Genetic detection of two novel LRP5 mutations in patients with familial exudative vitreoretinopathy

doi:10.21203/rs.3.rs-2431551/v1

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Research Article

Genetic detection of two novel LRP5 mutations in patients with familial exudative vitreoretinopathy

https://doi.org/10.21203/rs.3.rs-2431551/v1

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Journal Publication

published 29 Nov, 2023

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Objective

To identify causative genetic mutations by targeted exome sequencing in 9 independent pedigrees with familial exudative vitreoretinopathy (FEVR) and characterize the novel pathogenic mutations by molecular dynamics simulation.

Methods

Clinical data were collected from 9 families with FEVR. The causative genes were screened by targeted next-generation sequencing (TGS) and verified by the Sanger sequencing. In silico analyses (SIFT, Polyphen2, Revel, Mutation taster, and GERP++) were carried out to evaluate the pathogenicity of the mutations. Molecular dynamics was simulated to predict the alterations of protein conformation and flexibility transformation on pathogenesis.

Results

A 44% overall detection rate was achieved with four mutations including c.4289delC:p.Pro1431Argfs*8, c.2073G > T:p.Trp691Cys, c.1801G > A:p.Gly601Arg in LRP5 and c.633T > A:p.Tyr211* in TSPAN12 in 4 unrelated probands. Based on in silico analysis and ACMG standard, two of them, c.4289delC:p.Pro1431Argfs*8 and c.2073G > T:p.Trp691Cys of LRP5 were identified as novel pathogenic mutations. According to a molecular dynamics simulation, both mutations altered the secondary structure and spatial conformation, thus compromising its stability and flexibility.

Conclusion

Two novel genetic variants of the LRP5 gene were found to contribute to FEVR in this study, enriching the mutation spectrum of this condition. The impact of these two mutations on protein structure was validated by molecular dynamics simulation, further evidencing their pathogenicity.

Familial Exudative Vitreoretinophay (FEVR)

Genetic

Mutation

Retina

LRP5

Molecular dynamics simulation

Familial exudative vitreoretinopathy (FEVR) is a monogenic genetic disease and highly heterogeneous with autosomal dominant and recessive inheritance patterns. In addition, X-linked inheritance has also been reported. ¹ Currently, LRP5 and the other four genes FZD4, TSPAN12, ZNF408, and NDP in the Wnt signaling pathway, have been found in about 50% of FEVR cases.^2–5

The symptoms of FEVR are varied including a lack of perfusion of the peripheral retina, neovascularization, vitreoretinal traction, macular displacement, retinal folds, and retinal detachment (RD). ^6–8 Since the penetrance of the disease is incomplete and the fundus manifestations are comparable to those of other retinal diseases such as high myopia or retinopathy of prematurity (ROP), the disorder may easily be undiagnosed or misdiagnosed. ^9–11 Therefore, fundus imaging combined with genetic screening is crucial in diagnosis and genetic counseling.

To date, although multiple studies have confirmed the pathogenicity of mutations in FEVR patients using second generation sequencing technologies and in silico analysis, few attempts have been conducted to analyze the conformational changes of protein structure at the atomic or molecular level. The effects of conformational changes caused by mutant proteins have been reported in other ophthalmologic studies using molecular dynamics (MD) simulation, ^12–16 revealing the importance of MD simulation in understanding the functional mechanisms of proteins and the molecular basis of diseases. ¹⁷

Given this, we combined the targeted next-generation sequencing (TGS) panel with MD simulation to characterize the novel pathogenic mutations in nine Chinese families with clinically diagnosed FEVR, attempting to provide additional evidence for exploring the pathogenicity of FEVR mutation at the dynamic molecular level.

Study subjects and clinical examinations

Data from patients with clinically diagnosed FEVR at Ningxia Eye Hospital, Ningxia Hui Autonomous Region People Hospital were collected. The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Ningxia Hui Autonomous Region People Hospital (No. 2020-KY-GZR019). The informed consent was signed by each participant or the guardians in the study.

