CN111593117A - Application of CRYBB3 gene mutation as Marfan syndrome diagnosis marker - Google Patents

Abstract

The invention discloses application of CRYBB3 gene mutation as a Marfan syndrome diagnosis marker. The invention discovers that c.75+1G > A mutation can cause Marfan syndrome for the first time through a second-generation sequencing technology. The research result of the invention can be used for screening carriers of Marfan syndrome pathogenic gene mutation at early stage to provide a good prenatal and postnatal care guidance on one hand, and can provide a molecular diagnosis basis for Marfan syndrome patients on the other hand.

Description

Application of CRYBB3 gene mutation as Marfan syndrome diagnosis marker

Technical Field

The invention belongs to the field of medical diagnosis, and relates to application of CRYBB3 gene mutation as a Marfan syndrome diagnosis marker.

Background

Marfan syndrome (MFS is one of the most common autosomal dominant hereditary diseases, and French pediatrician in the eighteenth century describes that the MFS is mainly characterized by the defects of three major systems of bones, eyes and cardiovascular systems for the first time, fingers are slender due to the involvement of bones and are in the shape of spider fingers (toes), so the MFS is also called spider finger (toe) syndrome (arachnodactony), the prevalence rate is 1/3000-1/5000, more than 25% of sporadic cases, sudden rupture of aortic dissection is the most major cause of death of MFS patients, and even for Marfan syndrome patients who are treated by operations and medicaments, various problems in daily life still need to be solved.

Early diagnosis and treatment of MFS is an effective way to reduce morbidity and mortality. At present, the diagnosis of MFS is mainly based on clinical standards, and the detection of molecular level is significant. Especially for children whose clinical phenotype is not yet fully manifested, asymptomatic family orthologs or prenatal diagnosis, molecular biological diagnosis is the best way to provide evidence of early diagnosis.

However, some patients who are not diagnosed clearly often show only one or two systemic diseases, and the clinical manifestations can not meet the current MFS diagnosis standard, however, the patients suspected of Marfan syndrome also need genetic testing to help the further diagnosis for early treatment.

Disclosure of Invention

One of the objects of the present invention is to provide a gene mutation for diagnosing Marfan's syndrome.

Another object of the present invention is to provide the use of the above-mentioned gene mutation.

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

the invention provides a gene mutation for diagnosing Marfan syndrome, wherein the gene mutation is a mutation on CRYBB3 gene.

Further, the mutation is c.75+1G > a.

According to still another aspect of the present invention, there is also provided a reagent for detecting a mutation in a gene as described above.

The reagent includes a reagent which can be used in the method for detecting a mutation in a gene of the present invention.

Genetic mutations or variations can be detected by certain methods known to those skilled in the art, including, but not limited to, DNA sequencing; primer extension assay comprising cell mutationSpecific nucleotide incorporation assays and cell mutation specific primer extension assays (e.g., cell mutation specific PCR, cell mutation specific Ligation Chain Reaction (LCR), and nick-LCR); mutation-specific oligonucleotide hybridization assays (e.g., oligonucleotide ligation assays); a cleavage protection assay in which protection from a cleavage agent is used to detect mismatched bases in a nucleic acid duplex; analysis of the binding of the MutS protein; electrophoretic analysis comparing the molecular mobility of variant and wild-type nucleic acids; denaturing gradient gel electrophoresis (DGGE, as in, for example, Myers et al (1985) Nature 313: 495); analysis of rnases cleavage at mismatched base pairs; analyzing chemical or enzymatic cleavage of heteroduplex DNA; mass spectrometry (e.g., MALDI-TOF); genetic Bit Analysis (GBA); 5' nuclease assay (e.g., TaqMan^TM) (ii) a And assays employing molecular beacons. Some of these methods are discussed in more detail below.

Detection of mutations or variations in a target nucleic acid can be achieved by molecular cloning and sequencing of the target nucleic acid using techniques well known in the art. Alternatively, amplification techniques such as Polymerase Chain Reaction (PCR) can be used to amplify a target nucleic acid sequence directly from a preparation from genomic DNA. The nucleic acid sequence of the amplified sequence can then be determined and variations identified therefrom. Amplification techniques are well known in the art, for example, the polymerase chain reaction is described in Saiki et al, Science 239: 487, 1988; U.S. Pat. nos. 4,683,203 and 4,683,195.

