Molecular diagnostics for retinitis pigmentosa

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Clinica Chimica Acta 313 Ž2001. 209–215 www.elsevier.comrlocaterclinchim

Molecular diagnostics for retinitis pigmentosa Kwun Yan Yeung, Larry Baum, Wai Man Chan, Dennis S.C. Lam, Alvin K.H. Kwok, Chi Pui Pang ) Department of Ophthalmology and Visual Sciences, The Chinese UniÕersity of Hong Kong, Hong Kong, China Received 20 February 2001; accepted 25 June 2001

Abstract Background: At least 1 million people worldwide have retinitis pigmentosa ŽRP., making it relatively common among the inherited forms of blindness. Mutations in many genes may cause RP. The most common known mutation, Pro347Leu in rhodopsin, is found in no more than about 1% of unrelated patients, implying the impracticality of a diagnostic test which would screen only for a few, common mutation sites. Conclusions: Ongoing discovery and study of RP genes makes it feasible to consider a molecular diagnostic test which would screen coding regions of all known RP genes by a mutation detection method such as conformation-sensitive gel electrophoresis followed by sequencing. The parallel development of RP genetic knowledge and treatments such as gene therapy will make such tests both possible and necessary. q 2001 Elsevier Science B.V. All rights reserved. Keywords: RP; Diagnosis; Gene therapy; Rhodopsin; Review

1. Prevalence Retinitis pigmentosa is among the most common inherited forms of blindness, affecting about 1 in every 4000 people in all ethnic groups worldwide. 2. Clinical features and prognosis This heterogeneous group of retinal degenerative diseases is characterized by similar clinical findings, but is linked to at least 36 different genetic loci w1,2x. The course is intractable, with no effective treatment at present. ) Corresponding author. Department of Ophthalmology, The Chinese University of Hong Kong, and Hong Kong Eye Hospital, 3rF, 147K Argyle Street, Kowloon, Hong Kong, China. Tel.: q852-27623169; fax: q852-27159490. E-mail address: [email protected] ŽC.P. Pang..

The typical history is the onset of progressive poor night vision Žoccasionally poor peripheral vision. in the first or second decade of life. Central visual loss is rarely the presenting symptom Žexcept with inverse RP or cone-rod dystrophy., and visual acuity is usually preserved until the later stages. These symptoms are easily understood because in classic RP, rod degeneration precedes the onset of cone degeneration. Ocular findings comprise atrophic changes of the retinal pigment epithelium ŽRPE. followed by appearance of melanin-containing structures in the retinal vascular layer and around Muller cells causing a ¨ ‘bone-spicule’ like pigmentation. Typical fundus appearance includes the triad of Ž1. attenuated arterioles, Ž2. bone-spicule pigmentation Žmostly paravenular and midperipheral., and Ž3. waxy pallor of the optic disc. Myopia is relatively common. Subcapsular posterior cataract and destruction of the

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vitreous often occur in patients older than 40 years. In some early cases, electroretinography ŽERG. can help with the diagnosis. There is a reduced amplitude of the scotopic and photopic b-wave and a delay in time between the flash of light and peak of the b-wave Ždelayed implicit time.. When monitoring the disease by ganzfeld ERG, RP patients lose, on average, 16–18.5% of the remaining amplitude per year w3x. In spite of these common phenotypic features, the term RP actually consists of a group of clinically heterogeneous disorders. Significant differences are found in the onset of symptoms, progression of visual loss, degree of morphological changes, and association with systemic disease. RP is also a component of over 30 syndromes, most of which show autosomal recessive inheritance w4x. Extraocular abnormalities can be found in Usher syndrome Ždeafness., Bardet–Biedl syndrome Žpolydactyly, obesity, mental retardation, and hypogenitalism. and Refsum disease Žpolyneuritis and ataxia. w5x. 3. Methods of detection and treatment RP commonly presents as night blindness, or difficulty of visual adaptation to dim lighting conditions, usually by early to mid-adulthood. Diagnosis is currently by examination of the retinal appearance, supplemented by electroretinography to test retinal response in dim light Žscotopic ERG.. Characteristics are reduced peripheral vision, narrowing of retinal arterioles, and appearance of dots or patches of dark pigment in the retina w5x. Often the optic nerve and patches of the retina become pale. The scotopic ERG amplitude is reduced. With few exceptions, such as Refsum disease, there is no treatment for RP. Some symptomatic relief may be derived from cataract extraction w6x and carbonic anhydrase inhibition, if there is macular edema w7x. Currently, the only treatment for RP with some effect is administration of vitamin A w8–10x. It helps to delay the progression of visual loss in some cases. In one report, oral vitamin A supplementation was shown to slow the rate of retinal degeneration in adult patients with the common forms of retinitis pigmentosa in a randomized, controlled, doublemasked trial w8x. This conclusion was based on measuring the rate of ERG amplitude decline.