All probands and family members underwent detailed ophthalmic examinations, including best corrected visual acuity (BCVA), slit-lamp biomicroscopy, funduscopy, ultra wide-field (UWF) fundus photography (UWFFP, Daytona P200T, OPTOS, Fife, UK), and fundus fluorescein angiography (FFA; Spectralis HRA + OCT, Heidelberg Engineering GmbH, Heidelberg, DE). Patients with avascular peripheral retina and neovascularization, brush-like vessels, retinal detachment (RD), vitreous hemorrhage, severe subretinal exudates, and macular ectopia were diagnosed with FEVR. ¹⁸ The disease stage was determined in patients of FEVR in each eye using Trese's staging system. ¹⁹ Meanwhile, the basic information, including gender, age, birth history, family history, and other clinical data of the patients and family members, was gathered. Notably, the patients with a history of premature delivery or other retinal vascular diseases with similar clinical manifestations were excluded.

Mutation identification and in silico analysis

Targeted high-throughput sequencing was conducted using the next-generation sequencer Illumina Hiseq 2500 (Illumina, San Diego, California, USA), and the variants were confirmed by Sanger sequencing. The read sequences were matched to the human reference genome hg19 (GRCH37) with the Burrows-Wheeler aligner version 0.7.10 (BWA) (http://bio-bwa.sourceforge.net/) program. The information and frequency on each variant were queried against public databases including the dbSNP (https://www.ncbi.nlm.nih.gov/SNP/), EXac (https://gnomad.broadinstitute.org/), GenomAD (https://gnomad.broadinstitute.org/), and 1000genomes (https://www.internationalgenome.org/). Pathogenicity and protein function of variants were predicted using SIFT (http://sift.jcvi.org), Mutation taster (http://www.mutationtaster.org), Polyphen-2 (http://genetics.bwh.harvard.edu/pph2), REVEL (https://ngdc.cncb.ac.cn/biocode/tools/BT004461), and GERP++ (http://mendel.stanford.edu/SidowLab/downloads/gerp).²⁰ Each variant was further filtered in databases such as Clinvar (https://www.ncbi.nlm.nih.gov/clinvar/) and HGMD (http://www.biobaseinternational.com/product/hgmd) to confirm whether it had previously been reported. Segregation studies were analyzed among the family members^21–22 and the American College of Medical Genetics and Genomics (ACMG) ²³ guidance was adopted for the potential deleteriousness.

The Three-dimensional Structure Modeling

The three-dimensional (3D) structures of wildtype and mutations of LRP5 were created by Alphafold2 (https://alphafold.ebi.ac.uk) under multiple sequence alignment using amino acid sequence obtained from the universal protein resource (http://www.uniprot.org;Entry:O75197). ²⁴ The protein model was scored via the frame aligned point error function (FAPE), ²⁵ based on neural network training. The structure of the dynamic simulation was provided by the top-ranked result, and PyMOL software (https://pymol.org/2/) was then used to show the protein structure.²⁶

Molecular Dynamic Simulation

To predict the stability and spatial conformational change of the whole protein at the atomic level, a 200ns molecular dynamics (MD) simulation of wildtype and novel mutations was performed with the GROMACS 2019.6 package (https://www.gromacs.org) utilizing Amber14sb all-atom force field. ²⁷ The wildtype and mutant protein structures underwent solvation in a three-points (TIP3P) water model, in a cubic water box with a separation of 1 Å between the protein structure and the box edges, using the periodic boundary conditions to avoid edge effects. ^{28, 29} The system was neutralized by Na + and Cl- ions, then relaxed by the steepest descent algorithm and the conjugate gradient algorithm within 50,000 steps. The coulomb force and van der Waals interactions were calculated based on a cut-off distance of 1.4nm. Each system was then equilibrated with NVT (isochoric-isothermal) and NPT (isothermal-isobaric) ³⁰ ensembles. During the 200ns simulation, root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (RG) data were saved for analysis. Hydrogen bond number (HBNUM) and secondary structure calculations were carried out by the dictionary secondary structure pattern (DSSP) algorithm. ³¹ The ploting of trajectories of Cα atoms of proteins was conducted using Origin 85 (https://www.originlab.com/origin).

