Article Text

Trio-based whole-exome sequencing reveals mutations in early-onset high myopia
  1. Lu Ye1,
  2. Yi-Ming Guo1,
  3. Yi-Xin Cai2,
  4. Junhan Wei1,
  5. Juan Huang1,
  6. Jiejing Bi1,
  7. Ding Chen3,4,
  8. Fen-Fen Li3,4,
  9. Xiu-Feng Huang2
  1. 1Shaanxi Eye Hospital, Xi’an People’s Hospital (Xi'an Fourth Hospital), Affiliated People’s Hospital of Northwest University, Xi'an, Shaanxi, China
  2. 2Zhejiang Provincial Clinical Research Center for Pediatric Disease, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
  3. 3National Clinical Research Center for Ocular Diseases, Wenzhou Medical University Eye Hospital, Wenzhou, Zhejiang, China
  4. 4State Key Laboratory of Ophthalmology Optometry and Visual Science, Wenzhou Medical University Eye Hospital, Wenzhou, Zhejiang, China
  1. Correspondence to Dr Xiu-Feng Huang; hxfwzmc{at}


Purpose Myopia, especially high myopia (HM), represents a widespread visual impairment with a globally escalating prevalence. This study aimed to elucidate the genetic foundations associated with early-onset HM (eoHM) while delineating the genetic landscape specific to Shaanxi province, China.

Methods A comprehensive analysis of whole-exome sequencing was conducted involving 26 familial trios displaying eoHM. An exacting filtration protocol identified potential candidate mutations within acknowledged myopia-related genes and susceptibility loci. Subsequently, computational methodologies were employed for functional annotations and pathogenicity assessments.

Results Our investigation identified 7 genes and 10 variants associated with HM across 7 families, including a novel mutation in the ARR3 gene (c.139C>T, p.Arg47*) and two mutations in the P3H2 gene (c.1865T>C, p.Phe622Ser and c.212T>C, p.Leu71Pro). Pathogenic mutations were found in syndromic myopia genes, notably encompassing VPS13B, TRPM1, RPGR, NYX and RP2. Additionally, a thorough comparison of previously reported causative genes of syndromic myopia and myopia risk genes with the negative sequencing results pinpointed various types of mutations within risk genes.

Conclusions This investigation into eoHM within Shaanxi province adds to the current understanding of myopic genetic factors. Our results warrant further functional validation and ocular examinations, yet they provide foundational insights for future genetic research and therapeutic innovations in HM.

  • Genetics
  • Optics and Refraction

Data availability statement

No data are available.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See:

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.


  • Prior genetic studies have explored the genetic underpinnings of early-onset high myopia (eoHM) in the Chinese population. Yet, the variable genetic landscape and demographic composition across China’s provinces, including unexplored regions like Shaanxi, complicate these genetic explorations.


  • Our investigation identified 7 genes and 10 variants associated with HM across 7 families, including a novel mutation in the ARR3 gene and 2 mutations in the P3H2 gene.


  • Our study’s meticulous examination of eoHM families from the Shaanxi province addresses a crucial gap in myopia genetic research in this specific region.


Myopia is a leading cause of global visual impairment, with its prevalence rapidly increasing worldwide, especially in East Asia.1 High myopia (HM), an extreme form of myopia characterised by a refractive error of ≤−6 dioptre (D) (or axial length (AL) ≥26 mm), signifies a critical aspect within the myopia spectrum.2 In 2000, around 163 million individuals were diagnosed with HM, accounting for 2.7% of the global population, and forecasts suggest a staggering climb to nearly 1 billion people with HM by 2050.3 Moreover, individuals with HM face escalated risks of developing additional ocular complications, such as cataracts, glaucoma, retinal detachment and chorioretinal degeneration, positioning HM as a primary driver of irreversible blindness.4 5

Genetic studies on HM, especially early-onset HM (eoHM), offer a crucial approach for unravelling its genetic mechanisms. Although almost 200 genetic loci have been found to be associated with myopia by genome-wide association study, those risk variants mostly carry low risk.6 Importantly, a subset of genes for secondary syndromic myopia overlaps with those for common myopia.6 Notably, whole-exome sequencing (WES) has proven powerful to identify rare variants. The application of WES in HM or eoHM has led to the identification of several genes associated with myopia. These genes encompass various categories: autosomal dominant (AD) genes such as ZNF644,7 SCO2,8 SLC39A5,9 P4HA2,10 BSG11 and CCDC11112; autosomal recessive (AR) genes such as P3H2,13 LRPAP114 and CTSH15; and X-linked genes namely OPN1LW16 and ARR3.17 Such genetic insights offer valuable pathways toward comprehending the genetic landscape of myopia, particularly HM and its early onset.

