaaagt ataattaccc tcagtcacac aaattagtta acagtgtatc tgtgaataca 840
gattgtgtcc agcaacgagg atctccacat gaaagtgtag aggatggcag aaagtcacag 900
tgtgatgatg cttcagagcc actcaatagc tataaatatg atgcttataa aaatattgac 960
aaaaactcat tcaacattat gggtgactgg ccttcatctg attctttagc tcagagggaa 1020
cacagatcaa gaatgccaaa gactggttta agtgagccca acctagaaat tggaacaaat 1080
gacaaaatga atgaaatatc cttatctaca gcacatgagg cctgttgggg cacaagctct 1140
caaaaactaa agacattggg tagctccaat ctaggaagtt ctgaaatgct gctcagtgaa 1200
atgacctgtg agagtcagac ttgtctaagt aaaaagagtc atgggcaaca cacatcgttg 1260
cctcttactt ttaccaatag tgcaccaact gtttctggag tagtagaacc acaaacgtta 1320
gctgaatgtc aagagcaaat gcctaagaga gaccctggaa aagaagtagg catgtgcacc 1380
cagactgaac cacaggattt tgctctttta tggaaaatag aaaagaataa aattagcatt 1440
tcagattcta tcaaagtatt aacaggaaga ttagatggat ttaagccgaa agttttcaat 1500
attaacacaa aatcagacgt tcaagaagca attccatata gagtaatgta tgataaaagc 1560
acgtttgttg aagaaagtga gcttaccagt gcagatgaat ctgaaaatct taacattctt 1620
tgtaaactgt ttggatcctt ttcattagaa gccctgaaag acttatatga gaggtgcaat 1680
aaagatatta tttgggccac aagccttttg ttggattctg aaactaagtt atgtgaggat 1740
acagagtttg agaatttcca aaaatcgtgt gatggatcac aaattgggcc tttttctctg 1800
gggttgaatt tgaaagaaat tattagccaa agaggaactt tagagaattc taattctcct 1860
gtgccagagt ttagccatgg gattggtatt agtaacgctg actcacagtc tacttgtgat 1920
gcagaaagag gaaactcaga gcaggcggaa atgagagctg tcactcctga aaaccatgaa 1980
tcgatgacaa gtatatttcc cagtgctgct gtgggtctaa agaataataa tgacatactt 2040
cctaacagcc aggaagaact tttatatagc agtaagcagt cctttccagg tattctaaaa 2100
gctactactc ctaaagatat gagtgaaaca gaaaaaaacc tagtagtcac agagactgga 2160
gacaacatac attctccttc acatttctct gatattttta actttgtatc tagtacttca 2220
aatcttgaat taaatgaaga aatttatttt actgattctc ttgaaataaa gagaaatgaa 2280
aattttccaa aggattatgt gaaattttca gatgaagaag aatttatgaa tgaagatgag 2340
aaggaaatga aggaaattct aatggcagga agtagtttat cagctggagt tagtggggaa 2400
gataaaaccg agatattgaa tcccactcca gcgatggcca aatctctgac catagactgt 2460
ctggaattgg cattaccccc tgaactggct tttcaactta atgaattagt tggtcctgtt 2520
ggtattgatt cagcccaaga gacgatgaag atattatctc tgaaaaagaa gaaaatattt 2580
tatctttatc tttgaagcat ctagagttca ctgaagagaa gaatcttgat gtaaccaaag 2640
aaacaatgtt acctgagaat gttgcatatc tctctaatgc agatttaaac aaaagaagaa 2700
aagaaataag tgatatgaat cctagcattc aaagtgcttt aattctggaa actccacaca 2760
tgtatttttc tgactctgaa agcaaactac aggcaacaga caaaagtgaa aacgagcaaa 2820
tagaaatggt ggctgtaaaa gggtatagta aaactgacac agatagttct atggagagag 2880
tatcacctag tacttgctgt agtgaaaata atcaagaaga ctgtgatctt gcaaatagtg 2940
gaccacttca aaatgaaaaa tcctcacctg gtgaaatagt ggaagaaaga gcaacagtaa 3000
cgaaaaaagc ctttgggaaa caaaaaagca aatcgacttt ggaaaagttc ccaagacatg 3060
agctatcaaa ttttgttggt gactggccag ttgataagac tattggtcag aggacaaaaa 3120
ggaacagaaa aactgaaaaa acttcatccg tacaaagcga caaaaagtat aattaccctc 3180
agtcacacaa attagttaac agtgtatctg tgaatacaga ttgtgtccag caacgaggat 3240
ctccacatga aagtgtagag gatggcagaa agtcacagtg tgatgatgct tcagagccac 3300
tcaatagcta taaatatgat gcttataaaa atattgacaa aaactcattc aacattatgg 3360
gtgactggcc ttcatctgat tctttagctc agagggaaca cagatcaaga atgccaaaga 3420
ctggtttaag tgagcccaac ctagaaattg gaacaaatga caaaatgaat gaaatatcct 3480
