中国汉族散发性圆锥角膜患者中的5个新型ZNF469基因突变

Five novel ZNF469 gene mutations in sporadic keratoconus patients in the Han Chinese population

摘要

目的 圆锥角膜(keratoconus，KC)是一种进行性角膜扩张疾病，其病因包括遗传、环境、生物力学和炎症反应等方面。本研究旨在筛查中国汉族人群中散发性KC患者的潜在致病突变基因。 方法 纳入25例散发性KC患者和50例无关人群匹配的健康对照，鉴定汉族人群中散发性KC患者的潜在致病突变基因。通过Sanger测序和全外显子测序(whole exome sequencing，WES)对锌指蛋白469(zinc finger protein 469，ZNF469)基因突变进行分析。同时，采用生物信息学方法分析探讨ZNF469基因对KC发病的影响。 结果 在KC患者中检测到5种新的错义杂合变异。新发现的2种复合杂合变异c.8986G>C(p. E2996Q)、c.11765A>C(p. D3922A)和c.4423C>G(p. L1475V)、c.10633G>A(p. G3545R)被确定为KC的潜在致病基因突变。 结论 ZNF469基因的突变可能参与了中国汉族KC患者的发病，突变位点可能有助于今后KC患者及其家属的遗传筛查。

Abstract

Objective Keratoconus (KC) is a progressive corneal ectasia disorder, arising from a myriad of causes including genetic predispositions, environmental factors, biomechanical influences, and inflammatory reactions. This study aims to identify potential pathogenetic gene mutations in patients with sporadic KC in the Han Chinese population. Methods Twenty-five patients with primary KC as well as 50 unrelated population-matched healthy controls, were included in this study to identify potential pathogenic gene mutations among sporadic KC patients in the Han Chinese population. Sanger sequencing and whole-exome sequencing (WES) were used to analyze mutations in the zinc finger protein 469 (ZNF469) gene. Bioinformatics analysis was conducted to explore the potential role of ZNF469 in KC pathogenesis. Results Five novel heterozygous missense variants were identified in KC patients. Among them, 2 compound heterozygous variants, c.8986G>C (p. E2996Q) with c.11765A>C (p. D3922A), and c.4423C>G (p. L1475V) with c.10633G>A (p. G3545R), were determined to be possible pathogenic factors for KC. Conclusion Mutations in the ZNF469 gene may contribute to the development of KC in the Han Chinese population. These mutation sites may provide valuable information for future genetic screening of KC patients and their families.

Graphical abstract

关键词

圆锥角膜 / ZNF469基因 / 突变 / Sanger测序 / 中国汉族 / keratoconus / ZNF469 gene / mutation / Sanger sequencing / Han Chinese population

Key words

keratoconus / ZNF469 gene / mutation / Sanger sequencing / Han Chinese population

Keratoconus (KC) represents a highly frequent ectatic disorder characterized by corneal thinning and ectasia, abnormal astigmatism, myopia, or vision loss^[1]. KC often takes place between 20 and 40 years of age and always involves bilateral eyes. KC incidence is affected by race and region, ranging from 0.05% to 0.23%^[2], and the male-female ratio of KC in Asia is 0.9-2.5꞉1^[3]. The treatment methods for KC begin with not rubbing eyes and wearing glasses or contact lenses, 10% to 20% of KC patients eventually need corneal transplantation^[4]. It represents a major corneal transplantation indication worldwide.

Environmental and genetic factors are associated with KC susceptibility^[5]. Eye rubbing, eye compression, atopy, and sun exposure are identified to have an important effect on the environmental etiology of KC^[6]. Additionally, several genes are suggested to be associated with KC, such as visual system homeobox 1 (VSX1) gene^[7-8], transforming growth factor-beta (TGF-β) gene^[9], superoxide dismutase 1 (SOD1) gene^[10-11], lysyl oxidase (LOX) gene^[12-13], interleukin-1 (IL-1) gene^[14-15], microRNA 184 (miR184) gene ^[16], and zinc finger protein 469 (ZNF469) gene^[17]. Decreased central corneal thickness (CCT) together with corneal protrusion represents the major features of KC^[18]. Single-nucleotide polymorphisms (SNPs) 100 kb upstream in ZNF469 show the highest relationship to CCT^[19]. ZNF469 homozygous mutations can induce brittle cornea syndrome type 1. However, they are not the genetic factors related to KC^[5].

All the exons providing guidelines for protein-encoding within one genome are called the exome, while whole exome sequencing (WES) is an approach to sequence exome. It can identify protein-coding region variations of different genes, not just several genes^[20]. This study aims to identify potential pathogenetic gene mutations in patients with sporadic KC in the Han Chinese population.

1 Materials and methods

1.1 Ethics statements

This study was conducted following the Institutional Review Board of the Third Xiangya Hospital, Central South University (No.2018-S400). Every participant provided his/her informed consent.

1.2 Subject recruitment

Twenty-five KC patients were recruited at the Department of Ophthalmology, the Third Xiangya Hospital, Central South University. Fifty unrelated population-matched healthy controls were also included.

