CHAPTER 28 – ABC TRANSPORTERS AND HUMAN EYE DISEASE

CHAPTER 28 – ABC TRANSPORTERS AND HUMAN EYE DISEASE CHAPTER 28 – ABC TRANSPORTERS AND HUMAN EYE DISEASE CHAPTER 28 – ABC TRANSPORTERS AND HUMAN EYE DISEASE CHAPTER 28 – ABC TRANSPORTERS AND HUMAN EYE DISEASE CHAPTER 28 – ABC TRANSPORTERS AND HUMAN EYE DISEASE CHAPTER 28 – ABC TRANSPORTERS AND HUMAN EYE DISEASE CHAPTER 28 – ABC TRANSPORTERS AND HUMAN EYE DISEASE

577 28 CHAPTER ABC TRANSPORTERS AND HUMAN EYE DISEASE RANDO ALLIKMETS INTRODUCTION Human ATP-binding cassette (ABC) transporter genes have emerged as increasingly important players in inherited diseases Out of approximately 50 known human genes (see Chapter 3), at least 15 have been associated with a disease phenotype (Dean et al., 2001) The widespread impact of ABC transporters on human health was anticipated due to the vital function of these proteins in all cell types This chapter will focus on two ABC genes, ABCA4 and ABCC6, which are both involved in diseases of the eye Diseases of the retina include a wide spectrum of photoreceptor-affecting phenotypes, which have been mapped to over 120 loci on the human genome (RetNet™ Retinal Information Network; http://www.sph.uth.tmc.edu/ Retnet/home.htm) Currently, less than half of the causal genes have been identified, although substantial progress has been made in determining the genetic basis of monogenic eye disorders Mutations in new genes responsible for some form of retinal degeneration are identified on a regular basis However, the vast majority of these genes are involved in rare phenotypes in a limited number of patients When the ABC transporter gene ABCA4 (formerly known as ABCR) was cloned and characterized in 1997 as the causal gene for autosomal recessive Stargardt disease (Allikmets et al., 1997a), it seemed as if just another missing link was added to the extensive table of genetic ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9 determinants of rare monogenic retinal dystrophies Now, more than three years later, mutations in the ABCA4 gene continue to emerge as one of the predominant determinants of a wide variety of retinal degeneration phenotypes The discovery of the association between mutations in the ABCC6 gene and an eye phenotype (Bergen et al., 2000; Le Saux et al., 2000; Ringpfeil et al., 2000; Struk et al., 2000) added a second gene to the list of ABC transporters that are involved in retinal disorders ABCA4 IN RETINAL DYSTROPHIES Several laboratories independently described ABCA4 in 1997 as the causal gene for Stargardt disease (STGD1 (MIM 248200)) (Allikmets et al., 1997a; Azarian and Travis, 1997; Illing et al., 1997) Autosomal recessive STGD (arSTGD) is a juvenile-onset macular dystrophy associated with rapid central visual impairment, progressive bilateral atrophy of the foveal retinal pigment epithelium, and characteristic frequent appearance of orange-yellow flecks around the macula and/or the midretinal periphery (Figure 28.1) There is no definitive evidence of genetic heterogeneity of arSTGD; all families segregating the disorder have been linked to the ABCA4 locus on human chromosome 1p13–p22 (Anderson et al., 1995; Kaplan et al., 1993) Consequently, the role of the Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved 578 ABC PROTEINS: FROM BACTERIA TO MAN Figure 28.1 Fundus photographs of patients with Stargardt disease (A) and age-related macular degeneration (AMD) (B) Note macular dystrophy and characteristic orange-yellow flecks around the macula and the midretinal periphery in the case of Stargardt macular dystrophy, and degeneration of the macula and drusen (yellowish deposits around the macula) in the case of AMD ABCA4 gene in arSTGD has not been disputed, even despite a relatively low (usually ϳ60%) mutation detection rate of ABCA4 in STGD patients (Lewis et al., 1999; Maugeri et al., 1999; Rivera et al., 2000; Simonelli et al., 2000) Subsequently, several cases were reported where ABCA4 mutations segregated with retinal dystrophies of a substantially different phenotype, such as autosomal recessive cone–rod dystrophy (arCRD) (Cremers et al., 1998; Rozet et al., 1998) and autosomal recessive retinitis pigmentosa (arRP) (Cremers et al., 1998; MartinezMir et al., 1998; Rozet et al., 1999) arCRD and arRP have been characterized as groups of genetically heterogeneous diseases where several loci have been implicated by linkage (RetNet™) Clinical heterogeneity of these disorders further complicates the assessment of genetic determinants for each disease entity Cone–rod dystrophy is characterized by more prominent cone degeneration, in comparison with rod degeneration, which is distinguished by more distinctive reduction of the photopic