ABSTRACT Keratoconus (KC) is a common degenerative corneal disease, and heredity plays a key role in its development. Although few genes are known to cause KC, a large proportion of

disease-causing genes remain to be revealed. Here, we report the identification of _TUBA3D_ as a novel gene linked to KC. Using whole-exome sequencing of a twins pedigree, a novel de novo

mutation (c.31 C > T, p.Gln11stop) in _TUBA3D_ gene was identified. A screening performed in 200 additional unrelated patients with KC revealed another two mutations (c.201insTT,

p.Val68Leufs*2; c.*2 G > A) in two patients. _TUBA3D_ was expressed highly in the cornea, and the twins had lower _TUBA3D_ expression and higher UPA and MMP1 expressions than the normal

thinning, which is characteristic of KC corneas. Our study showed that _TUBA3D_ is a new gene that causes KC, thus supporting the evidence that this protein has an additional function into

the human cornea. SIMILAR CONTENT BEING VIEWED BY OTHERS TARGETED NEXT-GENERATION SEQUENCING ANALYSIS IN ITALIAN PATIENTS WITH KERATOCONUS Article 29 April 2024 A MULTI-ANCESTRY GWAS OF

FUCHS CORNEAL DYSTROPHY HIGHLIGHTS THE CONTRIBUTIONS OF LAMININS, COLLAGEN, AND ENDOTHELIAL CELL REGULATION Article Open access 06 April 2024 RESEQUENCING OF CANDIDATE GENES FOR KERATOCONUS

REVEALS A ROLE FOR EHLERS–DANLOS SYNDROME GENES Article 19 March 2021 INTRODUCTION Keratoconus (KC) is a common degenerative corneal disease with a worldwide prevalence of approximately

1:20001,2. Onset usually occurs during adolescence. KC can affect patients lifelong. KC is characterized by corneal ectasia, thinning, and a cone-shaped protrusion, thus resulting in reduced

vision, irregular astigmatism, and corneal scarring1. It affects both genders and all ethnicities2. The limited availability of medical treatments makes KC become a significant clinical

problem worldwide and a leading indication for corneal transplantation3,4. Despite the visual and social effects of KC, its underlying biochemical and cellular basis is poorly understood. A

large number of studies show a strong familial predisposition in KC development, and genetic factors play a key role in its development5,6,7. In most published studies, the inheritance

pattern of KC is autosomal dominant with incomplete penetrance or variable expressivity8,9,10,11. Detection and identification of the pathogenic gene is both a research hotspot and a

challenge in this field. To date, more than 20 genes/loci identified by genetic mapping or candidate gene screening have been reported to be associated with KC5,6,7. However, most of the

results cannot be repeated in other studies. Only a small number of gene mutations were confirmed to be pathogenic, such as _VSX1_, _TGFBI_, and _mir184_ 7,12,13,14. These reported

pathogenic genes explain only a small percentage of KC7,13,15, and the etiology of most patients is still unknown. To gain further understanding of KC, we focused on patients who did not

carry a defined genetic mutation in any of the previously reported genes. Our group collected the pedigree of one pair of monozygotic twins. The twins had KC, but their parents did not. This

situation suggested that the inheritance pattern of this family was either autosomal recessive or de novo mutation. Twins are an excellent material for genetic studies of human traits and

diseases. This twin pedigree provides valuable samples and is an effective way to detect the pathogenic genes of KC. First, we excluded the reported candidate gene mutation by sequencing.

Then, we conducted exome sequencing on four core members of this family (the twins and their parents) to find a new candidate gene. Through whole-exome sequencing (WES), we identified a de

novo heterozygous nonsense mutation in a tubulin gene, tubulin alpha 3d _(TUBA3D)_. A screening of this gene in 200 patients with KC found additional two unrelated patients with different

mutations in the _TUBA3D_ gene. We further demonstrated that the mutant protein of _TUBA3D_ was unstable and could lead to human corneal fibroblast cells (HTK) performing higher matrix

metalloproteinases (MMPs) expression and oxidative stress. Our findings identify a new disease gene underlying KC and provide insight into _TUBA3D_ dysfunction in human corneal degeneration.