In the context of its heterogeneous aetiology, it is valuable to elucidate the contribution of the reported myopia-associated genes in eoHM cases. Several genetic studies have investigated the genetic basis of eoHM in the Chinese population. For example, Liu et al performed WES in 67 Tujia Chinese patients with eoHM, pinpointing mutations in three genes (ARR3, SLC39A5 and NDUFAF7).18 Another study recruited 27 eoHM families from the Ningxia Hui Autonomous Region, uncovering mutations in four genes (CSMD1, PARP8, ADAMTSL1 and FNDC3B).19 Wang et al investigated 14 eoHM genes in a sizeable cohort and revealed ARR3 as the most common cause of Mendelian eoHM.20 However, current genetic research on myopia across various provinces in China is insufficient, particularly in Shaanxi province, which is known for its high incidence of the condition (54.9% prevalence of myopia).21 Compared with other regions in China, the complex geographical, cultural environment and demographic composition of Shaanxi pose significant challenges yet also demand a more profound level of research into myopia.22 Especially in Xi'an, the capital city of Shaanxi province, the whole city-level prevalence of total myopia was 57.1% and the figure for HM was 1.0%.21

In the current study, we recruited 26 familial trios with eoHM from Shaanxi province. Employing a blend of WES and comprehensive genetic analysis, we aim to decipher the genetic predisposition of eoHM from Shaanxi province. Additionally, we conducted a genotype–phenotype correlation analysis, aimed at furnishing insights into the mutation spectrum and the clinical characteristics of eoHM patients in Shaanxi province.

Materials and methods

Study subjects

This study comprised a cohort of 26 families in the Shaanxi province. Peripheral blood samples were collected from all patients and their parents.

An overview of the recorded refractive error and ocular AL measurements for each patient within the familial cohorts is summarised in table 1. To augment the visual understanding, online supplemental figure 1 provides a consolidated pedigree depiction coupled with pertinent clinical particulars for all 26 families. The recruitment of patients adhered to specific inclusion criteria: (1) an age not exceeding 6 years; (2) manifestation of a refractive error equal to or exceeding −6 D and/or an AL surpassing 26.00 mm and (3) the absence of any documented ocular pathologies or systemic ailments apart from HM. This stringent set of criteria was meticulously devised to ensure the enrolment of participants that accurately represented cases of eoHM, minimising any confounding variables that could potentially impede the genetic predisposition analysis.

Table 1

Clinical data for 26 probands with eoHM

Whole-exome sequencing

The WES process was carried out using the Exome Enrichment Agilent SureSelectXT Human All Exon V6 Kit (Agilent Technologies, USA), adhering to well-established protocols. DNA fragments underwent sequencing on Illumina HiSeq 6000 Analyzers, and Illumina libraries were methodically generated through the Hiseq6000 sequencing platform, all in strict accordance with the manufacturer’s stipulations. To ensure the integrity of the data, a combination of local realignments, quality control measures and variant calling was meticulously performed employing the Genome Analysis Toolkit. Sequencing reads were precisely aligned to the human reference genome (hg19) via the BWA-MEM software. The culmination of WES yielded an average read depth of approximately 100X, thus robustly guaranteeing extensive coverage.

Variant filtering

To identify potential candidate variants associated with HM, we meticulously executed a stringent filtering strategy. This strategy places a premium on rare variants situated within genes with established implications in ocular diseases and HM, as visually depicted in online supplemental figure 1. Synonymous variants were preemptively excluded; Variants boasting a global minor allele frequency surpassing 0.02 across datasets, such as the Genome Aggregation Database (gnomAD), were omitted from further analysis. Variants not demonstrating heterozygosity within all AD families were removed. Similarly, variants lacking homozygosity within all AR families were also discarded. Cosegregation analysis was conducted meticulously to examine the inheritance patterns of these variants throughout all family members. For missense mutations, pathogenicity was imputed if they were projected to be pathogenic by no fewer than three of the five computational methods encompassing SIFT, PolyPhen-2-HVAR, Mutation Taster, CADD and REVEL. For splicing mutations, multiple tools were employed to evaluate their impact and potential significance.