Clinical Manifestations

General data, visual acuity results, fundus photographs, and FFA of 8 affected individuals including proband and first-degree relatives from 4 families were shown in Table 1. Among probands, there were 3 males and 1 female with a mean age of 12.25 years old (range, 7 to 17 years), while there were 1 male and 3 female of affected family members with a mean age of 43.25 years old (range, 42 to 45 years). The visual acuity ranged from finger count before the eye to 1.0. Fundus manifestations varied significantly among individuals even of the same family, 3/4 of the probands manifested as stage 3 while their parents were stage 2. Specifically, the FFA of the probands in three families exhibited RD in either of the eyes except for the proband from family 1 whose fundus only showed dilated blood vessels and fluorescein leakage, while their parents were consistent in showing no perfusion area, increased vascular branching and stained vascular walls. (Fig. 1).

Mutations Detected By Targeted Genetic Sequencing

Four mutations were identified in four out of nine (44%) families (Table 2). Among them, two heterozygous mutations (c.4289delC:p.Pro1431Argfs*8 and c.2073G > T:p.Trp691Cys) located in LRP5 were novel while the other two mutations (c.1801G > A:p.Gly601Arg in LRP5 and c.633T > A:p.Tyr211* in TSPAN12) have been reported previously. ^{32, 33}

The novel mutation c.4289delC (p.Pro1431Argfs*8) in LRP5 resulted in the proline (Pro) at position 1431 being replaced by arginine (Arg) and premature termination at position 1437 after eight frameshift amio acids, which eventually led to a truncated protein product (Fig. 2A). The second novel mutation c.2073G > T (p.Trp691Cys) was found in family 2 and resulted in the tryptophn (Trp) at position 691 being replaced by cysteine (Cys) (Fig. 2A). These two mutations were absent in other healthy family members but detected in the probands and affected relatives.

Two mutations in LRP5 (c.1801G > A:p.Gly601Arg) and TSPAN12 (c.633T > A: p.Tyr211*) identified in families 3 and 4, respectively, have previously been reported as pathogenic. The heterozygous missense mutation c.1801G > A of LRP5 caused glycine (Gly) at position 601 replaced by arginine (p.Gly601Arg) (Fig. 2A). The heterozygous missense mutation c.633T > A of TSPAN12 caused nonsense mutations in amino acids (p.Tyr211*), resulting in a truncation of more than 30% of the protein (Fig. 2A).

Pathogenicity Analysis

Two mutations in LRP5 (c.4289delC: p.Pro1431Argfs*8 and c.2073G > T: p.Trp691Cys) and one mutation in TSPAN12 (c.633T > A:p.Tyr211*) were not observed in databases such as dbSNP, ExAC or 1000genomes, while the minimum allele frequency (MAF) of the mutation (c.1801g > A:p.Gly601Arg) was 0.0000083, Two of the mutations (c.2073G > T:p.Trp691Cys and c.1801g > A:p.Gly601Arg) were predicted to be disease-causing or damaging by in silico analysis methods SIFT, Polyphen-2, REVEL (scored 0.994, and 0.965 respectively), mutation taster and GERP++ (scored 4.11, and 4.13 respectively). The remaining two mutations (c.4289delC:p.Pro1431Argfs*8 and c.633T > A:p.Tyr211*) were predicted to be disease-causing or damaging by mutation taster and GERP++ (scored 4.53 and 5.68 respectively) (Table 2). All four variants were cosegregated with patients among family members and the sites are conserved in various species from humans to zebrafish (Fig. 2B). Therefore, all of these variations were considered pathogenic according to the ACMG standard.

Molecular Dynamic Simulation And Data Analysis

RMSD (root mean square deviation) and RMSF (root mean square fluctuation) are common measures of protein spatial variations in an MD simulation. RMSD describes the molecule’s overall discrepancy with respect to a reference conformation. The RMSD values of the wildtype and two mutations of LRP5 were depicted in Fig. 3A. Throughout the simulation, the structure of p.Trp691Cys was more stable than the wildtype and p.Pro1431Argfs*8 (P = 0.000, ＜0.05) while the backbone atoms of the three proteins may be maintained around 1.5nm.

RMSF, on the other hand, is a per atom quantity describing the atom’s variation over the whole trajectory. The RMSF values of the three proteins were shown in Fig. 3B that the highly active sites of wildtype were 10–27, 1318–1387, 1513–1520, and 1606–1615, while those of p.Trp691Cys were 14–15, 1331–1333, and 1486–1489, and those of p.Pro1431Argfs*8 were 1–28 and 1261–1385, indicating large differences in highly flexible sites among the three proteins. The flexible region of the p.Trp691Cys ensemble was smaller than that of the wildtype, and the overall structure was more convergent and rigid, while the p.Pro1431Argfs*8 ensemble showed more divergence and flexibility at the 1261–1385 residues. The changes in the active regions of the two mutant proteins were also shown in the 3D structure diagram in Fig. 3C. These results indicated that the two mutations had notable alterations in the LRP5 protein structure.

