PMC7195602_01
Genealogy of the affected family. Solid symbols indicate affected subjects. Arrow indicates the proband.
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Five affected family members in two generations (three siblings and two sons of the proband) were identified (Figure 1). The proband (subject II-5, Figure 1), was a 59-year-old woman who had consulted for decreased vision over the previous 20 years and drooping eyelids. Four available healthy relatives (individuals II-1, II-9, II-10, and III-3 in Figure 1) were demonstrated to carry wild-type GSN sequences.
Phenotypic appearance of FAF affected subjects. Clinical images of subjects II-2 (A), II-5 (B), and II-6 (C). Cutis laxa, drooping eyelids, and hyperpigmented skin are evident.
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FAF such as cutis laxa and masklike facies were evident in this patient. Figure 2 shows the facial appearance of subjects II-5, II-2, and II-6.
Slit-lamp photographs of affected subjects II-2, II-5, and II-6. Multiple central and peripheral lattice-pattern lines and thin dots with centripetal distribution are evident at the subepithelium and anterior corneal stroma. On retroillumination, branching refractile lines were evident in all three patients. A: Right eye. B: Left eye.
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On slit-lamp corneal examination, all three affected patients exhibited bilateral subepithelial haze with pronounced central and peripheral lattice-pattern lines involving the visual axis (Figure 3A-F) which are typical LCD features.
Slit-lamp photographs of subjects III-1 and III-2. Incipient corneal changes corresponding to lattice lines are observed predominantly in the right eye of both subjects. A: Right eye. B: Left eye.
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However, the examination of the cornea disclosed subtle changes consisting of bilateral, thin branching refractile, whitish lines and dots located subepithelially and at the anterior stroma in the central and peripheral corneas in both subjects (Figure 4).
Histopathological findings in skin from FAF II-5 subject. A:Histological sections from the eyelid skin biopsy, stained with hematoxylin and eosin and Congo red, showing thin skin with mild orthokeratotic hyperkeratosis, papillomatosis, mild inflammatory infiltrate composed of perivascular lymphocytes in the superficial dermis and focally pigmentary incontinence. Dermal appendices appear normal. Congo red staining indicates amyloid deposits composed of internal degradation fragments produced during the aberrant processing in the hair follicles (arrows). B: Eyelid skin biopsy shows fibroconnective tissue, skeletal muscle, and vessels (arrows). The Congo red staining is positive in the walls of the vessels.
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The Congo red stain showed abundant deposition of amyloid material in the basement membranes of hair follicles and in blood vessel walls (Figure 5A,B).
Partial Sanger sequencing of exon 12 of the GSN gene. A heterozygous T>G transversion at nucleotide 1631 was demonstrated in affected patients (A-D). This variant predicts the novel p.Met544Arg substitution at the GSN protein. E: A control DNA sequence is shown.
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This variant was confirmed with Sanger sequencing in DNA from all available relatives affected with FAF (Figure 6A-C), as well as in DNA from the two younger individuals with mild lattice corneal dystrophy and no evidence of systemic disease (Figure 6).
In silico pathogenicity predictions for the novel GSN (c.1631T>G; p.Met544Arg) variant identified in this study. A: Variant impact was assessed using the Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), MutationTaster, and Protein Variation Effect Analyzer (PROVEAN) algorithms. B: In silico prediction of the effects on the protein stability produced by the p.Met544Arg replacement on GSN. Algorithm predictions are based on changes in folding free energy (DeltaDeltaG in Kcal/mol). Protein stability predictions were performed using the crystal structure of calcium-free human gelsolin. C: Alignment of the amino acid sequence of gelsolin across different species. The shaded column with the red mark indicates the position of the mutated residue identified in this study. As observed, methionine 544 is strictly conserved among numerous vertebrate species.
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Four different algorithms used in this study (SIFT, PolyPhen-2, MutationTaster, and PROVEAN) predicted that the p.Met544Arg is deleterious, possibly damaging, or disease causing (Figure 7A). Additionally, three different prediction tools based on changes in folding free-energy (DUET, SDM, and CUPSAT) predicted that this variant reduces the structural stability of the GSN protein (Figure 7B). Finally, the amino acid sequence comparison showed that methionine 544 is strictly conserved among GSN proteins from numerous vertebrate species (Figure 7C).