Rod Photoreceptor Responses in Children With Smith-Lemli-Opitz Syndrome FREE

PATIENTS WITH Smith-Lemli-Opitz syndrome (SLOS) (Online Mendelian Inheritance in Man [OMIM] 270400) have low serum cholesterol and detectable 7-dehydrocholesterol and 8-dehydrocholesterol concentrations due to a deficiency of the enzyme Δ7-dehydrocholesterol reductase.^{1 - 6} The mechanisms by which the biochemical abnormalities cause the SLOS phenotype of microcephaly, mental retardation, facial dysmorphia, genital and limb abnormalities, poor growth, and failure to thrive remain unknown. Some SLOS-affected individuals have ptosis and cataracts.^{7 - 8}

Cholesterol, an important component of the lipid bilayer of cell membranes, affects the fluidity of membranes and the movement of proteins within the membranes.^{9 - 12} Rhodopsin is the membrane-bound receptor in retinal rod cells.¹³ Cholesterol concentration seems to affect the folding and unfolding of rhodopsin in the outer segment disk membrane as it participates in the transduction cascade.¹¹ Regeneration of rhodopsin is less complete and slower in rod outer segment membranes with higher or lower cholesterol concentrations than found in control disc membranes.^{9 - 12 ,14} Phototransduction is initiated by capture of photons by rhodopsin, which then diffuses in the disc membrane to activate transducin, which then activates phosphodiesterase resulting in involvement of cyclic guanosine monophosphate and closure of the channels in the plasma membrane.¹⁵ Deactivation proceeds through another series of molecular steps whereby the cell recovers the ability of the channels to close in response to light.¹⁵ The kinetics of the molecular processes involved in activation, and probably deactivation, are governed by the rate of encounter of the proteins involved in the transduction processes.¹⁶

The steps involved in visual transduction can be described precisely.^{15 ,17 - 18} Furthermore, these processes can be investigated in patients using noninvasive electroretinographic(ERG) procedures.^{19 - 21} We used contemporary ERG procedures to study the kinetics of activation and deactivation of phototransduction in rods of children with SLOS.

Data of 13 patients, including 3 sibling pairs (patients 2 and 3, 4 and 5, and 11 and 12), with biochemically confirmed SLOS are summarized in Table 1. At the ages studied, normal scotopic ERG response parameters are mature.¹⁹ Cholesterol precursors that are not present in healthy individuals were detectable in all of the SLOS-affected patients (Table 1). All patients received cholesterol supplementation, although compliance was inconsistent in some patients. The median spherical equivalent was +1.00 diopter (D) (range, −0.50 to +3.5 D). Patients 1 and 3 had tiny nuclear cataracts in both eyes. Patients 3, 11, and 12 had ptosis. The study conformed to the principles outlined by the Declaration of Helsinki and was approved by the Children's Hospital Committee on Clinical Investigation, Boston, Mass. Informed written consent was obtained from parents prior to the child's participation in the study. Healthy control subjects (n = 26) were aged 8 to 52 years (median age, 22 years).²⁰

Electroretinographic testing was done at the time of other investigations under light inhalation anesthesia that does not alter ERG responses.²² Procedures for preparation of the patient and data acquisition were performed as previously described.²⁰

Rod photoresponse characteristics were estimated from the a-waves by means of the Hood and Birch²¹ formulation of the Lamb and Pugh^{17 - 18} model of the biochemical processes involved in the activation of rod phototransduction. The main parameters of this model are S and R_mp3. Sometimes referred to as a sensitivity parameter, ²¹ S summarizes the kinetics of the series of processes from photon capture up to, and including, closure of the channels in the plasma membrane of the photoreceptor.¹⁸ In other words, the molecular mobilities of the proteins in the transduction cascade are a determinant of S. R_mp3 is the amplitude of the saturated rod response²¹ and represents the number of channels available for closure by light. A curve-fitting routine (MATLAB, fimins subroutine; The Math Works, Inc, Natick, Mass) was used to determine the best-fitting values of S, R_mp3, and t_d, a brief delay, in the following equation:

(1) (I, t) = {1 − exp[−0.5 I S (t-t_d)²]}R_mp3.

In this equation, I is the flash in estimated number of isomerizations per rod per flash. Approximately 8.5 isomerizations per rod per flash are produced by 1 scotopic troland second.²³ Fitting of the model was restricted to a maximum of 20 milliseconds after stimulus onset. For the 26 controls, the mean (SD) value of S is 10.15 (1.58) seconds⁻² and that of R_mp3 is 391 (79) µV.

The kinetics of recovery of the rod cell's response to light were evaluated using a paired flash paradigm^{20 ,24 - 27} in 8 patients (patients 1-3, 8, and 10-13). Recovery, termed "deactivation, "is accomplished by a series of molecular processes whereby the channel's and rod's circulating current are restored to a response-ready state.¹⁵ At 7 selected interstimulus intervals (2-120 seconds) after a test flash, a probe flash was presented. Between each test-probe pair, 2 minutes in the dark was allowed. The amplitude of the response to the probe was expressed as a percentage of amplitude of the response to the test flash alone. For 8 controls, amplitude is 50% when the median interstimulus interval is 3 seconds (range, 2-5 seconds) and 100% at the 120-second interstimulus interval.

The b-wave stimulus-response data were fit by the function

(2) V/V_max = I/(I + σ)

as previously described.^{20 ,28} The main parameters in this equation are V, the b-wave amplitude produced by flash intensity I; V_max, the saturated b-wave amplitude; and log σ, the flash intensity that evokes a half-maximum response amplitude. For controls, the mean (SD) value of log σ is −0.84(0.10) log scotopic troland seconds and that of V_max is 378 (57)µV.

In an analysis reminiscent of Granit, ^{29 - 30} the ERG waveform is considered the sum of the photoreceptor and postreceptoral retinal responses.^{31 - 32} Equation 1 modeled the rod photoresponse, sometimes called "P₃." The photoresponse was digitally subtracted from the ERG waveform to obtain P₂, which is thought to represent mainly the on-bipolar cell response, but also activity in other second- and third-order retinal neurons.^{31 - 36} In an analysis similar to that using equation 2 for the b-wave, the P₂ stimulus-response function was fit with

(3) P₂/P_2max = I/(I + k_p2),

where P_2max is the saturated amplitude and k_p2 is the semisaturation constant. Thus, 1/k_p2 is a measure of sensitivity.

For each patient, data from 1 eye were analyzed. The patients' and controls' ERG parameters were compared (t test). In addition, individual patients' results were compared with the prediction interval for controls.³⁷

Sample a- and b-wave records from 3-year-old patient 4 are shown in Figure 1. The mean values of the parameters calculated from the a-wave (S and R_mp3) and b-wave (log σ and V_max) were significantly lower in patients with SLOS (Table 2).

The rod photoresponse parameters, R_mp3 and S, for each patient are plotted in Figure 2. In every case of SLOS, S was below the normal mean value and most (10 of 13) were below the 95% prediction limit for normal (Figure 2A). Thus, the kinetics of the activation of phototransduction, represented by S, are slower than normal in patients with SLOS. As for R_mp3, only 1 (patient 3) has a value below the limits of normal (Figure 2B).

Results of the paired flash test of deactivation are shown in Figure 3. With decreasing time after the test flash, as the sample records for patient 2 illustrate (Figure 3A), the amplitude of the response to the probe flash decreased. In every patient, the interstimulus interval (Figure 3B) at which the response was 50% of the dark-adapted response amplitude was longer (range, 6-16 seconds; median, 10.5 seconds) than in any control subject (range, 2-5 seconds; median, 3 seconds). Thus, the kinetics of deactivation were significantly delayed in the patients (Table 2).^{15 ,24 ,26 - 27}

Sample P₂ records are shown in Figure 4A. The parameters of the P₂ stimulus-response function, P_2max and log k_p2, calculated by the fit of equation 3 to the P₂ stimulus-response data (Figure 4B), are shown in Figure 4C and D. All but patient 6 had log k_p2 values below the 95% prediction limit of normal, whereas only 4 had P_2max below the 95% prediction limit. The patients' mean values of log k_p2 and P_2max differ significantly from those of the controls (Table 2).

Receptoral and postreceptoral deficits in SLOS sensitivity (S and log k_p2) and saturated amplitude (R_mp3 and P_2max)were compared and the results are shown in Figure 5. For both sensitivity and saturated amplitude, receptoral and postreceptoral deficits are correlated (sensitivity, r = 0.64, P = .02; saturated amplitude, r = 0.78, P = .003, respectively). Deficits appear more marked in postreceptoral than receptoral sensitivity(Figure 5A). Deficits in receptoral and postreceptoral saturated amplitudes are minimal (Figure 5B). Low sensitivity at the receptor can cause low postreceptoral sensitivity with little change in saturated amplitude.^{31 - 32 ,38}

None of the ERG parameters (Table 2) was correlated with the concentrations of cholesterol, 7-dehyrocholesterol, 8-dehydrocholesterol, or with the ratio of cholesterol to total sterol levels(Table 1). The ERG parameters of the 4 patients with cholesterol concentrations less than 100 mg/d L did not differ significantly from those of the other 9 patients. None of the ERG parameters varied with age.

The kinetics of rod activation (Figure 2) and deactivation (Figure 3)are slowed significantly in these children with SLOS. The mean value of S, which summarizes the kinetics of the proteins involved in the activation of phototransduction, is only 61% of normal. Possibly the basis for the slow kinetics, as in the experimental cell membranes, ^{11 ,14} is a low cholesterol concentration in the outer segment membranes. The retina of the rat model of SLOS has a low cholesterol concentration.⁹

Although statistically lower in SLOS-affected patients than in controls, the saturated amplitude of the rod response, R_mp3, is robust at 84% of normal. Thus, the number of channels available for closure by light and length of the outer segment must not be reduced drastically in SLOS-affected patients. A small decrease in the outer segment length and a consequent decrease in the level of rhodopsin available for photon capture are unlikely to explain the proportionately larger decrease in S. Disorder in the cholesterol-deficient lipid bilayer and impaired mobility of proteins in the outer segment membranes are 2 possible, nonmutually exclusive explanations for low S.

None of the sterol levels (Table 1) were correlated with any ERG parameter. Plasma cholesterol level accounts for only 33% of the overall severity score in patients with SLOS.³⁹ Furthermore, as does the brain, the retina makes its own cholesterol by the same biosynthetic pathway as in other tissues. Accordingly, cholesterol levels in peripheral blood are not necessarily an indicator of cholesterol levels in the retina. As in the rat model of SLOS, the deficit in the cholesterol concentration in the retina may not be as severe as in the serum.⁹

The slow kinetics in SLOS are evidence of significant impairment of a membrane-bound signaling system, namely, rhodopsin in the rod outer segment membranes. Whether the retinal dysfunction is progressive or is associated with visual impairment is not yet known. For now, we recommend that the vision and retinal status of children with SLOS be monitored. In the quest to learn more about SLOS, investigation of other membrane-bound signaling systems involved in development^{40 - 41} and neural processes may be warranted.

Corresponding author: Anne B. Fulton, MD, 300 Longwood Ave, Boston, MA 02115 (e-mail: anne.fulton@tch.harvard.edu).

Submitted for publication November 19, 2002; final revision received May 23, 2003; accepted June 20, 2003.

This study was supported in part by grants EY 10597 (Drs Hansen and Fulton) and M01 RR02172 to Children's Hospital, Boston, from the National Eye Institute, National Institutes of Health, Bethesda, Md.