RESULTS CLINICAL FEATURES The proband and her twin sister, aged 23 years old, were born from an uneventful pregnancy. Seven years ago, the proband began vision loss with no cause. Five

months ago, her visual acuity decreased significantly. She came to the hospital for medical treatment and was diagnosed with bilateral KC. Both her eyes had corneal ectasia, thinning, and a

cone-shaped protrusion with Vogt’s striae and Fleischer’s ring. Signs of videokeratography showed the typical characteristics of KC (Fig. 1B). Clinical examination of her twin sister and

parents showed that her twin sister was a KC patient too, but their parents were not. The pedigree of this family is shown in Fig. 1A, and it suggests that the inheritance pattern of the

twins’ family was either autosomal recessive or de novo mutation. EXOME SEQUENCING DETECTS A NONSENSE VARIANT IN _TUBA3D_ To reveal the underlying genetic defect in this family, we initially

screened three reported candidate genes (_VSX1_, _TGFBI_, and _mir184_) using Sanger sequencing. No pathological mutation was found in any of these knows genes. Thus, we conducted WES on

four core members of this family (the twins and their parents) to find a new candidate gene. The average sequencing depth and coverage of our analysis were 62.85× and 99.96%, respectively.

With the data analysis and variants, as well as the filtering strategy described in the methods section, neither a gene with a homozygous variant nor a gene with a compound heterozygous

variant was identified. To detect the de novo variations, we compared the sequencing results from the twins to their parents, and one gene (_TUBA3D_) with de novo variants was identified.

Sanger sequencing was conducted to confirm the results obtained by WES. Finally, the de novo mutation (NM_080386.3: c.31 C > T, p.Gln11stop in _TUBA3D_) in the twins was confirmed by

Sanger sequencing (Fig. 1A). This mutation was a true de novo mutation as it was absent in her parents (Fig. 1A). The mutation was not found in the 200 unrelated health controls and absent

from the dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/), HapMap, 1000 Genomes (http://www.1000genomes.org), NHLBI Exome sequencing project databases, and ExAC database. This variant was

located in a highly conserved domain and led to a truncated protein (Fig. 2A). It was considered “disease causing” as predicted by Sorting Intolerant from Tolerant (SIFT)16 and Mutation

Taster (Table 1)17. These findings suggest that it is a candidate mutation. MUTATIONAL SCREENING IN ADDITIONAL KC PATIENTS To examine the mutation frequency of _TUBA3D_ in KC, we screened

200 unrelated sporadic KC patients. We identified two additional patients with KC who had heterozygous _TUBA3D_ mutations (Table 1). One mutation was c.201insTT (p.Val68Leufs*2), which is an

insertion mutation that changes the reading frame (i.e., grouping of the codons) and results in a completely different translation from the original. This mutation was predicted to alter

highly conserved amino acids among different species (Fig. 2A) and considered “disease causing” as predicted by Mutation Taster (Table 1). It was also not found in 200 unrelated health

controls and was absent from the dbSNP, HapMap, 1000 Genomes, and NHLBI Exome sequencing project databases. The other mutation was c.*2 G > A, which is located in the 3′UTR of _TUBA3D_,

and it was absent in the 200 unrelated health controls. To further identify the putative pathogenicity of these mutations, we computed the physico-chemical parameters and predicted the

tertiary structure of mutant and wildtype proteins. The physico-chemical parameter changes of the mutant protein are shown in Table 2. Compared with the wildtype, the mutant proteins have a

lesser number of amino acids, have a changed aliphatic index and hydropathicity, and are unstable (Table 2). The tertiary structures of mutant and wildtype proteins are illustrated in Fig. 

2B. As the c.31 C > T mutant protein sequence is too short, its tertiary structures cannot be predicted. These additional mutations of _TUBA3D_ in other patients and the predicted

changing of mutant proteins support the finding that _TUBA3D_ is a disease-causing gene. _TUBA3D_ EXPRESSION ANALYSIS IN HUMAN TISSUES To determine whether or not _TUBA3D_ was expressed in

the cornea or other tissues and cell lines, we examined _TUBA3D_ expression by PCR in human cornea, sclera, peripheral blood, human corneal epithelial cells (HCEC), and HTK. The agarose gel

result showed that _TUBA3D_ was expressed in all the above tissue and cell lines (Fig. 3A). Western blot analysis confirmed that _TUBA3D_ was expressed in both cornea and HTK (Fig. 3B). We

also examined the expressions of _TUBA3D_ and MMP genes _UPA_, _UPAR_, _MMP1_, and _MMP9_ in the blood samples of the twins and their parents. The results are shown in Fig. 3C. Compared with

the normal parents, the twins had lower _TUBA3D_ expression and higher _UPA_ and _MMP1_ expressions. THE C.31 C > T (GLN11STOP) AND C.201INSTT (VAL68LEUFS*2) MUTATIONS LEAD TO _TUBA3D_

