Corneal histomorphology and electron microscopic observation of R124L mutated corneal dystrophy in a relapsed pedigree

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(wù)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(wù)風險。分析和評價企業(yè)短期償債能力的財務(wù)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(quán)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。”劉勇剛先生謂“此文呼吁朝廷選用‘天下之將’，賦以重權(quán)，‘便宜從事，不煩廟堂之論’，可稱剴切之論。”[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(quán)委托統(tǒng)帥各一人，足以了事。似此有見于北宋兵權(quán)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。”[2]54而且，在宋太祖時期就有“杯酒釋兵權(quán)”一事，終宋一代都對統(tǒng)領(lǐng)兵權(quán)的武將有所忌憚，想要把兵權(quán)全權(quán)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?qū)I(yè)，裝修完全從自身的需求出發(fā)，隨意改變房屋結(jié)構(gòu)和功能。