Whole-Exome Sequencing of 21 Families: Candidate Genes for Early-Onset High Myopia

High myopia is the most severe and pathological form of myopia. It occurs when the spherical refractive error exceeds –6.00 spherical diopters (SDs) or the axial length (AL) of the eye is greater than 26 mm. This article focuses on early-onset high myopia, an increasingly common condition that affects children under 10 years of age and can lead to other serious ocular pathologies. Through the genetic analysis of 21 families with early-onset high myopia, this study seeks to contribute to a better understanding of the role of genetics in this disease and to propose candidate genes. Whole-exome sequencing studies with a panel of genes known to be involved in the pathology were performed in families with inconclusive results: 3% of the variants found were classified as pathogenic, 6% were likely pathogenic and the remaining 91% were variants of uncertain significance. Most of the families in this study were found to have alterations in several of the proposed genes. This suggests a polygenic inheritance of the pathology due to the cumulative effect of the alterations. Further studies are needed to validate and confirm the role of these alterations in the development of early-onset high myopia and its polygenic inheritance.


Introduction
Myopia is the most common eye disorder in the world [1].From a physiological point of view, it involves a refractive error where the light rays entering the eye focus on a point in front of the retina, leading to decreased visual acuity.This refractive error is due to a postnatal axial elongation of the eye in the process of emmetropization [2,3].

Introduction
Myopia is the most common eye disorder in the world [1].From a physiological point of view, it involves a refractive error where the light rays entering the eye focus on a point in front of the retina, leading to decreased visual acuity.This refractive error is due to a postnatal axial elongation of the eye in the process of emmetropization [2,3].Although myopia is usually considered a benign condition that can be corrected with the use of glasses, contact lenses or refractive surgery, it is becoming a public health concern due to its increasing prevalence in younger populations and its progression to its most severe and pathological form, high myopia (HM) (Figure 1).HM is defined as a spherical refractive error exceeding -6.00 spherical diopters (SD) or an axial length (AL) greater than 26 mm [4,5].
This study focuses on early-onset HM (EoHM), which occurs in children under 10 years of age [3].Through an examination of the disease from a genetic perspective, this analysis seeks to contribute to a better understanding of the role of genetics in its development.
Figure 1.Schematic optics of the eye.In emmetropic eyes, the parallel rays of a distant object are focused precisely on the photoreceptors located on the retina.Myopic eyes have longer axial length, and the image of a distant object falls in front of the photoreceptors and cannot be brought into focus by accommodation.The myopic eye can become highly myopic, accentuating this defocus considerably.Modified from Morgan et al. [5].
HM increases the susceptibility to other ocular complications that have a significant impact on quality of life and can lead to irreversible vision loss such as cataracts, glaucoma, retinal detachment and macular degeneration.It is therefore important to diagnose myopia and its progression to HM for early prevention and intervention before the onset of more severe pathological manifestations.In addition, HM can appear as one of the first clinical manifestations of conditions such as Marfan or Stickler syndromes, retinal disorders or congenital stationary night blindness (CSNB), allowing for the early diagnosis and treatment of these diseases [4].
The prevalence of myopia is highest in East Asia, affecting 90% of high school graduates.The reported prevalence of myopia among Caucasians in Europe is 30.6% and that of HM is 2.7%.Based on current trends, the study by Holden et al., estimates that the number of people affected will significantly increase worldwide by 2050, as may be observed in Figure 2 [4,6].This study focuses on early-onset HM (EoHM), which occurs in children under 10 years of age [3].Through an examination of the disease from a genetic perspective, this analysis seeks to contribute to a better understanding of the role of genetics in its development.
HM increases the susceptibility to other ocular complications that have a significant impact on quality of life and can lead to irreversible vision loss such as cataracts, glaucoma, retinal detachment and macular degeneration.It is therefore important to diagnose myopia and its progression to HM for early prevention and intervention before the onset of more severe pathological manifestations.In addition, HM can appear as one of the first clinical manifestations of conditions such as Marfan or Stickler syndromes, retinal disorders or congenital stationary night blindness (CSNB), allowing for the early diagnosis and treatment of these diseases [4].
The prevalence of myopia is highest in East Asia, affecting 90% of high school graduates.The reported prevalence of myopia among Caucasians in Europe is 30.6% and that of HM is 2.7%.Based on current trends, the study by Holden et al., estimates that the number of people affected will significantly increase worldwide by 2050, as may be observed in Figure 2 [4,6].The etiology of myopia and HM is heterogeneous and multifactorial, involving a combination of environmental (external), microenvironmental (internal) and genetic factors [4].
Currently, the most studied environmental factor believed to protect individuals from developing myopia is the time spent outdoors [4].Conversely, several studies have linked the progression of myopia to the time spent in near work and exposure to artificial The etiology of myopia and HM is heterogeneous and multifactorial, involving a combination of environmental (external), microenvironmental (internal) and genetic factors [4].
Currently, the most studied environmental factor believed to protect individuals from developing myopia is the time spent outdoors [4].Conversely, several studies have linked the progression of myopia to the time spent in near work and exposure to artificial light [3,5].In the case of EoHM, however, given the young age of the patients, environmental factors are not considered to have a significant influence on the development of myopia.This suggests that there is a greater genetic burden involved in EoHM, supporting a genetic approach to the disease [3].
Microenvironmental factors, such as oxidative stress, oxidation and angiogenesis, can induce or accentuate EoHM.Given its high oxygen demand and direct exposure to natural light, the retina is particularly vulnerable to oxidative stress.In addition, patients with EoHM have an imbalance in the oxidative/antioxidative status of the retina, suggesting that oxidative stress plays a direct role in the development of this pathology [3].
Genetic factors, as explained above, play a critical and major role in the development of EoHM.Genetic information initiates and regulates the processes involved in emmetropization, including the regulation of microenvironmental factors and visual feedback:

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Visual feedback driven by optical defocus.The defocus signal is detected by the retina and triggers a multilayered signaling cascade involving a large number of coding and regulatory genes, sequentially affecting the retina, retinal pigment epithelium (RPE), choroid, sclera and its extracellular matrix (ECM).In this last step of the cascade, changes occur in the composition of the ECM and are reflected in axial elongation (Figure 3).Alterations in these genes and their function can disrupt emmetropization and result in excessive axial length [2].The classification of genetic alterations affecting visual feedback may be divided into two main groups: those involved in the retina, affecting the function of photoreceptors, RPEs, and ON and OFF bipolar neurons; and those that occur in the sclera, affecting the composition and metabolism of the ECM [5].
The role of genetic factors in myopia is supported by several studies.It has been observed that children with a family history of myopia have longer axial lengths and a higher risk of developing myopia during childhood compared to the average population [4].Due to differences in study design and methods of analysis, heritability estimates in the literature range from 15% to 98%; however, the true heritability of myopia is probably between 60% and 80%.
Genome-wide association studies (GWASs) and segregation analyses have identified more than 26 loci and 400 genes involved in refractive error and the development of common myopia [4,7].Fewer genes have been associated with EoHM: 11 genes with autosomal dominant inheritance have been identified for its non-syndromic form (ZNF644, SCO2, SLC39A5, CCDC111, P4HA2, BSG, CPSF1, NDUFAF7, TNFRSF21, XYLT and DZIP1); four genes with autosomal recessive inheritance (LRPAP1, CTSH, LEPREL1 and LOXL3); and two X-linked genes (ARR3 and OPN1LW).Other studies have associated The classification of genetic alterations affecting visual feedback may be divided into two main groups: those involved in the retina, affecting the function of photoreceptors, RPEs, and ON and OFF bipolar neurons; and those that occur in the sclera, affecting the composition and metabolism of the ECM [5].
The role of genetic factors in myopia is supported by several studies.It has been observed that children with a family history of myopia have longer axial lengths and a higher risk of developing myopia during childhood compared to the average population [4].Due to differences in study design and methods of analysis, heritability estimates in the literature range from 15% to 98%; however, the true heritability of myopia is probably between 60% and 80%.
Most of the variants studied, especially in common myopia, carry a low risk and can be prevalent in the general population, contributing to a small extent and cumulatively to the overall risk.Therefore, it is postulated that the inheritance of myopia and high myopia follows a polygenic pattern, in which several genes together contribute to the manifestation of the disease [8].
In this study, next-generation sequencing (NGS) was used to sequence the whole exome of 21 families diagnosed with EoHM.Whole-exome sequencing makes it possible to study alterations and their involvement in a larger number of genes, thus allowing for the diagnosis and proposal of new candidate causal genes for EoHM in patients.