cone b-wave amplitude than the scotopic (rod b-wave) amplitude in the electroretinogram (ERG) Conversely, retinitis pigmentosa affects predominantly rod photoreceptors; the scotopic ERG is more severely reduced than the photopic ERG, and patients present with night blindness and loss of peripheral vision In all studies, disease-associated ABCA4 alleles have revealed an extraordinary heterogeneity (Allikmets et al., 1997a; Fishman et al., 1999; Lewis et al., 1999; Maugeri et al., 1999; Rozet et al., 1998; Simonelli et al., 2000) (Figure 28.2) The current tally of all ABCA4 alleles suggests over 400 disease-associated ABCA4 variants (R Allikmets, unpublished data), allowing comparison of this gene to one of the bestknown members of the ABC superfamily, the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan et al., 1989) (see Chapter 29) What makes ABCA4 an even more difficult diagnostic target than CFTR is that the most frequent disease-associated ABCA4 alleles (e.g G1961E, G863A/delG863, and A1038V) have been described in ϳ10% of STGD patients across all populations studied, whereas the delF508 allele of CFTR accounts for close to 70% of all cystic fibrosis alleles (Zielenski and Tsui, 1995) Based on these findings, several investigators have proposed a model that suggests a direct correlation between the continuum of disease phenotypes and residual ABCA4 activity/function (Allikmets, 1999; Lewis et al., 1999; Maugeri et al., 1999; Shroyer et al., 1999; van Driel et al., 1998) (Figure 28.3) According to the predicted effect on the ABCA4 transport function, Maugeri et al (1999) classified ABCA4 mutant alleles as ‘mild’, ‘moderate’ and ‘severe’ Different combinations of these were predicted to result in distinct phenotypes in a continuum of disease manifestations, the severity of disease manifestation being inversely proportional to the residual ABCA4 activity (Figure 28.3) ABC TRANSPORTERS AND HUMAN EYE DISEASE ABCA4 2273 STGD ABCC6 1503 PXE *Missense mutation *Nonsense mutation *Deletion–insertion–splicing mutation Figure 28.2 Mutations in ABCA4 and ABCC6 genes Schematic representation of mutation spectrum is shown for ABCA4 in Stargardt disease (STGD) and for ABCC6 in pseudoxanthoma elasticum (PXE) Note the high prevalence of evenly distributed missense alleles in ABCA4 and C-terminal distribution of mainly deleterious mutations in ABCC6 The positions of the predicted transmembrane segments and the two NBDs in each gene are also indicated Phenotype Normal Normal or AMD STGD CRD RP ABCA4 activity Genotype Allele D2177N G1961E IVS36 ϩ1G>A Allele delG863/ G863A R681X IVS36 ϩ1G>A 1847 delA L541P A1038V G1961E L541P A1038V 1847 delA Mutation: mild–moderate–severe Figure 28.3 Genotype/phenotype model for ABCA4 Modified from van Driel et al (1998), Maugeri et al (1999), and Shroyer et al (1999) In addition, several studies have identified frequent complex alleles in both STGD and CRD patients (Lewis et al., 1999; Maugeri et al., 1999; Rivera et al., 2000) The most prominent of these are L541P/A1038V and R943Q/ G863A/delG863 Recently, in an extension of their earlier study, the laboratory of Frans Cremers has determined the major role of mutant ABCA4 alleles in arCRD (Maugeri et al., 2000) This groundbreaking discovery of the major genetic component in a prominent fraction of retinal disease distinguishes autosomal recessive CRD as a disorder caused predominantly by genetic defects in one gene This finding argues against the former assumption that arCRDs represent a genetically heterogeneous entity similar to arRP (RetNet™) The same study suggests that we revisit our current knowledge on the molecular genetics of arRP The prediction that ABCA4 alleles are responsible for ϳ8% of arRP (Maugeri et al., 2000), making it the most prominent cause of the autosomal recessive form of retinitis pigmentosa, seems reasonable and is currently under further investigation 579 580 ABC PROTEINS: FROM BACTERIA TO MAN ABCA4 IN AGE-RELATED MACULAR DEGENERATION (AMD) The summarized data presented in the previous sections establish allelic variation in ABCA4 as the most prominent cause of retinal dystrophies with Mendelian inheritance patterns The latest estimates suggest the carrier frequency of ABCA4 alleles in the general population is ϳ5% (Maugeri et al., 1999; Yatsenko et al., 2001; R Allikmets, unpublished observation) This brings us to the hottest topic of ophthalmic genetics – the role of heterozygous ABCA4 alleles in a complex trait, age-related macular degeneration (AMD, also designated ARMD2 (MIM 153800)) AMD, as a typical late-onset complex disorder, is caused by a combination of genetic and environmental factors (Figure 28.1B) Its prevalence increases