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Home , Choroideremia, Macular degeneration, Rab escort protein 1, Retinitis pigmentosa

Exp. Eye Res. (2002) 74, 371±381 doi:10.1006/exer.2001.1126, available online at http://www.idealibrary.com on

MacularPigmentandLuteinSupplementationinChoroideremia

JACQUE L. DUNCANa, TOMAS S. ALEMANa, LEIGH M. GARDNERa,ELAINEDECASTROa, DANIEL A. MARKSa, JESSICA M. EMMONSa,MICHELLEL.BIEBERa, JANETD.STEINBERGa,JEANBENNETTa,EDWINM.STONEb,IANM.MACDONALDc, ARTUR V. CIDECIYANa, MAUREEN G. MAGUIREa AND SAMUEL G. JACOBSONa* aDepartment of , Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, U.S.A., bDepartment of Ophthalmology, University of Iowa Hospitals and Clinics, Iowa City, IA, U.S.A. and cDepartmentofOphthalmology,UniversityofAlberta,Edmonton,Alberta,Canada

(Received St. Louis 14 September 2001 and accepted in revised form 18 October 2001)

Choroideremia is an incurable X-linked retinal degeneration caused by in the encoding escort -1. A group of clinically de®ned and genotyped patients were studied to determine: (1) the degree of rod and cone dysfunction and structural abnormality in the central and the level of macular pigment; and (2) the response of macular pigment and foveal vision to a 6 month trial of supplementation with oral (at 20 mg per day). Rod and cone-mediated function was measured with dark-adapted static perimetry; in vivo retinal structure was determined with optical coherence tomography; and macular pigment optical density was measured with heterochromatic ¯icker photometry. In this cohort of patients (ages 15±65 years), both rod- and cone-mediated central function declined with age as did central retinal thickness. Macular pigment levels did not differ between patients and male control subjects. Supplementation of oral lutein in a subset of patients led to an increase in serum lutein and macular pigment levels; absolute foveal sensitivity did not change. It is concluded that macular pigment density can be augmented by oral intake of lutein in patients with . There was no short-term change in the central vision of the patients on the supplement, but long-term in¯uences of lutein supplementation on disease natural history warrant further study. # 2002 Elsevier Science Ltd. Key words: carotenoids; choroideremia; cone; lutein; macular pigment; perimetry; REP-1; retinal degeneration; rod.

1. Introduction macular pigment (MP) and lutein supplementation in patients with pigmentosa (RP) or Usher Choroideremia (CHM) is an X-linked progressive were performed recently (Aleman et al., retinal degeneration affecting photoreceptors, retinal 2001). Augmenting MP, the yellowish carotenoid pigment epithelial (RPE) cells and (MacDo- complex principally composed of lutein and zeax- nald et al., 1997; Syed et al., 2001). The molecular anthin, may ameliorate central retinal dysfunction/ basis for CHM is in the gene encoding Rab degeneration through a number of proposed actions, escort protein-1 (REP-1) (Cremers et al., 1990; Merry such as ®ltering shorter wavelengths of light and pre- et al., 1992; van Bokhoven et al., 1994). To date, venting photochemical damage, possibly quenching mainly loss of function mutations have been identi®ed singlet oxygen, and stabilizing ( in CHM patients (MacDonald et al., 1998). Facility of et al., 1997; Landrum et al., 1997; Hammond, clinical diagnosis and greater understanding of Wooten and Snodderly, 1998; Russell, 1998; Cho, molecular cause in CHM, however, have not yet led Hung and Seddon, 1999; Schalch, Dayhaw-Barker to the treatment of this serious retinal disease. As in and Barker, 1999; Bernstein et al., 2001; Crabtree many inherited retinal degenerations, CHM patients et al., 2001; Hammond, Wooten and Curran-Celen- lose until only a limited central tano, 2001; Yemelyanov, Katz and Bernstein, 2001). island of function remains; this island eventually is Lower risk for age-related has also lost to the disease (McCulloch and McCulloch, been associated with higher dietary intake and serum 1948; KaÈrnaÈ, 1986). levels of non- A carotenoids (Seddon et al., Prompted by the intriguing report of short-term 1994; Snodderly, 1995; Beatty et al., 1999; Bone et al., improvement in central vision after lutein supple- 2000) and this has led to the speculation of wider mentation in inherited retinal degenerations (Dagne- value to other central photoreceptor-RPE disease lie, Zorge and McDonald, 2000), a series of studies of (Dagnelie et al., 2000). In patients with RP or , as a group, MP was normal; and it could be * Address correspondence to: Samuel G. Jacobson, Scheie Eye Institute, 51 N. 39th Street, Philadelphia, PA 19104, U.S.A. E-mail: augmented in many but not all of the patients by [email protected] supplemental lutein taken orally over a 6 month

