August 1999
Volume 40, Issue 9
Free
Retina  |   August 1999
Tubby-like Protein 1 Homozygous Splice-Site Mutation Causes Early-Onset Severe Retinal Degeneration
Author Affiliations
  • Charles A. Lewis
    From the Riverview Medical Center, Carrabelle, Florida;
  • Iván R. Batlle
    Oftalmologia Especializada, Santo Domingo, Dominican Republic; the
  • Karla G. R. Batlle
    Oftalmologia Especializada, Santo Domingo, Dominican Republic; the
  • Poulabi Banerjee
    Departments of Genetics and Development and Psychiatry, Columbia Genome Center, College of Physicians and Surgeons at Columbia University and New York Psychiatric Institute, New York; the
  • Artur V. Cideciyan
    Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia; and
  • Jiancheng Huang
    Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia; and
  • Tomás S. Alemán
    Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia; and
  • Yijun Huang
    Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia; and
  • Jurg Ott
    Rockefeller University, Laboratory of Statistical Genetics, New York, New York.
  • T. Conrad Gilliam
    Departments of Genetics and Development and Psychiatry, Columbia Genome Center, College of Physicians and Surgeons at Columbia University and New York Psychiatric Institute, New York; the
  • James A. Knowles
    Departments of Genetics and Development and Psychiatry, Columbia Genome Center, College of Physicians and Surgeons at Columbia University and New York Psychiatric Institute, New York; the
  • Samuel G. Jacobson
    Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia; and
Investigative Ophthalmology & Visual Science August 1999, Vol.40, 2106-2114. doi:
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      Charles A. Lewis, Iván R. Batlle, Karla G. R. Batlle, Poulabi Banerjee, Artur V. Cideciyan, Jiancheng Huang, Tomás S. Alemán, Yijun Huang, Jurg Ott, T. Conrad Gilliam, James A. Knowles, Samuel G. Jacobson; Tubby-like Protein 1 Homozygous Splice-Site Mutation Causes Early-Onset Severe Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 1999;40(9):2106-2114.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To characterize the disease expression of an autosomal recessive human retinal degeneration associated with a mutation in TULP1 (tubby-like protein 1), a gene with currently unknown function.

methods. Homozygotes and heterozygotes from an extended Dominican kindred with a TULP1 splice-site gene mutation (IVS14+1,G→A) were studied clinically and with visual function tests. Sequence analysis of TULP1 was also performed in unrelated patients with severe retinal degeneration from a North American clinic population.

results. Homozygotes had nystagmus, visual acuity of 20/200 or worse, color vision disturbances, bull’s eye maculopathy, and peripheral pigmentary retinopathy. Younger patients had a relatively wide extent of kinetic visual fields; older patients had only peripheral islands. No rod function was measurable by psychophysics in any of the patients; markedly reduced cone function was detectable across the visual field of younger patients and in the remaining peripheral islands of older patients. Rod and cone electroretinograms (ERGs) were not detectable using standard methods; microvolt-level cone ERGs were present in some patients. Heterozygotes had normal visual function. No putative pathogenic sequence changes in TULP1 were observed in North American patients with comparably severe retinal phenotypes, mainly in the diagnostic category of Leber congenital amaurosis.

conclusions. This TULP1 splice-site mutation in homozygotes causes early-onset, severe retinal degeneration involving macular and peripheral cones and rods. The constellation of phenotypic findings suggests that the TULP1 gene product is critically important for normal photoreceptor function and may play a role in retinal development.