To test and correlate the effects of such therapy with the genotype of the disease in order to discover what type of patient would be the best candidate for vitamin A therapy, two contrasting mouse models of RP have been treated w11x. Transgenic mice with a rhodopsin ŽRHO. Thr 17 Met mutation or a Pro 347 Ser mutation were fed a diet containing either a normal or an elevated amount of vitamin A. Based on the rate of ERG amplitude decline and histologic evaluation, vitamin A supplementation conferred therapeutic benefit in the case of the Thr 17 Met but not the Pro 347 Ser RHO mutation. A theory for this differential effect is that the Pro 347 Ser mutation destabilizes rhodopsin, and a high concentration of 11-cis retinal Žwhich is derived from vitamin A and which rhodopsin must bind to function properly. may bind to and increase the fraction of stable rhodopsin. Pro 347 Ser rhodopsin shows deficient trafficking to the rod outer segments but normal binding of 11-cis retinal, thus vitamin A should not help in these mutations. Some other mutant rhodopsins are also not destabilized; these are called class I mutations. Others, like Thr 17 Met , are produced at lower levels than the wild type, accumulate in the endoplasmic reticulum, and bind 11-cis retinal variably or not at all; these are class II mutants w12,13x. But because vitamin A treatment specifically addresses the class II RHO mutations, RP patients must first be genetically identified to determine whether they are candidates for this therapy. If this conclusion is confirmed in humans, this would be an example of pharmacogenomics in the treatment of RP.

4. Genetics: mode of inheritance and genes Autosomal dominant ŽADRP. represents 15–20% of all cases; autosomal recessive ŽARRP. comprises 20–25% of cases; X-linked recessive ŽXLRP. makes up 10–15%, and the remaining 40–55% of cases, in which family history is absent, are called simplex ŽSRP. w14x. However, these frequencies have varied in different studies w15–18x. For each mode of segregation, multiple genes have been identified ŽTable 1.. Genetic studies to date have identified at least 36 chromosomal loci associated with RP, with mutations identified in at least 19 genes that cause forms of non-syndromic RP w1,2x. Of these, five are pre-

K.Y. Yeung et al.r Clinica Chimica Acta 313 (2001) 209–215 Table 1 Genes and loci identified in retinitis pigmentosaa Inheritance pattern

Generlocus

Chromosome

ADRP

RP18 RHO RDSrPeripherin RP9 RP10 RP1 NRL RP13 RP17 CRX RP11 RPE65 ABCR RP19 RHO b-Subunit cGMP PDE a-Subunit cGMP PDE cGMP-gated channel TULP1 CralBP RPGR RP2 RP6 ROM1

1q 3q 6p 7p 7q 8q 14q 17p 17q 19q 19q 1p 1p 1p 3q 4p 5q 4p 6p 15q Xp Xp Xp 11q

ARRP

XLRP

Digenic RP

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Hong Kong Chinese w22,23x. In RHO, we identified one nonsense mutation, 5211delC , predicted to replace the negatively charged final 22 amino acids containing six phosphorylation sites by a 32 amino acid positively charged nonsense sequence with only two phosphorylatable residues. These changes may destroy the C-terminal localization signal and lead to mis-sorting of rhodopsin. Two missense changes were found: Ala 299 Ser and Pro 347 Leu . Each sequence alteration was found in one RP patient. Two controls also had Ala 299 Ser, suggesting this is a rare case of a RHO missense alteration that does not lead to RP. The expected frequency of rhodopsin mutations among all RP patients in this population is less than 7% Ž2r101s 2.0%, 95% confidence interval: 0.2– 7.0%.. In RP1, we identified one subject with a mutation, R 677 X . This is the most common mutation found in Americans. Another truncation was found, R 1933 X , but in a control rather than an RP patient. This suggests that a normal level of the C-terminal is not essential for normal eye function. So far this truncation is known to exist only in Chinese.