tatctacagc acatgaggcc tgttggggca caagctctca aaaactaaag acattgggta 3540
gctccaatct aggaagttct gaaatgctgc tcagtgaaat gacctgtgag agtcagactt 3600
gtctaagtaa aaagagtcat gggcaacaca catcgttgcc tcttactttt accaatagtg 3660
caccaactgt ttctggagta gtagaaccac aaacgttagc tgaatgtcaa gagcaaatgc 3720
ctaagagaga ccctggaaaa gaagtaggca tgtgcaccca gactgaacca caggattttg 3780
ctcttttatg gaaaatagaa aagaataaaa ttagcatttc agattctatc aaagtattaa 3840
caggaagatt agatggattt aagccgaaag ttttcaatat taacacaaaa tcagacgttc 3900
aagaagcaat tccatataga gtaatgtatg ataaaagcat gtttgttgaa gaaagtgagc 3960
ttaccagtgc agatgaatct gaaaatctta acattctttg taaactgttt ggatcctttt 4020
cattagaagc cctgaaagac ttatatgaga ggtgcaataa agatattatt tgggccacaa 4080
gccttttgtt ggattctgaa actaagttat gtgaggatac agagtttgag aatttccaaa 4140
aatcgtgtga tggatcacaa attgggcctt tttctctggg gttgaatttg aaagaaatta 4200
ttagccaaag aggaacttta gagaattcta attctcctgt gccagagttt agccatggga 4260
ttggtattag taacgctgac tcacagtcta cttgtgatgc agaaagagga aactcagagc 4320
aggcggaaat gagagctgtc actcctgaaa accatgaatc gatgacaagt atatttccca 4380
gtgctgctgt gggtctaaag aataataatg acatacttcc taacagccag gaagaacttt 4440
tatatagcag taagcagtcc tttccaggta ttctaaaagc tactactcct aaagatatga 4500
gtgaaacaga aaaaaaccta gtagtcacag agactggaga caacatacat tctccttcac 4560
atttctctga tatttttaac tttgtatcta gtacttcaaa tcttgaatta aatgaagaaa 4620
tttattttac tgattctctt gaaataaaga gaaatgaaaa ttttccaaag gattatgtga 4680
aattttcaga tgaagaagaa tttatgaatg aagatgagaa ggaaatgaag gaaattctaa 4740
tggcaggaag tagtttatca gctggagtta gtggggaaga taaaaccgag atattgaatc 4800
ccactccagc gatggccaaa tctctgacca tagactgtct ggaattggca ttaccccctg 4860
aactggcttt tcaacttaat gaattatttg gtcctgttgg tattgattca g 4911
Claims (8)
1. A mutated N4BP2 Gene associated with non-syndromic cleft lip and palate, characterized in that the mutated N4BP2 Gene comprises a non-frameshift deletion mutation site 4:40133481 as well as 40133489delAAGATATTT in exon 13 and a missense mutation site 4:40123905T > G and a missense mutation site 4:40122958C > T in exon 9 respectively compared with the unmutated N4BP2 Gene with Gene bank accession No. NM-018177.6.
2. The mutated N4BP2 gene related to non-syndromic cleft lip and palate according to claim 1, wherein the nucleotide sequence of exon 13 of N4BP2 after said mutation is shown in SEQ ID No. 3.
3. The mutated N4BP2 gene related to non-syndromic cleft lip and palate according to claim 1, wherein the nucleotide sequence of exon 9 of N4BP2 after said mutation is shown in SEQ ID No.4 and SEQ ID No. 5.
4. Use of the mutated N4BP2 gene associated with non-syndromic cleft lip and palate disease as defined in any one of claims 1 to 3 in the manufacture of a diagnostic kit for detecting non-syndromic cleft lip and palate disease.
5. A mutant N4BP2 associated with non-syndromic cleft lip and palate, characterized in that said mutant N4BP2 comprises p.lys1531_ Phe1533del, p.thr1076met and p.phe1392val amino acid mutation sites compared to the unmutated N4BP2 of Gene bank accession No. NP _ 060647.2.
6. Use of the mutated N4BP2 associated with non-syndromic cleft lip and palate disease of claim 5 in the manufacture of a diagnostic kit for detecting non-syndromic cleft lip and palate disease.