Results and Discussion
The results of this study were obtained from an ongoing project seeking to identify new genes responsible for EoHM in a sample of families from a tertiary hospital in Spain.This project also aims to evaluate the implementation of NGS and its relevance to the management of patients with EoHM.A total of 21 probands (33.33% female [7/21] and 66.66% male [14/21]) and nine affected relatives with EoHM from 21 unrelated families were recruited based on their phenotype (Table 1) and the inclusion criteria indicated in the Materials and Methods.
The ages of the probands range from 6 to 80, yet they all share the common characteristic of exhibiting HM before the age of 10, thus constituting a unified group for genetic analysis.It is worth noting that 62% of the subjects in this study have not yet reached the age of 10 (Figure 4).were recruited based on their phenotype (Table 1) and the inclusion criteria indicated in the Materials and Methods.The ages of the probands range from 6 to 80, yet they all share the common characteristic of exhibiting HM before the age of 10, thus constituting a unified group for genetic analysis.It is worth noting that 62% of the subjects in this study have not yet reached the age of 10 (Figure 4).The average spherical equivalent for the probands is −12.044diopters in the right eye and −11.499 diopters in the left eye.Table 2 shows the descriptive statistics, including the mean and standard deviation of the refractive outcomes in this cohort.Figure 5 depicts the clinical characteristics of the EoHM patients based on fundoscopic examination.33% of them had not developed yet any features (normal).The most prevalent phenotypic feature is diffuse chorioretinal atrophy, present in 19% of the cases alone and adding a total of 38% accompanied by other features, followed by a tesselated fundus, with 9%.The average spherical equivalent for the probands is −12.044diopters in the right eye and −11.499 diopters in the left eye.Table 2 shows the descriptive statistics, including the mean and standard deviation of the refractive outcomes in this cohort.Figure 5 depicts the clinical characteristics of the EoHM patients based on fundoscopic examination.33% of them had not developed yet any features (normal).The most prevalent phenotypic feature is diffuse chorioretinal atrophy, present in 19% of the cases alone and adding a total of 38% accompanied by other features, followed by a tesselated fundus, with 9%.For the gender-based difference analysis, the patients were divided into two groups: more severe EoHM and less severe EoHM, obtaining the following frequencies (Table 3).For the gender-based difference analysis, the patients were divided into two groups: more severe EoHM and less severe EoHM, obtaining the following frequencies (Table 3).Since we have frequencies lower than 5, a Fisher's exact test was performed, resulting in a p-value of 0.6557.Given that this p-value is greater than 0.05, there is insufficient evidence to reject the null hypothesis (it can be found in Materials and Methods section).Thus, there is no association between severity and gender; they are independent variables.
The results obtained were evaluated considering the limitations of exome sequencing such as the low capture efficiency in the sequencing of certain regions, the inability to analyze regulatory regions far from the exons, the lack of previous scientific studies of yet unknown genes and the inability to conduct functional studies of all the variants found.
As one of the objectives of this work was to identify new genes responsible for EoHM and exome sequencing implies the possibility of finding variants in genes not previously related to ocular pathology, we considered variants classified as pathogenic (P), likely pathogenic (LP) and variants of uncertain significance (VUS) not previously associated with HM, but that are involved in the regulation of emmetropization.
We found 83 variants that may be involved in the development of EoHM.Two (3%) were classified as P, five (6%) was LP and 74 (91%) was VUS (Figure 6).The American College of Medical Genetics (ACMG) criteria for classifying pathogenic variants may be observed in Table A1.Since we have frequencies lower than 5, a Fisher's exact test was performed, resulting in a p-value of 0.6557.Given that this p-value is greater than 0.05, there is insufficient evidence to reject the null hypothesis (it can be found in Materials and Methods section).Thus, there is no association between severity and gender; they are independent variables.
The results obtained were evaluated considering the limitations of exome sequencing such as the low capture efficiency in the sequencing of certain regions, the inability to analyze regulatory regions far from the exons, the lack of previous scientific studies of yet unknown genes and the inability to conduct functional studies of all the variants found.
As one of the objectives of this work was to identify new genes responsible for EoHM and exome sequencing implies the possibility of finding variants in genes not previously related to ocular pathology, we considered variants classified as pathogenic (P), likely pathogenic (LP) and variants of uncertain significance (VUS) not previously associated with HM, but that are involved in the regulation of emmetropization.
We found 83 variants that may be involved in the development of EoHM.Two (3%) were classified as P, five (6%) was LP and 74 (91%) was VUS (Figure 6).The American College of Medical Genetics (ACMG) criteria for classifying pathogenic variants may be observed in Table A1.The alterations found in the families included in the study and their relationship to EoHM are detailed below.

OFT-00074 Family
Candidate genes on Table 4.One of the proposed causal alterations for EoHM in this family was classified as pathogenic and affects the splicing of the TRPM1 gene.Its associated pathology is night blindness, congenital stationary (complete), 1C and autosomal recessive [9].It encodes a permeable calcium cation channel, mainly expressed in retinal bipolar cells.Because CSNB and EoHM are associated and cases of EoHM have been observed in CSNB patients with altered TRPM1, the involvement of this gene in its development has been proposed, although the cause of this association is not yet known [10][11][12].It has been hypothesized that TRPM1 plays a role in embryonic development by influencing synaptic activity, optic nerve formation, photoreceptor arrangement and ON bipolar cell function [13].In the Gene Ontology Resource database, this gene is associated with visual perception and the cellular response to light stimulus (Table A2) [14].The alterations found in the families included in the study and their relationship to EoHM are detailed below.

OFT-00074 Family
Candidate genes on Table 4.One of the proposed causal alterations for EoHM in this family was classified as pathogenic and affects the splicing of the TRPM1 gene.Its associated pathology is night blindness, congenital stationary (complete), 1C and autosomal recessive [9].It encodes a permeable calcium cation channel, mainly expressed in retinal bipolar cells.Because CSNB and EoHM are associated and cases of EoHM have been observed in CSNB patients with altered TRPM1, the involvement of this gene in its development has been proposed, although the cause of this association is not yet known [10][11][12].It has been hypothesized that TRPM1 plays a role in embryonic development by influencing synaptic activity, optic nerve formation, photoreceptor arrangement and ON bipolar cell function [13].In the Gene Ontology Resource database, this gene is associated with visual perception and the cellular response to light stimulus (Table A2) [14].
Other VUS alterations detected in this family included that of ARHGEF18, which codes for a guanine nucleotide exchange factor (GEF) that directly controls the activation of Rho GTPases and is involved in retinal development and degeneration [15].This gene is a key regulator of RhoA-Rock2 signaling, which is crucial for the maintenance of polarity in the vertebrate retinal epithelium.In addition, ARHGEF18 is required to maintain apico-basal polarity, tight junction localization and cortical actin, thus shaping the morphology of these cells [16].This gene is associated with retinitis pigmentosa 78 [9].
KDM6B encodes a lysine-specific demethylase that demethylates histone H3 (epigenetic control) and other non-histone proteins.It is involved in processes of organogenesis and retinal development at the amacrine, horizontal and ganglion cell level, allowing the focusing or defocusing signal to correctly reach the brain from the retina [17].
The selected candidate genes did not have AD inheritance and were not found in homozygosity, suggesting the possibility of a cumulative effect of the proposed alterations as a cause of EoHM in the affected individuals.Other VUS alterations detected in this family included that of ARHGEF18, which codes for a guanine nucleotide exchange factor (GEF) that directly controls the activation of Rho GTPases and is involved in retinal development and degeneration [15].This gene is a key regulator of RhoA-Rock2 signaling, which is crucial for the maintenance of polarity in the vertebrate retinal epithelium.In addition, ARHGEF18 is required to maintain apico-basal polarity, tight junction localization and cortical actin, thus shaping the morphology of these cells [16].This gene is associated with retinitis pigmentosa 78 [9].
KDM6B encodes a lysine-specific demethylase that demethylates histone H3 (epigenetic control) and other non-histone proteins.It is involved in processes of organogenesis and retinal development at the amacrine, horizontal and ganglion cell level, allowing the focusing or defocusing signal to correctly reach the brain from the retina [17].
The selected candidate genes did not have AD inheritance and were not found in homozygosity, suggesting the possibility of a cumulative effect of the proposed alterations as a cause of EoHM in the affected individuals.Het: heterozygous; AR: autosomal recessive.

OFT-00097 Family
Candidate genes on Table 5.The proband in this family had a variant inherited from his mother of the most altered gene identified in this study, HSPG2.This is a gene with incomplete penetrance [18], encoding perlecan, a large multidomain proteoglycan.It binds and cross-links to ECM components and molecules on the cell surface, allowing it to interact with laminin, prolargin, type IV collagen, FGFBP1, FBLN2, FGF7 and transthyretin and maintain the endothelial barrier function in the vascular ECM.HSPG2 also promotes the activity of growth factors such as FGF2, thus stimulating endothelial growth and fibroblast regeneration [15].The study by Wan et al., includes it as one of the new candidate genes for EoHM [19].
Other altered genes affecting the structure of the sclera were also observed, such as COL9A2, a collagen associated with Stickler syndrome, which, being a heterozygous variant, may have contributed to HM in conjunction with the other alterations without developing the full syndrome [20].Fibulin1 (FBLN1) is a glycoprotein incorporated into a fibrillar ECM [15], which is expressed in fibroblasts of the sclera, enabling cell-matrix interactions, and is involved in the regulation of ocular growth [21].
CACNA1F presents a pathogenic alteration in homozygosis that adds a stop codon.The protein expressed is a multi-pass transmembrane that functions as an alpha-1 subunit of the voltage-dependent calcium channel and mediates the entry of calcium ions into the cell.CACNA1F is associated with several X-linked eye disorders including CSNB type 2A and Aland Island eye disease [15].The gene is involved in the cone and rod response and was identified in a study of patients with CSNB as predominant in those who also had EoHM [22].
CSMD1, another of the most altered genes found in this study, is located at the MYP10 locus and is mainly expressed in the peripheral retina and in the area surrounding Het: heterozygous; AR: autosomal recessive.