a

An update of all genes involved in retinal diseases can be found in Retnet: http:rrwww.sph.uth.tmc.edurRetNetr.

dominantly associated with ADRP, eight with ARRP, two with XLRP, and four with more than one form w2x. Among so many different RP genes, mutations in no single gene cause more than a 10th of all RP cases. The gene whose mutations may cause the most cases is rhodopsin, contributing to about 10% of RP, mostly ADRP w19,20x. New evidence suggests the RPGR gene may contribute to a similar proportion of RP cases, causing most XLRP w21x.

5. RHO and RP1 mutations in Chinese To shed more light on the range of mutations in RP genes in different populations, we have screened by CSGE all coding exons and splice sites of RHO and RP1 for sequence changes in 101 unrelated RP patients Žof any pattern of inheritance. and 190 normal controls over 60 years of age, who were all

6. Common mutations as diagnostic markers Genetic testing of family members of RP patients may identify those relatives who will develop RP. It may also help in the choice of appropriate treatment, although therapeutic options are currently very limited. As more and better options are developed, identification of the responsible mutation will become increasingly useful for a patient’s treatment. The most commonly known RP-causing mutation is RHO Pro 347 Leu , accounting for 3.6–5% of ADRP patients w10,19x, or about 0.5–1.0% of all unrelated RP patients. It has been found in Europe w24x, Asia w25x, and Africa w26x, and has arisen independently more than once. However, the low proportion of this mutation among RP families, even among ADRP families specifically, makes the screening of relatives for this one known mutation almost useless. However, could a screen for several common mutations detect the cause of a majority of RP cases? Other common RP mutations include RP1 Arg 677 ter, responsible for about 4% of ADRP w27,28x, and RHO Pro 23 His , which is only found in North

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America but causes about 9% of ADRP there w19x. Several other mutations in rhodopsin may each cause about 1% of ADRP in the U.S. w19x, but further studies would be needed to confirm the prevalence of these mutations in other populations. The top 10 most common known RP mutations cause only about a quarter of ADRP in the U.S., and less elsewhere in the world due to the absence of Pro 23 His w19x. Thus, a practical molecular diagnostic protocol for RP must screen entire genes, not just known mutation sites.

7. Merits and pitfalls of molecular tests for early detection of RP Many methods are available to screen genes for mutations. These include single strand conformation polymorphism analysis w29x, conformation sensitive gel electrophoresis w30x, enzymatic or chemical cleavage of mismatched base pairs w31,32x, differential unfolding of homoduplexes and heteroduplexes by denaturing gradient gel electrophoresis ŽDGGE. w33,34x, denaturing high performance liquid chromatography ŽDHPLC. w35x, and direct DNA sequencing. However, SSCP is not reliable with fragments of greater than about 200 bp, and the sensitivity is estimated to range from about 60% to 95% w29,31, 36–38x. DGGE is highly sensitive, but requires the use of GC clamps in one of the primers for each PCR product and a considerable effort to optimize the conditions for analysis of a given gene w33,34, 39,40x. CSGE detects heterozygous mutations with over 90% sensitivity in PCR products up to 800 bp. We have recently optimized the protocol to allow non-radioactive, high throughput CSGE ŽHTCSGE. of about 700 samples per gel with a running time of 9 h, or 1.3 samples per minute w41x. Homozygous mutations can be detected by mixing PCR products with a wild-type product. Using such high throughput methods, it is possible to screen the coding sequences of all known RP genes. Assuming an average of 20 PCR primer pairs to amplify the coding regions and splice sites of each of 20 genes, about 800 PCR product gel loadings would be needed to screen for both heterozygous and homozygous mutations. Thus, seven subjects could be initially screened on eight CSGE gels. One technician may be able to screen seven RP index patients

