INTRODUCTION

Corneal dystrophy (CD) with R124L mutation is a familial, primary, binocular autosomal dominant genetic disease

. According to the 2015 International Classification of Corneal Dystrophies (IC3D) and based on the affected anatomic site, CD is sub-classified: epithelial/sub-epithelial,epithelial-stromal, stromal, and endothelial dystrophy

. CD with R124L mutation belongs to epithelial-stromal CD

. The gene mutation sites of CD have been confirmed to be in the transforming growth factor B-induced (

or

)gene on chromosome 5q31

. The diagnostic basis of CD mainly includes clinical observation of lesion location and morphology, histopathological changes, and genetic testing and typing results

.

Current treatments for CD include phototherapeutic keratectomy (PTK) and keratoplasty

. After surgery,the symptoms could be improved or relieved and vision could be improved. Some patients may inevitably suffer from aggravation or recurrence of corneal opacity

. The pathological changes of R124L mutant CD are reported to be mainly caused by accumulation of abnormal substances in the anterior stroma

. However, the corneal pathological changes of recurrent cases have not been reported. In this study, a Chinese CD family with R124L mutation who had a history of consanguineous marriage was enrolled. The pathological changes of cornea tissues with recurrence after keratoplasty were observed by electron microscopy and histopathological analysis. The sites of gene mutations were detected. Our findings may provide evidence for the pathological changes of recurrent CD caused by R124L.

本工程現(xiàn)已運行1年多的時間，運行過程中，尚未發(fā)現(xiàn)地表隆起、樁的變形，周邊地基土變形等現(xiàn)象，目前使用正常，表明本方案的合理性。

SUBJECTS AND METHODS

The 14 patients had recurrent eye pain, eye abrasion and tearing, accompanied by redness and progressive vision loss since childhood. After keratoplasty, the patients IV7, IV9, and V2 (Figure 1) had CD relapse at 33.5±3.0mo. For the corneal morphology of recurrent cases, it was observed that the corneal epithelium was rougher and the corneal roughness was more obvious. The corneal epithelium was eroded into clumps and the deposition was thicker and closer to the corneal surface(Figure 2). The clinical manifestations of the pedigree patients are shown in Table 1. All patients were found to have R124L mutation in the

gene (heterozygous mutation in exon 4 c.418 G＞T; Figure 3).

Some studies have reported that the R124L mutation of CD has an early onset age and often has recurrent corneal epithelial erosion from childhood, with obvious symptoms and rapid progress

. Later, there may be corneal punctuated or map-like turbidity, which may gradually expand and merge,leading to vision loss and blindness

. Surgical treatments such as PTK or keratoplasty can be performed. Nevertheless,the disease often relapses within several years after the operation. When there is CD recurrence, PTK or keratoplasty can be performed again

. Therefore, the patients with CD recurrence in this study underwent PKP or LKP again.

Routine ophthalmological examinations, including visual acuity examination, slit lamp microscope examination, and,intraocular pressure and fundus examination were performed.

Peripheral blood (10 mL each) was collected from 14 patients with CD. The blood was anticoagulated with EDTA. DNA was extracted from peripheral blood with DNA extraction kit (Qiagen, Santa Clara, CA, USA). Gene sequencing was performed with BigDye terminator V3.1 cycle sequencing kit (Qiagen, Santa Clara, CA, USA) on an ABI 31300XL analyzer (ABI, Foster City, CA). Sequence Scanner V1.0 software was used for analyzing sequencing data, with reference to NCBI GeneBank(NM_000358 for

).

The corneal specimens were fixed with 2%paraformaldehyde glutaraldehyde, rinsed with phosphoric acid buffer, fixed with 4% osperium tetroxide, dehydrated with ethanol step by step, fixed with epoxy resin, made into ultrathin sections, stained with lead uranium acetate and lead citrate, and were observed under transmission electron microscope (TEM).

The corneal tissues were fixed with 0.4 g/L paraformaldehyde, dehydrated with graded ethanol, transparent with xylene, embedded with paraffin,and cut into 5 μm sections. Hematoxylin-eosin (HE) staining,Alcian blue staining, Periodic acid-Schiff (PAS) staining,Congo red staining, and Masson’s trichrome staining were carried out respectively according to routine procedures.The sections were observed and photographed under a light microscope.

