CN113151438A - Application of reagent for detecting biomarkers in preparation of kit - Google Patents
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Abstract
The present invention provides use of a reagent for detecting a biomarker for preparing a kit for diagnosing Stargardt. With reference sequence SEQ ID NO:1, said biomarker comprising at least one of: c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37 del.
Description
Technical Field
The present invention relates to the field of biotechnology, in particular to the use of reagents for detecting biomarkers in the preparation of kits, and more particularly to non-diagnostic methods, genotyping systems for nucleic acid typing.
Background
Stargardt disease (STGD) is a retinal macular dystrophy disease which is usually seen in adolescents, has progressive decline of central vision in both eyes, is inherited from Autosomal Recessive Inheritance (ARI), has the incidence rate of about 1/10,000 and has the vision of 0.1 or below. Most lesions are located at the level of the Retinal Pigment Epithelium (RPE), eventually leading to photoreceptor cell death. The macula part is a round or oval pigment epithelium atrophy focus and pigment disorder.
STGD is highly genetically heterogeneous, and its pathogenic genes are mainly locked to ABCA4(ATP-BINDING cassete, SUBFAMILY a, MEMBER 4, ABCA4), the 4th MEMBER of the a subtype in the ATP-BINDING CASSETTE family, encoding PHOTORECEPTOR cell-SPECIFIC ATP-BINDING TRANSPORTERs on the RETINA (phototoreceptitor-SPECIFIC ATP-BINDING cassete TRANSPORTERs, RETINA-specfic, ABCR), and are therefore also referred to as ABCR. The ABCA4 gene is located on chromosome 1p22.1, contains 50 exons, and encodes 2273 amino acids. The ABCA4 mutation may also contribute to the pathogenesis of other inherited retinal degenerative diseases (HRD), such as Cone/rod dystrophy (cordi) and Retinitis Pigmentosa (RP), which are also high risk causative factors for advanced age-related macular degeneration (AMD).
Currently, the application of gene replacement therapy to treat HRD such as STGD has entered clinical trial, such as treatment of STGD (ABCA4 gene mutation, NCT01367444) and USH1B subtype patients of USH (MYO7A gene mutation, NCT 01062) with equine infectious anemia virus (ELAV) vector. Meanwhile, the transplantation of RPE cells derived from embryonic stem cells has also been applied to the clinical trial study of STGD. For such HRD diseases, the definitive genetic and clinical diagnosis of patients is the first prerequisite for the implementation of gene or stem cell therapy, and the identification of causative genes is the basis for the development of HRD therapy, so molecular genetic studies have become the first focus of attention.
In summary, the improvement of the new STGD genotype is important for the development and improvement of a new STGD gene detection technology.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following problems:
the human monogenic hereditary diseases are often related to factors such as human families, regions and the like, with the development of plans such as a human genome plan and a plan of going out of africa, the research on the migration, tracing and tracking of different human families begins to be developed from different monogenic hereditary diseases as entry points, and the expansion and discovery of different monogenic hereditary disease types are the basis of the research.
The improvement of the STGD new genotype can not only guide marriage and childbirth, prevent in advance, help prenatal diagnosis, clinical early diagnosis and differential diagnosis, but also reveal the pathophysiological mechanism of diseases, provide new theoretical basis and material basis for drug screening, and provide scientific theoretical basis for forthcoming gene or stem cell treatment.
To this end, in a first aspect of the invention, the invention proposes the use of a reagent for detecting a biomarker for the preparation of a kit for the diagnosis of STGD. According to an embodiment of the invention, the biomarker is a nucleic acid molecule represented by the reference sequence SEQ ID NO:1, said biomarker comprising at least one of: c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37 del. According to the embodiment of the invention, the nucleotide sequence of SEQ ID NO. 1 is the same as the nucleotide sequence of the coding region of the wild-type ABCA4 gene, the base of 371 th position of the nucleotide sequence of SEQ ID NO. 1 is guanine, and the inventor finds that the nucleic acid molecule with the deletion of the nucleotide at the corresponding position is a biomarker and is closely related to STGD, and forms a new genotype of the STGD; 1 nucleotide sequence of the nucleotide sequence of SEQ ID NO. 1, wherein the base at the 6788 th site is guanine, the inventor finds that the base at the corresponding site is mutated, and the nucleic acid molecule mutated into adenine is a biomarker closely related to STGD and forms a new genotype of STGD; the base of 681 th position of the nucleotide sequence of SEQ ID NO. 1 is thymine, the inventor finds that the base of the nucleotide at the corresponding position is mutated, the nucleic acid molecule mutated into guanine is a biomarker and is closely related to STGD, and a new genotype of the STGD is formed; the 5509 th base of the SEQ ID NO. 1 nucleotide sequence is cytosine, the inventor finds that the base of the nucleotide at the corresponding position is mutated, the nucleic acid molecule mutated into thymine is a biomarker and is closely related to STGD, and a novel genotype of Stargardt is formed; the inventor finds that all the nucleic acid molecules which are deleted from the corresponding positions of the No. 37 exon of the SEQ ID NO. 1 nucleotide sequence are biomarkers, are closely related to STGD, and form a novel genotype of STGD. According to the embodiment of the invention, the kit can judge whether the biomarker exists through the reagent, and further judge whether the detected biological sample belongs to the STGD new genotype.
In a second aspect of the invention, the invention provides a non-diagnostic method for typing nucleic acids. According to an embodiment of the invention, the method comprises, obtaining at least a portion of genomic sequence information of the subject; comparing at least a portion of the genomic sequence information to a reference sequence to determine whether the genomic sequence has at least one of the following biomarker combinations: c.371delG and c.6118C > T combination, c.6788G > A and c.5300T > C combination, c.681T > G and c.5882G > A combination, c.5509C > T and c.6563T > C combination, and EX37del and c.4537_4538insC combination, wherein the reference sequence comprises at least the amino acid sequence of SEQ ID NO: 371, 6118, 6788, 5300, 681, 5882, 5509, 6563, No. 37 exon, and 4537 to 4538 locus information in 1. The non-diagnostic method for nucleic acid typing according to the embodiment of the invention can effectively determine whether the genome of the subject contains the site mutation, and further carry out subsequent related scientific research.
In a third aspect of the invention, a genotyping method is provided. According to an embodiment of the invention, the method comprises: (1) extracting a nucleic acid sample of a biological sample for sequence determination of the nucleic acid sample; (2) determining the sequence of the nucleic acid sample so as to perform sequence alignment of the nucleic acid sample; (3) comparing the sequence of the nucleic acid sample to the sequence of SEQ ID NO:1, and judging whether the nucleic acid sample sequence has a combination of c.371delG and c.6118C > T, a combination of c.6788G > A and c.5300T > C, a combination of c.681T > G and c.5882G > A, a combination of c.5509C > T and c.6563T > C or a combination of EX37del and c.4537_4538insC, and further judging whether the nucleic acid molecule is a mutant type. The method provided by the embodiment of the invention is based on a sequencing means, and can be used for simply and quickly judging whether the ABCA4 gene in a biological sample genome to be tested has the following mutations compared with the wild ABCA 4: deletion of guanine at 371 th site and mutation of cytosine at 6118 th site into thymine, or mutation of guanine at 6788 th site into adenine and mutation of thymine at 5300 th site into cytosine, or mutation of thymine at 681 th site into guanine and mutation of guanine at 5882 th site into adenine, or mutation of cytosine at 5509 th site into thymine and mutation of thymine at 6563 th site into cytosine, deletion of exon 37 th site and insertion of cytosine between 4537 th site and 4538 th site.
In a fourth aspect of the invention, a genotyping system is provided. According to an embodiment of the present invention, the system comprises a nucleic acid sample extraction device for extracting a nucleic acid sample from a biological sample; a nucleic acid sequence determining device connected to the nucleic acid extracting device for analyzing the nucleic acid sample to determine the nucleic acid sequence of the nucleic acid sample; and a judging device connected to the nucleic acid sequence determining device, and judging whether the nucleic acid molecule is mutant or not based on comparison between the nucleic acid sequence of the nucleic acid sample and a reference genome to judge whether the nucleic acid sequence has a combination of c.371delG and c.6118C > T, a combination of c.6788G > A and c.5300T > C, a combination of c.681T > G and c.5882G > A, a combination of c.5509C > T and c.6563T > C, or a combination of EX37del and c.4537_4538 insC. The system according to the embodiment of the invention can perform the genotyping method, and can simply and quickly judge whether the ABCA4 gene in the genome of the biological sample to be tested has the following mutations compared with the wild-type ABCA 4: deletion of guanine at 371 th site and mutation of cytosine at 6118 th site into thymine, or mutation of guanine at 6788 th site into adenine and mutation of thymine at 5300 th site into cytosine, or mutation of thymine at 681 th site into guanine and mutation of guanine at 5882 th site into adenine, or mutation of cytosine at 5509 th site into thymine and mutation of thymine at 6563 th site into cytosine, deletion of exon 37 th site and insertion of cytosine between 4537 th site and 4538 th site.