0014-4835/02/030371‡11 $35.00/0 # 2002 Elsevier Science Ltd. 372 J. L. DUNCAN ET AL. period. Central vision was unchanged in these patients Macular pigment optical density was measured to short-term lutein supplementation (Aleman et al., with heterochromatic ¯icker photometry (Werner, 2001). Donnelly and Kleigl, 1987; Hammond, Wooten and The present study extends this work to CHM, a Snodderly, 1997; Bone et al., 2000) using an LED- molecularly and clinically more homogeneous retinal based MP densitometer (Wooten et al., 1999; degeneration. First, details of central retinal rod and Hammond and Caruso-Avery, 2000). Details of this cone-mediated function and in vivo retinal structure methodology in patients with retinal degeneration were investigated in a group of genotyped CHM have recently been provided (Aleman et al., 2001). As patients; then was questioned if MP optical density in this earlier study of inherited retinal disease, 58 and serum carotenoids were normal in CHM; lastly, a eccentricity was used as a reference in many patients subset of patients were studied over a 6 month period to minimize secondary effects due to degeneration by while they were supplemented with lutein to deter- limiting testing to the more healthy retina; all patients mine if baseline serum carotenoid, MP and vision were able to perform matches reliably at this location. could be modi®ed. Supplementation with Lutein A subset of seven patients with CHM participated in 2. Materials and Methods a 6 month pilot trial of oral lutein supplementation. Subjects and Mutation Analysis There was no placebo control group and no attempt was made to mask the patient as to the content of the Patients with CHM (n ˆ 13) and subjects with supplement. Dietary information was provided by normal vision (n ˆ 40) participated in the study. All patients at the baseline through the Health Habits and patients were examined and diagnosed clinically. History Questionnaire (HHHQ) developed by the Mutations in the CHM gene were identi®ed with single National Cancer Institute (Block et al., 1986); and strand conformation polymorphism (SSCP) analysis the data were analysed using the HHHQ Diet System and direct sequencing (Nesslinger et al., 1996)as Analysis Software (Block et al., 1994). Following two described previously (MacDonald et al., 1998). baseline visits (separated by no more than 1 month), Informed consent was given by all subjects; institu- subjects supplemented their diet with a commercially tional approval was obtained and the tenets of the available form of lutein at 20 mg per day (TWIN Declaration of Helsinki were followed. Laboratories Inc., NY, U.S.A.). Subjects were instructed to take the lutein supplement with dinner, presuming this meal contained the most and Evaluation of the Macula in CHM: Function, Structure would enhance absorption of the supplement (Kha- and Macular Pigment chick, Beecher and Smith, 1995). A further visit Central rod- and cone-mediated function was occurred 6 months after starting the supplement. measured with two-color (500 and 650 nm stimuli, Baseline and follow-up visits included a clinical 1.78 diameter, 200 msec duration) dark-adapted examination, fasting (overnight) venous blood sample static perimetry. Sensitivity was determined at ®xation for serum carotenoids, and measurements of MP and at 28 intervals along the horizontal meridian density and absolute sensitivity at the fovea with a across the central 208 (Jacobson et al., 1986, 2000). 650 nm target (Aleman et al., 2001). In all but one These pro®les were analysed for photoreceptor-media- patient, both eyes were able to be studied. tion; rod- or cone-mediated sensitivities were averaged across the central 208 and plotted against the age of Statistical Analyses the patient. For the central rod averages, only those pro®les were included in which all detectable sensi- SAS (version 8.00) statistical software was used to tivities using the 500 nm stimulus were rod-mediated perform data analyses. Mean values from the two (n ˆ 9); for central cone averages, only those pro®les baseline visits were used in describing the study were included in which all detectable sensitivities with groups and in calculating the change after supple- the 650 nm stimulus were cone-mediated (n ˆ 13). mentation with lutein. The average of the measure- Macular structure was quanti®ed by the in vivo ments from each eye was also used to establish technique of optical coherence tomography (OCT); the person-speci®c characteristics. t-tests comparing principles of OCT (Huang et al., 1991; Hee et al., means and signi®cance levels for correlation coef®- 1995) and the present techniques (Jacobson et al., cients involving data from two eyes of the same 1998, 2000) have been published. Horizontally and person were performed using a robust variance vertically oriented scans crossing ®xation were estimator to accommodate the correlation between obtained in all subjects. Retinal thickness at ®xation eyes (Zeger and Liang, 1986). Inter-session variability was measured with a computer algorithm (Hee et al., was assessed with signed and absolute differences of 1996) and averaged results from at least three central measurements between the ®rst and second baseline scans were used for the data analyses. visit. Inter-session differences were examined for the CHOROIDEREMIA AND LUTEIN SUPPLEMENTATION 373 correlation between eyes of the same patient; no range of central retinal thicknesses observed pattern of consistent (direct or inverse correlation) or (Fig. 2(A)±(D)). It was ®rst inquired whether, on signi®cant correlation was observed between the eyes. average, the foveal thickness in the CHM patients Therefore, analyses of inter-session variability treated differed from that in a group of normal men. The the data from each eye as independent observations. average thickness in the patients (mean + S.D.