Nineteen (76%) KC patients had bilateral lesions, while 72.7% of those with affected eyes were defined as completion stage of KC according to Chinese consensus^[2]. Slit-lamp examination, visual acuity, confocal microscopy, anterior segment optical coherence tomography, and corneal topography were all performed on all subjects. KC was diagnosed based on a history of impaired vision caused by progressive abnormal astigmatism, as well as symptoms such as thinning of the corneal stroma, apical conical corneal protrusion, corneal scarring, with/without striae within the posterior stroma (Vogt’ striae), and epithelial iron pigment deposition (Fleischer ring). Patients with concurrent atopy KC or allergy because of trauma, refractive surgery, Ehlers-Danlos syndrome, or Down syndrome were not included in this study.

1.3 DNA collection and WES

After receiving informed consent, peripheral venous blood samples were obtained from the study group with the diagnosis of KC, according to an experienced ophthalmologist. For this assay, genomic DNA (gDNA) from peripheral blood was isolated with a Sompura Blood DNA kit (Magen, China) in line with specific instructions. DNA was later analyzed with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., USA). The qualified gDNA samples were randomly fragmented by Q800R Sonicator and the size of the library fragments was mainly distributed between 200 bp and 500 bp. After process of end-repairing, A-tailing reactions, and adaptor ligation, the DNA fragments were amplified via ligation-mediated PCR. Purification and hybridization to the exome array for enrichment were performed. Enriched DNA samples were indexed and sequenced on a Next Seq500 sequencer (Illumina, San Diego, CA, USA) with 150 cycles of single end reads, according to the manufacturer’s protocols.

1.4 Mutation screening and data analysis

The ZNF469 (NM_001127464) gene was sequenced with Next Seq 500 (Illumina, San Diego, CA, USA). Table 1 shows how the coding regions of ZNF469, including exons, intron-exon junctions, and promoter regions, were amplified using PCR and corresponding primers. Agarose gel electrophoresis was used to validate the PCR, which was then followed by sequencing with the NextSeq High Output Reagent Cartridge V2 (Illumina).

The annotations of the sequencing data are the variation of exons and the introns in the 10 bp upstream and downstream of the exon. This variation includes missense, nonsense, synonym, frameshift, whole frame, clipping, and so on. Ensembl, Human Gene Mutation Database (HGMD), single nucleotide polymorphism database (dbSNP), Exome Sequencing Project 6500 (ESP6500), Exome Aggregation Consortium (ExAC), and other population databases were employed for labeling SNPs and low-frequency benign variants. The average depth threshold was more than 200× for the interested regions, which added to the data reliability and high quality. Polymorphism Phenotyping v2 (PolyPhen-2), Sorting Intolerant from Tolerant (SIFT), MutationTaster software, Functional Analysis Through Hidden Markov Models (FATHMM), and Genomic Evolutionary Rate Profiling (GERP) analyses were employed for predicting potential variant-induced effects. This work further categorized potential variants by following American College of Medical Genetics (ACMG) guidelines^[21].

1.5 Statistical analysis

SPSS 26.0 was employed for statistical analysis. Normally distributed continuous variables were expressed as mean±standard deviation, and categorical variables as quantity (percentage).

2 Results

2.1 Clinical data

KC patients included 9 female (36%) and 16 male (64%) cases, while healthy controls included 21 (42%) female and 29 (58%) male, all of whom were of Han Chinese ethnicity. The age of KC patients was 14 to 72 years, and healthy controls was 21 to 61 years. Table 2 shows the characteristics of the 25 KC patients. The average age of the 25 KC patients was (25.5±13.1) years, and the average maximum keratometric reading for the affected eyes was (67.2±10.9) diopters (D). The average thinnest point pachymetry was (385.5±84.2) µm and (374.1±92.6) µm in the right and left eyes, respectively.

Detailed clinical characteristics of patients with the ZNF469 gene variants are presented in Table 3. The proband 1 from family 1 is a 23-year-old woman whose cornea can be seen a cone-like change at the apex with corneal scarring under slit lamp examination and joints were hypermotility (Figure 1). Ophthalmologic examination using corneal topography showed corneal thinning and increased keratometry. The central corneal thickness was detected to be 365 μm (Figure 2A). Besides, the corneal stroma has discontinuous nerve fibers and longitudinal or oblique striae as observed by a confocal microscope examination (Figure 2B). Anterior segment photograph of proband 2 demonstrates cone-like change and Vogt’ striae (Figure 3A). Corneal tomographic imaging demonstrates significant inferior steepening and high oblique astigmatism with diffuse stromal thinning (Figure 3B). Confocal microscopy observation shows tortuous nerve fibers, stromal haze, and dark bands (Figure 3C).