with age; among persons 75 years and older, mild or early forms occur in nearly 30% and advanced forms in about 7% of the population (Klein et al., 1992; Vingerling et al., 1995) Consequently, various forms of AMD affect over 10 million individuals in the United States alone In 1997, results of a joint study of four laboratories suggested an association of heterozygous ABCA4 alleles with the AMD phenotype (Allikmets et al., 1997b) This ‘classical’ casecontrol study of 167 AMD patients and 220 controls found ABCA4 alterations in 16% of patients that were interpreted as associated with the disease phenotype because they were found in less than 1% of controls Most alterations resulted in rare missense mutations, some of which had also been found in STGD1 patients (Allikmets et al., 1997b) Subsequently, several reports disputed the conclusions of this study, stating that they were unable to replicate these findings and, therefore, to confirm the association (De La Paz et al., 1999; Guymer et al., 2001; Stone et al., 1998) Problems with replication of an association study of a complex disease are not unexpected and discussion of the topic is beyond the scope of this review (see, for example, Long and Langley, 1999; O’Donovan and Owen, 1999) In short, difficulties arise mainly due to small sample size in studies of rare variants with modest effect on a complex trait Our hypothesis-generating finding that heterozygous ABCA4 mutations may increase susceptibility to AMD was recently tested by an expanded collaborative study including 15 centers in Europe and North America (ABCR Consortium; Allikmets, 2000) In this study, the two most common AMD-associated variants, G1961E and D2177N, were genotyped in 1218 unrelated AMD patients and 1258 reportedly unaffected, matched controls Together, these two non-conservative amino acid changes were found in one allele of ABCA4 in 40 patients (ϳ3.4%) and in 12 controls (ϳ0.95%), a statistically significant difference (p Ͻ 0.0001) (Allikmets, 2000) The risk of AMD was estimated to be increased about threefold in carriers of D2177N and about fivefold in carriers of G1961E In the context of common complex disorders, this represents an important contribution to the disease load Since AMD affects millions of people worldwide and the described mutations represent only two out of thirteen reported earlier (Allikmets et al., 1997b), the number of people at increased risk of developing age-related maculopathy as carriers for variant ABCA4 alleles is substantial Finally, the following comments are offered on the meta-analysis of published data on the two most frequent ABCA4 variants (Table 28.1) It is apparent that the main reason for the controversial interpretation of the data is the small sample size in individual studies If analyzed separately, none of the smaller studies, with the exception of Allikmets et al (1997b), yields statistically significant results A substantial increase in the sample size, as in the Consortium study, or in the proposed metaanalysis, results in a substantial increase of power of statistical analysis Resulting p values, as well as relative risk estimates, leave no doubt that the association is statistically significant It is noteworthy that the relative risk estimates calculated from the meta-analysis are slightly increased compared to the Consortium study (Allikmets, 2000) and are estimated at over for the D2177N mutation and at approximately for the G1961E variant These analyses clearly demonstrate the critical need for large cohorts of cases and matched controls for association studies of rare alleles Considering all available data, heterozygous ABCA4 alleles are estimated to increase susceptibility to AMD in about 8–10% of all cases However, this estimate has to be viewed with caution, since the analysis of ABCA4 variation in AMD is far from complete It should be remembered that even in Stargardt disease approximately 30–40% of disease-associated ABC TRANSPORTERS AND HUMAN EYE DISEASE TABLE 28.1 META-ANALYSIS OF PUBLISHED DATA ON TWO ABCA4 ALLELES Study D2177N Case p Control G1961E Case Control p Allikmets et al (1997b) ABCR Consortium (Allikmets, 2000) Guymer et al (2001) De La Paz et al (1999) 7/167 21/1189 1/220 8/1258 0.012 0.005 6/167 19/1218 0/220 4/1258 0.006 0.0008 7/544 2/164 4/689 0/56 0.1 0.55 5/544 N/A 3/689 N/A 0.16 N/A Total Odds ratio (95% CI) 37/2064 13/2223 0.0002 3.1 (1.6–5.9) 30/1900 7/2167 Ͻ0.0001 5.0 (2.2–11.3) N/A, not applicable; p values were calculated from the one-sided Fisher’s exact test, and odds ratios were calculated from the exact conditional hypergeometric distribution ABCA4 alleles go undetected (Allikmets, 1999) In addition, as emphasized above, founder