CN104745591B

CN104745591B - CYP4V2 gene mutant and application thereof

Info

Publication number: CN104745591B
Application number: CN201310749646.5A
Authority: CN
Inventors: 阴正勤; 孟晓红; 金鑫; 徐海伟; 黎其友; 李世迎
Original assignee: First Affiliated Hospital of TMMU
Current assignee: First Affiliated Hospital of TMMU
Priority date: 2013-12-31
Filing date: 2013-12-31
Publication date: 2020-04-03
Anticipated expiration: 2033-12-31
Also published as: CN104745591A

Abstract

The invention discloses a CYP4V2 gene mutant and application thereof, in particular to a separated nucleic acid for coding the CYP4V2 mutant, a separated polypeptide, a method for screening a biological sample susceptible to primary crystalline retinal degeneration, a system for screening the biological sample susceptible to primary crystalline retinal degeneration and a kit for screening the biological sample susceptible to primary crystalline retinal degeneration. Wherein the isolated nucleic acid encoding the CYP4V2 mutant has substantial homology to SEQ ID NO: 1 compared to 1, with the c.413+2T > G mutation. By detecting whether the novel mutant exists in a biological sample, whether the biological sample is susceptible to primary crystalline retinal degeneration can be effectively detected.

Description

CYP4V2 gene mutant and application thereof

Technical Field

The invention relates to a CYP4V2 gene mutant and application thereof. In particular, the invention relates to isolated nucleic acids encoding CYP4V2 mutants, isolated polypeptides, methods of screening biological samples for susceptibility to primary crystalline retinal degeneration, systems for screening biological samples for susceptibility to primary crystalline retinal degeneration, kits for screening biological samples for susceptibility to primary crystalline retinal degeneration, constructs, and recombinant cells.

Background

Bietti's crystalloid retinal degeneration (BCD for short) is a rare hereditary blindness-causing disease, the worldwide incidence rate is about 1/24000, the high-incidence population is Chinese, and the incidence rate in China is about 1/4000. BCD patients usually develop diseases in 20-30 years and become blind in 40-50 years, so that the BCD patients become major eye diseases threatening the visual function and visual quality of young and middle-aged people all over the world at present, the life quality of the patients is seriously affected finally, and huge burden is brought to families and society. BCD is a blinding hereditary eye disease caused by degeneration of retinal photoreceptor cells (including cone cells and rod cells) and retinal pigment epithelial cells, accompanied by progressive choroidal vascular atrophy, sclerosis. The genetic mode of BCD is mainly autosomal recessive (auto-receptive BCD, ARBCD for short).

The genetic aspects of primary BCD are less studied at present, and the mechanisms associated with pathogenesis remain unclear. Thus, the research on primary BCD, especially on its pathogenic gene, is still in depth.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. To this end, it is an object of the present invention to propose a method that enables efficient screening of biological samples susceptible to primary crystalline retinal degeneration (BCD).

The present invention is completed based on the following work of the inventors: the inventor determines a new pathogenic mutation of primary crystal-like retinal degeneration, namely c.413+2T > G mutation of CYP4V2 gene, by exome sequencing, Sanger sequencing verification and protein structure analysis.

Furthermore, according to a first aspect of the present invention, there is provided an isolated nucleic acid encoding a CYP4V2 mutant. According to an embodiment of the invention, the nucleic acid is identical to SEQ ID NO: 1, compared with the wild-type CYP4V2 gene, the mutant has c.413+2T > G mutation, namely, the 413 th cleavage site TT mutation of the cDNA of the mutant CYP4V2 gene is TG. According to the embodiments of the present invention, the inventors have determined a mutant of CYP4V2 gene, which is closely related to the onset of primary crystalline retinal degeneration, so that whether a biological sample is susceptible to primary crystalline retinal degeneration can be effectively detected by detecting the presence or absence of the mutant in the biological sample.

According to a second aspect of the invention, an isolated polypeptide is provided. According to an embodiment of the invention, the isolated polypeptide has the amino acid sequence as shown in SEQ ID NO: 23, or a pharmaceutically acceptable salt thereof. By detecting whether the polypeptide is expressed in a biological sample, whether the biological sample is susceptible to primary crystalline retinal degeneration can be effectively detected.

According to a third aspect of the invention, there is provided a method of screening a biological sample susceptible to primary crystalline retinal degeneration. According to an embodiment of the invention, the method comprises the steps of: extracting a nucleic acid sample from the biological sample; determining the nucleic acid sequence of the nucleic acid sample; the nucleic acid sequence of the nucleic acid sample is identical to the sequence shown in SEQ ID NO: 1, having the c.413+2T > G mutation is indicative of the biological sample being susceptible to primary crystalline retinal degeneration. By the method for screening a biological sample susceptible to primary crystalline retinal degeneration according to the embodiment of the present invention, a biological sample susceptible to primary crystalline retinal degeneration can be effectively screened.

According to a fourth aspect of the present invention, there is provided a system for screening a biological sample susceptible to primary crystalline retinal degeneration. According to an embodiment of the invention, the system comprises: a nucleic acid extraction device for extracting a nucleic acid sample from the biological sample; a nucleic acid sequence determining device connected with the nucleic acid extracting device and used for analyzing the nucleic acid sample so as to determine the nucleic acid sequence of the nucleic acid sample; and a determination device connected to the nucleic acid sequence determination device so as to determine the sequence of the nucleic acid sample based on the sequence of the nucleic acid sample as set forth in seq id NO: 1, and whether or not it has the c.413+2T > G mutation, determining whether the biological sample is susceptible to primary crystalline retinal degeneration. By using the system, the method for screening the biological sample susceptible to the primary crystalline retinal degeneration can be effectively implemented, so that the biological sample susceptible to the primary crystalline retinal degeneration can be effectively screened.

