ACTA 0 P H T H A L M 0 L O G I CA 72 (1994) 678-682

Age differences of visual field impairment and mutation spectrum

in Danish choroideremia patients

Thomas Rosenberg' and Marianne Schwartz2

National Eye Clinic for the Visually Impaired', Copenhagen, Section of Clinical Genetics2, Department of Clinical Genetics, Rigshospitalet,

University of Copenhagen, Copenhagen, Denmark

Abstract. Visual prognosis is a crucial theme in the councelling of individuals affected by a progressive reti- nal dystrophy. Unfortunately prognostic predictions are hampered by large interindividual differences in disease courses even within well defined nosological entities. Ten patients from 8 families affected by choroideremia were studied. The clinical signs in our patients were rather uniform. Deterioration of the peripheral visual fields typically began in the second decade of life, and pro- gressed during the following one or two decades. Ester- man transformation of peripheral visual field meas- urements was chosen as the best single indicator of visual impairment. Noticeable age differences in residual visual fields among patients were demonstrated. The age dif- ference between the mildest and the severest cases amounted to 25 years. One of the expectations of the ex- ploration of disease genes, is the potential predictive value of mutation identification with regard to pheno- typic variability. Different presumed causative mutations were identified. Nevertheless, all the mutations are pre- dicted to cause premature stops during translation, re- sulting in a non-functional or missing protein. Conse- quently, the observed age variation in the photopic visual field degradation must be due to still unrecognized fac- tors, either constitutional and/or environmental.

Key words: choroideremia - mutation - phenotype - retinal dystrophy - visual impairment - visual field.

Choroideremia is a hereditary dystrophy of the re- tina and the choroid, characterized by night blind- ness from early childhood on, and severe visual im-

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pairment in early adult life caused by a progressive constriction of the visual fields. Eventually, com- plete blindness occurs, 30-50 years after the debut (Kurstjens 1965; K h a 1986). A distinct ophthal- moscopical appearance constitutes the principal clinical difference between choroideremia and re- tinitis pigmentosa, which shows similar visual symptoms.

The disease pattern follows a rather uniform course, from more or less uncharacteristic fundus changes in the initial stage towards a pathogno- monic late appearance. Sorsby et al. (1952) de- scribed the range of manifestations, and deli- neated three stages based on the ophthalmoscopic appearance. An initial stage, a second stage, and a terminal stage was also delimited by Kurstjens (1965).

Choroideremia is an X chromosome-linked dis- ease, and heterozygous females show characteristic morphological changes, easily recognized by oph- thalmoscopy (Goedbloed 1942; Waardenburg 1942; Westerlund 1956).

The choroideremia gene (CHM) has been cloned (Cremers et al. 1990; Merry et al. 1992; van Bok- hoven et al. 1994a), and mutation analyses have demonstrated point mutations as well as deletions in affected members of several choroideremia families (van den Hurk et al. 1992; Schwartz et al. 1993; van Bokhoven et al. 199413).

We investigated the occurrence of choroidere-

mia in the Danish population. Twenty-one families were encountered with a total of 37 affected men. Meticulous genealogical studies failed to demon- strate interfamilial relations between our pedi- grees. Different disease-causing mutations were identified in 10, possibly in 11, out of 12 investi- gated families. The present study was intended to investigate genotype-phenotype relations in order to improve prognostic councelling.

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Patients and Methods

During a 30-year period, 23 affected males from 17 families underwent examination in our clinic. The syndromic phenotypes of 3 patients from 2 families with cytogenetically identified muta- tions have been described earlier (Rosenberg et al. 1986,1987). The diagnostic criteria of choroidere- mia included a history of night blindness which was confirmed by dark adaptation analysis if possible. The fundus appearance which formed a major differential diagnostic sign was documented by fundus photography. The diagnosis was sup- ported by standardized visual field measurements with a Goldmann perimeter, object IV/4e, and dark adapted full field electroretinography. Fi- nally, ophthalmoscopical carrier signs in mothers, sisters or daughters of the affected were con- sidered as verification of the diagnosis of the prob- and.

