A novel mutation of RPGR in a Chinese family with X-linked retinitis pigmentosa

INTRODUCTION

聽說蒂納茲家族在曼杜里亞還買下另外一個釀酒合作社——Cantine San Giorgio，擁有一個有6層樓，21米高，用于存放木桶的高塔，當地稱為Torre Vinaria，重力原理讓葡萄酒從不銹鋼桶流入下層的容器中，從而免受炎熱氣候的負面影響，遺憾的是，本次行程太匆忙，最后沒能去成Cantine San Giorgio，就得趕往下一個酒莊。

So far, more than 80 genes have been identified which are associated with RP, including 3 genes (

,

,and

) for X-linked retinitis pigmentosa (XLRP)

.Among all the related genes of XLRP, mutations account for over 70% in

and approximately 8% to 15% in

,respectively

. Up to now, 261 mutaions including deletion translocation, point muations (frameshit, nonsence, splice site)and deep intronic mutations has been detected of

(http://www.hgmd). What’s more, the majority of variants occurr in the region of

. Due to

mutations,variable expression and penetrance, even the manifesting of female carriers, it is a challenge to identify the underlying genetic defect and provide appropriate genetic counseling for the individuals with RP

.

R etinitis pigmentosa (RP), is the most prevalent form of inherited retinal degeneration, which represent a spectrum of eye disorders that primarily resulted by retinal photoreceptor cells malfunctioning, and ultimately leading to blindness

. In common, at the initial stages of the disease, male patients exhibit nyctalopia. With progressive photoreceptor degeneration, visual field reduces gradually and eventually reaches blindness in midlife. According to previous reports, in general, there were between 1/3000 and 1/4000 cases of RP. Commonly, the mode of inheritance of this disease can be autosomal dominant (30%-40%), autosomal recessive(50%-60%) and X-linked manner (5%-15%)

.

Herein, we reported a case of Chinese family, consisted of 20 numbers, in which 4 individuals with a novel hemizygous mutation c.2865G＞A p.W955X in

gene and 8 carrierswere investigated. Based on genetic analysis, pedigree analysis and comprehensively clinical features, we characterized the phenotypic manifestation associated with the mutation.

SUBJECTS AND METHODS

Meanwhile, it is noting that ORF15, which is the terminal exon of the

containing repetitive glutamic acid and glycinerich sequence, the product that is considered to assist in regulating molecules between the inner and outer segments

.Breuer

described that the special sequence region creates a mutational hotspot, the incidence rate has reached 60% roughly, and majority of these occurs in the region between codons 801 and 1070. The novel mutation in this study was further validating the region of high mutation.

The family, which is a 4 generation pedigree,comprised 4 affected individuals (Figure 1). The proband IV-1 and family members (II-5, III-3, III-5, III-8, III-9, IV-2, IV-3,IV-4, IV-5, IV-6), totally 11, underwent a complete ophthalmic examination. It includes best-corrected visual acuity (BCVA),slit lamp biomicroscopy, intraocular pressure (IOP), cycloplegic refraction by compound tropicamide, detailed fundus photography, full-field electroretinogram (ERG) according to International Society for Clinical Electrophysiology of Vision(ISCEV) standards and optical coherence tomography (OCT),that were performed by ophthalmologists.

Peripheral blood sample of 20 subjects was collected into sample tube which contains ethylenediamine tetraacetic acid (EDTA). Genomic DNA was extracted from peripheral blood leukocytes. Primers were designed from GenBank (OMIM 312610) and adopted the published exon 15 sequence (Table 1)

. The reaction mixture was set up with high fidelity Taq polymerase (Invitrogen,USA). Cycling conditions of the PCR were conducted as follows: (95℃ 30s, 57.5℃ 45s, 72℃ 45s) ×32 cycles, 72℃5min (Bio-Rad, USA). The products were sequenced by company (TSINGKE, China) and visualized by the Finch TV software (Geospiza, USA). The presence of the mutations in

gene was assessed by comparing the patient’s sequence with the reference sequence (Figure 2).