Rg values, which were used to examine the distance or fluctuation from c-α-atoms to the center of mass, were 4.34 Å, 4.25 Å, and 4.45 Å for the wildtype and two mutants, respectively (Fig. 3C). C-α-atoms fluctuated more in the p.Pro1431Argfs*8 mutant (P = 0.000, < 0.05), and less in the p.Trp691Cys mutant (P = 0.000, < 0.05) compared with the wildtype (Fig. 3D), indicating that the mutation may increase the stability of p.Trp691Cys and the flexibility of p.Pro1431Argfs*8.

HBNUM values, an index to reflect hydrogen bond numbers and protein structural stability, indicated that they had similar structural stability in p.Trp691Cys and wildtype, while the stability of p.Pro1431Argfs*8 was lower due to the absence of C-terminus structures (Fig. 3E). For intramolecular interactions through hydrogen bonds, as shown in Fig. 3F, the Trp691 formed hydrogen bonds with Ser700 in the wildtype, while the Cys691 of p.Trp691Cys formed hydrogen bonds with Ala677, Ala679, and Ser700. In the p.Pro1431Argfs*8 mutant, the Pro1431 was transformed into Arg, which formed hydrogen bonds with Tyr719, Glu721, and Thr737. The alterations in hydrogen bond distribution certainly contributed to the structural and conformational changes.

Then selected the last 50ns of the simulation for DSSP analysis and visualized the 3D structures and active sites by PyMOL (Figs. 4A and B) after the final structure reached convergence during the simulation. The DSSP results indicated the numbers of coils, turn, and a-helix increased but the number of b-sheet, bend, and 3-helix decreased in p.Trp691Cys, compared with those in the wildtype (Figs. 4C). The numbers of all secondary structures of p.Pro1431Argfs*8 were lower than those of wildtype (Figs. 4C).

Table 1

Summary of general Information and phenotype characteristics of affected individuals from 4 FEVR families
Family	Patients	Gender	Age at Initial visit	BCVA		FEVR Stage		FFA
				OD	OS	OD	OS
Family 1	II2:	F	9y	0. 1	0. 4	2	2	OU: Dilated blood vessels; fluorescein leakage
	I2:	F	42y	1. 0	1. 0	2	2	OU: no perfusion area; increased vascular branches; stained vascular wall
Family 2	II1:	M	17y	1. 0	0. 6	2	3	OD: fluorescein leakage; no perfusion area OS: postoperative of RD
	I2:	F	45y	0. 8	0. 8	2	2	OU: no perfusion area; increased vascular branches
Family 3	II2:	M	7y	0. 2	FC/BE	2	3	OD:paraoptic disc and temporal retinal atrophy, fluorescein leakage OS: RD and retinal fold
	I1:	M	43y	1. 0	1. 0	2	2	OU: no perfusion area; increased vascular branches; stained vascular wall
Family 4	II2:	M	16y	0. 5	0. 7	3	2	OD: RD and fluorescein leakage OS: no perfusion area; increased vascular branches; fluorescein leakage
	I2:	F	43y	1. 0	1. 0	2	2	OU: no perfusion area; increased vascular branches; fluorescein leakage

BCVA, Best corrected visual acuity; F, Female; FC/BE, finger count before the eye; FFA, Fundus fluorescein angiography; M, Male; OD, right eye; OS, left eye; OU, both eyes; RD: retinal detachment.

Table 2

Computational analysis of causative mutations in four probands with FEVR
Family	proband	Gene	ORF and Amino acid changes	Allele Status	MAF	SIFT	Polyphen-2	REVEL	Mutation taster	GERP ++	ACMG	Genetic Model	Ref
Family 1	II2	LRP5	c.4289delC: p.Pro1431Argfs*8	Het	NA	NA	NA	NA	DC	4. 53	Pathogenic	AD	Novel
Family 2	II1	LRP5	c.2073G > T: p.Trp691Cys	Het	NA	D	PrD	0. 994	DC	4. 11	Pathogenic	AD	Novel
Family 3	II2	LRP5	c.1801G > A: p.G601R	Het	0. 0000083	D	PrD	0. 965	DC	4. 13	Pathogenic	AD	Reported
Family 4	II2	TSPAN12	c.633T > A: p.Tyr211*	Het	NA	NA	NA	NA	DC	5. 68	Pathogenic	AD	Reported

Notes: In silico analyses: SIFT, Polyphen-2, REVEL, and GERP ++.

