Four genes have been identified that when mutated convey a macular dystrophy phenotype (Table 243-2). The gene causing Best disease VMD2 encodes bestrophin, a tetraspan transmembrane protein of unknown function. Both the Doyne ( EFEMP1 ) and Sorsby ( TIMP3 ) genes encode extracellular matrix proteins. The function of EFEMP1, the EGF-containing fibrillin-like extracellular matrix protein 1, is unknown, while tissue inhibitor of metalloproteinase-3 (TIMP3) is one of three known proteins that inhibit matrix metalloproteinases. The Stargardt macular dystrophy gene, ABCR , encodes an ATP-binding cassette transporter that appears to transport a key chemical component of the visual cycle across the rim of the photoreceptor disc membrane in the outer segments. Each of the four genes is expressed in the retina; VMD2 specifically expressed in the RPE, and ABCR is exclusively expressed in photoreceptors.
Table 243-2: Inherited Macular Dystrophy |Favorite Table|Download (.pdf) Table 243-2: Inherited Macular Dystrophy
|Disease ||Inheritance ||MIM ||Genome location ||Gene ||Protein |
|Best ||AD ||153700 ||11q13 || VMD2 ||bestrophin |
|Doyne ||AD ||126600 ||2p16 || EFEMP1 ||EGF-containing fibrillin-like extracellular matrix protein 1 |
|Sorsby ||AD ||136900 ||22q12.1-q13.2 || TIMP3 ||tissue inhibitor of metalloproteinase-3 |
|Stargardt ||AR ||248200 ||1q21-p22.1 || ABCR ||ABCR or rim protein |
The gene for Best vitelliform macular dystrophy (BMD) (MIM 153700) was mapped to 11q1396 (Table 243-2). This location was confirmed in multiple populations and the genetic interval refined.97–101 Interestingly, haplotype analysis identified a 37-year-old male who appeared to represent nonpenetrance.102 Physical mapping identified several genes in the genetic interval.103 Independently, two groups identified the VMD2 gene, which is mutated in Best disease.
In one study, meiotic recombination mapping in a single large Swedish pedigree narrowed the Best locus to ≈800 kb. Sequence scanning of shotgun libraries prepared from nine PAC clones detected a high degree of similarity with a retina-specific human cDNA. The gene contained 11 exons and appeared to span at least 16 kb of genomic sequence.104 The 1755-bp open reading frame predicted a 585-amino-acid protein of 68 kDa with homology to the RFP (Arg-Phe-Pro) protein family. The protein was named bestrophin to acknowledge that mutations within the gene result in Best macular dystrophy.104
Five missense amino acid substitutions were identified in bestrophin among six independent families, including both a W93C mutation that segregated with meiotic recombinants in the Swedish family and a Y227N alteration in a Dutch kindred. This latter mutation has also been described elsewhere.104,105 The VMD2 gene is expressed selectively in the RPE of the adult mouse and human eye; the only other identified expression was in the Sertoli cells of the mouse testis. Neither the function of bestrophin nor that of any related RFP family members can be inferred from the amino acid sequence. However, based upon the known BMD pathology, including the abnormal accumulation of lipofuscin in RPE cells associated with progressive macular degeneration, Petrukhin et al.104 suggested that bestrophin may mediate the transport or the metabolism of an essential component of lipofuscin granules. Considering a correlation between the expression pattern of human VMD2 and its rodent homologue and the tissue distribution of the long chain fatty acid docosahexanoic acid (DHA), bestrophin might be involved in either the transport or the metabolism of polyunsaturated fatty acids in the human retina.104 Interestingly, the VMD2 gene overlaps in a tail-to-tail manner with another gene. The VMD2 gene contains a 174-bp region of antisense sequence complementary to the 3′ UTR of the brain-specific form of ferritin heavy-chain mRNA.104 Oxidative decomposition of polyunsaturated fatty acids in the retina can be catalyzed by ferrous iron,106 and iron-induced damage to the outer segments results in the accumulation of lipofuscin granules in RPE cells.107