An analysis of the data using SPSS18.0 software was carried out. Two-tailed Student’s

-test was used to determine whether there were significant differences between two groups. The criterion for statistical significance was

＜0.05.

As a result of the detailed family history and the clinical presentation of the patients, an X-linked genetic defect was inferred. Hence, direct Sanger sequence was conducted to verify the possible diseasecausing mutations of

and

(data not shown). In the event, no variants were identified, whole exome sequencing(WES) would be conducted. Fortunately, a novel mutation(c.2865G＞A p.W955X) of

was identified in the proband, which was not reported in either public databases.What’s more, mutated individuals and carriers can be classified separately according to the same criteria.

In

, there is a novel nonsense mutation W955X(c.2865G＞A) that leads to premature termination codons,resulting in truncated proteins.

Among 20 participating individuals, 4 affected patients with hemizygous mutation and 8 carries with heterozygous mutation were confirmed eventually (Table 2). Unfortunately, it’s difficult to obtain the clinical data of II-1 and III-1, because of living so far away. There was, however, a detailed medical history that had shown signs of visual dysfunction of RP.

打造智慧教室，利用移動互聯(lián)網、云計算、物聯(lián)網、大數據等前沿信息技術，開展課程線上線下的混合式教學，提供個性化師生服務和智能化管理，將物理環(huán)境和虛擬環(huán)境有機融合，實現多元化交互教學的學習環(huán)境。構建線上資源數據庫，綜合計量經濟學教學內容、測試題目、考評標準等。數據庫還需引入人工智能技術，比如當學生線上測試時出現較多錯誤時，數據庫可以自行調低測試難度；對學生在學習中提出的各種疑問，通過問題中的關鍵詞使用機器應答的形式，給出特定范圍內可量化的標準答案，若學生仍未能從機器回答中獲得滿意解答，可連線到教師端人工解答。

The patient (IV-1), a 10-year-old boy, he exhibited typical manifestations of RP. Such as a significant decline in visual acuity (OD: +0.25 DS, -0.75 DC×5°=0.4; OS: -0.50 DS,-1.50 DC×170°=0.4), with initial symptoms being nyctalopia,which presented in the decade of his life. Bilateral fundus changes revealed tilted optic disc, optic disc drusen, myopic maculopathy, macular atrophy, even hyperpigmented deposits in the periphery. There was a drastic reduction in the amplitude of rods and cones on ERGs. During OCT imaging, it was a common to see the degeneration and loss of the outer retinal bands in the peripheral retina, especially in regions near ellipsoid zone (EZ) as well as the inner/outer segments (IS/OS),while relative structure and function was conserved sparing of the central macula (Figure 3C).

圍繞四個重大平臺 全力做好用地保障（張龍明等） .......................................................................................1-27

According to previous researches, the mutational hotspot of the RPGR

has been reported to cause pathological myopia (PM) in Asians almost exclusively

. Zhang

even suggested that PM appears may be a distinct phenotype that associated with ORF15 nonsense mutations (c.2833G＞T p.E945X). There is a possibility that the rehabilitation of cell degeneration may be involved in PM in XLRP patients

. In comparison, patients with RP manifested a refractive error ranging from +1.0 DS to -5.0 DS (media -2.22 D), in which the percent of subjects with moderate myopia is 50%, without high myopia. Meanwhile, myopia was also performed in normal individuals (III-9) and female carriers (IV-4, IV-5, IV-6), in which just only one carrier (IV-4) has moderate myopia and severe astigmatism. In a word, the incidence of PM in affected male and female carriers was lower than ever reports

.Except for myopia, esotropia and exotropia are also obvious association with BCVA. In this study, BCVA deterioration of affected subjects did not correlate with increasing of age, that conclusion was disagree with previous report

. Small sample is to a great extent a limitation of this study.

