CN111304318B

CN111304318B - Application of substance for detecting MIP gene mutation in diagnosis of panda cataract

Info

Publication number: CN111304318B
Application number: CN202010263770.0A
Authority: CN
Inventors: 由玉岩; 柏超; 王伟; 贾婷; 刘学锋; 尹彦强; 普天春; 卢岩; 夏茂华; 陈玉村; 王利勤; 修云芳; 杜洋; 马涛; 刘彦晖; 张成林; 李晓光
Original assignee: Beijing Zoo
Current assignee: Beijing Zoo
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2022-06-07
Anticipated expiration: 2040-04-07
Also published as: CN111304318A

Abstract

The invention discloses an application of a substance for detecting MIP gene mutation in diagnosing cataracts of pandas. The invention discovers a gene mutation related to the giant panda cataract, namely, the 686 th nucleotide of MIP gene is mutated from G to A, so that the 229 th nucleotide of the encoded MIP protein is mutated from serine residue to asparagine residue, the mutation exists in Ginie of the giant pandas suffering from the cataract, and the mutation does not exist in the rest giant pandas, thereby showing that the mutation can be used for diagnosing the giant panda cataract or evaluating the risk of suffering from the cataract.

Description

Application of substance for detecting MIP gene mutation in diagnosis of panda cataract

Technical Field

The invention relates to the application of a substance for detecting MIP gene mutation in diagnosing panda cataract in the field of animal medicine.

Background

Most cataracts are associated with aging, environmental factors or genetic factors. Approximately one third of cataracts have a significant genetic component, some due to disruption of normal lens development, some due to mutations in the lens proteins themselves or other proteins required for normal lens physiology, including blockade of oxidative stress pathways. The cumulative response of oxidative damage can also gradually damage the lens, or lead to accumulation of pigment or aggregation of lens proteins, which are the main components for maintaining lens transparency and refraction. However, genetic mutations affect lens proteins, as well as mutations affecting structural proteins of the connexin and myelin precursor families, gap connexins, or stress response components. Some mutations affect transcription factors expressed in the lens, such as FOXE3, HSF4, MAF, MIP, and PITX3, because these mutations have the potential to disrupt the activity of many downstream genes.

Cataracts are a common cause of vision impairment and blindness in humans, other primates and companion animals, and most cataract studies have therefore focused on these species or murine disease models. However, containment of large wild animals tends to have a longer life span than wild congeners, and cataract development is also prominent with age. More than 20% of the elderly pandas (Ailuropoda melanoleuca) in china suffer from cataracts, which have a significant impact on their quality of life. Panda cataract prevalence increases with age of animals, which severely affects their health and well-being. However, few studies have focused on the cause and treatment of cataracts in pandas. Due to the special shape of the giant panda lens, the curvature of the lens is difficult to measure in vitro, and the lens cannot be manufactured and operated. Therefore, the causes of cataract, prevention strategies and treatment methods of captive pandas are awaiting further research.