+ ˆ Means of inter-session differences and person-speci®c 182.5 + 59.3 mm, n ˆ 11) and normals (mean + variables were compared with independent t-tests S.D. ˆ l66.9 + 14.7 mm, n ˆ 13) were similar; there with adjustment for unequal variance when indi- was no statistically signi®cant difference between the cated. The Wilcoxon rank sum test was used to two groups (t-test; P ˆ 0.41). Inspection of the cross- compare highly skewed distributions. Proportions sectional images in the CHM patients, however, were compared using chi-square tests with the exact suggested greater complexity than revealed by the computation of the P values. averaged data. There was an apparent relationship to age. When OCT thickness in the central fovea was plotted against age of the patient (Fig. 2(E)), there was 3. Results a strong correlation (Spearman correlation coef®cient, Clinical and molecular details of the CHM patients r ˆÀ0.86; P 5 0.0001) indicating a decline with in this study are listed (Table I). The identi®ed the age of the patient. There was no such relationship mutations in the CHM gene would be predicted to in normals (r ˆÀ0.23; P ˆ 0.44). In this sample of lead to a non-functional REP-1. was 20/ CHM patients, there was also greater than normal 30 or better in one or both eyes in all but two patients. retinal thickness in some of the younger individuals; The extent of kinetic visual ®eld measured with the with more advanced disease stage and age, thickness V-4e test target was within normal limits in two of became equal to normal, and ®nally it was reduced three patients between ages 15 and 22 years. In the below normal (Fig. 2(E)). These surprising foveal second through fourth decades of life, most patients thickness data among the patients accounted for the had central and peripheral islands separated by mid- similarity between patients and normals when only peripheral . Later ages showed only a small averaged results were compared. central island of vision. For this kinetic perimetry A measure of central cone function (dark-adapted target, there was increasing loss of visual ®eld extent sensitivity, 650 nm target, at ®xation) was also plotted with patient age (Spearman correlation coef®cient, for the CHM patients (Fig. 2(F)) and a decline with r ˆÀ0.85; P 5 0.0001). age was found (Spearman correlation coef®cient, r ˆÀ0.87; P ˆ 0.0001), suggesting a relationship between the central-most structure and cone-mediated Macular Function and in vivo Structure sensitivity. What are the function and structure of the central retina in patients with CHM? Representative results of Macular Pigment in Patients with Choroideremia two color dark-adapted perimetry across the central 208 of visual ®eld in three CHM patients indicate that In anticipation of the lutein supplementation trial in there can be substantial rod-mediated function near CHM patients, the authors asked a number of questions ®xation although this decreases with increasing about MP in CHM. Does MP level in CHM differ from eccentricity (Fig. 1(A)). The average sensitivity of normal? Representative spatial pro®les of MP optical rod-mediated loci in the central ®eld (excluding the density in two normal male subjects and three CHM value at ®xation) was calculated and plotted against patients (P1, P5, and P8) are shown (Fig. 3). MP age (Fig. 1(B)). Average rod sensitivity declined with peaked within the central 18 and declined with retinal increasing age (Spearman correlation coef®cient, eccentricity in all normal subjects and patients; from r ˆÀ0.78; P ˆ 0.01). Long-/middle-wavelength (L/ the examples shown it is evident that there was a range M) cone function peaked at ®xation and decreased of measurable MP densities. As a group, the mean MP with eccentricity (Fig. 1(C)). Similar to central rod- density (measured with the conventional 18 diameter mediated function, average L/M cone-mediated func- stimulus) was comparable between the CHM patients tion declined with age (Spearman correlation coef®- (mean + S.D. ˆ 0.23 + 0.18, n ˆ 11) and normal cient, r ˆÀ0.94; P 5 0.0001). The slopes of these male subjects (0.27 + 0.08, n ˆ 13), and there were rod- and cone-mediated functions vs age were not no statistically signi®cant differences. Mean MP density signi®cantly different (slope difference estimate, levels in both CHM patients and normal male subjects À0.005; 95 % con®dence interval, À0.02 to 0.01; in the current study were within the normal range of P ˆ 0.52) suggesting a relatively equal reduction of other published data (that included both sexes) using these parameters with increasing age in the central the same instrumentation and target (Wooten et al., ®eld of this group of CHM patients. 1999; Hammond and Caruso-Avery, 2000; Aleman Foveal structure in the CHM patients was assessed et al., 2001). in vivo with OCT (Fig. 2). Cross-sectional retinal Are MP measurements made in CHM patients images in four representative patients illustrate the more variable on successive sessions than those 374

