A Novel Nonsense Mutation(c.1499C>G) in CRB1 Caused Leber Congenital Amaurosis-8 in a Chinese Family and Literature Review

Background:Leber’s congenital amaurosis (LCA) is a severe hereditary retinopathy disease that is characterized by early and severe reduction of vision,nystagmus,sluggish or absent pupillary responses. To date, the pathogenesis of LCA remains unclear,and the majority cases are caused by autosomal recessive inheritance. In this study, we explored the mutation in the Crumbs homologue 1(CRB1) gene in a Chinese family with LCA. Methods:We conducted comprehensive ocular examinations and collected 5 ml of blood samples from members of a Chinese family with LCA. The pathogenic gene was identi�ed by capturing and sequencing the related genes of ocular diseases. Results: We found a nonsense mutation(c.1499C>G) in the 6th exon of CRB1 in a Chinese family with LCA,which predicted a change of the protein p.S500X,may lead to loss of gene function. Conclusions: This study reported a novel mutation(c.1499C>G,p.S500X) of the CRB1 gene occurred in a Chinese family with LCA, thus expanding the spectrum of CRB1 mutations causing LCA. And we summarize the 76 mutations reported so far in CRB1 that caused LCA8.


Introduction
Since Theodore Leber rst described Leber's congenital amaurosis (LCA) 152 years ago(in 1869), We have obtained a great deal of information about LCA both in terms of clinical characteristics and molecular genetics.LCA, a rare but important juvenile retinal dystrophy, is an inherited retinal disorder most often diagnosed in infancy in the rst 6 months of life and characterized by the presence of nystagmus, poor visual acuity (VA), and a severely reduced or nondetectable electroretinogram [1,2]. In the worldwide,the prevalence of LCA is 1/81000 to 1 /30000 in newborn babies.Though the incidence is low, it also causes blindness in 20% of school-age children and accounts for approximately 5% of all hereditary retinopathy [3,4]. LCA is currently described into 21 types according to the pathogenic genes, with autosomal recessive inheritance as the dominant. LCA8 is caused by homozygous or compound heterozygous mutation in the CRB1 gene (604210) on chromosome 1q31.