一天，愛德華多很晚才從街上回家，看到克里斯蒂安的黑馬拴在木樁上。老大穿著他那身最體面的衣服在院子里等他。女人捧著馬黛茶罐進進出出。克里斯蒂安對愛德華多說：“我要到法里亞斯那兒去玩。胡利安娜就留給你啦；如果你喜歡她，你就派她用場吧。”

償債能力分析，是指對企業(yè)償還各種債務的能力和保障程度所作的分析，通過這種分析可以有效地揭示出企業(yè)存在的財務風險。分析和評價企業(yè)短期償債能力的財務指標主要有流動比率、速動比率等。

RESULTS

The pedigree diagram of the family with a history of consanguineous marriage is shown in Figure 1. There were 59 members (14 patients with CD and 45 healthy people) of 4 generations in this family, all of whom were Han Chinese. Among the 14 patients with CD,1 patient (2 eyes) underwent penetrating keratoplasty (PKP)and CD recurred 36mo after the operation. The right eye of this patient underwent PKP again because of recurrence.One patient (1 eye) underwent PKP and CD recurred 30mo after the operation. One patient (2 eyes) underwent lamellar keratoplasty (LKP) and CD recurred 33mo after surgery. After recurrence, binocular LKP was performed again.

The study was carried out in accordance with the principles of the Declaration of Helsinki and was approved by the Weifang Eye Hospital Ethics Committee.Each patient provided the signed informed consent.

還有如《將帥》篇，論選將的重要性。秦觀深感北宋兵權輪換制的流弊，提出“臣以為西北二邊，宜各置統(tǒng)帥一人……進退賞罰，盡付其手，得以便宜從事?！眲⒂聞傁壬^“此文呼吁朝廷選用‘天下之將’，賦以重權，‘便宜從事，不煩廟堂之論’，可稱剴切之論?！盵4]而在這一點上，朱剛先生論述：“唯《將帥》篇謂西北二邊之事，朝廷不必詳其細節(jié)，宜全權委托統(tǒng)帥各一人，足以了事。似此有見于北宋兵權集中之流弊，但此策亦早見于蘇軾的賢良進卷，秦觀只承其說而已?！盵2]54而且，在宋太祖時期就有“杯酒釋兵權”一事，終宋一代都對統(tǒng)領兵權的武將有所忌憚，想要把兵權全權委托一名統(tǒng)帥，只能說是不切實際之言。

HE staining showed that there were 5 to 6 layers of epithelial cells in normal corneal tissue with regular arrangement and uniform thickness of the pre-elastic layer (Figure 4A).Masson’s trichrome staining showed regular arrangement of matrix collagen fiber bundles (Figure 4B). However, in the corneal tissue of recurrent cases, the thickness of corneal epithelium was uneven and the arrangement of epithelial cells was disordered (Figure 4C, 4D). The normal columnar structure of basal cells and the structure of the anterior elastic layer disappeared. Instead, a large amount of red staining substances were observed. Congo red staining was positive(Figure 4E). Orange red was observed in the sub-epithelial and anterior stroma. Masson’s trichrome staining was positive(Figure 4F). The sub-epithelial and anterior stroma was stained red and the stromal collagen fibers were stained blue.PAS staining was positive (Figure 4G). Red substance was seen in the sub-epithelial and anterior stroma of the cornea.Alcian blue staining was negative and no specific staining was observed.

商品住宅交付時多為毛坯房，絕大多數(shù)業(yè)主不具備裝修專業(yè)知識或選擇的裝修公司不夠專業(yè)，裝修完全從自身的需求出發(fā)，隨意改變房屋結構和功能。特別是在一些不好的戶型中，容易出現(xiàn)大改動，易造成房屋結構的破壞，留下質量安全隱患。部分裝修公司自身技術不過關，又缺乏有效的監(jiān)管，裝修完不久就出現(xiàn)吊頂開裂、墻磚脫落等問題，業(yè)主無處維權。

劉劍文解釋道，在個人所得中，有勞動所得（積極所得），也有非勞動所得（消極所得）。前者比如工薪所得、勞務所得、稿酬所得和特許權使用費所得，而后者比如利息、股息、紅利所得、偶然所得等，而按照目前的稅制，只需繳納20%的稅。