TABLE 1 Clinical and molecular characteristics of the patients

Age at Kinetic visual Lutein trial Patient ®rst visit Visual ®eld Extent participant number (years) Position Mutation Predicted effect Eye acuity* (V-4e){ (age at baseline)

1 15 Intron 9 1274 ‡ 1 G to A anomalous splicing and RE 20/20 À0.75 96.6N truncated gene product LE 20/20 À0.25 2{ 18 Exon 6 829 C to T R267X RE 20/20 À2.50 À 0.25  045 74.5 Y (30) LE 20/20 À3.00 À 1.00  145 3 22 Exon 6 829 C to T R267X RE 20/20 ‡0.75 À 0.50  090 89.6 Y (22) LE 20/20 ‡0.50 À 0.75 Â090 4 23 Exon 5 579_587 del 9 ins 8 E183fsX196 RE 20/20 À0.50 À 0.75  095 28.9 Y (29) LE 20/20 À0.50 À 0.25  085 5{ 23 Exon 6 829 C to T R267X RE 20/20 À2.25 À 1.00  068 69.0 Y (36) LE 20/20 À2.75 À 0.50  102 631 } RE 20/20 À5.00 À 1.00  090 63.4 Y (36) LE 20/20 À4.75 À 0.75  085 7 33 Exon 6 838 C to T R270X RE 20/30 À9.00 À 1.25  180 5.0 Y (45) LE 20/25 À9.25 À 1.25  045 8 34 Intron 2 146 ‡ 1 G to A anomalous splicing and RE 20/20 À1.75À 0.50  100 59.4 Y (34) truncated gene product LE 20/50 À2.25 9 34 Exon 14 1693 A to T R555X RE 20/400 À2.00 À 1.00  060 27.5N LE 20/30 ‡0.50 À 1.25  135 10{ 46 Exon 13 1614_1618 del TGTT H529fsX535 RE 20/50 À7.50 0.2N LE 20/50 À7.50 11 48 Exon 6 787 C to T R253X RE 20/40 À6.50 À 0.50  090 17.6N LE 20/25 À6.75 À 0.50  090 12 58 Exon 6 838 C to T R270X RE 20/25 ‡0.50 À 2.00  090 0.3N LE 20/20 ‡0.25 À 1.00 Â090 13{ 65 Exon 13 1614_1618 del TGTT H529fsX535 RE 20/40 ‡4.25 À 0.50  110 5.2N LE 1/200 ‡4.25 À 0.50  035 .L UCNE AL. ET DUNCAN L. J.