Individual III-3, available records of the oldest of patients were found at the age of 31. Initial symptoms include poor visual acuity (BCVA OD: 0.4; OS: 0.3), which typically appears in the third decade of life, compared to others. He diagnosed with RP and displayed typical disease characteristics like bone-spicule pigment deposition. Temporal yellow dots in fundus and flat or barely extinguishing ERG were also found. A significant alteration in the organization of the retina, characterized by multiple low reflective cystic spaces and distortion of layers,was also seen in both eye by OCT (Figure 3A). Meanwhile,the results of III-8 ophthalmic examination showed poor visual acuity (OU 0.4) and ametropia (OD: -5.00 DS, -0.75 DC×180°; OS: -4.75 DS, -0.75 DC×180°). The funduscopic,structural and functional changes were also detected, consist with affected individuals (Figure 3B). Exams of both eyes of patients were normal in terms of intraocular pressure and anterior segment (Table 2).

Combined with their family history, genetic testing and detailed ophthalmicexamination were performed to confirm

mutation carrier status. Then we looked into the clinical features of carriers. In summary, individuals with heterozygous mutation had various symptoms, ranging from completely normal condition to slight or mild retinal changes to obvious complaints. Compared to 4 male patients (III-3, III-8, IV-1, IV-2), the visual function of 6 female carries (II-5, III-5, IV-3, IV-4, IV-5, IV-6), was much better. All female carriers (age range from 6 to 35y), had a minimum of 20/50 vision in one eye (BCVA ranged from 0.4 to 1.0). Three female carriers (IV-4, IV-5, IV-6) suffered from myopia, which the power of spherical equivalent (SE) range was -1.0 to -8.75 diopters (D). What’s more, one of the female carriers (IV-4) had high myopia (SE OD -7.5 D, OS -8.75 D).Anisometropia, denoted by ＞1.5 D of spherical degree or ＞1.0 D of cylindrical degree, was observed in 2 female carriers(IV-4, IV-6). In addition, half of female carriers (II-5, IV-4)who harbor poor visual acuity suffered esotropia or exotropia,this might also be a factor to influence BCVA.

又譬如，據人民網報道，北京市土地儲備中心密云縣分中心原主任任明信（已被判刑13年），也是以購房缺錢為由向開發(fā)商陳勃羽借款的。陳勃羽在法庭作證時說，他理解任明信就是要讓他出錢，自己雖不情愿但又不敢得罪他。即便任明信真還錢，他也不敢要，畢竟業(yè)務上要靠他幫忙。

中國農村家庭生育二孩的成本核算及分攤機制研究——基于川渝兩地的實地調研....................................................................................................................邱德勝 李詩韻 王志章（8）

DISCUSSION

The development of DNA sequencing technology has made genetic testing more accessible to patients. It is conventional that scholars are passionately interested in next-generation sequencing (NGS) to explore the novel causative gene even unknown mutations

. Surely, it offers great benefits to diagnosis. At the same time, it also has limitations, such as long time consuming, high costing and false error. Herein, we used the direct Sanger sequence and it was reliable and costeffective to focus on the hot point of mutation. History and clinical characteristics had further revealed the true identity of the disease. As a consequence, the result forcefully suggested that the direct sequence is still an effective detection method of special diseases.

Remarkably, it was noting that a total of 66.7% (8 of 12 eyes) of eyes of female carriers exhibited patchy pigment clumping in the peripheral retina, however, in 4 of the 12 eyes with carriers,the fundus appeared fairly normal (Figure 4). Additionally,there was a dramatic reduction in rod and cone amplitude for affected males, while 83.3% of female carriers were relatively stable, within a relatively normal range (Figure 5). What’s more, 2 female carriers subjects (33.3%) had abnormal OCT findings, such as slight disruption of IS/OS or thinning of the outer retinal layer. Negative traits of OCT were detected from 4 carriers (III-5, IV-3, IV-5, IV-6). The detailed clinical features are summarized in Table 2.

A cone-rod or cone dystrophies due to mutations in the

gene, in which a truncation mutant tends to override endogenous RPGR proteins to affect photoreceptor function.Consistent with previous notion, multiple cystics and distortion of outer layers (external limiting membrane, EZ and IS/OS)were detected in the peripheral retina of affected male subjects,with relative structure of IS/OS line sparing of the macula.In terms of ERG, it showed minimal cone response and extinguished rod response. The results of all those revealed extensive loss of photoreceptors, especially in the peripheral retina rather than the macula, which could account for the remaining visual acuity and flat ERG response.

