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

I NTRODUCTION

Human ATP-binding cassette (ABC)

trans-porter 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

dis-eases of the eye

Diseases of the retina include a wide spec-trum of photoreceptor-affecting phenotypes,

which have been mapped to over 120 loci on

the human genome (RetNet™ Retinal

Informa-tion 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

deter-mining the genetic basis of monogenic eye

dis-orders 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 (for-merly known as ABCR) was cloned and

charac-terized 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

determinants of rare monogenic retinal dystro-phies Now, more than three years later,

muta-tions 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

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 impair-ment, 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

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28

CHAPTER

ABCA4 gene in arSTGD has not been disputed,

even despite a relatively low (usually ⬃60%)

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

pheno-type, such as autosomal recessive cone–rod

dys-trophy (arCRD) (Cremers et al., 1998; Rozet

et al., 1998) and autosomal recessive retinitis

pig-mentosa (arRP) (Cremers et al., 1998;

Martinez-Mir et al., 1998; Rozet et al., 1999) arCRD and

arRP have been characterized as groups of

genet-ically heterogeneous diseases where several loci

have been implicated by linkage (RetNet™)

Clinical heterogeneity of these disorders further

complicates the assessment of genetic

determi-nants for each disease entity Cone–rod

dystro-phy is characterized by more prominent cone

degeneration, in comparison with rod

degenera-tion, which is distinguished by more distinctive

reduction of the photopic cone b-wave

ampli-tude than the scotopic (rod b-wave) ampliampli-tude in

the electroretinogram (ERG) Conversely,

retini-tis pigmentosa affects predominantly rod

photo-receptors; the scotopic ERG is more severely

reduced than the photopic ERG, and patients

present with night blindness and loss of

periph-eral 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 best-known 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 ⬃10% 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 investiga-tors have proposed a model that suggests a direct correlation between the continuum of disease phenotypes and residual ABCA4

activ-ity/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

func-tion, 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 dis-ease manifestation being inversely proportional

to the residual ABCA4 activity (Figure 28.3)

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.

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

predic-tion that ABCA4 alleles are responsible for

⬃8% 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

ABCA4

ABCC6

STGD

PXE

*Missense *Nonsense *Deletion–insertion–splicing

mutation mutation mutation

1503 2273

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.

D2177N

Mutation: mild – moderate –severe

G1961E IVS36

⫹1G>A

IVS36

⫹1G>A

delG863/

G863A

1847 delA R681X

L541P A1038V G1961E

L541P A1038V

1847 delA

ABCA4

activity

Genotype Allele 1

Allele 2

Figure 28.3 Genotype/phenotype model for ABCA4 Modified from van Driel et al (1998), Maugeri et al.

(1999), and Shroyer et al (1999).

ABCA4 IN

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 ⬃5%

(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 labo-ratories suggested an association of

heterozy-gous ABCA4 alleles with the AMD phenotype

(Allikmets et al., 1997b) This ‘classical’

case-control 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

alter-ations resulted in rare missense mutalter-ations,

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

repli-cate 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,

difficul-ties 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

(⬃3.4%) and in 12 controls (⬃0.95%), a

sta-tistically significant difference (p⬍ 0.0001) (Allikmets, 2000) The risk of AMD was esti-mated to be increased about threefold in carriers

of D2177N and about fivefold in carriers of G1961E In the context of common complex dis-orders, this represents an important contribu-tion 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 develop-ing age-related maculopathy as carriers for

vari-ant ABCA4 alleles is substvari-antial.

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

ana-lyzed separately, none of the smaller studies,

with the exception of Allikmets et al (1997b),

yields statistically significant results A sub-stantial increase in the sample size, as in the Consortium study, or in the proposed meta-analysis, 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 signif-icant It is noteworthy that the relative risk esti-mates calculated from the meta-analysis are slightly increased compared to the Consortium study (Allikmets, 2000) and are estimated at over 3 for the D2177N mutation and at approx-imately 5 for the G1961E variant

These analyses clearly demonstrate the criti-cal 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

suscep-tibility 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

ABCA4 alleles go undetected (Allikmets, 1999).