According to a fifth aspect of the invention, there is provided a kit for screening a biological sample susceptible to primary crystalline retinal degeneration. According to an embodiment of the invention, the kit comprises: a reagent suitable for detecting a CYP4V2 gene mutant, wherein the sequence shown in SEQ ID NO: 1 compared with the CYP4V2 gene mutant, the CYP4V2 gene mutant has c.413+2T > G mutation. With the kit according to an embodiment of the present invention, a biological sample susceptible to primary crystalline retinal degeneration can be effectively screened.

According to a sixth aspect of the invention, the invention also proposes a construct. According to an embodiment of the invention, the construct comprises the isolated nucleic acid encoding a CYP4V2 mutant as described above. Therefore, the recombinant cells obtained by transforming the receptor cells with the constructs of the present invention can be effectively used for screening drugs for treating primary crystalline retinal degeneration.

According to a seventh aspect of the present invention, there is also provided a recombinant cell. According to an embodiment of the invention, the recombinant cell is obtained by transforming a recipient cell with the construct described above. According to some embodiments of the present invention, using the recombinant cells of the present invention, drugs for treating primary crystalline retinal degeneration can be effectively screened.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a schematic diagram of a system and its components for screening a biological sample susceptible to primary crystalline retinal degeneration, in accordance with an embodiment of the present invention, wherein,

FIG. 1I is a schematic diagram of a system for screening biological samples susceptible to primary crystalline retinal degeneration according to an embodiment of the present invention,

FIG. 1II is a schematic view of a nucleic acid extracting apparatus according to an embodiment of the present invention,

FIG. 1III is a schematic view of a nucleic acid sequence determination apparatus according to an embodiment of the present invention;

FIG. 2 shows a family diagram of a BCD patient's family according to one embodiment of the present invention;

FIG. 3 illustrates a fundus image of a proband in the BCD patient family of FIG. 2, according to one embodiment of the present invention;

FIG. 4 shows a representative Sanger sequencing validation peak plot of the c.413+2T > G mutation sites of the CYP4V2 gene for the proband, normal human within the family, and normal control outside the family of the BCD patients shown in FIG. 2, according to one embodiment of the present invention, wherein,

FIG. 4I is a representative Sanger sequencing verification peak diagram of c.413+2T > G mutation sites of CYP4V2 gene of proband according to an embodiment of the present invention,

FIG. 4II is a graph of a representative Sanger sequencing verification peak of the c.413+2T > G mutation site of the CYP4V2 gene of a normal human in a family according to an embodiment of the present invention,

FIG. 4III is a representative Sanger sequencing validation peak plot of the c.413+2T > G mutation site of the CYP4V2 gene of an out-of-home normal control according to an embodiment of the present invention;

FIG. 5 shows patient II of the BCD family of patients of FIG. 2, according to one embodiment of the present invention: 3 and out-of-home normal control cDNA c.413+2T > G mutation sites Sanger sequencing validated peak plots.

Detailed Description

The present invention will now be described with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

CYP4V2 gene mutant

According to a first aspect of the present invention, there is provided an isolated nucleic acid encoding a mutant CYP4V 2. According to an embodiment of the invention, the nucleic acid is identical to SEQ ID NO: 1 compared to 1, with the c.413+2T > G mutation. The expression "nucleic acid encoding a CYP4V2 mutant" as used herein refers to a nucleic acid substance corresponding to a gene encoding a CYP4V2 mutant, i.e., the type of nucleic acid is not particularly limited, and may be any polymer including deoxyribonucleotides and/or ribonucleotides corresponding to a gene encoding a CYP4V2 mutant, including but not limited to DNA, RNA or cDNA. According to a specific example of the present invention, the aforementioned nucleic acid encoding the CYP4V2 mutant is DNA. According to the embodiment of the present invention, the inventors have determined a mutant of CYP4V2 gene, which is closely related to the onset of primary crystalline retinal degeneration, so that whether a biological sample is susceptible to primary crystalline retinal degeneration can be effectively detected by detecting the presence of the mutant in the biological sample, and whether an organism is susceptible to primary crystalline retinal degeneration can be effectively predicted by detecting the presence of the mutant in the organism.

For the purposes of the present description and claims, reference to nucleic acids will be understood by those skilled in the art to include virtually either or both of the complementary strands. For convenience, in the present specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. For example, reference is made to SEQ ID NO: 1, actually including its complement. One skilled in the art will also appreciate that one strand may be used to detect the other strand and vice versa.

The nucleic acid encoding the CYP4V2 mutant is a novel pathogenic mutation of a pathogenic gene CYP4V2 of primary crystal-like retinal degeneration, which is determined by the inventor through exome sequencing, Sanger sequencing verification and protein structure analysis. This pathogenic mutation site has not been mentioned in the prior art.

Wherein, the cDNA of the wild type CYP4V2 gene has a nucleotide sequence shown as follows:

ATGGCGGGGCTCTGGCTGGGGCTCGTGTGGCAGAAGCTGCTGCTGTGGGGCGCGGCGAGTGCCCTTTCCCTGGCCGGCGCCAGTCTGGTCCTGAGCCTGCTGCAGAGGGTGGCGAGCTACGCGCGGAAATGGCAGCAGATGCGGCCCATCCCCACGGTGGCCCGCGCCTACCCACTGGTGGGCCACGCGCTGCTGATGAAGCCGGACGGGCGAGAATTTTTTCAGCAGATCATTGAGTACACAGAGGAATACCGCCACATGCCGCTGCTGAAGCTCTGGGTCGGGCCAGTGCCCATGGTGGCCCTTTATAATGCAGAAAATGTGGAGGTAATTTTAACTAGTTCAAAGCAAATTGACAAATCCTCTATGTACAAGTTTTTAGAACCATGGCTTGGCCTAGGACTTCTTACAAGTACTGGAAACAAATGGCGCTCCAGGAGAAAGATGTTAACACCCACTTTCCATTTTACCATTCTGGAAGATTTCTTAGATATCATGAATGAACAAGCAAATATATTGGTTAAGAAACTTGAAAAACACATTAACCAAGAAGCATTTAACTGCTTTTTTTACATCACTCTTTGTGCCTTAGATATCATCTGTGAAACAGCTATGGGGAAGAATATTGGTGCTCAAAGTAATGATGATTCCGAGTATGTCCGTGCAGTTTATAGAATGAGTGAGATGATATTTCGAAGAATAAAGATGCCCTGGCTTTGGCTTGATCTCTGGTACCTTATGTTTAAAGAAGGATGGGAACACAAAAAGAGCCTTCAGATCCTACATACTTTTACCAACAGTGTCATCGCTGAACGGGCCAATGAAATGAACGCCAATGAAGACTGTAGAGGTGATGGCAGGGGCTCTGCCCCCTCCAAAAATAAACGCAGGGCCTTTCTTGACTTGCTTTTAAGTGTGACTGATGACGAAGGGAACAGGCTAAGTCATGAAGATATTCGAGAAGAAGTTGACACCTTCATGTTTGAGGGGCACGATACAACTGCAGCTGCAATAAACTGGTCCTTATACCTGTTGGGTTCTAACCCAGAAGTCCAGAAAAAAGTGGATCATGAATTGGATGACGTGTTTGGGAAGTCTGACCGTCCCGCTACAGTAGAAGACCTGAAGAAACTTCGGTATCTGGAATGTGTTATTAAGGAGACCCTTCGCCTTTTTCCTTCTGTTCCTTTATTTGCCCGTAGTGTTAGTGAAGATTGTGAAGTGGCAGGTTACAGAGTTCTAAAAGGCACTGAAGCCGTCATCATTCCCTATGCATTGCACAGAGATCCGAGATACTTCCCCAACCCCGAGGAGTTCCAGCCTGAGCGGTTCTTCCCCGAGAATGCACAAGGGCGCCATCCATATGCCTACGTGCCCTTCTCTGCTGGCCCCAGGAACTGTATAGGTCAAAAGTTTGCTGTGATGGAAGAAAAGACCATTCTTTCGTGCATCCTGAGGCACTTTTGGATAGAATCCAACCAGAAAAGAGAAGAGCTTGGTCTAGAAGGACAGTTGATTCTTCGTCCAAGTAATGGCATCTGGATCAAGTTGAAGAGGAGAAATGCAGATGAACGCTAA(SEQ ID NO：1)，

the encoded protein has the following amino acid sequence:

MAGLWLGLVW QKLLLWGAAS ALSLAGASLV LSLLQRVASY ARKWQQMRPIPTVARAYPLVGHALLMKPDG REFFQQIIEY TEEYRHMPLL KLWVGPVPMV ALYNAENVEVILTSSKQIDKSSMYKFLEPW LGLGLLTSTG NKWRSRRKML TPTFHFTILE DFLDIMNEQA NILVKKLEKH INQEAFNCFFYITLCALDII CETAMGKNIG AQSNDDSEYV RAVYRMSEMI FRRIKMPWLW LDLWYLMFKE GWEHKKSLQILHTFTNSVIA ERANEMNANE DCRGDGRGSA PSKNKRRAFL DLLLSVTDDE GNRLSHEDIR EEVDTFMFEGHDTTAAAINW SLYLLGSNPE VQKKVDHELD DVFGKSDRPA TVEDLKKLRY LECVIKETLR LFPSVPLFARSVSEDCEVAG YRVLKGTEAV IIPYALHRDP RYFPNPEEFQ PERFFPENAQ GRHPYAYVPF SAGPRNCIGQKFAVMEEKTI LSCILRHFWI ESNQKREELG LEGQLILRPS NGIWIKLKRR NADER(SEQ ID NO：2)。

the CYP4V2 gene mutant discovered by the inventor is compared with the sequence shown in SEQ ID NO: 1, compared with the wild-type CYP4V2 gene, the mutant has c.413+2T > G mutation, namely, the 413 th cleavage site TT mutation of the cDNA of the mutant CYP4V2 gene is TG. Thus, the encoded product has a splice mutation compared to wild-type CYP4V2, i.e. the mutation is due to the c.413+2T > G mutation.

It is known that the CYP4V2 gene encodes 19.28kp in full length, contains 11 exons, and encodes a protein of 525 amino acids, which belongs to the CYP450 family and plays an important role in fatty acid metabolism. At present, people have little cognition on the CYP4V2 gene in vivo, and no report related to the CYP4V2 gene and the c.413+2T > G mutation thereon as the pathogenic mutation of primary crystalloid retinal degeneration is found yet.

According to a second aspect of the invention, an isolated polypeptide is provided. According to an embodiment of the invention, the isolated polypeptide has the amino acid sequence as shown in SEQ ID NO: 23, and the amino acid sequence shown in the specification:

MAGLWLGLVWQKLLLWGAASALSLAGASLVLSLLQRVASYARKWQQMRPIPTVARAYPLVGHALLMKPDGREFFQQIIEYTEEYRHMPLLKLWVGPVPMVALYNAENVEYWKQMALQEKDVNTHFPFYHSGRFLRYHESTSKYIGSETKKTHPPRSITLLFLHHSLCLRYHLINSYGEEYWCSKQEAFRVCPCSLLNELDDISKNKDALALAASLVPYVERRMGTQKEPSDPTYFYQQCHRLTGQLNERQFRLGREWQGLCPLQKETQGLSILAFKCDMNRREQAKSDRYSRRSSHLHVRGARYNCSCNKLVLIPVGFHPRSPEKSGSFIGERVWEVAPSRYSRRPEETSVSGMCYKGDPSPFSFCSFICPPCTVRLLSGRLQSSKRHKSRHHSLCIAQRSEILPQPRGVPAYAVLPRECTRAPSICLRALLCWPQELYRSKVCCDGRKDHSFVHPEALLDRIQPEKRRAWSRRTVDSSSKTWHLDQVEEEKCRSTX(SEQ ID NO：23)。

according to some embodiments of the invention, the polypeptide is encoded by the aforementioned isolated nucleic acid encoding a CYP4V2 mutant. Whether the biological sample is susceptible to primary crystalline retinal degeneration can be effectively detected by detecting whether the polypeptide is expressed in the biological sample, and whether the biological sample is susceptible to primary crystalline retinal degeneration can be effectively predicted by detecting whether the polypeptide exists in an organism.