* Best corrected visual acuity. { Patients 2 and 5 are siblings; Patients 13 and 10 are uncle and nephew, respectively. { Average of both eyes, expressed as a percentage of normal mean of V-4e target; 2 S.D. below normal equals 90 %. } No mutation detected to date. RE, right eye; LE, left eye; bp, ; del, deletion; ins, insertion. CHOROIDEREMIA AND LUTEIN SUPPLEMENTATION 375

FIG. 1. Central rod- and cone-mediated visual function in choroideremia as a function of age. (A,C) Horizontal pro®les in the dark-adapted state (500 nm stimulus, A; 650 nm stimulus, C) from three representative patients at different disease stages. Connected symbols are patient data. Broken lines de®ne + 2S.D. from the mean normal for each test condition (Jacobson et al., 1991). (B, D) Average sensitivity derived from the pro®les exempli®ed in (A) and (C) plotted against age of the subject at the time of testing. Solid lines are the linear regression ®t to the patient data and dashed lines de®ne the 95 % prediction interval of a group of normals (of both sexes) for each test condition (B, n ˆ 21, age range, 14±42 years; D, n ˆ 5, age range, 25±51 years). Normal data in (C) and (D) were obtained in the dark during the cone plateau phase following a bright adapting exposure (Jacobson et al., 1991). made in normals? Inter-session variation in MP, as normals; a test for difference between patient and illustrated by the pro®les in normal subjects and normal results at all eccentricities was not signi®- patients of Fig. 3, was quanti®ed by determining cant. To examine the potential issue of systematic mean absolute differences between MP densities learning effects, signed differences between the visits measured on two visits separated by less than 1 were calculated; there was no substantial increase month. At the four retinal eccentricities tested, the between the visits for the four stimuli in patients or mean differences (S.D.) for each stimulus in CHM normals. Mean differences were: 0.00 and 0.00 at patients were as follows: 0.178:0.04 (0.05); 0.58: 0.178;0.00 and À0.01 at 0.58;0.02 and 0.01 at 0.05 (0.06); 1.08:0.05 (0.05); and 2.08:0.05 1.08; and 0.02 and À0.01 at 2.08, for CHM patient (0.05). For the normal male subjects, the differences and normal eyes, respectively. were: 0.178:0.03 (0.01); 0.58:0.02 (0.01); 1.08: Is there a relationship between MP density (at 0.58 0.03 (0.02); and 2.08:0.04 (0.01). The absolute eccentricity) and serum level or dietary intake of value of the differences in CHM patients between lutein in CHM patients? Mean serum lutein and baseline sessions was within the expected range of dietary lutein were not signi®cantly different in the 376 J. L. DUNCAN ET AL.

FIG. 2. Central retinal structure and foveal function in choroideremia. (A±D) Vertical OCT scans split at the fovea to permit comparison of central retinal thickness in different patients. OCT images are displayed with the logarithm of re¯ectivity mapped to a gray scale (lower left). Numbers on the gray scale allow comparison with images using pseudocolor displays (1, white; 2, red; 3, yellow; 4, green; 5, blue; and 6, black; Jacobson et al., 2000; Aleman et al., 2001). (E) OCT thickness as a function of age of the choroideremia patients. (F) Foveal sensitivity (650 nm, 1.78, dark-adapted) as a function of age in choroideremia. Dashed lines de®ne the 95 % prediction interval of a group of normal male subjects for each test condition (E, n ˆ 13, age range, 21±57 years; F, n ˆ 8, age range, 26±47 years). patients compared with normal subjects (Wilcoxon retinal function, correlation was found with MP rank test, P ˆ 0.41 for serum lutein, P ˆ 0.18 for density in CHM patients (Spearman correlation coef®- dietary lutein). Neither serum nor dietary lutein cient, r ˆ 0.73, P ˆ 0.0006). Using the OCT measure- correlated with MP density in CHM patients (Spear- ment of central retinal thickness as the structural man correlation coef®cient, r ˆÀ0.12, P ˆ 0.64; parameter, there was also a strong relationship with and r ˆ 0.09, P ˆ 0.72); in normal subjects there MP density (r ˆ 0.66, P ˆ 0.003). This suggests that was a signi®cant correlation of MP density level with the MP level may be related to the stage of disease in serum lutein (r ˆ 0.52, P ˆ 0.007) but MP was not the CHM patients. correlated with dietary lutein (r ˆ 0.13, P ˆ 0.53). Does macular disease severity, estimated by func- Effects of Lutein Supplementation tional and structural measures, relate to MP density in CHM patients (n ˆ 7) between the ages of 22 and CHM patients? Using the absolute threshold to a 45 years participated in a pilot trial of the effects of 650 nm target at ®xation as the measure of central oral lutein supplementation on MP and central vision. CHOROIDEREMIA AND LUTEIN SUPPLEMENTATION 377