Lens fiber gene (MIP) encodes the most abundant connective membrane protein in the mature lens and plays a crucial role in maintaining the normal structure and internal circulation of the lens. Different mutations often trigger different cataract phenotypes, suggesting that MIPs have multiple physiological functions that mediate transmembrane transport of water channel molecules and some small neutral solutes. As an integral membrane protein, it accounts for over 45% of the total membrane protein of lens fiber cells, and plays a role in the formation of gap junctions and tissue crystallins. In this way, MIP can play an important role in maintaining lens transparency by reducing the space between fibers and is necessary for the proper focusing properties of the lens. During cataract formation and aging, selective proteolysis of MIPs occurs, which may modulate the MIP channels and their adhesion properties. In addition, mutations in the MIP gene were associated with hereditary cataract in mice (CATFR), Lens Opacity (LOP), HFI, TOHM and two human cataracts. The different cataract phenotypes caused by mutation indicate that the aquaporins have different functions in the crystalline lens and complex pathogenic mechanisms. Previous studies have shown that most mutations in the transmembrane protein (AQP0) disrupt its normal trafficking, resulting in retention of the protein in the cytoplasm rather than on the plasma membrane. This can lead to loss of water channel function, which may be the cause of cataract formation. MIP gene mutations disrupt water balance or lens permeability leading to cataract in mice. MIPs also play an important role in intercellular adhesion of lens fibroblasts. Previous cataract studies have shown that most MIP mutations reduce protein expression, compromising normal protein localization. However, detailed mechanisms based on specific mutations remain hidden behind the phenotypic heterogeneity, requiring further investigation. The transmembrane protein (AQP0) is primarily responsible for permeability and transparency in the lens fiber membrane. Since there is no vascular system in the lens, it relies heavily on this delivery system to maintain normal intercellular water flow and maintain lens clarity and stability. MIP mutations in humans, wild-type mice and knockout mice have been shown to induce bilateral cataracts. This information further supports the conclusion that this protein plays an important role in normal lens development. In addition to functioning as a water channel, MIPs also function as structural and adhesion molecules, which are necessary to maintain lens transparency. MIP mutations in human, mouse and knockout mouse models have been shown to induce bilateral cataracts, further highlighting the important role of this protein for normal lens growth.

Disclosure of Invention

The invention aims to solve the technical problem of how to diagnose the cataracts of pandas.

In order to solve the above technical problems, the present invention provides, in the first place, any one of the following uses of a substance for detecting MIP gene mutation:

x1, preparing a product for diagnosing or assisting in diagnosing cataracts of pandas;

x2, preparing a product for evaluating or assisting in evaluating the risk of developing cataract of the pandas to be tested;

x3, preparing a product for evaluating or assisting in evaluating the risk of developing cataract of the panda offspring to be tested;

the MIP gene is mutated into nucleotide of 686 position of the MIP gene from G to A (namely, the 80 position of the sequence 1 in the sequence table is mutated from G to A).

In the above application, the substance for detecting MIP gene mutation comprises a primer pair capable of amplifying a DNA fragment containing the MIP gene mutation.

In the application, the primer pair can be composed of two single-stranded DNAs shown as sequences 3 and 4 in a sequence table.

The substance for detecting MIP gene mutation can be composed of the primer pair and other reagents except the primer required for PCR amplification.

The invention also provides any one of the following applications of the substance for detecting MIP protein mutation:

X2, preparing and evaluating or assisting in evaluating the risk product of the pandas to be detected for suffering from cataract;

x3, preparing a product for evaluating or assisting in evaluating the risk of the panda offspring suffering from cataract;

the MIP protein is mutated into a MIP protein with the 229 th site from a serine residue to an asparagine residue (namely, the 27 th site of a sequence 2 in a sequence table is mutated from a serine residue to an asparagine residue).

The substance for detecting a mutation in the MIP protein may be a substance capable of specifically recognizing the MIP protein or a mutation in said MIP protein.

As above, the product may be a kit.

The invention also provides a kit, which comprises the substance for detecting MIP gene mutation or the substance for detecting MIP protein mutation.

The kit can have any one of the following uses:

y1, diagnosis or auxiliary diagnosis of cataracts of pandas;

y2, evaluating the risk of the pandas to be detected suffering from cataract;

y3, evaluating the risk of the offspring of the pandas to be detected to suffer from cataract.

The kit can also comprise a carrier which is recorded with a1) or a2) or a3) as follows:

a1) the following a11) or a 12):

a11) if the genome of the panda to be detected contains the MIP gene mutation, the panda to be detected has or is candidate to have cataract;

a12) If the genome of the panda to be detected does not contain the MIP gene mutation, the panda to be detected does not suffer from cataract or candidate does not suffer from cataract;

a2) the following a21) or a 22):

a21) if the genome of the panda to be detected contains the MIP gene mutation, the risk of the panda to be detected suffering from cataract is higher than that of a normal panda;

a22) if the genome of the panda to be detected does not contain the MIP gene mutation, the risk of the panda to be detected suffering from cataract is not higher than that of a normal panda;

a3) the following a31) or a 32):

a31) if the genome of the panda to be detected contains the MIP gene mutation, the risk of cataract of the offspring of the panda to be detected is higher than that of the normal panda;

a32) if the genome of the panda to be detected does not contain the MIP gene mutation, the risk of cataract of the offspring of the panda to be detected is not higher than that of the normal panda.