Two lines of research, one into rodent single-gene obesities 1 and the other into genetic causes of human retinitis pigmentosa (RP) 2 have recently intersected. The result is the discovery that mutations in TULP1 (tubby-like protein 1) can cause forms of human autosomal recessive RP. 3 4 5  
Tubby (tub) is an autosomal recessive murine disease with maturity-onset increase in body weight accompanied by insulin resistance and abnormal glucose tolerance. 6 Tub mice, known to be the same as rd5, also have retinal and cochlear degeneration. 7 8 9 10 11 The mutation associated with tub/rd5 is a G→T transversion that abolishes a donor splice-site, leading to the replacement of the carboxyl-terminal 44 amino acids with approximately 20 amino acids. 12 13  
A family of tubby genes is now recognized and includes human TUB (homologue of mouse tub), TULP1, TULP2, and TULP3. The carboxyl-terminal end of tubby gene family members is conserved, and there are related proteins in lower animals and plants. TUB (human chromosome 11p15.4) is expressed in many tissues, but TULP1 (6p21.3) has been found mainly in the retina. TULP2 (19q13.1) is highly expressed in testis but minimally in retina. 14 15 TULP3 (12p13.2) has recently been described and is expressed in the eye. 15 The function of these proteins is currently unknown. 
For several years, we have investigated an autosomal recessive retinal degeneration in large pedigrees living in the Dominican Republic. A genomic search for linkage led to the identification and refinement of a locus on chromosome 6p, 16 17 designated RP14. We recently identified the molecular cause of retinal degeneration in the Dominican families as a splice-site mutation (IVS14+1,G→A) in the TULP1 gene. 3 Simultaneous with our report, two other studies noted the pathogenicity of TULP1 gene mutations by candidate gene screening in patients with RP. 4 5  
None of the reports to date have given detailed descriptions of the human disease phenotypes associated with TULP1 gene mutations. To increase understanding of the pathophysiology of TULP1-related human retinal degeneration, we studied the disease expression in family members of the large Dominican kindred with the IVS14+1,G→A TULP1 mutation. This extensive family provides an opportunity to estimate the natural history of the disease in a cross-sectional investigation. Once the phenotype was established, the TULP1 gene was screened for mutations in a sample of North American patients with comparable clinical presentations and unknown genotype. Results from the present study can serve as a template for comparison: with the phenotypes of other human TULP1-associated retinal degenerations to determine whether there is allele specificity, with work on the tub/rd5 mouse, and with ongoing in vitro and genetically engineered animal studies attempting to elucidate the role of TULP1 in the retina. 18  
Methods
Subjects
Patients included in this study were from the extended Dominican kindreds in which RP14 was first mapped to chromosome 6p21.3 and later identified to have a TULP1 gene mutation. 3 16 17 A further group of unrelated patients had DNA samples screened for mutations in TULP1. Consent for all procedures was obtained from subjects after the nature of the studies had been explained. The research procedures were in accordance with institutional guidelines and the Declaration of Helsinki. 
Phenotype Analyses
Clinical Examinations.
A history of medical diseases was obtained by interview of the patients and their families. Because of the association of the tub gene with obesity, body weight and height were measured in the patients and body fat determined (Body Fat Analyzer Model HBF-300; Omron Healthcare, Vernon Hills, IL). Because of the association of cochlear degeneration with the tub gene, a portable hearing test 19 (3000 ± 90 Hz; 35 ± 3 dB) was used to screen patients for hearing loss (Physician HearPen; Starkey Laboratories, Eden Prairie, MN), and balance was assessed with tandem walking and standing on one leg with eyes closed. 20  
Two types of ocular examination were performed: screening exams in 1992 and 1993 and more complete assessments in 1998. In 1992 and 1993, the examinations were to confirm the diagnosis of retinal degeneration and enable molecular genetic analyses to proceed. These examinations occurred in the remote villages of the Dominican Republic where the kindreds lived. Visual acuity was estimated with near vision cards and fundus appearance assessed with indirect ophthalmoscopy. In 1998, patients were transported to an ophthalmology office in Santo Domingo and had a complete eye examination and the following tests: Goldmann kinetic perimetry, Farnsworth D-15 color panel, fundus photography, dark-adapted static threshold perimetry, and electroretinography (ERG). 
Perimetry.
Kinetic perimetry was performed with a Goldmann perimeter using V-4e and I-4e test targets; visual field extent was quantified. 21 Static threshold perimetry in the dark-adapted state (>2 hours) was performed using an automated perimeter (Humphrey Field Analyzer, San Leandro, CA) and techniques similar to those previously described. 22 23 24 Chromatic stimuli were presented by affixing filters (blue, Wratten 47; or red, Wratten 26) to goggles worn over the test eye; normal controls and heterozygotes required an additional 2-log-unit neutral density filter because of the lower thresholds of these subjects. Profiles of dark-adapted sensitivity at 2o intervals were measured in the central 60o along the horizontal meridian. In patients with absolute central scotomas on kinetic perimetry, function was assessed with a customized 24o × 24o grid of nine loci (12o spacing) positioned in the peripheral field. Homozygotes who retained central but unstable fixation and those with relatively stable eccentric fixation loci were asked to look toward their finger which was positioned inside the perimeter bowl at the desired fixation locus. The degree of eccentric fixation was determined using the Goldmann kinetic perimeter. The patient was asked to fixate a large target that was moved on the perimeter bowl until the corneal light reflex was centered on the pupil, viewed through the telescope; the final location of the target was measured and taken as the eccentricity of fixation. Rod and cone mediation of stimuli and normal values at the test locations were determined in controls in the fully dark-adapted state (n = 6) and during the cone plateau after a 99% bleach (n = 2). 23 All red results are reported after application of a 5.5-dB correction which was the mean dark-adapted extramacular sensitivity difference between blue and red stimuli in normal subjects. Equal sensitivities (−6 to +6 dB, based on 2 SD) to blue and red stimuli were thus considered rod mediated; based on results in normal subjects acquired at cone plateau, higher sensitivity to red than blue with a red>blue difference of 20 dB (12–28 dB, based on 2 SD) was considered cone mediated. 