In a fifth aspect of the invention, a kit is provided. Primers, probes and/or antibodies for detecting at least one of the following biomarkers according to embodiments of the present invention: c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37 del. The kit according to the embodiment of the invention can specifically mark the target sequence of the sample to be detected, and further judge the nucleotide sequence of the target site and the wild ABCA4 gene in the sample to be detected, namely SQ ID NO:1, whether at least one of the following mutations has occurred: the 371 th base lacks guanine, 6778 th base is mutated from guanine to adenine, 681 th base is mutated from thymine to guanine, 5509 th base is mutated from cytosine to thymine.
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 is a schematic flow diagram of a non-diagnostic method for nucleic acid typing according to an embodiment of the present invention;
FIG. 2 is a schematic flow diagram of a genotyping method according to an embodiment of the invention;
FIG. 3 is a schematic flow chart of determining the sequence of the nucleic acid sample according to an embodiment of the present invention;
FIG. 4 is a block diagram of a genotyping system according to an embodiment of the invention;
FIG. 5 is a block diagram of a nucleic acid sequence determination apparatus according to an embodiment of the present invention;
FIG. 6 is a ten STGD family spectrogram according to an embodiment of the present invention;
fig. 7 is a right fundus oculi rendering, fundus Autofluorescence (AF), and macular Optical Coherence Tomography (OCT) images of an STGD Family predecessor according to an embodiment of the present invention, wherein the first row is image acquisition of an STGD-Family1 normal control, and the second to sixth rows are image acquisition of STGD-Family1 to STGD-Family5 predecessors, respectively. The first column is fundus color photograph, and an arrow indicates an OCT scanning position; the second column is an AF image; the third column is macular OCT, AF and macular OCT can complement each other to reflect the damage of retina photoreceptor cell layer and RPE structure and function, the second and the third columns of white arrows show that the macula area ellipsoid area (EZ) zone and outer limiting membrane (ELM) are interrupted and disappear in the macular lesion area, and the area of interruption and disappearance is consistent with the abnormal AF area;
FIG. 8 is a left eye fundus image, AF and macular OCT image of an STGD Family member predecessor, in which the first row is the image acquisition of an STGD-Family1 normal contrast, the second to sixth rows are the image acquisition of STGD-Family1 to STGD-Family5 predecessors, respectively, the first column is the fundus color photograph, and the arrow indicates the OCT scanning position, according to an embodiment of the present invention; the second column is an FAF image; the third column is macular OCT, AF and macular OCT can be supplemented to reflect the damage of retina photoreceptor cell layer and RPE structure and function, the binocular fundus is basically consistent with AF, except that F4 predecessor person binocular fundus color photograph and AF type are different;
fig. 9 shows Electroretinogram inspection results of STGD pedigree provenance according to the embodiment of the invention, wherein the first to sixth rows are Full-Field Electroretinograms (FERGs) and graph electroretinograms (PERGs) of normal control, STGD-Family1 to STGD-Family5, respectively, the first column is dark 0.01ERG, the second column is dark 10.0ERG, the third column is bright 30Hz flicker ERG, the fourth column is bright 3.0ERG, and the fifth column is PERG. According to ERG typing, F1 and F4 belong to type 1, and F2, F3 and F5 belong to type 3;
FIG. 10 is a qPCR validation of STGD-Family5 ABCA4 mutant EX37del according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In a first aspect of the invention, the invention proposes the use of a reagent for detecting a biomarker for the preparation of a kit for the diagnosis of Stargardt. According to an embodiment of the invention, the biomarker is a nucleic acid molecule represented by the reference sequence SEQ ID NO:1, said biomarker comprising at least one of: c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37 del. According to one embodiment of the invention, the reference sequence SEQ ID NO 1 is the sequence of wild-type ACBA4 gene, and the inventors confirmed that the mutations of ACBA4 gene c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37del are closely related to STGD, so that whether the biological sample has STGD disease phenotype with higher probability can be deduced by detecting whether the biological sample has the mutation of the above-mentioned sites. c.371delg substitution of arginine for leucine at codon 124 of ACBA4, which after insertion of 30 wrong amino acids at this position, leads to premature termination of ABCA4 coding and translation (p.arg124leufsx30); large fragment deletion of EX37del at exon 37 of ACBA4 (complete deletion or partial sequence deletion of exon 37) resulted in significant reduction of exon 37 mRNA.
According to an embodiment of the invention, the reagent is further for detecting at least one of the following biomarkers: c.6118C > T, c.4537_4538insC, c.5300T > C, c.5882G > A and c.6563T > C. According to the embodiment of the invention, the reagent of the invention can be further used for detecting c.6118C > T, c.4537_4538insC, c.5300T > C, c.5882G > A or c.6563T > C, and the 5 mutations are also related to STGD. c.6118c > T has been reported to replace arginine by a stop codon at exon 44 codon 2040 of ACBA4 (p.arg2040ter), resulting in premature termination of the amino acid coding; c.4537-4538 insC is an insertion mutation at exon 30 of the ACBA4 gene, with cytosine (C) inserted at positions 4537 and 4538, resulting in a frame shift of the codon, glutamine mutated to proline at codon 1513, and an amino acid coding early termination after insertion of 41 mis-translated amino acids after proline, resulting in a truncation of the protein (p.gln 1513profsxsx41).
According to an embodiment of the invention, the reagent is further for detecting at least one of the following combinations of biomarkers: c.371delG and c.6118C > T, c.6788G > A and c.5300T > C, c.681T > G and c.5882G > A, c.5509C > T and c.6563T > C, and EX37del and c.4537_4538 insC. According to the embodiment of the invention, the mutations appear at the same time in pairs, which indicates that the sample to be detected has higher risk of suffering from Stargardt disease, namely: the detection of c.371delG and c.6118C > T, or c.6788G > A and c.5300T > C, or c.681T > G and c.5882G > A, or c.5509C > T and c.6563T > C, or EX37del and c.4537_4538insC, and the detection of c.371delG, c.6788G > A, c.681T > G, c.5509C > T or EX37del alone is not sufficient to indicate that the sample to be detected has a high risk of suffering from STGD diseases, but can be used as a reference factor for detecting that the sample has Stargardt diseases.
According to the specific embodiment of the invention, the gene ABCA 417 pathogenic mutation is found in the STGD family by adopting the target sequence capture of a specific gene and the NGS technology and through Sanger sequencing verification and mutation and clinical phenotype cosegregation verification, wherein 5 of the pathogenic mutations are new mutations found for the first time in the research: c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37 del. The nonsense mutation c.6118c > T was detected simultaneously in two STGD families, of which two were detected as homozygous mutations. The pathogenic mutations are summarized in table 1.
