Retinal Function in Carriers of Bardet-Biedl Syndrome

Objective  To test the hypothesis that carriers of Bardet-Biedl syndrome have abnormal rod-mediated responses.

Methods  Parents (n = 26) of children with Bardet-Biedl syndrome (BBS), who are obligate carriers of BBS, consented to scotopic, full-field electroretinography(ERG). Responses were recorded over a 4 to 5 log unit range, up to a maximum stimulus of approximately +3.6 log scotopic troland seconds. The parameters of activation of the rod photoresponse, S (sensitivity parameter) and R_mp3(amplitude of the saturated rod response), were derived by fitting a transduction model to the ERG a-waves. For the b-wave, the stimulus producing a half maximum response (log 𝛔) and the saturated amplitude (V_max) were determined. The model of the rod photoresponse was subtracted from the intact ERG to demonstrate a corneal positive potential (P₂)_, and log k_P2 and P_2max were determined. The carriers' ERG responses were compared with those of healthy control subjects (n = 26).

Main Outcome Measures  Sensitivity (S, log 𝛔, and log k_P2) and saturated amplitude (R_mp3, V_max, and P_2max) of receptoral and postreceptoral response components.

Results  All parents had decreased P₂ sensitivity, and most (15 [60%] of 26) had decreased b-wave sensitivity. The rod photoresponse sensitivity and the saturated amplitudes of the rod cell response, b-wave and P₂, did not differ significantly between carriers and controls.

Conclusions  Diminished P₂ sensitivity is characteristic of the carriers of BBS. The site of the primary defect in the BBS rod pathway appears to be proximal to the outer segments, most likely before the rod–bipolar cell synapse.

BARDET-BIEDL SYNDROME (BBS) (OMIM 209900) is an autosomal recessive condition. The cardinal features are retinal degeneration, obesity, mild mental retardation, learning disabilities, hypogonadism, and postaxial polydactyly.^1-4 Renal anomalies are common.³

Bardet-Biedl syndrome is both phenotypically and genetically heterogeneous. Clinical diagnosis is based on the presence of 4 of the 5 cardinal features. Even in the same family, affected individuals may have different combinations of these features.⁵ Six different BBS genes have been mapped to chromosomes 11q13 (BBS1), ⁶ 16q21 (BBS2), ⁷ 3p13-12(BBS3), ⁸ 15q22.3-23(BBS4), ⁹ 2q31 (BBS5), ¹⁰ and 20p12 (BBS6).^11,12 Four of the BBS genes (BBS1, BBS2, BBS4, and BBS6) have been identified.^11-15 The predicted BBS1, BBS2, and BBS4 proteins do not show similarity to any known proteins. BBS6 is caused by a defect in the MKKS gene^11,12 and has properties that are homologous to chaperonins, proteins involved in the folding and transport of membrane proteins. BBS1, BBS2, and BBS4 genes are not similar to chaperonins.

Retinal dysfunction in BBS is well recognized.^16-19 However, even in early childhood, analysis of retinal function in BBS is frustrated by the attenuation of ERG responses. This precludes an informative analysis of the physiologic processes in many individuals with BBS. An anatomical study of a BBS retina showed good preservation of the inner retina while the photoreceptor layer was degenerated, suggesting that the photoreceptors are the primary retinal site of BBS.²⁰

Carriers of BBS are at increased risk for obesity, hypertension, and diabetes mellitus, ^21,22 as well as renal disease.^23,24 In one BBS family, renal manifestations in carriers were inherited as an autosomal dominant trait that was mapped to the BBS2 locus.²⁴ Although there has been little discussion of an increased risk of retinal disease among BBS carriers, 2 carriers with maculopathy have been reported.^17,21 Thus, there is some evidence that individuals who are heterozygous for BBS may have retinal abnormalities.