Patient and public involvement

Patients diagnosed with eoHM, along with their immediate family members, were actively engaged in this research endeavour. The participation of these patients encompassed the donation of peripheral blood specimens, which facilitated genetic analysis through WES to procure familial genetic data. Furthermore, comprehensive clinical data, including ophthalmic parameters such as visual acuity, AL and fundus photography, were meticulously collected from the probands. This multidimensional involvement was integral to the acquisition and interpretation of both genetic and clinical data, thereby enriching the depth and breadth of our investigative efforts.


In our current investigation, we included a cohort comprising 26 families with eoHM. Within this cohort, a total of 16 females (61.5%) and 10 males (38.5%) were represented (table 1). The average age at which HM was diagnosed stood at 2.67±1.30 years (mean±SD), whereas the mean age of examination was calculated as 4.08±1.55 years. Remarkably, a substantial majority of probands, accounting for 24 individuals (92.3%), were below the age of seven at the time of examination. Analysis of the probands’ eyes revealed an average spherical equivalent (SE) of −8.96±3.13 dioptres (D) in the right eyes and −8.23±4.13 D in the left eyes. Within this cohort, 17 patients (65.4%) showcased HM in both eyes, whereas the remaining subjects (34.6%) manifested HM in a solitary eye. Furthermore, premature ocular development was observed in patients with eoHM, substantiated by the mean AL of 25.2±1.64 mm in the right eyes and 25.5±1.34 mm in the left eyes. Fundus photography revealed characteristic retinal indications associated with HM, including a leopard-print fundus, retinal vascular attenuation and peripheral degenerative zones.

A novel mutation (c.139C>T, p.Arg47*) was identified in the non-syndromic high-myopia gene, ARR3. Prior research has elucidated that this gene adheres to an unconventional X-linked inheritance pattern, termed X-linked female-limited.23 According to this pattern, female family members carrying heterozygous mutations manifest the phenotype while hemizygous male family members do not. In the current family, the father, a hemizygous individual, remains unaffected by HM, whereas the heterozygous proband (the daughter) presents the corresponding phenotype. It is noteworthy that the proband’s grandmother also exhibits HM. These observations provide compelling evidence supporting the associations between the identified ARR3 mutation and the observed high-myopia phenotype, affirming its role within this familial context. Furthermore, our investigation has unveiled pathogenic mutations, specifically c.1865T>C (p.Phe622Ser) and c.212T>C (p.Leu71Pro), in the P3H2 gene (formerly known as LEPREL1). These mutations, carried separately by parents, manifest eoHM when passed on to offspring.

Furthermore, we detected pathogenic mutations in six syndromic myopia genes, including VPS13B, TRPM1, RPGR, NYX and RP2 in six subjects (table 2). Figure 1 lucidly portrays the clinical parameters aligned with myopia in probands. An overarching pattern emerges among these individuals, characterised by an early-onset age, accelerated AL elongation, elevated myopic spherical refractive error, and predominantly synchronous bilateral initiation (online supplemental table 1). These mutations are established precursors of syndromic myopia conditions, such as HM accompanied by cataract and vitreoretinal degeneration (MIM:614292) and congenital stationary night blindness type 1C (MIM:613216), where HM serves as an initial manifestation. Figure 2 provides a visual representation via Sanger validation of these mutations.

Table 2

Identification of one non-syndromic myopia and six syndromic variants in this study

Figure 1

Clinical parameters associated with myopia in the sequencing proband of the positive group. AL, axial length; K1, flat meridian; K2, steep meridian; SE, spherical equivalent.

Figure 2

Identification of one non-syndromic myopia and six syndromic variants in this study. ‘M’ denotes mutation. Left to right: Sequences from affected individual with identified mutation, sequences from unaffected control, pedigree plots of mutations.