This study observed the tenets of the Declaration of Helsinki. And it was approved by the Ethics Committee of Children’s Hospital of Hebei Province. Twenty participating individuals agreed study and signed informed consent.

在公路工程建設過程中，施工單位應該堅持依法監(jiān)督的原則，增強自身的誠信理念與服務意識，注重做好質量安全環(huán)節(jié)的監(jiān)督與管理工作。為達到這一目標，施工單位需要注重提高質量安全監(jiān)管隊伍的專業(yè)素質與監(jiān)管水平。因此，施工單位應該注重定期組織培訓活動，既豐富監(jiān)管人員的專業(yè)知識，提高其綜合素質水平，在執(zhí)行監(jiān)管行為的過程中，要嚴格遵循相關規(guī)章制度，全面保障質量安全監(jiān)管工作的成效。同時，施工單位可以加強文化建設，營造質量安全至上的良好精神氛圍，打造公路工程的質量安全監(jiān)督文化，增強施工人員和質量安全監(jiān)管人員的責任感，使其自覺地采取行動，落實質量控制工作和安全監(jiān)督工作，推進公路工程建設獲得質的發(fā)展。

Some scholars believed that patients with XLRP who have frameshift mutations or nonsense mutations within exons 1 to 14 may develop severe clinical features, it is possibly because of this reason that truncated protein from the RPGR

variant transcript interacts relatively well with other proteins

.However, in the current study, not only mutated males but also some carried females with the W955X (c.2865G＞A)mutation had a more significantly severe visual impairment,more varied fundus (including tilted optic disc, bone-spicule pigment deposition, retinal arteriole attenuation and optic disc pallor, even retinal atrophy), faster loss of ERG amplitudes and more thinning of OCT than patients with the mutations in exon 1 to 14 that described in some past reports

. In summary,we found a mutation in the RPGR

region that may have potentially more deleterious clinical features.

Meanwhile, the identical mutation was confirmed in his consanguineous brother (IV-2). He showed poor visual acuity,with mild myopia and myopic astigmatism (OD: -2.50 DS,-2.5 DC×5°=0.3; OS: -2.5 DS, -2.50 DC×175°=0.3). Myopic maculopathy, Bull’s eye, attenuated arteries and bone-spicule hyperpigmentation in retina of fundus were also discovered beginning in the first decade, accompanied by obviously flat ERG and atrophy of outer retinal bands at the (para)fovea of OCT (Figure 3D).

Based on mutagenesis analysis, an exon 15 mutation in

gene has been identified. The gene, which is located on the X chromosome and approximately accounts for 5%-15% of all cases of RP

. Commonly, multiple isoforms of RPGR were detected in functional performance. The RPGR

is one of isoforms, which consists of 15 exons coding 1152-aa protein,enriches in retina and is thought to facilitate the connecting and trafficking of rod and cone photoreceptors

. Previous analysis revealed that the RPGR

variants are associated with significantly dysfunction of retinal defects, ranging from RP to cone-rod or macular degeneration

. In the present study, nearly all the patients suffered from visual disturbance and tended to be worse with increasing age. That was consistent with previous reports

.

Compare with 4 patients, carriers showed a wide range of clinical features. ERG and fundus showed abnormalities in 16.7% and 66.7% of female carriers, respectively, which is lower than previous reports

. Additionally, normal layers of OCT were also more apparent in female carriers. Variability of phenotypic severity in female carriers is attributed to a variety of factors, such as age, environment, skewed X-activation, and genetic modifiers

. As discussed above, RP exhibits a higher degree of phenotypic heterogeneity and variability. Nanda

advocated female carriers who suffer from severe visual impairment and display a retinal phenotype should be considerate to process therapeutic intervention. Specifically,phenotypic follow-up of female carriers must be conducted for a longer period of time

. Hence, it’s significant to discover novel RP mutations, generalize phenotype and assess the process, so that patients can undergo genetic therapy treatment timely

.