Under TEM, it was observed that the epithelium microvilli of recurrent cornealspecimens reduced or disappeared. The shape of basal cells varied (Figure 4I). There was swelling of cell mitochondria(Figure 4J). There were vacuoles in the cytoplasm (Figure 4K). The nuclei were hyperchromatic, enriched, and crescentshaped, or became fragmented, distorted, contracted, or even disappeared. Typical apoptotic features were observed (Figure 4L). Desmosomal junctions between some cells were reduced or disappeared. The boundaries between the anterior elastic layer and basal cells were blurred or disappeared (Figure 4M).A large number of high-density abnormal substances were dispersed and accumulated, and no obvious fibrous structure was observed in the depositions (Figure 4N).

DISCUSSION

The characteristics of the CD family in this study were as follows. First, it has been reported that the earliest onset age was generally around 7 years old

. In this study, the youngest age of onset in this family was 1 year old. To our knowledge,this is the youngest reported age of onset. Second, this family was with a history of consanguineous marriage.

The gene mutation of CD and the pathological changes of cornea have been reported previously

. This paper is the first to report the pathological changes and TEM ultrastructure of the corneal graft with recurrent CD. In the CD classification in 2015, R124L mutation is classified as epithelial-basal CD. This paper was also one of the few families with R124L mutations reported with a history of consanguineous marriage.

In this study, gene sequencing found heterozygous gene mutation at the R124L locus in all patients, which was consistent with previous findings

. The R124L mutation is also a common mutation type. In other atypical cases,R125H, R555Q, R555W and a few mutations at the R124C,G623D, H572R and H626P sites have also been reported

.Our study also found that the CD of this pedigree had high incidence, early onset age, severe condition and high recurrence rate. A possible explanation is the genetic features of consanguineous marriage. The mutation from the polar hydrophilic arginine to the non-polar hydrophobic leucine may change the polarity and hydrophilicity of amino acids,thus changing the three-dimensional structure of protein and contributing to the severity of the disease.

Fourteen patients with CD from a family with a history of consanguineous marriage in China were enrolled.Among them, the ratio of male to female was 6:8. The youngest was 1 year old, and the oldest was 69 years old. The 14 patients underwent routine ophthalmological examination and peripheral blood collection. The diseased corneal tissues were collected from patients of IV7 and V2 (Figure 1) who had recurrence after keratoplasty and underwent keratoplasty again. For control, 3 pieces of normal corneal tissues donated by Weifang Eye Hospital were collected.

通過使用二元亮度差分方程(圖5)獲得了地面真實角點,檢測結果得到了一定的改進?？赡苁且驗椴捎枚M制方法,非均勻平坦區(qū)域之間的角點可以得到更高的USAN,并且超過亮度差閾值。

In this study, HE staining clearly showed the scope and location of recurrent lesions, including uneven thickness of corneal epithelial cells, disordered cell arrangement, swelling of some corneal epithelial cells and disappearance of the normal cylindrical structure of basal cells. It is reported that PAS staining could be positive in CD with R124L mutations

.In this study, corneal tissue sections with recurrent lesions were stained with Congo red, showing lamellar orange color in the sub-epithelial and anterior stroma of the cornea, which confirmed that the extracellular substance was amyloidosis.Masson’s trichrome staining of the corneal epithelium and anterior stroma showed red staining, indicating that the extracellular depositions were acidic and microfibrous.Therefore, in this relapsed CD family with R124L mutation,the corneal depositions were extracellular amyloid fibrin. This was consistent with previous studies

.

Histopathological examination serves as the gold standard for diagnosis

. HE staining can clarify the scope, size and shape of the lesion. Specific staining can reveal specific components(intrinsic and extrinsic) of the lesion and can give a rough idea of the nature of its depositions. TEM can reveal the pathological changes at the cellular/ultrastructural level. In this study, TEM examination displayed that there were reduced or disappeared epithelium microvilli of recurrent corneal specimens. The basal cell morphology in the epithelium was different. Mitochondria in cells were swelling. Vacuoles were observed in the cytoplasm. Corneal histopathological and TEM ultrastructural changes of the recurrent cases further confirmed that the recurrent CD caused by R124L mutation had characteristic structural changes, which might be related to clinical symptoms and gene mutations

.