Photopic visual field measurements were chosen as the best measure of visual impairment. The re- sidual peripheral area of the visual fields were quantified by the method of Esterman (1968). A transparent Esterman grid was placed on the Goldmann chart and the number of points entirely within the residual field was counted for each eye separately. The scores from the right and left eye were averaged, representing the percentage of preserved peripheral field with the Goldmann IV/ 4e object.

Blood samples were obtained from 12 of the above mentioned families with non-syndromic choroideremia.

Molecular genetic techniques including DNA isolation, Southern blotting, polymerase chain re- action (PCR) amplification, and single strand con- formation analysis (SSCA) have been described previously (van den Hurk et al. 1992; Schwartz et al. 1993; van Bokhoven et al. 199413).

* JH * BO

Peri p hero I visual field %

10 c

a FK

i A KW 1 9

10 20 30 40 50 Age-Years

Fg. 1. Esterman scores of residual peripheral visual fields re- lated to the age at examination in 10 patients with cho- roideremia.

Results

Esterman scores were obtained from 10 patients belonging to 8 families. Fig. 1 shows the preserved peripheral visual fields in percentage (Esterman scores) as a function of the age at examination. From Fig. 1 it appears that some of our cases MS, LN, FK, and KW had a severe course with final dis- appearance of peripheral visual fields between age 20 and 30 years. Cases TN, UH, and CN seemingly had a lower decay m-te leading to an extinct pe- ripheral field between age 40 and 50 years. Finally, case PU who was examined only once at 37 years of age had lost only 25% of his peripheral visual field. Expressed as age difference at the observed or extrapolated total disappearance of the peripheral visual fields, the interindividual difference amounted to 25 years between the most severely affected and the relatively mildy affected cases.

Causative mutations were detected in 10, pos- sibly 11, of the 12 families with non-syndromic cho- roideremia. In one family (patient MS) a de novo mutation was observed, while in the other families the mutation was confirmed as being present in the carrier mothers.

A broad spectrum of mutations was found (Table 1). In five cases frameshifts occurred by an insertion of 1 nucleotide (patients BO and JS), a deletion of 2 nucelotides (FK and CN) or a deletion of 4 nucleotides 0. In addition, nonsense muta- tions were detected in patients LH and TN leading to premature stop codons in exons 7 and 12, re-

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Table I. Mutations in the CHM gene in Danish choroideremia pa- tients.

Predicted effect Patient I Exon 1 Mutation

PU BO, JH, KW FK LN

FC TN UH

JS

J W

CN MS

intron 4 5 5 7 8 intron 11 12 12 13

14 1-15

- 1 4 T - G insertion (A) deletion (AG) C - T insertion (C)

C - A deleted deletion (?TGT) deletion (IT) deleted

- 2 A - G

splice error frameshift frameshift

frameshift splice error

frameshift

Arg - stop

cys - stop

frameshift frameshift no gene product

In case JS, FC, and JW the clinical data were u n f t for the calculation of an Esterman score.

spectively. In 2 other patients (PU and FC) single base substitutions in intron sequences were found. In patient FC an aberrant splicing of the CHM pri- mary transcript is predicted, whereas it is un- known whether the mutation in patient PU will lead to errors in the splicing reaction (van Bok- hoven 1994b). In patient UH a deletion of exon 12 was found, and in patients MS the whole CHM gene was deleted.

Discussion

Throughout half a century, following the first de- scription of choroideremia (Mautner 1872), it was considered a developmental and stationary condi- tion (Wolff 1930), until a.0. Bedell (1937) estab- lished the present view of a progressive choroidal and retinal dystrophy.

A considerable chronological variation among patients has been described with respect to age of onset and the age of final vision loss (Kurstjens 1965; Karna 1986; Sorsby et al. 1952; McCulloch 1969; Pameyer et al. 1960). However, most meas- ures of severity are unsuited for quantification and therefore unfit for a grading of disease progress. This applies to the fundoscopic appearance. Nor is the debut of choroideremia suitable for a grading of severity due to the early and insidious onset of the symptoms. Visual acuity remains normal in most patients until the final stage is reached, as

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opposed to dark adaptation, which often is se- verely diminished at the patient's first visit for oph- thalmological examination, rendering both measures unsuitable. ERG demonstrates early ex- tinction of the rod responses, while the cone-medi- ated potentials are better preserved (Bounds & Johnston 1955).