DEGRADATION AND HIGHER MMPS EXPRESSION To examine whether or not the identified mutations affected _TUBA3D_ stability, we constructed plasmids with c.31 C > T (Gln11stop) and c.201insTT

(Val68Leufs*2) mutations expressed in HTK cells. Immunofluorescence (Supplementary Fig. S1) and Western blot (Fig. 4B) all revealed that wildtype-expressing cells detected the _TUBA3D_–HA

fusion protein expression, but the c.31 C > T (Gln11stop)- and c.201insTT (Val68Leufs*2)-expressing cells showed no signal of HA, which suggested that these mutations affected _TUBA3D_

stability and led to _TUBA3D_ degradation. To test whether or not the mutant proteins affected the MMPs, we examined the expressions of genes _UPA_, _UPAR_, _MMP1 MMP2_, _MMP3_, _MMP9_,

_MMP10_, _MMP12_, _MMP13_, _TIMP1_, and _TIMP2_ using quantitative real-time PCR (qRT-PCR) (Fig. 4A) and Western blot (Fig. 4B). The results showed that c.31 C > T (Gln11stop)- and

c.201insTT (Val68Leufs*2)-expressing cells had high _UPA_, _UPAR_, _MMP1_, _MMP3_, and _MMP13_ expressions. Therefore, we further examined the extracellular matrix (ECM) protein (including

Collagen I, IV, VI and Fibronectin) expression levels in HTK cells after transfecting with mutant _TUBA3D_. The results showed that Collagen IV, VI and Fibronectin protein levels drop

slightly in c.31 C > T (Gln11stop)- and c.201insTT (Val68Leufs*2)-expressing cells compared with wildtype cells (Fig. 5). These results indicated that the mutant proteins of _TUBA3D_

could lead to HTK cells performing higher MMP expressions and affect the extracellular matrix. However, there was no significant change in cell morphology after transfecting with mutant

_TUBA3D_ (Fig. 6A). THE C.31 C > T (GLN11STOP) AND C.201INSTT (VAL68LEUFS*2) MUTATIONS LEAD TO HTK CELLS PERFORMING HIGHER OXIDATIVE STRESS To examine whether or not the identified

mutations affect the oxidative stress of HTK cells, we measured the reactive oxygen species (ROS) and SOD1, SOD2 expression levels of the wildtype, c.31 C > T (Gln11stop), and c.201insTT

(Val68Leufs*2) mutation cells. The results showed that c.31 C > T (Gln11stop) and c.201insTT (Val68Leufs*2) mutation cells had significantly higher ROS levels (Fig. 6B), lower SOD1 

expression levels (Fig. 6C) than the wildtype cells. We also measured the cell survival of the HTK cells post transfection with and without antioxidant NAC (N-acetyl-L-cysteine). The

relative cell survival rate of HTK cells were decreased after transfecting with mutant _TUBA3D_, and were recovered at 48 h, 72 h by adding NAC (Fig. 6D). These results suggested that the

mutant proteins of _TUBA3D_ could result in the decrease of SOD1 expression and lead to HTK cells performing higher oxidative stress, and affect cell survival. DISCUSSION In this study,

using exome sequencing of a twin pedigree and directly screening 200 unrelated patients with KC, we identified _TUBA3D_ as a novel gene linked to KC and found that it accounted for 1% of KC

cases. To date, our functional studies provide initial evidence of the association of this gene with KC. _TUBA3D_ is located on chromosome 2q21.1, encodes a member of the alpha tubulin

family, which is a major component of microtubules, and maintains cellular structure and function in intracellular transport18. This gene is highly expressed in normal human cornea, as

indicated by qRT-PCR analysis, which suggests that _TUBA3D_ could have an important role in the maintenance of corneal structure and function. In this study, a de novo heterozygous mutation

in the _TUBA3D_ gene (c.31 C > T) was identified in the proband patient and her twin sister. By screening additional sporadic patients with KC, we identified two unique heterozygous

mutations in _TUBA3D_ (c.201insTT and c.*2 G > A) in two unrelated patients. None of these mutations was found in the 200 Chinese healthy controls. The mutations c.31 C > T and

c.201insTT were novel based on their absence in all the databases. They were predicted to alter highly conserved amino acids among different species and considered “disease causing” as

predicted by Mutation Taster. These mutations were also predicted to change the physico-chemical parameters and the tertiary structure of protein tremendously. These findings support