Clinical Manifestation
This study was performed in agreeent with the declaration of Helsinki. It was reviewed by the research unit's professional ethics committee and informed consent was obtained and signed by the investigator.
The proband( gure 1,A, 2),a 2-years-old girl came to the hospital on account of her parents complained that she could not accurately grasp things. Her both eyes are performance as a horizontal pendulum nystagmus and was unable to comply with the detailed eye examination. Sequencing chromatograms:the proband show a homozygous mutation in CRB1 gene: nucleotide 1499 changed from cytosine C to guanine G (c.1499C G) homozygous mutation( gure 1,B) .Under the guidance of the paediatrician, the opportunity for examination was obtained through oral anesthesia.On examination,her eyes are in normal position, the cornea and lens are clear, fundoscopy showed the color of the optic disc in both eyes was light, and the blood vessels from both eyes were thin and narrow( gure 1,C). The pigmentation of the retina at the posterior pole was peppery and salt-like, and the macular area was a mass of lesions with a lot of pigmentation. Her parents and sister underwent detailed eye examinations(the results show in Table 1), including binocular corrected visual acuity, slit lamp examination, fundus photography, macular and optic disc OCT scanning, electroretinogram(ERG),which showed normal results( gure 1,D). For LCA, the criteria are: signs of blindness or severe visual impairment from birth or within the rst year of life,an ERG reduction of more than 50%, and congenital nystagmus [5].Fundus examinations could reveal diagnostic clues, including peripheral pigmentary retinopathy, central maculopathy with or without bull's eye pattern, or even macular atrophy. And, indispensable, molecular con rmation is needed.
In our study,the proband's eye examinations and genetic tests were consistent with the diagnosis of LCA. The homozygous mutation in the 6th exon of CRB1: nucleotide 1499 changed from cytosine C to guanine G (c.1499C G), resulting in nonsense mutation of amino acids (p.S500X) which hasn't been reported before. Through genealogical analysis,the proband's parents and sister had heterozygous variation at this site.According to the ACMG(American College of Medical Genetics and Genomics) guidelines, the mutation was preliminarily determined to be pathogenic:PVS1+PM2+PM3_Supporting(hom). reported, including heparin, sulfatides, integrins, dystroglycan, nidogen, and bulin.In neurexin the G domain is known to bind neurexophilins, α-latrotoxin and neuroligins [7,8].
Another anatomical feature of LCA includes decreased thickness in different layers, especially in the outer nuclear layer (ONL), loss of integrity in the ellipsoid zone, and disorganized macular atrophy [9]. Unfortunately, the proband we reported was too young to cooperate with optical coherence tomography (OCT) and ERG examination, so we could not analysis the clinical features of these two aspects.
enrolled 404 Spanish cases in study, 114 of which suffered from LCA and 290 from EORP(early-onset RP). Their study revealed that 11% of Spanish patients carried mutations in CRB1, ranging from 9% of EORP to 14% of LCA cases. And more than three quarters of the mutations identi ed have been rst described in their study [13].
Liping Yang et,al[6] through 18cases presenting with LCA to identify disease-causing mutations.They report compound heterozygous mutations of the CRB1 gene which included three novel heterozygous mutations: c.3059delT (p.M1020SfsX1), c.3460T>A (p.C1154S), and c.4207G>C (p.E1403Q). Hosono, K., et al reports the mutations of LCA and inherited retinal dystrophy(IRD) associated genes in 34 Japanese families, which is the frst to conduct a next generation sequencing(NGS) based molecular diagnosis of a large Japanese LCA cohort, achieved a detection rate of approximately 56%. Their results show that the most frequently mutated genes were CRB1, NMNAT1, and RPGRIP1 [14].In recently, Zhu, L., et al. [15]enrolled 37 patients with strictly de ned LCA in a cohort of IRD in ten years(2009-2019). Their results revealed that CRB1 gene occupied a greater proportion(27%) associated LCA in the western Chinese population.
CRB1 mutation is a common cause of LCA, and related mutations include missense mutation, nonsense mutation, insertion, deletion and splicing. The following Table 2 lists the mutations in LCA caused by CRB1 which including mutation types, sites,corresponding amino acid changes and regions in recent years. These results are for readers' veri cation and reference. Up to now, a total of 76 certain CRB1 mutations caused LCA.Furthermore,it has been reported that mutations in the CRB1 are responsible for 7.4%-27% of LCA in different populations.The pathogenic mutations were mainly missense, deletion and nonsense mutations, including insertion and splicing, which re ected the richness of mutation types( Table 3). The mutation sites of LCA8 were mainly concentrated in exon 6, 7 and 9 of CRB1, and the clear pathogenic sites were found in all exons except exon 4, 5 and 10, indicating the universality of mutation regions( Table 4). The reported cases involved more than 10 countries and regions, including China, England, Japan, Spain and Italy, which also showed that the global coverage of LCA caused by CRB1 is extensive.