OFT-00097 Family
Candidate genes on Table 5.The proband in this family had a variant inherited from his mother of the most altered gene identified in this study, HSPG2.This is a gene with incomplete penetrance [18], encoding perlecan, a large multidomain proteoglycan.It binds and cross-links to ECM components and molecules on the cell surface, allowing it to interact with laminin, prolargin, type IV collagen, FGFBP1, FBLN2, FGF7 and transthyretin and maintain the endothelial barrier function in the vascular ECM.HSPG2 also promotes the activity of growth factors such as FGF2, thus stimulating endothelial growth and fibroblast regeneration [15].The study by Wan et al., includes it as one of the new candidate genes for EoHM [19].
Other altered genes affecting the structure of the sclera were also observed, such as COL9A2, a collagen associated with Stickler syndrome, which, being a heterozygous variant, may have contributed to HM in conjunction with the other alterations without developing the full syndrome [20].Fibulin1 (FBLN1) is a glycoprotein incorporated into a fibrillar ECM [15], which is expressed in fibroblasts of the sclera, enabling cell-matrix interactions, and is involved in the regulation of ocular growth [21].
CACNA1F presents a pathogenic alteration in homozygosis that adds a stop codon.The protein expressed is a multi-pass transmembrane that functions as an alpha-1 subunit of the voltage-dependent calcium channel and mediates the entry of calcium ions into the cell.CACNA1F is associated with several X-linked eye disorders including CSNB type 2A and Aland Island eye disease [15].The gene is involved in the cone and rod response and was identified in a study of patients with CSNB as predominant in those who also had EoHM [22].
CSMD1, another of the most altered genes found in this study, is located at the MYP10 locus and is mainly expressed in the peripheral retina and in the area surrounding the macula.This gene plays a critical role in cone growth, including signal transduction and matrix adhesion, and has been proposed in multiple studies as a candidate gene for the development of EoHM [19,23].
Finally, an alteration in ADAMTSL1 is proposed as possibly contributing to the development of EoHM by encoding a thrombospondin motif metalloproteinase (ADAMTS).This protein may play important roles in the ECM, thus establishing a link to myopia [15].
In the case of this family, the most relevant alterations that may have influenced the EoHM of the probands are those present in the HSPG2, FBLN1 and CACNA1F genes, due to their inheritance pattern, although the other genes mentioned may also have contributed.Specifically, CSMD1 has been observed and proposed in this study and other related studies [19,23].the macula.This gene plays a critical role in cone growth, including signal transduction and matrix adhesion, and has been proposed in multiple studies as a candidate gene for the development of EoHM [19,23].
Finally, an alteration in ADAMTSL1 is proposed as possibly contributing to the development of EoHM by encoding a thrombospondin motif metalloproteinase (ADAMTS).This protein may play important roles in the ECM, thus establishing a link to myopia [15].
In the case of this family, the most relevant alterations that may have influenced the EoHM of the probands are those present in the HSPG2, FBLN1 and CACNA1F genes, due to their inheritance pattern, although the other genes mentioned may also have contributed.Specifically, CSMD1 has been observed and proposed in this study and other related studies [19,23].Het: heterozygous; Hemi: hemizygous; AD: autosomal dominant; AR: autosomal recessive; XL: Xlinked.* Gain of a stop codon, does not substitute for any amino acid.

OFT-00155 Family
Candidate genes on Table 6.In this family, we searched for alterations common to the two affected individuals, as well as including other genes, also shared with the unaffected sibling, reported in studies of EoHM or myopia.All the altered genes are involved in the maintenance of the sclera, with the exception of CACNA1F, as explained above, and COL9A3, a collagen described in Stickler syndrome, in which the most involved ocular structure is the choroid [15].The role of HSPG2 in the development of EoHM has been detailed above.
The LAMA1 gene is at the MYP2 locus and belongs to a family of structural glycoproteins in the ECM of the sclera and lens [15,25].It is one of the genes most associated with the development of EoHM, supported by several studies [25][26][27].Because it has an AR inheritance and is heterozygous, it is likely not the only altered gene causing EoHM in this family.There could be a cumulative effect enhanced by the other alterations indicated in Table 4, as most of these affect the sclera.For example, an alteration was observed in LAMA5, another glycoprotein in basement membranes with biological functions similar to LAMA1 [15].
Alterations were also found in the THBS2 and THBS1 genes, homotrimeric-disulfidelinked glycoproteins that mediate cell-cell and cell-matrix interactions.THBS1 can bind to fibrinogen, fibronectin, laminin, type-V collagen and alpha-V/beta-1 integrins [15].In a study of patterns of messenger RNA (mRNA) in sclera remodeling during the development of lens-induced myopia in Soricidae, changes were observed in the SPARC, THBS1, Het: heterozygous; Hemi: hemizygous; AD: autosomal dominant; AR: autosomal recessive; XL: X-linked.* Gain of a stop codon, does not substitute for any amino acid.

OFT-00155 Family
Candidate genes on Table 6.In this family, we searched for alterations common to the two affected individuals, as well as including other genes, also shared with the unaffected sibling, reported in studies of EoHM or myopia.All the altered genes are involved in the maintenance of the sclera, with the exception of CACNA1F, as explained above, and COL9A3, a collagen described in Stickler syndrome, in which the most involved ocular structure is the choroid [15].The role of HSPG2 in the development of EoHM has been detailed above.
The LAMA1 gene is at the MYP2 locus and belongs to a family of structural glycoproteins in the ECM of the sclera and lens [15,25].It is one of the genes most associated with the development of EoHM, supported by several studies [25][26][27].Because it has an AR inheritance and is heterozygous, it is likely not the only altered gene causing EoHM in this family.There could be a cumulative effect enhanced by the other alterations indicated in Table 4, as most of these affect the sclera.For example, an alteration was observed in LAMA5, another glycoprotein in basement membranes with biological functions similar to LAMA1 [15].
Alterations were also found in the THBS2 and THBS1 genes, homotrimeric-disulfidelinked glycoproteins that mediate cell-cell and cell-matrix interactions.THBS1 can bind to fibrinogen, fibronectin, laminin, type-V collagen and alpha-V/beta-1 integrins [15].In a study of patterns of messenger RNA (mRNA) in sclera remodeling during the development of lens-induced myopia in Soricidae, changes were observed in the SPARC, THBS1, THBS2, TNC and SPP1 genes, suggesting that these may play a role in increasing scleral sliding velocity [28].THBS2, TNC and SPP1 genes, suggesting that these may play a role in increasing scleral sliding velocity [28].Het: heterozygous; Hemi: hemizygous; AD: autosomal dominant; AR: autosomal recessive; XL: Xlinked.* Alterations shared with his brother (he has idiopathic motor nystagmus).

OFT-00175 Family
Candidate genes on Table 7.All the alterations found in the OFT-00175 family were classified as VUS.The proband had only one de novo alteration in HSPG2; the other alterations were inherited from his father.
The altered genes affecting the retina are PCDH15 and TRPM1.PCDH15 belongs to the cadherin superfamily, which encodes integral membrane proteins responsible for mediating calcium-dependent cell-cell adhesion.It plays an essential role in the maintenance of normal retinal and cochlear function [15].PCDH15 was proposed in the WES study by Wan et al., as a candidate gene related to EoHM [19], with an AR or digenic inheritance model.Digenic inheritance is that in which a mutation in two genes is necessary to cause a given phenotype.In this case, there were alterations in four genes.
Finally, BMPR2 is a member of the bone morphogenetic protein (BMP) family of transmembrane serine/threonine kinase receptors.Ligands for this receptor are members of the transforming growth factor (TGF)-β superfamily [15].This gene is also expressed in the retina but may have some relation to the development of EoHM given its association with the function of scleral fibroblasts in early myopia and incomplete penetrance [29].

OFT-00175 Family
Candidate genes on Table 7.All the alterations found in the OFT-00175 family were classified as VUS.The proband had only one de novo alteration in HSPG2; the other alterations were inherited from his father.
The altered genes affecting the retina are PCDH15 and TRPM1.PCDH15 belongs to the cadherin superfamily, which encodes integral membrane proteins responsible for mediating calcium-dependent cell-cell adhesion.It plays an essential role in the maintenance of normal retinal and cochlear function [15].PCDH15 was proposed in the WES study by Wan et al., as a candidate gene related to EoHM [19], with an AR or digenic inheritance model.Digenic inheritance is that in which a mutation in two genes is necessary to cause a given phenotype.In this case, there were alterations in four genes.
Finally, BMPR2 is a member of the bone morphogenetic protein (BMP) family of transmembrane serine/threonine kinase receptors.Ligands for this receptor are members of the transforming growth factor (TGF)-β superfamily [15].This gene is also expressed in the retina but may have some relation to the development of EoHM given its association with the function of scleral fibroblasts in early myopia and incomplete penetrance [29].THBS2, TNC and SPP1 genes, suggesting that these may play a role in increasing scleral sliding velocity [28].Het: heterozygous; Hemi: hemizygous; AD: autosomal dominant; AR: autosomal recessive; XL: Xlinked.* Alterations shared with his brother (he has idiopathic motor nystagmus).

