Gcap Mutations And Retina Disease

The GCAP1 gene has been linked to autosomal dominant cone or cone-rod dystrophies (adCD, adCORD) in several unrelated families (Figure 7), and the GCAP2 gene was recently linked to autosomal dominant RP in a single small family (Sato et al., 2005). The Y99C mutation is located adjacent to the EF3-hand motif (Payne et al., 1998) and three mutations (I143NT, E155G, L151F) have been described in or adjacent to the EF4-hand motif (Wilkie et al., 2001; Nishiguchi et al., 2004; Sokal et al., 2004; Jiang et al., 2005). A fifth mutation (P50L) is located between

Figure 7. Sequence alignment and positions of mutations associated with disease. Functional EF-hand motifs are shaded. Positions of mutations are indicated by vertical arrows. Conserved residues are printed white on black or gray background. In GCAP1, Y99C is located adjacent to EF3 and I143NT, E155G, L151F are located in EF4. For GCAP2, G157R is located in EF4

Figure 7. Sequence alignment and positions of mutations associated with disease. Functional EF-hand motifs are shaded. Positions of mutations are indicated by vertical arrows. Conserved residues are printed white on black or gray background. In GCAP1, Y99C is located adjacent to EF3 and I143NT, E155G, L151F are located in EF4. For GCAP2, G157R is located in EF4

EF1 and EF2, and does not affect Ca2+ binding (Newbold et al., 2001). A single identified mutation in GCAP2 is located within the EF4-hand motif (Sato et al., 2005) (Figure 7).

6.1. The GCAP1(Y99C) Mutation

Patients with autosomal dominant cone dystrophy have reduced visual acuity and experience a complete loss of color vision. In a four-generation British family (27 members, 7 affected), dominant cone dystrophy was mapped to chromosome 6p21.1, the locus of the GCAP1/GCAP2 gene array (Payne et al., 1998). A missense mutation (A^G) at codon 99 in exon 2 of the gene encoding GCAP1 (GUCA1A) was identified. The mutation replaced Tyr at position 99 with Cys (Y99C), a change that was absent in over 200 unrelated controls. The same mutation was later identified independently in two ancestrally related families (Downes et al., 2001a). The Y99C mutation in GCAP1 has been reported to cause both cone-rod dystrophy and isolated macular dysfunction (Michaelides et al., 2005). The authors suggested that the phenotypic variation indicated an intrafamilial heterogeneity of retinal dysfunction that can be observed in persons harboring the same mutation. The residue Tyr99 immediately precedes the EF3-hand motif, one of three functional EF-hand high affinity Ca2+-binding sites in GCAPs. Residues flanking the hydrophilic Ca2+-binding loop are invariantly hydrophobic (Falke et al., 1994). Substitution by a smaller polar Cys leads to changes in the orientation of the N-terminal a-helix and a decrease in affinity for Ca2+. When Tyr99 was replaced by Trp, a hydrophobic residue, biological activity of mutant GCAP1 was unchanged (Sokal et al., 1999). Analysis of the EF3-hand motif Ca2+ binding kinetics with the Y99W mutant (W3Cys-), exploiting the intrinsic Trp fluorescence of Trp99, showed a significant increase in the Trp fluorescence intensity of W3-GCAP1(w-) in the presence of high Ca2+, reflecting a conformational change (Sokal et al., 1999). The change in fluorescence provided a convenient tool to measure the kinetics of Ca2+ binding using a stopped flow setup. The results showed that GCAP1 rapidly binds Ca2+ (k > 2x108M-1s-1) and rapidly loses its bound Ca2+(k-: = 72s-1 at 37oC) at the EF3-hand motif, thereby displaying thermodynamic and kinetic properties that are compatible with its involvement early in the phototransduction response. Thus, the EF3-hand motif is a key region for conversion of GCAP1 from activator to inhibitor consistent with mutations in this region being causative of cone dystrophy.

6.2. The GCAP1(P50L) Mutation

The GCAP1(P50L) mutation was also suggested to be associated with autosomal cone dystrophy (Downes et al., 2001a; Newbold et al., 2001). Amino acid residue Pro50 is located in a variable region between the EF1-hand and EF2-hand motifs (Figure 7), and not conserved in other GCAPs. We found that biochemical properties of recombinant GCAP1(P50L) were largely identical to WT GCAP1, activating photoreceptor GC at [Ca2+] < 100 nM and inhibiting it at micromolar concentrations (Sokal et al., 2000). We also found a reduced capacity to bind free Ca2+ which can lead to changes in cone photoreceptor Ca2+homeostasis (Sokal et al., 2000). Newbold et al. demonstrated that the P50L mutant was less stable and more susceptible to proteolysis (Newbold et al., 2001). The biochemical mechanism leading to dominant cone dystrophy with this mutation in GCAP1 is unclear.