According to a third aspect of the invention, there is provided a method of screening a biological sample susceptible to primary crystalline retinal degeneration. According to an embodiment of the invention, the method comprises the steps of:

extracting a nucleic acid sample from the biological sample. According to the embodiment of the present invention, the type of the biological sample is not particularly limited as long as a nucleic acid sample reflecting the presence or absence of the mutation in the biological sample CYP4V2 can be extracted from the biological sample. According to an embodiment of the present invention, the biological sample may be at least one selected from human blood, skin, subcutaneous tissue, preferably peripheral blood. Therefore, the sampling and detection can be conveniently carried out, so that the efficiency of screening the biological sample susceptible to the primary crystalline retinal degeneration can be further improved. The term "nucleic acid sample" as used herein is to be understood broadly according to embodiments of the present invention and may be any sample that reflects the presence or absence of a mutation in CYP4V2 in a biological sample, such as whole genomic DNA extracted directly from a biological sample, a portion of the whole genome that includes the sequence encoding CYP4V2, total RNA extracted from a biological sample, or mRNA extracted from a biological sample. According to one embodiment of the invention, the nucleic acid sample is whole genomic DNA. Therefore, the source range of the biological sample can be expanded, and various information of the biological sample can be determined simultaneously, so that the efficiency of screening the biological sample susceptible to primary crystal-like retinal degeneration can be improved. In addition, according to an embodiment of the present invention, for using RNA as the nucleic acid sample, extracting the nucleic acid sample from the biological sample may further include: extracting an RNA sample from the biological sample, preferably the RNA sample is mRNA; and obtaining a cDNA sample by reverse transcription reaction based on the obtained RNA sample, the obtained cDNA sample constituting a nucleic acid sample. Thus, the efficiency of screening a biological sample susceptible to primary crystalline retinal degeneration using RNA as a nucleic acid sample can be further improved.

Next, after obtaining the nucleic acid sample, the nucleic acid sample may be analyzed, thereby enabling determination of the nucleic acid sequence of the obtained nucleic acid sample. According to embodiments of the present invention, the method and apparatus for determining the nucleic acid sequence of the resulting nucleic acid sample are not particularly limited. According to embodiments of the present invention, the nucleic acid sequence of a nucleic acid sample may be determined by a sequencing method. Methods and apparatuses that may be used to perform sequencing according to embodiments of the present invention are not particularly limited. According to embodiments of the present invention, second generation sequencing techniques may be employed, as well as third generation and fourth generation or more advanced sequencing techniques. According to a specific example of the present invention, the nucleic acid sequence may be sequenced using at least one selected from Hiseq2000, SOLiD, 454, and a single molecule sequencing device. Therefore, by combining the latest sequencing technology, the higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved, so that the characteristics of high throughput and deep sequencing of the sequencing devices can be utilized to further improve the efficiency of detecting and analyzing the nucleic acid sample. Therefore, the accuracy and the precision of the subsequent analysis of the sequencing data can be improved. Thus, according to embodiments of the present invention, determining the nucleic acid sequence of the nucleic acid sample may further comprise: firstly, aiming at the obtained nucleic acid sample, constructing a nucleic acid sequencing library; and sequencing the obtained nucleic acid sequencing library so as to obtain a sequencing result consisting of a plurality of sequencing data. According to some embodiments of the invention, the resulting nucleic acid sequencing library may be sequenced using at least one selected from Hiseq2000, SOLiD, 454, and single molecule sequencing devices. In addition, according to embodiments of the present invention, the nucleic acid sample may be screened to enrich for the CYP4V2 exon, and the screening enrichment may be performed before, during, or after the sequencing library is constructed. According to one embodiment of the present invention, constructing a nucleic acid sequencing library for a nucleic acid sample further comprises: carrying out PCR amplification on a nucleic acid sample by using CYP4V2 exon specific primers; and constructing a nucleic acid sequencing library aiming at the obtained amplification products. Thus, CYP4V2 exon can be enriched by PCR amplification, thereby further improving the efficiency of screening biological samples susceptible to primary crystalline retinal degeneration. According to an embodiment of the present invention, the sequence of the CYP4V2 exon-specific primer is not particularly limited, and according to a preferred embodiment of the present invention, the CYP4V2 exon-specific primer has the sequence of SEQ ID NO: 7 and 8:

a forward primer: 5'-AGATTCGCCTCCTCCCACCTCAC-3' (SEQ ID NO: 7);

reverse primer: 5'-ACCTGGACTCTTGGCCTCTTGACG-3' (SEQ ID NO: 8).

The inventors surprisingly found that amplification of the CYP4V2 exon can be significantly efficiently accomplished in a PCR reaction system by using the above primers. Note that, these SEQ ID NOs: 7 and SEQ ID NO: the nucleotide sequence shown in 8 was unexpectedly obtained by the present inventors after having performed a hard work.

With regard to the methods and procedures for constructing sequencing libraries for nucleic acid samples, those skilled in the art may make appropriate selections based on different sequencing techniques, and with regard to the details of the procedures, see the manufacturers of sequencing instruments such as the protocols provided by Illumina, see, for example, the Multiplexing Sample Preparation Guide (Part # 1005361; Feb2010) or Paired-End Sample Preparation Guide (Part # 1005063; Feb2010), incorporated herein by reference. The method and apparatus for extracting a nucleic acid sample from a biological sample according to an embodiment of the present invention are not particularly limited, and may be performed using a commercially available nucleic acid extraction kit.

It should be noted that the term "nucleic acid sequence" used herein is to be understood in a broad sense, and may be complete nucleic acid sequence information obtained by assembling sequencing data obtained by sequencing a nucleic acid sample, or may be nucleic acid sequences directly obtained by using sequencing data (reads) obtained by sequencing a nucleic acid sample, as long as the nucleic acid sequences contain coding sequences corresponding to CYP4V 2.