Table 1: pathogenic mutation of STGD family ABCA4 gene
Of the 17 mutations identified, 5 were the first to be proposed in this application, 2 of which were truncation mutations, leading to premature termination of the amino acid translation process during translation of the mRNA, which leads to disruption of protein structure and function, and large deletion mutations. The large fragment heterozygosis deletion (EX37del) of the STGD-Family 537 exon is carried out, qPCR verification is carried out on the No. 37 exon, and the result shows that the expression quantity of mRNA of the paternal No. 37 exon of the proband and the proband is obviously lower than that of a normal control, and the difference has statistical significance (p is less than 0.01); the expression level of exon 37 mRNA of proband mother is not different from that of normal control, and has no statistical significance (p > 0.05). The remaining two missense mutations are predicted to be pathogenic according to pathogenicity prediction criteria. In STGD F1, two complex heterozygous mutations associated with the disease were detected: mutation c.6190g > a and splicing mutation c.1937+1G > a. Missense mutation c.6190g > a at exon 45 resulted in the substitution of threonine for alanine at codon 2064 (p.ala2064thr), reported in chinese and estonia populations. In F2, two compound heterozygote mutations of c.2424C > G and c.1761-2A > G were detected in proband (F2-II-3). The nonsense mutation c.2424c > G is located in exon 16, and tyrosine was replaced by a stop codon (p.tyr808ter) at codon 808, resulting in premature termination of the amino acid coding of the ABCA4 gene. In F3, proband (F3-II-2) detected two compound heterozygous mutations c.371delG and c.6118C > T. The new mutation c.371delg, in which 30 wrong amino acids were inserted after the substitution of arginine by leucine at codon 124, resulted in the premature termination of ABCA4 coding and translation (p.arg124leufsx30). Another nonsense mutation, c.6118c > T, has been reported to replace arginine at exon 44 codon 2040 with a stop codon (p.arg2040ter), resulting in premature termination of the amino acid coding. In F4, two reported mutations, c.6118C > T and c.3385C > T, were found in the proband (F4-II-1). The nonsense mutation c.6118c > T was detected twice in this study. The missense mutation c.3385c > T is located at exon 23 codon 1129 with cystine replacing arginine (p.arg 1129cys). In F5, a large fragment deletion of exon 37 (EX37del) was detected in proband (F5-II-1). The other is an insertion mutation at the position of exon 30, cytosine (C) is inserted in the positions 4537 and 4538 (c.4537 — 4538insC), which results in the frame shift of the codon, glutamine is mutated to proline at the 1513 position of the codon, and the amino acid coding is terminated early after 41 mis-translated amino acids are inserted after proline, resulting in protein truncation (p.gln1513profsx41), which is reported in the literature. In F6, the homozygote missense mutation c.1924t > a was detected, which we have previously reported. In F7, proband F7-II-2 carries the homozygous splicing mutation c.6816+1G > A, also reported in STGD patients in China. F8 detected two mutations that could be related to the disease, a new missense mutation at exon 49 c.6788G > A and a reported missense mutation at exon 37 c.5300T > C. Two mutations c.10695t > a and c.449t > G were detected by F9. c.10695t > a is a missense mutation reported in exon 42, with a glutamic acid substituted for glycine at codon 1961 (p.g 1961e). c.449T > G is a newly reported mutation in this study, with the tyrosine replaced by a stop codon at codon 227 (p.Y227Ter) and the amino acid encoding a premature stop. In F10, two missense mutations c.6563t > C and c.5509c > T led to the development of this family disease.
According to an embodiment of the invention, the reagent is a primer set having the nucleotide sequence of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11, wherein the nucleotide sequence having the sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3 for detecting c.371delg, said primer having the nucleotide sequence shown in SEQ ID NO: 4 and SEQ ID NO: 5 for detecting EX37del, having the nucleotide sequence shown in SEQ ID NO: 6 and SEQ ID NO: 7 is used for detecting c.6788G > A, and the primer has the nucleotide sequence shown in SEQ ID NO: 8 and SEQ ID NO: 9 for detecting c.681t > G, said primer having the nucleotide sequence shown in SEQ ID NO: 10 and SEQ ID NO: 11 for the detection of c.5509C > T. According to the application of the embodiment, the reagent can specifically detect at least one of the following mutations: c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37del, and the mutation is specifically marked by specifically complementary pairing with a nucleic acid target of a sample to be detected; the mutation is detected by detecting secondary products of nucleic acid targets, such as peptide chains, proteins, amino acid residues and the like, so as to assist in detecting the mutation, and whether the biological sample is a Stargardt high-incidence sample or not is deduced.
Wild-type human ACBA4 sequence:
the nucleic acid or nucleic acid sequence underlined in bold italics in the above sequence is 371, 681, 37 exon, 5509, 6788, respectively.
Detection of the upstream primer sequence of c.371delG:
5’-GGCAGGAGAATAGCGTGAAC-3’(SEQ ID NO:2)
detection of c.371delG downstream primer sequence:
5’-CTGTGGAGGAAGAGGCATCT-3’(SEQ ID NO:3)
upstream primer sequence for detection of EX37 del:
5’-TTCCGAGATGAATTATTCCGTGAGTG-3’(SEQ ID NO:4)
downstream primer sequence for detection of EX37 del:
5’-CAGCAGGAGCAGTGCCACAA-3’(SEQ ID NO:5)
detecting the upstream primer sequence of c.6788G > A:
5’-GGCATATCTGAGCCTTGGAG-3’(SEQ ID NO:6)
detecting the sequence of a downstream primer of c.6788G > A:
5’-GTGGGTGTAGGGTGCTGTTT-3’(SEQ ID NO:7)
detecting the upstream primer sequence of c.681T > G:
5’-AAACCCCTCCCTTACCACAC-3’(SEQ ID NO:8)
detecting the sequence of the downstream primer of c.681T > G:
5’-TAGGACGTGGGTGTCTTTCC-3’(SEQ ID NO:9)
detecting the sequence of the upstream primer of c.5509C > T:
5’-TAACCCTCCCAGCTTTGGAC-3’(SEQ ID NO:10)
detecting the sequence of the downstream primer of c.5509C > T:
5’-TGTCCTGTGAGAGCATCTGG-3’(SEQ ID NO:11)
exon 37:
GTGTGGTTTAATAACAAAGGCTGGCATGCCCTGGTCAGCTTTCTCAATGTGGCCCACAACGCCATCTTACGGGCCAGCCTGCCTAAGGACAGGAGCCCCGAGGAGTATGGAATCACCGTCATTAGCCAACCCCTGAACCTGACCAAGGAGCAGCTCTCAGAGATTACAGT(SEQ ID NO:12)
in the present specification and claims, where reference is made to nucleic acids, it will be understood by those skilled in the art that the actual inclusion of either strand or the complementary double strand is intended to be convenient, although in most cases only one strand will be present in the specification and claims, but in fact the other strand complementary thereto will be disclosed. For example, reference to SEQ ID NO 1 actually includes the complementary sequence thereof. It will be understood by those skilled in the art that, in the case of one strand, the other strand can be deduced on the basis of the base-complementary pairing rules, and in the case of one strand, the other strand can be detected on the basis of the base-complementary pairing rules.
In a second aspect of the invention, the invention provides a non-diagnostic method for typing nucleic acids. According to an embodiment of the invention, with reference to fig. 1, the method comprises:
s100, obtaining at least one part of the genome sequence information of the subject. According to the embodiment of the invention, at least a part of the genome sequence of the subject can be obtained by sequencing, and the whole genome of the subject can be sequenced, the ACBA4 gene can be sequenced, and other Stargardt related genes can be sequenced. The sequencing can be a sequencing mode which can obtain nucleic acid sequence information, such as first-generation sequencing, second-generation sequencing, third-generation sequencing, single cell sequencing, more advanced sequencing technology and the like.
S200, comparing at least a portion of the genomic sequence information to a reference sequence to determine whether the genomic sequence has at least one of the following biomarker combinations: c.371delG and c.6118C > T combination, c.6788G > A and c.5300T > C combination, c.681T > G and c.5882G > A combination, c.5509C > T and c.6563T > C combination, and EX37del and c.4537_4538insC combination, wherein the reference sequence comprises at least the amino acid sequence of SEQ ID NO: 371, 6118, 6788, 5300, 681, 5882, 5509, 6563, EX37, 4537 to 4538 site information in 1. According to the embodiment of the invention, whether the ACBA4 gene of the sample to be detected has mutation at the site is determined, so as to further judge whether the sample to be detected is from a Stargardt high-risk patient.
The non-diagnostic method for nucleic acid typing can be used for drug screening, establishing and perfecting a drug screening system of Stargardt disease, efficiently detecting the genotype of a Stargardt disease model, and judging the genotype of the disease model before and after the drug is used by detecting whether mutation occurs at the site, wherein if the drug is useful, ACBA4 of the disease model is wild type, and if the disease model still has the mutation, the indication of drug ineffectiveness or low efficiency is provided.
In a third aspect of the invention, a genotyping method is provided. According to an embodiment of the invention, with reference to fig. 2 and 3, the method comprises the following steps:
s1000, extracting a nucleic acid sample of the biological sample so as to determine the sequence of the nucleic acid sample. According to an embodiment of the present invention, the biological sample may be selected from at least one of cells, tissues, organs, blood, skin, subcutaneous tissues of human or animal, and the "biological sample" is not particularly limited as long as it can be a nucleic acid sample extracted to a subject. According to one embodiment of the invention, the biological sample is human peripheral blood, and the peripheral blood is taken conveniently and quickly without causing huge trauma to a subject. The nucleic acid sample is to be understood in a broad sense, and may be any sample capable of reflecting whether the ACBA4 gene in the biological sample has a mutation of a combination of c.371delG and c.6118C > T, a combination of c.6788G > A and c.5300T > C, a combination of c.681T > G and c.5882G > A, a combination of c.550C > T and c.65T > C or a combination of EX37del and c.4537_4538insC, for example, a whole genome DNA directly extracted from the biological sample, a part of the whole genome containing the coding sequence of the ACBA4 gene, total RNA extracted from the biological sample, or mRNA extracted from the biological sample. According to an embodiment of the invention, the nucleic acid sample is whole genomic DNA. Therefore, the source range of the biological sample can be expanded, and a plurality of kinds of information of the biological sample can be determined simultaneously, so that the detection efficiency can be improved. In addition, for using RNA as the nucleic acid sample, extracting the nucleic acid sample from the biological sample may further comprise: the efficiency of detection using RNA as a nucleic acid sample can be further improved by extracting an RNA sample, preferably mRNA, from a biological sample, and obtaining a cDNA sample by a reverse transcription reaction based on the obtained RNA sample, the obtained cDNA sample constituting a nucleic acid sample. It will be appreciated by those skilled in the art that with the development of sequencing technology, no nucleic acid extraction step may be performed prior to sequencing, e.g. single cell sequencing does not require nucleic acid extraction from a cell sample for high throughput sequencing of nucleic acids within a cell sample, and therefore this step is aimed at pre-processing of nucleic acid sequences in order to obtain a biological sample.
