The cohort

We used data from children referred to the tertiary pediatric ophthalmology center at University Eye Clinic in Ljubljana between 2010 and 2022 who met the following inclusion criteria: age up to and including 15 years and HM equal to or exceeding SE -5.0 D before the age of 10 years. We excluded children who were highly likely to have developed HM as a result of another eye condition, e.g., retinopathy of prematurity, congenital glaucoma, secondary glaucoma after early surgery for congenital cataract, other types of secondary glaucoma, and exacerbation or development of HM as a complication of surgical treatment of other ocular pathology. Children with HM who underwent genetic analysis as part of segregation analysis but were not the probands were excluded. A flowchart of children who participated in the study is presented in Fig 1.

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Fig 1. The flowchart outlining participant enrollment, exclusion, and attrition.

https://doi.org/10.1371/journal.pone.0313121.g001

Written informed consent was obtained from the parents of all participating children. The research adhered to the tenets of the Declaration of Helsinki and was approved by the Republic of Slovenia National Medical Ethics Committee (case number 0120-284/2022/3).

Genetic testing

The results of the genetic tests were reviewed at the Clinical Institute of Genomic Medicine (CIGM) and the Genetic Clinic of the Children’s Clinic in Ljubljana. Patients with non-syndromic high myopia (HM) underwent exome sequencing (ES). In syndromic HM cases, molecular karyotyping was performed first, with ES used subsequently if no causative mutation was identified. All genetic testing was performed as part of routine diagnostics. The DNA was isolated from peripheral blood samples using the Qiagen Mini kit (Qiagen, Valencia, CA). Microarray analysis was performed using the oligonucleotide array Agilent Technologies 4 × 180 K, according to the manufacturer’s instructions. The array images were acquired using the Agilent laser scanner G2565CA. The image files were quantified using the Agilent Feature extraction software and analyzed with the Agilent Cytogenomics 5.3 software (Agilent Technologies). Interpretation of the microarray results was performed according to the ACMG and ClinGen guidelines [11], taking into account publicly available databases (DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources https://decipher.sanger.ac.uk/), Database of Genomic Variants (DGV) (http://dgv.tcag.ca/dgv/app/home), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), ClinGen (http://dbsearch.clinicalgenome.org/search/)), and the in-house database of detected variants [12]. Exome sequencing was performed using a standardized series of procedures, starting with an in-solution capture of exome sequences (TruSight One, TruSight Exome, and Nextera Coding Exome capture kits, Agilent SureSelect Human All Exon v2, Agilent SureSelect Human All Exon v5 capture kits). This was followed by sequencing on Illumina MiSeq or Illumina HiSeq 2500 platform. Exome data analysis was conducted using an internally developed pipeline along with various bioinformatics approaches, including phenotype-driven gene targets and trio design, as previously described [13, 14]. Briefly, each variant was annotated based on data such as chromosomal location, zygosity, transcript information, predicted effects on protein structure and function, and local and global frequency data from The Genome Aggregation Database (gnomAD) and the Slovenian Genomic Database. In silico predictions and previous associations with diseases were sourced from the HGMD, LOVD, and ClinVar databases, as well as through literature searches. Variants were categorized as pathogenic, likely pathogenic, or variant of uncertain significance according to the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology standards for interpreting sequence variants [15]. Variants classified as pathogenic or likely pathogenic were labeled causative if they were associated with patient phenotypes.

In patients with isolated HM and those with HM and other ocular involvement, an eye disorder gene panel comprising 549 genes was used (S1 Table). For patients with HM and systemic involvement, this panel was supplemented with a phenotype-based gene panel generated using a web tool (https://www.kigm.eu/generator/). This tool facilitated tracking genes associated with clinical signs and symptoms using HPO nomenclature [13, 14]. If results were negative, an untargeted interpretation of all exome variants was performed.

Phenotyping

The children were examined by a pediatric ophthalmologist and a clinical geneticist and categorized into three groups based on their clinical presentation: HM with systemic involvement, HM with other ocular involvement, and isolated HM. Children with HM and systemic features with or without additional ocular pathologies were classified in HM with systemic involvement group, while the HM with other ocular involvement group included children in which HM was accompanied with additional pathologies limited to the eye (e.g., retinal dystrophies, strabysmus, anisometric amblyopia, nystagmus, …).