6.3. The GCAP1(E155G) Mutation

An A464G transition in the GUCA1A gene, not seen in 200 normal controls, was identified in a large dominant cone dystrophy pedigree of 67 individuals, of whom 33 were found to be affected (Wilkie et al., 2001). The age at onset of decreased visual acuity and color vision defects was 8-24 years. This transition changes amino acid Glu155 in the EF4-hand motif to Gly, and is predicted to affect Ca2+ coordination at the EF4-hand motif. Ca2+ binding at the EF4-hand motif does not affect structural changes of GCAP1 to the same extent as it does at the EF3-hand motif, as shown measuring intrinsic fluorescence as a function of Ca2+ using a Trp at position 142 (Sokal et al., 1999). However, it is expected to exert similar dominant effects on GC1 stimulation as does GCAP1(Y99C). The residue Glu155 of GCAP1 is invariant in all GCAPs (Palczewski et al., 2004); an invariant Glu at position 12 of the EF-hand loop, contributing both of its side-chain oxygen atoms to the metal-ion coordination, has been shown to be essential for Ca2+ coordination (Nakayama et al., 1992; Falke et al., 1994).

6.4. The I143NT Mutation

A novel GCAP1 mutation, I143NT (substitution of Ile at codon 143 by Asn and Thr) affecting the EF4-hand Ca2+-binding loop was identified in a heterozygote father and son with autosomal dominant cone degeneration. Both patients had much greater loss of cone function versus rod function; previous histopathologic evaluation of the father's eyes at autopsy (age 75 years) showed no foveal cones and only a few, scattered cones remaining in the peripheral retina. Biochemical analysis showed that the GCAP1-I143NT mutant adopted a conformation susceptible to proteolysis, and the recombinant mutant protein inhibited GC only partially at high Ca2+ concentrations. Properties of the GCAP1-I143NT mutant protein suggest that it is incompletely inactivated by high Ca2+ concentrations as should occur with dark adaptation.

6.5. The GCAP1(L151F) Mutation

GCAP1(L151F) mutations affecting the EF4-hand motif linked to dominant cone dystrophy and cone-rod dystrophy were identified in two unrelated families. In one family (Sokal et al., 2004), affected family members experienced dyschro-matopsia, hemeralopia, and reduced visual acuity by the second to third decade of life. Electrophysiology revealed a non-recordable photopic response with later attenuation of the scotopic response. GCAP1-L151F stimulation of photoreceptor GC was not completely inhibited at high physiological [Ca2+], consistent with a lowered affinity for Ca2+ binding to the EF4-hand motif. These results showed that a conservative L151F mutation in the EF4-hand motif of GCAP1 is associated with adCORD. Although there is a conservative substitution, molecular dynamics suggests a significant change in Ca2+ binding to the EF4-hand and EF2-hand motifs and changes of the shape of L151F-GCAP1.

In a second family (Jiang et al., 2005) of 24 individuals at risk for disease in a five generation family, 11 members were affected. Clinical presentations included photophobia, color vision defects, central acuity loss, and legal blindness with advanced age. The disease phenotype was observed in the second and third decades of life and segregated in an autosomal dominant fashion. An electroretinographic analysis was consistent with cone dystrophy. Mutational analysis and direct sequencing revealed a C451T transition in GUCA1A, corresponding to a L151F mutation in GCAP1.

6.6. The GCAP2(G157R) Mutation

All GCAP1 mutations identified to date and linked to dystrophy are in high-affinity Ca2+-binding sites (exception P50L), affecting association/dissociation of Ca2+. Recently, a G157R mutation in the EF4-hand motif of GCAP2 (Figure 7) was reported to be associated with autosomal dominant RP in 3 families of Japanese origin (Sato et al., 2005). Gly157 is a key conserved residue in the EF4-hand motif, and its replacement by R is expected to influence Ca2+ binding, similar to that observed for the EF3/4-hand motif mutations in GCAP1. The mutation is a strong candidate for causing dominant RP for the following reasons. First, in GCAP2, the EF4-hand motif is more important for Ca2+-dependent stimulation of GC than the EF3-hand motif that contributes to Ca2+ sensitivity of GC only weakly (Olshevskaya et al., 1999a). For example, a GCAP2(Y104C) mutation, corresponding to GCAP1(Y99C) linked to disease, has no effect on Ca2+ sensitivity (Dizhoor et al., 1998). Second, the mutation is predicted to affect severely the affinity of Ca2+ to the EF4-hand motif. Third, GCAP2 is expressed predominantly in rods, thus mutations will affect rods consistent with an RP-like phenotype. Fourth, the G157R mutation segregated with RP in Japanese patients and was not present in over 100 control subjects.

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