FIG. 3. Spatial macular pigment pro®les and their variation in normal subjects and choroideremia patients. (A) Two normal subjects, open symbols, and (B) three patients (P1, P5, and P8), ®lled symbols, tested on two different visits (symbols connected with dashed lines denote the second visit). Insets are the frequency distributions of macular pigment density for the 0.58 eccentricity target in each group.

Mean MP densities at four eccentricities at baseline attempt try to de®ne further the response to supple- and after 6 months of lutein supplementation in four mentation in normal subjects vs patients. We plotted normal male subjects (Fig. 4(A)) and the CHM the difference in MP density in each eye between patients (Fig. 4(B)) are shown. Mean MP at 0.178 average baseline and after supplementation at 0.178 retinal eccentricity increased between baseline and vs 0.58 eccentricities (Fig. 4(F)). post-supplementation by 0.09 in normal subjects Vertical and horizontal lines in Fig. 4(F) de®ne the (P ˆ 0.0026) and by 0.08 in the patients 95th percentile for differences within patients (P 5 0.0001). Patients also showed signi®cant between the two baseline values. For the normal increases in MP of 0.07 at 0.58 (P ˆ 0.0007); 0.06 subjects, 3/8 (37.5 %) eyes showed no change in MP at 1.08 (P ˆ 0.005); and 0.04 at 2.08 (P ˆ 0.0009). with supplementation at either central target Normal subjects showed no statistically signi®cant (Fig. 4(F), lower left quadrant). When there was a differences at 0.58,1.08 and 2.08 eccentricities response, it was an increase in MP density only at (P ˆ 0.56, 0.35 and 0.66, respectively). 0.178 (5/8 or 62.5%; Fig. 4(F), upper left quadrant). The distribution of change between the two baseline For the patients, 6/13 (46.1 %) eyes showed no MP values for the most central targets (0.178 and 0.58 increase in MP with either target. When there was a eccentricities) for each patient eye was then compared response (7/13 or 53.9 %), it could be an increase in with the distribution of change between baseline and 6 MP with the 0.178 (4/13), the 0.58 (2/13) target or months post-supplementation (Fig. 4(C) and (D)). both (1/13). If response to lutein supplementation is Overlap was present between the distributions of MP de®ned by this statistical criterion using the two differences at baseline versus post-supplementation, central or peak MP measures, then both normal and but there was a signi®cant shift towards higher MP patient groups had non-responders and responders. density for each target (P 5 0.0001 for 0.l78 target, Were there any predictive factors that would help and P ˆ 0.017 for 0.58 target). Comparison of serum determine which patients would respond to lutein lutein levels at baseline versus post-supplementation supplementation with increased MP and which would showed a shift in the distribution towards higher levels not? If a retinal responder was de®ned as an (P ˆ 0.03). individual with at least one eye showing a statistically The observation from the spatial pro®les (Fig. 4(A) signi®cant increment in MP level (for one or both and (B)) that normal subjects tended to change only at central targets) post-supplementation, then ®ve of the the 0.178 eccentricity while CHM patients showed a seven patients were responders. Accepting that the response at this and other eccentricities led to the numbers of patients are small, it was asked whether 378 J. L. DUNCAN ET AL.