Statistics

Thirty-seven children who underwent genetic testing were included in the analysis. Demographic data (age and sex) and refractive error are presented as average value ± standard deviation (SD). Basic calculation software was used to perform statistical analysis. Statistical calculations were based on 36 probands, as two children were siblings sharing the same genetic variant. The cohort was divided into four groups based on the result of the genetic testing: those with confirmed genetic causes for HM, those with variants of uncertain significance (VUS), those with (likely) pathogenic genetic variants related to a syndromic phenotype but not previously linked to HM development, and those lacking identifiable genetic variants to explain their clinical phenotype. Percentages of genetically confirmed probands were calculated and presented, divided in systemic, ocular involvement and isolated high myopia group.

Diagnostic yield of the study

The diagnostic yield of our study was higher than expected at 61.1%. Similar studies in this field report a proportion of genetically confirmed HM cases of around 20% and up to 80% in the case of specific subject selection [10, 16–19]. The high diagnostic yield of our study is most likely due to the sample selection. Namely, we included children with HM who were treated in a tertiary paediatric ophthalmology centre, so a higher proportion of pathological conditions can be expected than in the general population of children of the same age. As a result, it is difficult to extrapolate the results of the survey to the general population of children in Slovenia. This suggests that the proportion of genetically explainable cases of HM in children is probably lower in the general population. However, given the high diagnostic yield, it can be argued that referral of children with HM treated in tertiary-level ophthalmology outpatient clinics for genetic testing is reasonable, as this is likely to clarify the aetiology of their clinical condition.

HM with systemic involvement and HM with other ocular involvement

The percentage of participants in groups with systemic involvement or with ocular involvement varies widely between studies. While Marr et al. (2001) reported 92% of HM cases associated with systemic or ocular features, the hospital-based sampling limits the study’s generalizability [20]. In contrast, other investigators have reported a significantly lower percentage of HM cases with systemic or ocular involvement, ranging from 27% to 44% [10, 21]. In particular, Marfan syndrome and Stickler syndrome, in which HM is a common clinical manifestation, occurred frequently in this group. Notably, the potential for Stickler syndrome to initially present as isolated HM highlights the difficulty of phenotypic classification, as other ocular features may manifest later [22].

In our study, 50% of children had HM with systemic involvement, which is significantly higher than the 15% reported by Haarman et al. [10]. The high proportion of systemic disabilities in our sample reflects the elevated prevalence of these conditions among children treated in an at-risk outpatient clinic. On the other hand, our result is very similar to that of Marr J.E. et al., who found 54% of participants to have an underlying systemic disease with or without other eye problems [20]. In the same study, a similar percentage was found to have HM with other eye problems (e.g., lens subluxation, coloboma, retinal dystrophy, anisometropic amblyopia), namely 38% compared to 38.9% in our study.

Among the syndromes in which HM is common, Stickler’s and Knobloch’s syndromes are the most frequently described in the literature, which is also consistent with the results of our study [10, 17, 18]. In our sample, we also found HM associated with several other syndromes, and it is particularly interesting that, in contrast to the aforementioned studies, we reported three children (13.6% of genetically confirmed HM cases) with Pitt-Hopkins syndrome.

It is also worth noting that our study describes cases of HM in individuals with variants in genes that have not yet been clearly associated with the development of myopia (TRPV4, TUBA1A, SBDS, KIDINS220).

The TRPV4 gene encodes for a transient receptor potential (TRP) channel that is expressed in all ocular tissues and is critical for maintaining proper ocular physiology. Disturbances in its expression are mainly associated with skeletal dysplasia and neuromuscular disorders, such as congenital distal spinal muscular atrophy, which was the main phenotype of the proband 3, in addition to HM. While myopia has not been previously reported in individuals with TRPV4-related disorders, its potential role in the development of eye diseases such as glaucoma and retinopathy has already been described [23].

TUBA1A encodes for α-tubulin, a major component of microtubules, which are involved in essential cellular processes of intracellular transport, cell division and neuronal migration. Pathogenic variants in TUBA1A have been shown to cause a neurodevelopmental syndrome, characterized by structural brain malformations, congenital microcephaly, developmental delay, intellectual disability, and epilepsy (OMIM: 611603) [24]. Proband 6 carries a de novo heterzygous pathogenic variant c.641G>A, p.(Arg214His) in TUBA1A. Her phenotype includes hypotonia, severe global developmental delay, intellectual disability, epileptic encephalopathy, acquired microcephaly, and HM. Tubulin-related genes are essential for brain and eye development. TUBA1A is expressed in the developing eye and has been shown to be essential for the optic nerve regeneration in the zebrafish model [25]. Patients with pathogenic TUBA1A variants and various eye anomalies such as coloboma (PMID: 26130693, PMID: 38502138), microphthalmia and cataracts (PMID: 26294046) have been reported, demonstrating the importance of this gene in eye development, structure and function [26–28].