Limitations of this study: First, this study only observed the corneal tissues of recurrent cases and did not compare them with the corneal tissues after the first operation. Second, only one family with a history of consanguineous marriage was studied. The sample size should be expanded in future study.

In this Chinese recurrent CD family with R124L gene mutation, the corneal epithelium of the recurrent cases was rougher. The recurrent specimens suggested extracellular amyloid fibrin in the cornea. Early diagnosis of CD is based on slit lamp examination and clinical symptoms. The advanced cases of CD are classified and diagnosed according to corneal involvement, anatomical site, histopathological features, genetic pattern and genetic testing results. Although consanguineous marriage is now relatively rare in China, it still exists in relatively remote and economically underdeveloped parts of the country. Thus, earlier screening of patients and their families of consanguineous marriage should be performed.

Supported by the National Natural Science Foundation of China (No.81770955); the Major Clinical Research Project of Shanghai Shenkang Hospital Development Center (No.SHDC2020CR1043B); the Project of Shanghai Xuhui District Science and Technology (No.2020-015);the Project of Weifang Science and Technology Bureau(No.2021RKX160).

None;

None;

None;

None;

None;

None;

None;

None.

1 Atia R, Jouve L, Georgon C, Laroche L, Borderie V, Bouheraoua N. Imaging of reis-Bückler corneal dystrophy.

2019;42(1):105-107.

2 Jun I, Ji YW, Choi SI, Lee BR, Min JS, Kim EK. Compound heterozygous mutations in TGFBI cause a severe phenotype of granular corneal dystrophy type 2.

2021;11:6986.

3 Moshirfar M, Bennett P, Ronquillo Y. Corneal Dystrophy. In:

Treasure Island (FL): StatPearls Publishing; 2022.

4 Cai JQ, Zhu LR, Zha Y, Kang QY. TGFBI gene mutation analysis in Chinese families with corneal dystrophies.

2016;20(7):388-392.

5 Zhang YL, Ying JL, Zhou WP, Zhu LR, Cai JQ. Analyses of TGFBI gene mutation spectrum in four Chinese families with corneal dystrophy.

2015;95(2):116-119.

6 Chao-Shern C, DeDionisio LA, Jang JH, Chan CC, Thompson V, Christie K, Nesbit MA, McMullen CBT. Evaluation of TGFBI corneal dystrophy and molecular diagnostic testing.

2019;33(6):874-881.

7 Nagano C, Nozu K, Yamamura T,

. TGFBI-associated corneal dystrophy and nephropathy: a novel syndrome?

2019;8(1):14-17.

8 Weiss JS, M?ller HU, Lisch W,

. The IC3D classification of the corneal dystrophies.

2008;27(Suppl 2):S1-S83.

9 Lisch W, Weiss JS. Clinical and genetic update of corneal dystrophies.

2019;186:107715.

10 Matthaei M, Hribek A, Clahsen T, Bachmann B, Cursiefen C,Jun AS. Fuchs endothelial corneal dystrophy: clinical, genetic,pathophysiologic, and therapeutic aspects.

2019;5(4):151-175.

11 Lang SJ, B?hringer D, Reinhard T. Penetrating limbokeratoplasty for gelatinous corneal dystrophy.

2019;236(2):169-172.

12 Vinciguerra P, Vinciguerra R, Randleman JB, Torres I, Morenghi E,Camesasca FI. Sequential customized therapeutic keratectomy for reis-Bücklers’ corneal dystrophy: long-term follow-up.

2018;34(10):682-688.

13 Sridhar U, Tripathy K, Bansal Y. Repeated phototherapeutic keratectomy (PTK) followed by PTK with photorefractive keratectomy for anterior granular corneal dystrophy.

2020;68(12):3038.

14 Zeng L, Zhao J, Chen YJ, Shang JM, Aruma A, Zhou XT. Multiple phototherapeutic keratectomy treatments in a Chinese pedigree with corneal dystrophy and an R124L mutation: a 20-year observational study.

2019;19(1):191.

15 Chiou AG, Florakis GJ, Copeland RL, Williams VA, McCormick SA,Chiesa R. Recurrent meesmann’s corneal epithelial dystrophy after penetrating keratoplasty.

