Whole-exome sequencing identifies LRIT3 mutations as a cause of autosomal-recessive complete congenital stationary night blindness

Abstract

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disorder. Two forms can be distinguished clinically: complete CSNB (cCSNB) and incomplete CSNB. Individuals with cCSNB have visual impairment under low-light conditions and show a characteristic electroretinogram (ERG). The b-wave amplitude is severely reduced in the dark-adapted state of the ERG, representing abnormal function of ON bipolar cells. Furthermore, individuals with cCSNB can show other ocular features such as nystagmus, myopia, and strabismus and can have reduced visual acuity and abnormalities of the cone ERG waveform. The mode of inheritance of this form can be X-linked or autosomal recessive, and the dysfunction of four genes (NYX, GRM6, TRPM1, and GPR179) has been described so far. Whole-exome sequencing in one simplex cCSNB case lacking mutations in the known genes led to the identification of a missense mutation (c.983G>A [p.Cys328Tyr]) and a nonsense mutation (c.1318C>T [p.Arg440(∗)]) in LRIT3, encoding leucine-rich-repeat (LRR), immunoglobulin-like, and transmembrane-domain 3 (LRIT3). Subsequent Sanger sequencing of 89 individuals with CSNB identified another cCSNB case harboring a nonsense mutation (c.1151C>G [p.Ser384(∗)]) and a deletion predicted to lead to a premature stop codon (c.1538_1539del [p.Ser513Cysfs(∗)59]) in the same gene. Human LRIT3 antibody staining revealed in the outer plexiform layer of the human retina a punctate-labeling pattern resembling the dendritic tips of bipolar cells; similar patterns have been observed for other proteins implicated in cCSNB. The exact role of this LRR protein in cCSNB remains to be elucidated.

Figure 1

LRIT3 Mutations and the Associated Electroretinographic Phenotype (A) Family A (left) and family B (right), each with an affected individual who is a compound heterozygote for mutations in LRIT3, and cosegregation analysis in available family members. The cCSNB-19018-affected index individual (II.1 of family A) used for whole-exome next-generation sequencing is marked with a red arrow. The mother of I.2 of family A is deceased, which is highlighted by a crossed line. Females and males are depicted by circles and squares, respectively. Filled and unfilled symbols indicate affected and unaffected status, respectively. ERGs of a normal (“unaffected”) subject are compared with those of individuals II:1 from family A and II:1 from family B. Normal ERGs have a rod-driven waveform, a mixed (combined) rod and cone response elicited by a bright flash of light, and two cone-driven responses at different frequencies of stimulation (1 and 29 Hz). The ERGs from affected individuals II:1 from family A and II:1 from family B are typical of cCSNB and differ from a normal ERG as follows: there is a mixed response with a preserved negative component of the waveform (a-wave [from photoreceptors]) but a reduced positive component (b-wave [from postreceptoral retinal activity]), a cone-driven 1 Hz response with an unusual waveform, a cone-driven 29 Hz flicker ERG with a typical broadened trough and a mildly delayed implicit time, and no detectable rod-driven waveform. (B) LRIT3 (RefSeq NM_198506.3) structure containing four coding exons. Different mutations identified in persons with cCSNB are depicted. Filled and unfilled boxes represent coding regions and UTRs of LRIT3, respectively. The start codon is indicated by an arrow. (C) The translated amino acid sequence of LRIT3 (RefSeq NP_940908.3), predicted domains, and the different LRIT3 alterations of two persons with cCSNB are indicated. The exact start and end of the domains vary depending on the different prediction programs used.

Figure 2

LRIT3 Immunohistochemistry in Human Retinal Sections (A) LRIT3 signal (green) in the OPL double labeled with an ON bipolar cell marker against Goα (red) was detected in the human retina by confocal microscopy. A strong signal with the human LRIT3 antibody was detected in a punctate manner at presumable dendritic tips of the ON bipolar cells (16 stacks of the confocal-microscopy image were chosen). The following abbreviations are used: PHR, photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; and GCL, ganglion cell layer. (B) A 3.5× zoom of (A) focuses on LRIT3 staining at presumable dendrites of the bipolar cells (three stacks of the confocal-microscopy image were chosen). The scale bar represents 20 μm.

Figure 3

Genes Underlying CSNB Different forms of CSNB in humans are classified according to their electroretinographic features, mode of inheritance, clinical phenotype, and mutated genes. Affected individuals discussed herein show a complete type of Schubert-Bornschein ERG. Genes are indicated in italics and are underlined. Chromosomal locations are given in parentheses. The following abbreviations are used: cCSNB, complete CSNB; icCSNB, incomplete CSNB; AR, autosomal recessive; and AD, autosomal dominant.