2.2 WES and bioinformatics analysis

According to the results of WGS, 2 of 25 KC patients carried the 5 novel sequence variants in the ZNF469 gene c.6503C>T (p. P2168L), c.8986G>C (p. E2996Q), c.11765A>C (p. D3922A), c.4423C>G (p. L1475V), and c.10633G>A (p. G3545R), which are summarized in Table 4. Sanger sequencing was later carried out among 50 normal subjects to avoid false-positive results. Figure 4 displays the sequencing chromatograms of the 5 mutations, all within the exonic region of ZNF469. These variants were missense mutations without indel, nonsense mutation, or frameshift, which were not previously mentioned in KC cases. Next, we collected peripheral venous blood samples and performed NGS from the immediate family members of the 2 patients who carried the five novel mutations in ZNF469. Figure 5A shows one of the 3 mutations (c.11765A>C) of the patient II:1 was from the father, and the other 2 mutations (c.6503C>T and c.8986G>C) were from her mother. Figure 5B shows a mutation (c.4423C>G) of the patient II:3 was from his mother, and the other one (c.10633G>A) was a novel variant.

The SIFT, Polyphen-2, Mutation Assessor, FATHMM, and GERP analyses of variants are shown in Table 5. The prediction analyses of SIFT showed that the 4 variants, p. P2168L, p. E2996Q, p. D3922A, and p. L1475V, were damaged, while variant p. D3922A might have been damaged by Polyphen-2. The variants, p. P2168L, p. E2996Q, p. D3922A, p.L1475V, and p. G3545R were found to be neutral, medium, medium, low, and neutral, respectively, by Mutation Assessor analysis. The FATHMM showed that the 3 variants, p. E2996Q, p. D3922A, and p.L1475V were damaged, and p. P2168L and p. G3545R were tolerable. Additionally, p. E2996Q and p. D3922A variants were predicted to be conserved by GERP. According to these analyses, p. E2996Q, p. D3922A, and p. L1475V were damaged and could not be tolerated, while p. P2168L and p. G3545R could be tolerated. As a result, p. E2996Q, p. D3922A, and p. L1475V heterozygote coding variants may be associated with the pathogenesis of KC disease.

3 Discussion

KC is genetically complex, yet its main contributing factors remain unclear. As revealed by many articles, ZNF469 gene mutations induce brittle cornea syndrome^[18-19] and contribute to the pathogenicity of KC disease^{[17, 22-24]}. However, Karolak, et al^[25] showed that ZNF469 was not the causative gene of KC in Polish cases. Lucas, et al^[26] demonstrated that uncommon ZNF469 variants did not promote KC susceptibility in Europeans. Thus, the expression of ZNF469 varied among different races and regions.

In our study, 5 novel missense heterozygote variants were detected in 2 of 25 sporadic KC patients: c.6503C>T, c.8986G>C, c.11765A>C, c.4423C>G as well as c.10633G>A inducing amino acid substitutes P2168L, E2996Q, D3922A, L1475V, and G3545R, respectively. These 5 mutations have never been reported. Identifying 5 possible novel ZNF469 mutations among 8% of KC cases demonstrated the possible function of ZNF469 in KC among Han Chinese ethnicity. Bioinformatics analysis supported the pathogenicity of the c.8986G>C, c.11765A>C and c.4423C>G variants. Novel 2 compound heterozygous variants c.8986G>C, c.11765A>C, and c.4423C>G, c.10633G>A were identified as the genetic causes of KC.

The ZNF469 gene locates in 16q24.2. The major parts of the two-exon gene ZNF469 are 5 typical C-terminal C2H2 zinc-finger domains^[18]. The link between ZNF469 and CCT has been demonstrated repeatedly in genome-wide association studies (GWAS)^[27]. ZNF469 has low homology to some collagen, indicating that it is a potential extra-nuclear regulator or transcription factor for collagen fiber synthesis and organization^[28], and ZNF469 functional mutations may affect corneal collagen fibril homogeneity as well as regularity^[29]. As a result, ZNF469 abnormalities can cause corneal fragility and fragility. Nonetheless, the most likely pathogenic alleles among KC cases are missense variants, which may have a less negative impact on protein activity than the truncated ZNF469 mutation, which is closely linked to brittle cornea syndrome^[19]. Bao, et al^[30] generated a *znf469* mutant zebrafish via CRISPR/Cas9 and observed reduced corneal stromal thickness, disorganized collagen fibers, and impaired extracellular matrix (ECM)-related pathways, suggesting a key role of ZNF469 in corneal structural maintenance. However, definitive evidence supporting a major role of ZNF469 in human KC pathogenesis remains lacking. The role and functional studies of ZNF469, such as gene-related pathways within the human cornea, should be expanded.

In conclusion, We conclude that 5 novel ZNF469 variants viz.c.8986G>C (p. E2996Q), c.11765A>C (p. D3922A), c.4423C>G (p. L1475V), c.6503C>T (p. P2168L) as well as c.10633G>A (p. G3545R) among sporadic KC cases are the critical factors promoting the pathogenic mechanism among KC cases of Han Chinese ethnicity. Two compound heterozygous ZNF469 variants (c.8986G>C and c.11765A>C; c.4423C>G and c.10633G) might be the pathogenic causes in families with KC disorders.

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基金资助

the National Natural Science Foundation(82271057)

the Natural Science Foundation of Hunan Province(2023JJ30818)

RIGHTS & PERMISSIONS

(Open access):This is an open access article under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND4.0) (https://creativecommons.org/licenses/by-nc-nd/4.0/).