FIG. 4. Effect of lutein supplementation on macular pigment, serum lutein and foveal sensitivity (A,B) Mean macular pigment spatial pro®les for normal subjects and patients at baseline (circles) and after 6 months of lutein supplementation (triangles). Error bars, S.E.M. (C±E) Distribution of differences in macular pigment (0.178and 0.58 eccentricities) and serum lutein (mgmlÀ1) between baseline visits (black bars) and between mean baseline and 6 month post-supplementation values (hatched). (F) Change in macular pigment at 0.178 vs 0.58 eccentricities after supplementation. Baseline inter-session variability (95 % con®dence limits) is de®ned by the horizontal and vertical lines. Normal subjects, open circles; CHM patients, ®lled circles. (G) Foveal sensitivity (650 nm, 1.78, dark-adapted) in patients at baseline and after 6 months of lutein supplementation. The diagonal line represents no change. Lines connecting symbols are from the two eyes of the same patient, when performed. the two groups differed in any obvious way? Respon- ing eyes showed no major difference in average foveal der vs non-responder groups did not differ in average sensitivity (29.1vs29.2 dB) or average retinal age (32 vs 35 years); mean baseline serum lutein thickness (223 vs 198 mm). values were similar (0.15 vs 0.21 mgmlÀ1); and An important question is whether there was any mean serum lutein increases were not dramatically difference in absolute threshold (650 nm stimulus) at different (200 % vs 175 %, respectively). Considering ®xation between baseline and post-supplementation. central disease severity, responding and non-respond- Foveal thresholds were little changed between visits at CHOROIDEREMIA AND LUTEIN SUPPLEMENTATION 379 baseline and after 6 months of lutein supplementation patients. There are histopathological reports in two (Fig. 4(G)). Pre- and post-supplementation results patients with late stages of CHM of extensive inner were highly correlated (r ˆ 0.96, P 5 0.0001); a retinal gliosis and a preretinal membrane (Ghosh and test for difference was not signi®cant (P ˆ 0.50). McCulloch, 1980). A thin preretinal membrane could in theory alter foveal architecture leading to the OCT observations in the younger CHM patients, but this 4. Discussion was not evident. It is interesting to note that there is CHM has been recognized as clinically distinct from also a lack of normal foveal architecture in forms of other retinal degenerations for more than half a albinism, which share with CHM pigmenta- century (reviewed in McCulloch and McCulloch, tion disturbances in RPE and choroid (Fulton, Albert 1948; KaÈrnaÈi, 1986); and, for the past decade, the and Craft, 1978). Whether there is a developmental molecular cause has been known to be mutations in component to the OCT observations of the central the gene encoding REP-1, part of the complex retina of younger CHM patients is an intriguing but membrane traf®cking pathways of cells (reviewed in speculative possibility. Alory and Balch, 2001). As the molecular pathology Macular pigment levels have not been reported of CHM is better understood, longstanding questions previously in CHM patients. In the cohort of CHM about disease expression may be answered. For patients, if analysed as a group, MP was found to be example, the early funduscopic feature in CHM of within normal limits. It is important to note that the abnormal melanin pigmentation in retinal pigmented validity of the assumptions implicit in the use of the epithelium (RPE) and choroid in hemizygotes and current heterochromatic ¯icker photometry technique heterozygotes (McCulloch and McCulloch, 1948) to estimate MP was not explicitly proven in the could result from defective transport of patients. Speci®cally, it was assumed that the differ- in RPE and choroidal melanocytes secondary to ence in sensitivity to the blue and green stimuli at the Rab27a hypoprenylation (Pereira-Leal, Hume and level of L/M-cone outer segments was invariant across Seabra, 2001). The disease has been suggested to the central retinal locations. Theoretically, outer lead from de®cient transport, to light retinal degeneration could affect the relative abun- damage at the level of the RPE, and then secondary dance and photopigment density of L and M cones photoreceptor and choroidal disease (Stenmark and differentially across the regions tested, although there Olkkonen, 2001). REP-1, however, has been localized has been no such evidence to date. Generation of in photoreceptors (van den Hurk et al., 1997; psychophysical MP absorption spectra in CHM Bernstein and Wong, 1998; Syed et al., 2001), so patients remains to be performed in future studies. albinotic-appearing RPE and choroid may be only part Were there measurable effects of oral supplement- of