alleles in some ethnic groups can seriously affect the analysis, suggesting large, multicenterbased studies of matched cases and controls as the only alternative method to achieve statistical significance Consorted study design also helps to minimize the confounding effect of population stratification, the most serious reason for spurious associations (Allikmets, 2000) FUNCTIONAL STUDIES OF ABCA4 The ABCA4 protein was first described in the 1970s as an abundant component of photoreceptor outer segment disk rims (Papermaster et al., 1976, 1978) Hence, it was called a Rim protein (RimP) for the following 20 years Only in 1997 was the gene encoding RimP cloned and characterized as a member of the ABC transporter superfamily, suggesting a transport function of some substrate in photoreceptor outer segments (Allikmets et al., 1997a; Illing et al., 1997) All-trans-retinal, the isoform of rhodopsin chromophore, was identified as a potential substrate of ABCA4 by its ability to stimulate ATP hydrolysis by the purified reconstituted ABCA4 protein in vitro, suggesting that retinal could also be the physiological substrate for ABCA4 (Sun et al., 1999) Studies of Abca4 knockout mice fully support this hypothesis, and it has been proposed that ABCA4 is a ‘flippase’ for the protonated complex of all-trans-retinal and phosphatidylethanolamine (N-retinylidene-PE) (Weng et al., 1999) Mice lacking the functional Abca4 gene demonstrated delayed dark adaptation, increased all-trans-retinal following light exposure, elevated phosphatidylethanolamine (PE) in rod outer segments, accumulation of the protonated Schiff base complex of N-retinylidene-PE, and striking deposition of a major lipofuscin fluorophore in retinal pigment epithelium (RPE) Based on these findings, it was suggested that the ABCA4-mediated retinal degeneration may result from ‘poisoning’ of the RPE caused by A2-E accumulation, with secondary photoreceptor degeneration due to loss of the RPE support role (Weng et al., 1999) A2-E, a pyridinium bis-retinoid, is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine, and has been characterized as one of the major components of retinal pigment epithelial lipofuscin Accumulation of lipofuscin in the macular region of RPE is characteristic of aging eyes and is the hallmark of both STGD1 and AMD Together, these findings define ABCA4 as the ‘rate-keeper’ of retinal transport in the visual cycle, as illustrated in the proposed model shown in Figure 28.4A ABCA4 is apparently not absolutely essential for this process, since individuals completely lacking the functional protein (e.g some arRP patients) maintain some eyesight for several years Over time, however, even mild dysfunction of ABCA4 affects the vision irreparably (Figure 28.4B) Most recently, intriguing data that fully support ABCA4 involvement in AMD were obtained from studies of Abca4(ϩ/Ϫ) heterozygous mice (Mata et al., 2001) A phenotype similar to that seen in Abca4 knockouts (A2E accumulation in the RPE, etc.) 581 582 ABC PROTEINS: FROM BACTERIA TO MAN ABCR prRDH PE Opsin All-transretinal 11-cis-retinal Rod outer segment Disk phagocytosis Retinoid recycling Retinal pigment epithelial cell Lysosome A Mutant ABCR prRDH PE Opsin Rod outer segment ‘Poisoned’ RPE cell B All-transretinal 11-cis-retinal Disk phagocytosis A2-E Retinoid recycling Lysosome Figure 28.4 Model for ABCA4 function in the visual cycle A, Normal visual cycle in the case of functional ABCA4 Photoactivation of rhodopsin (orange arrow) results in the hydrolysis and release of all-trans-retinal into the photoreceptor outer segment disk membrane ABCA4 either transports and/or presents the all-trans-retinal or its complex with phosphatidylethanolamine (RAL-PE) to retinol (continued) ABC TRANSPORTERS AND HUMAN EYE DISEASE WT R1898H G1961E D2177N ATPase activity (% of wt basal) was described in heterozygous mice, but its manifestation occurred at a slower, age-related, rate The distinct, AMD-resembling phenotype in the Abca4(ϩ/Ϫ) mouse model suggests that humans heterozygous for ABCA4 mutations may be predisposed to A2E accumulation and concomitant retinal or macular disease (Mata et al., 2001) Remarkable allelic heterogeneity of the ABCA4 gene has substantially complicated genetic analysis of its involvement in retinal disease, especially in the AMD complex trait In a situation where a modest effect of a mutation can only be estimated by association analysis, the crucial question of the functional significance of a particular sequence variant often remains unanswered Recent data from photoaffinity labeling and ATPase activity experiments from Jeremy Nathans’ laboratory has dramatically advanced our knowledge in this field by determining