In summary, we reported the clinical features of individuals with RP and carriers caused by a novel mutation (c.2865G＞A p.W955X) in

, and broadened the spectrum of mutations,which is valuable for provide future genetic counselling and specific gene therapy.

（1）人類活動導致了水土流失。隨著人類科學技術的不斷進步，城市建設手段越來越先進，建設速度也越來越快，與此同時，不斷擴張的城市范圍，對自然環(huán)境造成了極大的破壞，大量的植被消失導致植物的蒸騰作用減弱，從而造成了水土流失的現象，進而使河流中的泥沙含量大幅度上升，這又導致河床高度上升，河水溢出河道造成了洪水災害。

We thank all the individuals for participating in this project.

引理為偽BCI-代數X的一個猶豫模糊濾子的充要條件是對任意γ ∈ P([0,1]), 要么 ?, 要么為X的濾子.

Sun HH and Chen L conceived the idea; Sun HH, Zhao JC, Yang SL, Shi JD, and Wei YS collected clinical message and performed the experiments; Sun HH, Wang JC performed data analyses; Sun HH, Gu F and Chen L wrote the manuscript. All authors approved the manuscript.

Supported by Natural Science Foundation of Hebei Province (No.H2021316006); Hebei Provincial the Ministry of Health Research Fund for Medical Sciences(No.20200638).

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1 Hamel C. Retinitis pigmentosa.

2006;1:40.

2 Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa.

2006;368(9549):1795-1809.

3 Tee JJL, Smith AJ, Hardcastle AJ, Michaelides M. RPGR-associated retinopathy: clinical features, molecular genetics, animal models and therapeutic options.

2016;100(8):1022-1027.

4 Campochiaro PA, Mir TA. The mechanism of cone cell death in retinitis pigmentosa.

2018;62:24-37.

5 Zhang ZM, Dai HH, Wang L, Tao TC, Xu J, Sun XW, Yang LP, Li GL.Novel mutations of RPGR in Chinese families with X-linked retinitis pigmentosa.

2019;19(1):240.

6 Sharon D, Sandberg MA, Rabe VW, Stillberger M, Dryja TP, Berson EL.

and

mutations and clinical correlations in patients with X-linked retinitis pigmentosa.

2003;73(5):1131-1146.

7 Stone EM, Andorf JL, Whitmore SS, DeLuca AP, Giacalone JC, Streb LM, Braun TA, Mullins RF, Scheetz TE, Sheffield VC, Tucker BA.Clinically focused molecular investigation of 1000 consecutive families with inherited retinal disease.

2017;124(9):1314-1331.8 Tsang SH, Sharma T. X-linked retinitis pigmentosa.

2018;1085:31-35.

9 Vervoort R, Lennon A, Bird AC, Tulloch B, Axton R, Miano MG,Meindl A, Meitinger T, Ciccodicola A, Wright AF. Mutational hot spot within a new RPGR exon in X-linked retinitis pigmentosa.

2000;25(4):462-466.

10 Churchill JD, Bowne SJ, Sullivan LS, Lewis RA, Wheaton DK, Birch DG, Branham KE, Heckenlively JR, Daiger SP. Mutations in the X-linked retinitis pigmentosa genes RPGR and RP2 found in 8.5% of families with a provisional diagnosis of autosomal dominant retinitis pigmentosa.

2013;54(2):1411-1416.

11 Appelbaum T, Becker D, Santana E, Aguirre GD. Molecular studies of phenotype variation in canine RPGR-XLPRA1.

2016;22:319-331.

12 Chiang JPW, Lamey TM, Wang NK, Duan J, Zhou W, McLaren TL, Thompson JA, Ruddle J, De Roach JN. Development of highthroughput clinical testing of RPGR ORF15 using a large inherited retinal dystrophy cohort.

2018;59(11):4434-4440.

13 Levy SE, Boone BE. Next-generation sequencing strategies.

2019;9(7):a025791.

