Novel LOXL3-associated stickler syndrome-like phenotype: a case report.

PURPOSE: To report the case of a young boy with early onset high myopia (eoHM), foveal hypoplasia and skeletal dysplasia due to a homozygous LOXL3 pathogenic variant. Atypically, this was from a paternal uniparental isodisomy (UPiD) of chromosome 2. CLINICAL CASE: Four-year-old boy with several months history of holding items close to his face was found to have reduced visual acuity 6/30 in both eyes, bilateral vitreous syneresis, foveal hypoplasia and bilateral high myopia (-8.50D). A skeletal survey showed spondylo-epi-metaphyseal dysplasia. Whole-exome sequencing (WES) revealed a homozygous LOXL3 variant c.1448_1449del, p.(Thr483Argfs*13), inherited through paternal UPiD of chromosome 2. CONCLUSION: To our knowledge, this is the first reported case of LOXL3-associated eoHM, foveal hypoplasia and mild skeletal dysplasia due to the rare phenomenon of paternal UPiD of chromosome 2. This case further delineates the phenotype associated with LOXL3 pathogenic variants and supports truncating LOXL3 pathogenic variants being associated with a phenotypic spectrum; from isolated eoHM through to a Stickler syndrome-like phenotype.

De novo variation in ARID1B gene causes Coffin-Siris syndrome 1 in a Chinese family with excessive early-onset high myopia.

Coffin-Siris syndrome (CSS) is a rare autosomal dominant inheritance disorder characterized by distinctive facial features, hypoplasia of the distal phalanx or nail of the fifth and additional digits, developmental or cognitive delay of varying degree, hypotonia, hirsutism/hypertrichosis, sparse scalp hair and varying kind of congenital anomalies. CSS can easily be misdiagnosed as other syndromes or disorders with a similar clinical picture because of their genetic and phenotypic heterogeneity. We describde the genotype-phenotype correlation of one patient from a healthy Chinese family with a novel genotype underlying CSS, who was first diagnosed in the ophthalmology department as early-onset high myopia (eoHM). Comprehensive ophthalmic tests as well as other systemic examinations were performed on participants to confirm the phenotype. The genotype was identified using whole exome sequencing, and further verified the results among other family members by Sanger sequencing. Real-time quantitative PCR (RT-qPCR) technology was used to detect the relative mRNA expression levels of candidate genes between proband and normal family members. The pathogenicity of the identified variant was determined by The American College of Medical Genetics and Genomics (ACMG) guidelines. STRING protein-protein interactions (PPIs) network analysis was used to detect the interaction of candidate gene-related proteins with high myopia gene-related proteins. The patient had excessive eoHM, cone-rod dystrophy, coarse face, excessive hair growth on the face, sparse scalp hair, developmental delay, intellectual disability, moderate hearing loss, dental hypoplasia, patent foramen ovale, chronic non-atrophic gastritis, bilateral renal cysts, cisterna magna, and emotional outbursts with aggression. The genetic assessment revealed that the patient carries a de novo heterozygous frameshift insertion variant in the ARID1B c.3981dup (p.Glu1328ArgfsTer5), which are strongly associated with the typical clinical features of CSS patients. The test results of RT-qPCR showed that mRNA expression of the ARID1B gene in the proband was approximately 30% lower than that of the normal control in the family, suggesting that the variant had an impact on the gene function at the level of mRNA expression. The variant was pathogenic as assessed by ACMG guidelines. Analysis of protein interactions in the STRING online database revealed that the ARID1A protein interacts with the high myopia gene-related proteins FGFR3, ASXL1, ERBB3, and SOX4, whereas the ARID1A protein antagonizes the ARID1B protein. Therefore, in this paper, we are the first to report a de novo heterozygous frameshift insertion variant in the ARID1B gene causing CSS with excessive eoHM. Our study extends the genotypic and phenotypic spectrums for ARID1B-CSS and supplies evidence of significant association of eoHM with variant in ARID1B gene. As CSS has high genetic and phenotypic heterogeneity, our findings highlight the importance of molecular genetic testing and an interdisciplinary clinical diagnostic workup to avoid misdiagnosis as some disorders with similar manifestations of CSS.

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.

Novel mutations of the X-linked genes associated with early-onset high myopia in five Chinese families.