In an independent study, a physical map consisting of YAC108 and PAC109 contigs in combination with gene identification approaches103,110 isolated several genes in the candidate interval. Northern blot analysis showed that one of these nine novel genes was expressed exclusively in the RPE as a single 2.4-kb transcript.105 The corresponding gene, VMD2 , contained 11 exons in an open reading frame of 1755 bp predicting a 585-amino-acid protein of 67.7 kDa. Ten missense amino acid mutations and 1 amino acid deletion mutation were identified in 12 other families with Best disease, and the alterations cosegregated with the disease.105
As mentioned above, the 3′ UTR of VMD2 mRNA contains a region of antisense complementary to the 3′ end of the brain-specific form of ferritin mRNA. Ferritin is an iron-storage protein that is important in the prevention of the deleterious oxidation of the ferrous form of iron within cells. The antisense regulation of ferritin and/or bestrophin expression might contribute to the pathogenesis of other forms of macular degeneration.105
Mutation analysis of the VMD2 gene in 13 other families segregating Best macular dystrophy identified 7 distinct mutations in 9 families.111 These included a novel frameshift mutation (1574delCA), the first to be identified in the 3′ one-third of the gene, and two missense amino acid substitutions (W93C and R218C) reported previously.104,105 Three of the seven families in which a VMD2 mutation was confirmed showed a nonpenetrant individual; one of these three families segregated the R28C alteration and two families segregated the E300D mutation.111 However, in 4 of 13 families no disease-associated alterations were detected.
Similarly, an independent study detected 15 different missense mutations in 19 of 22 families with BMD.112 In both VMD2 mutation detection studies, the identified missense amino acid substitutions appeared to cluster in certain regions of the protein. Five mutations—G299E, E300D, D301E, F3055, and T3071—lie close to one another, suggesting that this region of the protein may also have an important function.111,112 This domain might be exposed to the outer surface of the protein because it is highly negatively charged and may be involved in binding a positively charged molecule such as a ligand substrate.111 All currently identified variants VMD2 are displayed on the VMD2 mutation database Web site (www.uni-wuerzburg.de/humangenetics/vmd2.html).
EFEMP1 (Genbank Nm 004105)
The gene for Doyne honeycomb retinal dystrophy (MIM 126600) was mapped to 2p16113,114 (Table 243-2). Refined genetic mapping and physical analysis narrowed the region to 3 Mb.115 A positional candidate approach identified a gene, EFEMP1 , in which a single amino acid substitution (R345W) segregated with Malattia Leventinese or DHRD.116
Thirty-three families including 131 affected individuals were genotyped with 19 short-tandem repeat (STR) markers from the critical interval.116 Haplotype sharing in 23 Swiss families who were believed for geographic reasons to share a common ancestor revealed a smaller interval. After physical mapping for verification of the genetic map and evaluation of candidate genes, six genes were screened for coding sequence alterations. Three genes were present in retinal cDNA libraries and mapping within the narrowest genetic interval. After genomic structure characterization, these genes were screened in a large cohort of families segregating either Doyne honeycomb retinal dystrophy or Malattia Leventinese. A mutation in the EFEMP1 gene, a C > T transition predicting a R345W missense amino acid substitution, was identified in 161 of the 162 patients tested, while no alteration was found in 477 control unaffected individuals or in 494 unrelated patients with AMD.116 The one patient from one discordant family was found, on reexamination, to have a phenotype more consistent with AMD. One patient was shown to be homozygous for the R345W mutation; that patient's phenotype was similar to that observed in the heterozygous state, suggesting a true dominant allele.116 Thus, a single amino acid substitution causes all known cases of DHRD. This may represent a founder effect or potentially a hypermutable base.