Accordingly, we conducted an electroretinographic (ERG) study of the rod and rod-mediated responses in 26 obligate carriers, the parents of children with BBS. Would there be significant retinal dysfunction in individuals heterozygous for BBS? Would the ERG results disclose abnormal retinal processes that might be clues to the function of the BBS protein in the retinal cells and to the mechanisms involved in photoreceptor demise?²⁰

Twenty-six parents of children with BBS participated. The parents were assumed to be obligate carriers with one BBS gene mutation. Each parent had 1 to 3 children with BBS, whose features included retinal degeneration and at least 3 of the following: mild mental retardation, postaxial polydactyly, obesity, and hypogonadism. Sixteen parents (8 families) were of European ancestry. Ten (6 families) were from Puerto Rican families with mutations of the BBS1 gene.¹³ Subjects were aged 26 to 50 years (median, 41 years) at the time of the ERG test and 15 were women. Except for a 30-year-old father with maculopathy since childhood and visual acuity of 20/400, all other parents were free of ocular and visual complaints and had normal fundi on ophthalmoscopy. Corrected visual acuities were 20/25 or better. Spherical equivalents ranged from +6.75 to –6.50 diopters (median Plano). The study conformed to the principles outlined by the Declaration of Helsinki and was approved by the Children's Hospital (Boston, Mass) committee on clinical investigation. Informed written consent was obtained from all subjects prior to participation. Healthy control subjects (n = 26), 25 of whom had been included in prior reports, ^25,26 were aged 8 to 52 years (median, 22 years).

The left pupil was dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride. After 30 minutes of dark adaptation and instillation of 0.5% procaracaine, a Burian-Allen (Hansen Ophthalmic Development Lab, Solon, Iowa) bipolar electrode was placed on the left cornea. A ground electrode was placed on the skin over the mastoid. Responses to strobe flashes were differentially amplified (AC-coupled 1-1000 Hz bandpass; 1000 gain), displayed on an oscilloscope, and stored on disk for later analysis. An adjustable voltage window was used to reject responses contaminated by artifacts. Two to 16 responses were averaged in each stimulus condition. The interstimulus interval ranged from 2 to 60 seconds.

Blue (Wratten 47B, λ<510 nm; Eastman Kodak Co, Rochester, NY) strobe stimuli (Novatron of Dallas, Dallas, Tex) were delivered through a 41-cm integrating sphere, controlled in intensity by calibrated neutral-density filters, and ranged from dim flashes that evoked a small b-wave (<15 µV) to those that saturated the a-wave amplitude in controls.²⁶ The unattenuated flash, measured with a detector (S350; UDT Instruments, Baltimore, Md) placed at the position of the subject's cornea, was 3.82 log µW/cm² per flash. The scotopic troland value of the stimulus was calculated by taking each subject's pupillary diameter into account.²⁶

Rod photoresponse characteristics were estimated from the a-waves by means of the Hood and Birch²⁷ formulation of the Lamb and Pugh^28,29 model of the biochemical processes involved in the activation of rod phototransduction. The main parameters of this model are S and R_mp3. S is a sensitivity parameter and R_mp3 is the amplitude of the saturated rod response.²⁷ A curve fitting routine was used to determine the best-fitting values of S, R_mp3, and t_d (a brief delay), in the following equation:

(1) R (I, t) = (1 − exp [−0.5 I S (t − t_d)²]) R_mp3

In this equation, I is the flash in the estimated number of isomerizations per rod per flash. Approximately 8.5 isomerizations per rod per flash are produced by 1 scotopic troland second.³⁰ Fitting of the model was restricted to the leading edge of the a-wave response or to a maximum of 20 milliseconds after stimulus onset. All 3 parameters were free to vary. For controls, the mean ± SD value of S is 10.15 ± 1.58 seconds^-2 and that of R_mp3 is 391 ± 79 µV.

The b-wave stimulus-response function,

(2) V/V_max = I / (I + 𝛔),

was fit to the b-wave amplitudes of each subject by means of an iterative procedure that minimized the mean square deviation of the data from the equation.²⁶ In this equation, V is the b-wave amplitude produced by flash intensity I, and V_max is the saturated b-wave amplitude. The flash intensity that evokes a half-maximum response amplitude is 𝛔. Thus, 𝛔 is the semisaturation constant, and 1/𝛔 is a measure of sensitivity. The stimulus-response function was fit up to the higher intensities, at which a-wave intrusion occurs.³¹ For controls, ²⁶ the mean ± SD value of log 𝛔 (half maximum response) was –0.84 ± 0.10 log scotopic troland seconds, and that of V_max was 378 ± 57 µV.