Electroretinography.
Full-field ERGs were performed with a computer-based portable electrodiagnostic system (EPIC-3000; LKC Technologies, Gaithersburg, MD) and Burian–Allen bipolar contact lens electrodes (Hansen Ophthalmics, Iowa City, IA) in 16 homozygotes, 2 heterozygotes, and 5 normal subjects (ages, 22–35 years). A handheld Ganzfeld (Kurbisfeld) with stroboscope and background light was used to deliver the following four stimuli: dim white flash, dark-adapted (>2 hours), to elicit a rod ERG b-wave (minimum normal amplitude, 160 μV; maximum normal implicit time, 92 msec); bright white flash, dark-adapted, to elicit a mixed rod and cone ERG a-wave (minimum normal amplitude, 112 μV) and b-wave (minimum normal amplitude, 310 μV); and light-adapted (>9 minutes) cone ERGs elicited with 1 Hz bright white flashes on a white background (minimum normal b-wave amplitude, 70 μV); and 29 Hz bright white flashes on the same background (minimum normal peak-to-peak amplitude, 65 μV; maximum normal timing, 29.5 msec). In patients with no recordable responses to these stimuli, a special protocol was used to resolve smaller signals. In this protocol, the stimulus was a 29-Hz bright white flash presented without background (after approximately 10 minutes of light adaptation). Responses were recorded with an amplifier bandwidth of 1 to 70 Hz, digitized at 5 kHz, and averaged over >200 cycles. Off-line, ERGs were detrended by subtracting a third-order polynomial fit to data; peak-to-peak amplitude and timing of the small signals in patients were estimated by fitting a 29-Hz sinusoid to the data and allowing the amplitude and the phase to vary. Photoelectric artifact was ignored in all data manipulations by discarding regions in the immediate neighborhood of the stimulus. 
PCR Reactions and Sequence Analysis
Patients (n = 25) with severe infantile or childhood onset of retinal degeneration were included in this part of the study. The patients were examined by one of the authors (SGJ) and carried the clinical diagnoses of Leber congenital amaurosis (LCA) or early-onset RP (simplex, multiplex, or autosomal recessive). Patients had a history of visual disturbance in the first months or years of life, usually with nystagmus, reduced visual acuity, nondetectable ERGs and funduscopic evidence of pigmentary retinopathy. These patients were previously determined not to have RPE65 or CRX gene mutations 25 (Jacobson, unpublished data, 1999). Blood samples were obtained and DNA extracted. 16  
The exons of TULP1 were PCR amplified using the primers designed from adjacent intron sequences 50 to 100 bp from the splice site using the PRIMER program. 26 The primer sequences and the genomic sequence of TULP1 have been deposited with GenBank under the accession numbers AF034919–AF034923. For all exons, 100 ng genomic DNA was amplified in a 40-μl reaction with 0.2 μM of each primer and 0.2 μM of each dNTP. PCR amplification was performed using a program with cycling conditions (94°C for 1 minute 15 seconds; 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds; 72°C for 1 minute; and 72°C for 8 minutes) in a DNA engine tetrad (PTC-225: MJ Research, Watertown, MA). For exons 2 and 3, 10% dimethyl sulfoxide was added to the PCR reactions. Exons 1 through exon 14 were PCR amplified using 1 U Taq DNA polymerase (Gibco, Grand Island, NY) at a MgCl2 concentration of 1.8 mM, and exon 15 was amplified using 1 U at a MgCl2 Taq DNA polymerase (Gibco) concentration of 1.5 mM. The exons and the exon–intron junctions were sequenced with the primers described above using an automated sequencer (model 373A; ABI). Alignment was performed using software (Sequencher, ver. 3; Gene Codes). 
Results
The homozygotes and heterozygotes in this study are shown in the pedigrees of Figure 1 . Pedigrees A and B have been greatly simplified from far more extensive pedigrees including several hundred people who can be traced to two founders born in the early 1800s. Many patients in the present study are multiply related to the founders, but some of the consanguinity could not be readily illustrated. Of the 64 living members depicted in Figure 1 , 52 were genotyped; several heterozygotes and normal relatives were genotyped but are not shown. Forty-two family members shown were assessed for phenotype with either screening tests or more extensive evaluations or both: 27 of 29 homozygotes, 13 of 21 heterozygotes, and 2 normal subjects. 
Homozygotes were in general good health. Body habitus was assessed because of the association of the tub gene with obesity in mice. 6 None of the homozygotes was obese, 27 as defined by a body mass index (BMI; weight(kilograms)/height(meters)2 of greater than 30 kg/m2. Mean BMI in male homozygotes was 22.3 kg/m2 (n = 12; range, 15.5–26.4), and mean body fat was 12.2% (n = 10; range, 6–19). Female homozygotes had a mean BMI of 23.4 kg/m2 n = 12; range, 17.4–28.4) and mean body fat of 25.3% (n = 10; range, 12–33). Results of screening tests for hearing were normal in 23 of 24 homozygotes tested, and balance was normal in all 24. 
Ocular Examinations