The EFEMP1 gene was originally isolated through differential screening of a cDNA library constructed from a patient with Werner syndrome.117 It was previously mapped to 2p16.118,119 The gene encoded the protein S1-5, which was found to be overexpressed in senescent fibroblasts and induced by growth arrest of young cells through depletion. The protein belongs to the family of extracellular matrix glycoprotein known as fibrillins and contains multiple EGF (epidermal growth factors) modules. A related family member EFEMP2 was recently mapped to chromosome 11q13,119 a gene-rich region that also contains the major locus for Bardet-Biedl syndrome, BBS1.120
TIMP3 (Genbank Nm 000362)
The gene for Sorsby fundus dystrophy (MIM 136900) was initially mapped to chromosome 22q13121 (Table 243-2). This same genomic region contained the gene for tissue inhibitor of metalloproteinases-3 (TIMP3) which was known to play a pivotal role in remodeling the extracellular matrix. As a positional candidate gene, TIMP3 was screened for mutations in two families segregating Sorsby fundus dystrophy.122 Two mutations, S181C and W168C, introduce an additional cysteine residue in the C-terminal region of the mature protein.122
The tissue inhibitors of metalloproteinases (TIMPs) are a group of zinc-binding endopeptidases involved in the degradation of the extracellular matrix. TIMP3 encodes a 188-amino-acid mature polypeptide with a 23-residue signal peptide.123,124 The nucleotide sequence and deduced translational product of the TIMP3 cDNA are highly similar to the TIMP1 and TIMP2 gene products, including 12 conserved cysteine residues at the same relative position. TIMP3 is encoded by 5 exons extending over approximately 55 kb of genomic DNA.125 TIMP3 localizes to the extracellular matrix in both its glycosylated and unglycosylated forms.126 The NH2-terminal domain is responsible for the metalloproteinase inhibitor activity while the C-terminal domain is important for mediating the specific functions of the molecule.
The finding of the TIMP3 mutation in Sorsby fundus dystrophy focused attention on the metabolism of the extracellular matrix and its control in other macular dystrophies.122 A comparison of amino acid sequences of TIMP1, 2, and 3 demonstrates 12 conserved cysteine residues at the same relative position that are implicated in the correct protein folding by forming 6 intrachain disulfide bonds.122 The finding of different mutations resulting in changes to cysteine in two original families with Sorsby fundus dystrophy suggests that intramolecular disulfide bond formation may prevent proper folding of the three-dimensional proteins.122 Interestingly, two other Sorsby associated TIMP3 mutations, one in a German-Czech family S156C127 and the other in a Finnish family G166C,128 resulted in amino acid substitutions to cysteine. Sixteen families with Sorsby have been reported to segregate a S156C mutation,129,130 of which 15 were from diverse parts of the British Isles.128
The gene for Stargardt macular dystrophy (MIM 248200) was mapped initially to 1p21-p13 in eight French kindreds.131 Four additional families with what was deemed a later onset macular dystrophy were shown to link to the same region, thus supporting by genetic analysis the long-held clinical suspicion that these phenotypes represented allelic variants of the same mutant gene.132
In a large study of 47 families, mostly North American outbred kindreds, Anderson et al. confirmed linkage of STGD to 1p markers with a lod score of 32.7 and demonstrated the genetic homogeneity of this disorder.133 Twenty-two disease chromosomes showed informative crossover events between the disease locus and flanking polymorphic markers, thus refining the STGD locus to a 4-cM interval. Haplotype analysis identified four cases of nonpenetrant offspring, each of whom carried two STGD alleles identical with an affected sibling.133
Physical analysis with an STS content mapping approach delineated a YAC contig containing STGD-linked markers that spanned approximately 31 cM and completely encompassed the 4-cM critical interval delineated by historic recombinants. This landmark mapping approach yielded a physical map that excluded the positional candidate genes such as RPE65 and the α-subunit of cone transducin.133 The ABCR gene was mapped to the YAC contig encompassing the genetic (4-cM) critical interval.134
