Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

(Invitrogen) and the plasmids from positive clones were sequenced to ensure the correct mutation was introduced and that there were no PCR errors.

2.2. Expression and Purification of the Recombinant gC1q Domain

Escherichia coli LMG194 cells harbouring the above recombinant plasmids were grown in LB medium containing 100 mg of ampicillin at 37°C with agitation at 220 rpm. The expression of the recombinant gC1q domain (wildtype and mutant) were induced at 18°C overnight by addition of 0.02% arabinose when the absorbance at 600 nm (A600) reached 0.4-0.6. Cells were harvested by centrifugation and resuspended in 50 mM Tris-HCl buffer, pH8.0, containing 150 .mM NaCl and 10% glycerol with proteinase inhibitors (Roche). The cells were disrupted by a French press and centrifuged at 13000 rpm for 30 min to collect the supernatant. The fusion proteins were affinity purified with Ni-NTA superflow (QIAGEN) according to the manufacturer’s instructions.

2.3. Sucrose Gradient Sedimentation

150 ml of purified gC1q domain protein was applied to 4.2 ml 5-20% sucrose gradient in 50 mM Tris-HCl buffer (pH8.0) containing 150 mM NaCl. Protein standards were applied to parallel gradients: chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), catalase (250 kDa) and urease (272 kDa). The gradients were spun for 22 hr at 4°C in a Sorvall TH-641 rotor at 50,000 rpm and 23 fractions were sequentially collected from the bottom of the gradient and analysed by their UV absorbance at 280 nm and western blotting.

2.4. Determination of the Native Molecular Size of gC1q Domain

Purified gC1q domain and non-denatured protein markers of known molecular size (Sigma) were separated in 6, 8, 10 and 12% polyacrylamide (PAGE) gels under nondenaturing conditions. Gels were stained with Coomassie Brilliant Blue R250. The Relative Mobility (Rf) of each protein compared to the tracking dye was determined on a set of gels with various polyacrylamide concentrations; 100[Log (Rf ¥ 100)] values were plotted against the gel concentration as percentages and the negative slopes of the plots were plotted against the known molecular weights of the standards. This produced a linear plot and the molecular size of the unknown protein was determined by its negative slope.

2.5. SDS-polyacrylamide Gel Electrophoresis (PAGE) and Blotting

The samples were loaded on a 12% SDS-PAGE gel for electrophoresis. Samples were boiled for 5 min before loading as required. Gels were stained with Coomassie Brilliant Blue R250 or transferred to nitrocellulose membranes. For the blotting, membranes were blocked in 5% non-fat dried milk in PBS and primary antibody (anti-His tag, Invitrogen) was used at 1 : 300 dilution. Anti-mouse HRP-conjugated secondary antibody (Amersham) was used at 1 : 5000 dilution. Bound antibody was visualised using ECL kit (Amersham).

In a thermostability analysis of C1QTNF5 gC1q domain, the wildtype (Ser163) gC1q domain was incubated at different temperatures (60°C-84°C) in sample buffer for 10 min and run on a 12% non-denaturing polyacrylamide gel electrophoresis (PAGE) gel. The wildtype gC1q domain is stable at temperatures up to 70°C, but starts to degrade or aggregate at higher temperatures, so that most has degraded at 80°C (Fig. 7.4c).

4. DISCUSSION

The short-chain collagen C1QTNF5, like other members of this protein family, such as collagen VIII and X, includes both a short-chain collagen and a gC1q domain, which is thought to be necessary for trimerisation and “zippering” of the collagen trimer. The gC1q domain contains a group of highly conserved aromatic amino acids which are proposed to be involved in this trimerisation step (Brass et al., 1992). In the related protein collagen X, this aromatic motif is critical for the interaction of the gC1q domains (Chan et al., 1999). Deletion of the same aromatic motif in C1QTNF5 also shows loss of trimerisation (unpublished data). Collagen X exists as a major trimer and as a high molecular weight multimer (Frischholz et al., 1998), while EMILIN initially assembles into trimers but also forms large multimers (Mongiat et al., 2000). The results presented here suggest that the native form of C1QTNF5 gC1q domain also exists as a trimer but a hexameric form is also detectable so that the possibility of higher order multimers or a hexagonal lattice, as found in some other members of this protein family, is not excluded. C1QTNF5 is predicted to be secreted by retinal pigment epithelial (RPE) cells so that one possible role in late-onset retinal degeneration is in cell adhesion to the underlying Bruch’s membrane. Further work is required to establish whether or not the 163Arg mutation compromises such cell adhesion by assay in transfected RPE cells.