The limited ocular examinations in 1992 and 1993 of the visually impaired family members showed they had nystagmus, abnormal visual acuity and visual fields, and retinal degeneration. Table 1 lists clinical data from the more recent and complete ocular examinations. By history, homozygotes had childhood onset of night blindness; relatives reported that night vision problems were evident in these affected people by age 3 and as young as 10 months of age. The homozygotes had reduced visual acuity and nystagmus. The nystagmus appeared to be pendular with both horizontal and rotary components. Color vision with a Farnsworth D-15 panel was either unmeasurable or abnormal without a specific axis of confusion. Most patients had myopic refractive errors. Slit lamp examinations showed vitreous condensations and cellularity in a minority of patients; the one patient with cataracts, a 42-year-old man (A, VI-5), had only minimal nuclear sclerotic changes. Applanation tonometry was normal in all subjects. 
Retinal appearance of homozygotes of different ages is shown in Figure 2 . A 9-year-old boy (A, VII-16; Fig. 2A ) and 10-year-old girl (A, VII-14) had attenuated retinal vessels and minimal pigmentary retinopathy, mainly beyond the vessel arcades. An annulus of yellow deposits in the central 10o was present in many of the patients in the second and third decades of life (for example, A, VII-15; A, VII-1; and B, VI-4, Figs. 2B 2C 2D ); these patients also had pigmentary disturbances, including bone spicule-like pigment, from the vessel arcades into the peripheral retina. Bull’s eye macular lesions and peripheral pigmentary retinopathy were present in the remainder of homozygotes, as exemplified in a 42-year-old man (A, VI-5; Fig. 2E ). Another 42-year-old (A, V-5; Fig. 2F ) had a bull’s eye macular lesion in one eye and geographic atrophy (diameter of approximately 10o) in the central retina of the other eye. The two heterozygotes examined, both in the fourth decade of life, had normal visual acuity, visual fields, and ERGs (see later results) but showed many (10–20) small drusenlike lesions in the central retina (not shown). 
Optic disc appearance was not normal in the homozygotes. Disc pallor confined to the temporal sector (Fig. 2G) was evident on clinical examination and fundus photographs of seven patients (age range, 9–42). Stereo-viewing of the photographs of these optic discs showed some excavation of the temporal sector. Most of the remaining patients had a more generalized waxy pallor of the discs. Patient A, VII-10 had apparent elevation of the disc margins, presumably from optic disc drusen. The ratio of the average optic disc diameter to the disc–macula distance was calculated from measurements on fundus photographs of 11 homozygotes. The results ranged from 2.28 to 2.69, all within normal limits and unlike those reported in patients with optic nerve hypoplasia. 28  
Visual Function
Representative results of kinetic perimetry in patients at different disease stages are illustrated in Figures 3A 3B 3C 3D 3E 3F , and the extent of visual field in 15 homozygotes and two heterozygotes is graphically summarized (Fig. 3G) . The two heterozygotes we examined had normal kinetic fields to targets V-4e and I-4e (Fig. 3A) . Three of six homozygotes under the age of 18 years showed either a normal or near normal extent of visual field with the V-4e target (Fig. 3B) , whereas the other three had reductions in field extent with residual field being somewhat elliptical in shape (Fig. 3C) . There was no detection of the I-4e target in any of the 15 homozygotes tested. Homozygotes more than 18 years of age had more reduced extent of field; large central scotomas were detectable (Fig. 3D) , or there were only measurable islands of peripheral vision (Figs. 3E 3F) . Visual field extent (average of both eyes) plotted against age indicates substantial but impaired extent of field in the first two decades of life and a decline in later decades. Best fit exponential to the data from homozygotes is shown (VF = 10[−0.44(age−8.2)], VF as fraction of mean normal, age in years). 
Two-color dark-adapted perimetry in 12 homozygotes who could be studied with this technique showed there was no measurable rod function and only severely impaired cone function (Fig. 4) . Dark-adapted profiles in the central 60o of normal subjects and a heterozygote are illustrated in Figure 4A . Both heterozygotes showed rod mediation of the chromatic stimuli and normal sensitivity (Fig. 4A) . Representative results are shown for two of the eight younger homozygotes (ages, 9–22 years) who could perform this psychophysical task (Figs. 4B 4C) . Profiles in a 9-year-old (Fig. 4B) and 18-year-old (Fig. 4C) indicate no measurable rod sensitivity. There was cone-mediated detection of both colors and a minimum of 2 log units of cone sensitivity loss, compared with normal results obtained at cone plateau. Peripheral islands of vision detected on kinetic perimetry were examined in four older homozygotes (ages, 25, 26, 30, and 42 years) with absolute central scotomas and relatively stable eccentric fixation. There was no measurable rod function in these patches of vision (data shown are average of measurements from nine loci); cone-mediated sensitivity was detectable but reduced by more than 2 log units. Heterozygotes tested similarly for peripheral function had rod-mediation and normal rod sensitivity (Fig. 4D)
Rod, mixed, and cone ERGs in a normal subject, a heterozygote, and a homozygote are compared in Figure 5 A. Both heterozygotes had ERGs that were normal in amplitude and timing. All 16 homozygotes had nondetectable ERGs under these conditions. Using a special protocol, however, two of the homozygotes showed microvolt-level cone flicker ERGs (1.3 and 0.6 μV for A, VII-15 and A, VII-16, respectively; normal amplitude, >72μV) which were delayed in implicit time (Fig. 5B)
Candidate Gene Screening with TULP1
All 15 exons of TULP1 and the exon–intron junctions in the 25 unrelated patients with LCA or early-onset RP were screened. No putative pathogenic sequence changes were observed in any of the patients. 
Discussion
Central Visual Abnormalities, an Early Feature of This Splice-Site TULP1 Mutation
The disease associated with the IVS14+1,G→A TULP1 gene mutation is an early-onset, severe, retina-wide degeneration. Central visual dysfunction and nystagmus are characteristic findings at all stages of the disease. In the first decade of life, there is no measurable rod function and severely impaired cone function throughout the retina. A decline in this residual cone vision occurs over the ensuing decade such that by the third and fourth decades of life, only islands of peripheral cone-mediated vision remain. At the later stages of disease, the ophthalmoscopic appearance is that of severe pigmentary retinopathy with maculopathy. 