The KIDINS220 gene encodes a scaffolding transmembrane protein that controls neuronal cell survival, differentiation into exons and dendrites, and synaptic plasticity. De novo loss-of-function variants involving the last 2 exons (29 and 30) of KIDINS220 have been associated with spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO) (OMIM #617296) [29]. The phenotype of the proband 15 in our study with the de novo likely pathogenic variant (c.4053+1G>T) corresponded to SINO syndrome, and the ocular symptoms included nystagmus, strabismus, amblyopia, high myopia, and astigmatism. Interestingly, a similar ophthalmologic phenotype was also observed in her identical twin sister with SINO syndrome, who did not participate in the study because her myopia was below the threshold for inclusion in the study. Expression studies during zebrafish and Xenopus laevis embryogenesis have demonstrated dynamic expression of KIDINS220 in the eye region. Additionally, ophthalmologic phenotypes, including nystagmus, reduced vision, and often esotropia, possibly secondary to hypermetropia, have already been described in individuals with SINO syndrome, indicating a role for the gene in eye function [29]. Although pathogenic variants in the KIDINS220 have not yet been linked to the development of myopia and therefore cannot be considered causative for this phenotype, the number of individuals SINO syndrome is still small, suggesting that the complete spectrum of ophthalmologic phenotypes may not yet be fully understood.

In the group with only ocular involvement, we observed 6 cases of (probably) pathogenic variants in genes associated with retinal dystrophies (RPGR; N = 2, CACNA1F; N = 2, RP2, NDP), which corresponds to 27.3% of all genetically confirmed cases of HM. This is a lower but comparable proportion to the study by Haarman et al. in which pathogenic variants in genes associated with retinal dystrophies were found in 39% of genetically confirmed HM cases (FAM161A, GUCY2D, PDE6H, CACNA1F, NYX, RPGR and TRPM1) [10]. A novel pathogenic NDP variant was identified in a patient with Norrie’s disease. However, a variant affecting the same amino acid site but replacing it with a different amino acid has been previously reported in a patient with Norrie’s disease [30]. The RP2 variant found is the most frequently reported pathogenic variant in the RP2 gene in patients with X-linked retinitis pigmentosa [31, 32]. Causal alterations in the CACNA1F gene are responsible for incomplete congenital stationary night blindness 2A (OMIM #300071) and retinal dystrophy (OMIM #300476). Both variants found in our study have already been described in the literature [33, 34]. Pathogenic variants in the RPGR gene are a known cause of X-linked retinal dystrophy in male patients, which also includes X-linked cone and rod dystrophy 1 (OMIM #304020) and retinitis pigmentosa 3 (OMIM #300029).

In addition, we identified a pathogenic heterozygous variant in the ZEB1 gene that is a known cause of posterior polymorphous corneal dystrophy 3 (OMIM #609141) and Fuchs endothelial corneal dystrophy 6 (OMIM #613270).

Isolated form of HM

Our finding of a low prevalence of isolated high myopia (HM) aligns with the 8% reported by Marr et al. [20]. They concluded that the diagnosis of isolated HM can only be made by a process of elimination and that in the case of apparently isolated HM in the first 10 years of life, possible systemic disease should be considered rather than isolated HM, given the significantly higher incidence of ocular and systemic abnormalities associated with HM [20].

We identified a pathogenic variant in the ARR3 gene occurring in female siblings, a variant previously reported in female myopia patients in two large families [35–37]. Mutations in ARR3 are recognized as a common cause of isolated HM with X-linked inheritance and female-restricted HM [10, 18].

Strengths, limitations, and relevance of the study

The study’s limited sample size, selective participant recruitment, and short duration necessitate cautious interpretation of findings, particularly regarding broader epidemiological implications. A large-scale, national study is needed to obtain a more accurate picture of HM genetic background in the Slovenian pediatric population. This study should include a broader spectrum of children with HM and reduce potential sampling biases seen in our tertiary-care setting.