the effect of close to 40 ABCA4 mutations (Sun et al., 2000) Thus, they demonstrated that both ABCA4 variants analyzed in the Consortium study (Allikmets, 2000), G1961E and D2177N, affect the protein’s ATPase activity in vitro (Figure 28.5) The mutant G1961E protein, produced following the transfection of human embryonic kidney (293) cells with cloned cDNA, exhibits several-fold lower binding of 8-azido-ATP and dramatic inhibition of ABCA4 ATPase activity by retinal as compared to the wild-type protein The D2177N variant had no effect on 8-azido-ATP binding, but exhibited a reproducible elevation in ATPase activity relative to the wild-type protein (Sun et al., 2000) Consequently, the ABCA4 variants considered to be associated with the AMD phenotype are not anonymous single nucleotide polymorphisms (SNPs), but rather mutations affecting ABCA4 function These results will also challenge several suggestions that G1961E, the mutation most frequently found in STGD and AMD patients, is indeed a benign variant in linkage disequilibrium with another diseasecausing mutation (Fishman et al., 1999; Guymer et al., 2001) 350 300 250 200 150 100 50 0 20 40 60 All-trans -retinal (µM) Figure 28.5 Effect of retinal on ATP hydrolysis by AMD-associated ABCA4 mutations Modified from Sun et al (2000) Note drastic inhibition of ATPase activity by the G1961E variant and elevation of the activity by the D2177N mutation, as compared to the wild type Another issue that has been clarified is that of the functional significance of the G863A/ delG863 variant This variant is the most common single allele among STGD patients in northern Europe, and is also present in approximately 3% of the general population (Maugeri et al., 1999) Although Maugeri et al (1999) classified this variant as a ‘mild’ mutation, its role in retinal pathology has been disputed because of its high (Ͼ1%) frequency in the general population The studies of Sun and colleagues (2000) clearly demonstrate a profound biochemical defect caused by either version of this mutation Finally, both mutations found in the ‘German’ complex allele, L541P and A1038V, analyzed independently and in combination, render the ABCA4 protein defective (Sun et al., 2000) In summary, functional studies fully support the proposed genotype/phenotype model of ABCA4, and offer several tools to advance our knowledge about the role of ABCA4 in chorioretinal disease Figure 28.4 (continued) dehydrogenase (prRDH) on the cytosolic face of the disk After reduction to all-trans-retinol the retinoid continues the visual cycle The processed, RAL-PE free, disks are phagocytosed and digested by the retinal pigment epithelial cell B, Altered cycle in the case of mutant ABCA4 Note accumulation of N-retinylidene-PE in rod outer segment disks and deposition of A2E in the retinal pigment epithelium (RPE) The accumulation of retinoids in phagolysosomes of the RPE leads to permanent A2E deposits followed by the RPE cell death and degeneration of photoreceptors 583 584 ABC PROTEINS: FROM BACTERIA TO MAN ABCC6 AND PSEUDOXANTHOMA ELASTICUM Pseudoxanthoma elasticum (PXE; MIM 264800) is a rare autosomal recessive (or dominant) disorder affecting the skin, eyes and cardiovascular system, with considerable morbidity and mortality The disease affects the elastic fibers of affected organs, which become progressively calcified The eyes are involved, displaying the characteristic appearance of angioid streaks, which result from fractures in Bruch’s membrane, an elastin-rich sheath beneath the retina As a result of fragmentation of this membrane, the blood vessels in the back of the eye break, resulting in bleeding and neovascularization Consequently, the affected individuals experience progressive loss of visual acuity, which can be severe, although entire loss of vision is extremely rare Thus, PXE has been considered as a prototypic heritable connective tissue disorder affecting the elastic fiber system Recently, PXE was linked to mutations in the ABCC6 gene by four independent groups (Bergen et al., 2000; Le Saux et al., 2000; Ringpfeil et al., 2000; Struk et al., 2000) Genetic linkage analyses in various multiplex families have failed to suggest locus heterogeneity and therefore ABCC6 seems to be the only gene underlying the PXE phenotype The ABCC6 gene consists of a total of 31 exons dispersed within ϳ73 kb of DNA on chromosome 16p13.1; the corresponding mRNA, ϳ6 kb, encodes a polypeptide of 1503 amino acids (Belinsky and Kruh, 1999; Kool et al., 1999) (see also Chapter 21) ABCC6 is predicted to consist of three transmembrane regions comprising five, six and six transmembrane-spanning segments, respectively (Figure 28.2) The majority of identified