the expression and CHM could also have a primary ation of lutein on serum carotenoids and MP in CHM? effect on photoreceptors. As in many retinal degener- Supplementation increased the serum levels of lutein ations, the CHM disease sequence is likely to be above baseline in all CHM patients and this is consistent complex and there may be a cascade of Rab-associated with the earlier observations in patients with other defects due to relative loss of REP-1 vs REP-2 activity inherited retinal degenerations and studies in normal (Cremers et al., 1994; Alory and Balch, 2000, 2001; subjects (Hammond et al., 1997; Landrum et al., 1997; Detter et al., 2000; Pereira-Leal et al., 2001). Aleman et al., 2001; Hininger et al., 2001). Macular As a prelude to a pilot intervention with oral lutein pigment levels increased from baseline in the CHM in CHM patients, details of macular function and patients as a group. If changes in MP greater than the structure and macular pigment in CHM were sought. 95th percentile of changes between baseline sessions How do the results compare with those in the are considered to be evidence of an effect of supple- literature? There are considerable published data on mentation, then about 54 % of the eyes would be central changes and visual acuity in CHM (for considered as responding whereas 46 % were not example, McCulloch, 1969; KaÈrnaÈ, 1986; Sieving, responding. Oral lutein intake over 6 months thus did Nieffennegger and Berson, 1986; Grover et al., 1998; not assure a measurable increase in MP. This was also Roberts et al., 2001). The visual acuity results in the the experience in RP and Usher syndrome (Aleman cohort of CHM patients generally concur with those in et al., 2001) and re¯ects observations in some groups the literature. Rod- and cone-mediated function across of normal subjects (Hammond et al., 1997; Landrum the central retina in CHM has not been speci®cally et al., 1997). The factors that were examined were not explored; a decades-long decline was found in both predictive of whether or not there would be a response. photoreceptor systems. Foveal retinal thickness by Whether other techniques of measuring MP would OCT became reduced with age and this paralleled a detect higher percentages of responders to supplement- loss of foveal cone sensitivity. An unexpected obser- ation awaits further study (Bernstein et al., 1998; vation was a greater than normal foveal thickness in Berendschot et al., 2000; Delori et al., 2001; Ermakov, some of the younger CHM patients. These patients all McClane and Gellerman, 2001). had excellent visual acuity and were not ®xating No short-term bene®t on foveal dark-adapted eccentrically. There was no in the function was found in this pilot study. This mirrors 380 J. L. DUNCAN ET AL. the observations that were made in RP and Usher Cho, E., Hung, S. and Seddon, J. M. (1999). Nutrition syndrome (Aleman et al., 2001). Considering the (Berger, J. W., Fine, S. L. and Maguire, M. G., Eds.) In Age-Related Macular Degenerationpp 57±67. Mosby Year progressive loss of macular structure and function Book, Inc: St. Louis, MO, U.S.A. and macular pigment in CHM, it would seem Crabtree, D. V., Ojima, I., Geng, X. and Adler, A. (2001). worthwhile to determine if there is long-term bene®t Tubulins in the primate retina: evidence that xantho- to central vision of this supplement. phylls may be endogenous ligands for the paclitaxel- binding site. Bioorg. Med. Chem. 9, 1967±76. Cremers, F. P. M., Armstrong, S. A., Seabra, M. C., Brown, Acknowledgements M. S. and Goldstein, J. L. (1994). REP-2, a Rab escort protein encoded by the choroideremia-like gene. J. Biol. This work was supported by The Chatlos Foundation, Inc., Chem. 269, 2111±7. National Institutes of Health (EY-05627; EY-13203); Cremers, F. P. M., van de Pol, D. J. R., van Kerkhoff, L. P. M., Foundation Fighting Blindness, Inc.; Macula Vision Wieringa, B. and Ropers, H.-H. (1990). Cloning of a Research Foundation; F. M. Kirby Foundation; The Macular gene that is rearranged in patients with choroideremia. Disease Foundation; and the Mackall Trust. S. G. Jacobson is Nature 347, 674±6. an RPB Senior Scienti®c Investigator; J.B. and A.V.C. Dagnelie, G., Zorge, I. S. and McDonald, T. M. (2000). Cideciyan are RPB Special Scholars. The authors thank Mr Lutein improves visual function in some patients with D. Hanna, Dr J. Huang, Ms K. Mejia, Ms E. Dale, Ms R-J. retinal degeneration: a pilot study via the internet. Chen and Ms K. McTaggart for help with data collection and Optometry 71, 147±64. analyses. Mr G-S. Ying provided invaluable assistance with Delori, F. C., Goger, D. G., Hammond, B. R., Snodderly, D. M. statistical analyses. and Burns, S. A. (2001). 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