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，利用移動互聯網、云計算、物聯網、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，將物理環境和虛擬環境有機融合，實現多元化交互教學的學習環境。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，畢竟業務上要靠他幫忙。

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