Mutations in KCNJ13 cause autosomal-dominant snowflake vitreoretinal degeneration

Abstract

Snowflake vitreoretinal degeneration (SVD, MIM 193230) is a developmental and progressive hereditary eye disorder that affects multiple tissues within the eye. Diagnostic features of SVD include fibrillar degeneration of the vitreous humor, early-onset cataract, minute crystalline deposits in the neurosensory retina, and retinal detachment. A genome-wide scan previously localized the genetic locus for SVD to a 20 Mb region flanked by D2S2158 and D2S2202. This region contains 59 genes, of which 20 were sequenced, disclosing a heterozygous mutation (484C > T, R162W) in KCNJ13, member 13 of subfamily J of the potassium inwardly rectifying channel family in all affected individuals. The mutation in KCNJ13, the gene encoding Kir7.1, was not present in unaffected family members and 210 control individuals. Kir7.1 localized to human retina and retinal pigment epithelium and was especially prevalent in the internal limiting membrane adjacent to the vitreous body. Molecular modeling of this mutation predicted disruption of the structure of the potassium channel in the closed state located immediately adjacent to the cell-membrane inner boundary. Functionally, unlike wild-type Kir7.1 whose overexpression in CHO-K1 cells line produces highly selective potassium current, overexpression of R162W mutant Kir7.1 produces a nonselective cation current that depolarizes transfected cells and increases their fragility. These results indicate that the KCNJ13 R162W mutation can cause SVD and further show that vitreoretinal degeneration can arise through mutations in genes whose products are not structural components of the vitreous.

Figure 1

Sequence Tracings and Structural Changes of the Normal and R162W Kir7.1 Proteins (A) Sequence tracings of normal and predicted structure of the normal and 484C > T KCNJ13 gene resulting in the arginine-to-tryptophan (R162W) amino acid change. (B) An overview of the normal human Kir7.1 tetramer built by homology modeling. Magenta cylinders and yellow arrows indicate α helices and β strands, respectively. R162 residues in each monomer are shown as red spheres. The estimated location of the membrane is shown by two blue lines. (C) Detail of R162 and its interactions. (D) Detail of W162 interface area of the Kir7.1 monomer. Residues predicted to undergo a significant change of conformation because of the R162W mutation are shown in red. (E) Predicted structural changes for residues K164, P163, and R166 due to the R162W mutation. Wild-type and mutant side chains are shown in green and red, respectively. Yellow arrows show the direction of the change.

Figure 2

Electrophysiological Properties of Normal and Mutant Rat Kir7.1 Expressed in CHO Cells The top line show examples of currents measured in Kir7.1 (A) or R162W mutant Kir7.1 (B) transfected CHO cells in response to voltage-clamp steps from the holding potential −50 mV to voltages between −115 mV and 65 mV. The middle-left (2.5 K) and -right (150K) panels show single-experiment examples of I-V relations measured in cells expressing Kir 7.1 (A) and R162W mutant Kir7.1 (B). The mean current measured isochronally between 250 ms and 450 ms of the 500 ms pulse is shown. The net Ba²⁺ inhibited current differs between the normal and mutant Kir7.1 both in the shape of the curve and its reversal potential (the point at which it crosses the abscissa). The bottom-left panels show superimposed traces from the middle panels, and the bottom-right panels show mean data comparing normal ([A], n = 10 cells from three experiments) and R162W mutant ([B], n = 13 cells per three experiments) Kir7.1. In the R162W mutant, the 2.5K current has lost its potassium selectivity (Er = −9 mV), has a less pronounced inwardly rectifying quality, and appears qualitatively similar to the 150K current in both normal and mutant molecules. Transfected cells showing increased leak current (I > 800 pA at 50 mV; n = 1 and n = 136 for cells transfected with Kir 7.1 and R162W mutant, respectively) were omitted from analysis.

Figure 3

Localization of Kir7.1 in the Retina and RPE (A) Control section without anti-Kir7.1 antibody. (B) Retinal section stained with antibody to Kir7.1 showing localization to the internal limiting membrane (ILM), nerve fiber layer (NFL), inner nuclear layer (INL), inner plexiform layer (IPL), and retinal pigment epithelium (RPE). These retinal layers are indicated by the arrows from top to bottom of (B). (C) DAPI-stained retinal section showing the INL and ONL. (D) Autofluorescence showing the RPE. (E) Immunofluorescent staining showing Kir7.1, especially in the ILM and RPE. (F) Merged images of DAPI, autofluorescence, and Kir7.1 images showing colocalization of Kir7.1 within the RPE, INL, and ONL.