S2000, determining the sequence of the nucleic acid sample so as to carry out sequence alignment of the nucleic acid sample.
S2100, the ACBA4 gene was first amplified using ACBA 4-specific primers so as to obtain an amplification product of the ACBA4 gene, and the ACBA 4-specific primers are not particularly limited.
According to the embodiment of the invention, the detection efficiency can be further improved by extracting, amplifying and enriching the ACBA4 gene.
S2200, adding the amplification product to a linker to obtain a sequencing library, and selecting a desired linker according to a sequencing technology to construct the sequencing library.
S2300, sequencing the sequencing library so as to determine the sequence of the nucleic acid sample. The sequencing library is utilized to obtain the sequence information of the nucleic acid sample, the sequence of the nucleic acid sample can be determined in a sequencing mode, and the sequencing technology can be the technology which can perform high-throughput and high-depth sequencing, such as first-generation sequencing, second-generation sequencing, third-generation sequencing, single-molecule sequencing, single-cell sequencing and the like, and can also be the combined use of the technologies, so that the rapid, efficient and accurate sequencing effect can be achieved.
S3000, and finally comparing the sequence of the nucleic acid sample with SEQ ID NO:1, comparing to judge whether the nucleic acid sample sequence has at least one of the following combinations: c.371delG and c.6118C > T combination, c.6788G > A and c.5300T > C combination, c.681T > G and c.5882G > A combination, c.5509C > T and c.6563T > C combination and EX37del and c.4537_4538insC combination, and further judging whether the sequence of the nucleic acid sample is mutant, namely, the sequence of the nucleic acid sample is compared with the wild-type ACBA4 gene, and if the sequence of the nucleic acid sample has the following mutations, the biological sample is mutant: deletion of guanine at position 371 and mutation of cytosine at position 6118 into thymine, or mutation of guanine at position 6788 into adenine and mutation of thymine at position 5300 into cytosine, or mutation of thymine at position 681 into guanine and mutation of guanine at position 5882 into adenine, or mutation of cytosine at position 5509 into thymine and mutation of thymine at position 6563 into cytosine, deletion or partial deletion of exon 37 and insertion of cytosine between positions 4537 and 4538.
In a fourth aspect of the invention, a genotyping system is provided. Referring to fig. 4, the present system includes, according to an embodiment of the present invention: a nucleic acid sample extraction device 100 for extracting a nucleic acid sample from a biological sample; a nucleic acid sequence determination device 200, wherein the nucleic acid sequence determination device 200 is connected to the nucleic acid extraction device 100 and is used for analyzing the nucleic acid sample so as to determine the nucleic acid sequence of the nucleic acid sample; a judging means 300, said judging means 300 being connected to said nucleic acid sequence determining means 200, and determining the sequence of said nucleic acid sample based on the sequence of said nucleic acid sample and the sequence of SEQ ID NO:1 to determine whether the nucleic acid sequence has at least one of the following combinations: c.371delG and c.6118C > T combination, c.6788G > A and c.5300T > C combination, c.681T > G and c.5882G > A combination, c.5509C > T and c.6563T > C combination and EX37del and c.4537_4538insC combination, and further judging whether the nucleic acid molecules are mutant, namely, the sequence of the nucleic acid sample is aligned with the wild-type ACBA4 gene, and if the sequence of the nucleic acid sample has the following mutations, the biological sample is mutant: deletion of guanine at position 371 and mutation of cytosine at position 6118 into thymine, or mutation of guanine at position 6788 into adenine and mutation of thymine at position 5300 into cytosine, or mutation of thymine at position 681 into guanine and mutation of guanine at position 5882 into adenine, or mutation of cytosine at position 5509 into thymine and mutation of thymine at position 6563 into cytosine, deletion or partial deletion of exon 37 and insertion of cytosine between positions 4537 and 4538.
According to an embodiment of the present invention, the nucleic acid sample extraction apparatus 100 is used to extract a nucleic acid sample of a biological sample in order to perform sequence determination of the nucleic acid sample. According to an embodiment of the present invention, the biological sample may be a cell, tissue, organ, blood, or the like of a human or an animal, and the "biological sample" is not particularly limited as long as a nucleic acid sample can be extracted to a subject. According to one embodiment of the invention, the biological sample is human peripheral blood, and the peripheral blood is taken conveniently and quickly without causing huge trauma to a subject. The nucleic acid sample is DNA, RNA, cDNA or polymer, and the nucleic acid sample contains the nucleotide sequence similar to the nucleotide sequence shown in SEQ ID NO:1, matched information. It will be appreciated by those skilled in the art that with the development of sequencing technology, no nucleic acid extraction step may be performed prior to sequencing, e.g. single cell sequencing does not require nucleic acid extraction from a cell sample for high throughput sequencing of nucleic acids within a cell sample, and therefore this step is aimed at pre-processing of nucleic acid sequences in order to obtain a biological sample.
The nucleic acid sequence determination apparatus 200 is used for determining the sequence of the nucleic acid sample so as to perform sequence alignment of the nucleic acid sample. According to an embodiment of the present invention, referring to fig. 5, the nucleic acid sequence determination apparatus 200 further includes a target gene amplification unit 210 for independently amplifying at least one of the ACBA4 genes using ACBA 4-specific primers, respectively, so as to obtain at least one of amplification products of the ACBA4 gene; a sequencing library constructing unit 220, wherein the sequencing library constructing unit 220 is connected with the target gene amplification unit 210, and the amplification product is added into a joint so as to obtain a sequencing library; a sequencing unit 230, said sequencing unit 230 being connected to said sequencing library construction unit 220 for sequencing said sequencing library as input in order to obtain sequencing data of said nucleic acid sample. As mentioned above, the ACBA4 specific primer is not particularly limited, and the sequencing method can be used for determining the sequence of the nucleic acid sample, and the sequencing technology can be a first-generation sequencing technology, a second-generation sequencing technology, a third-generation sequencing technology, a single-molecule sequencing technology, a single-cell sequencing technology, and the like, which can perform high-throughput and high-depth sequencing, or a combination of the above technologies, so as to achieve a rapid, efficient, and accurate sequencing effect.
In a fifth aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit comprises: primers, probes and/or antibodies for detecting at least one of the following biomarkers: c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37 del.
According to an embodiment of the invention, the kit further comprises: primers, probes and/or antibodies for detecting at least one of the following biomarkers: c.6118C > T, c.4537_4538insC, c.5300T > C, c.5882G > A and c.6563T > C. The kit according to the embodiment of the invention comprises a probe for specifically recognizing the mutation site, and a primer containing the mutation site is amplified. The probe is used for directly positioning, identifying and marking the site, and judging whether the site generates the mutation or not by judging whether the probe can generate base complementary pairing with the site or not. In addition, primer sequences for amplifying target sequences including the above mutation sites may be included. The amplification is to quantitatively amplify and extract fragments of the obtained nucleic acid molecules based on the base complementary pairing principle by using the primers and the obtained nucleic acid molecules. Labels such as a linker, a fluorescent group, a biotin group, a tag and the like are added to the primer, so that preparation can be carried out for other technical means such as sequencing or positioning of subsequent nucleic acid molecules.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Examples
1. Family pedigree characteristics of ten Stargardt diseases
Ten STGD family information were collected in total for this study. STGD F1, STGD F2, STGD F3, and STGD F6 families had two or more patients, and the remaining families had only one patient per patient. The analysis of the simplified family spectrogram (shown in fig. 6) shows that: the proband the patient carry two allelic mutations of the gene ABCA4, and the two allelic mutations are from father or mother; the clinical phenotypes of the parents were normal and were carriers of the gene ABCA 4; there is no continuous inheritance phenomenon in the family pathogenic system spectrogram. The genetic characteristics conform to the characteristics of Autosomal Recessive Inheritance (ARI).
2. Ten Stargardt disease pedigree clinical phenotype profiles
The report divides patients into two categories, the first category being adult onset patients (age at which symptoms first appear ≧ 17 years) and the second category being pediatric onset patients (age at which symptoms first appear <17 years).