Why is there a close resemblance between L-ORD and age-related macular degeneration? The most likely explanation is the common occurrence of a thick sub-RPE deposit in both conditions, which impairs transport of nutrients between choroid and RPE and/or adhesion between RPE and its basement membrane. The molecular basis of C1QTNF5 Ser163Arg pathogenicity could firstly be due to lack of wildtype protein (haploinsufficiency), since the mutant protein is unstable and readily aggregates in vitro (Hayward et al., 2003), so that a 50% reduction in functional C1QTNF5 is likely. Alternatively, this study shows that multimerisation is apparently normal in the 163Arg mutant, so that formation of heteromultimers of wildtype and mutant monomers, which are found in vitro (data not shown) could substantially reduce the amount of normal multimer. For example, assuming equal expression and random oligomerisation of subunits, the amount of functional trimer could be as little as 12.5% of normal, if the mutant monomer de-stabilises or disrupts the function of all heterotrimers. The mutation could therefore exert a dominant-negative effect on C1QTNF5 function. Further work is required to distinguish between these alternative disease models.

5. ACKNOWLEDGEMENTS

We gratefully acknowledge an RD2004 Young Investigator Travel Award from National Eye Institute, NIH to Dr. Xinhua Shu, and the financial support of the Foundation Fighting

Blindness, Macula Vision Research Foundation, Macular Disease Foundation, NIH/NEI, and Medical Research Council.

6. REFERENCES

Brass, A., Kadler, K.E., Thomas, J.T., Grant, M.E., Boot-Handford, R.P., 1992, The fibrillar collagens, collagen VIII, collagen X and the C1q complement proteins share a similar domain in their C-terminal noncollagenous regions, FEBS 303:126-128.

Chan, D., Freddi, S., Weng, Y.M., Bateman, J.F., 1999, Interaction of collagen a1 (X) containing engineered NC1 mutations with normal a1 (X) in vitro, J. Biol. Chem. 274:13091-13097.

Crabb, J.W., Miyagi, M., Gu, X., Shadrach, K., West, K.A., Sakaguchi, H., Kamei, M., Hasan, A., Yan, L., Rayborn, M.E., Salomon, R.G., Hollyfield, J.G., 2002, Drusen proteome analysis: an approach to the etiology of age-related macular degeneration, Proc Natl Acad Sci U S A. 99:14682-14687.

Ferris, F.L., Fine, S.L., Hyman, L., 1984, Age related macular degeneration and blindness due to neovascular maculopathy, Arch. Ophthalmol. 102:1640-1642.

Green, W.R., Enger, C., 1993, Age-related macular degeneration histopathologic studies: the 1992 Lorenz E. Zimmerman Lecture, Ophthalmology 100:1519-1535.

Frischholz, S., Beier, F., Girkontaite, I., Wagner, K., Poschl, E., Turnay, J., Mayer, U., von der Mark, K., 1998, Characterization of human type X procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells, J Biol. Chem. 273:4547-4555.

Hammond, C.J., Webster, A.R., Snieder, H., Bird, A.C., Gilbert, C.E., Spector, T.D., 2002, Genetic influence on early age-related maculopathy: a twin study, Ophthalmology. 109:730-736.

Hayward, C., Shu, X., Cideciyan, A.V., Lennon, A., Barran, P., Zareparsi, S., Sawyer, L., Hendry, G., Dhillon, B., Milam, A.H., Luthert, P.J., Swaroop, A., Hastie, N.D., Jacobson, S.G., Wright, A.F., 2003, Mutation in a shortchain collagen gene, C1QTNF5, results in extracellular deposit formation in late-onset retinal degeneration: a genetic model for age-related macular degeneration, Hum Mol Genet. 12:2657-2667.