mutations reside in the COOH-terminal half of the protein, affecting primarily the intracellular domains In contrast to the ABCA4 gene, the majority of defects are deleterious mutations resulting in premature termination of translation, or mutations affecting the consensus splice sites, which are predicted to result in out-offrame deletions in the mRNAs (Figure 28.2) A particularly common allele carries a nonsense mutation R1141X, which has been independently described in families of various ethnic backgrounds (Bergen et al., 2000; Le Saux et al., 2000; Ringpfeil et al., 2000; Struk et al., 2000) The endogenous function of ABCC6 is currently unknown Initially, ABCC6 (also referred to as MRP6) was classified as a member of the multiple drug resistance-associated protein subgroup because of its homology to MRP1 (ABCC1), which has been well characterized as a transmembrane efflux pump primarily transporting amphipathic anticancer drugs, as well as glutathione, glucuronide and sulfate conjugated compounds (Borst et al., 1999; Leslie et al., 2001) (see Chapter 19) It was suggested, therefore, that the function of ABCC6 could also relate to cellular detoxification (Belinsky and Kruh, 1999) More recently, however, the substrate specificity of ABCC6 has been shown to be quite different from ABCC1 and other MRP-like proteins, and the only substrate demonstrated so far is BQ123, a small anionic peptide (Madon et al., 2000) ABCC6 appears different from all other proteins of this subgroup also by its reported localization on both lateral and canalicular membranes of hepatocytes (Madon et al., 2000) although this finding requires confirmation (see Chapter 21) The expression of ABCC6 predominantly in the liver and kidney – organs not affected in PXE – raises the question of the relationship between the ABCC6 mutations and the manifestations in PXE affecting the elastic fibers As a hypothesis, one could propose that the absence of functional ABCC6 results in accumulation of certain metabolic compounds, resulting in progressive calcification of elastic fibers This information, together with clinical observations suggesting environmental, hormonal and/or dietary modulation of the disease, raises the intriguing possibility that PXE is a primary metabolic disorder at the environment–genome interface (Uitto et al., 2001) PERSPECTIVES The scientific progress in determining the role of the ABCA4 gene in retinal pathology has been remarkable We have significantly expanded our knowledge of the extensive range of phenotypes caused by various combinations of ABCA4 mutations ABCA4 research has led to the formation of multicenter studies, encompassing large cohorts of ethnically diverse samples Currently, ABCA4 is described as the transporter of N-retinylidene-PE, and there is an in vitro system(s) to study functional implications of all mutations Finally, there is a mouse model that accurately reproduces many ABC TRANSPORTERS AND HUMAN EYE DISEASE features of the human disorders Most recent advances in the ABCA4 research include the generation of ABCR350 microarrays (Allikmets et al., 2001), which, by containing all genetic variations of the ABCA4 gene, can be used for systematic screening of patients with any and all ABCA4-associated pathology Nevertheless, much more is yet to be accomplished in ABCC6 research The generation and characterization of Abcc6 knockout mice should provide important clues as to the endogenous cellular function of this MRP-related transporter With ABCA4, however, our efforts should now move to the next stage of research, directed towards finding therapeutic solutions for ABCA4-mediated chorioretinal disease by either improving the transport function of ABCA4 or by preventing accumulation of toxic products resulting from ABCA4 malfunction Immediate areas of research may include gene therapy and determining synergistic activators for ABCA4 It is highly likely that even a slight improvement of ABCA4 function could delay the onset of related pathology and improve the quality of life of those individuals affected ACKNOWLEDGMENTS The author sincerely appreciates the work of all collaborators and colleagues involved in the research of the ABCA4 gene, and excellent technical assistance by J Tammur Support by the Ruth and Milton Steinbach Fund, Research to Prevent Blindness Career Development Award, and NIH Grant EY-13435 is gratefully acknowledged REFERENCES Allikmets, R (1999) Molecular genetics of agerelated macular degeneration: current status Eur J Ophthalmol 9, 255–265 Allikmets, R (and the International ABCR Screening Consortium) (2000) Further evidence for an association of ABCR alleles with 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