Based on the results of electroretinograms (FERG and PERG), patients were classified into type 3, which is currently the most common classification standard.
ERG 1: the clinical manifestation is the lightest, the damage of the electrophysiological function of the retina is only limited to a macular area, the photopic and scotopic reactions of the FERG are normal, and the PERG is abnormal;
ERG 2: the clinical manifestations are moderate, the damage of the electrophysiological functions of the retina has affected the cone function of the whole retina except the macular area, the photopic response of FERG is abnormal, and PERG is abnormal;
ERG 3: the clinical manifestations are the most serious, the damage of the electrophysiological function of the retina not only affects the macular area, but also affects the functions of the cone and rod of the whole retina, and the photopic and scotopic vision response and PERG of FERG are abnormal.
AF is specifically classified into type 3 according to the manifestation of the patient's fundus AF:
AF1 type: the clinical visual function impairment is the least, the low fluorescence is limited in the fovea centralis of the macula, the fluorescence with or without high signal or low signal is in the side of the fovea centralis, and the background fluorescence is homogeneous and uniform.
AF2 type: in the clinical aspect, the function is damaged moderately, the low fluorescence of the macula lutea central fovea is limited, the fluorescence of high signals or low signals beside the central fovea extends to the outside of the blood vessel arch ring, and the background fluorescence is heterogeneous and uneven.
AF3 type: the clinical visual function is most seriously damaged, the posterior pole part is spread with a plurality of low-signal fluorescence, the center is concave or has no high-signal or low-signal fluorescence, and the background fluorescence is heterogeneous and uneven.
The fundus can be classified into three types according to the performance of the fundus of a patient.
Type 1: atrophy of the macular area with or without yellow spots.
Type 2: lesions in the macular area extend to the vascular arch or nasal side to the optic disc.
Type 3: the lesion exceeds the vascular arch of the macular area and extends to the whole posterior pole.
The patient's age at the earliest symptoms, Best Corrected Visual Acuity (BCVA), AF and ERG characteristics were combined to classify the severity of the disease into mild, moderate and severe grade 3, as shown in Table 2.
Table 2: disease staging of STGD
Light and heavy: as long as the standards of more than or equal to 2 are met; BCVA, best corrected vision; FAF, retinal autofluorescence; ERG, full field electroretinogram.
The ten ancestral patients and the patients mainly complain about the progressive decline of the vision of the centers of the eyes, the onset age is 3-20 years old, the earliest patient is 3 years old when symptoms appear, and the family members find that the patients are very close to watching TV. They exhibited a series of more typical STGD ophthalmic symptoms. The clinical characteristics of the ten STGD family probands are summarized in table 3, including the age of treatment, sex, age of onset, earliest symptoms, best corrected vision, etc., and fundus images for ophthalmic examination, AF, OCT, FERG, PERG, etc. Fundus photographs, AF images, and OCT images of the first five probands are shown in fig. 7 (right eye) and fig. 8 (left eye); the results of the FERG and PERG examinations are shown in FIG. 9 (right eye).
Table 3: summary of clinical features of STGD family member predecessor
M, female; f, male; BCVA, best corrected vision; r, right eye, L, left eye; AF, fundus autofluorescence; FERG, full field ERG; PERG, Pattern ERG; mfERG, multifocal ERG.
STGD F1, STGD F4, and STGD F9 probands were all patients with STGD adulthood onset (age of first onset of symptoms ≥ 17 years) and exhibited mild clinical phenotype. The STGD F1, STGD F4, and STGD F9 probands all retained poor central vision by age 28: f1 the BCVA of the right eye and the BCVA of the left eye of the proband is 0.25 and 0.30 respectively; f4 the first person has slightly poor BCVA in both eyes, the right eye is 0.20, and the left eye is 0.15; f9 proband both eyes were 0.20. Fundus photographs of the right eye of the proband F1, F4 showed a typical macular region of oval atrophy, similar to the "bronze" appearance, characterized by a "bull's eyes" with a ring of yellowish white spots deposited around the atrophy around the upper and lower vascular arches (fig. 7: first column). The eyes of the proband F1 and the right eye fundus AF of the proband F4 are shown: the yellow spot area can see elliptical low fluorescence accompanied by a circle of uniform and homogeneous high fluorescence signal, which is corresponding to yellowish white lipofuscin spots, the background fluorescence is uniform, and AF is AF1 type; the fundus color photograph and AF of the left eye of the patient with the F4 probation are both 2 types, and the focus extends to the outside of the blood vessel arch ring. Except that the fundus color photographs of both eyes of the proband are different from the AF type of F4, the fundus and AF types of both sides of the other proband and the patient are identical. The STGD F10 patient began to develop symptoms at the age of 14, had good vision correction for both eyes at 0.5/0.4, and had fundus color photograph and AF of type 2. SD-OCT examinations of four groups of probands F1, F4, F9 and F10 all showed marked thinning of the neuroepithelial layer of the macular degeneration area, thinning of the ONL layer, interruption and disappearance of the macular region ellipsoid area (EZ) zone and outer limiting membrane (ELM) in the macular degeneration area, and the area where the interruption disappeared coincided with the abnormal AF area (fig. 7, 8). This cohort of probands (F1, F4, F9 and F10) showed normal FERG with clinically significant reduction or undetectable P-ERG, belonging to ERG1 type (fig. 9). The disease was graded as mild according to clinical presentation, and other patients in these families also exhibited a relatively mild STGD disease phenotype.
STGD F2, F3, F5, F6, F7 and F8 patients are all childhood STGD patients (age of onset ≤ 10 years). They complain of a decline in central vision at the age of 3-10 years. The patient was classified as severe. They reported poor vision (BCVA ≦ 0.10), reading and school difficulties. Probands F3 and F8 were least aged and were found to have poor vision at the ages of 3 and 5, respectively. The vision loss of the F3 proband was first discovered by his family at the age of 3, when he was watching television at a particularly close distance. At the time of 9 years old visit, the atrophic changes of the retina and choroid in the macular area of both eyes surpassed the vascular arch (type 2 fundus) in the macular area (fig. 7 and 8). The AF images showed local low fluorescence signals in the macula, extending to the anterior and nasal side of the vascular arch to the optic disc (AF 2 type) (fig. 7, 8). SD-OCT scans showed complete loss of EZ and ELM, mainly manifested by atrophic thinning of the retinal neuroepithelium, RPE, and underlying choroid. The P-ERG and photopic ERGs (LA 3.0 and LA 3.030hz) of F3 and F8 probands were undetectable, and the scotopic ERGs (DA 0.01 and DA 10.0) showed severe drop, belonging to ERG3 type (FIG. 9). There were numerous extensive atrophic changes in the retinal neuroepithelium, RPE of F2, F5, F6 and F7 probands, ranging beyond the vascular arch to the posterior pole (type 3 fundus) (fig. 7, 8). In addition, fundus photographs of F6 proband showed a very large amount of pigmentation behind the retina, easily confounding the diagnosis with RP, which we have previously reported. The AF image is 3 type (fig. 7 and 8). SD-OCT found macular retinal neuroepithelium and extensive RPE atrophy. F5 probands showed more severe neuroretinal thinning, degenerative atrophy of RPE and choriocapillaris on SD-OCT images (fig. 7, 8). F2, F5, F6 and F7 probands P-ERG and light and dark adaptation ERGs were all significantly reduced or undetectable, belonging to ERG3 type (fig. 9).
3. Screening for pathogenic mutations
Genome DNAs of ten pedigree probands (F1-II-2, F2-II-3, F3-II-1, F4-II-1, F5-II-1, F1-II-2, F2-II-3, F3-II-1, F4-II-1 and F5-II-1) are obtained by using a genome extraction kit, and target region capture is carried out by using specific primers of known HRD pathogenic genes, and amplification, library building and sequencing analysis are carried out.
And analyzing 103 the variation conditions of the genes of the exon region, the boundary region of the exon and the intron and the non-coding region at the 5 'end and the 3' end of the known HRD pathogenic gene according to the sequencing result, and performing high-throughput capture sequencing on the target sequence. The coverage rate of the target sequence achieved by sequencing is more than 98.5%, the coverage rate of the target region after more than 10 times of reading is 90.0-92.7%, and the average coverage rate after more than 20 times of reading is 81.9-86.0%. The 100% exon base pair coverage depth is more than 200 x.
We first screened SNVs and INDEL information in raw sequencing data captured at the target region, and then screened biallelic mutations that fit the ARI inheritance rules for further analysis. Finally, the result is compared with the database such as dbSNP 132, HapMap Project, 1000Genome Project, YH database, etc., to eliminate the known pathogenic gene mutation and screen out the possible pathogenic mutation. All selected mutation sites need to be checked by Sanger sequencing to eliminate false positives, and whether cosegregation characteristics are shown in family members is checked.