The ABC (ATP-binding cassette) superfamily includes genes whose products are transmembrane proteins involved in energy-dependent transport of a wide spectrum of substrates across membranes.135,136 Many diseases caused by mutations in members of this superfamily result in defects in the transport of specific substrates (Table 243-3).137–147 ABCR is expressed in the retina and encodes an ATP-binding cassette (ABC) transporter gene, thus making ABCR an excellent positional candidate for STGD. Indeed, both compound heterozygote and homozygote ABCR mutations were identified in STGD patients.133 A high percentage of missense mutations was observed, implying that most STGD patients have at least one allele that retains partial function.134
Table 243-3: Inherited Human Disease Associated with Mutations in ABC Transporters |Favorite Table|Download (.pdf) Table 243-3: Inherited Human Disease Associated with Mutations in ABC Transporters
|Disease ||MIM ||Gene |
|Cystic fibrosis; CBAVD ||219700 ||Cystic fibrosis transmembrane conductance regulator (CFTR) |
|Adrenoleukodystrophy ||300100 || ALD |
|Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) ||601820 ||Sulfonylurea receptor (SUR) |
|Progressive familial intrahepatic cholestasis (PFIC2) ||601847 ||Bile salt export pump (BSEP) |
|PFIC3 ||602347 ||Multidrug-resistant (MDR3); P-glycoprotein 3 (PGY3 |
|Dubin-Johnson syndrome; hyperbilirubinemia II ||237500 ||Canalicular multispecific organic anion transporter (CMOAT; MRP2) |
|HLA class I deficiency ||170260 ||Transporter associated with antigen processing (TAP2) |
|STGD1, RP19, arCRD, AMD ||248200 || ABCR |
|Tangier, Familial HDL deficiency ||205400 || ABC1 |
|Pseudoxanthoma elasticum (PXE) ||264800 || ABCC6 |
The ABCR gene includes 50 coding exons that spans a genomic region of approximately 150 kb.148–150 The open reading frame of 6819 bp encodes a predicted 2273-amino-acid protein of approximately 220 kDa.134 The ABCR mRNA is specific to the retina and approximately 8 kb in length.134 The ABCR protein localizes to the disc membrane of the outer segments of the photoreceptors.151, 151a ABCR is identical to Rim protein.151–153 Rim protein was initially purified from frog and bovine rod outer segments and shown to be localized to the rims of the disc.154,155 It comprises approximately 1 to 3 percent of the rod outer segment membrane proteins and binds nucleotides.156 The estimated mole ratio of ABCR to rhodopsin is approximately 1:120.151
Retinaldehyde (retinal) stimulates ATP hydrolysis by purified and reconstituted ABCR, suggesting that ABCR may transport retinal in an energy-dependent (ATP) manner.157 All-trans-retinal stimulates the ATPase activity of ABCR, three to fourfold with a half-maximal effect at 10 to 15 μM. Both 11-cis and 13-cis retinal show similar activity; by contrast, among 37 structurally diverse nonretinoid compounds studied, including 9 previously characterized substrates or sensitizers of another ABC transporter P-glycoprotein, only 4 show significant ATPase stimulation when tested at 20 μM.157 The dose-response curves of these four compounds indicate multiple binding sites or interactions with ABCR. Thus, these data suggest that retinoids, and most likely retinal, are the natural substrate for transport by ABCR in rod outer segments.157
The knockout mouse orthologue of ABCR , abcr, has been constructed.158 Homozygous mice lacking abcr show delayed dark adaptation, increased all-trans-retinal following light exposure, elevated phosphatidylethanolamine (PE) in outer segments, accumulation of the protonated Schiff base complex of all-trans-retinal and PE (N-retinylidene-PE), and deposition of a major lipofuscin fluorophore (N-retinylidene-N-retinylethanolamine or A2-E) in RPE.158 These data suggest that ABCR functions as an outwardly directed flippase for N-retinylidene-PE.158 A model for ABCR function, photoreceptor degeneration, and visual loss in the ABCR-mediated diseases has been proposed as a three-step process: (a) protonated N-retinylidene-PE accumulates in the outer segments due to loss of the flippase activity; (b) isomers of A2-E build up in the RPE lysosomes resulting in progressively impaired digestion of phagocytosed photoreceptor outer segments and ultimate dissolution of cellular membranes; and (c) photoreceptors die due to loss of RPE support functions.158a
The observations of increasing ATPase activity of reconstituted ABCR in the presence of all-trans-retinal was explained by the flippase model in the following way: The increase in ATPase activity was seen only when ABCR was reconstituted in the presence of PE. Thus, it was suggested that all-trans-retinal reacted with PE to form N-retinylidene-PE, the substrate for the ABCR flippase.157,158
Clinical Spectrum of ABCR Mutations.