14 Zhang QH, Giacalone JC, Searby C, Stone EM, Tucker BA, Sheffield VC. Disruption of RPGR protein interaction network is the common feature of RPGR missense variations that cause XLRP.

2019;116(4):1353-1360.

15 Hong DH, Pawlyk BS, Adamian M, Sandberg MA, Li TS. A single,abbreviated RPGR-ORF15 variant reconstitutes RPGR function

.

2005;46(2):435-441.

16 Koyanagi Y, Akiyama M, Nishiguchi KM,

. Genetic characteristics of retinitis pigmentosa in 1204 Japanese patients.

2019;56(10):662-670.

17 Kurata K, Hosono K, Hayashi T, Mizobuchi K, Katagiri S, Miyamichi D, Nishina S, Sato M, Azuma N, Nakano T, Hotta Y. X-linked retinitis pigmentosa in Japan: clinical and genetic findings in male patients and female carriers.

2019;20(6):E1518.

18 Megaw RD, Soares DC, Wright AF. RPGR: Its role in photoreceptor physiology, human disease, and future therapies.

2015;138:32-41.

19 Breuer DK, Yashar BM, Filippova E,

. A comprehensive mutation analysis of

and

in a North American cohort of families with X-linked retinitis pigmentosa.

2002;70(6):1545-1554.

20 Fahim AT, Bowne SJ, Sullivan LS, Webb KD, Williams JT, Wheaton DK, Birch DG, Daiger SP. Allelic heterogeneity and genetic modifier loci contribute to clinical variation in males with X-linked retinitis pigmentosa due to RPGR mutations.

2011;6(8):e23021.

21 Sandberg MA, Rosner B, Weigel-Difranco C, Dryja TP, Berson EL.Disease course of patients with X-linked retinitis pigmentosa due to RPGR gene mutations.

2007;48(3):1298-1304.

22 Wu ZJ, Hiriyanna S, Qian HH, Mookherjee S, Campos MM, Gao C, Fariss R, Sieving PA, Li TS, Colosi P, Swaroop A. A long-term efficacy study of gene replacement therapy for RPGR-associated retinal degeneration.

2015;24(14):3956-3970.

23 Sheng XL, Li ZL, Zhang XF, Wang J, Ren HW, Sun YB, Meng RH,Rong WN, Zhuang WJ. A novel mutation in retinitis pigmentosa GTPase regulator gene with a distinctive retinitis pigmentosa phenotype in a Chinese family.

2010;16:1620-1628.

24 Jones BW, Pfeiffer RL, Ferrell WD, Watt CB, Marmor M, Marc RE. Retinal remodeling in human retinitis pigmentosa.

2016;150:149-165.

25 Sanchez Tocino H, Diez Montero C, Villanueva Gómez A, Lobo Valentin R, Montero-Moreno JA. Phenotypic high myopia in X-linked retinitis pigmentosa secondary to a novel mutation in the RPGR gene.

2019;40(2):170-176.

26 Talib M, van Schooneveld MJ, Van Cauwenbergh C,

. The spectrum of structural and functional abnormalities in female carriers of pathogenic variants in the RPGR gene.

2018;59(10):4123-4133.

27 Nanda A, Salvetti AP, Clouston P, Downes SM, MacLaren RE.Exploring the variable phenotypes of RPGR carrier females in assessing their potential for retinal gene therapy.

(

)2018;9(12):E643.

28 Cehajic Kapetanovic J, McClements ME, Martinez-Fernandez de la Camara C, MacLaren RE. Molecular strategies for

gene therapy.

(

) 2019;10(9):674.

29 Cehajic-Kapetanovic J, Xue K, Martinez-Fernandez de la Camara C,

. Initial results from a first-in-human gene therapy trial on X-linked retinitis pigmentosa caused by mutations in RPGR.

2020;26(3):354-359.

30 Giacalone JC, Andorf JL, Zhang QH,

. Development of a molecularly stable gene therapy vector for the treatment of

-associated X-linked retinitis pigmentosa.

2019;30(8):967-974.