The nystagmus and reduced visual acuity but relatively wide visual fields in young TULP1 homozygotes indicate that central visual abnormalities are a prominent early feature of the disease expression. This early central visual loss is probably because of central retinal photoreceptor maldevelopment, dysfunction, or degeneration as part of the generalized retinopathy. LCA, for example, is typified by early nystagmus and reduced visual acuity. 29 30 Signs of maculopathy, such as annuli of yellow deposits, bull’s eye appearance, and atrophic lesions, eventually become visible by ophthalmoscopy and point to significant central retinal degenerative disease at the photoreceptor and retinal pigment epithelial level. 
Do the optic disc changes seen on ophthalmoscopy in some of the patients suggest this TULP1 mutation causes a complicated disease expression involving not only photoreceptors but also more proximal retinal cells or the optic nerve? The most parsimonious explanation for optic disc findings in this TULP1 mutation is that they are a secondary manifestation of a primary photoreceptor disease expression. Optic disc changes in retinal degenerations have traditionally been ascribed to the retinopathies. 31 Temporal pallor of the optic disc has been reported in retinal degenerations with early maculopathy such as the cone–rod dystrophies. 32  
Brief accounts of three other patients representing two different TULP1 genotypes are in the literature. Two compound heterozygotes (Arg420Pro, Phe491Leu) in the fourth decade of life were described as having visual acuity of less than 20/200, only central islands of visual field remaining, and essentially no detectable ERGs. 4 A homozygote with the IVS14–6,C→A mutation, also in the fourth decade of life, was reported to have constricted visual fields and no detectable ERG. 5 No descriptions of phenotype were provided for another homozygote (Lys489Arg), 5 a compound heterozygote (Ile459Lys, IVS2+1,G→A), 4 and other patients with heterozygous changes considered pathogenic. 4 5 At present, the limited information on patients other than those with the IVS14+1,G→A TULP1 mutation prevents discussion about possible allele specificity. 
It is notable, however, that the patients found to have TULP1 mutations by candidate gene screening were from populations with the clinical diagnosis of RP. 4 5 The retinal degeneration resulting from the IVS14+1,G→A TULP1 mutation is an atypical form of RP. 33 It is not a cone–rod dystrophy; although there are macular lesions, early unmeasurable rod function with residual impaired cone function weighs against this diagnostic category. 34 35 A clinical category that may describe the disease better is LCA, 29 30 a genetically heterogeneous set of diseases 2 with early age of onset of severe retinal degeneration with nystagmus, visual acuity loss, and undetectable ERGs. The possibility that the two previous screenings of patients with RP for TULP1 mutations 4 5 may not have included patients with LCA led us to examine a group of non-Dominican patients fitting into this disease category. Mutations in TULP1 were not the cause of LCA in this sample of patients, although we cannot exclude the possibility that coding sequence variants account for a small fraction of LCA mutations in other samples or that mutations in the regulatory sequences of TULP1 contribute to the pathologic course of LCA. These findings, taken together with results of the other published candidate gene screenings, 4 5 extend the conclusion that TULP1 is a relatively rare cause of autosomal recessive retinal degeneration. 
Relationship of TULP1-Associated Human Disease to theTub Mouse Phenotype
The exact function is not yet known for any of the tubby family of genes, members of which currently include tub, TUB, TULP1, TULP2, and TULP3. 12 13 14 15 It is of interest that the causative mutations in tub leading to the mouse phenotype and TULP1 leading to the disease in the patients in this study are at the identical donor splice site. 3 Both mutations would be expected to alter the evolutionarily conserved carboxyl-terminal end of these related molecules. From our results, only the retinal degeneration was known to be shared by the patients and the tub mouse. The patients were not obese and had no major impairment of hearing or balance; more extensive testing was not possible on site in the Dominican Republic. 
How does the tub/rd5 retinal degeneration compare with that in patients with the splice-site TULP1 mutation? Early and prominent photoreceptor disease is a shared feature. The murine homozygous phenotype involves a progressive retina-wide rod and cone degeneration; abnormally distorted outer segments are found at 3 weeks, the earliest time studied. 7 8 9 10 11 The inner retina has been described as normal. 7 8 Similar to human RP 36 and other animal models of inherited retinal degeneration, 37 38 39 photoreceptor cell death in tub/rd5 occurs by an apoptotic mechanism. 18  
It is of interest that a recent study found that retinal expression of the tub gene in mice occurs during embryogenesis only in retinal ganglion cells and postnatally only in photoreceptors. 40 TULP1 may also play some role in human retinal development and the splice-site mutation could lead to a developmental defect manifesting clinically as visual acuity loss and nystagmus. The profound and early photoreceptor degeneration bespeaks a critical functional role for TULP1 in these cells postnatally or possibly during embryogenesis. Further studies, such as those of genetically engineered murine models of TULP1-associated disease, should help elucidate disease mechanisms. Considering the unique features of structure, function, and development of the primate central retina 41 42 and the early central visual losses in patients in this study, investigations to localize precisely the gene product of TULP1 in human adult and fetal retinal tissue should also be of value. 
 