7. A PCR primer composition for detecting the N4BP2 gene of the non-frameshift deletion mutation related to the non-syndromic cleft lip and palate as described in claims 1-2, wherein the PCR primer composition consists of an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2.
8. Use of the PCR primer composition of claim 7 for detecting the mutated N4BP2 gene associated with non-syndromic cleft lip and palate disease in the preparation of a diagnostic kit for detecting non-syndromic cleft lip and palate disease.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102084000A (en) * | 2008-02-01 | 2011-06-01 | 总医院有限公司 | Use of microvesicles in diagnosis, prognosis and treatment of medical diseases and conditions |
| CN103937804A (en) * | 2014-04-21 | 2014-07-23 | 哈尔滨医科大学 | Congenital aniridia disease-causing gene, kit for detecting congenital aniridia disease-causing gene and application of gene |
| CN104651400A (en) * | 2015-02-13 | 2015-05-27 | 山东大学 | Method for conditional knockout of Lox13 gene of mouse and application |
| CN105803054A (en) * | 2014-12-31 | 2016-07-27 | 天津华大基因科技有限公司 | Kit and use thereof in detection of orofacial clefts related genes |
| US20170137885A1 (en) * | 2014-05-22 | 2017-05-18 | The Scripps Research Institute | Gene expression profiles associated with sub-clinical kidney transplant rejection |
| CN108384789A (en) * | 2018-05-10 | 2018-08-10 | 哈尔滨医科大学 | With the TYR genes after the relevant mutation of 1 type of eye skin albinism and its purposes in gene diagnosis |
| JP2018143178A (en) * | 2017-03-06 | 2018-09-20 | 国立大学法人三重大学 | Genetic risk detection method for cardiovascular disease |
| CN110699446A (en) * | 2019-11-07 | 2020-01-17 | 南京医科大学附属口腔医院 | SNP marker rs3174298 related to non-syndrome cleft lip and palate diagnosis and application thereof |
| CN111334513A (en) * | 2020-02-13 | 2020-06-26 | 南京医科大学附属口腔医院 | Non-syndromic cleft lip related low-frequency/rare mutation and detection method thereof |
-
2020
- 2020-12-04 CN CN202011417022.XA patent/CN112458104A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102084000A (en) * | 2008-02-01 | 2011-06-01 | 总医院有限公司 | Use of microvesicles in diagnosis, prognosis and treatment of medical diseases and conditions |
| CN103937804A (en) * | 2014-04-21 | 2014-07-23 | 哈尔滨医科大学 | Congenital aniridia disease-causing gene, kit for detecting congenital aniridia disease-causing gene and application of gene |
| US20170137885A1 (en) * | 2014-05-22 | 2017-05-18 | The Scripps Research Institute | Gene expression profiles associated with sub-clinical kidney transplant rejection |
| CN105803054A (en) * | 2014-12-31 | 2016-07-27 | 天津华大基因科技有限公司 | Kit and use thereof in detection of orofacial clefts related genes |
| CN104651400A (en) * | 2015-02-13 | 2015-05-27 | 山东大学 | Method for conditional knockout of Lox13 gene of mouse and application |
| JP2018143178A (en) * | 2017-03-06 | 2018-09-20 | 国立大学法人三重大学 | Genetic risk detection method for cardiovascular disease |
| CN108384789A (en) * | 2018-05-10 | 2018-08-10 | 哈尔滨医科大学 | With the TYR genes after the relevant mutation of 1 type of eye skin albinism and its purposes in gene diagnosis |
| CN110699446A (en) * | 2019-11-07 | 2020-01-17 | 南京医科大学附属口腔医院 | SNP marker rs3174298 related to non-syndrome cleft lip and palate diagnosis and application thereof |
| CN111334513A (en) * | 2020-02-13 | 2020-06-26 | 南京医科大学附属口腔医院 | Non-syndromic cleft lip related low-frequency/rare mutation and detection method thereof |
Non-Patent Citations (4)
| Title |
|---|
| ELIZABETH J. LESLIE等: "Association studies of low-frequency coding variants in nonsyndromic cleft lip with or without cleft palate.", 《AMERICAN JOURNAL OF MEDICAL GENETICS, PART A》 * |
| LESLIE EJ等: "Homo sapiens NEDD4 binding protein 2 (N4BP2), transcript variant 1, mRNA", 《GENBANK DATABASE》 * |
| 周水勇等: "河南地区96例非综合征性唇腭裂患者TGF-α基因TaqI位点多态性观察", 《山东医药》 * |
| 耿宏 等: "遗传因素及环境因素对唇腭裂畸形发生的影响", 《中国临床康复》 * |
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