OFT-00175 Family
Candidate genes on Table 7.All the alterations found in the OFT-00175 family were classified as VUS.The proband had only one de novo alteration in HSPG2; the other alterations were inherited from his father.
The altered genes affecting the retina are PCDH15 and TRPM1.PCDH15 belongs to the cadherin superfamily, which encodes integral membrane proteins responsible for mediating calcium-dependent cell-cell adhesion.It plays an essential role in the maintenance of normal retinal and cochlear function [15].PCDH15 was proposed in the WES study by Wan et al., as a candidate gene related to EoHM [19], with an AR or digenic inheritance model.Digenic inheritance is that in which a mutation in two genes is necessary to cause a given phenotype.In this case, there were alterations in four genes.
Finally, BMPR2 is a member of the bone morphogenetic protein (BMP) family of transmembrane serine/threonine kinase receptors.Ligands for this receptor are members of the transforming growth factor (TGF)-β superfamily [15].This gene is also expressed in the retina but may have some relation to the development of EoHM given its association with the function of scleral fibroblasts in early myopia and incomplete penetrance [29].Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00178 Family
Candidate genes on Table 8.In this family, variants inherited from the mother and father were found affecting both scleral and retinal development, which could cumulatively cause EoHM.
Genes involved in the identified retinal development included PCDH15 and CSMD1, discussed above.LRP2, another gene in this group, encodes a multi-ligand endocytic receptor, has a role in cell signaling and has been associated with Donnai-Barrow and Stickler syndromes, both of which are characterized by EoHM [15].This gene was found to have a necessary function for normal eye growth through inactivation of Lrp2 in the mouse forebrain (including the neural retina and retinal and ciliary pigment epithelia), resulting in a 40% greater axial elongation compared to controls.Bipolar, photoreceptor and retinal ganglion cells were also affected [30].
The proband had two heterozygous alterations in the MAP3K1 gene, one inherited from his mother and the other from his father, known as double heterozygous or compound heterozygous.This gene is known to be involved in eye development, with high expression in the retina.Its low expression in the mouse retina affects vascularization, RPE, photoreceptor loss and early degeneration [31,32].
LAMA4 is another gene associated with alterations affecting the sclera.Expression changes in the HIF-1α/miR-150-5p/LAMA4/p38 MAPK axis have been observed in ECM degradation of scleral fibroblasts under hypoxic conditions, leading to the pathological progression of HM.An increased expression of LAMA4 has been observed in patients with HM [33].
At the scleral level, excessive PLG expression has been reported in HM patients.Here, plasmin (in its active form) can degrade fibrin and convert potential matrix metalloproteinases (pro-MMPs) into active MMPs, capable of destroying the ECM, thus reducing scleral stiffness and making it unable to maintain its necessary stiffness, strength and elasticity.Plasmin also participates in other processes involved in the pathogenesis of HM such as tissue remodeling and angiogenesis [34].
VASH1 enables actin-binding and metallocarboxypeptidase activity.It is involved in the negative regulation of angiogenesis, the migration of blood vessel endothelial cells and proteolysis [15] and is expressed in the neural retina, sclera, choroid and RPE [35].
Several of these alterations may play a significant role in EoHM.Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00178 Family
Candidate genes on Table 8.In this family, variants inherited from the mother and father were found affecting both scleral and retinal development, which could cumulatively cause EoHM.
Genes involved in the identified retinal development included PCDH15 and CSMD1, discussed above.LRP2, another gene in this group, encodes a multi-ligand endocytic receptor, has a role in cell signaling and has been associated with Donnai-Barrow and Stickler syndromes, both of which are characterized by EoHM [15].This gene was found to have a necessary function for normal eye growth through inactivation of Lrp2 in the mouse forebrain (including the neural retina and retinal and ciliary pigment epithelia), resulting in a 40% greater axial elongation compared to controls.Bipolar, photoreceptor and retinal ganglion cells were also affected [30].
The proband had two heterozygous alterations in the MAP3K1 gene, one inherited from his mother and the other from his father, known as double heterozygous or compound heterozygous.This gene is known to be involved in eye development, with high expression in the retina.Its low expression in the mouse retina affects vascularization, RPE, photoreceptor loss and early degeneration [31,32].
LAMA4 is another gene associated with alterations affecting the sclera.Expression changes in the HIF-1α/miR-150-5p/LAMA4/p38 MAPK axis have been observed in ECM degradation of scleral fibroblasts under hypoxic conditions, leading to the pathological progression of HM.An increased expression of LAMA4 has been observed in patients with HM [33].
At the scleral level, excessive PLG expression has been reported in HM patients.Here, plasmin (in its active form) can degrade fibrin and convert potential matrix metalloproteinases (pro-MMPs) into active MMPs, capable of destroying the ECM, thus reducing scleral stiffness and making it unable to maintain its necessary stiffness, strength and elasticity.Plasmin also participates in other processes involved in the pathogenesis of HM such as tissue remodeling and angiogenesis [34].
VASH1 enables actin-binding and metallocarboxypeptidase activity.It is involved in the negative regulation of angiogenesis, the migration of blood vessel endothelial cells and proteolysis [15] and is expressed in the neural retina, sclera, choroid and RPE [35].
Several of these alterations may play a significant role in EoHM.teinases (pro-MMPs) into active MMPs, capable of destroying the ECM, thus reducing scleral stiffness and making it unable to maintain its necessary stiffness, strength and elasticity.Plasmin also participates in other processes involved in the pathogenesis of HM such as tissue remodeling and angiogenesis [34].
VASH1 enables actin-binding and metallocarboxypeptidase activity.It is involved in the negative regulation of angiogenesis, the migration of blood vessel endothelial cells and proteolysis [15] and is expressed in the neural retina, sclera, choroid and RPE [35].
Several of these alterations may play a significant role in EoHM.Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00191 Family
Candidate genes on Table 9.The PER3 gene belongs to the period gene family, which encodes components of the circadian rhythms of locomotor activity, metabolism and behavior [15].It has a role in the negative regulation of transcription by RNA polymerase II [14].Circadian rhythm genes are associated with refractive error.PER3 is located near the MYP14 locus and is expressed in ON and OFF bipolar cells [36].
COL11A1, also associated with Stickler and Marshall syndromes, may only present a phenotype in the eye, such as EoHM [15,37].As for FRMPD1, the protein it encodes directly interacts with Gpsm2 (G-protein signaling modulator 2) and is necessary for the optimization of the rod-to-bipolar synaptic transmission when Gαt is present at the synapse [38].
In the two affected individuals of this family, the effect of the alteration in COL11A1 predominates, making it the primary factor in the pathogenesis of the disease given its inheritance pattern, classification, extensive evidence in the literature and association with Stickler and Marshall syndromes.Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00191 Family
Candidate genes on Table 9.The PER3 gene belongs to the period gene family, which encodes components of the circadian rhythms of locomotor activity, metabolism and behavior [15].It has a role in the negative regulation of transcription by RNA polymerase II [14].Circadian rhythm genes are associated with refractive error.PER3 is located near the MYP14 locus and is expressed in ON and OFF bipolar cells [36].
COL11A1, also associated with Stickler and Marshall syndromes, may only present a phenotype in the eye, such as EoHM [15,37].As for FRMPD1, the protein it encodes directly interacts with Gpsm2 (G-protein signaling modulator 2) and is necessary for the optimization of the rod-to-bipolar synaptic transmission when Gαt is present at the synapse [38].
In the two affected individuals of this family, the effect of the alteration in COL11A1 predominates, making it the primary factor in the pathogenesis of the disease given its inheritance pattern, classification, extensive evidence in the literature and association with Stickler and Marshall syndromes.Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00209 Family
Candidate genes on Table 10.The candidate genes for EoHM in this family include GLB1.The protein it encodes (galactosidase beta 1) is present in human choroidal endothelial cells and acts as a receptor for elastin-derived peptides (EDPs).Elevated levels of circulating EDP do not affect retinal function in mice but increase the expression and deposition of collagen IV in the RPE/choroid complex [39].COL9A1 is associated with Stickler syndrome [15]; KDM6B has been discussed above.Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00209 Family
Candidate genes on Table 10.The candidate genes for EoHM in this family include GLB1.The protein it encodes (galactosidase beta 1) is present in human choroidal endothelial cells and acts as a receptor for elastin-derived peptides (EDPs).Elevated levels of circulating EDP do not affect retinal function in mice but increase the expression and deposition of collagen IV in the RPE/choroid complex [39].COL9A1 is associated with Stickler syndrome [15]; KDM6B has been discussed above.Het: heterozygous; AR: autosomal recessive.

OFT-00217 Family
Candidate genes on Table 11.The proband in family OFT-00217 inherited alterations from both her mother and father.AGRN is located at the MYP14 locus, encoding agrin, a large proteoglycan with multiple isoforms.It contains several laminin G domains, a Kazal-like serine protease inhibitor and epidermal growth factor.The study by Zheng et al., [40] found that AGRN is involved in baseline refractive development, demonstrating that this gene interacts with EGR1, which is implicated in refractive development and regulates synaptic physiology in the retina [15,40].Several studies of different populations have associated rare or infrequent heterozygous alterations in AGRN with the development of HM [40].
The GRM6 gene is involved in ON synaptic transmission in bipolar cells, controlling the release of dopamine, which has been suggested as a factor in ocular growth.Mutations in this gene cause CSNB type 1B.HM is often seen in CSNB type 1B patients with altered GRM6.A link between GRM6 and susceptibility to HM has been suggested in a number of studies [41].
Alterations in the CNTN6 gene may be involved in the development of EoHM, as contactin-6 is responsible for the correct development and maintenance of the central nervous system, particularly in axonal projection, branching and synapses.Meguro et al., associated an altered expression of this gene affecting GABA receptor levels or synaptogenesis with HM [42].FRMD4B is also involved at the retinal level, through direct interaction of the protein it encodes with cytohesin-3, and functions as a scaffolding protein.Cytohesin-3 plays an important role in insulin, epidermal and nerve growth factor signaling, with a recent study [42] suggesting that the FRMD4B-cytohesin-3 complex affects cell junction dynamics in the retina and contributes to the regulation of photoreceptor cell growth and development [42].
LRP1 affects the sclera, encoding protein 1 of the low-density lipoprotein receptor family of cytokines and growth factors, and is involved in several cellular processes, even the negative regulation of gene expression [14].LRP1 deficiency leads to the disruption of TGF-β and may result in abnormal remodeling of the ECM of the developing eye, making this gene a candidate for HM [43].
Further studies are needed to demonstrate the direct involvement of these genes with EoHM for an accurate diagnosis, but for now, they are good candidates.Het: heterozygous; AR: autosomal recessive.