In addition, as emphasized above, founder

alleles in some ethnic groups can seriously affect

the analysis, suggesting large,

multicenter-based studies of matched cases and controls as

the only alternative method to achieve

statis-tical significance Consorted study design also

helps to minimize the confounding effect of

population stratification, the most serious

rea-son for spurious associations (Allikmets, 2000)

The ABCA4 protein was first described in the

1970s as an abundant component of

photorecep-tor 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

charac-terized 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

chro-mophore, was identified as a potential substrate

of ABCA4 by its ability to stimulate ATP

hydrol-ysis 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 adap-tation, increased all-trans-retinal following light

exposure, elevated phosphatidylethanolamine (PE) in rod outer segments, accumulation of the

protonated Schiff base complex of

N-retinyli-dene-PE, and striking deposition of a major lipo-fuscin 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 photore-ceptor degeneration due to loss of the RPE

sup-port 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

stud-ies of Abca4( ⫹/⫺) heterozygous mice (Mata et al., 2001) A phenotype similar to that seen in Abca4

knockouts (A2E accumulation in the RPE, etc.)

TABLE28.1 META-ANALYSIS OF PUBLISHED DATA ON TWOABCA4ALLELES

(Allikmets, 2000)

(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.

prRDH

All-trans-retinal

11-cis-retinal

Opsin PE

Rod outer segment

Retinal pigment epithelial cell

Disk

recycling Lysosome

A

Mutant ABCR prRDH

All-trans-retinal

11-cis-retinal

Opsin Rod outer

segment

‘Poisoned’

RPE cell

Disk phagocytosis

Retinoid recycling Lysosome

A2-E PE

B

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)

was described in heterozygous mice, but its

manifestation occurred at a slower, age-related,

rate The distinct, AMD-resembling phenotype

in the Abca4(⫹/⫺) 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

signifi-cance of a particular sequence variant often

remains unanswered Recent data from

photo-affinity labeling and ATPase activity

experi-ments 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

ana-lyzed 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

trans-fection 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

com-pared 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

disease-causing mutation (Fishman et al., 1999; Guymer

et al., 2001).

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 approxi-mately 3% of the general population (Maugeri

et al., 1999) Although Maugeri et al (1999)

clas-sified this variant as a ‘mild’ mutation, its role in retinal pathology has been disputed because of its high (⬎1%) frequency in the general popula-tion 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

chorio-retinal 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.

350 300 250

150

50 0

All-trans -retinal (µM)

R1898H G1961E D2177N WT

100 200

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.

ABCC6 AND

Pseudoxanthoma elasticum (PXE; MIM 264800)

is a rare autosomal recessive (or dominant)

dis-order affecting the skin, eyes and cardiovascular

system, with considerable morbidity and

mor-tality 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

mem-brane, 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

experi-ence 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

disor-der 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

there-fore ABCC6 seems to be the only gene

under-lying the PXE phenotype The ABCC6 gene

consists of a total of 31 exons dispersed within

⬃73 kb of DNAon chromosome 16p13.1; the

cor-responding mRNA, ⬃6 kb, encodes a

polypep-tide of 1503 amino acids (Belinsky and Kruh,

1999; Kool et al., 1999) (see also Chapter 21)

ABCC6 is predicted to consist of three

trans-membrane regions comprising five, six and six

transmembrane-spanning segments,

respec-tively (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

transla-tion, or mutations affecting the consensus splice

sites, which are predicted to result in

out-of-frame deletions in the mRNAs (Figure 28.2) A

particularly common allele carries a nonsense

mutation R1141X, which has been

independ-ently 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 cur-rently 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 sug-gested, therefore, that the function of ABCC6 could also relate to cellular detoxification (Belinsky and Kruh, 1999) More recently, how-ever, the substrate specificity of ABCC6 has been shown to be quite different from ABCC1 and other MRP-like proteins, and the only sub-strate demonsub-strated 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

mani-festations in PXE affecting the elastic fibers

As a hypothesis, one could propose that the absence of functional ABCC6 results in accu-mulation of certain metabolic compounds, resulting in progressive calcification of elastic fibers This information, together with clinical observations suggesting environmental, hor-monal and/or dietary modulation of the dis-ease, raises the intriguing possibility that PXE

is a primary metabolic disorder at the

environ-ment–genome interface (Uitto et al., 2001).

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, encom-passing 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

impli-cations of all mutations Finally, there is a mouse model that accurately reproduces many

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

impor-tant 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

The author sincerely appreciates the work of all

collaborators and colleagues involved in the

research of the ABCA4 gene, and excellent

tech-nical 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

acknowl-edged

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