Myopia stands as a pivotal focus in ophthalmic research, especially in the realm of HM, which has garnered significant attention due to its escalating prevalence. The emergence of HM is a result of a complex interplay between environmental influences and genetic factors.24 Present investigations underscore strategies aimed at curbing myopia prevalence, such as reducing screen time and encouraging outdoor activities, as potential measures to mitigate its progression.25 Numerous genetic studies have been conducted to understand the genetic underpinnings of myopia, including molecular genetic studies, family aggregation studies and twins studies.26 These investigations have uncovered a substantial genetic predisposition to HM, identifying numerous pathogenic genes or susceptibility loci strongly associated with its development.

In contrast to proband-centred WES, trios-based WES offers a more comprehensive approach. This method not only enables the identification of gene variants and the in-depth assessment of their pathogenicity but also facilitates the exploration of gene variants based on the principles of cogenetic segregation. This approach enhances the establishment of a robust gene–phenotype relationship mapping network and allows for a more thorough investigation into the inheritance patterns of mutated genes. Besides, given the limited genetic research targeting myopia and the imperative for myopia research within the distinct population of Shaanxi province, we have enrolled a cohort of 26 individuals from the Shaanxi region, characterised by eoHM diagnoses predating school age in our study. By employing a trios-based WES approach within this cohort, we aim to delve deeper into the genetic landscape of eoHM, elucidating the interplay between genetic variants and the manifestation of this condition within a familial context.

In our study, we identified a novel missense mutation (c.139C>T: p.Arg47*) in the ARR3 gene of the proband. It is intriguing to observe that the father of the proband, a visually normative individual, carries a hemizygous mutation in the ARR3 gene while the proband’s grandmother is afflicted by eoHM. This constellation of findings bolsters the hypothesis of an X-linked female-limited inheritance pattern in this specific case. ARR3 exhibits a unique expression pattern,27 28 primarily confined to the retina, is crucial for modulating retina-specific signalling mechanisms. Its role in desensitising photoactivated G protein-coupled receptors, with a distinct preference for opsins.29 30 Pathogenic mutations in ARR3 have been inextricably linked with myopia, a condition distinguished by X-linked female-limited inheritance. ARR3 holds a unique position as the second identified instance of an X-linked female-restricted ailment.17 Within a diagnostic WES cohort, the frequency of pathogenic variants in the ARR3 gene has risen to 5%, making it a relatively common trigger for HM.31 Furthermore, when focusing on eoHM, ARR3 stands out as the most frequently implicated gene, accounting for approximately 3.1% of Mendelian eoHM cases.20

The P3H2 gene, also known as LEPREL1, encodes prolyl 3-hydroxylase 2 (P3H2), an essential 2-oxoglutarate-dependent dioxygenase responsible for catalysing the hydroxylation of collagens.32 Prolyl hydroxylation represents a pivotal post-translational modification process primarily associated with fibril-forming collagens, including collagens I, II, IV and V.33 Consequently, the inactivation of P3H2 due to impaired collagen hydroxylation is likely the underlying mechanism for zonular instability, cataract development and an increased susceptibility to retinal tears and detachment in individuals harbouring recessive LEPREL1 mutations.34 Worth noting is the substantial eye enlargement resulting from the disruption of the inner limiting membrane in chick embryos, suggesting that recessive LEPREL1 mutations causing defects in the inner limiting membrane may contribute to paediatric HM.35

P3H2 exhibits a broad expression across diverse ocular structures, encompassing the iris, lens and trabecular meshwork.36 Notably, mutations within the P3H2 gene have been linked to the development of non-syndromic severe myopia, accompanied by the onset of cataracts and vitreoretinal degeneration.34 In our research, we detected previously unreported mutations in P3H2 (c.1865T>C:p.Phe622Ser and c.212T>C:p.Leu71Pro) that were inherited from carrier parents and culminated in the onset of HM in the offspring. These novel mutations require comprehensive investigation to elucidate their functional implications.

Furthermore, our exploration led to the identification of several genetic mutations relevant to syndromic HM, such as the c.2405_2406delAG mutation in the RPGR gene. This detected mutation in the RPGR gene is particularly noteworthy for its location within exon ORF15, a region known for its high recurrence of hotspot mutations.37 Remarkably, it has been postulated that mutations clustered towards the 3' terminus of exon ORF15 in RPGR are frequently associated with cone-rod degeneration.38 Within the context of a specific family, the uncle, on positive gene sequencing, showcased a complete absence of light perception and typified retinitis pigmentosa on fundus evaluation. These genetic screening results offer valuable insights that could lead to the development of preventive measures and early preparedness for prospective fundus pathology in children grappling with this ailment.