1998;17(5):566-570.

16 Zhao F, Liu Y, Guan T. Analysis of

gene mutations in three Chinese families with corneal dystrophy.

2019;2019:6769013.

17 Liang QF, Sun XG, Jin XY.

gene mutation in a Chinese pedigree with Reis-Bücklers corneal dystrophy.

2012;32(1):74-80.

18 Tanhehco TY, Eifrig DE Jr, Schwab IR, Rapuano CJ, Klintworth GK. Two cases of Reis-Bücklers corneal dystrophy (granular corneal dystrophy type III) caused by spontaneous mutations in the

gene.

2006;124(4):589-593.

19 Piao MZ, Zhou XT, Wu LC, Chu RY. Arg555Gln mutation of

gene in geographical-type Reis-Bücklers corneal dystrophy in a Chinese family.

2012;40(3):1149-1155.

20 Yang QN, Zhao YW, Guo LH, Yan NH, Liu XY, Cai SP. Arg124Cys mutation of the

gene in a Chinese pedigree of Reis-Bücklers corneal dystrophy.

2011;4(3):235-238.

21 Ma K, Liu G, Yang Y, Yu M, Sui RF, Yu WH, Chen XM, Deng YP, Yan NH, Cao GQ, Liu XY.

gene mutation analysis in a Chinese pedigree of Reis-Bücklers corneal dystrophy.

2010;16:556-561.

22 Li DD, Qi YH, Wang L, Lin H, Zhou N, Zhao LM. An atypical phenotype of Reis-Bücklers corneal dystrophy caused by the G623D mutation in TGFBI.

2008;14:1298-1302.

23 Wheeldon CE, de Karolyi BH, Patel DV, Sherwin T, McGhee CNJ,Vincent AL. A novel phenotype-genotype relationship with a TGFBI exon 14 mutation in a pedigree with a unique corneal dystrophy of Bowman’s layer.

2008;14:1503-1512.

24 Venkatraman A, Hochart G, Bonnel D, Stauber J, Shimmura S, Rajamani L, Pervushin K, Mehta JS. Matrix-assisted laser desorption ionization mass spectrometry imaging of key proteins in corneal samples from lattice dystrophy patients with TGFBIH626R and TGFBI-R124C mutations.

2019;13(1):e1800053.

25 Trufanov SV, Tekeeva LY, Salovarova EP, Bag RZ, Sukhanova EV.Corneal dystrophies.

2018;134(5):118-125.

26 de Sousa Peixoto R, Mutch S, Eason J, Jaakson K, Haamer E,Maharajan VS. Association of unilateral lattice corneal dystrophy on slit lamp and bilateral confocal microscopy features with H572R mutation in the TGFBI gene.

2019;33(12):1973-1975.

27 Wang LY, Zhao CC, Zheng T, Zhang Y, Liu HR, Wang X, Tang XL,Zhao BW, Liu P. Torin 1 alleviates impairment of TFEB-mediated lysosomal biogenesis and autophagy in TGFBI (p.G623_H626del)-linked thiel-behnke corneal dystrophy.

2022;18(4):765-782.

28 Li DD, Qi YH, Han Q, Lin H, Zhao LM, Zhang CM. Analysis of TGFBI gene mutation in a Chinese family with atypical Reis-Buckler corneal dystrophy.

2009;26(3):245-248.

29 Qiu WY, Zheng LB, Pan F, Wang BB, Yao YF. New histopathologic and ultrastructural findings in Reis-Bücklers corneal dystrophy caused by the Arg124Leu mutation of TGFBI gene.

2016;16(1):158.

30 Nishino T, Kobayashi A, Mori N, Yokogawa H, Sugiyama K.

imaging of reis-Bücklers and thiel-behnke corneal dystrophies using anterior segment optical coherence tomography.

2020;14:2601-2607.

31 Liang QF, Pan ZQ, Sun XG, Baudouin C, Labbé A. Reis-Bücklers corneal dystrophy: a reappraisal using

and

imaging techniques.

2014;51(4):187-195.

32 Reda AM, Saad El-Din SA. Rare stromal corneal dystrophic diseases in Oman: a clinical and histopathological analysis for accurate diagnosis.

2020;13(2):70-75.