Figure 1.
 
Pedigrees of the extended kindreds from the Dominican Republic simplified to show the patients involved in this study. Patient numbers correspond to those in Table 1 . Inset (dotted rectangle) shows consanguineous marriages of homozygotes with their affected offspring. Three heterozygotes in Pedigree A are the same people shown in Pedigree B; B, IV-4 is A, IV-1; B, IV-9 is A, V-13; and B, IV-7 is A, V-11. Circles, females; squares, males; slash through symbol, deceased. Filled symbols, homozygotes for the TULP1 gene mutation; half-filled symbols, heterozygotes.
Figure 1.
 
Pedigrees of the extended kindreds from the Dominican Republic simplified to show the patients involved in this study. Patient numbers correspond to those in Table 1 . Inset (dotted rectangle) shows consanguineous marriages of homozygotes with their affected offspring. Three heterozygotes in Pedigree A are the same people shown in Pedigree B; B, IV-4 is A, IV-1; B, IV-9 is A, V-13; and B, IV-7 is A, V-11. Circles, females; squares, males; slash through symbol, deceased. Filled symbols, homozygotes for the TULP1 gene mutation; half-filled symbols, heterozygotes.
Table 1.
 
Clinical Characteristics of the Homozygotes
Table 1.
 
Clinical Characteristics of the Homozygotes
Pedigree No. Age at Visit (years) Sex Visual Acuity* Refraction, †
A VII-16 9 M 20/400 −4.00 −2.50× 180
A VII-14 10 F CF plano−1.00× 180
A VII-15 15 F 20/200 −4.00−3.00× 180
B VII-1 16 F 20/200 −8.75
A VII-13 16 F CF −4.25
A VII-10 17 M 20/200 −1.00
A VII-12 18 F 20/400 −2.00−0.50× 180
A VII-1 22 F 20/200 −3.50
A VII-9 25 M CF −7.25
A VI-2 26 F CF −6.50
B VI-4 27 F CF −2.75
B VI-3 30 M HM −10.50
A VII-6 31 M HM −9.50
A V-6 35 F 20/400 −0.25−0.50× 180
A V-5 42 M LP −2.25
A VI-5 42 M 20/400 −11.00
Figure 2.
 
Fundus photographs illustrating different disease stages in six homozygotes (A through F) and optic disc appearance in three homozygotes (G). For two patients in (G), the optic disc of the right eye is shown (left and middle) and for the third patient, the left eye is illustrated (right).
Figure 2.
 
Fundus photographs illustrating different disease stages in six homozygotes (A through F) and optic disc appearance in three homozygotes (G). For two patients in (G), the optic disc of the right eye is shown (left and middle) and for the third patient, the left eye is illustrated (right).
Figure 3.
 