In an analysis reminiscent of that of Granit, ^32,33 the ERG waveform is considered to be the sum of the photoreceptor and postreceptoral retinal responses.^34,35 Equation 1 modeled the rod photoresponse, sometimes called P₃. The photoresponse was digitally subtracted from the ERG waveform to obtain P₂ (positive potential), which is thought to represent mainly the on-bipolar cell response, but also activity in other second- and third-order retinal neurons.^34-39 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.

The on-bipolar cells have their own G-protein cascade. To evaluate the kinetics of the G-protein cascade, ^38,39 the latency at which P₂ reached 50 µV was noted. In normal retinas, this latency, plotted as a function of stimulus intensity on log-log coordinates, is a linear function³⁸ with a slope of about–0.2. For the 26 controls, the mean (SD) slope was –0.216 ± 0.05. Departures from this relationship indicate dysfunction of the on-bipolar cells' G-protein cascade.³⁸

The carriers' and controls' ERG parameters were compared (t test). The data from the BBS carrier with maculopathy were excluded from the t tests. A P value less than or equal to .01 was required for statistical significance. In addition, results of individual carriers were compared with the prediction interval for controls.²⁶ The prediction interval gives the range of values within which results from individuals in the healthy population are expected to fall.⁴⁰

Sample a- and b-wave results from a 42-year-old BBS carrier who had response parameters close to the median values are shown in Figure 1. In Figure 2, rod photoresponse parameters S and R_mp3, calculated from the a-waves, and log 𝛔 and V_max, calculated from the b-waves, are shown for every BBS carrier (N = 26).

The parameters of the rod photoresponse, S and R_mp3, fell within the limits of normal (Figure 2, upper panels), with the exception of R_mp3 for the carrier with maculopathy (filled triangle). Although S and R_mp3 were below the normal mean in most BBS carriers, the carriers' and controls' values did not differ significantly (Table 1).Among the BBS carriers, b-wave log 𝛔 was broadly distributed, with 15 (60%) of the points below the 95% prediction limit; log 𝛔 differed significantly between carriers and controls (Table 1). There was considerable overlap of V_max in BBS carriers and controls, and the difference between carriers and controls was not significant (Table 1). No parameter varied significantly with age or spherical equivalent. No parameter differed significantly with sex or ancestry (European vs Puerto Rican).

Subtraction of the rod photoresponse from the intact ERG (Figure 3A) yielded a family of P₂ (Figure 3B). The slope of the log P₂ latency function from a 41-year-old BBS carrier was determined (Figure 3C). The slopes of the log P₂ latency function did not differ between BBS carriers and controls (Figure 3D and Table 1).

The P₂ stimulus-response functions are summarized in Figure 4. In every BBS carrier, log k_P2 was at or below the 95% prediction limit (Figure 4B), and the difference between BBS carriers and controls was significant (Table 1). On the other hand, the saturated amplitude, P_2max, was similar in BBS carriers and controls (Figure 4C and Table 1). The ratio of the amplitudes of the saturated postreceptoral response, P_2max, and receptoral response, R_mp3, did not differ between BBS carriers and controls (t₄₉= −0.28, P = .78).

There is significant retinal dysfunction, specifically decreased log k_P2, among individuals who are heterozygous for BBS. Nonetheless, with the exception of the individual with maculopathy, the carrier state was not associated with retinal disease or visual disability in these 26- to 50-year-olds.

