Low Incidence of Retinitis Pigmentosa Among Heterozygous Carriers of a Specific Rhodopsin Splice Site Mutation

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1 Low Incidence of Retinitis Pigmentosa Among Heterozygous Carriers of a Specific Rhodopsin Splice Site Mutation Philip J. Rosenfeld, Lauri B. Hahn, Michael A. Sandberg, Thaddeus P. Dryja, and Eliot L. Berson Purpose. To determine whether a rhodopsin splice donor site mutation at the 5' end of intron 4 is a cause of autosomal dominant retinitis pigmentosa. Methods. Heterozygous carriers of the same rhodopsin splice site mutation in two pedigrees were identified using single-strand conformation polymorphism analysis. Twelve heterozygous carriers were evaluated by ophthalmoscopy, Goldmann kinetic visual fields, dark adaptation thresholds, and full-field electroretinograms including rod intensity-response functions. Clinical findings from the heterozygous carriers of the splice site mutation were compared with those from heterozygous carriers from a separate family with a known recessive rhodopsin null mutation, Glu249X. Results. Analysis of DNA from 48 members of two pedigrees revealed 25 heterozygous carriers of the splice site mutation, ranging in age from 14 to 82 years. There were no homozygotes with the rhodopsin splice site mutation. Of the 25 heterozygous carriers, 24 were asymptomatic. Eleven asymptomatic heterozygotes were examined, including four older than 65 years of age. They were found to have normal fundi, full visual fields, and slightly elevated final rod dark adaptation thresholds. Their rod electroretinographic b-wave amplitudes were slightly diminished over the full range of blue light intensities. Rod a-wave implicit times were slightly but significantly prolonged in response to the brightest blue flash of light These subtle abnormalities in rod function were similar to those found in asymptomatic heterozygous carriers of the recessive Glu249X mutation. Only one of the 25 heterozygous carriers of the splice site mutation had symptoms and signs of retinitis pigmentosa. Conclusions. Because 96% of these heterozygous carriers do not have retinitis pigmentosa, it is unlikely that this mutation in intron 4 is a dominant allele. The subtle abnormalities of rod function found in asymptomatic carriers are similar to those found in heterozygous carriers of a recessive rhodopsin allele. The one heterozygous carrier with retinitis pigmentosa probably has a second mutation in the rhodopsin gene or has a defect or defects in another gene that causes his disease. Invest Ophthalmol Vis Sci. 1995;36: Doth dominant and recessive mutations in the rhodopsin gene causing retinitis pigmentosa (RP) have been described. 1 2 It is usually clear from an inspection of a pedigree whether a given rhodopsin mutation results in a dominant or recessive allele, but in some From the Herman-Gund Laboratory for the Study of Retinal Degenerations and the Taylor Smith laboratory, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts. Supported in part by National Eye Institute, grants EY00169 and EY08683, The George Gund Foundation, The Foundation Fighting Blindness (Baltimore), and- an unrestricted grant from Research to Prevent Blindness to the Harvard Medical School Department of Ophthalmology. Submitted for publication April 21, 1995; revised June 13, 1995; accepted June 14, l-'mpiietaiy interest category: N. Reprint requests: Eliot L. Berson, Harvard Medical School, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA instances it is difficult to make this distinction. This article focuses on one such problematic mutation, a G-to-T transversion at the consensus splice donor site of intron 4 (position 4335). In a previous article, 2 we interpreted this mutation as a recessive allele because it was found heterozygously in an asymptomatic 28- year-old woman with no family history of RP. The patient had the same mild rod functional abnormalities seen in the heterozygous carriers of an indisputable recessive allele (Glu249X). Since publication of our article, another family has been described in which some members carry this same splice site mutation heterozygously. 3 ' 4 The oldest of these heterozygous carriers (76 years of age) had symptoms and signs of RP, but the younger heterozy Investigative Ophthalmology & Visual Science, October 1995, Vol. 36, No. Copyright Association for Research in Vision and Ophthalmology