Given that the emergence of HM predominantly materialises through the interplay of polygenic microvariations coupled with environmental influences, the cumulative impact of these polygenic microvariations plays a pivotal role in the onset of HM. Our study also includes an examination of documented mutations in HM risk genes, encompassing TTN, RBM25, MST1 and ANGPT1. These genes hold pivotal roles in steering ocular AL elongation, modulating scleral thickness, influencing choroidal blood flow and orchestrating other factors that are germane to the onset of HM. The identification of pathogenic mutations in these genes promises to significantly enrich our understanding of the pathogenesis of HM.19

Crucially, our study is uniquely focused on eoHM families originating from the densely populated Shaanxi province, characterised by a notably high prevalence of HM prevalence in the northwest region. Hitherto, the landscape has been marked by a dearth of holistic genetic studies dedicated to myopia within this specific region. Ergo, our study assumes a vanguard position in charting the genetic spectrum of HM germane to the Shaanxi population, thereby bridging a pivotal gap in our fund of knowledge. Furthermore, the scarcity of research on eoHM in the Chinese populations, especially comprehensive WES analysis within eoHM pedigrees like ours, underscores the novelty and importance of your research. We expect that our research findings will substantially enrich the current body of knowledge in myopia research, providing valuable insights that can guide future studies and inform policy decisions in the field of myopia.

Despite the significant strides achieved by our study in elucidating the genetic foundations of eoHM within Shaanxi population, several challenges and limitations persist. Our sample size, while informative, remains relatively small, potentially limiting our capacity to detect low frequency or rare variants. To enhance the depth of our findings and bolster statistical robustness, future studies should aim to expand participant numbers. Furthermore, while WES effectively captures coding regions, it is crucial to acknowledge the potential influence of non-coding regions on eoHM. Broader sequencing coverage encompassing these non-coding areas is essential for a more comprehensive understanding of the genetic landscape contributing to eoHM. Moreover, additional functional investigations and ex vivo experimental validations are indispensable to corroborate the myriad HM risk genes spotlighted in our study. We need to further demonstrate the correlation between these gene mutations and changes in clinical parameters.

In summary, our study’s meticulous examination of eoHM families from the Shaanxi province addresses a crucial gap in myopia genetic research in this specific region. By consolidating and integrating our findings, we aspire to provide a valuable reference for genetic counselling and personalised treatment approaches.

Data availability statement

No data are available.

Ethics statements

Patient consent for publication

Ethics approval

This study involves human participants and was approved by Ethics Committee of Xi’an People’s Hospital (Xi’an Fourth Hospital): No.: 20230011. Participants gave informed consent to participate in the study before taking part.


Supplementary material

  • Supplementary Data

    This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.


  • LY and Y-MG contributed equally.

  • Contributors L,Y designed, funded and supported the article and wrote the initial manuscript; Y-M,G participated in the writing and subsequent proofreading of the original manuscript and participated in the collection, analysis and processing of the data; Y-X,C contributed to the graphical visualisation and writing of the project, and J,W, J,H and JJ,B participated in the data collection, processing and manuscript proofreading of the project. D,C and F-F,L helped with the writing and graphical visualisation of the article and assisted in coordinating the entire process. X-F,H was involved in the design and overall planning of the project and controlled the entire process, evaluating and revising the final manuscript writing and proofreading.

    Besides, L,Y and X-F,H is responsible for the overall content as guarantor.

  • Funding This work was supported by Xi'an Medical Research-Discipline Capacity Building Project (23YXYJ0002), Key R&D Plan of Shaanxi Province: Key Industrial Innovation Chain (Cluster)—Social Development Field (No.2022ZDLSF03-10), Natural Science Foundation Project of Zhejiang Province (LWY20H120001), Medical and Health Project of Major Scientific and Technological Innovation of Wenzhou City (ZY2019012) and National Natural Science Foundation of China (82201229).

  • Competing interests None declared.

  • Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.