_TUBA3D_ as a disease-causing gene of KC. Proteolytic phenomena contribute to the pathogenesis of KC, and MMPs were reported to be overexpressed in KC19,20,21. The identification of _TUBA3D_

as a KC-causing gene raises the intriguing question of how mutations in a cytoskeleton gene that is expressed in the cornea could lead to a cornea-specific phenotype. _TUBA3D_ encodes a

cytoskeleton gene that has not been reported to be related to any other disease. To investigate the pathogenic mechanism of _TUBA3D_, we conducted function analysis through constructed

plasmids with c.31 C > T (Gln11stop) and c.201insTT (Val68Leufs*2) mutations and expressed them in HTK cells. Our results showed that these mutations could affect _TUBA3D_ stability and

lead to _TUBA3D_ degradation. Moreover, c.31 C > T (Gln11stop)- and c.201insTT (Val68Leufs*2)-expressing cells had higher MMPs _(UPA_, _UPAR_, _MMP1_, _MMP3_, _MMP13)_ expressions and ROS

levels than the wildtype cells. _TUBA3D_ is a major component of microtubules, and it maintains cellular structure and function in intracellular transport18. Mutation of this gene could

lead to protein degradation, which could affect the normal function of microtubules. Conversely, overexpressed MMPs could lead to proteolytic phenomena, which are a well-known pathogenesis

of KC19. Oxidative stress was also reported to be involved in the pathogenesis of KC22,23. In summary, mutations of _TUBA3D_ could lead to protein degradation, MMPs overexpression, and high

oxidative stress, thus reducing the amount of extracellular matrices within corneas. These changes undoubtedly play a major role in stromal thinning and Bowman’s layer/basement membrane

breaks, which are characteristic of KC corneas. To the best of our knowledge, this study is the first to identify _TUBA3D_ mutations in cases of KC. Our results indicate that the mutation

frequency of _TUBA3D_ in the population of Chinese patients with KC is 1.0% (two independent cases among 200). Our functional characterization also supports the hypothesis that _TUBA3D_ is a

new causative gene of KC and provides new insights into the molecular mechanisms underlying KC. Therefore, the mutational screening of _TUBA3D_ should be considered for patients with KC to

ensure proper molecular diagnosis. METHODS SUBJECT RECRUITMENT AND CLINICAL EXAMINATION The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics

Committee of Shandong Eye Institute (Qingdao, China). Written informed consent was obtained from all participants (or guardians). Patients diagnosed with KC were recruited from Qingdao Eye

Hospital, Shandong Eye Institute (Qingdao, China). The diagnosis of KC was based on clinical examination (corneal stromal thinning, Vogt’s striae, Fleischer’s ring, Munson’s sign, signs of

videokeratography, and refractive errors). In total, the family of one pair of monozygotic twins and 200 sporadic KC patients were collected. The pedigree (Fig. 1A) suggested that the

inheritance pattern of these twins’ family was either autosomal recessive or de novo mutation. Two hundred unrelated healthy individuals of Chinese origin were used as control. Peripheral

blood samples from all participating individuals were collected in EDTA tubes. Genomic DNA was extracted with Blood DNA Kit (Tiangen Biotech Co., Beijing, China). WES AND DATA ANALYSIS We

conducted WES in all the members of this family (the twins and their parents) at Novogene (Beijing, China) to identify the causal gene. The SureSelect Human All ExonV5 Kit (Agilent

Technologies, USA) was used for exome capture. The IlluminaHiseq. 2500 platform (Illumina Inc., San Diego, CA, USA) was employed for the genomic DNA sequencing of the twins and their

parents. The sequencing reads were mapped to the reference genome (UCSC hg19) using the Burrows–Wheeler Aligner software24. Samtools25 and Picard (http://broadinstitute.github.io/picard)

were utilized to sort bam files and conduct duplicate marking to generate a final bam file, respectively. Samtools mpileup and bcftools were used to perform variant calling and to identify

SNP/indels. ANNOVAR26 was used to conduct annotation. Variants were common (>0.5%) in dbSNP, HapMap, and the 1000 Genomes Project were filtered. Variants that were not present in any of

the above databases were considered novel. Only SNPs and indels occurring in exons or located in canonical splicing sites were further analyzed, and nonsynonymous SNPs were submitted to

SIFT16 and Polymorphism Phenotyping version 2 (PolyPhen-2)27, Mutation Taster17, and CADD28 for functional prediction. Among the four software programs, two showed that the variant was not

benign and could be retained. Given the characteristics of the pedigree, homozygous, compound heterozygous, or de novo variations were considered to be candidate causal variations29.