Abbreviations: ACMG, American College of Medical Genetics and Genomics; AD, autosomal dominant inheritance; D, damaging/ deleterious; DC, disease-causing; Het, heterozygous; MAF, minimum allele frequency; NA, not available; ORF, open reading frame; PrD, probably damaging. Ref

In this study, 3 mutations of LRP5 and 1 of TSPAN12 in four unrelated FEVR families were found to be pathogenic according to the ACMG guidelines. Two of the LRP5 mutations, c.4289delC (p.Pro1431Argfs*8) and c.2073G>T (p.Trp691Cys), were novel mutations with extensive changes in protein stability and flexibility according to MD simulation. It is reported that approximately 50% of FEVR patients harbored deleterious variants, ³⁴ among which LRP5 accounts for about 12% - 25% and TSPAN12 for about 3% - 10%, ^{2, 35-39}and that the mutation frequency of the same gene varies in different ethnicities. In this study, the total detection rate of all mutations was 44% (4/9), which was consistent with previous reports. ³⁴Of all the FEVR patients, 75% (3/4) were caused by the LRP5 mutation, whereas 25% (1/4) were by the TSPAN12 mutation. In the same region of China (Ningxia), Zou. et al. ³³ reported that four out of eight probands (50%, 4/8) harbored mutations in TSPAN12 (75%, 3/4) and FZD4 (25%, 1/4) genes, but none carried LRP5 mutations. When combining the data of both studies, it showed that LRP5 accounted for 37.5% (3/8) and TSPAN12 for 50% (4/8) of FEVR patients in Ningxia. As a result, it was hypothesized that the LRP5 and TSPAN12 gene were the more prevalent causal genes in the Ningxia population, but it was also necessary to rule out the possibility that the limited sample size contributed to the selection bias. Therefore, it needs to be verified with a sizable sample in subsequent investigations.

As reported previously, the LRP5 gene mutations lead to the inactivation of the Wnt signaling pathway, thus reducing retinal angiogenesis and triggering FEVR lesions. ^{40, 41}In this study, we found that three mutations of LRP5 were pathogenic including the novel missense mutation c.2073G>T (p.Trp691Cys) located in the highly conserved third YWTD-EGF-β-propeller domains. Studies of LRP6 suggest that the third YWTD-EGF repeats are the region to bind with ligands of the Wnt signal pathway. ⁴² Considering the high similarity between LRP5 and LRP6 proteins, ⁴²it is reasonable to presume that the c.2073G>T (p.Trp691Cys) mutation in LRP5 may affect the Wnt signaling pathway by disrupting the binding of extracellular ligands to YWTD-EGF-β-propeller domains. Another frameshift mutation of LRP5, c.4289delC (p.Pro1431Argfs*8), was first identified in this study, which resulted in a premature termination of the protein at position 1437 and generated a truncated protein that may pathogenic due to the loss of function.

To elaborate the pathogenic mechanism at the dynamic molecular level, we employed alphafold2 to model the 3D structure of LRP5 for the first time and implemented 200ns MD simulation to analyze the changes in RMSD, RMSF, RG, HBNUM, and DSSP values in wildtype and mutant proteins. These indicators confirmed that the p.Trp691Cys and p.Pro1431Argfs*8 ‎mutations triggered FEVR manifestation by altering the overall conformation, which may have an impact on the ligand binding of the LRP5 protein. A large number of studies and experiments have shown that the ligand binding region of proteins opened to interact with ligands due to the activity of flexible regions, which was closely related to various biological processes such as molecular docking, catalytic activity, and transportation of small molecules. ^43-50