Discussion
LCA is the earliest and most severe hereditary retinopathy, in which the function of cone-rod cells in both eyes is completely lost at birth or within one year after birth, leading to congenital blindness in infants. The majority cases are caused by autosomal recessive inheritance.Typical characteristics of LCA includes:early and severe reduction of vision associated with nystagmus,photophobia, sluggish or absent pupillary responses, nger pressure on eyeballs;fundus appearance, ranging from normal, maculopathy, to typical RP-like abnormalities; and electroretinogram showed that A and B waves were at and even severely reduced to non-detectable. It also can be accompanied by keratoconus, hyperopia, developmental delay and nervous system abnormalities et.al. [32] In some cases/reports,there are many similar clinical features between LCA and early-onset RP and even the diagnosis is ambiguous [33].Early-onset RP,usually, is considered as a relatively milder form,which patients do not have a congenital onset of visual impairment.We could distinguish the following phenotypes: LCA,early onset retinal degeneration;RP, presence of preservation of the para-arteriolar retinal pigment epithelium and Coats-like vasculopathy [34].
So far,21 pathogenic genes associated with LCA have been reported. CRB1 belongs to LCA8.CRB1 gene maps to chromosome 1q31.3,is composed of 12 exons,the longest isoform consists of 1,406 amino acids. This gene encodes a protein which is similar to the Drosophila crumbs protein and localizes to the inner segment of mammalian photoreceptors. In Drosophila crumbs localizes to the stalk of the y photoreceptor and may be a component of the molecular scaffold that controls proper development of polarity in the eye [35],and CRB1 has been found to be important in maintaining cellular polarity [36].
In the mouse retina, CRB1 is expressed in the inner segment of the photoreceptors and Muller cells to maintain adequate morphogenesis and polarity in retinal development [37]. Therefore, CRB1 gene mutations often lead to a variety of retinal dystrophy, including retinitis pigmentosa (RP), LCA ,macular dystrophy and so on. Approximately 9-17% of LCA cases have been related to CRB1 mutations, especially which are higher in the Chinese population [38,39].A wide variety of visual acuity was noted in patients with mutations in CRB1,ranging from 20/30 to NLP [10,40].
Among LCA,RPE65 mutations were almost always associated with normal macular thickness, as assessed by OCT, whereas CRB1 mutations were associated with reduced retinal thickness and a coarsely laminated retina.Fundus abnormalities were more heterogeneous in carriers of CRB1 mutations. In fact, Some scholars observed salt-and-pepper retinal dystrophy in younger patients and subsequently massive spicular and not nummular pigmentation at the posterior pole, which was reported to be a phenotypic feature of carriers of CRB1 mutations [11].Saloni Walia et.al. [41] through a multicentere retrospective observational study with 169 patients of LCA found that mutations in RPE65(LCA-Type Ii) and CRB1(LCA-8) may be associated with a relatively better VA in early life compared with other gene mutations. And, onset of the symptoms of LCA after the age of 1 year is also associated with an overall better VA prognosis.

Conclusions
LCA is one of the earliest and most severe forms of inherited IRD, the patients suffer from severe visual impairment during childhood, with their vision continuously deteriorating, the nal outcome of which usually is complete loss of vision by their thirties or forties [42].Therefore, it is very important to nd an effective treatment. Albert M et,al. provided an entirely new dimension in ocular therapeutics for gene-therapy to LCA2, patients with LCA2 who received AAV2.hRPE65v2 by subretinal injection showed evidence of improvement in retinal function,in the pupillary light re ex, reduction in nystagmus . This clinicaltrials approaches to the treatment of LCA and possibly other forms of retinal degeneration [43].
Although much is still unknown about the pathogenesis of LCA. However, with the improvement of next-generation sequencing technology and the application of various molecular biological means, the research on corresponding cell functions, the identi cation of gene subtypes and the establishment of animal models have greatly promoted our understanding of LCA. These latest advances provide a steady stream of evidence for a better understanding and treatment of LCA in the future. And may be useful for faster gene diagnosis, prenatal testing, the development of potential gene therapies, and for improving the understanding of the molecular pathogenesis of LCA.

Declarations
Ethics approval and consent to participate This study was approved by the Ethics Committee of A liated Hospital of Yunnan University. All experimental protocols were approved by the A liated Hospital of Yunnan University,and methods were carried out in accordance with relevant guidelines and regulations.All participants were informed about the purpose of the protocol and signed consent forms. The guardian(parent) of the patients consented to participation of the study.

Consent to publish
Written informed consent was obtained from the guardian(parent) of the patients,and they consented to publication of the study. The guardian (parent) of the patients consented for their medical information to be published.

Availability of data and materials
The relevant data were generated during this study and included in this article. And raw sequence data were not applicable to share in this article as no datasets were generated during the current study. The corresponding author Liwei Zhang (drzhangliwei@163.com) should be contacted if someone wants to request the data from this study.