_TUBA3D_ SEQUENCING AND GENOTYPING Sanger sequencing was performed to confirm and segregate the obtained results through whole-exome sequencing and to screen the _TUBA3D_ gene in 200

unrelated sporadic KC patients. Primers were designed to amplify all five coding fragments and the intron–exon boundaries of the _TUBA3D_ gene (NM_080386.3, see Supplementary Table S1).

Every target fragment was amplified using Taq DNA polymerase (Takara, Dalian, China). The products were purified with alkaline phosphatase (Shrimp) (Takara, Dalian, China) and exonuclease I

(Takara, Dalian, China), and subjected to direct DNA sequencing using the BigDye™ Terminator v3.1 Cycle Sequencing kit and ABI PRISM 3730 sequencer (Applied Biosystems Inc., USA). Sequences

were aligned and analyzed using the DNASTAR software package (DNASTAR Inc., USA). The novel mutations of _TUBA3D_ were also genotyped in 200 unrelated healthy controls using high-resolution

melt (HRM) analysis or Sanger sequencing. The primers used for genotyping the mutations of _TUBA3D_ are shown in Supplementary Table S2. For HRM, the amplification assays were performed

using the Type-it HRM PCR Kit (QIAGEN, Germany) on the Rotor-Gene Q Real-Time PCR system (QIAGEN, Germany). The results were obtained and analyzed using the Rotor-Gene Q Series Software

(version 2.1.0). The Sanger sequencing method is the same as the above description. PROTEIN STRUCTURE AND FUNCTION PREDICTION Multiple protein sequence alignment among various species was

carried out by MEGA software30. To further identify the putative pathogenicity of the variants, MutationTaster (http://www.mutationtaster.org/) was applied to test the possible effect of

amino acid substitution on protein function31. The physico-chemical parameters of mutant and wild-type proteins were computed by ProtParam tool (http://web.expasy.org/protparam/). The

tertiary structure of mutant proteins was predicted by the Swiss-Model workspace (http://swissmodel.expasy.org)32. REVERSE TRANSCRIPTION-PCR Total RNA was prepared from venous blood (1 ml)

of the family members using the RNA isolation kit for mammalian blood (Tiangen, Beijing, China). Total RNA was prepared from normal human corneas and cell samples using the NucleoSpin RNA II

System (Macherey-Nagel, Duren, Germany). cDNA was synthesized from RNA using a commercial kit (PrimeScriptTM RT Reagent Kit (Perfect Real Time); Takara, Dalian, China). Expressions of the

_TUBA3D_ gene and the MMP genes (urokinase-type plasminogen activator, _UPA_; urokinase-type plasminogen activator receptor, _UPAR_; matrix metallopeptidase 1, _MMP1_; matrix

metallopeptidase 2, _MMP2_; matrix metallopeptidase 3, _MMP3_; matrix metallopeptidase 9, _MMP9_; matrix metallopeptidase 10, _MMP10_; matrix metallopeptidase 12, _MMP12_; matrix

metallopeptidase 13, _MMP13_; TIMP metallopeptidase inhibitor 1, _TIMP1_; and TIMP metallopeptidase inhibitor 2, _TIMP2_) were measured by qRT-PCR and normalized to

glyceraldehyde-3-phosphate dehydrogenase (_GAPDH_). Primer sequences of the genes used for qRT-PCR are shown in Supplementary Table S3. _TUBA3D_–HA FUSION CONSTRUCTS AND EXPRESSION IN HUMAN

CORNEAL FIBROBLAST CELLS A telomerase-infected, extended-lifespan human corneal fibroblast cell line (HTK) were cultured in DMEM/F12 medium (Corning, USA) containing 10% fetal bovine serum

(Gibco, USA) at 37 °C with 5% CO2. The wildtype, c.31 C > T (p.Glu11Stop), and c.201insTT (p.Val68Leufs*2) CDS sequences of the _TUBA3D_ gene with the hemagglutinin (HA) tag at the