OFT-00217 Family
Candidate genes on Table 11.The proband in family OFT-00217 inherited alterations from both her mother and father.AGRN is located at the MYP14 locus, encoding agrin, a large proteoglycan with multiple isoforms.It contains several laminin G domains, a Kazal-like serine protease inhibitor and epidermal growth factor.The study by Zheng et al. [40] found that AGRN is involved in baseline refractive development, demonstrating that this gene interacts with EGR1, which is implicated in refractive development and regulates synaptic physiology in the retina [15,40].Several studies of different populations have associated rare or infrequent heterozygous alterations in AGRN with the development of HM [40].
The GRM6 gene is involved in ON synaptic transmission in bipolar cells, controlling the release of dopamine, which has been suggested as a factor in ocular growth.Mutations in this gene cause CSNB type 1B.HM is often seen in CSNB type 1B patients with altered GRM6.A link between GRM6 and susceptibility to HM has been suggested in a number of studies [41].
Alterations in the CNTN6 gene may be involved in the development of EoHM, as contactin-6 is responsible for the correct development and maintenance of the central nervous system, particularly in axonal projection, branching and synapses.Meguro et al., associated an altered expression of this gene affecting GABA receptor levels or synaptogenesis with HM [42].FRMD4B is also involved at the retinal level, through direct interaction of the protein it encodes with cytohesin-3, and functions as a scaffolding protein.Cytohesin-3 plays an important role in insulin, epidermal and nerve growth factor signaling, with a recent study [42] suggesting that the FRMD4B-cytohesin-3 complex affects cell junction dynamics in the retina and contributes to the regulation of photoreceptor cell growth and development [42].
LRP1 affects the sclera, encoding protein 1 of the low-density lipoprotein receptor family of cytokines and growth factors, and is involved in several cellular processes, even the negative regulation of gene expression [14].LRP1 deficiency leads to the disruption of TGF-β and may result in abnormal remodeling of the ECM of the developing eye, making this gene a candidate for HM [43].
Further studies are needed to demonstrate the direct involvement of these genes with EoHM for an accurate diagnosis, but for now, they are good candidates.Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00223 Family
Candidate genes on Table 12.In the analysis of this family, we studied the alterations present in the two affected individuals.The protein encoded by USH2A is abundantly expressed in the macular and peripheral retina, mainly by photoreceptors.Alterations in this gene have been associated with Usher syndrome type IIa and retinitis pigmentosa.In a WES study of 20 patients with EoHM, USH2A was identified as a candidate gene, with pathogenic variants in four participants [19].ALKBH5 enables mRNA N6-methyladenosine dioxygenase activity, is involved in the response to hypoxia [15], and a decrease in its expression has been observed in individuals with EoHM [44].
This study proposes the involvement of both alterations in the development of EoHM.Het: heterozygous; AR: autosomal recessive.

OFT-00253 Family
Candidate genes on Table 13.In this family, we detected four genes that had not been observed in the other families studied.CEP290 encodes the centrosomal protein 290, which is highly expressed in the retina of individuals with HM, being predominant in the retinal photoreceptors.Wan et al., proposed this gene as a candidate for causing EoHM [19].
CPSF1 is known to play a role in mRNA processing; however, the relationship between CPSF1 and human eye diseases, including myopia, remains unknown.Results from Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00223 Family
Candidate genes on Table 12.In the analysis of this family, we studied the alterations present in the two affected individuals.The protein encoded by USH2A is abundantly expressed in the macular and peripheral retina, mainly by photoreceptors.Alterations in this gene have been associated with Usher syndrome type IIa and retinitis pigmentosa.In a WES study of 20 patients with EoHM, USH2A was identified as a candidate gene, with pathogenic variants in four participants [19].ALKBH5 enables mRNA N6-methyladenosine dioxygenase activity, is involved in the response to hypoxia [15], and a decrease in its expression has been observed in individuals with EoHM [44].Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00223 Family
Candidate genes on Table 12.In the analysis of this family, we studied the alterations present in the two affected individuals.The protein encoded by USH2A is abundantly expressed in the macular and peripheral retina, mainly by photoreceptors.Alterations in this gene have been associated with Usher syndrome type IIa and retinitis pigmentosa.In a WES study of 20 patients with EoHM, USH2A was identified as a candidate gene, with pathogenic variants in four participants [19].ALKBH5 enables mRNA N6-methyladenosine dioxygenase activity, is involved in the response to hypoxia [15], and a decrease in its expression has been observed in individuals with EoHM [44].
This study proposes the involvement of both alterations in the development of EoHM.Het: heterozygous; AR: autosomal recessive.

OFT-00253 Family
Candidate genes on Table 13.In this family, we detected four genes that had not been observed in the other families studied.CEP290 encodes the centrosomal protein 290, which is highly expressed in the retina of individuals with HM, being predominant in the retinal photoreceptors.Wan et al., proposed this gene as a candidate for causing EoHM [19].
CPSF1 is known to play a role in mRNA processing; however, the relationship between CPSF1 and human eye diseases, including myopia, remains unknown.Results from This study proposes the involvement of both alterations in the development of EoHM.

OFT-00253 Family
Candidate genes on Table 13.In this family, we detected four genes that had not been observed in the other families studied.CEP290 encodes the centrosomal protein 290, which is highly expressed in the retina of individuals with HM, being predominant in the retinal photoreceptors.Wan et al., proposed this gene as a candidate for causing EoHM [19].
CPSF1 is known to play a role in mRNA processing; however, the relationship between CPSF1 and human eye diseases, including myopia, remains unknown.Results from several studies suggest that mutations in CPSF1 may be a new cause of EoHM [40,45,46].OPN4 encodes melanopsin, a photoreceptor opsin expressed in retinal ganglion and amacrine cells.These are necessary for correct refractive development and protection from myopia progression, as they are involved in the eye's response to myopigenic stimuli, acting in part through dopaminergic mechanisms [47].
The proband was double heterozygous for the MYOM1 gene, which is located at the MYP2 locus and is a structural constituent of the cytoskeleton thought to integrate the thin and thick filaments and confer elasticity to the M-band of the sarcomere in striated muscle.Alterations in this gene have been associated with the development of HM [48].several studies suggest that mutations in CPSF1 may be a new cause of EoHM [40,45,46].OPN4 encodes melanopsin, a photoreceptor opsin expressed in retinal ganglion and amacrine cells.These are necessary for correct refractive development and protection from myopia progression, as they are involved in the eye's response to myopigenic stimuli, acting in part through dopaminergic mechanisms [47].
The proband was double heterozygous for the MYOM1 gene, which is located at the MYP2 locus and is a structural constituent of the cytoskeleton thought to integrate the thin and thick filaments and confer elasticity to the M-band of the sarcomere in striated muscle.Alterations in this gene have been associated with the development of HM [48].Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00268 Family
Candidate genes on Table 14.The proband, his mother (supposedly unaffected) and grandmother (affected) presented two alterations in the CSMD1 gene that compromise its function and could be the cause of EoHM.Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00268 Family
Candidate genes on Table 14.The proband, his mother (supposedly unaffected) and grandmother (affected) presented two alterations in the CSMD1 gene that compromise its function and could be the cause of EoHM.several studies suggest that mutations in CPSF1 may be a new cause of EoHM [40,45,46].OPN4 encodes melanopsin, a photoreceptor opsin expressed in retinal ganglion and amacrine cells.These are necessary for correct refractive development and protection from myopia progression, as they are involved in the eye's response to myopigenic stimuli, acting in part through dopaminergic mechanisms [47].
The proband was double heterozygous for the MYOM1 gene, which is located at the MYP2 locus and is a structural constituent of the cytoskeleton thought to integrate the thin and thick filaments and confer elasticity to the M-band of the sarcomere in striated muscle.Alterations in this gene have been associated with the development of HM [48].Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.

OFT-00268 Family
Candidate genes on Table 14.The proband, his mother (supposedly unaffected) and grandmother (affected) presented two alterations in the CSMD1 gene that compromise its function and could be the cause of EoHM.Het: Heterozygous.

OFT-00332 Family
Candidate genes on Table 15.Most of the genes proposed in this family may have AD inheritance and affected individuals at different levels: retina (ZNF644 and ARHGEF15), choroid (CFH) and sclera (HSPG2 and LRP1), as well as CPSF1, which is known to be involved in the pathology although its exact cause is unknown.ZNF644 encodes a zinc finger transcription factor in the RPE, regulating genes involved in ocular development.An alteration in this gene may therefore impact the structure of the eye, leading to the progression of EoHM.It has been related to the non-syndromic form of EoHM in several studies with an AD model of inheritance [15,49,50].ARHGEF15, a guanine nucleotide exchange factor specific for RhoA, has been found to activate VEGF-induced Cdc42, promoting retinal angiogenesis [51].leading to the progression of EoHM.It has been related to the non-syndromic form of EoHM in several studies with an AD model of inheritance [15,49,50].ARHGEF15, a guanine nucleotide exchange factor specific for RhoA, has been found to activate VEGF-induced Cdc42, promoting retinal angiogenesis [51].
CFH has antioxidant effects and regulates caspase-dependent apoptosis in retinal pigment epithelial cells under oxidative stress.This gene also blocks the pro-inflammatory effects of malondialdehyde, a major product of lipid peroxidation, and protects against oxidative stress in vivo in mice.Statistically significant higher levels of CFH have been observed in HM patients versus mild myopia and control groups, being greater in eyes with choroidal atrophy and its neovascularization, suggesting that it plays a key role in the development of myopia [52].

OFT-00403 Family
Candidate genes on Table 16.All three family members shared a single candidate variant, TSG101, which has a role in cell growth and differentiation and may act as a negative growth regulator.In the study by Le et al., the loss of TSG101 severely altered the polarity of the RPE, forming irregular aggregates with a non-polarized distribution of cell adhesion proteins and the activation of epidermal growth factor receptor signaling [53].In this particular case, the variant has a low allele frequency in the overall population and is synonymous; in an in silico splicing analysis using Alamut Visual 2.15 software, it was found unaltered by the variant.TSG101 could therefore be a candidate for EoHM; however, further studies of this gene and its variants with other genes are necessary to determine the cause of the pathology.CFH has antioxidant effects and regulates caspase-dependent apoptosis in retinal pigment epithelial cells under oxidative stress.This gene also blocks the pro-inflammatory effects of malondialdehyde, a major product of lipid peroxidation, and protects against oxidative stress in vivo in mice.Statistically significant higher levels of CFH have been observed in HM patients versus mild myopia and control groups, being greater in eyes with choroidal atrophy and its neovascularization, suggesting that it plays a key role in the development of myopia [52].