Both clinical and genetic studies suggested that Stargardt disease and late-onset FF are part of the same clinical spectrum. Indeed, ABCR mutations were identified in early onset STGD, as well as late onset FF.94,134,159–162 Late-onset disease appears to correlate with milder mutant ABCR alleles.94,160 It has been noted that most STGD-associated ABCR mutant alleles were missense mutations134 and that there is no instance of STGD that had two apparent null alleles, suggesting retention of partial function of at least one mutant allele.134 Indeed three groups have reported that null alleles at the ABCR locus are associated with a more severe clinical phenotype similar to retinitis pigmentosa.163–165 In fact, ABCR double-null mutations are responsible for the RP19 locus.166 A fourth retinal dystrophy phenotype, a severe combined cone-rod dystrophy, has also been associated with specific combinations of ABCR mutant alleles.164 In fact, ABCR mutations account for 80% of cone-rod dystrophy families in one study.164a Some pedigrees segregate both STGD and retinitis pigmentosa,94,165 or both RP19 and cone-rod dystrophy,164 depending upon the paired combinations of ABCR mutant alleles in the nuclear families.
A large genotype/phenotype analysis identified ABCR mutations and correlated the age-of-onset of 150 families with STGD. Interestingly, in a pseudodominant STGD family, sibs with identical compound heterozygous mutations were concordant for the half-decade of onset of visual symptoms.94 These observations suggested that the selected combination of specific ABCR mutant alleles determines the age at onset of this disease phenotype.94 Analysis of 53 families in which both ABCR mutant alleles were identified enabled a correlation between the position in the ABCR protein of the combination of mutant alleles and age-of-onset of the disease. These genotype/phenotype correlations suggested that missense amino acid substitutions located in the N-terminal one-third of ABCR seem to be associated with earlier onset of disease and may represent misfolding alleles.94
The study of 40 Western European patients with Stargardt disease identified one mutation, 2588G > C, in 15 of 40 (37.5 percent) patients.161 This mutation shows linkage disequilibrium with a rare polymorphism, 2828G > A, suggesting a founder effect. Interestingly, the 2588 G > C mutation not only causes a substitution of an alanine for glycine at amino acid residue 863 (G863A), but also affects the splicing at the 3′ splice site 3 bp downstream in exon 17.161 Thus, the resulting mutant ABCR proteins either lack Gly863 or contain the missense mutation G863A. The authors hypothesized that this 2588G > C alteration was a mild mutation that causes STGD only in combination with another severe ABCR mutation. This concept was supported by the observation that the accompanying ABCR mutations in at least 5 of 8 STGD patients were potentially null (severe) and that a combination of two mild (G863A) mutations was not observed among 68 STGD patients.161 This linkage disequilibrium has been confirmed independently in a North American population of mostly European descent.167
Another striking observation about mutations at the ABCR locus was the high frequency of complex alleles, that is, more than one mutation on a single disease chromosome.94 In 10 (7 percent) of the 150 European-American families, 2 different mutations were identified on a single chromosome, resulting in several different complex alleles.94 These data emphasize the importance of segregation analysis of all ABCR variants in families with STGD.94 The frequency of ABCR complex alleles is similar to that reported with another ABC transporter, the gene CFTR responsible for cystic fibrosis.168
ABCR Heterozygous Mutations and Susceptibility to AMD.
Given the several phenotypic similarities between STGD and ARM, including the accumulation of drusen in and under the RPE and the frequent progressive atrophy of the macular RPE, the hypothesis that some ABCR alterations may be associated with AMD was tested.169 The 50 coding exons of ABCR were screened in 167 unrelated AMD patients and 220 racially matched population controls. The cohort consisted of 96 AMD patients from Boston and 71 from Utah. Thirty-three patients (20 percent) had wet AMD and 80 percent had the dry form. Thirteen different AMD-associated alterations in ABCR were detected among 26 AMD patients (16 percent).169 Most were missense mutations, but two changes were deletions representing frameshifts and one was at a splice donor site.169 The AMD-associated alterations were scattered throughout the coding sequence of ABCR, although more were located toward the 3′ end.169 Interestingly, most were missense mutations located outside of conserved functional domains such as the ATP-binding cassette regions169 (Fig. 243-2). Among the Utah and Boston AMD patients, similar fractions of variants in AMD patients were observed (13 of 71 and 13 of 96, respectively). Furthermore, there was a similar distribution of the common mutant alleles G1961E (2 and 4) or D2177N (5 and 2) among the two cohorts. Interestingly, three AMD-associated ABCR variants169 had been identified previously in families with STGD (R1898H, G1961E, 6519del11).134 Subsequently, four other AMD-associated ABCR mutations (E471K, R1129L, 5196+1 G > A, and L1970F) were identified in unrelated STGD families.94,169 More impressively, 21 percent of 145 STGD families report a positive family history of AMD in the ancestral lineage of direct descent to the STGD-affected sibship.94
Diagram of the ABCR gene with STGD and AMD alterations. Transmembrane domains predicted by hydropathy plot are shown as black bars, and the ATP-binding domains are shown as hatched bars below. Arrows indicate alterations identified in both STGD1 and AMD patients shown mutations; D = deletions; S = splice donor site mutations; X = stop codon-generating mutation. The number at the right signifies the last codon.
