Background: High myopia (HM) is a leading cause of blindness that has a strong genetic predisposition. However, its genetic and pathogenic mechanisms remain largely unknown. Thus, this study aims to determine the genetic profile of individuals from two large Chinese families with HM and 200 patients with familial/sporadic HM. We also explored the pathogenic mechanism of HM using HEK293 cells and a mouse model.Methods: The participants underwent genome-wide linkage analysis and exome sequencing. Visual acuity, electroretinogram response, refractive error, optical parameters and retinal rod cell genesis were measured in knockout mice. Immunofluorescent staining, biotin-labelled membrane protein isolation and electrophysiological characterisation were conducted in cells transfected with overexpression plasmids.Results: A novel HM locus on Xp22.2-p11.4 was identified. Variant c.539C>T (p.Pro180Leu) inGLRA2gene was co-segregated with HM in the two families. Another variant, c.458G>A (p.Arg153Gln), was identified in a sporadic sample. TheGlra2knockout mice showed myopia-related phenotypes, decreased electroretinogram responses and impaired retinal rod cell genesis. Variants c.458G>A and c.539C>T altered the localisation of GlyRα2 on the cell membrane and decreased agonist sensitivity.Conclusion: GLRA2was identified as a novel HM-causing gene. Its variants would cause HM through altered visual experience by impairing photoperception and visual transmission.

Nance-Horan Syndrome (NHS) (OMIM: 302350) is a rare X-linked developmental disorder characterized by bilateral congenital cataracts, with occasional dental anomalies, characteristic dysmorphic features, brachymetacarpia and mental retardation. Carrier females exhibit similar manifestations that are less severe than in affected males. Here, we report a four-generation Chinese family with multiple affected individuals presenting Nance-Horan Syndrome. Whole-exome sequencing combined with RT-PCR and Sanger sequencing was used to search for a genetic cause underlying the disease phenotype. Whole-exome sequencing identified in all affected individuals of the family a novel donor splicing site mutation (NM_198270: c.1045 + 2T > A) in intron 4 of the gene NHS, which maps to chromosome Xp22.13. The identified mutation results in an RNA processing defect causing a 416-nucleotide addition to exon 4 of the mRNA transcript, likely producing a truncated NHS protein. The donor splicing site mutation NM_198270: c.1045 + 2T > A of the NHS gene is the causative mutation in this Nance-Horan Syndrome family. This research broadens the spectrum of NHS gene mutations, contributing to our understanding of the molecular genetics of NHS.

Recent studies have uncovered that noncoding sequence variants may relate to Axenfeld-Rieger syndrome (ARS), a rare developmental anomaly with genetic heterogeneity. However, how these genomic regions are functionally and structurally associated with ARS is still unclear. In this study, we performed genome-wide linkage analysis and whole-genome sequencing in a Chinese family with ARS and identified a heterozygous deletion of about 570 kb (termed LOH-1) in the intergenic sequence between paired-like homeodomain transcription factor 2 ( PITX2 ) and family with sequence similarity 241 member A. Knockout of LOH-1 homologous sequences caused ARS phenotypes in mice. RNA-Seq and real-time quantitative PCR revealed a significant reduction in Pitx2 gene expression in LOH-1 ^–/– mice, while forkhead box C1 expression remained unchanged. ChIP-Seq and bioinformatics analysis identified a potential enhancer region (LOH-E1) within LOH-1. Deletion of LOH-E1 led to a substantial downregulation of the PITX2 gene. Mechanistically, we found a sequence (hg38 chr4:111,399,594–111,399,691) that is on LOH-E1 could regulate PITX2 by binding to RAD21, a critical component of the cohesin complex. Knockdown of RAD21 resulted in reduced PITX2 expression. Collectively, our findings indicate that a potential enhancer sequence that is within LOH-1 may regulate PITX2 expression remotely through cohesin-mediated loop domains, leading to ARS when absent.