Kinetic perimetry with V-4e and I-4e test targets in a heterozygote (A) and five homozygotes of different ages (B through F). Black is absolute scotoma. (G) Graph of visual field extent (average of two eyes) as a fraction of normal mean (V-4e target) versus age in 15 homozygotes (filled circles) and 2 heterozygotes (unfilled circles). Dotted lines denote 2 SD below normal mean. Solid line is best fit exponential to the data of homozygotes.
Figure 3.
 
Kinetic perimetry with V-4e and I-4e test targets in a heterozygote (A) and five homozygotes of different ages (B through F). Black is absolute scotoma. (G) Graph of visual field extent (average of two eyes) as a fraction of normal mean (V-4e target) versus age in 15 homozygotes (filled circles) and 2 heterozygotes (unfilled circles). Dotted lines denote 2 SD below normal mean. Solid line is best fit exponential to the data of homozygotes.
Figure 4.
 
Two-color (red, R; blue, B) dark-adapted static threshold perimetric profiles in a heterozygote (A) and homozygotes (B, C). Lower limits of normal sensitivities (mean − 2SD) in the dark-adapted state are shown in (A). Dotted lines in B, C denote normal dark-adapted cone sensitivity to the red stimulus measured during the cone plateau after a full bleach. Hatched bar marks location of the physiologic blind spot. N, nasal field; T, temporal field. (D) Summary of two-color dark-adapted perimetry results in patches of peripheral vision (centered near 30–40o along the horizontal meridian) of four homozygotes and two heterozygotes. Hatching shows range of normal results under fully dark-adapted conditions and at cone plateau. Dotted lines show hypothetical regions of sensitivity loss when both stimuli are detected by rods (rod mediation) or by cones (cone mediation).
Figure 4.
 
Two-color (red, R; blue, B) dark-adapted static threshold perimetric profiles in a heterozygote (A) and homozygotes (B, C). Lower limits of normal sensitivities (mean − 2SD) in the dark-adapted state are shown in (A). Dotted lines in B, C denote normal dark-adapted cone sensitivity to the red stimulus measured during the cone plateau after a full bleach. Hatched bar marks location of the physiologic blind spot. N, nasal field; T, temporal field. (D) Summary of two-color dark-adapted perimetry results in patches of peripheral vision (centered near 30–40o along the horizontal meridian) of four homozygotes and two heterozygotes. Hatching shows range of normal results under fully dark-adapted conditions and at cone plateau. Dotted lines show hypothetical regions of sensitivity loss when both stimuli are detected by rods (rod mediation) or by cones (cone mediation).
Figure 5.
 
(A) Representative rod, mixed cone and rod, and cone ERG results in a normal subject, a heterozygote, and a homozygote. (B) Cone flicker ERGs (29 Hz) in a normal subject, a heterozygote, and two homozygotes, obtained with a special protocol for small signals. Dashed lines show 29-Hz sinusoids fitted to the data; arrows depict implicit times. Calibrations are to the right and below the responses.
Figure 5.
 
(A) Representative rod, mixed cone and rod, and cone ERG results in a normal subject, a heterozygote, and a homozygote. (B) Cone flicker ERGs (29 Hz) in a normal subject, a heterozygote, and two homozygotes, obtained with a special protocol for small signals. Dashed lines show 29-Hz sinusoids fitted to the data; arrows depict implicit times. Calibrations are to the right and below the responses.
The authors thank Dr. Grant Liu for his generous help with interpreting the neuro-ophthalmological findings in the patients; and David Hanna, Kai Zhao, Leigh Gardner, Jason Christopher, and Dr. Rodrigo Montemayor for their help with the studies. 
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Figure 1.
 
Pedigrees of the extended kindreds from the Dominican Republic simplified to show the patients involved in this study. Patient numbers correspond to those in Table 1 . Inset (dotted rectangle) shows consanguineous marriages of homozygotes with their affected offspring. Three heterozygotes in Pedigree A are the same people shown in Pedigree B; B, IV-4 is A, IV-1; B, IV-9 is A, V-13; and B, IV-7 is A, V-11. Circles, females; squares, males; slash through symbol, deceased. Filled symbols, homozygotes for the TULP1 gene mutation; half-filled symbols, heterozygotes.
Figure 1.
 
Pedigrees of the extended kindreds from the Dominican Republic simplified to show the patients involved in this study. Patient numbers correspond to those in Table 1 . Inset (dotted rectangle) shows consanguineous marriages of homozygotes with their affected offspring. Three heterozygotes in Pedigree A are the same people shown in Pedigree B; B, IV-4 is A, IV-1; B, IV-9 is A, V-13; and B, IV-7 is A, V-11. Circles, females; squares, males; slash through symbol, deceased. Filled symbols, homozygotes for the TULP1 gene mutation; half-filled symbols, heterozygotes.
Figure 2.
 