The kit can also comprise a carrier which is recorded with the following b1) or b2) or b 3):

b1) the following b11) or b 12):

b11) if the genome of the panda to be detected contains the MIP protein mutation, the panda to be detected has or is candidate to have cataract;

b12) if the genome of the panda to be detected does not contain the MIP protein mutation, the panda to be detected does not suffer from cataract or candidate does not suffer from cataract;

b2) The following b21) or b 22):

b21) if the genome of the panda to be detected contains the MIP protein mutation, the risk of suffering cataract of the panda to be detected is higher than that of the normal panda;

b22) if the genome of the panda to be detected does not contain the MIP protein mutation, the risk of the panda to be detected suffering from cataract is not higher than that of a normal panda;

b3) the following b31) or b 32):

b31) if the genome of the panda to be detected contains the MIP protein mutation, the risk of cataract of the offspring of the panda to be detected is higher than that of the normal panda;

b32) if the genome of the panda to be detected does not contain the MIP protein mutation, the risk of cataract of the offspring of the panda to be detected is not higher than that of the normal panda.

The kit can be composed of the substance for detecting MIP gene mutation or the substance for detecting MIP protein mutation, and can also be composed of the substance for detecting MIP gene mutation or the substance for detecting MIP protein mutation and the carrier which is described as a1), a2), a3), b1), b2) or b 3).

In the present invention, the cataract may be age-related cataract induced by gene mutation.

The inventor of the invention screens a gene mutation related to giant panda cataract by a method combining a functional candidate gene screening method and bioinformatics analysis, namely, the 686 nucleotide of the MIP gene is mutated from G to A, so that the 229 th nucleotide of the encoded MIP protein is mutated from serine residue to asparagine residue, the mutation exists in the giant pandas suffering from the cataract, and the mutation does not exist in the rest giant pandas, which indicates that the mutation can be used for diagnosing the giant panda cataract or evaluating the risk of suffering from the cataract.

Drawings

Fig. 1 jenni right eye of female panda.

FIG. 2 identification of Ginie MIP gene mutation in panda. Sequence tracking was performed over a 16bp region at the site of mutation, and 5 individuals unaffected by this site (top) were compared to jane individuals (bottom), showing the heterozygote c.686g > a mutation with an arrow.

FIG. 3 effect of highly conserved serine to aspartic acid substitution at the COOH end of MIP. (a) Multiple alignments of the highly conserved sequence of 21 amino acid residues in 5 homologous genes of MIP (giant panda (Jini), mouse, cow, bat and human) showed that panda S229N substitution affected serine residues conserved in all species (corresponding to human residue S229N). (b) The overall decrease in hydrophobicity was confirmed by a primary scale analysis of the human protein with allelic mutation (S229N) (boxed).

Figure 4 is based on the structural prediction of the missense mutation S229N in panda MIP protein. (a) Structural alterations using Discovery Studio Visualizer visualization. (b) The interaction between amino acid chains was predicted using the streamer and modeler.

FIG. 5 is a structural model of MIP amino acids. The upper diagram: a structural homology model of wild-type human MIP amino acids is shown. The following figures: structural changes of the gieni MIP mutant amino acids are shown. Due to the c.686g > a mutation, the COOH-terminal amino acid of the MIP is truncated.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The experimental procedures in the following examples are all conventional ones unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

Example 1 discovery of Gene mutation associated with cataracts of pandas

Materials (I) and (II)

Giant panda with cataract: jini, born in 1993, was bred in captivity from females in Beijing zoo. Cataract developed in 2015 as shown in fig. 1. Unilateral eye disease, characterized by the early stage of cortical cataract, with lenticular opacity around the cortex, feathery opacity. Vision deteriorates.