2 Rhodopsin Splice Site Mutation 2187 FIGURE 1. Two pedigrees that segregate the intron 4 splice donor site mutation. Family D586 has no propositus because there are no affected persons; an arrow points to the first carrier identified in this pedigree. The propositus in family 6965 is designated by an arrow. Stars denote family members who underwent genotype analysis. Crosses denote family members who were examined clinically as well. Pedigree symbols with a black circle indicate heterozygous carriers. The age of each family member as of April 1994 is listed below each symbol. Pedigree #D586 I II III Pedigree #6965 I IV 6 OK3D0DD 57 I gous carriers (46 and 21 years of age) were asymptomatic and had full visual fields and no evidence of a pigmentary retinopathy. Psychophysical, electroretinographic, and retinal densitometric testing of the asymptomatic heterozygous carriers showed slight rod abnormalities. The authors concluded that the rod abnormalities in the asymptomatic younger patients represented the early stages of RP and that this splice site mutation was a dominant cause of RP. 4 We have now identified a third family with many members who are heterozygous carriers of this splice donor site mutation in intron 4 of the rhodopsin gene. We have examined heterozygotes of various ages from this large family, as well as the 72-year-old asymptomatic heterozygote who is the father of the 28-year-old woman first identified with this mutation. 2 The current study was performed to clarify whether this mutation is a dominant cause of RP. MATERIALS AND METHODS Ascertainment of Patients Two families with the splice donor site mutation in intron 4 of the rhodopsin gene were identified. The heterozygous carrier (N88) from the first family (BGL #D586) was identified among a group of 117 unrelated persons with no personal or family history of RP who provided DNA samples as normal volunteers. 2 The propositus from the second family (BGL #6965) was identified from among 100 unrelated symptomatic patients with isolate RP from the United States or Canada. The study was conducted with the approval of the institutional review boards of Harvard Medical School and the Massachusetts Eye and Ear Infirmary. Tenets of the Declaration of Helsinki were followed, and informed consent was obtained from all participants. Each subject involved in the study was questioned directly by telephone or in person regarding visual symptoms before DNA analysis. Leukocyte nuclei were prepared from 10 to 50 ml of venous blood drawn from each family member or control subject who agreed to participate as described previously. 5 These nuclei were stored at 70 C before DNA purification and analysis. Screening for the Splice Site Mutation in Intron 4 of the Rhodopsin Gene The rhodopsin gene sequences were screened for mutations using single-strand conformation polymorphism (SSCP) analysis. 11 The oligonucleotide sequences and experimental conditions for polymerase chain reaction (PCR)-mediated amplification of exon 4 and the flanking intron sequences have been described. 7 PCR-amplified DNA was directly sequenced as previously described. 8 Ophthalmologic, Electroretinographic, and Psychophysical Testing We clinically evaluated members from both families with the rhodopsin splice site mutation. To reduce ascertainment bias that might underrepresent the proportion of clinically affected carriers, we targeted the elder carriers for clinical examination because they would be most likely to show signs or symptoms of RP. Most persons who agreed to participate underwent an ocular examination, Goldmann kinetic perimetry, final rod dark adaptation testing, and full-field electro-