4. Determination of possible pathogenic mutations in the STGD pedigree ABCA4 Gene
By adopting target sequence capture and NGS technology of specific genes and Sanger sequencing verification and mutation and clinical phenotype coseparation verification, 417 pathogenic mutations of the gene ABCA are found in an STGD family, wherein 5 of the pathogenic mutations are new mutations found for the first time in the research: c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37 del. The nonsense mutation c.6118c > T was detected simultaneously in two STGD families, of which two were detected as homozygous mutations. The causative mutations are summarized in Table 1.
Of the 17 mutations identified, 5 were reported for the first time, 2 of them were truncation mutations, which resulted in premature termination of the amino acid translation process during mRNA translation, resulting in disruption of protein structure and function. The other is large fragment deletion mutation, which is confirmed by RT-qPCR (figure 10), and the primer sequence is shown as SEQ ID NO: 4 and SEQ ID NO: 5. The large fragment heterozygosis deletion (EX37del) of the STGD-Family 537 exon is carried out, qPCR verification is carried out on the No. 37 exon, and the result shows that the expression quantity of mRNA of the paternal No. 37 exon of the proband and the proband is obviously lower than that of a normal control, and the difference has statistical significance (p is less than 0.01); the expression level of exon 37 mRNA of proband mother is not different from that of normal control, and has no statistical significance (p > 0.05). The remaining two missense mutations were predicted to be pathogenic according to our previous four pathogenicity prediction criteria.
In STGD F1, two complex heterozygous mutations associated with the disease were detected: mutation c.6190g > a and splicing mutation c.1937+1G > a. Missense mutation c.6190g > a at exon 45 resulted in the substitution of threonine for alanine at codon 2064 (p.ala2064thr), reported in chinese and estonia populations. In F2, two compound heterozygote mutations of c.2424C > G and c.1761-2A > G were detected in proband (F2-II-3). The nonsense mutation c.2424c > G is located in exon 16, and tyrosine was replaced by a stop codon (p.tyr808ter) at codon 808, resulting in premature termination of the amino acid coding of the ABCA4 gene. In F3, proband (F3-II-2) detected two compound heterozygous mutations c.371delG and c.6118C > T. The new mutation c.371delg, in which 30 wrong amino acids were inserted after the substitution of arginine by leucine at codon 124, resulted in the premature termination of ABCA4 coding and translation (p.arg124leufsx30). Another nonsense mutation, c.6118c > T, has been reported to replace arginine at exon 44 codon 2040 with a stop codon (p.arg2040ter), resulting in premature termination of the amino acid coding. In F4, two reported mutations, c.6118C > T and c.3385C > T, were found in the proband (F4-II-1). The nonsense mutation c.6118c > T was detected twice in this study. The missense mutation c.3385c > T is located at exon 23 codon 1129 with cystine replacing arginine (p.arg 1129cys).
In F5, a large fragment deletion of exon 37 (EX37del) was detected in proband (F5-II-1). The other is an insertion mutation at the position of exon 30, cytosine (C) is inserted in the positions 4537 and 4538 (c.4537 — 4538insC), which results in the frame shift of the codon, glutamine is mutated to proline at the 1513 position of the codon, and the amino acid coding is terminated early after 41 mis-translated amino acids are inserted after proline, resulting in protein truncation (p.gln1513profsx41), which is reported in the literature. In F6, the homozygote missense mutation c.1924t > a was detected, which we have previously reported. In F7, proband F7-II-2 carries the homozygous splicing mutation c.6816+1G > A, also reported in STGD patients in China. F8 detected two mutations that could be related to the disease, a new missense mutation at exon 49 c.6788G > A and a reported missense mutation at exon 37 c.5300T > C. Two mutations c.10695t > a and c.449t > G were detected by F9. c.10695t > a is a missense mutation reported in exon 42, with a glutamic acid substituted for glycine at codon 1961 (p.g 1961e). c.449T > G is a newly reported mutation in this study, with the tyrosine replaced by a stop codon at codon 227 (p.Y227Ter) and the amino acid encoding a premature stop. In F10, two missense mutations c.6563t > C and c.5509c > T led to the development of this family disease.
5. Association analysis of Stargardt disease gene mutation and clinical phenotypic characteristics
As mentioned above, Null variants refer to splicing mutations that lead to mRNA splicing errors or truncation mutations that lead to premature termination of the translation process of amino acids during translation of mRNA. Such mutations result in premature termination of the amino acid coding, thereby producing truncated proteins. However, studies have shown that such truncated proteins are virtually absent in vivo, because such mutations mediate mRNA degradation (NMD), block mRNA degradation, and increase the expression level of the mRNA of the mutant gene, but still cannot synthesize the truncated protein, and in combination with blocking protein degradation, the protein cannot be synthesized and expressed. Truncation mutations can result in complete loss of function of the protein, and thus are highly pathogenic. It is generally accepted that two mutations of a gene are independent and overlapping in pathogenicity, and therefore both mutations should be considered simultaneously in analyzing the degree of pathogenicity of the mutation. In this study, children-onset Stargardt patients (F2, F3, F5, F6, F7, and F8) had a more severe clinical phenotype, characterized by early severe visual function loss, and destruction of retinal cone rod function; on the contrary, in the adult-onset Stargardt patients (F1, F4, and F9), visual function was well preserved, and in addition to the destruction of the photoreceptor function in the macular region, the whole retinal cone rod cell function was also well preserved. In addition, childhood onset of Stargardt is predominantly present in the severe forms of ABCA4 genotype: both mutations were null varients (F2, F3, F5 and F7) or homozygous (F6). However, the adult onset STGD (F1, F4 and F9) genotype was among the missense mutations + null variant, indicating that a portion of ABCA4 protein function was preserved. The F8 genotype is two missense mutations, but the clinical phenotype is severe, which indicates that partial missense mutations can also cause severe damage to the function of the protein.
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 are not necessarily intended to 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. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> seventy-fourth group military hospital of China people liberation army
<120> use of reagent for detecting biomarker in preparation of kit
<130> PIDC3204684
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 6821
<212> DNA
<213> Artificial Sequence
<220>
<223> wild type human ACBA4 sequence
<400> 1
atgggcttcg tgagacagat acagcttttg ctctggaaga actggaccct gcggaaaagg 60
caaaagattc gctttgtggt ggaactcgtg tggcctttat ctttatttct ggtcttgatc 120
tggttaagga atgccaaccc actctacagc catcatgaat gccatttccc caacaaggcg 180
atgccctcag caggaatgct gccgtggctc caggggatct tctgcaatgt gaacaatccc 240
tgttttcaaa gccccacccc aggagaatct cctggaattg tgtcaaacta taacaactcc 300
atcttggcaa gggtatatcg agattttcaa gactcctcat gaatgcacca gagagccagc 360
accttggccg tatttggaca gagctacaca tcttgtccca attcatggac accctccgga 420
ctcacccgga gagaattgca ggaagaggaa tacgaataag ggatatcttg aaagatgaag 480