Fundus photographs illustrating different disease stages in six homozygotes (A through F) and optic disc appearance in three homozygotes (G). For two patients in (G), the optic disc of the right eye is shown (left and middle) and for the third patient, the left eye is illustrated (right).
Figure 2.
 
Fundus photographs illustrating different disease stages in six homozygotes (A through F) and optic disc appearance in three homozygotes (G). For two patients in (G), the optic disc of the right eye is shown (left and middle) and for the third patient, the left eye is illustrated (right).
Figure 3.
 
Kinetic perimetry with V-4e and I-4e test targets in a heterozygote (A) and five homozygotes of different ages (B through F). Black is absolute scotoma. (G) Graph of visual field extent (average of two eyes) as a fraction of normal mean (V-4e target) versus age in 15 homozygotes (filled circles) and 2 heterozygotes (unfilled circles). Dotted lines denote 2 SD below normal mean. Solid line is best fit exponential to the data of homozygotes.
Figure 3.
 
Kinetic perimetry with V-4e and I-4e test targets in a heterozygote (A) and five homozygotes of different ages (B through F). Black is absolute scotoma. (G) Graph of visual field extent (average of two eyes) as a fraction of normal mean (V-4e target) versus age in 15 homozygotes (filled circles) and 2 heterozygotes (unfilled circles). Dotted lines denote 2 SD below normal mean. Solid line is best fit exponential to the data of homozygotes.
Figure 4.
 
Two-color (red, R; blue, B) dark-adapted static threshold perimetric profiles in a heterozygote (A) and homozygotes (B, C). Lower limits of normal sensitivities (mean − 2SD) in the dark-adapted state are shown in (A). Dotted lines in B, C denote normal dark-adapted cone sensitivity to the red stimulus measured during the cone plateau after a full bleach. Hatched bar marks location of the physiologic blind spot. N, nasal field; T, temporal field. (D) Summary of two-color dark-adapted perimetry results in patches of peripheral vision (centered near 30–40o along the horizontal meridian) of four homozygotes and two heterozygotes. Hatching shows range of normal results under fully dark-adapted conditions and at cone plateau. Dotted lines show hypothetical regions of sensitivity loss when both stimuli are detected by rods (rod mediation) or by cones (cone mediation).
Figure 4.
 
Two-color (red, R; blue, B) dark-adapted static threshold perimetric profiles in a heterozygote (A) and homozygotes (B, C). Lower limits of normal sensitivities (mean − 2SD) in the dark-adapted state are shown in (A). Dotted lines in B, C denote normal dark-adapted cone sensitivity to the red stimulus measured during the cone plateau after a full bleach. Hatched bar marks location of the physiologic blind spot. N, nasal field; T, temporal field. (D) Summary of two-color dark-adapted perimetry results in patches of peripheral vision (centered near 30–40o along the horizontal meridian) of four homozygotes and two heterozygotes. Hatching shows range of normal results under fully dark-adapted conditions and at cone plateau. Dotted lines show hypothetical regions of sensitivity loss when both stimuli are detected by rods (rod mediation) or by cones (cone mediation).
Figure 5.
 
(A) Representative rod, mixed cone and rod, and cone ERG results in a normal subject, a heterozygote, and a homozygote. (B) Cone flicker ERGs (29 Hz) in a normal subject, a heterozygote, and two homozygotes, obtained with a special protocol for small signals. Dashed lines show 29-Hz sinusoids fitted to the data; arrows depict implicit times. Calibrations are to the right and below the responses.
Figure 5.
 
(A) Representative rod, mixed cone and rod, and cone ERG results in a normal subject, a heterozygote, and a homozygote. (B) Cone flicker ERGs (29 Hz) in a normal subject, a heterozygote, and two homozygotes, obtained with a special protocol for small signals. Dashed lines show 29-Hz sinusoids fitted to the data; arrows depict implicit times. Calibrations are to the right and below the responses.
Table 1.
 
Clinical Characteristics of the Homozygotes
Table 1.
 
Clinical Characteristics of the Homozygotes
Pedigree No. Age at Visit (years) Sex Visual Acuity* Refraction, †
A VII-16 9 M 20/400 −4.00 −2.50× 180
A VII-14 10 F CF plano−1.00× 180
A VII-15 15 F 20/200 −4.00−3.00× 180
B VII-1 16 F 20/200 −8.75
A VII-13 16 F CF −4.25
A VII-10 17 M 20/200 −1.00
A VII-12 18 F 20/400 −2.00−0.50× 180
A VII-1 22 F 20/200 −3.50
A VII-9 25 M CF −7.25
A VI-2 26 F CF −6.50
B VI-4 27 F CF −2.75
B VI-3 30 M HM −10.50
A VII-6 31 M HM −9.50
A V-6 35 F 20/400 −0.25−0.50× 180
A V-5 42 M LP −2.25
A VI-5 42 M 20/400 −11.00
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