3 2188 Investigative Ophthalmology & Visual Science, October 1995, Vol. 36, No. 11 O o II III i rlv n Wild-type Mutant Wild-type -Mutant Wild-type A Mutant Wild-type FIGURE 2. Segregation of the rhodopsin donor splice site mutation within two families as shown by single-strand conformation polymorphism (SSCP) analysis. The SSCP analyses of family members are located beneath their respective pedigree designation. The arrows on the left of the autoradiograph designate the position of the single-strand DNA fragments that encode a wild-type sequence. The wild-type sequence is represented by the normal control in the left lane. The arrows on the right depict the single-strand DNA fragments with the G-to-T transversion at the 5' splice donor site of intron 4 within the rhodopsin gene. (A) SSCP analysis of pedigree #BGL-D586. (B) SSCP analysis of pedigree #BGI^6965. B Mutant retinography, including a rod electroretinogram (ERG) intensity amplitude profile. One person (82 years of age) only underwent an ocular examination. Goldmann kinetic visual fields were measured for each eye using the V-4-e white stimulus on a white background of 31.5 apostilbs and by bringing the test lights from nonseeing to seeing areas. Visualfieldswere digitized, and areas were quantified in degrees squared; an average area of both eyes was obtained for each person. Rod dark adaptation thresholds were measured after 45 minutes of dark adaptation with an 11 white test light fixated 7 above the fovea in the Goldmann-Weekers dark adaptometer as previously described. 9 Full-field ERG testing was performed using a bipolar contact lens electrode placed on the topically anesthetized cornea after 45 minutes of dark adaptation. 110 After repeat dark adaptation for 45 minutes, the rod b-wave amplitude versus retinal illuminance function was obtained as described. 2 '" In addition, a- wave implicit (peak) time was measured in ERG responses to the brightest blue flash (i.e., 2.1 log scot td -sec) obtained as part of the intensity amplitude profile. Results from the group of asymptomatic heterozygous carriers of the splice site mutation (n = 11) and from the group of asymptomatic carriers of the Glu249X mutation (n = 4) were compared to the results from a normal control group (n = 19). The data for the maximum rod ERG amplitude and the retinal illuminance for a half-maximal ERG (sigma; a) were converted to the log scale to approximate a normal distribution for statistical analyses. Visual field area, log maximum rod b-wave amplitude, and log sigma (a) were each regressed on diagnostic category (splice versus control or Glu249X versus control) adjusting for age. The log maximum rod b-wave amplitude was regressed on diagnostic category, age, and the cross-product of diagnostic category by age. The cross-product term tested whether the effect of age on b-wave amplitude depended on the diagnostic category. Rod a-wave implicit time was regressed on diagnostic category, adjusting for both age and pupil diameter. The mean value for the log elevation of dark-adapted threshold was compared to zero for each patient group by t-test. Statistical analyses were performed with JMP, version 3.1 (SAS Institute, Cary, NC). RESULTS Figure 1 identifies the heterozygous carriers of the splice site mutation in two pedigrees. In these two pedigrees, 48 family members were screened and 25 heterozygous carriers were identified (age range, 14 to 82 years). Of these 25 heterozygous carriers, only one reported symptoms of nightblindness, loss of visual acuity, and loss of peripheral vision; that person was diagnosed with RP during his adolescence. Of the 24 asymptomatic carriers, 11 were examined by us, and all had normal results on fundus examination. It is noteworthy that the five oldest asymptomatic carriers from these two pedigrees (ages 66, 70, 71, 72, and 82 years) showed no evidence of intraretinal pigment, retinal vessel attenuation, or optic disc pallor. Representative SSCP analyses from some members of both pedigrees are shown in Figure 2. Figure 2A shows the SSCP analyses of the asymptomatic propositus from pedigree #D586 and her asymptomatic 72-year-old father; both are heterozygous carriers of

4 Rhodopsin Splice Site Mutation 2189 TABLE l. Comparison of Asymptomatic Heterozygous Carriers of the Intron-4 Splice Donor Site Mutation and the Glu249X Mutation to Normal Controls Parameter Normal Controls (n = 19) Intron 4 Splice Site Mutation Carriers (n = //) (V-value Versus Controls) Glu249X Mutation Carriers (n = 4) (P-value Versus Controls) Age (years) 34.6 ± ± 6.1 (P = 0.031) 40.5 ± 10.2 (NS) Visual field (degrees 2 ) ± ± 389 (NS)* ± 164 (NS)* Final rod dark adaptation threshold elevation (log units)f 0.52 ± 0.09 (P< 0.001)t 0.67 ± 0.13 (P= 0.014)t Log maximum rod ERG amplitude 2.58 ± ± 0.06 (P= 0.004)* 2.57 ± 0.04 (NS)* Log a 0.14 ± ± 0.08 (P = 0.003)* 0.64 ± 0.07 (P= 0.005)* a-wave implicit times (msec) 26.0 ± ± 0.9 (P< 0.001) ± 0.9 (P= 0.03)H Values are mean ± SEM. * Adjusting for age. fthe mean final dark adaptation threshold for normal individuals is 1.70 log microapostilbs. 