for mutations in known RP genes within about two weeks from the time of blood drawing to obtaining the sequence containing the probable causative mutation. To determine the sensitivity and specificity of this screen, we can estimate conservatively that PCR will fail to produce the desired products about 10% of the time, and that CSGE and sequencing will each fail to detect mutations 10% of the time. False positives should not be a problem because mutations will be confirmed by a repeated PCR and direct DNA sequencing. Known RP genes will only contain a fraction of all RP mutations, and if that fraction is, say, half, then this screen would have a sensitivity of 90% = 90% = 90% = 50% s 36%. The specificity would depend on the ability to differentiate harmless sequence changes from disease-causing mutations. For genes such as rhodopsin, in which the spectrum of sequence changes in both RP patients and controls has been extensively studied, the difference will be clear because every amino acid sequence change reported causes RP, with only three exceptions, Val 104 Ile w42x, Phe 220 Leu w19x, and Ala 299 Ser w23x. Therefore, the specificity would be nearly 100%. However, in newly discovered genes the possibility of false positives is greater. For example, all diseasecausing mutations so far published for the RP1 gene have been truncations, and nearly all subjects reported to have truncations also had RP. But we have found a truncation in RP1 ŽArg 1933ter . in an elderly normal control subject w22x, suggesting that not all truncations in RP1 are disease-causing mutations. If this nonsense polymorphism had been first found in the course of a mutation screen of RP patients, it might be falsely called a disease-causing mutation. Therefore, thorough study of the RP gene mutation spectra in patients and controls in multiple ethnic groups will be needed to increase the specificity of mutation screening to an acceptable level.

8. The need for new treatments Clinical genetic testing for RP mutations will really become useful only when effective treatments tailored to different genetic defects have been developed. Treatments being explored include diltiazem

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w43x, transplantation of normal photoreceptors and RPE cells w44–48x, secretion by retinal cells of photoreceptor trophic factors from genes introduced via viral vectors w49–53x, and delivery to mutant photoreceptors and RPE cells of replacement genes or ribozymes in viral vectors w53–61x. Transplantation of sheets of rods into the subretinal space of RP model mice, whose rods had all died but some of whose cones were still alive, kept a fraction of cones alive which otherwise would have died w48x. This result suggests that rods may produce a cone trophic factor w62x. Another hypothesis is that the death of rods decreases retinal consumption of oxygen but not its supply, resulting in an increased concentration in the retina to toxic levels, which damage cones w63x. Thus, either transplantation of rods, or the discovery and use of a cone trophic factor, may, while letting rods die, keep cones alive. Since cones are responsible for central vision in humans, such a partial treatment would be satisfactory. Gene therapy for RP can take several approaches Žsee Ref. w53x for an excellent review of gene therapy for eye diseases.: production of trophic factors to delay or prevent secondary photoreceptor death or dysfunction, delivery of deficient genes to correct ARRP or XLRP, or blocking expression of mutant proteins to prevent ADRP. The trophic factors CNTF and FGF have been tried with some success to delay photoreceptor death in animal models of RP. The CNTF gene, administered by adenovirus or adenoassociated virus ŽAAV. into the subretinal space, led to the production of CNTF by retinal cells and partial neuroprotection in rodent models of RP w49,50,53x. FGF-2 delivered by AAV slowed the rate of photoreceptor degeneration w51x, though ERG amplitudes were not significantly affected w52x. More direct gene therapy approaches target the mutated gene itself. Replacement of the deleted peripherinrrds gene in rds mice via an AAV vector results in some preservation of retinal structure and electrophysiological response w61x. Replacement of the gene for the b-subunit of rod cGMP phosphodiesterase ŽPDEb ., which is mutated in rd mice, reduced the photoreceptor death in these mice w59x. These positive results suggest the potential to ameliorate the symptoms of any recessive RP by viral vector-mediated replacement of the defective gene.

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To block the toxic effects of dominant mutant proteins, ribozymes have shown promise. These RNA enzymes specifically cut mutant RNA while leaving intact the RNA transcribed from the normal allele w53,60,64x. Ribozymes specifically directed against the Pro 23 His RHO RNA, when delivered to photoreceptor cells by an AAV vector, slow the rate of photoreceptor degeneration in rats with this mutation w60x. Alternatively, ribozymes may cut both alleles, and the normal gene may be introduced simultaneously to replace the native normal RNA w65x. Diltiazem is a calcium- and cGMP-gated channel blocker widely used in cardiology. High concentrations of cGMP accumulate in rods of rd mice. Intraperitoneal injection of diltiazem reduced the death of rods in these mice w43x. This drug may therefore be considered for testing in humans with PDEb mutations.

Acknowledgements We thank The Industrial Support Fund, Hong Kong, for financial support.

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