Lactoferrin Glu561Asp facilitates secondary amyloidosis in the cornea

Abstract

Aim: To elucidate the pathogenic mechanism of amyloid formation in corneal amyloidosis with trichiasis.

Methods: Ophthalmological examination was performed in nine patients to determine secondary corneal amyloidosis with trichiasis. Congo red staining and immunohistochemistry using anti-human lactoferrin antibody were used for biopsied corneal samples. For genetic analyses, single strand conformation polymorphism (SSCP), direct DNA sequence analysis, and polymerase chain reaction (PCR) induced mutation restriction analysis (IMRA) were employed to detect lactoferrin gene polymorphism.

Results: All patients had had trichiasis at least for 1 year, and all amyloid-like deposits were found in one eye with trichiasis. Ophthalmological examination revealed that eight patients showed gelatinous type of amyloid deposition and one showed lattice type of amyloid deposition. Studies of biopsied corneal samples with Congo red stain revealed positive staining just under the corneal epithelial cells. Immunoreactivity of anti-human lactoferrin antibodies was recognised in all tissues with positive Congo red staining. Lactoferrin gene analysis revealed that seven patients were heterozygotic and two were homozygotic for lactoferrin Glu561Asp. The frequency of the polymorphism in the patients was significantly different from that in 56 healthy control subjects.

Conclusion: Lactoferrin Glu561Asp is a key polymorphism related to facilitating amyloid formation in corneal amyloidosis with trichiasis.

Figure 1

Amyloid deposits in the cornea of patients 1 (A) and 9 (B).

Figure 2

Histochemical analysis for the excised cornea, (A) and (B) Congo red staining and an anti-human lactoferrin antibody staining of the sample from patient 8. (C) An anti-human lactoferrin antibody staining of the corneal epithelium of patient 8. (D) Immunohistochemistry using an anti-human lactoferrin antibody for cilia in patient 9. The arrow indicates the positive stained mass stuck on the cilia. Magnification ×100.

Figure 3

Detection of the lactoferrin Glu561Asp gene by means of PCR-IMRA, M represents the 100 bp DNA size marker; C represents the data from control subject; Numbers 1–9 correspond the patient numbers given in table 1. Analyses for lactoferrin Glu561Asp were performed via PCR-IMRA as described. When allele for lactoferrin Glu561Asp only exists, the digestion bands (158 bp and 28 bp) are observed.

Figure 4

Structural feature of the human lactoferrin Glu561. (A) A ribbon diagram of human lactoferrin. The region depicted in close up (B) is indicated by a rectangle. (B) The main chain backbone is shown in grey. The residues, which involve the charged or hydrophobic interaction network in this region, are shown as a ball and stick model. The Asp mutated from Glu561 is shown in purple.