17 X Versus zero value and adjusting for age. a = retinal illuminance (scot.td.-sec.) required for 50% maximum amplitude. U Adjusting for age and pupil diameter. NS = not significant. the mutation as shown by the presence of both wildtype and mutant bands on the autoradiogram. The mutant band corresponds to the G-to-T transversion at position 4335 of the splice donor site within intron 4 of the rhodopsin gene; this mutation was confirmed by direct PCR-DNA sequencing of the amplified DNA region as previously shown. 2 Figure 2B shows the SSCP analyses of the propositus with RP from pedigree #6965, his asymptomatic mother, and his asymptomatic son; all show the same wild-type and mutant bands seen in pedigree #D586. Direct PCR sequencing of this amplified DNA identified the same G-to-T transversion at position 4335 identified in pedigree #D586 (data not shown). SSCP analysis and direct sequencing of the entire coding region and all intron-exon boundaries of the rhodopsin gene from this propositus with RP failed to identify any additional mutations (data not shown). Figure 3 illustrates full-field ERG responses from a normal control subject, the symptomatic propositus with RP in pedigree #6965, his asymptomatic mother, and his asymptomatic son. The two asymptomatic heterozygous carriers show slighdy diminished rod b-wave amplitudes in response to 0.5-Hz dim blue light and 0.2-Hz bright blue light, but normal amplitudes to 0.5- Hz and 30-Hz white light. In contrast, the heterozygous propositus, who is symptomatic with RP, has nondetectable rod responses to the 0.5-Hz dim blue light and markedly diminished responses to both the 0.5- Hz and 30-Hz white light. The cone isolated responses to 30-Hz white light are comparable in amplitude to the 0.5-Hz responses (normally from rods and cones), suggesting that this patient has no detectable rod function. Figure 4 depicts the rod ERG intensity-amplitude functions for a normal control group (n = 19) and for the group of asymptomatic heterozygous carriers of the rhodopsin splice site mutation (n = 11). At each light intensity, the mean rod b-wave amplitude of the carrier group is less than the control group. The carrier group had a smaller maximum rod b-wave amplitude than the control group after adjusting for age (Table 1; P = 0.004). Moreover, these carriers showed a decline in this maximum amplitude with increasing age that was significantly different from zero (P = 0.05) but not significantly different from the decline seen in the control group (P = 0.063). Figure 5 illustrates this variation of the maximum rod b-wave amplitude with age. The two groups also differ in the amount of light required to elicit a half-maximal response (Fig. 4, vertical dashed lines); the carrier group re-

5 2190 Investigative Ophthalmology 8c Visual Science, October 1995, Vol. 36, No. 11 III-23 Heterozygote Ago 45 FIGURE 3. Full-field electroretinograms of heterozygous carriers of the rhodopsin splice site mutation. Full-field electroretinograms are shown from an unaffected control (27 years of age), an asymptomatic heterozygote (11-12, 66 years of age) who is the mother of the affected propositus, an asymptomatic heterozygote (FV-20, 14 years of age) who is the son of the propositus, and the propositus with RP (III- 23, 45 years of age) from pedigree #6965. The electroretinograms show rod responses to 0.5-Hz dim blue light, rodisolated responses to 0.2-Hz bright blue light (2.1 log scot td -sec), mixed rod-cone responses to 0.5-Hz white light, and cone responses to 30-Hz white light. The rod isolated responses were obtained as part of the rod ERG intensity-amplitude functions. The stimulus onset is noted by vertical dotted lines (left and middle columns) and short vertical solid lines (right column). Two or three consecutive sweeps are superimposed. The oblique arrows pointing to the bright blue light responses identify the a-wave peaks; implicit times were 26 msec for the control and 30 msec for the two asymptomatic carriers. Under these test conditions, normal amplitudes are ^100 //V (0.5-Hz dim blue flash), s=350 fi\ (0.5-Hz white flash), and ^50 (xv (30-Hz white flicker). A calibration symbol is located beneath each column for the electroretinograms in that column. We