Finally, after determining the nucleic acid sequence of the nucleic acid sample, comparing the nucleic acid sequence of the resulting nucleic acid sample with the nucleic acid sequence of seq id NO: 1. If there is a c.413+2T > G mutation in the resulting nucleic acid sequence, this indicates that the biological sample is susceptible to primary crystalline retinal degeneration. Thus, by the method of screening a biological sample susceptible to primary crystalline retinal degeneration according to an embodiment of the present invention, a biological sample susceptible to primary crystalline retinal degeneration can be efficiently screened. According to an embodiment of the invention, the nucleic acid sequence is identical to SEQ ID NO: 1, the method and apparatus for performing the comparison is not particularly limited, and may be operated by any conventional software, and according to the specific embodiment of the present invention, the comparison may be performed by SOAP software.

It should be noted that the use of the "method for screening a biological sample susceptible to primary crystalline retinal degeneration" according to the embodiment of the present invention is not particularly limited, and may be used, for example, as a screening method for non-diagnostic purposes.

System and kit for screening biological samples susceptible to primary crystalloid retinal degeneration

According to a fourth aspect of the present invention, there is provided a system for efficiently carrying out the above method for screening a biological sample susceptible to primary crystalline retinal degeneration.

Referring to fig. 1, the system 1000 for screening a biological sample susceptible to primary crystalline retinal degeneration according to an embodiment of the present invention includes a nucleic acid extraction device 100, a nucleic acid sequence determination device 200, and a determination device 300.

According to an embodiment of the present invention, the nucleic acid extraction apparatus 100 is used to extract a nucleic acid sample from a biological sample. As described above, according to the embodiment of the present invention, the type of the nucleic acid sample is not particularly limited, and for using RNA as the nucleic acid sample, the nucleic acid extraction apparatus further includes an RNA extraction unit 101 and a reverse transcription unit 102, wherein the extraction unit 101 is used for extracting the RNA sample from the biological sample, and the reverse transcription unit 102 is connected to the RNA extraction unit 101 for performing a reverse transcription reaction on the RNA sample to obtain a cDNA sample, and the obtained cDNA sample constitutes the nucleic acid sample.

According to an embodiment of the present invention, the nucleic acid sequence determining apparatus 200 is connected to the nucleic acid extracting apparatus 100, and is configured to analyze the nucleic acid sample to determine the nucleic acid sequence of the nucleic acid sample. As indicated above, sequencing methods can be used to determine the nucleic acid sequence of a nucleic acid sample. Thus, according to one embodiment of the present invention, the nucleic acid sequence determination apparatus 200 may further include: a library construction unit 201 and a sequencing unit 202. The library construction unit 201 is used for constructing a nucleic acid sequencing library aiming at a nucleic acid sample; the sequencing unit 202 is connected to the library construction unit 201 and is configured to sequence the nucleic acid sequencing library to obtain a sequencing result consisting of a plurality of sequencing data. As described above, CYP4V2 exon can be enriched by PCR amplification, and the efficiency of screening biological samples susceptible to primary crystalline retinal degeneration can be further improved. Thus, the library construction unit 201 may further comprise a PCR amplification module (not shown) in which CYP4V2 exon-specific primers are arranged to perform PCR amplification on the nucleic acid sample using the CYP4V2 exon-specific primers, according to an embodiment of the present invention, the CYP4V2 exon-specific primers have the amino acid sequence as shown in SEQ ID NO: 7 and 8. According to an embodiment of the invention, the sequencing unit 202 may comprise at least one selected from the group consisting of hipseq 2000, SOLiD, 454 and a single molecule sequencing device. Therefore, by combining the latest sequencing technology, the higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved, so that the characteristics of high throughput and deep sequencing of the sequencing devices can be utilized to further improve the efficiency of detecting and analyzing the nucleic acid sample. Therefore, the accuracy and the precision of the subsequent analysis of the sequencing data are improved.

According to an embodiment of the present invention, the judging means 300 is connected to the nucleic acid sequence determining means 200, and is adapted to compare the nucleic acid sequence of the nucleic acid sample with the nucleic acid sequence of SEQ ID NO: 1 to determine whether the biological sample is susceptible to primary crystalline retinal degeneration. Specifically, the nucleic acid sequence based on the nucleic acid sample is identical to the nucleic acid sequence of SEQ ID NO: 1, and whether the biological sample has the c.413+2T > G mutation, and judging whether the biological sample is susceptible to primary crystalline retinal degeneration. As previously mentioned, according to one embodiment of the invention, the nucleic acid sequence of the nucleic acid sample is identical to SEQ ID NO: 1, having the c.413+2T > G mutation is indicative of a biological sample being susceptible to primary crystalline retinal degeneration. As previously described, according to embodiments of the invention, the nucleic acid sequence is identical to SEQ ID NO: 1, the device for performing the comparison is not particularly limited, and may be operated by any conventional software, and according to the specific example of the present invention, the comparison may be performed by SOAP software.

Thus, the method for screening a biological sample susceptible to primary crystalline retinal degeneration can be effectively carried out using this system, and a biological sample susceptible to primary crystalline retinal degeneration can be effectively screened.

According to a fifth aspect of the invention, there is provided a kit for screening a biological sample susceptible to primary crystalline retinal degeneration. According to an embodiment of the present invention, the kit for screening a biological sample susceptible to primary crystalline retinal degeneration comprises: a reagent suitable for detecting a CYP4V2 gene mutant, wherein the sequence shown in SEQ ID NO: 1 compared with the CYP4V2 gene mutant, the CYP4V2 gene mutant has c.413+2T > G mutation. With the kit according to an embodiment of the present invention, a biological sample susceptible to primary crystalline retinal degeneration can be effectively screened. In this context, the term "reagent suitable for detecting a mutant CYP4V2 gene" is to be understood in a broad sense, i.e., a reagent for detecting a gene encoding CYP4V2, or a reagent for detecting a polypeptide mutant CYP4V2, for example, an antibody recognizing a specific site may be used. According to one embodiment of the invention, the agent is a nucleic acid probe or primer, preferably the nucleic acid probe or primer has the sequence as shown in SEQ ID NO: 7-8. Thus, a biological sample susceptible to primary crystalline retinal degeneration can be efficiently screened.