The target nucleic acid sequence can also be amplified using the ligase chain reaction, which is known in the art. See, e.g., Wu et al, Genomics 4: 560-569(1989). In addition, a technique known as allele-specific PCR can also be modified and used to detect somatic mutations (e.g., substitutions). See, e.g., Ruano and Kidd (1989) Nucleic acids research 17: 8392, adding a solvent to the mixture; McClay et al (2002) Analytical biochem.301: 200-206. In certain embodiments of this technique, a mutation-specific primer is used, wherein the 3' terminal nucleotide of the primer is complementary to (i.e., capable of specific base-pairing with) a particular variation in the target nucleic acid. If the specific variation is not present, no amplification product is observed. Mutation (e.g., substitution) can also be detected using an Amplification-hindered mutation system (ARMS). ARMS are described, for example, in European patent application publication No.0332435, and Newton et al, Nucleic Acids Research, 17: 7,1989.

Other methods useful for detecting mutations or variations (e.g., substitutions) include, but are not limited to, (1) mutation-specific nucleotide incorporation assays, such as single-base extension assays (see, e.g., Chen et al (2000) Genome Res.10: 549-557; Fan et al (2000) Genome Res.10: 853-860; Pastinen et al (1997) Genome Res.7: 606-614; and Ye et al (2001) hum.mut.17: 305-316); (2) mutation-specific primer extension assays (see, e.g., Ye et al (2001) hum. mut.17: 305-316; and Shen et al Genetic Engineering News, Vol.23, 3/15 2003), including somatic mutation-specific PCR; (3) 5' nuclease assay (see, e.g., De La Vega et al (2002) BioTechniques 32: S48-S54 (described in TaqMan^TMAn assay); ranade et al (2001) Genome Res.11: 1262-1268; and Shi (2001) clin. chem.47: 164-172); (4) assays using molecular beacons (see, e.g., Tyagi et al (1998) NatureBiotech.16: 49-53; and Mhlanga et al (2001) Methods 25: 463-71); and (5) oligonucleotide ligation assays (see, e.g., Grossman et al (1994) Nuc. acids Res.22: 4527-4534; patent application publication No. US2003/0119004A 1; PCT International publication No. WO 01/92579A 2; and U.S. Pat. No. 6,027,889).

Mutations or variations can also be detected by mismatch detection methods. Mismatches refer to hybridized nucleic acid duplexes that are not 100% complementary. The lack of complete complementarity may be due to deletion, insertion, inversion, or substitution. An example of a Mismatch Detection method is the Mismatch Repair Detection (MRD) assay, described, for example, in Faham et al, proc.natl acad.sci.usa 102: 14717-14722(2005) and Faham et al, hum.mol.genet.10: 1657-1664(2001). Another example of mismatch cleavage technology is rnase protection methods, described in detail in Winter et al, proc.natl.acad.sci.usa, 82: 7575, 1985, and Myers et al, Science 230: 1242, 1985. For example, the methods of the invention can involve the use of labeled ribonucleic acid probes that are complementary to a human wild-type target nucleic acid. The ribonucleic acid probe is annealed (hybridized) with a target nucleic acid derived from a tissue sample, and subsequently digested with the enzyme rnase a, which is capable of detecting some mismatches in the double-stranded RNA structure. If RNase A detects a mismatch, it cleaves at the site of the mismatch. Thus, when the annealed RNA preparations are separated on an electrophoretic gel matrix, if RNase A has detected and cleaved mismatches, a smaller RNA product is seen than the RNA probe and the full length double stranded RNA of mRNA or DNA. The riboprobe need not be the full length of the target nucleic acid, but can be part of the target nucleic acid, provided it encompasses the location suspected of having the variation.

In a similar manner, a DNA probe can be used to detect mismatches, e.g., via enzymatic or chemical cleavage. See, e.g., Cotton et al, proc.natl.acad.sci.usa, 85: 4397, 1988; and Shenk et al, proc.natl.acad.sci.usa, 72: 989, 1975. Alternatively, mismatches may be detected by a shift in electrophoretic mobility of the mismatched duplex relative to the matched duplex. See, e.g., Cariello, Human Genetics, 42: 726, 1988. A target nucleic acid suspected of containing a variation can be amplified prior to hybridization using a ribonucleic acid probe or a DNA probe. Southern hybridization can also be used to detect changes in a target nucleic acid, particularly if the change is a gross rearrangement, such as a deletion and an insertion.