Background: Stickler syndrome is a multisystemic disorder characterized by ophthalmological and non‐ophthalmological abnormalities, frequently misdiagnosed due to high clinical heterogeneity. Stickler syndrome type I (STL1) is predominantly caused by mutations in the COL2A1 gene.Methods: Exome sequencing and co‐segregation analysis were utilized to scrutinize 35 families with high myopia, and pathogenic mutations were identified. Mutant COL2A1 was overexpressed in cells for mechanistic study. A retrospective genotype–phenotype correlation analysis was further conducted.Results: Two novel pathogenic mutations (c.2895+1G>C and c.3505G>A (p.Val1169Ile)) and two reported mutations (c.1597C>T (p.Arg533*) and c.1693C>T (p.Arg565Cys)) in COL2A1 were identified causing STL1. These mutations are all in the G‐X‐Y triplet, and c.2895+1G>C contributed to aberrant RNA splicing. COL2A1 mutants tended to form large aggregates in the endoplasmic reticulum (ER) and elevated ER stress. Additionally, mutations c.550G>A (p.Ala184Thr) and c.2806G>A (p.Gly936Ser) in COL2A1 were found in high myopia families, but were likely benign, although c.2806G>A (p.Gly936Ser) is on G‐X‐Y triplet. Moreover, genotype–phenotype correlation analysis revealed that mutations in exon 2 mainly contribute to retinal detachment, whereas mutations in the collagen alpha‐1 chain region of COL2A1 tend to cause non‐ophthalmologic symptoms.Conclusion: This study broadens the COL2A1 gene mutation spectrum, provides evidence for ER stress caused by pathogenic COL2A1 mutations and highlights the importance of non‐ophthalmological examination in clinical diagnosis of high myopia.

Rafiq syndrome (RAFQS) is a rare autosomal recessive disorder that is classified as a type II congenital disorder of glycosylation (CDG-II), and caused by MAN1B1 gene mutation. To date, 24 pathogenic MAN1B1 mutations have been reported in association with MAN1B1-CDG. However, the underlying pathogenic mechanisms remain poorly understood. In this study, we recruited a consanguineous family from Pakistan with multiple affected individuals exhibiting mild facial dysmorphism, developmental delay, and intellectual disability. Utilizing exome sequencing and homozygosity mapping, we identified a novel MAN1B1 mutation (c.772_775del) that co-segregated with RAFQS in this family. Analysis of public single-cell transcriptomic data revealed that MAN1B1 is predominantly expressed in dorsal progenitors and intermediate excitatory neurons during human brain development. Knockdown of Man1b1 in primarily cultured mouse excitatory neurons disrupted axon growth, dendrite formation, and spine maturation, and could not be rescued by truncated variants identified in the family. Furthermore, in utero, electroporation experiments revealed that Man1b1 knockdown in the murine cortex impaired neural stem cells’ proliferation and differentiation, as well as cortical neuron migration. Collectively, these findings elucidate a critical role for MAN1B1 in the etiology of RAFQS and demonstrate that loss-of-function mutation in MAN1B1 disrupt neuro-developmental processes, providing mechanistic insights into the pathogenesis of this disorder.

Background: High myopia (HM) refers to an eye refractive error exceeding −5.00 D, significantly elevating blindness risk. The underlying mechanism of HM remains elusive. Given the extensive genetic heterogeneity and vast genetic base opacity, it is imperative to identify more causative genes and explore their pathogenic roles in HM. Methods: We employed exome sequencing to pinpoint the causal gene in an HM family. Sanger sequencing was used to confirm and analyse the gene mutations in this family and 200 sporadic HM cases. Single-cell RNA sequencing was conducted to evaluate the gene’s expression patterns in developing human and mouse retinas. The CRISPR/Cas9 system facilitated the gene knockout cells, aiding in the exploration of the gene’s function and its mutations. Immunofluorescent staining and immunoblot techniques were applied to monitor the functional shifts of the gene mutations at the cellular level. Results: A suspected nonsense mutation (c.C172T, p.Q58X) in CCDC66 was found to be co-segregated with the HM phenotype in the family. Additionally, six other rare variants were identified among the 200 sporadic patients. CCDC66 was consistently expressed in the embryonic retinas of both humans and mice. Notably, in CCDC66-deficient HEK293 cells, there was a decline in cell proliferation, microtube polymerisation rate and ace-tubulin level. Furthermore, the mutated CCDC66 failed to synchronise with the tubulin system during Hela cell mitosis, unlike its wild type counterpart. Conclusions: Our research indicates that the CCDC66 variant c.C172T is associated with HM. A deficiency in CCDC66 might disrupt cell proliferation by influencing the mitotic process during retinal growth, leading to HM.