In addition, 5 pandas were selected as controls from Chongqing, Chengdu and Beijing zoos. The information for each panda is as follows:

Panda name	Year of birth	Source	Disease states	Sex
					Girl and old jacket	1999 year	Chongqing zoo	Healthy panda without cataract	Female
Freezing point	Year 2000	Chengdu Zoo	Health without cataract	Male sex
					Ya A	1990 s	Chengdu Zoo	Health without cataract	Female
Le Le	In 1986	BEIJING ZOO	Cataract patients	Female
					Jane	1993	BEIJING ZOO	Cataract patients	Female
New star	In 1982	Chongqing zoo	Cataract patients	Female

Wherein, happy: in 2011, there are two types of cataract, including left-eye cortical cataract and right-eye nuclear cataract, which are rare.

The new star: cataract onset in 2010, a highly mature stage of cortical cataract, secondary glaucoma (clinically known as phakic glaucoma). Clouding of the lens, dislocation into the anterior chamber, leukoplakia. The hardness of the lens nucleus is II. The two sides of the cornea are basically atrophied, the vision is poor, the movement is slow and the expression is dull.

The samples used hereinafter were all those collected from the panda normal physical examination.

Second, detection method

According to the previous report, 11 highly homologous cataract candidate genes of animals are selected for mutation screening, and the 11 genes are CRYAB gene, CRYBA1 gene, CRYBB1 gene, CRYGC gene, HSPB6 gene, HSPB7 gene, HSPB9 gene, GJA3 gene, AQP3 gene, MIP gene and HSF4 gene. The genomic DNA of each panda was amplified by PCR using the primers for these genes shown in Table 1.

The PCR reaction systems (total volume 25. mu.l) were all: 2 XPCR mix 12.5. mu.l (containing Taq DNA polymerase, dNTPs, MgCl₂Reaction buffer, enhancer and optimizer for PCR reaction and stabilizer) (Tiangen Biochemical technology (Beijing) Ltd., product No. KT201), 1. mu.l each of forward primer and reverse primer, 2. mu.l (20ng) of genomic DNA, and the balance of water. And (3) PCR reaction conditions: denaturation at 95 ℃ for 5 min; then 34 cycles of annealing at 95 ℃ for 30s at the corresponding annealing temperature for 30s and annealing at 72 ℃ for 30s are carried out; finally, extension was carried out at 72 ℃ for 10 minutes.

The resulting PCR products were sequenced using an ABI 3730 automated sequencer (PE Biosystems, Foster City, Calif.). Sequencing results were analyzed using Chromas 2.33 and compared to reference sequences in NCBI databases.

TABLE 1 PCR primers for screening candidate Gene mutations

Third, sequence analysis

Mutation analysis: a new mutation was identified by sequencing 11 candidate genes from each panda (FIG. 2). The mutation exists in MIP exon 4 of giant panda 'Jini', namely c.686G > A, namely the 686 th nucleotide in the 4 th exon of MIP gene is mutated from G to A (namely the 80 th nucleotide in sequence 1 in the sequence table is mutated from G to A, and the sequence 1 is a sequence of Jini), and the mutation causes the 229 th serine residue of the MIP protein coded by the MIP gene to be replaced by the asparagine residue (S229N) (namely the 27 th serine residue in sequence 2 in the sequence table is replaced by the asparagine residue, and the sequence 2 is a sequence of Jini). The mutation is located in the PCR amplified product of MIP-4 by the primer pair consisting of sequence 3 and sequence 4 in Table 1. The mutation is only present in pandas named "ginie" and is a heterozygous mutation, with no mutation in c.686g > a of MIP in the remaining healthy panda individuals.

Fourth, bioinformatics analysis

The inventors selected the MIP amino acid sequences of other mammals for conservation analysis, aligned the sequences of each species with the panda MIP amino acid sequences, and found that the amino acids at the mutation positions in guinie MIPs are highly conserved in the other three species (fig. 3, CLC Main Workbench Software). The S229N mutation in jannie is located in exon 4, the intracellular COOH domain of the transmembrane MIP protein.