Restriction Fragment Length Polymorphism (RFLP) probes to the target nucleic acid or surrounding marker genes can be used to detect variations, such as insertions or deletions. Insertions and deletions can also be detected by cloning, sequencing and amplification of the target nucleic acid. Single Strand Conformation Polymorphism (SSCP) analysis can also be used to detect mutated base variants of cells. See, e.g., Orita et al, proc.natl.acad.sci.usa 86: 2766. 2770, 1989, and Genomics, 5: 874-879, 1989. SSCP can be modified to detect cellular mutations. SSCP identifies base differences by changes in electrophoretic mobility of single-stranded PCR products. The double stranded PCR product may be denatured by heating or otherwise generating a single stranded PCR product. Single-stranded nucleic acids can refold or form secondary structures that are dependent in part on the base sequence. The different electrophoretic mobilities of the single-stranded amplification products are related to the base sequence differences at the SNP positions. Denaturing Gradient Gel Electrophoresis (DGGE) distinguishes SNP alleles on the basis of different sequence-dependent stability and melting properties inherent in polymorphic DNA and the corresponding differences in electrophoretic migration patterns in denaturing gradient gels.

Microarrays may also be used to detect gene mutations or variations. Microarray refers to a multiplexing technique that typically uses an array of thousands of nucleic acid probes that hybridize under high stringency conditions to, for example, a cDNA or cRNA sample. Probe-target hybridization is typically detected and quantified by detecting a fluorophore, silver, or chemiluminescent labeled target to determine the relative abundance of nucleic acid sequences in the target. In a typical microarray, probes are attached to a solid surface by covalent bonds to a chemistry matrix (via epoxy silane, amino silane, lysine, polyacrylamide, etc.). Solid surface refers to, for example, glass, silicon wafers, or microscopic beads. Various microarrays are commercially available, including those manufactured by Affymetrix and Illumina, for example.

Another method for detecting cell or gene mutations is based on mass spectrometry. Mass spectrometry exploits the unique mass of each of the four nucleotides of DNA. Nucleic acids potentially containing mutations can be analyzed unambiguously by mass spectrometry by measuring mass differences of nucleic acids with cellular mutations. MALDI-TOF (matrix assisted laser Desorption ionization-time of flight) mass spectrometry techniques can be used to determine molecular weights with extreme accuracy, such as those of nucleic acids containing cellular mutations. Numerous approaches to nucleic acid analysis based on mass spectrometry have been developed. Exemplary mass spectrometry-based methods include primer extension assays, which can also be used in combination with other approaches, such as traditional gel-based formats and microarrays.

Further, the reagent comprises specific amplification primers for the gene mutation sites described above.

Further, the reagent also comprises dNTPs, Taq enzyme and Mg²⁺And PCR reaction buffer solution and other conventional reagents for PCR amplification reaction.

The invention provides the use of a mutation in a gene as hereinbefore described in the preparation of a reagent as hereinbefore described. The skilled person designs specific amplification primers or specific detection probes according to the upstream and downstream sequences of the gene mutation site. Methods for designing primers and probes are routine in the art.

The invention also provides the application of the gene mutation in preparing Marfan syndrome diagnostic products.

The invention also provides the application of the reagent in preparing Marfan syndrome diagnostic products.

Further, the product comprises a kit, a chip or test paper.

In particular, the product comprises the aforementioned agent.

Further, the diagnostic product diagnoses whether the individual has Marfan syndrome by detecting the presence or absence of the aforementioned genetic mutation in the sample.

In a particular embodiment of the invention, the sample source is blood.

The invention also provides a Marfan syndrome diagnostic product which comprises a reagent for detecting whether the gene mutation exists.

Further, the diagnostic product comprises a kit, a chip, or a strip.

The term "diagnosis" as used herein refers to the identification or classification of a molecular or pathological state, disease or condition. "diagnosis" may also refer to the classification of a particular subtype of disease, for example by molecular characteristics (e.g., a subpopulation of patients characterized by nucleotide variation in a particular gene or nucleic acid region).

Compared with the prior art, the invention has the beneficial effects that:

the gene mutation provided by the invention can distinguish Marfan syndrome patients from normal people, so that the mutation can be used as a biomarker for clinically and auxiliarily diagnosing Marfan syndrome.

