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

Abstract

Aim: To investigate the histological characteristics and ultrastructure of recurrent Chinese R124L mutated corneal dystrophy after keratoplasty.

Methods: The subjects were enrolled from a Chinese family of corneal dystrophy with R124L heterozygous gene mutation and with a history of consanguineous marriage. Normal corneal samples were used as controls.

Results: In this family, 2 patients (3 eyes) underwent penetrating keratoplasty (PKP) and 2 patients (4 eyes) underwent lamellar keratoplasty (LKP). They had recurrence at 33.5±3.0 (range 30-36)mo after keratoplasty. Among them, 1 patient (1 eye) underwent PKP again and 1 patient (2 eyes) underwent LKP again. In the R124L mutated recurrent corneal dystrophy, the corneal turbidity was mainly distributed from the upper corneal cortex to the anterior stroma; the corneal epithelium surface was rougher and more uneven; and, the corneal erosions were larger. Hematoxylin-eosin staining showed that the thickness of the corneal epithelium was uneven; the arrangement of the epithelial cells was disordered; and, some corneal epithelial cells were swollen. The results of Congo red staining, Masson's trichrome staining and Periodic acid-Schiff staining were positive, while that of Alcian blue staining was negative. Under a transmission electron microscope, deposition of high electron density substances between epithelial and basal cells, and, apoptosis of basal cells were observed. Many high electron density depositions were observed in the sub-epithelial and anterior corneal matrix.

Conclusion: In the Chinese family of recurrent corneal dystrophy with R124L gene mutation, the corneal epithelia of the recurrent cases are rougher, and the corneal depositions are extracellular amyloid fibrin.

Keywords: R124L mutation; corneal dystrophy; electron microscope observation; pathology.

Figure 1. The pedigree diagram of the CD family with R124L mutation

III5 was closely related to III6. IV7 underwent binocular penetrating keratoplasty (PKP). There was relapse in both eyes at 36mo after PKP and PKP was performed again on the right eye after recurrence. IV9 underwent binocular lamellar keratoplasty (LKP). Binocular LKP was performed for V2. The patient had CD recurrence at 33mo after the operation and LKP was performed again on both eyes after the recurrence. V25 was treated with PKP for the right eye, and CD recurred at 30mo after surgery.

Figure 2. Anterior segment photography

A-F: Images of the right eye of IV7 in the family. IV7 had relapse 3y after PKP, and underwent PKP again 27y after PKP. He was followed up 6y after the second PKP. A: Image at 1d after secondary PKP (2013-03). The corneal graft was transparent. The suture was present. B: Corneal graft transparency at 2y and 1mo after secondary PKP (2015-04). C: Image at 3y after secondary PKP (2016-03). Spotty grayish white opacity was observed in the cornea graft. D: Image at 5y and 10mo after secondary PKP (2019-01). Corneal epithelium was rough and eroded into clumps. The cornea was uneven. The deposition was coarse and thick. E: Image at 5y and 11mo after secondary PKP (2019-02). The picture showed conjunctival hyperemia, corneal edema and erosion. F: Image at 6y after secondary PKP (2019-03). The gray and white deposition was fused to flake on the surface of the cornea graft. G-J: Images of the left eye of IV7 in the family. G: Gray and white substance deposition in the center of the corneal graft at 9y after PKP (2013). H: Image at 11y after PKP (2015). I: Image at 12y after PKP (2016). J: Image at 15y after PKP (2019). The image showed that the gray and white deposition in the central cornea gradually increased and thickened with time and the corneal surface was rough. PKP: Penetrating keratoplasty.

Figure 3. Direct sequencing analysis of exon 4 of TGFBI gene

The mutation c.418 G>T resulted in Arg124Leu (R124L; indicated by the arrow).

Figure 4. Staining of corneal tissue

A, B: Normal corneal tissue sections. A: HE staining of normal corneal tissue showed that there were 5-6 layers of corneal epithelial cells with regular arrangement and the thickness of the pre-elastic layer was uniform (HE staining ×40). B: Masson's trichrome staining of normal cornea showed that the matrix collagen fiber bundles were orderly arranged (Masson's trichrome staining ×200). C-H: Histological staining of the corneal tissue of the recurrent cases. C, D: HE staining of the corneal tissue of the recurrent cases, showing uneven thickness of the corneal epithelium, disordered cell arrangement, and, disappearance of the normal cylindrical structure of the basal cells and the pre-elastic layer structure. A large number of abnormal substances were observed (C, HE staining ×100. D, HE staining ×400). E: PAS staining. Red substance was observed in the sub-epithelial and anterior stroma of the cornea (PAS staining ×100). F: The corneal tissue was stained with Congo red, and the sub-epithelial and anterior corneal stroma were flecked orange (Congo red staining ×100). G: Masson's trichrome staining. The sub-epithelial and anterior corneal stroma were red and the matrix collagen fibers were blue (Masson's trichrome staining ×100). H: Alcian blue staining. No specific staining was observed (Alcian blue staining ×100). I-N: Transmission electron microscopy (TEM) of the recurrent corneal epithelial cells. I, J: It was found that the microvilli of the recurrent corneal epithelial cells were reduced or disappeared. The basal cells were different in morphology and irregular in arrangement, and a few of them were columnar (4J, ×2500, 4K, ×6000). K: The cytoplasmic mitochondria were swelled and vacuolated (×15 000), the nuclei were hyperchromatic, and the chromatin in the nucleus was rough and concentrated. L: The typical apoptotic feature was observed (×15 000). M: Desmosomal junctions between some cells were reduced or disappeared (×15 000). N: The boundary between the anterior elastic layer and basal cells was blurred or disappeared. No obvious fibrous structure was observed in the depositions (×30 000). HE: Hematoxylin-eosin; PAS: Periodic acid-Schiff.