C-terminal were synthesized directly and subcloned into the pcDNA3.1( + ) expression vector (Invitrogen, USA), respectively. The expression vectors were transfected in six-well plates using

the Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s protocol. After transfection, the cells were collected at 72 or 48 hours to measure the mRNA/protein levels

of HA, _TUBA3D_, MMPs and ECM proteins, or the ROS and related genes expression levels respectively. IMMUNOFLUORESCENCE STAINING AND FLOW CYTOMETRY ANALYSIS For immunofluorescence staining,

the cells were fixed in 4% paraformaldehyde in phosphate-buffered solution (PBS) for 15 min, followed by three PBS washes. The fixed cells were incubated with 0.1% Triton X-100 in PBS for 5 

min, blocked in 5% BSA, and incubated with HA probe antibody (sc-805, Santa Cruz Biotechnology) overnight before incubation with the fluorescence-conjugated secondary antibody (Invitrogen,

USA) for 1 hour. Images were obtained using an Eclipse TE2000-U confocal laser scanning microscope (Nikon, Tokyo, Japan) after counterstaining with 4′,6-diamidino-2-phenylindole. The ROS

levels were measured using a Reactive Oxygen Species Assay Kit (Beyotime, China). Flow cytometry analysis was performed according to the manufacturer’s protocols, and the results were

obtained. Cell survival of the HTK cells post transfection with and without antioxidant (NAC, 0.1 mM, Sigma-Aldrich) was measured using WST-1 Cell Proliferation and Cytotoxicity Assay Kit

(Beyotime, China). WESTERN BLOT Total protein was prepared from each group using radioimmunoprecipitation assay (RIPA) buffer (Galen, Beijing, China) and quantified. Western blot analyses

were performed as we described previously33. For each sample, the levels of proteins of interest were normalized to that of GAPDH. Primary antibodies included HA probe (sc-805, Santa Cruz

Biotechnology), a3C Tubulin antibody (sc-134240, Santa Cruz Biotechnology), UPA antibody (ab169754, Abcam), UPAR antibody (ab103791, Abcam), MMP1 antibody (ab52631, Abcam), MMP3 antibody

(ab52915, Abcam), MMP13 antibody (ab39012, Abcam), superoxide dismutase 1(SOD1) antibody (MABC684, Merck-Millipore), superoxide dismutase 2 (SOD2) antibody (ab68155, Abcam), collagen I

antibody (ab138492, Abcam), collagen IV antibody (ab6598, Abcam), collagen VI antibody (ab182744, Abcam), fibronectin antibody (ab32419, Abcam) and anti-GAPDH antibody (KC-5G5, Kangchen,

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Vis. Sci._ 55, 8031–8043 (2014). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We are greatly indebted to the people who participated in this research. This

work was supported by the National Natural Science Foundation of China (grant numbers 81370989, 81570821, and 81500763); National Basic Research Program of China (grant number 2013CB967004);

Taishan Top Scholarship Program (grant number 20081148); Shandong Provincial Excellent Innovation Team Program; and Young and Middle-aged Scientists Research Awards Fund of Shangdong

Province (grant number BS2015YY014). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye

Institute, Shandong Academy of Medical Sciences, Qingdao, 266071, China Xiao-dan Hao, Peng Chen, Yang-yang Zhang, Wei-yun Shi & Hua Gao * Shandong Eye Hospital, Shandong Eye Institute,

Shandong Academy of Medical Sciences, Jinan, 250021, China Su-xia Li, Wei-yun Shi & Hua Gao Authors * Xiao-dan Hao View author publications You can also search for this author inPubMed 

Google Scholar * Peng Chen View author publications You can also search for this author inPubMed Google Scholar * Yang-yang Zhang View author publications You can also search for this author

inPubMed Google Scholar * Su-xia Li View author publications You can also search for this author inPubMed Google Scholar * Wei-yun Shi View author publications You can also search for this

author inPubMed Google Scholar * Hua Gao View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS X.D.H. and H.G. designed the research. X.D.H.,

P.C. and Y.Y.Z. performed the experiments. X.D.H., P.C. and S.X.L. analyzed the data and participated in the discussion. X.D.H. and H.G. wrote and revised the paper. W.Y.S. analyzed the data

and participated in the revision of the paper. CORRESPONDING AUTHOR Correspondence to Hua Gao. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare that they have no competing

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Hao, Xd., Chen, P., Zhang, Yy. _et al._ De novo mutations of _TUBA3D_ are associated with keratoconus. _Sci Rep_ 7, 13570 (2017).

https://doi.org/10.1038/s41598-017-13162-0 Download citation * Received: 21 April 2017 * Accepted: 19 September 2017 * Published: 19 October 2017 * DOI:

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