The secondary structure and active sites of the protein were altered by the changes in hydrogen bonds, and this in turn modified the protein's spatial conformation, making the entire protein more rigid or flexible, were also observed. Following a 200ns MD simulation of p.Trp691Cys, replacing Trp691 with Cys691 induced additional hydrogen bond formation among adjacent residues in the protein. These hydrogen bonds would increase the rigidity of the second structure and decrease the flexibility of the protein. For the p.Pro1431Argfs*8 mutation, premature termination at position 1437 resulted in the reduction of the overall secondary structures and hydrogen bond number. Simultaneously, the positively polarized His1432 was converted to Thr with neutral polarity and Glu1437 with negative polarity to Ser with neutral polarity. Such a mutation may destroy the salt bridge formed between the original residues, causing regions 1261-1385 to be more active and to pull away from their original position. Consequently, the reduction of hydrogen bond number and the destruction of the salt bridge led to a decrease in protein stability.

In terms of the relationship between genotype and phenotype, our study revealed that probands and their parents exhibit different clinical phenotypes despite sharing the same pathogenic mutation. The parents of patients with severe FEVR typically displayed mild manifestations, in agreement with the previous report by Gilmour ⁵¹. Similarly, probands of different families harbored the same variant may have different fundus performances of the disorder. In this study, proband 4 carrying TSPAN12 mutation (c.633T>A:p.Tyr211*) showed retinal detachment, while the same mutation detected by Zou et al ³³ resulted in milder conditions as vitreous hemorrhage and peripheral avascular area or vascular exudate. Furthermore, another study ³² reported that younger FEVR patients usually displayed more severe phenotypes than older patients. Gilmour et al ⁵¹ speculated that individuals carrying the same mutation often present different phenotypes due to the influence of epigenetics and environmental factors. They found that even minor changes in the oxygen content or intrauterine conditions, such as PaO2 alteration or exposure to certain drugs, may have a considerable impact on patients who harbor FEVR mutations.

Nevertheless, the study was still limited by its relatively small sample size and sequencing technology, thus additional genes or mutations such as some deep intronic mutations or copy number variants involved in FEVR have not been detected. Furthermore, to verify two novel mutations(c.2073g>t:p.Trp691Cys and c.4289delc:p.pro1431Argfs*8), laboratory testing in vitro or in animals should be conducted. In the future, targeted next-generation sequencing combined with whole exome sequencing applied in larger samples will be considered in our studies, as well as laboratory testing.

Taken together, combining mutation screening and molecular dynamics simulation, two novel mutations of the LRP5 gene (c.2073g>t:p.Trp691Cys and c.4289delc:p.pro1431Argfs*8) responsible for FEVR were identified. This study expanded the mutation spectrum of FEVR and offered new insights for the genetic investigation of patients with FEVR.

1. Ethics approval and consent to participate: The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Ningxia Hui Autonomous Region People Hospital (No. 2020-KY-GZR019). The informed consent was signed by each participant or the guardians in the study.

2. Consent for publication: Not applicable.

3. Availability of data and materials: The datasets generated and/or analysed during the current study are available in the [National Library of Medicine] repository, [https://www.ncbi.nlm.nih.gov/sra/PRJNA922952].

4. Competing interests: The authors declare that they have no competing interests.

5. Funding: National Natural Science Foundation of China (82060182), Ningxia Natural Science Foundation (2021AAC03301); Fundamental Research Funds for the Central Universities ( 31920200027).

6. Authors' contributions: Li JY and Wang CJ collected the data and wrote the main manuscript. Zhang SH, Cai B, Pan B, Sun CH, Qi XL, Ma CM, Fang W and Jin KX analyzed and interpreted the patient data regarding the FEVR disease. Bi XJ, Jin ZB and Zhuang WJ designed the work and substantively revised the article. All authors read and approved the final manuscript.

7. Acknowledgements: We gratefully thank all the patients and family members for their participation as well as the support provided by the funding of the study.

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No competing interests reported.

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Genetic detection of two novel LRP5 mutations in patients with familial exudative vitreoretinopathy

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Abstract

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Methods

Results

Conclusion

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Introduction

Materials And Methods

Study subjects and clinical examinations

The Three-dimensional Structure Modeling

Molecular Dynamic Simulation

Results

Clinical Manifestations

Mutations Detected By Targeted Genetic Sequencing

Pathogenicity Analysis

Molecular Dynamic Simulation And Data Analysis

Discussion

Declarations

References

Additional Declarations

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