OFT-00403 Family
Candidate genes on Table 16.All three family members shared a single candidate variant, TSG101, which has a role in cell growth and differentiation and may act as a negative growth regulator.In the study by Le et al., the loss of TSG101 severely altered the polarity of the RPE, forming irregular aggregates with a non-polarized distribution of cell adhesion proteins and the activation of epidermal growth factor receptor signaling [53].In this particular case, the variant has a low allele frequency in the overall population and is synonymous; in an in silico splicing analysis using Alamut Visual 2.15 software, it was found unaltered by the variant.TSG101 could therefore be a candidate for EoHM; however, further studies of this gene and its variants with other genes are necessary to determine the cause of the pathology.Het: heterozygous.

OFT-00429 Family
Candidate genes on Table 17.The proband in this family had two deletions in the HSPG2 gene found in previous families.As the alterations are close to each other, it may be observed that they are in cis, which means they were inherited from the same parent.
This individual also had an altered LRPAP1 gene, which encodes a chaperone of LRP1, inhibits its degradation and influences the activity of TGF-β.An LRP1 deficiency Het: heterozygous.

OFT-00429 Family
Candidate genes on Table 17.The proband in this family had two deletions in the HSPG2 gene found in previous families.As the alterations are close to each other, it may be observed that they are in cis, which means they were inherited from the same parent.
This individual also had an altered LRPAP1 gene, which encodes a chaperone of LRP1, inhibits its degradation and influences the activity of TGF-β.An LRP1 deficiency and up-regulation of TGF-β have been observed in individuals with EoHM.TGF-β plays an important role in scleral ECM remodeling in myopia, and LRPAP1 has been linked to EoHM in several studies [43,54].
MMP9 is another gene involved in scleral ECM composition, implicated in the breakdown of type IV and V collagens [15,55].Higher levels of MMP9 have been found to reduce scleral elastin, making it more prone to deformation under intraocular pressure, linking it to the development of myopia and HM [55,56].
Finally, affecting the retina, we observed alterations in the FLRT3 gene, which is involved in various signaling pathways and has been related to the development of the central nervous system and the eye due to its expression during ocular development in mouse embryos.It has also been associated with the development of HM in Central European families [57].Het: heterozygous.

OFT-00429 Family
Candidate genes on Table 17.The proband in this family had two deletions in the HSPG2 gene found in previous families.As the alterations are close to each other, it may be observed that they are in cis, which means they were inherited from the same parent.
This individual also had an altered LRPAP1 gene, which encodes a chaperone of LRP1, inhibits its degradation and influences the activity of TGF-β.An LRP1 deficiency and up-regulation of TGF-β have been observed in individuals with EoHM.TGF-β plays an important role in scleral ECM remodeling in myopia, and LRPAP1 has been linked to EoHM in several studies [43,54].
MMP9 is another gene involved in scleral ECM composition, implicated in the breakdown of type IV and V collagens [15,55].Higher levels of MMP9 have been found to reduce scleral elastin, making it more prone to deformation under intraocular pressure, linking it to the development of myopia and HM [55,56].
Finally, affecting the retina, we observed alterations in the FLRT3 gene, which is involved in various signaling pathways and has been related to the development of the central nervous system and the eye due to its expression during ocular development in mouse embryos.It has also been associated with the development of HM in Central European families [57].

OFT-00474 Family
Candidate genes on Table 18.All the alterations selected as candidate genes in this proband were inherited from his father.The importance of LAMA1 and FRMPD1 in the development of EoHM has been discussed above.The only novel gene is PRIMPOL, a DNA primase-polymerase that facilitates DNA damage tolerance by mediating

OFT-00474 Family
Candidate genes on Table 18.All the alterations selected as candidate genes in this proband were inherited from his father.The importance of LAMA1 and FRMPD1 in the development of EoHM has been discussed above.The only novel gene is PRIM-POL, a DNA primase-polymerase that facilitates DNA damage tolerance by mediating uninterrupted fork progression after UV irradiation and reinitiating DNA synthesis [15].PRIMPOL mRNA, which is expressed in many tissues including scleral fibroblasts, retinal epithelial and Müller cells, has been associated with the development of HM in several studies [58][59][60].uninterrupted fork progression after UV irradiation and reinitiating DNA synthesis [15].PRIMPOL mRNA, which is expressed in many tissues including scleral fibroblasts, retinal epithelial and Müller cells, has been associated with the development of HM in several studies [58][59][60].

OFT-00477 Family
Candidate genes on Table 19.The only candidate gene for the development of EoHM in this family was TSG101.This gene and its synonymous variant are the same as those observed in Family OFT-00403.Further study could be interesting to confirm or rule out its role in the pathology.Het: heterozygous.

OFT-00506 Family
Candidate genes on Table 20.In this family, we identified a single candidate alteration in the COL9A3 gene.This gene was also altered in family OFT-00155 and has an AD inheritance pattern, making it a good candidate.Het: heterozygous.

OFT-00477 Family
Candidate genes on Table 19.The only candidate gene for the development of EoHM in this family was TSG101.This gene and its synonymous variant are the same as those observed in Family OFT-00403.Further study could be interesting to confirm or rule out its role in the pathology.uninterrupted fork progression after UV irradiation and reinitiating DNA synthesis [15].PRIMPOL mRNA, which is expressed in many tissues including scleral fibroblasts, retinal epithelial and Müller cells, has been associated with the development of HM in several studies [58][59][60].

OFT-00477 Family
Candidate genes on Table 19.The only candidate gene for the development of EoHM in this family was TSG101.This gene and its synonymous variant are the same as those observed in Family OFT-00403.Further study could be interesting to confirm or rule out its role in the pathology.Het: heterozygous.

OFT-00506 Family
Candidate genes on Table 20.In this family, we identified a single candidate alteration in the COL9A3 gene.This gene was also altered in family OFT-00155 and has an AD inheritance pattern, making it a good candidate.Het: heterozygous.Het: heterozygous.

OFT-00506 Family
Candidate genes on Table 20.In this family, we identified a single candidate alteration in the COL9A3 gene.This gene was also altered in family OFT-00155 and has an AD inheritance pattern, making it a good candidate.uninterrupted fork progression after UV irradiation and reinitiating DNA synthesis [15].PRIMPOL mRNA, which is expressed in many tissues including scleral fibroblasts, retinal epithelial and Müller cells, has been associated with the development of HM in several studies [58][59][60].

OFT-00477 Family
Candidate genes on Table 19.The only candidate gene for the development of EoHM in this family was TSG101.This gene and its synonymous variant are the same as those observed in Family OFT-00403.Further study could be interesting to confirm or rule out its role in the pathology.Het: heterozygous.

OFT-00506 Family
Candidate genes on Table 20.In this family, we identified a single candidate alteration in the COL9A3 gene.This gene was also altered in family OFT-00155 and has an AD inheritance pattern, making it a good candidate.Het: heterozygous.Het: heterozygous.

OFT-00546 Family
Candidate genes on Table 21.The results for this family, like others in this study, suggest the cumulative effect of several altered genes involved in the development of EoHM inherited from both parents, although it includes genes that had not been observed in previous families.ABCA4 is expressed in the outer segments of the cone and rod photoreceptors of the retina and in the sclera, mediating the transport of an essential molecule, all-trans-retinal aldehyde, across the photoreceptor cell membrane upon activation following phototransduction.Mutations in ABCA4 can lead to multiple vision-related phenotypes, including retinitis pigmentosa, fundus flavimaculatus, cone-rod dystrophy and Stargardt disease.A variant in this gene responsible for myopia has recently been discovered, and has been proposed in the study by Wan et al., as a candidate gene for the development of EoHM [19].notypes, including retinitis pigmentosa, fundus flavimaculatus, cone-rod dystrophy and Stargardt disease.A variant in this gene responsible for myopia has recently been discovered, and has been proposed in the study by Wan et al., as a candidate gene for the development of EoHM [19].
LAMA2 encodes the alpha-2 subunit of laminin, a major component of the basement membrane, with an important role in connecting the collagen fibers of the sclera.This gene has been linked to refractive error and myopia in different ethnic populations [61,62].It may be that altered LAMA2 and LAMA5 have a greater effect on the development of EoHM than if they were isolated events, as observed in family OFT-00155t with LAMA1 and LAMA5.
Alterations were also found in the LTBP2 gene.This gene belongs to the family of latent TGF-β binding proteins, which are ECM proteins with a multidomain structure.It has been associated with Weill-Marchesani 3 and Stickler syndromes [15].

OFT-00586 Family
Candidate genes on Table 22.The alterations observed in this proband and her son affecting the development of EoHM primarily involve the retina.In addition to LTBP2 and TSG101, which were also altered in previous families, this family presented altered BICC1.BICC1, located at the MYP15 locus [63], encodes an RNA-binding protein that regulates expression [15] and has been associated with the development of HM in several studies among different populations [63,64].Alterations were also found in CNTN4, which encodes contactin-4, a glycosylphosphatidylinositol-anchored neuronal membrane protein that may play a role in the formation of axonal connections and arborization in LAMA2 encodes the alpha-2 subunit of laminin, a major component of the basement membrane, with an important role in connecting the collagen fibers of the sclera.This gene has been linked to refractive error and myopia in different ethnic populations [61,62].It may be that altered LAMA2 and LAMA5 have a greater effect on the development of EoHM than if they were isolated events, as observed in family OFT-00155t with LAMA1 and LAMA5.
Alterations were also found in the LTBP2 gene.This gene belongs to the family of latent TGF-β binding proteins, which are ECM proteins with a multidomain structure.It has been associated with Weill-Marchesani 3 and Stickler syndromes [15].

OFT-00586 Family
Candidate genes on Table 22.The alterations observed in this proband and her son affecting the development of EoHM primarily involve the retina.In addition to LTBP2 and TSG101, which were also altered in previous families, this family presented altered BICC1.BICC1, located at the MYP15 locus [63], encodes an RNA-binding protein that regulates expression [15] and has been associated with the development of HM in several studies among different populations [63,64].Alterations were also found in CNTN4, which encodes contactin-4, a glycosylphosphatidylinositol-anchored neuronal membrane protein that may play a role in the formation of axonal connections and arborization in the developing nervous system.CNTN4 has been associated with HM in the literature [42].Het: heterozygous; AD: autosomal dominant; AR: autosomal recessive.
In our analysis of families with EoHM, we found that most of the alterations identified were classified as VUS, reflecting the need for further studies to determine the pathogenicity of these variants and to confirm the involvement of the proposed genes in the pathology.Table A2 shows a summary of the different variants found in each family, its In our analysis of families with EoHM, we found that most of the alterations identified were classified as VUS, reflecting the need for further studies to determine the pathogenicity of these variants and to confirm the involvement of the proposed genes in the pathology.Table A2 shows a summary of the different variants found in each family, its classification, functional annotation and frequencies in the gnomAD global population.We also added the main pathways related to each gene.
The remaining 80.85% of genes are associated with various other pathologies.However, this does not imply that they cannot contribute to EoHM, as previously discussed, since they play a role in the development of the eye and ocular structures and have been considered candidates in prior studies.
Figure 7 illustrates that most of the identified variants are missense mutations, accounting for 83% of them.This can explain the substantial proportion of variants classified as VUS observed previously.Missense variants can have a variable impact on the function of the protein encoded by the gene by altering a single amino acid and, moreover, not all options are covered.In contrast, stop-gained variants are far more likely to result in a complete loss of protein function, so they are rarely classified as VUS.Notably, most alterations in the probands were primarily concentrated in the retina or sclera (Figure 8), an observation that coincides with the literature.
The main optical function of the crystalline lens is to transmit light, focusing it on the retina [65].The retina is a layer of photoreceptor cells and glial cells that captures incoming photons from the lens, it can discern whether the perceived image is blurred or not, and it transmits the photons along neuronal pathways as both electrical and chemical signals for the brain to perceive a visual picture [66].The choroid supplies the outer retina with nutrients and maintains the temperature and volume of the eye [67].And the sclera influences eye size, facilitating the excessive axial elongation that occurs during myopigenesis [68].If the variants impact genes that are expressed and have a function in ocular structures, their function may become compromised, leading to failures in processes such as visual acuity, light detection and regulation of ocular axial growth, resulting in a larger axial length in most of them.Among these, axial growth is particularly critical in the pathology under consideration.Notably, most alterations in the probands were primarily concentrated in the retina or sclera (Figure 8), an observation that coincides with the literature.influences eye size, facilitating the excessive axial elongation that occurs during myopigenesis [68].If the variants impact genes that are expressed and have a function in ocular structures, their function may become compromised, leading to failures in processes such as visual acuity, light detection and regulation of ocular axial growth, resulting in a larger axial length in most of them.Among these, axial growth is particularly critical in the pathology under consideration.The main optical function of the crystalline lens is to transmit light, focusing it on the retina [65].The retina is a layer of photoreceptor cells and glial cells that captures incoming photons from the lens, it can discern whether the perceived image is blurred or not, and it transmits the photons along neuronal pathways as both electrical and chemical signals for the brain to perceive a visual picture [66].The choroid supplies the outer retina with nutrients and maintains the temperature and volume of the eye [67].And the sclera influences eye size, facilitating the excessive axial elongation that occurs during myopigenesis [68].If the variants impact genes that are expressed and have a function in ocular structures, their function may become compromised, leading to failures in processes such as visual acuity, light detection and regulation of ocular axial growth, resulting in a larger axial length in most of them.Among these, axial growth is particularly critical in the pathology under consideration.
Several genes were found to be altered in more than one family, suggesting a stronger association with the shared pathology.Genes with alterations in two or more families were TRPM1, KDM6B, HSPG2, CACNA1F, CSMD1, LAMA1, COL9A3, LAMA5, THBS2, PCDH15, FRMPD1, AGRN, LRP1, USH2A, CPSF1, TSG101, FLRT3 and LTBP2.Specifically, HSPG2 was altered in six of the twenty-one families, while CSMD1 was altered in four, being the only candidate gene in several of them.
Although EoHM does not depend exclusively on the presence of alterations in these genes, it is influenced by the degree of penetrance, which was incomplete in some cases and unknown in others.Development of this pathology is also determined by the interaction and cumulative effect of these genes, with a genetic background affecting various levels of the multilayer signaling cascade (Figures 3 and 8).Some genetic alterations may not have a significant impact in isolation in healthy individuals, but may have a more pronounced effect in the case of probands when combined with other alterations, thereby contributing to the development of the disease.
In addition to their biological function, most of the genes we propose have also been identified as candidates for EoHM in independent studies, supporting their involvement here.

Materials and Methods
A combined ophthalmological and genetic study was performed by the Multidisciplinary Unit of Ophthalmogenetics (UMOG) of the La Paz University Hospital, in accordance with the principles of the Declaration of Helsinki, and was approved by the ethics committee.
The inclusion criteria in our study were the following: (1) bilateral myopia with a refractive error of ≤−6 diopters in at least one eye with onset before the age of 10 years; (2) inconclusive result in the massive sequencing study implementing a panel (OFT-v3-1) of 419 genes related to ophthalmological disorders with suspected genetic cause, 93 genes and regions related to the pathogenesis of EoHM or within loci related to EoHM; (3) absence of syndromic phenotype; and (4) absence of corneal disease or other ophthalmologic diseases leading to secondary high myopia.In addition, whenever possible, parents and other relatives of the patients were included in the study.
A total of 63 individuals from 21 unrelated families were recruited in accordance with the inclusion criteria, including 30 affected individuals aged between 6 and 80 years old.Once informed written consent was obtained from the probands and their parents or guardians, they entered the study and followed the workflow represented in Figure 9.The clinical ophthalmologic evaluation of patients and first-degree relatives who signed the consent form was performed by the Ophthalmology unit of Hospital La Paz (HULP-3576).Participants first underwent a complete ophthalmological evaluation, including bestcorrected visual acuity, refraction before and after cycloplegia, funduscopic examination, LA measurement, retinography and OCT imaging.
A genetic study was then performed on genomic DNA obtained from leukocytes isolated from a peripheral venous blood sample in the pre-analytical area of our institute using the Chemagic Magnetic Separation Module I (Chemagen, PerkinElmer, Waltham, MA, USA).Concentrations of the isolated DNA were quantified using a NanoDrop ND-1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).Library preparation was carried out using Nextera DNA Exome (Illumina DNA Prep with Enrichment) and IDT for Illumina DNA/RNA UD Indexes Set A, B, C or D, Tagmentation.Sequencing was performed on high-quality sequencers, HiSeq4000 and NovaSeq6000, capturing 19,433 genes using xGen™ Exome Research Panel v2 IDT.
The Clinical Bioinformatics team at the Institute of Medical and Molecular Genetics (INGEMM) then performed the first analysis of the sequences obtained using an analytical algorithm developed to identify single nucleotide polymorphisms (SNPs) and insertions and deletions of small DNA fragments (indels) within the exome capture regions.This process comprises a sample pre-processing step, alignment of reads to a reference genome, identification and functional annotation of variants, and filtering of those variants.All these steps employ open tools widely used in the scientific community as well as proprietary tools.In addition, the steps are robustly designed and include process parameters and adequate quality controls to deliver a reliable report on the variants in question.
After sequencing, basecall file conversion (BCL) to FASTQ files was performed using Illumina's demultiplexing software (bcl2fastq2 v2.20.0).The resulting DNA sequence reads were aligned and mapped to the human genome reference sequence (GRCh37/hg19) using bowtie2-align v2.0.6, after pre-processing and trimming using Trimmomatic v0.36 software.Realignment and recalibration of the reads were performed using the Genome The method followed for the statistical analysis was a Fisher's exact test to determine whether severity is dependent on gender, with the following hypotheses: Null Hypothesis (H 0 ): Severity and gender are independent variables.
Alternative Hypothesis (H 1 ): Severity and gender are dependent variables.The Fisher's exact test was conducted using the 'fisher.test()'function in RStudio.
Participants first underwent a complete ophthalmological evaluation, including bestcorrected visual acuity, refraction before and after cycloplegia, funduscopic examination, LA measurement, retinography and OCT imaging.
A genetic study was then performed on genomic DNA obtained from leukocytes isolated from a peripheral venous blood sample in the pre-analytical area of our institute using the Chemagic Magnetic Separation Module I (Chemagen, PerkinElmer, Waltham, MA, USA).Concentrations of the isolated DNA were quantified using a NanoDrop ND-1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).Library preparation was carried out using Nextera DNA Exome (Illumina DNA Prep with Enrichment) and IDT for Illumina DNA/RNA UD Indexes Set A, B, C or D, Tagmentation.Sequencing was performed on high-quality sequencers, HiSeq4000 and NovaSeq6000, capturing 19,433 genes using xGen™ Exome Research Panel v2 IDT.
The Clinical Bioinformatics team at the Institute of Medical and Molecular Genetics (INGEMM) then performed the first analysis of the sequences obtained using an analytical algorithm developed to identify single nucleotide polymorphisms (SNPs) and insertions and deletions of small DNA fragments (indels) within the exome capture regions.This process comprises a sample pre-processing step, alignment of reads to a reference genome, identification and functional annotation of variants, and filtering of those variants.All these steps employ open tools widely used in the scientific community as well as proprietary tools.In addition, the steps are robustly designed and include process parameters and adequate quality controls to deliver a reliable report on the variants in question.
The second analysis consisted of assessing the clinical significance of the variants found in the patients, relating them to their phenotype.To do so, we first filtered the data from bioinformatics using the criteria indicated in Table 25.

PM3
For recessive disorders, detected in trans with a pathogenic variant.

PM4
Protein length changes as a result of in-frame deletions/insertions in a non-repeat region or stop-loss variants.

PM5
Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been previously observed.

PP1
Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease.

PP2
Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease.

PP3
Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.)

PP5
Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

BP4
Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.)

BP6
Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.Positive regulation of MAP kinase activity, negative regulation of endothelial cell proliferation, negative regulation of cell-matrix adhesion, negative regulation of angiogenesis, collagen-containing extracellular matrix, fibronectin binding, integrin binding, extracellular matrix structural constituent, calcium ion binding, fibroblast growth factor binding, laminin binding, proteoglycan binding, transforming growth factor beta binding, fibrinogen binding, collagen V binding, response to hypoxia, sprouting angiogenesis, chronic inflammatory response, apoptotic process, inflammatory response, positive regulation of fibroblast migration, negative regulation of fibroblast growth factor receptor signaling pathway, positive regulation of extrinsic apoptotic signaling pathway via death domain receptors, positive regulation of reactive oxygen species metabolic process, extracellular region, extracellular space, extracellular matrix, fibrinogen complex NM_003247.Collagen-containing extracellular matrix, extracellular matrix structural constituent, glycosphingolipid binding, morphogenesis of an epithelial sheet, neuron projection development, establishment of epithelial cell apical/basal polarity, retinal blood vessel morphogenesis, positive regulation of integrin-mediated signaling pathway, extracellular region, extracellular space, extracellular matrix, collagen-containing extracellular matrix, protein complex involved in cell-matrix adhesion, laminin-1 complex, laminin-3 complex NM_152683.Photoreceptor inner segment, photoreceptor connecting cilium, neuronal cell body, collagen binding, visual perception, photoreceptor cell maintenance, sensory perception of light stimulus, extracellular region, USH2 complex Variants found in each family with its classification, functional annotation, frequencies in gnomAD global population and main pathways related to each gene affected.

Figure 1 .
Figure 1.Schematic optics of the eye.In emmetropic eyes, the parallel rays of a distant object are focused precisely on the photoreceptors located on the retina.Myopic eyes have longer axial length, and the image of a distant object falls in front of the photoreceptors and cannot be brought into focus by accommodation.The myopic eye can become highly myopic, accentuating this defocus considerably.Modified from Morgan et al. [5].

43 Figure 2 .
Figure 2. Graph representing the number of people estimated to develop myopia and HM for each decade from 2000 to 2050 [6].

Figure 2 .
Figure 2. Graph representing the number of people estimated to develop myopia and HM for each decade from 2000 to 2050 [6].

Figure 3 .
Figure 3. Representation of the multilayer signaling cascade that enables axial elongation.The signaling cascade order is represented by the numbers 1 to 4 Modified from Tedja et al. [7].

Figure 3 .
Figure 3. Representation of the multilayer signaling cascade that enables axial elongation.The signaling cascade order is represented by the numbers 1 to 4 Modified from Tedja et al. [7].

Figure 4 .
Figure 4. Representation of the proportions of probands younger and older than 10 years old.

Figure 4 .
Figure 4. Representation of the proportions of probands younger and older than 10 years old.

Figure 5 .
Figure 5. Clinical representation of probands.The proportions of features presented by the probands in this study are shown here, with their corresponding percentage.

Figure 5 .
Figure 5. Clinical representation of probands.The proportions of features presented by the probands in this study are shown here, with their corresponding percentage.

Figure 6 .
Figure 6.Representation of the variant pathogenicity found in possible candidate genes for EoHM.

Figure 6 .
Figure 6.Representation of the variant pathogenicity found in possible candidate genes for EoHM.

43 Figure 7 .
Figure 7. Representation of the functional annotation of variants and their proportion.

Figure 7 .
Figure 7. Representation of the functional annotation of variants and their proportion.

Figure 8 .
Figure 8. Representation of the affected ocular structures and their corresponding percentage .Retina structures are represented in blue, sclera structures in purple, choroid in red, crystalline lens in yellow, collagen in green and others in gray.

Figure 8 .
Figure 8. Representation of the affected ocular structures and their corresponding percentage.Retina structures are represented in blue, sclera structures in purple, choroid in red, crystalline lens in yellow, collagen in green and others in gray.

Table 1 .
Clinical evaluation of probands.
Results are presented as mean ± standard deviation.
Results are presented as mean ± standard deviation.

Table 4 .
Candidate genes of the OFT-00074 family.

Table 4 .
Candidate genes of the OFT-00074 family.

Table 5 .
Candidate genes of the OFT-00097 family.

Table 5 .
Candidate genes of the OFT-00097 family.

Table 6 .
Candidate genes of the OFT-00155 family.

Table 6 .
Candidate genes of the OFT-00155 family.

Table 7 .
Candidate genes of the OFT-00175 family.

ACMG Criteria ACMG Result Variant Zygosity Inheritance Total Families Model of In- heritance Gene Re- ported By
Het: heterozygous; Hemi: hemizygous; AD: autosomal dominant; AR: autosomal recessive; XL: X-linked.* Alterations shared with his brother (he has idiopathic motor nystagmus).

Table 7 .
Candidate genes of the OFT-00175 family.

Table 6 .
Candidate genes of the OFT-00155 family.

Table 7 .
Candidate genes of the OFT-00175 family.

Table 8 .
Candidate genes of the OFT-00178 family.

Table 8 .
Candidate genes of the OFT-00178 family.

Table 8 .
Candidate genes of the OFT-00178 family.

Table 9 .
Candidate genes of the OFT-00191 family.

Table 9 .
Candidate genes of the OFT-00191 family.

Table 10 .
Candidate genes of the OFT-00209 family.

Table 10 .
Candidate genes of the OFT-00209 family.

Table 11 .
Candidate genes of the OFT-00217 family.

Table 11 .
Candidate genes of the OFT-00217 family.

Table 12 .
Candidate genes of the OFT-00223 family.

Table 11 .
Candidate genes of the OFT-00217 family.

Table 12 .
Candidate genes of the OFT-00223 family.

Table 13 .
Candidate genes of the OFT-00253 family.

Table 13 .
Candidate genes of the OFT-00253 family.

Table 14 .
Candidate gene of the OFT-00268 family.

Table 14 .
Candidate gene of the OFT-00268 family.

Table 13 .
Candidate genes of the OFT-00253 family.

Table 14 .
Candidate gene of the OFT-00268 family.

Table 15 .
Candidate genes of the OFT-00332 family.

Table 15 .
Candidate genes of the OFT-00332 family.

Table 16 .
Candidate genes of the OFT-00403 family.

Table 16 .
Candidate genes of the OFT-00403 family.

Table 17 .
Candidate genes of the OFT-00429 family.

Table 17 .
Candidate genes of the OFT-00429 family.

Table 18 .
Candidate genes of the OFT-00474 family.

Table 18 .
Candidate genes of the OFT-00474 family.

Table 19 .
Candidate gene of the OFT-00477 family.

Table 20 .
Candidate gene of the OFT-00506 family.

Table 19 .
Candidate gene of the OFT-00477 family.

Table 18 .
Candidate genes of the OFT-00474 family.

Table 19 .
Candidate gene of the OFT-00477 family.

Table 20 .
Candidate gene of the OFT-00506 family.

Table 18 .
Candidate genes of the OFT-00474 family.

Table 19 .
Candidate gene of the OFT-00477 family.

Table 20 .
Candidate gene of the OFT-00506 family.

Table 21 .
Candidate genes of the OFT-00546 family.

Table 21 .
Candidate genes of the OFT-00546 family.

Table 24 .
Candidate genes of the OFT-00710 family.

Table 25 .
Filters used after the bioinformatic analysis.

Table A2 .
Cont.Collagen-containing extracellular matrix, amyloid-beta binding, calcium ion binding, extracellular matrix structural constituent conferring compression resistance, collagen V binding, angiogenesis, response to hypoxia, positive regulation of endothelial cell proliferation, inflammatory response, negative regulation of angiogenesis, extracellular region, extracellular space,

Table A2 .
Cont.Metalloendopeptidase activity, serine-type endopeptidase activity, extracellular matrix disassembly, collagen catabolic process, cellular response to reactive oxygen species, negative regulation of intrinsic apoptotic signaling pathway, negative regulation of cation channel activity, collagen-containing extracellular matrix, extracellular exosome, extracellular matrix organization, collagen catabolic process

Table A2 .
DNA binding, transcription corepressor activity, ubiquitin protein ligase binding, negative regulation of transcription by RNA polymerase II, regulation of cell growth, extracellular transport, negative regulation of epidermal-growth-factor-activated receptor activity, negative regulation of cell population proliferation Cont.Retinoid binding, 11-cis retinal binding, all-trans retinal binding, retinol transmembrane transporter activity, ABC-type transporter activity, visual perception, phototransduction, visible light, retinol transport, retinal metabolic process, photoreceptor cell maintenance, photoreceptor outer segment, photoreceptor disc membrane, rod photoreceptor disc membrane, ATPase-coupled intramembrane lipid transporter activityCollagen-containing extracellular matrix, extracellular matrix structural constituent, laminin binding, heparan sulfate proteoglycan binding, clustering of voltage-gated sodium channels, positive regulation of transcription by RNA polymerase II, extracellular region, collagen-containing extracellular matrix, extracellular exosome, neuromuscular junction development Maintenance of blood-brain barrier, structural molecule activity, extracellular matrix structural constituent, axon guidance, positive regulation of integrin-mediated signaling pathway, extracellular region, collagen-containing extracellular matrix, protein complex involved in cell-matrix adhesion

Table A2 .
Cont.DNA binding, transcription corepressor activity, ubiquitin protein ligase binding, negative regulation of transcription by RNA polymerase II, regulation of cell growth, extracellular transport, negative regulation of epidermal-growth-factor-activated receptor activity, negative regulation of cell population proliferation