In addition, the structural and functional impact of MIP mutations was predicted using Modeller 9.22(4) with MIP structure from Ovisaries (PDB: 2B6O) (5) as a modeling template. The structural changes caused by the S229N mutation are shown in fig. 4. In the wild type MIP protein, Ser229 forms a hydrogen bond with Ser231 (b in fig. 4). However, in the mutant, Asn229 is involved in two weak hydrogen bonds with Ser231 and Glu232, respectively (b in fig. 4). This molecular surface change may explain the dysfunction of mutant MIPs. These predictions suggest that the mutation may have an effect on protein structure or stability, which is associated with the development of cataracts. Meanwhile, Swiss predicts that the MIP structure also shows that 2 hydrogen bonds are added after MIP mutation, and the tensile force between 229 th amino acid and nearby amino acid is enhanced, so that the space structure of the protein is influenced, and the graph is shown in figure 5. Thus, substitution of asparagine residues for serine residues in ginie may lead to functional disorders of the MIP gene.

To date, 19 MIP gene mutations have been reported to cause cataracts, including 12 missense mutations, p.rj233k (Lin et al, 2007), p.v107i (Wang et al, 2010), p.rj187c (Wang et al, 2011), p.y177c (Yang et al, 2011), p.rj33c (Gu et al, 2007), p.dshir (Shentu et al, 2015), p.l170fs (Qin et al, 2016), p.g211r (Takahashi et al, 2017), p.g215d (Ding et al, 2014), p.g165d (Senthil et al, 2013), p.tjr 138r (Berry et al, 2000), p.eg (Berry 134al, 2000); two nonsense mutations p.r113 (Yu et al, 2014), p.y219 (Song et al, 2015); two frameshift mutations 638delG (Geyer et al, 2006) and c.682_683delAA (Long et al, 2018); an acceptor splice site mutation (IVS3-1G > A) (Jiang et al, 2009); one donor splice site mutation (c.606+1G > a) (Zeng et al, 2013); an initiation codon mutation (c.2t > C) (Xiao et al, 2011). Most MIP mutations occur predominantly in humans and mice.

The Major Intrinsic Protein (MIP) is a 28kDa transmembrane protein containing 263 amino acids (shield et al, 2001), also known as AQP0, which is a member of aquaporins, consisting of six transmembrane bilayer spanning domains (H1-H6), three extracellular loops (a, C and E), two intracellular loops (B and D) and an amino and carboxyl terminal constituting the intracellular domain (positions 220 to 263) (Chepelinsky et al, 2009). Several previous studies have shown that the AQP0COOH terminus is critical for lens development and transparency through interaction with calmodulin, the cytoskeletal proteins FILENSIN and CP49 and connexin 45.6. Cleavage of the intracellular COOH terminus decreases water permeability and enhances the adhesion properties of AQP0 on the extracellular surface, indicating a conformational change in the molecule. Possible aberrant splicing of MIP pre-mRNA can disrupt the normal COOH ends of AQP0 and lead to an imbalance in lens homeostasis, which is necessary to maintain transparency and ultimately leads to cataract formation. In addition, mutational analysis of AQP0 transcripts from the CATFR lens showed that CATFR mutations resulted in the substitution of the long terminal repeat for the COOH terminus of the MIP (AQP 0-LTR). The AQP0-LTR in CATFR accumulates in the subcellular compartment, disabling the stratification of mature fibroblasts into uniform concentric growth, eventually leading to cataracts.

The present inventors have discovered that panda cataracts caused by a novel missense mutation c.686g > a in exon 4 of the MIP gene result in serine to asparagine (p.s229n) in the COOH domain of the MIP. Predictive analysis has shown that substitutions are likely to alter the structure of the protein and disrupt interactions with sterically adjacent amino acid side chains, consistent with previous studies. These results indicate that the direct screening of MIP mutation by a pair of primers is an economical, effective, comprehensive and reliable molecular diagnosis method of cataract, and is helpful for predicting the risk of panda suffering from cataract or assisting in diagnosing panda cataract.