Jacobson, S.G., Cideciyan, A.V., Wright, E., Wright, A.F., 2001, Phenotypic marker for early disease detection in dominant late-onset retinal degeneration, Invest Ophthalmol Vis Sci. 42:1882-1890.

Kuntz, C.A., Jacobson, S.G., Cideciyan, A.V., Li, Z.Y., Stone, E.M., Possin, D., Milam, A.H., 1996, Sub-retinal pigment epithelial deposits in a dominant late-onset retinal degeneration, Invest Ophthalmol Vis Sci. 37:17721782.

Malek, G., Li, C.M., Guidry, C., Medeiros, N.E., Curcio, C.A., 2003, Apolipoprotein B in cholesterol-containing drusen and basal deposits of human eyes with age-related maculopathy. Am J Pathol. 162:413-425.

Milam, A.H., Curcio, C.A., Cideciyan, A.V., Saxena, S., John, S.K., Kruth, H.S., Malek, G., Heckenlively, J.R., Weleber, R.G., Jacobson, S.G., 2000, Dominant late-onset retinal degeneration with regional variation of subretinal pigment epithelium deposits, retinal function, and photoreceptor degeneration, Ophthalmology. 107:2256-2266.

Mongiat, M., Mungiguerra, G., Bot, S., Mucignat, M.T., Giacomello, E.G., Doliana, R., Colombatti, A., 2000, Self-assembly and supramolecular organization of EMILIN, J Biol Chem. 275:25471-25480.

Seddon, J.M., 2001, Age related macular degeneration. In Ryan, S.J. (ed), Retina (3rd edn), Mosb, St Louis, MO, pp. 1039-1050.

A recent study has revealed that mutations of the CYP4V5 gene, which encodes a member of the cytochrome p450 (CYP450) family proteins and is expressed abundantly in the retina, are the cause of this disease.11 The protein encoded by CYP4V5 is suggested to play a role in the lipid processing pathways,11 because some other members of the CYP450 proteins are implicated in lipid metabolism, and because cultured lymphocytes from patients with BCD showed abnormally high levels of triglyceride and cholesterol and absence of two fatty acid-binding proteins.12

We have examined the CYP4V5 gene in 8 separate Japanese patients with BCD and have identified a mutation in this gene in all. This would indicate that defects in this gene are the major cause of BCD.13 The patients had characteristic clinical features of BCD.13

3. PATIENTS AND METHODS

The procedures used in this study conformed to the tenets of the Declaration of Helsinki, and informed consent was obtained from each patient after an explanation of this study. Eight Japanese patients with BCD were examined. None of the other family members was affected, and to the best of our knowledge, the families were not related. Three unrelated patients were the offsprings of three consanguineous marriages. All individuals examined have been followed in the Departments of Ophthalmology, Nagoya University. Each patient received a complete ophthalmologic examination including best-corrected visual acuity, slit-lamp and fundus examination, fundus photography, Goldmann kinetic perimetry, fluorescein angiography, and electroretinography.

Genomic DNA was extracted from peripheral leukocytes. Exons 1 through 11 with flanking intron splice sites of the CYP4V5 gene were individually amplified by polymerase chain reaction (PCR), and the PCR products were purified and directly sequenced.

Standardized electroretinograms (ERGs) were elicited by ganzfeld stimuli after 30 minutes of dark-adaptation. The rod (scotopic) ERGs were elicited by a blue light at an intensity of 5.2 ¥ 10-3 cd/m2/sec. The mixed rod:cone single flash ERGs were elicited by a white stimulus at an intensity of 44.2 cd/m2/sec. The cone ERGs and the 30 Hz flicker ERGs were elicited by a white stimulus at an intensity of 4 cd/m2/sec and 0.9 cd/m2/sec, respectively, on a white background of 68 cd/m2.

