Clinical and genetic studies for a cohort of patients with Leber congenital amaurosis

Abstract

Purpose: Leber congenital amaurosis (LCA) is a group of early-onset retinal degenerative disorders, resulting in blindness in children. This study aimed to describe the clinical and genetic characteristics of a cohort of patients with LCA and to investigate the retinal vascular characteristics in LCA patients.

Methods: Fifty-two children with LCA were included in the study. All patients underwent detailed ocular examinations. Electroretinography (ERG) was used to evaluate the retinal function. Optical coherence tomography (OCT) was used to assess the structure change of the retina for those patients who were able to cooperate very well. Panel-based next-generation sequencing was performed to identify pathogenic variants in genes associated with LCA. Diameters of the retinal vessels were measured using the EVision AI screening system with an artificial intelligence (AI) technique. An ultrasound Doppler was used to evaluate hemodynamic parameters, including peak systolic velocity (PSV), resistive index (RI), and pulsatility index (PI), in the ophthalmic, central retinal, posterior ciliary, carotid, and internal carotid as well as external carotid arteries in 12 patients aged from 3 to 14 years.

Results: We detected 75 pathogenic variants from ten genes of RPGRIP1, CEP290, GUCY2D, LCA5, AIPL1, CRB1, RPE65, CRX, RDH12, and TULP1, including 29 novel and 36 previously reported variants in 52 affected children with LCA, with the highest detective rate in RPGRIP1 (26.9%). Fundus appearance is diverse in patients with LCA, ranging from normal to severe peripheral or central retinopathy. Retinal vasculature was evaluated in 12 patients with different gene variants, showing narrowed arteries with an average diameter of 43.6 ± 3.8 μm compared to that of 51.7 ± 2.6 μm in the normal controls (P < 0.001, n = 12). Meanwhile, their hemodynamic parameters were changed as well in the ophthalmic artery (OA), with a decreased PSV (P = 0.0132, n = 12) and slightly increased PI (P = 0.0488, n = 12) compared to the normal controls. However, the hemodynamic parameters did not change significantly in the other vessels.

Conclusions: Blood supply to the eyeball is predicted to be reduced in patients with LCA, presumably due to photoreceptor cell degeneration. The novel identified variants will expand the spectrum of variants in LCA-related genes and be useful for studying the molecular mechanisms of LCA.

Keywords: Gene; LCA; Retinal vasculature.

Fig. 1

Different manifestations of fundus and retinal structure in different causative genes of LCA. A(a) B(a) Patient 01 (5 months) and Patient 04 (6 years old) with the mutations of GUCY2D showed an almost normal fundus appearance. A(b) Patient 16 (2-year-old) with the mutations of RPGRIP1 showed attenuated vessels and mottled fundus. A(c, d) Patient 27 (1-year-old), 34 (5 months) with the mutations of CEP290 showed patchy retinal degeneration in the peripheral retina. A(e) Patient 45 with the mutations of LCA5 (3-year-old) showed thinner retina and periphery retinal pigment rearrangement. A(f) Patient 49 (5-year-old) with the mutations of RPE65 showed a white dot deposit. A(g) Patient 39 (3-year-old) with the mutations of CRB1 had the gold-foil reflection of macular, and the OCT image showed a coarsely laminated retina. A(h) Patient 51 (5-year-old) with the mutations of RDH12 showed attenuated vessels, macular atrophy, and bone-spicule pigmentation. OCT revealed marked atrophy of the outer retinal layer in the macular region, with the evident decline of the outer nuclear layer thickness and loss of the external limiting membrane and ellipsoid zone. B(b, c) Patient 13 (14 years old) with the mutations of RPGRIP1 showed a grey optic disc, attenuated vessels, retinal exudated, and retinal pigment deposited. Patients 24 (6 years old) showed retinal pigment rearrangement. B(d) Patient 44 (6 years old) with the mutations of LCA5 showed pale optic nerve and pigmentary changes. B(e) Patient 50 (5 years old) with the mutations of RPE65 showed mottled fundus. RPE atrophy, OCT scans confirmed that the peripheral outer retinal layers (myoid, ellipsoid, and outer segment layer) were thinned and disappeared in Figure B(b, c, d, e)

Fig. 2

The homology analysis of the proteins from five novel missense variants. (A–E) Multiple alignments of amino acids showed that leucine at position 172, 334 of GUCY2D, cysteine at position 698 of CRB1, threonine at position 1038 of CRB1, and leucine at position 58 of CEP290 were highly conserved among different species

Fig. 3

Three-dimension model construction for novel missense variations. The red dashed lines represent hydrogen bonds. (A) The model showed that a wild-type Leucine in GUCY2D was replaced by Proline at codon 172, which would make the connective hydrogen bands lost between the Leucine and Alanine at codon 168. (B) A wild-type Alanine in GUCY2D was replaced by Proline at codon 334. (C) A wild-type polar amino acid of Cysteine in CRB1 was replaced by a nonpolar amino acid of Phenylalanine at codon 698. Different programs predicted the Gibbs free energy (ΔΔG) and showed the protein was unstable. (D) A wild-type polar amino acid of Threonine in CRB1 was replaced by a nonpolar amino acid of an amino acid of Proline at codon 1038, disrupting the hydrogen bonding. (E) A large-size wild-type Leucine was replaced by a small-size Valine at codon 58 in CEP290 ΔΔG predicting the protein became unstable. All the above-mentioned amino acid substitutions may damage the stability of the protein structure and function

Fig. 4

Scatter diagram of vessel diameters of LCA patients (LCA) and controls (Ctrl). ***P < 0.001