report that 24 of 25 heterozygous carriers of the rhodopsin splice donor site mutation were asymptomatic. Of the 11 asymptomatic carriers who were examined by us, none showed ophthalmoscopic or visual field evidence of RP. This group of asymptomatic carriers had a rod abnormality identified by an increase in the dark adaptation threshold, a decrease in ERG b-wave amplitudes, an increase in the sigma value, and a prolongation of the a-wave implicit time. Even though the maximum rod b-wave amplitude decreased as a function of age in these asymptomatic carriers, the oldest tested carrier (72 years of age), who had the lowest ERG amplitude in this group, still had at least 10 times the rod ERG amplitude observed in the only symptomatic patient with RP (45 years of age). The findings of full visual fields, normal ophthalmologic examination results, and only mild rod functional abnormalities among the older asymptomatic carriers indicate that these persons do not have RP. Only 1 of the 25 carriers was symptomatic and showed signs of RP. One possible explanation is that the splice site mutation is inherited as a dominant allele with reduced penetrance. Other pedigrees with reduced penetrance of RP and unknown mutations have been reported. I2 ~ IS In these other studies, the lowest penetrance observed was approximately 50% by pedigree analysis. This is much higher than the 4% penetrance seen among our heterozygous carriers. The extremely low incidence of RP among our heterozygous carriers strongly suggests that the splice site mutation is not a dominant allele. A second possible explanation for the low frequency of disease among our carriers is that the splice site mutation is recessive and that the one patient with RP carries an undetected mutation in his other rhodopsin allele. A third possi- quires approximately one-half log unit more light than the control group (Table 1, log a). Table 1 summarizes the ocular findings for the controls, the asymptomatic splice mutation carriers, and the asymptomatic Glu249X mutation carriers. The mean age of the splice carrier group was significantly older than it was for the control group (P = 0.03). Visual field areas were full and nearly identical for all three groups. Both heterozygous carrier groups showed slight but significant elevations in final dark adaptation thresholds and a-wave implicit times. DISCUSSION LOG RETINAL ILLUMINANCE (SCOT.TD.-SEC.) FIGURE 4. Rod electroretinogram intensity amplitude profiles for wild-type controls and asymptomatic heterozygous carriers of the intron 4 splice donor site mutation. Mean log b-wave amplitude (± SEM) versus log retinal illuminance is plotted for 19 control subjects with wild-type rhodopsin alleles (open circles) and the 11 asymptomatic heterozygous carriers of the splice site mutation (closed squares). Vertical dashed lines designate the log retinal illuminances required for half-maximal responses (log a). The retinal illuminance required for half-maximal amplitude differed significantly between the control group and the carrier group (P = 0.003). scot td -sec. is scotopic troland-seconds.

6 Rhodopsin Splice Site Mutation nV -200nV which suggests that the splice donor site mutation, like the Glu249X mutation, is a recessive allele for RP with no clinically significant consequences for the heterozygous carrier other than for genetic counseling. It will be of interest to see if heterozygous carriers of other recessive alleles show the same rod functional abnormalities and if functional testing can aid in the detection of these carriers. C Normal Splice x x -100 iv 1.9 I I I I I I I Age (years) FIGURE 5. Effect of age on the maximum rod ERG b-wave amplitude for asymptomatic heterozygous carriers of the intron 4 splice donor site mutation and controls. The maximum rod ERG b-wave amplitude is plotted according to age for each member of the control group (n = 19; closed squares) and asymptomatic heterozygous carrier group (n = 11; crosses). The carrier group showed a decline in maximum amplitudes with increasing age (solid line) that was significantly different from zero (P = 0.05) but not significantly different (P = 0.063) from the decline seen in the control group (dashed line). bility is that our patient has a digenic form of RP and heterozygously carries a noncomplementing recessive mutation in an unlinked gene. To date, digenic RP of this type has only been shown to occur in patients who are double heterozygotes for peripherin RDS and R0M1 mutations," 1 but it is possible that other pairs of mutant genes also interact to cause retinal degeneration. A fourth possibility is that this patient has RP because of one or more defects in another gene and that the rhodopsin mutation does not cause his disease. One of these four possibilities may also apply to the one symptomatic heterozygous carrier with RP described by another group. 