It is to be noted that the features and advantages described in the method section for screening a biological sample susceptible to primary crystalline retinal degeneration described herein before are equally applicable to a system or a kit for screening a biological sample susceptible to primary crystalline retinal degeneration, and will not be described herein again.

Construct and recombinant cell

According to a sixth aspect of the invention, the invention also proposes a construct. According to an embodiment of the invention, the construct comprises the isolated nucleic acid encoding the CYP4V2 mutant as described above, i.e. the CYP4V2 gene mutant of the invention. Therefore, the recombinant cells obtained by transforming the receptor cells with the constructs of the present invention can be effectively used for screening drugs for treating primary crystalline retinal degeneration. The type of the recipient cell is not particularly limited, and may be, for example, an escherichia coli cell or a mammalian cell, and the recipient cell is preferably derived from a mammal.

The term "construct" as used in the present invention refers to a genetic vector comprising a specific nucleic acid sequence and capable of transferring the nucleic acid sequence of interest into a host cell to obtain a recombinant cell. According to an embodiment of the present invention, the form of the construct is not particularly limited. According to an embodiment of the present invention, it may be at least one of a plasmid, a phage, an artificial chromosome, a Cosmid (Cosmid), and a virus, and is preferably a plasmid. The plasmid is used as a genetic carrier, has the characteristics of simple operation, capability of carrying larger fragments and convenience for operation and treatment. The form of the plasmid is not particularly limited, and may be a circular plasmid or a linear plasmid, and may be either single-stranded or double-stranded. The skilled person can select as desired. The term "nucleic acid" used in the present invention may be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any particular limitation. For constructs used to construct recombinant cells, it is preferred that the nucleic acid be DNA, as DNA is more stable and easier to manipulate than RNA.

According to a seventh aspect of the present invention, there is also provided a recombinant cell. According to an embodiment of the invention, the recombinant cell is obtained by transforming a recipient cell with the construct described above. Thus, the recombinant cells of the invention are capable of expressing the mutants of the CYP4V2 gene carried by the constructs. According to some embodiments of the present invention, using the recombinant cells of the present invention, drugs for treating primary crystalline retinal degeneration can be effectively screened. According to the embodiment of the present invention, the kind of the recipient cell is not particularly limited, and may be, for example, an escherichia coli cell, a mammalian cell, and preferably, the recipient cell is derived from a non-human mammal.

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 determination of Primary Crystal-like retinal degeneration (BCD) causative mutations

1. Sample collection

In 2010, the inventor collected 1 BCD patient at chongqing, who had 5 family members, 4 family members participating in the study of the present invention, 3 patients (parents died) and proband daughter, all of which had signed informed consent, the family chart of the patient is shown in fig. 2, wherein □ represents a normal male, ■ represents a male patient, ● represents a female patient, and,

indicating normal male has passed, M indicates mutation, + indicates wild type, and ND (not detected) indicates that no gene detection was performed.

In this family, the proband (i.e., patient II: 3 in FIG. 2), female, was 44 years old with night blindness, with fundus examination showing typical crystalloid retinal degeneration changes, and FIG. 3 shows the fundus image of this BCD patient. As can be seen from fig. 3, the boundaries of the ocular fundus discs of both eyes of the patient are clear, the retinal pigment epithelium layer is subjected to diffuse atrophy, a large amount of crystalloid granule deposits can be seen in the posterior pole retina, the choroidal capillary atrophy and the retinal pigment epithelium atrophy. The phenotype of the crystal-like retinal degeneration in the family shows a recessive inheritance pattern, and the double-affinity proband daughter (i.e. the member shown in III: 1 in figure 2) of the patient is not diseased, is a heterozygous carrier, and 3 patients carry 2 allelic mutations and completely accord with the recessive inheritance characteristics of the autosome.

2. Identification of pathogenic mutations

The inventors sequenced the exome sequences of proband using the Sanger sequencing method using the hipseq 2000, SOLiD, 454 or single molecule sequencing device. The method comprises the following specific steps:

2.1DNA extraction

Peripheral blood of a patient in the family of the patient shown in FIG. 2 (i.e., patient II: 3 in FIG. 2) was collected, genomic DNA was extracted from a peripheral blood sample using a DNA extraction kit, and the concentration and purity of DNA were measured using a spectrophotometer to obtain the concentration and purity of each genomic DNAOD₂₆₀/OD₂₈₀Should be in the range of 1.7-2.0, and have a concentration of not less than 200 ng/microliter and a total amount of not less than 30 micrograms.

2.2 target region Capture and sequencing

Each genomic DNA sample was randomly fragmented into fragments of about 100-500 bp using a sonicator, and then ligated with adaptors at each end of the fragments according to the manufacturer's instructions (see: http:// www.illumina.com/Illumina/Solexa Standard library Specification, incorporated herein by reference in its entirety). The Library is purified, then is subjected to PCR amplification, is hybridized and enriched with a capture reagent, called SureSelect biological associated RNA Library (BAITS), and is subjected to PCR amplification, and the Library can be subjected to on-board sequencing after being detected to be qualified so as to obtain original sequencing data. Wherein the sequencing platform is HiSeq, the reading length is 35-100 bp, and the average sequencing depth of each sample is at least 50 x.