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<120> application of substance for detecting MIP gene mutation in diagnosis of giant panda cataract

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gtatactggg tgggcccaat cattggagga ggactaggca gtctcctcta cgactttctc 60

cttttccccc ggctcaagaa tgtttctgag agactctcta ttctcaaggg tgccaggccc 120

agtgactcca atggacaacc agagggcact ggggaacctg ttgaa 165

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Ser Ile Leu Lys Gly Ala Arg Pro Ser Asp Ser Asn Gly Gln Pro Glu

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Gly Thr Gly Glu Pro Val Glu

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Claims

1. Any one of the following uses of the substance for detecting MIP gene mutation:

The MIP gene is mutated into nucleotide 686 of the MIP gene from G to A.

2. Use according to claim 1, characterized in that: the substance for detecting MIP gene mutation comprises a primer pair capable of amplifying a DNA fragment containing the MIP gene mutation.

3. Use according to claim 2, characterized in that: the primer pair consists of two single-stranded DNAs shown as sequences 3 and 4 in the sequence table.

4. Any one of the following uses of a substance for detecting mutations in the MIP protein:

x1, preparing products for diagnosing or assisting in diagnosing cataracta of pandas;

the MIP protein is mutated from serine residue to asparagine residue at the 229 th position of the MIP protein.

5. A kit comprising the substance for detecting mutation of MIP gene according to any one of claims 1 to 3 or the substance for detecting mutation of MIP protein according to claim 4.

6. The kit of claim 5, wherein: the kit has any one of the following uses:

y1, diagnosis or auxiliary diagnosis of cataracts of pandas;

Y2, evaluating the risk of the pandas to be detected suffering from cataract;

7. The kit according to claim 5 or 6, characterized in that: the kit also comprises a carrier which is recorded with a1) or a2) or a3) as follows:

a1) the following a11) or a 12):

a11) the genome of a panda to be tested containing the MIP gene mutation of claim 1, wherein the panda to be tested has or is candidate for having cataract;

a12) if the genome of the panda to be tested does not contain the MIP gene mutation in the claim 1, the panda to be tested does not suffer from cataract or does not candidate to suffer from cataract;

a2) the following a21) or a 22):

a21) the genome of a panda to be tested contains the MIP gene mutation in the claim 1, and the risk of the panda to be tested suffering from cataract is higher than that of a normal panda;

a22) if the genome of the panda to be tested does not contain the MIP gene mutation in the claim 1, the risk of the panda to be tested suffering from cataract is not higher than that of the panda to be tested;

a3) the following a31) or a 32):

a31) if the genome of the panda to be tested contains the MIP gene mutation in the claim 1, the risk of cataract of the offspring of the panda to be tested is higher than that of the normal panda;

a32) If the genome of the panda to be tested does not contain the MIP gene mutation in the claim 1, the risk of cataract of the offspring of the panda to be tested is not higher than that of the offspring of the normal panda.

8. The kit of claim 5 or 6, wherein: the kit also comprises a carrier which is recorded with b1) or b2) or b3) as follows:

b1) the following b11) or b 12):

b11) the genome of a panda to be tested containing the MIP protein mutation of claim 4, wherein said panda to be tested has or is candidate for having cataract;

b12) if the genome of the panda to be tested does not contain the MIP protein mutation of claim 4, the panda to be tested does not suffer from cataract or is not candidate to suffer from cataract;

b2) the following b21) or b 22):

b21) the genome of a panda to be tested contains the MIP protein mutation in the claim 4, and the risk of the panda to be tested suffering from cataract is higher than that of a normal panda;

b22) if the genome of the panda to be tested does not contain the MIP protein mutation in the claim 4, the risk of the panda to be tested suffering from cataract is not higher than that of the panda to be tested;

b3) the following b31) or b 32):

b31) the genome of a panda to be tested contains the MIP protein mutation in the claim 4, and the offspring of the panda to be tested has higher risk of suffering cataract than the offspring of the normal panda;

b32) If the genome of the panda to be tested does not contain the MIP protein mutation of claim 4, the risk of cataract of the offspring of the panda to be tested is not higher than that of the offspring of the normal panda.