By detecting whether the subject carries the variation or not, the carrier of the variation can be detected, and the prenatal and postnatal care guidance and the genetic counseling are provided for the subject, so that the birth of the infant patient is reduced.

Provides possible drug treatment targets for human beings to overcome Marfan syndrome and promotes the research and development of innovative drugs.

Detailed description of the preferred embodiments

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press, 1989), or according to the manufacturer's recommendations.

Example 1 screening for genetic mutations in patients with Marfan syndrome and family members

Male, 37 years old, is admitted to the hospital for "intermittent chest distress and short breath for more than 4 months". After a patient catches a cold 4 months ago, the patient suffers from chest distress, short breath, incapability of lying down at night, cough, expectoration, fever, uncertain body temperature, poor appetite, reduced urine volume, no nausea, vomiting, dizziness and headache, and the patient is diagnosed in a local hospital to consider 'heart failure pneumonia', is given treatment for correcting heart failure, resisting infection and the like, and the symptoms are slightly improved and discharged. After discharge, the symptoms still have intermittent attacks, the nature degree is the same as before, the local hospital is respectively treated by infusion before 3 months and before half a month, the medicine such as digoxin hydrochlorothiazide warfarin is regularly taken orally after discharge, the symptoms of a patient are controlled poorly, the patient is diagnosed in the emergency treatment of the hospital, and the treatment such as diuresis, blood circulation and the like is given, so the patient can be treated by income diagnosis.

History of the past: before 1 year, because aortic dissection and aortic valve closure are not complete in Bental + Sun's + MVR + TVP operation in the Zhen Hospital, warfarin and digoxin are taken orally for treatment after operation, and digoxin is stopped taking medicine in the latter half of the operation. Deny the history of hypertension, diabetes and hyperlipemia. Deny food and drug allergy history.

Family history: the father had mitral insufficiency, aortic insufficiency and did mitral replacement and aortic valvuloplasty 7 years ago, 3 years ago, color heart hypercardia showed aortic sinus dilatation, aortic severe insufficiency and 3 years ago sudden death (details are not shown). A sister suddenly dies 1 year ago (not detailed). The color ultrasonography of the heart examined by the brother showed sinus widening (inner diameter 46mm) and no obvious abnormality in thoracic aorta CTA.

Physical examination: t36.5 ℃ P110 times/min R21 times/min BP 106/72 mmHg. Tall and thin body, clear spirit, coarse breathing sound of both lungs, and no smell or dry and wet rale. The heart rate is 110 times/minute, the rhythm is uniform, and the aortic valve auscultation area can hear the mechanical valve working sound. The abdomen is soft and there is no tenderness. The spine is bent sideways and the fingers are slender. There was no edema in both lower limbs.

Auxiliary inspection: chest CT: (1) sternum and heart present postoperative change, mitral valve area, aortic valve area high density, please combine the medical history; (2) aortic lesions, CTA exam suggested; (3) a small amount of pleural effusion on both sides with incomplete expansion of adjacent lung tissues; (4) the posterior basal sections of the two lung apices and the left lung inferior lobe are scattered in the cavity containing the air sac; (5) poor expansion of the upper tongue segment and the lower lobe of the left lung; (6) thickening the lobules of the right inferior pulmonary lobe at intervals with exudation, and suggesting a recent review; (7) dorsal pleura inferior to right inferior pulmonary lobe; the pleura between the two lateral lobes thickens.

Heart color Doppler ultrasound: LA 41mm, LV 75mm, RA 38mm, RV 19mm, EF 33.33% aortic dissection Bental + Sun's + MVR + TVP postoperative function is approximately normal; the left heart is enlarged, and the contraction and relaxation functions of the left ventricle are reduced; the right ventricular contraction function is reduced; aortic valve artificial mechanical valve mild insufficiency; the tricuspid valve is slightly incompetent.

Electrocardiogram (admission): sinus tachycardia, heart rate 104 times/minute, low and flat limb lead T wave, poor increment of V1-3 lead R wave and high voltage of left ventricle. BNP of 4330pg/ml, cTNI of 0.03ng/ml and INR of 1.83.

And (3) diagnosis:

1. acute attack of chronic heart failure, grade IV cardiac function, aortic insufficiency, mitral insufficiency after aortic valve replacement, and cardiac valvulopathy after mitral valve replacement.