aaacactgac actatttctc attaaaaaca tcggcctgtc tgactcagtg gtctaccttc 540
tgatcaactc tcaagtccgt ccagagcagt tcgctcatgg agtcccggac ctggcgctga 600
aggacatcgc ctgcagcgag gccctcctgg agcgcttcat catcttcagc cagagacgcg 660
gggcaaagac ggtgcgctat gccctgtgct ccctctccca gggcacccta cagtggatag 720
aagacactct gtatgccaac gtggacttct tcaagctctt ccgtgtgctt cccacactcc 780
tagacagccg ttctcaaggt atcaatctga gatcttgggg aggaatatta tctgatatgt 840
caccaagaat tcaagagttt atccatcggc cgagtatgca ggacttgctg tgggtgacca 900
ggcccctcat gcagaatggt ggtccagaga cctttacaaa gctgatgggc atcctgtctg 960
acctcctgtg tggctacccc gagggaggtg gctctcgggt gctctccttc aactggtatg 1020
aagacaataa ctataaggcc tttctgggga ttgactccac aaggaaggat cctatctatt 1080
cttatgacag aagaacaaca tccttttgta atgcattgat ccagagcctg gagtcaaatc 1140
ctttaaccaa aatcgcttgg agggcggcaa agcctttgct gatgggaaaa atcctgtaca 1200
ctcctgattc acctgcagca cgaaggatac tgaagaatgc caactcaact tttgaagaac 1260
tggaacacgt taggaagttg gtcaaagcct gggaagaagt agggccccag atctggtact 1320
tctttgacaa cagcacacag atgaacatga tcagagatac cctggggaac ccaacagtaa 1380
aagacttttt gaataggcag cttggtgaag aaggtattac tgctgaagcc atcctaaact 1440
tcctctacaa gggccctcgg gaaagccagg ctgacgacat ggccaacttc gactggaggg 1500
acatatttaa catcactgat cgcaccctcc gcctggtcaa tcaatacctg gagtgcttgg 1560
tcctggataa gtttgaaagc tacaatgatg aaactcagct cacccaacgt gccctctctc 1620
tactggagga aaacatgttc tgggccggag tggtattccc tgacatgtat ccctggacca 1680
gctctctacc accccacgtg aagtataaga tccgaatgga catagacgtg gtggagaaaa 1740
ccaataagat taaagacagg tattgggatt ctggtcccag agctgatccc gtggaagatt 1800
tccggtacat ctggggcggg tttgcctatc tgcaggacat ggttgaacag gggatcacaa 1860
ggagccaggt gcaggcggag gctccagttg gaatctacct ccagcagatg ccctacccct 1920
gcttcgtgga cgattctttc atgatcatcc tgaaccgctg tttccctatc ttcatggtgc 1980
tggcatggat ctactctgtc tccatgactg tgaagagcat cgtcttggag aaggagttgc 2040
gactgaagga gaccttgaaa aatcagggtg tctccaatgc agtgatttgg tgtacctggt 2100
tcctggacag cttctccatc atgtcgatga gcatcttcct cctgacgata ttcatcatgc 2160
atggaagaat cctacattac agcgacccat tcatcctctt cctgttcttg ttggctttct 2220
ccactgccac catcatgctg tgctttctgc tcagcacctt cttctccaag gccagtctgg 2280
cagcagcctg tagtggtgtc atctatttca ccctctacct gccacacatc ctgtgcttcg 2340
cctggcagga ccgcatgacc gctgagctga agaaggctgt gagcttactg tctccggtgg 2400
catttggatt tggcactgag tacctggttc gctttgaaga gcaaggcctg gggctgcagt 2460
ggagcaacat cgggaacagt cccacggaag gggacgaatt cagcttcctg ctgtccatgc 2520
agatgatgct ccttgatgct gctgtctatg gcttactcgc ttggtacctt gatcaggtgt 2580
ttccaggaga ctatggaacc ccacttcctt ggtactttct tctacaagag tcgtattggc 2640
ttggcggtga agggtgttca accagagaag aaagagccct ggaaaagacc gagcccctaa 2700
cagaggaaac ggaggatcca gagcacccag aaggaataca cgactccttc tttgaacgtg 2760
agcatccagg gtgggttcct ggggtatgcg tgaagaatct ggtaaagatt tttgagccct 2820
gtggccggcc agctgtggac cgtctgaaca tcaccttcta cgagaaccag atcaccgcat 2880
tcctgggcca caatggagct gggaaaacca ccaccttgtc catcctgacg ggtctgttgc 2940
caccaacctc tgggactgtg ctcgttgggg gaagggacat tgaaaccagc ctggatgcag 3000
tccggcagag ccttggcatg tgtccacagc acaacatcct gttccaccac ctcacggtgg 3060
ctgagcacat gctgttctat gcccagctga aaggaaagtc ccaggaggag gcccagctgg 3120
agatggaagc catgttggag gacacaggcc tccaccacaa gcggaatgaa gaggctcagg 3180
acctatcagg tggcatgcag agaaagctgt cggttgccat tgcctttgtg ggagatgcca 3240
aggtggtgat tctggacgaa cccacctctg gggtggaccc ttactcgaga cgctcaatct 3300
gggatctgct cctgaagtat cgctcaggca gaaccatcat catgtccact caccacatgg 3360
acgaggccga cctccttggg gaccgcattg ccatcattgc ccagggaagg ctctactgct 3420
caggcacccc actcttcctg aagaactgct ttggcacagg cttgtactta accttggtgc 3480
gcaagatgaa aaacatccag agccaaagga aaggcagtga ggggacctgc agctgctcgt 3540
ctaagggttt ctccaccacg tgtccagccc acgtcgatga cctaactcca gaacaagtcc 3600
tggatgggga tgtaaatgag ctgatggatg tagttctcca ccatgttcca gaggcaaagc 3660
tggtggagtg cattggtcaa gaacttatct tccttcttcc aaataagaac ttcaagcaca 3720
gagcatatgc cagccttttc agagagctgg aggagacgct ggctgacctt ggtctcagca 3780
gttttggaat ttctgacact cccctggaag agatttttct gaaggtcacg gaggattctg 3840
attcaggacc tctgtttgcg ggtggcgctc agcagaaaag agaaaacgtc aacccccgac 3900
acccctgctt gggtcccaga gagaaggctg gacagacacc ccaggactcc aatgtctgct 3960
ccccaggggc gccggctgct cacccagagg gccagcctcc cccagagcca gagtgcccag 4020
gcccgcagct caacacgggg acacagctgg tcctccagca tgtgcaggcg ctgctggtca 4080
agagattcca acacaccatc cgcagccaca aggacttcct ggcgcagatc gtgctcccgg 4140
ctacctttgt gtttttggct ctgatgcttt ctattgttat ccctcctttt ggcgaatacc 4200
ccgctttgac ccttcacccc tggatatatg ggcagcagta caccttcttc agcatggatg 4260
aaccaggcag tgagcagttc acggtacttg cagacgtcct cctgaataag ccaggctttg 4320
gcaaccgctg cctgaaggaa gggtggcttc cggagtaccc ctgtggcaac tcaacaccct 4380
ggaagactcc ttctgtgtcc ccaaacatca cccagctgtt ccagaagcag aaatggacac 4440
aggtcaaccc ttcaccatcc tgcaggtgca gcaccaggga gaagctcacc atgctgccag 4500
agtgccccga gggtgccggg ggcctcccgc ccccccagag aacacagcgc agcacggaaa 4560
ttctacaaga cctgacggac aggaacatct ccgacttctt ggtaaaaacg tatcctgctc 4620
ttataagaag cagcttaaag agcaaattct gggtcaatga acagaggtat ggaggaattt 4680
ccattggagg aaagctccca gtcgtcccca tcacggggga agcacttgtt gggtttttaa 4740
gcgaccttgg ccggatcatg aatgtgagcg ggggccctat cactagagag gcctctaaag 4800
aaatacctga tttccttaaa catctagaaa ctgaagacaa cattaaggtg tggtttaata 4860
acaaaggctg gcatgccctg gtcagctttc tcaatgtggc ccacaacgcc atcttacggg 4920
ccagcctgcc taaggacagg agccccgagg agtatggaat caccgtcatt agccaacccc 4980
tgaacctgac caaggagcag ctctcagaga ttacagtgct gaccacttca gtggatgctg 5040
tggttgccat ctgcgtgatt ttctccatgt ccttcgtccc agccagcttt gtcctttatt 5100
tgatccagga gcgggtgaac aaatccaagc acctccagtt tatcagtgga gtgagcccca 5160
ccacctactg ggtgaccaac ttcctctggg acatcatgaa ttattccgtg agtgctgggc 5220
tggtggtggg catcttcatc gggtttcaga agaaagccta cacttctcca gaaaaccttc 5280
ctgcccttgt ggcactgctc ctgctgtatg gatgggcggt cattcccatg atgtacccag 5340
catccttcct gtttgatgtc cccagcacag cctatgtggc tttatcttgt gctaatctgt 5400
tcatcggcat caacagcagt gctattacct tcatcttgga attatttgag aataaccgga 5460
cgctgctcag gttcaacgcc gtgctgagga agctgctcat tgtcttcccc cacttctgcc 5520
tgggccgggg cctcattgac cttgcactga gccaggctgt gacagatgtc tatgcccggt 5580
ttggtgagga gcactctgca aatccgttcc actgggacct gattgggaag aacctgtttg 5640
ccatggtggt ggaaggggtg gtgtacttcc tcctgaccct gctggtccag cgccacttct 5700
tcctctccca atggattgcc gagcccacta aggagcccat tgttgatgaa gatgatgatg 5760
tggctgaaga aagacaaaga attattactg gtggaaataa aactgacatc ttaaggctac 5820
atgaactaac caagatttat ccaggcacct ccagcccagc agtggacagg ctgtgtgtcg 5880
gagttcgccc tggagagtgc tttggcctcc tgggagtgaa tggtgccggc aaaacaacca 5940
cattcaagat gctcactggg gacaccacag tgacctcagg ggatgccacc gtagcaggca 6000
agagtatttt aaccaatatt tctgaagtcc atcaaaatat gggctactgt cctcagtttg 6060
atgcaattga tgagctgctc acaggacgag aacatcttta cctttatgcc cggcttcgag 6120
gtgtaccagc agaagaaatc gaaaaggttg caaactggag tattaagagc ctgggcctga 6180
ctgtctacgc cgactgcctg gctggcacgt acagtggggg caacaagcgg aaactctcca 6240
cagccatcgc actcattggc tgcccaccgc tggtgctgct ggatgagccc accacaggga 6300
tggaccccca ggcacgccgc atgctgtgga acgtcatcgt gagcatcatc agagaaggga 6360
gggctgtggt cctcacatcc cacagcatgg aagaatgtga ggcactgtgt acccggctgg 6420
ccatcatggt aaagggcgcc tttcgatgta tgggcaccat tcagcatctc aagtccaaat 6480
ttggagatgg ctatatcgtc acaatgaaga tcaaatcccc gaaggacgac ctgcttcctg 6540
acctgaaccc tgtggagcag ttcttccagg ggaacttccc aggcagtgtg cagagggaga 6600
ggcactacaa catgctccag ttccaggtct cctcctcctc cctggcgagg atcttccagc 6660
tcctcctctc ccacaaggac agcctgctca tcgaggagta ctcagtcaca cagaccacac 6720
tggaccaggt gtttgtaaat tttgctaaac agcagactga aagtcatgac ctccctctgc 6780
accctcgagc tgctggagcc agtcgacaag cccaggactg a 6821
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> detection of upstream primer sequence of c.371delG
<400> 2
ggcaggagaa tagcgtgaac 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> detection of downstream primer sequence of c.371delG
<400> 3
ctgtggagga agaggcatct 20
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> detection of upstream primer sequence of EX37del