PURPOSE: To report novel pathogenic variants of X-linked genes in five Chinese families with early-onset high myopia (eoHM) by using whole-exome sequencing and analyzing the phenotypic features. METHODS: 5 probands with X-linked recessive related eoHM were collected in Ningxia Eye Hospital from January 2021 to June 2022. The probands and their family members received comprehensive ophthalmic examinations,and DNA was abstracted from patients and family members. Whole-exome sequencing was performed on probands to screen the causative variants, and all suspected pathogenic variants were determined by Sanger sequencing and co-segregation analysis was performed on available family members. The pathogenicity of novel variants was predicted using silico analysis and evaluated according to ACMG guidelines. RT-qPCR was used to detect differences in the relative mRNAs expression of candidate gene in mRNAs available with the proband and family members in the pedigree 2. The relationship between genetic variants and clinical features was analyzed. RESULTS: All probands were male, and all pedigrees conformed to an X-linked recessive inheritance pattern. They were diagnosed with high myopia at their first visits between 4 and 7 years old. Spherical equivalent ranged between - 6.00D and - 11.00D.The five novel hemizygous variants were found in the probands, containing frameshift deletion variant c.797_801del (p.Val266Alafs*75) of OPN1LW gene in the pedigree 1, nonsense variant c.513G > A (p.Trp171Ter)of RP2 gene in the pedigree 2, missense variant c.98G > T (p.Cys33Phe) of GPR143 gene in the pedigree 3, frameshift deletion variant c.1876_1877del (p.Met626Valfs*22) of FRMD7 gene in the pedigree 4 and inframe deletion variant c.670_ 675del (p.Glu192_ Glu193del) of HMGB3 gene in the pedigree 5. All variants were classified as pathogenic or likely pathogenic by the interpretation principles of HGMD sequence variants and ACMG guidelines. In family 2, RT-qPCR showed that the mRNA expression of RP2 gene was lower in the proband than in other normal family members, indicating that such variant caused an effect on gene function at the mRNA expression level. Further clinical examination showed that pedigrees 1, 2, 3, and 4 were diagnosed as X-linked recessive hereditary eye disease with early-onset high myopia, including quiescent cone dysfunction, retinitis pigmentosa, ocular albinism, and idiopathic congenital nystagmus respectively. The pedigree 5 had eoHM in the right eye and ptosis in both eyes. CONCLUSION: In this paper,we are the first to report five novel hemizygous variants in OPN1LW, RP2, GPR143, FRMD7, HMGB3 genes are associated with eoHM. Our study extends the genotypic spectrums for eoHM and better assists ophthalmologists in assessing, diagnosing, and conducting genetic screening for eoHM.

De novo variation in EP300 gene cause Rubinstein-Taybi syndrome 2 in a Chinese family with severe early-onset high myopia.

BACKGROUND: Rubinstein-Taybi syndrome (RSTS) is characterized by distinctive facial features, broad and often angulated thumbs and halluces, short stature, and moderate-to-severe intellectual disability, classified into two types RSTS1 (CREBBP-RSTS) and RSTS2 (EP300-RSTS). More often, the clinical features are inconclusive and the diagnosis of RSTS is established in a proband with identification of a heterozygous pathogenic variant in CREBBP or EP300 to confirm the diagnosis. METHODS: In this study, to describe an association between the clinical phenotype and the genotype of a RSTS2 patient who was initially diagnosed with severe early-onset high myopia (eoHM) from a healthy Chinese family, we tested the proband of this family by whole exome sequencing (WES) and further verified among other family members by Sanger sequencing. Real-time quantitative PCR was used to detect differences in the relative mRNA expression of candidate genes available in the proband and family members. Comprehensive ophthalmic tests as well as other systemic examinations were also performed on participants with various genotypes. RESULTS: Whole-exome sequencing revealed that the proband carried the heterozygous frameshift deletion variant c.3714_3715del (p.Leu1239Glyfs*3) in the EP300 gene, which was not carried by the normal parents and young sister as verified by Sanger sequencing, indicating that the variant was de novo. Real-time quantitative PCR showed that the mRNA expression of EP300 gene was lower in the proband than in other normal family members, indicating that such a variant caused an effect on gene function at the mRNA expression level. The variant was classified as pathogenic as assessed by the interpretation principles of HGMD sequence variants and ACMG guidelines. According to ACMG guidelines, the heterozygous frameshift deletion variant c.3714_3715del (p.Leu1239Glyfs*3) in the EP300 gene was more likely the pathogenic variant of this family with RSTS2. CONCLUSIONS: Therefore, in this paper, we first report de novo heterozygous variation in EP300 causing eoHM-RSTS. Our study extends the genotypic spectrums for EP300-RSTS and better assists physicians in predicting, diagnosis, genetic counseling, eugenics guidance and gene therapy for EP300-RSTS.