3. Mutation detection, annotation, and database comparison

The raw sequencing data obtained above was processed using Illumina based software1.7, and after filtering for decontamination, aligned to the UCSC human reference genome (hg19, build37.1, http:// genome. sc. for unique sequence alignment to the genome) using SOAPaligner/SOAP2 (see: Li R, Li Y, Kristiansen K, et al, SOAP: short oligonucleotide alignment program. bioinformatics2008,24(5): 713; Li R, Yu C, Li Y, ea, SOAP2: an improved polynucleotide. for short analysis 2009,25(15): 1966. for 1967), which is incorporated herein by reference in its entirety. The genotype of the target region is then determined using SOAPsnp (see: Li R, Li Y, Fang X, Yang H, et al, SNPdetction for mapping parallel gene sequencing. genome Res2009,19(6):1124-1132, which is incorporated herein by reference in its entirety) to obtain Single Nucleotide Polymorphisms (SNPs) and insertion/deletion sequencing results for the patient.

Then, by filtering public databases such as dbSNP database (http:// hgdownloadload. cse. ucsc. edu/goldenPath/hg19/database/SNP135.txt. gz.), HapMap database (ftp:// ftp. ncbi. nlm. nih. gov/HapMap), thousand genome database (ftp:// ftp. 1000genome. ebi. ac. uk/vol1/ftp), Yanhuang database (http:// yh. genome. org. cn /), the synonymous mutations, non-coding region variations in the obtained sequencing results, and all known variations with allele frequency greater than 0.005 in the database were removed, and SIFT software was used to predict SNP functions, finally 3 nucleotide sites with possible pathogenic significance were obtained.

And (3) scanning the 3 de novo SNP loci in family members and genome DNA of a normal human control group, and finally determining that C.413+2T > G heterozygous mutation of the CYP4V2 gene causes deletion mutation of CYP4V2 protein, the gene mutation is coseparated with a disease phenotype in the autosomal recessive inheritance crystal-like retinal degeneration family, and the mutation is not detected in the normal control group. In conclusion, the CYP4V2 gene c.413+2T > G heterozygous mutation is presumed to be a pathogenic mutation of BCD.

Example 2: sanger sequencing verification of candidate CYP4V2 gene

A primer is designed aiming at the sequence of the CYP4V2 gene (exons 1-11), and the related sequence is obtained by the methods of PCR amplification, product purification and sequencing. The specific operation is as follows:

DNA extraction

Collecting peripheral blood of proband (i.e., patient II: 3 in FIG. 2) in the BCD family, extracting genomic DNA from peripheral blood leukocytes by conventional phenol-chloroform method, measuring DNA concentration and purity with spectrophotometer, and obtaining OD of genomic DNA of each sample₂₆₀/OD₂₈₀Are all between 1.7 and 2.0, the concentration is not less than 200 ng/microliter, and the total amount is not less than 30 micrograms.

2. Primer design

PCR reaction primer design reference human genome sequences are specified in table 1 below:

TABLE 1

Then, PCR reaction systems of the genome DNA samples are prepared and PCR reaction is carried out according to the following mixture ratio:

and (3) PCR reaction system:

system composition	Volume of
		DNA template (20 ng/microliter)	1 microliter
2 XGC buffer I (with addition of Mg)²⁺)	2.5 microliter
		2mM dNTP
	2 microliter

LA Taq enzyme (5U/microliter)	0.25 microliter
		Primer (100 ng/l) forward/reverse	Each 1 microliter
Deionized water	Adding to 25 microliter

Reaction conditions are as follows:

thus, PCR amplification products of the genomic DNA samples of each subject were obtained.

3. Sequencing

The PCR amplification products of the genomic DNA samples of each subject obtained in step 2 were directly subjected to DNA sequencing using the method of Sanger sequencing using the hipseq 2000, SOLiD, 454 or single molecule sequencing device.

Based on the sequencing result and further combined with CYP4V2c.413+2T > G protein structural analysis, the mutation is positioned on exon 3 of the CYP4V2 gene, the c.413 site TT is changed into TG, and cDNA sequencing analysis shows that 87 bases are deleted on exon 3 due to the abnormal shearing of mRNA of the shearing site at the 3' -end, wherein the 87 bases code for 29 amino acids, and the CYP4V2 gene protein structural truncation at the transcription level is abnormal, so that CYP4V2 is abnormally functional and causes diseases. From this, it was further shown that the CYP4V2 gene is a causative gene of BCD.

Example 3: sanger sequencing verification of pathogenic mutations

The CYP4V2 genes were examined in two additional patients (patients II: 1 and II: 2 in FIG. 2), proband daughter (III: 1 in FIG. 2) and 100 extrafamilial normal persons, respectively, in the pedigree described in example 1: the primer designed aiming at the CYP4V2 gene exon 3 in the embodiment 2 is utilized to obtain the related sequence of the mutation site through the methods of PCR amplification, product purification and sequencing, and the CYP4V2 gene and the correlation between the mutation and BCD are verified according to whether the sequence determination result belongs to the mutant type or the wild type.

The method comprises the following specific steps:

DNA extraction

According to the method for extracting DNA described in example 2, genomic DNA in peripheral venous blood of the prepared subjects was extracted and prepared, respectively, for use.

2.PCR reaction

The PCR reaction was performed using the primers and PCR conditions designed for exon 3 of the CYP4V2 gene in example 2. Thus, PCR amplification products of the genomic DNA samples of each subject were obtained.

3. Sequencing

The PCR amplification products of the genomic DNA samples of each subject obtained in step 2 were directly subjected to Sanger sequencing validation using the hisseq 2000, SOLiD, 454 or single molecule sequencing device. Representative Sanger sequencing verification peak diagrams of c.413+2T > G mutation sites of the CYP4V2 gene of proband in pedigree, normal persons in pedigree and normal control outside pedigree are shown in fig. 4.

As can be seen from FIG. 4, the CYP4V2 gene is homozygous for 413GG/GG in the wild type and heterozygous for 413GT/GC in the mutant type, and the mutation results in abnormal structural truncation of the CYP4V2 protein. In addition, it was verified that CYP4V2, c.413+2T > G heterozygous mutations did not appear to co-segregate for disease phenotype in the family, i.e. patients all carried the compound heterozygous mutation (2 alleles), whereas the unaffected family members (proband daughter) carried a single mutation (1 allele) that was pathogenic. Meanwhile, the inventors did not find the mutation site by examining 100 normal controls unrelated to the BCD family.