<400> 4
ttccgagatg aattattccg tgagtg 26
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> detection of downstream primer sequence of EX37del
<400> 5
cagcaggagc agtgccacaa 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> detection of upstream primer sequence of c.6788G > A
<400> 6
ggcatatctg agccttggag 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> detection of downstream primer sequence of c.6788G > A
<400> 7
gtgggtgtag ggtgctgttt 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> detection of upstream primer sequence of c.681T > G
<400> 8
aaacccctcc cttaccacac 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> detection of downstream primer sequence of c.681T > G
<400> 9
taggacgtgg gtgtctttcc 20
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> detection of the sequence of the upstream primer of c.5509C > T
<400> 10
taaccctccc agctttggac 20
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> detection of the downstream primer sequence of c.5509C > T
<400> 11
tgtcctgtga gagcatctgg 20
<210> 12
<211> 170
<212> DNA
<213> Artificial Sequence
<220>
<223> No. 37 exon sequence
<400> 12
gtgtggttta ataacaaagg ctggcatgcc ctggtcagct ttctcaatgt ggcccacaac 60
gccatcttac gggccagcct gcctaaggac aggagccccg aggagtatgg aatcaccgtc 120
attagccaac ccctgaacct gaccaaggag cagctctcag agattacagt 170
Claims (10)
1. Use of reagents for detecting biomarkers for the preparation of a kit for the diagnosis of Stargardt, characterized in that the marker is expressed as the reference sequence SEQ ID NO:1, said biomarker comprising at least one of:
c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37 del.
2. Use according to claim 1, wherein the reagent is further for detecting at least one of the following biomarkers:
c.6118C > T, c.4537_4538insC, c.5300T > C, c.5882G > A and c.6563T > C.
3. Use according to claim 1 or 2, characterized in that the reagent is further used for detecting at least one of the following combinations of biomarkers:
c.371delG and c.6118C > T combination, c.6788G > A and c.5300T > C combination, c.681T > G and c.5882G > A combination, c.5509C > T and c.6563T > C combination, and EX37del and c.4537_4538insC combination.
4. The use according to claim 3, wherein the reagent is a primer set having the sequence of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11, wherein the nucleotide sequence having the sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3 for detecting c.371delg, said primer having the nucleotide sequence shown in SEQ ID NO: 4 and SEQ ID NO: 5 for detecting EX37del, having the nucleotide sequence shown in SEQ ID NO: 6 and SEQ ID NO: 7 is used for detecting c.6788G > A, and the primer has the nucleotide sequence shown in SEQ ID NO: 8 and SEQ ID NO: 9 for detecting c.681t > G, said primer having the nucleotide sequence shown in SEQ ID NO: 10 and SEQ ID NO: 11 for the detection of c.5509C > T.
5. A non-diagnostic method for nucleic acid typing comprising:
obtaining at least a portion of genomic sequence information of a subject;
comparing at least a portion of the genomic sequence information to a reference sequence to determine whether the genomic sequence has at least one of the following biomarker combinations: c.371delG and c.6118C > T combination, c.6788G > A and c.5300T > C combination, c.681T > G and c.5882G > A combination, c.5509C > T and c.6563T > C combination, and EX37del and c.4537-4538 insC combination,
wherein the reference sequence comprises at least the sequence set forth in SEQ ID NO: 371, 6118, 6788, 5300, 681, 5882, 5509, 6563, EX37, 4537 to 4538 site information in 1.
6. A genotyping method, comprising:
(1) extracting a nucleic acid sample of a biological sample for sequence determination of the nucleic acid sample;
(2) determining the sequence of the nucleic acid sample so as to perform sequence alignment of the nucleic acid sample;
(3) comparing the sequence of the nucleic acid sample with SEQ ID NO. 1, and judging whether the sequence of the nucleic acid sample has at least one of the following combinations: c.371delG and c.6118C > T combination, c.6788G > A and c.5300T > C combination, c.681T > G and c.5882G > A combination, c.5509C > T and c.6563T > C combination and EX37del and c.4537_4538insC combination, thereby judging whether the nucleic acid molecule is mutant or not;
optionally, the gene is ACBA 4.
7. The method of claim 6, wherein the step (2) further comprises:
amplifying the ACBA4 gene by using an ACBA4 specific primer so as to obtain an amplification product of the ACBA4 gene;
adding the amplification product to a linker to obtain a sequencing library;
sequencing the sequencing library to determine the sequence of the nucleic acid sample.
8. A genotyping system, comprising:
a nucleic acid sample extraction device for extracting a nucleic acid sample from a biological sample;
a nucleic acid sequence determining device connected to the nucleic acid extracting device for analyzing the nucleic acid sample to determine the nucleic acid sequence of the nucleic acid sample;
a judging device connected to the nucleic acid sequence determining device for comparing the nucleic acid sequence of the nucleic acid sample with a reference genome to judge whether the nucleic acid sequence has at least one of the following combinations:
c.371delG and c.6118C > T combination, c.6788G > A and c.5300T > C combination, c.681T > G and c.5882G > A combination, c.5509C > T and c.6563T > C combination and EX37del and c.4537_4538insC combination, thereby judging whether the nucleic acid molecule is mutant or not;
optionally, the gene is ACBA 4;
optionally, the nucleic acid sequence determination apparatus further comprises:
a target gene amplification unit, which utilizes ACBA4 specific primers to amplify ACBA4 gene so as to obtain an amplification product of ACBA4 gene;
a sequencing library construction unit, wherein the sequencing library construction unit is connected with the target gene amplification unit, and the amplification product is added into a joint so as to obtain a sequencing library;
and the sequencing unit is connected with the sequencing library construction unit and performs sequencing by taking the sequencing library as an input so as to obtain the sequencing data of the nucleic acid sample.
9. A kit, comprising: primers, probes and/or antibodies for detecting at least one of the following biomarkers:
c.371delG, c.6788G > A, c.681T > G, c.5509C > T and EX37 del.
10. The kit of claim 9, further comprising: primers, probes and/or antibodies for detecting at least one of the following biomarkers:
c.6118C > T, c.4537_4538insC, c.5300T > C, c.5882G > A and c.6563T > C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202110193423.XA CN113151438A (en) | 2021-02-20 | 2021-02-20 | Application of reagent for detecting biomarkers in preparation of kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110193423.XA CN113151438A (en) | 2021-02-20 | 2021-02-20 | Application of reagent for detecting biomarkers in preparation of kit |
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| Publication Number | Publication Date |
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| CN113151438A true CN113151438A (en) | 2021-07-23 |
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|---|---|---|---|
| CN202110193423.XA Pending CN113151438A (en) | 2021-02-20 | 2021-02-20 | Application of reagent for detecting biomarkers in preparation of kit |
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| Country | Link |
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-
2021
- 2021-02-20 CN CN202110193423.XA patent/CN113151438A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 田露等: "国人 Stargardt 病患者 ABCA4 基因突变分析与表型特征", 眼科, vol. 25, no. 4, pages 219 - 224 * |
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