These findings suggest a model that predicts that some hypomorphic alleles, when present in the heterozygous state, result in phenotypic effects after prolonged periods of malfunction (>65 years). These deleterious consequences result from the cumulative effects of diminished transport of a critically important molecule. The buildup of a potentially toxic byproduct could also further exacerbate the disease. Based on the identification of heterozygous ABCR variants in AMD patients compared with controls, it was concluded that some mutations that cause recessive STGD might enhance susceptibility to AMD in the heterozygous state.169 We have never found more than one AMD-associated variant chromosome in ABCR or in any AMD proband, which is consistent with a dominant susceptibility locus.169 One prediction of this model is that those grandparents in STGD families who are heterozygous for an ABCR mutation may be susceptible to AMD. Evidence to support this prediction was published recently.170,171
This concept of phenotypic effects for heterozygous mutant alleles of genes responsible for recessive diseases is not novel. In fact, several published examples of susceptibility to multifactorial disease are associated with heterozygous mutations in a gene responsible for a recessive disease phenotype (Table 243-4).172–178 One example of an association between a monogenic disorder and a multifactorial disease is familial hypercholesterolemia (FH).172 Homozygotes for mutations in the gene encoding the LDL receptor develop extremely high serum-LDL levels and typically die of myocardial infarction as young adults. Heterozygotes develop moderately high LDL levels and manifest a multifactorial disorder, coronary artery disease, in their fourth or fifth decades.172 Cystic fibrosis is a common recessive disorder characterized by meconium ileus, pulmonary infection, and pancreatic insufficiency. Heterozygotes for mutation in CFTR, possibly the best-studied eukaryotic ABC-transporter gene, show an increased prevalence of chronic pancreatitis.173,174 Thus, in each of the examples listed in Table 243-4, heterozygous mutations in the causative single gene disease predispose the carrier individuals to express a multifactorial trait, whereas homozygous or compound heterozygous mutations in the same gene cause a classically recessive disorder, usually with an earlier onset and more definitive clinical phenotype.
Table 243-4: Recessive Disorders with Heterozygote Predisposition to Multifactorial Disease |Favorite Table|Download (.pdf) Table 243-4: Recessive Disorders with Heterozygote Predisposition to Multifactorial Disease
|Monogenic disease ||MIM ||Gene ||Multifactorial disease |
|Familial hypercholesterolemia ||143890 || LDLR ||Coronary artery disease |
|Cystic fibrosis ||219700 || CFTR ||Pancreatic insufficiency |
|Ataxia-telangiectasia ||208900 || ATM ||Breast cancer |
|α1-Antitrypsin deficiency ||107400 || AAT ||Chronic obstructive lung disease |
|Hyperlipoproteinemia ||238600 || LPL ||Ischemic heart disease |
|Stargardt disease ||248200 || ABCR ||Age-related macular degeneration |
The finding that heterozygous mutations in ABCR are associated with AMD created substantial excitement as the first substantive clue to a genetic and biologic causation of AMD, but also some controversy. Initially, two letters questioned the selection of patients and controls, as well as the statistical analysis.179,180 However, both further studies and additional statistical analysis of the original controls confirmed the initial interpretation.181
Three studies have provided additional data on allelic variation of ABCR in AMD.182–184 Each claims to reject an association between alterations in the ABCR gene and AMD, but, unfortunately, none of these reports reproduces the original study. Problems with replication are (a) patient selection and (b) efficiency of mutation detection. In all three studies, the patient population was enriched for individuals with the neovascular, or wet, form of AMD. For example, Stone et al. studied a cadre of individuals that purportedly contained 60 percent of patients with wet AMD, while the fraction of all 167 subjects in the original report169 who had wet AMD was 20 percent and those with dry AMD was 80 percent, closely resembling the proportion of the two forms of AMD in the general population. Thus, their report182 sustains the initial conclusion that variants of ABCR are rare in wet AMD.169 Similarly, a Japanese study183 included 87.5 percent of its 80 patients with the wet form of the disease, and an independent American study reported 80 percent of all patients with disciform disease.184