2. After aortic dissection. The patient has a history of aortic dissection, fathers (the heart color ultrasonography shows that the aortic sinus is expanded and the aortic valve is not closed fully) and sister sudden death, the patient screens the heart color ultrasonography to find that the aorta is widened, the body is thin and tall, the arm spread is larger than the height, the spinal lateral curvature and the fingers are slender, and the family is highly suspected to be a Marfan syndrome family. The patient and all healthy family members were screened for heart color ultrasound to find the brother and the large daughter with aortic broadening.

The complete exon gene sequence analysis is carried out on the patient, the brother and the daughter. The sequencing procedure was as follows:

(1) preparation of specimen DNA: collecting 3-5ml of peripheral blood of a patient, and extracting genome DNA.

(2) DNA sample testing the quality of the isolated genomic DNA was verified by using the following two methods in combination: a) DNA degradation and suspected RNA/protein contamination was verified by 1% agarose gel electrophoresis. b) By passing

3.0 Fluorometer ((Life Technologies, CA, USA) in the Qubit dsDNA hs analysis kit further accurate quantification of DNA sample concentration and purity.

(3) Library preparation and sequencing: exome sequences were enriched from genomic DNA using an Agilent SureSelect Human All Exon V6 liquid capture system. First, qualified genomic DNA was randomly fragmented into an average size of 180-280bp using a Covaris S220 sonicator. The remaining overhang is converted to blunt ends by exonuclease polymerase activity. Next, the DNA fragment is end-repaired and phosphorylated, then A-tailing is performed at the 3' end and ligated with adapters at the paired ends (Illumina). DNA fragments with adapter molecules connected at two ends are selectively enriched in PCR reaction. After the PCR reaction, the library was hybridized with biotin-labeled probes in solution, and then the exons were captured using magnetic beads with streptomycin. The captured library was enriched in a PCR reaction to add an index tag in preparation for sequencing. The product was purified using the AMPure XP system (Beckman Coulter, Beverly, USA) and quantified using the Agilent Bioanalyzer 2100 system. Finally, the DNA library was sequenced on the Illumina platform.

(4) Data information processing and mutation analysis refer to databases such as dbSNP, and the like, and look up patient gene mutation and other abnormalities by contrasting human reference genome GRCh37/hg 19. And (3) predicting the influence of the mutation on the function of the protein by using SIFT online prediction tools (http:// SIFT. icvi. org /) and software such as Polyphen-2 and the like.

The sequencing result shows that: the Pathogenic mutation sites (Pathogenic) are found by the whole exon sequencing of the PCR products of the patient, the brother and the daughter: 1G-to-A mutation (NM-004076: exon2: c.75+1G > A) exists in the splice site region of the No. 2 exon of the CRYBB3 gene at 1bp downstream of 75bp, and the mutation is predicted to influence splicing. According to the revised Ghent standard, three subjects can be diagnosed with Marfan syndrome.

In addition, the method described above was used to amplify NM-004076: exon2: c.75+1G > A of 601 enrolled healthy people who did not suffer from Marfan syndrome, and after amplification, sequencing analysis was performed, and the sequencing results showed that the NM-004076: exon2: c.75+1G > A mutation was not found in the control group.

Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. A genetic mutation for use in the diagnosis of marfan's syndrome, wherein said genetic mutation is a mutation in the CRYBB3 gene.

2. A genetic mutation as claimed in claim 1 wherein said mutation is c.75+1G > a.

3. A reagent for detecting a mutation in a gene according to claim 1 or 2.

4. The reagent of claim 3, wherein the reagent comprises an amplification primer specific for the site of the gene mutation.

5. The reagent of claim 4, wherein the reagent further comprises dNTPs, Taq enzyme, Mg²⁺And PCR reaction buffer.

6. Use of a mutation according to claim 1 or 2 for the preparation of an agent according to any one of claims 3 to 5.

7. Use of a mutation according to claim 1 or 2 or an agent according to any one of claims 3 to 5 for the preparation of a diagnostic product for equine fantastic syndrome.

8. Use according to claim 7, wherein the diagnostic product diagnoses whether the individual has Marfan syndrome by detecting the mutation according to claim 1 or 2 in the sample.

9. Use according to claim 8, wherein the sample source is blood.

10. A diagnostic product for Marfan syndrome, characterized in that it comprises the agent according to any one of claims 3 to 5.