4. RESULTS

A homozygous mutation in the CYP4V5 gene was found in the 8 patients with BCD.13 Seven patients had an IVS6-8_c.810del/insGC mutation with 17-bp deletion and 2-bp insertion that affected the IVS6 splice acceptor site probably resulting in an in-frame skipping of exon 7.11,13 The remaining patient had an L173W mutation in the gene.13

The clinical characteristics of the BCD patients are summarized in Table 8.1. The ages of the subjective onset of symptoms were between 35and 54-years-old. Four patients complained of night blindness. The first symptoms were nyctalopia in 3 patients, a reduction of central vision in 2, a disturbance of peripheral visual fields in 2, and 1 patient had no symptoms and his disease was detected during a health examination. The visual acuities of the patients ranged between 0.2 and 1.5 (Table 8.1).

* B-wave amplitudes from single white flash ERGs extracting mixed rod and cone responses. (normal range ≥314 mV).

ND, not determined.

A B

Figure 8.1. Fundus photographs and a fluorescein angiograms of patients with Bietti crystalline corneoretinal dystrophy associated with a mutation of the CYP4V5 gene (IVS6-8_c.810del/insGC). (A) right eye, case 3: numerous crystalline deposits scattered throughout the fundus and diffuse atrophy of the retinal pigment epithelium are seen. (B) right eye, case 5: atrophy of the retinal pigment epithelium and choriocapillaris loss at the posterior. The case number and the age (years) are indicated in each photograph.

Slit-lamp examination revealed crystalline deposits in the peripheral cornea in 5 patients. Each patient had a central, a paracentral, or a ring scotoma that was detected by Goldmann kinetic visual perimetry. The scotomas enlarged with age.

All patients characteristically had numerous, small retinal crystalline deposits concentrated in the posterior pole (Figure 8.1A). Fluorescein angiography showed varying degrees of RPE atrophy and choriocapillaris loss at the posterior pole and sometimes extending to the midperiphery (Figure 8.1B). The area of choriocapillaris loss enlarged with age.

Full-field ERGs showed different degrees of rod and cone dysfunction ranging from normal to severe reduction. The large variability in the amplitudes was noted even among patients carrying the same mutation and at a similar age (Figure 8.2). In patients with reduced ERG responses, both rod and cone ERG responses were reduced, and neither was

Figure 8.2. Full-field electroretinograms recorded from a normal subject and a patient with Bietti crystalline corneoretinal dystrophy associated with a mutation of the CYP4V5 gene. The arrows indicate the stimulus onset. The case number and the age (years) of each patient is noted under the case number in the left column.

predominant. There seemed to be no direct correlation between the degrees of reduction of the visual acuity and the degrees of reductions of the full-field ERG amplitudes. In a patient (case 2), the ERG responses recorded after a 17-year follow-up had decreased significantly (Figure 8.2).

5. DISCUSSION

We analyzed the CYP4V5 gene in 8 unrelated Japanese patients with BCD and identified mutations in the gene in all. Combining these findings with previous results that mutations in the CYP4V5 gene were found in 23 of 25 unrelated patients with BCD,11 we conclude that mutations in the CYP4V5 gene are the major cause of BCD.13

Seven of our 8 patients were found to have the IVS6-8_c.810del/insGC mutation in a homozygous state.13 In the previous study the same mutation was found in 7 of 8 unrelated Japanese BCD families as well as in 7 of 10 unrelated Chinese families.11 Thus, this mutation is considered to have a high incidence in the Japanese and Chinese populations. A founder effect rather than a mutational hot spot was considered for this mutation, because the mutation has never been identified in other populations including Caucasian.11,13 The founder of the mutation was likely to be a very old ancestor, because the region of the conserved linked markers extended less than 17.1 kb.13

All patients with the CYP4V5 gene mutations shared characteristic clinical features of BCD such as retinal crystalline deposits, RPE atrophy, and choriocapillaris loss. However, full-field ERGs showed remarkable variability in amplitudes even among patients with the same mutations and at a similar age. These observations would indicate the possibility that other genetic or environmental factors influenced the course of the disease.13