The Goldmann method of visual field measure- ments is a simple and reliable procedure, with ex- tended use as a standard in the evaluation of visual impairment. However, the presence of irregularly shaped scotomas in early choroideremia is a meth- odological impediment adding to the fluctuations observed. The transformation of the map of the re- sidual field into a single value by the Esterman technique is a good approximation to the degree of visual impairment. Unfortunately, Esterman grids do not include the central lo", which are often preserved for many years after a zero value is ob- tained by the Esterman method. Eccentric placing of the preserved area may introduce inaccuracy, and due to the often bizarre form of the field rem- nants an unrealistically low score is obtained. It may be argued that scotopic visual fields theoreti- cally would be more appropriate in reflecting rod photoreceptor death and that Goldmann field measurements might reflect cone losses as a purely secondary phenomenon. Even if so, we consider Goldmann field measurements as the most rele- vant appraisal of visual impairment due to visual field constriction.

The choroideremia gene product, Rab Escort Protein-1 (REP-1) (Andres et al. 1993; Farnsworth et al. 1991) is a component of the protein Rab- GGTase, an enzyme complex catalyzing the attach- ment of geranylgeranyl groups to Rab proteins. The Rab proteins constitute a large family of small GTP-binding proteins which are believed to con- trol various membrane vesicle transport mechan- isms by regulating the fusion events that underlie endocytosis and exocytosis (Takay et al. 1992; Pfef- fer 1992). An autosomal homologue of the CHM gene, designated CHML for choroideremia-like (Cremers et al. 1992, 1994) has been isolated. The corresponding protein, REP-2, might be able to compensate partially for the deficient REP-1 activ- ity (Cremers et al. 1992).

All causative mutations in the CHM gene identi- fied so far have been severe mutations (van den Hurk et al. 1992; Schwartz et al. 1993; van Bok- hoven et al. 1994b; Pascal 1993), leading to prema-

ture stops during translation. The synthesized pro- teins, if any, accordingly wil be C-terminally trun- cated. It has been shown that recombinant REP-1 protein that lacks the 70 C-terminal amino acids is inactive as a subunit of Rab GGTase (Cremers, un- published data). In consequence, choroideremia patients have no functional REP-1 protein. In other words, based on mutation analysis it may be anticipated that there is no coherence between age differences of visual field damage and the different CHM genotypes in choroideremia. Patient PU which presents an extremely mild course may be an exception from this statement. The T to G change at position - 14 of the acceptor splice site of intron 4 decreases the adherence to the splice site consensus sequence from 84% to 79%. Thus, it is unknown whether this mutation is causative lead- ing to errors in the splicing reaction. A reverse transcript PCR analysis of the CHM mRNA in this patient is in progress to investigate whether this in- tron mutation is indeed causative for choroidere- mia.

It is interesting to note that missense mutations leading to a malfunctioning or a partially inactive protein have not been found in choroideremia pa- tients so far. Therefore, it may be supposed that CHM missense mutations have no clinical effect at all or, alternatively give rise to very subtle changes and hitherto unrecognized phenotypes.

Since the marked interindividual differences in disease progress are unexplained by genotype, other determinants must be sought. As residual Rab GGTase activity was found in lymphoblasts from choroideremia patients with deletions of the entire CHM gene (Seabra et al. 1993), one possible factor might be a functional substitution by the autosomally coded REP-2 protein that closely re- sembles REP-1 (Cremers et al. 1992).

It might finally be concluded that despite ad- ding a definite improvement to the diagnosis of choroideremia, mutation analysis is of no relev- ance to the prediction of disease progress, still leaving the clinician with a demanding counselling task.

Acknowledgments

This work was supported by the Danish Eye Research Foundation (@enfonden), Cykelhandler P. Th. Ras- mussen og hustru Alma Rasmussens Mindelegat, Gerda og Aage Haensch Fond, and Hotelejer Carl Larsen og Hustru Nicoline Larsens Fond.

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Received on August 3rd, 1994.

Corresponding author: Thomas Rosenberg, MD National Eye Clinic for the Visually Impaired 1 Rymarksvej, DK-2900 Hellerup, Denmark.

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