Mutational investigation of 17 causative genes in a cohort of 113 families with nonsyndromic early-onset high myopia in northwestern China.

High myopia (HM) is a leading cause of visual impairment in the world. To expand the genotypic and phenotypic spectra of HM in the Chinese population, we investigated genetic variations in a cohort of 113 families with nonsyndromic early-onset high myopia from northwestern China by whole-exome sequencing, with focus on 17 known genes. Sixteen potentially pathogenic variants predicted to affect protein function in eight of seventeen causative genes for HM in fifteen (13.3%) families were revealed, including seven novel variants, c.767 + 1G > A in ARR3, c.3214C > A/p.H1072N, and c.2195C > T/p.A732V in ZNF644, c.1270G > T/p.V424L in CPSF1, c.1918G > C/p.G640R and c.2786T > G/p.V929G in XYLT1, c.601G > C/p.E201Q in P4HA2; six rare variants, c.799G > A/p.E267K in NDUFAF7, c.1144C > T/p.R382W in TNFRSF21, c.1100C > T/p.P367L in ZNF644, c.3980C > T/p.S1327L in CPSF1, c.145G > A/p.E49K and c.325G > T/p.G109W in SLC39A5; and three known variants, c.2014A > G/p.S672G and c.3261A > C/p.E1087D in ZNF644, c.605C > T/p.P202L in TNFRSF21. Ten of them were co-segregated with HM. The mean (± SD) examination age of these 15 probands was 14.7 (± 11.61) years. The median spherical equivalent was - 9.50 D (IQ - 8.75 ~ - 12.00) for the right eye and - 11.25 D (IQ - 9.25 ~ - 14.13) for the left eye. The median axial length was 26.67 mm (IQ 25.83 ~ 27.13) for the right eye and 26.25 mm (IQ 25.97 ~ 27.32) for the left eye. These newly identified genetic variations not only broaden the genetic and clinical spectra, but also offer convincing evidence that the genes ARR3, NDUFAF7, TNFRSF21, and ZNF644 contribute to hereditable HM. This work improves further understanding of molecular mechanism of HM.

Identification of a Novel Frameshift Variant of ARR3 Related to X-Linked Female-Limited Early-Onset High Myopia and Study on the Effect of X Chromosome Inactivation on the Myopia Severity.

X-linked myopia 26 (Myopia 26, MIM #301010), which is caused by the variants of ARR3 (MIM *301770), is characterized by female-limited early-onset high myopia (eo-HM). Clinical characteristics include a tigroid appearance in the fundus and a temporal crescent of the optic nerve head. At present, the limited literature on eo-HM caused by ARR3 mutations shows that its inheritance mode is complex, which brings certain difficulties to pre-pregnancy genetic counseling, pre-implantation genetic diagnosis, and prenatal diagnosis. Here, we investigated the genetic underpinning of a Chinese family with eo-HM. Whole exome sequencing of the proband revealed a novel frameshift mutation in ARR3 (NM_004312, exon10, c.666delC, p. Asn222LysfsTer22). Although the mode of inheritance of the eo-HM family fits the X-linked pattern of ARR3, the phenotypes of three patients deviate from the typical early-onset high myopia. Through X-chromosome inactivation experiments, the patient's different phenotypes can be precisely explained. In addition, this study not only enhanced the correlation between ARR3 and early-onset high myopia but also provided explanations for different phenotypes, which may inspire follow-up studies. Our results enrich the knowledge of the variant spectrum in ARR3 and provide critical information for preimplantation and prenatal genetic testing, diagnosis, and counseling.

Two novel variations in LRP2 cause Donnai-Barrow syndrome in a Chinese family with severe early-onset high myopia.

Donnai-Barrow syndrome (DBS) is a rare autosomal recessive disorder caused by mutation in the low density lipoprotein receptor-related protein 2 gene (LRP2). Defects in this protein may lead to clinical multiple organ malformations by affecting the development of organs such as the nervous system, eyes, ears, and kidneys. Although some variations on LRP2 have been found to be associated with DBS, early diagnosis and prevention of patients with atypical DBS remains a challenge for many physicians because of their clinical heterogeneity. The objective of this study is to explore the association between the clinical presentation and the genotype of a DBS patient who was initially diagnosed with early-onset high myopia (eoHM) from a healthy Chinese family. To this end, we tested the patient of this family via whole exome sequencing and further verified the results among other family members by Sanger sequencing. Comprehensive ophthalmic tests as well as other systemic examinations were also performed on participants with various genotypes. Genetic assessment revealed that two novel variations in LRP2, a de novo missense variation (c.9032G>A; p.Arg3011Lys) and a novel splicing variation (c.2909-2A>T) inherited from the father, were both carried by the proband in this family, and they are strongly associated with the typical clinical features of DBS patients. Therefore, in this paper we are the first to report two novel compound heterozygous variations in LPR2 causing DBS. Our study extends the genotypic spectrums for LPR2-DBS and better assists physicians in predicting, diagnosing, and conducting gene therapy for DBS.

Variants in HNRNPH1 are associated with high myopia in humans and ocular coloboma in zebrafish.

High myopia is one of the most common causes for blindness due to its associated complications. Genetic factor has been considered as the major cause for early-onset high myopia (eoHM), but exact genetic defects for most eoHM are yet to be identified. Through multi-step bioinformatics analysis of our in-house whole exome sequencing dataset from 6397 individuals, variants from 928 probands with eoHM were further compared with those from in-house controls as well as gnomAD database. The results showed that loss-of-function (LoF) variants in a novel gene HNRNPH1 were identified in two of 928 probands with eoHM but in none of 5469 probands with other eye conditions (p = 0.02). LoF variants in HNRNPH1 were extremely rare and intolerant, while two LoF variants in 928 eoHM were statistically higher than their frequency in gnomAD (p = 5.98 × 10-4 ). These two LoF variants, c.2dup/p.(M1?) and c.121dup/p.(Q41Pfs*20), were absent from existing database. Variants in HNRNPH1 have not been associated with any inherited eye disease before. Expression of HNRNPH1 was enriched in ganglion cell layer and inner nuclear layer in humans. Knockdown of hnrnph1 in zebrafish resulted in ocular coloboma. All these suggest that HNRNPH1 is potential contribution to eoHM when mutated.

Refractive Lens Exchange Surgery in Early-Onset High Myopia Patients With Partial Cataract.

Purpose: By reporting clinical characteristics and retinal image quality before and after refractive lens replacement surgery in early-onset high myopia (eoHM) patients presenting with partial cataract, we emphasized the need for an objective way to grade the severity of partial cataracts. Methods: This retrospective, consecutive case series included six Chinese patients (nine eyes). Analysis of previous medical records, visual acuity, optometry, retinal image quality, and axial length (AXL) before surgery and after surgery was performed. Results: Five females and one male (nine eyes) with a mean (± SD) age of 11.6 ± 7.9 years (range: 4-25 years) were included in this study. The preoperative spherical power ranged from -7.5 to -42 D. The mean follow-up time was 36 months (range: 24-48 months). Phacoemulsification was followed by in-the-bag implantation of intraocular lens. For patients who were under 6 years old, posterior capsulotomy + anterior vitrectomy were performed simultaneously. All surgeries were uneventful and no postoperative complications occurred during the entire follow-up period. All patients' uncorrected visual acuity improved by ≥2 lines postoperatively(Snellen acuity). LogMAR best-corrected visual acuity was improved at 24-month (P = 0.042) and endpoint (P = 0.046) follow-ups. Modulation transfer function cutoff frequency (MTFcutoff) and objective scatter index (OSI) was significantly improved at 12-month (P = 0.025, P = 0.038), 24-month (P = 0.005, P = 0.007) and endpoint (P = 0.005, P = 0.008) follow-ups. Postoperative AXL remained stable during 2-4 year follow-ups (P > 0.05). Conclusion: Refractive lens replacement surgery is safe and effective for improving functional vision in eoHM patients presenting with partial cataract. Retinal image quality could provide a useful and objective way to facilitate partial cataract severity evaluation and surgery decision making.

Next-Generation Sequencing Screening of 43 Families with Non-Syndromic Early-Onset High Myopia: A Clinical and Genetic Study.

Early-onset high myopia (EoHM) is a disease that causes a spherical refraction error of ≥-6 diopters before 10 years of age, with potential multiple ocular complications. In this article, we report a clinical and genetic study of 43 families with EoHM recruited in our center. A complete ophthalmological evaluation was performed, and a sample of peripheral blood was obtained from proband and family members. DNA was analyzed using a customized next-generation sequencing panel that included 419 genes related to ophthalmological disorders with a suspected genetic cause, and genes related to EoHM pathogenesis. We detected pathogenic and likely pathogenic variants in 23.9% of the families and detected variants of unknown significance in 76.1%. Of these, 5.7% were found in genes related to non-syndromic EoHM, 48.6% in genes associated with inherited retinal dystrophies that can include a syndromic phenotype, and 45.7% in genes that are not directly related to EoHM or retinal dystrophy. We found no candidate genes in 23% of the patients, which suggests that further studies are needed. We propose a systematic genetic analysis for patients with EoHM because it helps with follow-up, prognosis and genetic counseling.

Risk factors for early-onset high myopia after treatment for retinopathy of prematurity.

PURPOSE: To investigate the prevalence of myopia and high myopia and the risk factors for high myopia in infants at 3 years of age with retinopathy of prematurity (ROP). STUDY DESIGN: Retrospective, observational. METHODS: We retrospectively analyzed all 89 preterm infants (178 eyes) with medical records of ROP between October 2008 and March 2018 at Toho University Medical Center Omori Hospital; these infants had a birth weight of less than 1,500 g and were followed up at least until 3 years of age. Cycloplegic autorefraction was performed to measure refractive outcomes. Multivariate analysis was performed to determine the risk factors for early-onset high myopia at 3 years of age. RESULTS: The prevalence of myopia and high myopia was significantly higher in the treated group (59.7% and 17.9%, respectively) than in the untreated group (19.7% and 0%, respectively) (p<0.001). Spherical equivalent (SE) at age 3 was more myopic in the treatment group (-1.72 ± 3.53 D) than in the untreated group (0.54 ± 1.08 D) (p<0.001). In the sub-analysis of the treatment group, there was a significant correlation between SE at age 3 and the number of laser shots (R2 = 0.36, p<0.001). Multivariate logistic analysis showed that the number of laser shots was an independent risk factor for early onset high myopia (p<0.05). CONCLUSION: The number of laser shots is an independent risk factor for early onset high myopia, and preterm infants who have undergone laser treatment for severe ROP should be considered for early optical correction with cycloplegic refractive examination.

Differential methylation of microRNA encoding genes may contribute to high myopia.

Introduction: High myopia (HM), an eye disorder with a refractive error ≤-6.0 diopters, has multifactorial etiology with environmental and genetic factors involved. Recent studies confirm the impact of alterations in DNA methylation and microRNAs (miRNAs) on myopia. Here, we studied the combined aspects evaluating to the role of methylation of miRNA encoding genes in HM. Materials and Methods: From the genome-wide DNA methylation data of 18 Polish children with HM and 18 matched controls, we retrieved differentially methylated CG dinucleotides localized in miRNA encoding genes. Putative target genes of the highest-ranked miRNAs were obtained from the miRDB and included in overrepresentation analyses in the ConsensusPathDB. Expression of target genes was assessed using the RNA sequencing data of retinal ARPE-19 cell line. Results: We identified differential methylation of CG dinucleotides in promoter regions of MIR3621, MIR34C, MIR423 (increased methylation level), and MIR1178, MIRLET7A2, MIR885, MIR548I3, MIR6854, MIR675, MIRLET7C, MIR99A (decreased methylation level) genes. Several targets of these miRNAs, e.g. GNAS, TRAM1, CTNNB1, EIF4B, TENM3 and RUNX were previously associated with myopia/HM/refractive error in Europeans in genome-wide association studies. Overrepresentation analyses of miRNAs' targets revealed enrichment in pathways/processes related to eye structure/function, such as axon guidance, transcription, focal adhesion, and signaling pathways of TGF-ß, insulin, MAPK and EGF-EGFR. Conclusion: Differential methylation of indicated miRNA encoding genes might influence their expression and contribute to HM pathogenesis via disrupted regulation of transcription of miRNAs' target genes. Methylation of genes encoding miRNAs may be a new direction in research on both the mechanisms determining HM and non-invasive indicators in diagnostics.

Myopia-26, the female-limited form of early-onset high myopia, occurring in a European family.

BACKGROUND: Female-limited early-onset high myopia, also called Myopia-26 is a rare monogenic disorder characterized by severe short sightedness starting in early childhood and progressing to blindness potentially by the middle ages. Despite the X-linked locus of the mutated ARR3 gene, the disease paradoxically affects females only, with males being asymptomatic carriers. Previously, this disease has only been observed in Asian families and has not gone through detailed investigation concerning collateral symptoms or pathogenesis. RESULTS: We found a large Hungarian family displaying female-limited early-onset high myopia. Whole exome sequencing of two individuals identified a novel nonsense mutation (c.214C>T, p.Arg72*) in the ARR3 gene. We carried out basic ophthalmological testing for 18 family members, as well as detailed ophthalmological examination (intraocular pressure, axial length, fundus appearance, optical coherence tomography, visual field- testing) as well as colour vision- and electrophysiology tests (standard and multifocal electroretinography, pattern electroretinography and visual evoked potentials) for eight individuals. Ophthalmological examinations did not reveal any signs of cone dystrophy as opposed to animal models. Electrophysiology and colour vision tests similarly did not evidence a general cone system alteration, rather a central macular dysfunction affecting both the inner and outer (postreceptoral and receptoral) retinal structures in all patients with ARR3 mutation. CONCLUSIONS: This is the first description of a Caucasian family displaying Myopia-26. We present two hypotheses that could potentially explain the pathomechanism of this disease.

A novel deep intronic COL2A1 mutation in a family with early-onset high myopia/ocular-only Stickler syndrome.

PURPOSE: To identify the genetic defect causing early-onset high myopia (eoHM)/ocular-only Stickler syndrome (ocular-STL) in a large Chinese family. METHODS: Genomic DNA and clinical data from a four-generation family with eoHM/ocular-STL were collected. Whole-exome sequencing was performed on one affected member in initial screening. Linkage scan based on microsatellite markers was carried out initially from candidate loci associated with autosomal dominant eoHM and Stickler syndrome. Sanger sequencing was used to detect potential variants. The pathogenicity of candidate variants was evaluated using mini genes ex vivo. RESULTS: Eight patients and five unaffected members in the family participated in the study, in which the patients had high myopia with other variable ocular phenotypes but without extraocular abnormalities. Whole exome sequencing did not detect any potential pathogenic variant in all genes known to associate with the disease. The eoHM/ocular-STL in the family was mapped to markers around COL2A1 by candidate loci linkage scan, with a maximum lod score of 3.31 for D12S1590 at θ = 0. A novel deep intronic variant, c.86-50C > G in intron 1 of COL2A1, was detected by Sanger sequencing and co-segregated with eoHM/ocular-STL in the family. Ex vivo splicing test using mini genes confirmed that the variant created a new splicing acceptor 49 bp before the canonical splicing site of exon 2, resulted in addition of 49 bp fragment in the transcript (from c.86-49 to c.86-1) and premature termination. CONCLUSIONS: Linkage study, bioinformatics prediction, and ex vivo transcript analysis suggest a novel deep intronic variant adjacent to 5-prime of exon 2 of COL2A1, affecting exon 2 splicing, as a potential cause of ocular-STL in a large family. To our knowledge, this is the first report of an intronic variant around exon 2 as a cause of ocular-STL while a series of variants in the coding region of exon 2, a dispensable alternative-splicing exon for extraocular tissues, in COL2A1 have been reported to cause Stickler syndrome-related ocular phenotype alone.

Mutation screening of 17 candidate genes in a cohort of 67 probands with early-onset high myopia.

PURPOSE: To detect variants in 17 known potentially causative genes for non-syndromic myopia in 67 Tujia Chinese patients with early-onset high myopia (eo-HM). METHODS: DNA from 67 unrelated patients with early onset (<7 years old) high myopia (refraction error ≤ -6.00D or axial length > 26 mm) were subjected to whole-exome sequencing (WES). Variants in 17 candidate genes were analysed by multistep bioinformatics analysis. Subsequently, Sanger sequencing was used to verify identified candidate mutations and to assess available family members for co-segregation with myopia. RESULTS: A multistep systematic analysis of variants in 17 potentially causative genes for eo-HM revealed four novel pathogenic mutations and three potential pathogenic mutations in 4 of 17 genes in 7 of 67 (10.4%) probands. The pathogenic group included one missense mutation (c.100G > C, p.Asp34His) and one splice donor mutation (c.989 + 1G >A) in ARR3, one missense mutation (c.995C > A, p.Thr332Lys) in NDUFAF7 and one novel frameshift mutation (c.726dupA, p.Arg243fs*140) in SLC39A5. The potential pathogenic group included two missense mutations (c.3266A > G, p.Tyr1089Cys; c.913G > A, p.Glu305Lys) in ZNF644 and one missense mutation (c.960T > A, p.His320Gln) in NDUFAF7. Sequence changes were confirmed by Sanger sequencing; all had an allele frequency <0.01 in the 1000G, EVS, ExAC and gnomAD databases. Additionally, both the pathogenic and potentially pathogenic mutations were predicted to be damaging by SIFT, Polyphen-2, PROVEAN, MutationTaster2, CADD and REVEL except the p.Tyr1089Cys and p.Glu305Lys changes were predicted to be neutral by PROVEAN. CONCLUSION: Our research provides more evidence to support the hypothesis that mutations in ARR3, SLC39A5 and NDUFAF7 are disease-causing genes for eo-HM and broadens the eo-HM mutation spectrum among different ethnic groups. It also deepens understanding of the contributions of ARR3, SLC39A5, and NDUFAF7 to eo-HM.

Frequent mutations of RetNet genes in eoHM: Further confirmation in 325 probands and comparison with late-onset high myopia based on exome sequencing.

In our previous study, potential pathological mutations of RetNet genes were detected in 23.8% (71/298) of probands with early-onset high myopia (eoHM), based on whole exome sequencing (WES). The current study aimed to confirm this finding in an additional 325 probands with eoHM and to clarify its specificity by comparison of 195 probands with late-onset high myopia (loHM). Variants in the 234 RetNet genes were selected from whole-exome sequencing data and were filtered using multistep bioinformatics analyses. Potential pathological variants in 33 genes were detected in 76 of 325 (23.4%) probands with eoHM and 14 of 195 (7.2%) probands with loHM. Thirty-five of the 76 (46.1%) probands with eoHM had mutations in COL2A1, COL11A1, RPGR, and CACNAIF, while only 2/14 (14.3%) probands with eoHM were detected. The mutation frequency and spectrum of RetNet genes in the 325 probands with eoHM were similar to our previous study but were significantly different in 195 probands with loHM (P = 2 × 10-6 and 0.04). Data from eoHM and loHM strongly suggest that a significant proportion of eoHM is caused by mutations in RetNet genes. These results also provide initial genetic evidence that eoHM is different from loHM. The presence of mutations in 7.2% probands with loHM raises questions about pathogenicity and the variable manifestation of some mutations. The functional studies of the mutations in question and more extensive investigations of related phenotypes in the mutation carriers and their family members may provide valuable information to address these questions.

Trio-based exome sequencing arrests de novo mutations in early-onset high myopia.

The etiology of the highly myopic condition has been unclear for decades. We investigated the genetic contributions to early-onset high myopia (EOHM), which is defined as having a refraction of less than or equal to -6 diopters before the age of 6, when children are less likely to be exposed to high educational pressures. Trios (two nonmyopic parents and one child) were examined to uncover pathogenic mutations using whole-exome sequencing. We identified parent-transmitted biallelic mutations or de novo mutations in as-yet-unknown or reported genes in 16 probands. Interestingly, an increased rate of de novo mutations was identified in the EOHM patients. Among the newly identified candidate genes, a BSG mutation was identified in one EOHM proband. Expanded screening of 1,040 patients found an additional four mutations in the same gene. Then, we generated Bsg mutant mice to further elucidate the functional impact of this gene and observed typical myopic phenotypes, including an elongated axial length. Using a trio-based exonic screening study in EOHM, we deciphered a prominent role for de novo mutations in EOHM patients without myopic parents. The discovery of a disease gene, BSG, provides insights into myopic development and its etiology, which expands our current understanding of high myopia and might be useful for future treatment and prevention.