In the BBS carrier state, the activation of phototransduction in the rod outer segment, represented by S and R_mp3, is normal in almost all BBS carriers (Figure 2). Normal P_2max and slope of the log P₂ latency function (Figure 3) are evidence of normal bipolar cell function. The normal relationship between the saturated amplitude of the receptoral (R_mp3) and postreceptoral (P_2max) components is evidence of the integrity of the rod-bipolar synapse in the BBS carriers. In muscular dystrophy, that does alter the rod-bipolar synapse, owing to loss of the cytoskeletal protein, dystrophin. A pattern of low V_max and normal log 𝛔 is seen in these patients, ^41-43 whereas BBS carriers have the opposite: normal V_max and low log 𝛔. In view of this evidence for normal function of the rod-bipolar synapse as well as normal function of the rod outer segment and the bipolar cell, the site of action of the BBS carrier disorder must be between the outer segment and the synapse—specifically, the rod inner segment.

The observed ERG waveform is the sum of positive and negative potentials generated by the activity of receptoral and postreceptoral cells.^32-35 Signals are transmitted from receptor to postreceptoral cells. A deficit in the receptor's function becomes a deficit in the input to the bipolar cell that, in turn, is represented as an abnormality in the b-wave or P₂. For instance, a deficit in rod cell sensitivity (S) or saturated amplitude (R_mp3)results in a shift of log k_P2 to lower sensitivities while the saturated amplitude of the b-wave and P₂ remain robust, according to the dynamic model of Hood.³⁴ Although the model³⁴ does not deal explicitly with the inner segment, dysfunction in the inner segment is another candidate for abnormal input to the second-order cells. Among the BBS carriers, all had low log k_P2, but only 60% had low b-wave log 𝛔. Removal of competing signals by subtraction of P₃ appears to give a clearer representation of the bipolar cell activity.

Any of several presynaptic processes may be affected in BBS carriers. Exactly which of the processes is affected may be encoded by the specific BBS gene. After the activation of phototransduction, the current flows along the surface of the inner segment of the rod toward the synapse. The cellular processes that follow include the docking of vesicles on the synaptic ribbons, formation of synaptosomes for release across the synapse, and fusion with the postsynaptic membrane of the on-bipolar cell. The BBS genes may encode components involved in any of these steps. Possibly, the BBS genes encode components of the chaperonins complexes. In the instance of the BBS6 gene, which encodes protein homologous to chaperonins, ^11,12 the orderly folding and unfolding of proteins involved in inner segment and synaptic processes may be disrupted.

Mutations in different genes give rise to the BBS phenotype. At a molecular level, the various BBS gene products might act on different steps in the same biological processes, such as sequential steps of enzymatic, signaling, ligand-receptor, or other cellular pathways. With the exception of those of Puerto Rican ancestry who have BBS1 haplotypes, the BBS carriers studied herein were presumed to be genetically heterogeneous. The presence of the same ERG abnormality in all BBS carriers suggests that the several BBS genes are closely related in their function in the retina.

The loss of P₂ sensitivity is consistent with haplo-insufficiency in an autosomal recessive condition. Other autosomal recessive disorders of the retina have caused retinal dysfunction in carriers. For instance, individuals heterozygous for a splice-site mutation, G4335T, of the rhodopsin gene, had low V_max and low log 𝛔.⁴⁴ Asymptomatic carriers of Oguchi disease, an autosomal recessive condition caused by mutations in rhodopsin kinase, had mild ERG abnormalities, ⁴⁵ as did mice heterozygous for mutations⁴⁶ of the ABCR (adenosine triphosphate–binding cassette retina) gene, which encodes a protein in the rim of the outer segment discs and is associated with several autosomal recessive retinal degenerations, including Stargardt disease.

In summary, an ERG signature of the BBS carrier state is demonstrated. It is not commonly associated with retinal disease. The evidence that we obtained indicates that this abnormal ERG finding is a consequence of rod cell dysfunction proximal to the outer segments. We speculate that similar but more severe dysfunction afflicts patients with BBS, leading to destruction of the photoreceptor cell.

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

Submitted for publication September 24, 2002; final revision received February 12, 2003; accepted February 20, 2003.

This study was supported in part by grant EY10597 from the National Eye Institute, National Institutes of Health, Bethesda, Md, and grant M01RR02172 from the National Institutes of Health, Bethesda, to Children's Hospital, Boston, Mass.