Thus, it was further demonstrated that the CYP4V2 gene is a causative gene of BCD, and that the c.413+2T > G mutation of the CYP4V2 gene is a causative mutation of BCD.

Example 4: RT-PCR validation

The cDNA of patient II: 3, normal human in pedigree and normal control outside pedigree shown in FIG. 2 was tested according to the following procedure:

extraction of Total RNA by TRIzol method

20ml of fresh peripheral blood of patients II: 3 shown in FIG. 2, normal persons in the pedigree and normal control outside the pedigree was extracted and cultured at a tissue density of 100 mg/cell density of 1X 10⁷1ml of TRIzol was added, homogenized thoroughly, transferred to an EP tube, blown with a 1ml sample applicator to a clear liquid without cell clumps, mixed by inversion for 10 minutes at room temperature, followed by addition of a chloroform 1/5 volume (0.2ml, 1/5 as necessary for the total volume) and mixed by inversion for 10 minutes at room temperature, after 5 minutes at room temperature, centrifuged at 12000g for 15 minutes at 4 ℃, the supernatant aqueous phase (about 400. mu.l) was transferred to another 1.5ml EP tube, then an equal volume of isopropanol (about 400. mu.l) was added, mixed by centrifugation for 10 minutes at room temperature at 12000g for 10 minutes at 4 ℃ and the supernatant was discarded. Then, 1ml of ice-precooled 75% ethanol (DEPC water) was added, centrifuged at 4 ℃ and 7500g for 5 minutes, the supernatant was discarded,after air drying for 5-10 minutes (not completely dried), it was dissolved in DEPC water.

2. Two-step method RT-PCR

2.1. Taking a Fernentas kit as an example, 20ul of reaction system is used, RNA is centrifuged for a short time, 11ul of DEPC water and RNA (1ug) are put into an EP tube and placed on ice to enable the final concentration of RNA to be 0.5ug/ul, oligo (dt)1ul is added to be mixed evenly and gently, centrifugation is carried out for a short time, then the reaction solution is placed at 65 ℃ for 5min and placed on ice for 1min, and the following components are added in sequence after centrifugation for a short time: 4ul of 5 Xbuffer solution, 200ul of ribonuclease inhibitor, 1ul of 10mm dNTP, MIX2ul and 1ul of M-Mulu reverse transcriptase are mixed evenly and centrifuged for a short time, and finally the mixture is heated at 42 ℃, 60min and 70 ℃ for 5min, the reaction is stopped, and the mixture is placed on ice and PCR (product preservation at-20 ℃) is performed.

2.2.PCR reaction: the total volume was 20. mu.l, and doubled correspondingly if the system was 50. mu.l. The method comprises the following specific steps:

reaction system:

reaction conditions are as follows:

2. sequencing

The PCR amplification products obtained in step 2 were directly sequenced using HISEQ2000, SOLID, 454 or a single molecule sequencing device using Sanger sequencing method, and the sequencing results are shown in FIG. 5. From the results shown in FIG. 5, it is known that 87 bases (the portion shown in the box in FIG. 5) are deleted from exon 3 due to abnormal cleavage of mRNA at the cleavage site, and these 87 bases encode 29 amino acids, and the protein structure of the CYP4V2 gene at the transcription level is abnormally truncated, which causes the disease caused by abnormal CYP4V2 function. Thus, it was further shown that the CYP4V2 gene is a pathogenic gene for BCD, and CYP4V2c.413+2T > G mutation is a pathogenic mutation for BCD.

Example 5: detection kit

Preparing a detection kit comprising primers capable of detecting c.413+2T > G shear mutation of CYP4V2 gene for screening biological samples susceptible to primary crystalloid retinal degeneration, wherein the primers are specific primers of CYP4V2 gene exon, and the sequence of the primers is shown as SEQ ID NO: 7-8.

The method for screening the biological sample susceptible to primary crystallization-like retinal degeneration by using the kit comprises the following specific steps: extracting the DNA of a person to be tested according to the method of the step 1 of the embodiment 2, carrying out PCR reaction by taking the extracted DNA as a template and the exon-specific primer of the CYP4V2 gene, purifying the PCR product according to the conventional method in the field, sequencing the purified product, and then observing whether the sequence obtained by sequencing has c.413+2T > G mutation, so that whether the CYP4V2 gene mutant of the invention exists in the DNA of the person to be tested can be effectively detected, whether the person to be tested is susceptible to primary crystalline retinal degeneration can be effectively detected, and further, a biological sample susceptible to primary crystalline retinal degeneration can be screened from the person to be tested.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. An isolated nucleic acid encoding a CYP4V2 mutant, wherein the CYP4V2 mutant nucleic acid hybridizes to seq id NO: 1 compared with the wild-type CYP4V2 gene, namely TT of a 413 th shearing site of cDNA of the CYP4V2 mutant nucleic acid is mutated into TG, so that an encoded product has shearing mutation compared with the wild-type CYP4V2, wherein the amino acid sequence encoded by the CYP4V2 mutant nucleic acid is shown as SEQ ID NO: shown at 23.

2. The nucleic acid of claim 1, wherein the nucleic acid is DNA.

3. An isolated polypeptide having an amino acid sequence as set forth in SEQ ID NO: shown at 23.

4. The polypeptide of claim 3, wherein the polypeptide is encoded by the CYP4V2 mutant nucleic acid of claim 1.

5. A kit for screening a biological sample for susceptibility to primary crystalline retinal degeneration, comprising:

a reagent suitable for detecting a CYP4V2 mutant nucleic acid, wherein said CYP4V2 mutant nucleic acid is the mutant nucleic acid of claim 1 and said reagent is a nucleic acid probe.

6. A construct comprising the isolated nucleic acid encoding a CYP4V2 mutant according to claim 1.

7. A recombinant cell obtained by transforming a recipient cell with the construct of claim 6.