To make a statement about the lack of sequence variants in a gene in a cohort of patients, one must assure that the mutation scanning method employed will detect the substantial majority of all sequence changes. Regrettably, this was not true in all these studies.182–184 For ABCR , a valid standard for the efficiency of mutation screening is the fraction of mutations found in STGD patients, because ABCR is the gene exclusively responsible for classic recessive STGD.134 Stone et al. found ABCR mutations in a mere 19 percent of STGD chromosomes (82 of 430; Table 2 of reference182), while two other studies found ABCR mutations in 57 percent94 or 62 percent162 of STGD chromosomes. Furthermore, Stone et al. did not find any double-mutant chromosomes or complex alleles in their cadre of STGD patients, which have been reported at 7 percent of STGD chromosomes.94 Thus, the mutation detection rate even for STGD162,182 is much lower when compared to other laboratories.94,162 Similarly, in another laboratory, mutation detection was performed on only 20 percent of the coding exons and no noncoding regions.183 A third group184 did not find even the numerous common ABCR polymorphisms identified by multiple European and American laboratories,134,161,182 thus rendering the presented results effectively uninterpretable.
To investigate further the heuristic observation that heterozygous ABCR mutations may confer increased risk to AMD,169 the two most common AMD-associated ABCR alterations, G1961E and D2177N, were sought in a large collaborative study among 15 different centers in North America and Europe.185 These two mutations alone were screened in 1218 unrelated AMD patients of Caucasian origin and 1258 reportedly unaffected unrelated individuals as controls. These two abnormal sequence changes were found in one allele of ABCR in 40 AMD patients (≈3.4 percent) and 13 controls (≈0.9 percent); a statistically significant difference (P<0.0001).185 The results remain significant (P<0.0001) on the independent sample even after exclusion of the data from the previous pilot study.169 In AMD, the 4 percent frequency of these two mutations alone, G1961E and D2177N (≈4 percent), is a significant fraction in the context of a complex disorder. For comparison, 4 percent is greater than the frequency of all reported myocillin variants (2 to 4 percent) in 1703 glaucoma patients from 5 different populations.186 Furthermore, the myocillin mutations are heterozygous in a dominant disease, and thus may represent either later onset or variability of expression of the autosomal dominant juvenile-onset open-angle glaucoma.186
A Model for ABCR in Retinal Dystrophy and Degeneration.
Based on the findings of homozygous and compound heterozygous mutations of ABCR in STGD, FF, cone-rod dystrophy, RP19, and the finding of heterozygous ABCR mutations in AMD, a model of ABCR function has been derived.94,169,181,187 In this model (Fig. 243-3), the severity of the retinal dystrophy is inversely proportional to the residual ABCR activity. Severe mutations in both ABCR alleles result in complete loss of ABCR activity (two null alleles) and cause a retinitis pigmentosa phenotype. Two hypomorphic mutations, or a combination of a hypomorphic mutation with a null allele, can result in FF or STGD. A single, mild, heterozygous ABCR mutation acting over a prolonged time may enhance a susceptibility to AMD. This dominant susceptibility may result in AMD, if the proper interaction and exposure to environmental influences occur.187a
Phenotypes associated with ABCR mutations. At left, ABCR activity is represented by the filled triangle and depicts decreasing activity towards the top. Genotypes are denoted for both chromosomes. Null denotes mutations that lead to no functional protein products including frameshift, nonsense, and some splice and missense mutations. Hypo denotes hypomorphic alleles including most missense and splice mutations and some C-terminal frameshift and nonsense mutations. WT denotes wild-type or normal alleles. Arrows indicate association between genotypes and phenotypes; the bracket indicates that Stargardt disease and Fundus Flavimaculatus may be caused by either of the indicated genotypes.