4 The rod abnormalities identified in our asymptomatic heterozygous carriers (both young and old) are similar to the mild dysfunction identified in young and old carriers of the Glu249X mutation. Both mutations should result in an abnormally truncated protein that would be functionally inactive. The heterozygous carriers of these mutations would have only one functional rhodopsin allele and probably a decreased amount of rhodopsin in each photoreceptor. This is supported by the findings of slightly elevated rod psychophysical thresholds, by slightly reduced rod ERG responses, 2 ' 4 and by reduced rod visual pigment as measured by fundus reflectometry. 4 The asymptoi.iatic carrier groups with the splice site mutation and the Glu249X mutation appeared clinically similar, Key Words electroretinography, retinitis pigmentosa, rhodopsin, rod photoreceptor, splice donor site mutation Acknowledgments The authors thank Sumiko Miller, Sean Fitz-Gerald, and Kevin McDermott for their technical assistance. They also thank Jennifer Quinn, Carol Weigel-DiFranco, and Peggy Rodriguez for their help in coordinating this study. References 1. Dryja TP, McGee TL, Reichel E, et al. A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature. 1990;343: Rosenfeld PJ, Cowley GS, McGee TL, Sandberg MA, Berson EL, Dryja TP. A Null mutation in the rhodopsin gene causes rod photoreceptor dysfunction and autosomal recessive retinitis pigmentosa. Nature Genet. 1992; 1: Macke JP, Davenport CM, Jacobson SG, et al. Identification of novel rhodopsin mutations responsible for retinitis pigmentosa: Implications for the structure and function of rhodopsin. Am J Hum Genet. 1993; 53: Jacobson SG, Kemp CM, Cideciyan AV, Macke JP, Sung C-H, Nathans J. Phenotypes of stop codon and splice site rhodopsin mutations causing retinitis pigmentosa. Invest Ophthalmol Vis Sci. 1994; 35: Kunkel LM, Smith KD, Boyer SH, et al. Analysis of human Y-chromosome-specific reiterated DNA in chromosome variants. Proc Nail Acad Sci USA. 1977;74: Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics. 1989;5: Dryja TP, Hahn LB, Cowley GS, McGee TL, Berson EL. Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa. Proc Nail Acad Sci USA. 1991; 88: Yandell DW, Dryja TP. Direct genomic sequencing of alleles at the human retinoblastoma locus: Application to cancer diagnosis and genetic counseling. In: Furth M, Greaves M, eds. Cold Spring Harbor Symposium Series: Cancer Cells 7-Molecular Diagnostics of Human Cancer. Cold Spring Harbor: Cold Spring Harbor Press; 1989: Berson EL, Gouras P, Gunkel RD. Rod responses in

7 2192 Investigative Ophthalmology & Visual Science, October 1995, Vol. 36, No. 11 retinitis pigmentosa, dominantly inherited. Arch Ophthalmol. 1968; 80: Reichel E, Bruce AM, Sandberg MA, Berson EL. An electroretinographic and molecular genetic study of X-linked cone degeneration. Am J Ophthalmol. 1989; 108: Sandberg MA, Miller S, Berson EL. Rod electroretinograms in an elevated cyclic guanosine monophosphate-type human retinal degeneration. Invest Ophthalmol Vis Set. 1990;31: Berson E, Gouras P, Gunkel RD, Myrianthopoulos NC. Dominant retinitis pigmentosa with reduced penetrance. Arch Ophthalmol. 1969;81: Berson E, SimonofFE. Dominant retinitis pigmentosa with reduced penetrance: Further studies of the electroretinogram. Arch Ophthalmol. 1979;97: Jay M, Bird AC, Moore AN, Jay B. Nine generations of a family with autosomal dominant retinitis pigmentosa and evidence of variable expressivity from census records. JMed Genet. 1992; 29: Inglehearn CF, Carter SA, Keen TJ, et al. A new locus for autosomal dominant retinitis pigmentosa on chromosome 7p. Nature Genet. 1993; 4: Kajiwara K, Berson EL, Dryja TP. Digenic retinitis pigmentosa: Combined heterozygosity for mutations at the unlinked peripherin/rds and ROM1 loci in three families. Science. 1994: