ABCA4 Variants Research Articles

Late-Onset Stargardt Disease Due to Mild, Deep-Intronic ABCA4 Alleles.

To investigate the role of two deep-intronic ABCA4 variants, that showed a mild splice defect in vitro and can occur on the same allele as the low penetrant c.5603A>T, in Stargardt disease (STGD1). Ophthalmic data were assessed of 18 STGD1 patients who harbored c.769-784C>T or c.4253+43G>A in combination with a severe ABCA4 variant. Subjects carrying c.[769-784C>T; 5603A>T] were clinically compared with a STGD1 cohort previously published carrying c.5603A>T noncomplex. We calculated the penetrances of the intronic variants using ABCA4 allele frequency data of the general population and investigated the effect of c.769-784C>T on splicing in photoreceptor progenitor cells (PPCs). Mostly, late-onset, foveal-sparing STGD1 was observed among subjects harboring c.769-784C>T or c.4253+43G>A (median age of onset, 54.5 and 52.0 years, respectively). However, ages of onset, phenotypes in fundo, and visual acuity courses varied widely. No significant clinical differences were observed between the c.[769-784C>T; 5603A>T] cohort and the c.4253+43G>A or the c.5603A>T cohort. The penetrances of c.769-784C>T (20.5%-39.6%) and c.4253+43G>A (35.8%-43.1%) were reduced, when not considering the effect of yet unidentified or known factors in cis, such as c.5603A>T (identified in 7/7 probands with c.769-784C>T; 1/8 probands with c.4253+43G>A). Variant c.769-784C>T resulted in a pseudo-exon insertion in 15% of the total mRNA (i.e., ∼30% of the c.769-784C>T allele alone). Two mild intronic ABCA4 variants could further explain missing heritability in late-onset STGD1, distinguishing it from AMD. The observed clinical variability and calculated reduced penetrance urge research into modifiers within and outside of the ABCA4 gene.

Coexistence of GNAT1 and ABCA4 variants associated with Nougaret-type congenital stationary night blindness and childhood-onset cone-rod dystrophy.

A single variant (p.G38D) in the GNAT1 gene, encoding the rod-specific transducin α-subunit in phototransduction, has been reported only in one French family with Nougaret-type autosomal dominant congenital stationary night blindness (CSNB). We identified a Japanese family with Nougaret-type CSNB and cone-rod dystrophy (CORD). Five patients with CSNB and two patients with childhood-onset CORD were recruited. We performed a comprehensive ophthalmic examination including electroretinography (ERG). Disease-causing variants were identified by whole exome sequencing, with candidates confirmed by Sanger sequencing in nine family members. The GNAT1 variant (p.G38D) was identified in all four CSNB patients, whereas the two CORD patients carried biallelic truncated known ABCA4 variants as well as the GNAT1 variant. Clinically, no remarkable findings were observed in fuduscopy, fundus autofluorescence, or optical coherence tomography images from the CSNB patients. No response was detectable by rod ERG. The a-waves of standard and bright flash ERG were delayed and broadened rather than biphasic, and b/a-wave amplitude ratio was negative. Cone and 30-Hz flicker responses were normal, and overall, the ERG findings were compatible with previous descriptions of Nougaret-type CSNB. ERG of the CORD patients with macular atrophy showed non-recordable rod response and severely decreased standard flash, cone and 30-Hz flicker responses. This is the second report of a Nougaret-type CSNB family with the GNAT1 variant. Our novel findings suggest that coexistence of the GNAT1 and biallelic ABCA4 variants is associated with an overlapping phenotype with both Nougaret-type CSNB and CORD.

A case–control collapsing analysis identifies retinal dystrophy genes associated with ophthalmic disease in patients with no pathogenic ABCA4 variants

PurposeVariants in the ABCA4 gene are causal for a variety of retinal dystrophy phenotypes, including Stargardt disease (STGD1). However, 15% of patients who present with symptoms compatible with STGD1/ABCA4 disease do not have identifiable causal ABCA4 variants. We hypothesized that a case–control collapsing analysis in ABCA4-negative patients with compatible symptoms would provide an objective measure to identify additional disease genes. MethodsWe performed a genome-wide enrichment analysis of “qualifying variants”—ultrarare variants predicted to impact protein function—in protein-coding genes in 79 unrelated cases and 9028 unrelated controls. ResultsDespite modest sample size, two known retinal dystrophy genes, PRPH2 and CRX, achieved study-wide significance (p < 1.33 × 10−6) under a dominant disease model, and eight additional known retinal dystrophy genes achieved nominal significance (p < 0.05). Across these ten genes, the excess of qualifying variants explained up to 36.8% oF.A.ffected individuals. Furthermore, under a recessive model, the cone–rod dystrophy gene CERKL approached study-wide significance. ConclusionOur results indicate that case–control collapsing analyses can efficiently identify pathogenic variants in genes in non-ABCA4 retinal dystrophies. The genome-wide collapsing analysis framework is an objective discovery method particularly suitable in settings with overlapping disease phenotypes.

ABCA4 Gene Screening in a Chinese Cohort With Stargardt Disease: Identification of 37 Novel Variants.

Purpose: To clarify the mutation spectrum and frequency of ABCA4 in a Chinese cohort with Stargardt disease (STGD1). Methods: A total of 153 subjects, comprising 25 families (25 probands and their family members) and 71 sporadic cases, were recruited for the analysis of ABCA4 variants. All probands with STGD1 underwent a comprehensive ophthalmologic examination. Overall, 792 genes involved in common inherited eye diseases were screened for variants by panel-based next-generation sequencing (NGS). Variants were filtered and analyzed to evaluate possible pathogenicity. Results: The total variant detection rate of at least one ABCA4 mutant allele was 84.3% (129/153): two or three disease-associated variants in 86 subjects (56.2%), one mutant allele in 43 subjects (28.1%), and no variants in 24 subjects (15.7%). Ninety-six variants were identified in the total cohort, which included 62 missense (64%), 15 splicing (16%), 11 frameshift (12%), 6 nonsense (6%), and 2 small insertion or deletion (2%) variants. Thirty-seven novel variants were found, including a de novo variant, c.4561delA. The most prevalent variant was c.101_106delCTTTAT (10.5%), followed by c.2894A > G (6.5%) and c.6563T > C (4.6%), in STGD1 patients from eastern China. Conclusion: Thirty-seven novel variants were detected using panel-based NGS, including one de novo variant, further extending the mutation spectrum of ABCA4. The common variants in a population from eastern China with STGD1 were also identified.

Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides

PurposeUsing exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability. MethodsSequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects. ResultsIn 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects. ConclusionOur data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.

ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants

PurposeABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability. MethodsBy locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4. ResultsWe identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells. ConclusionNoncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders.

Highly Variable Disease Courses in Siblings with Stargardt Disease

To investigate intersibling phenotypic concordance in Stargardt disease (STGD1). Retrospective cohort study. Siblings with genetically confirmed STGD1 and at least 1 available fundus autofluorescence (FAF) image of both eyes. We compared age at onset within families. Disease duration was matched to investigate differences in best-corrected visual acuity (BCVA) and compared the survival time for reaching severe visual impairment (<20/200 Snellen or >1.0 logarithm of the minimum angle of resolution [logMAR]). Central retinal atrophy area was quantified independently by 2 experienced graders using semiautomated software and compared between siblings. Both graders performed qualitative assessment of FAF and spectral-domain (SD) OCT images to identify phenotypic differences. Differences in age at onset, disease duration-matched BCVA, time to severe visual impairment development, FAF atrophy area, FAF patterns, and genotypes. Substantial differences in age at onset were present in 5 of 17 families, ranging from 13 to 39 years. Median BCVA at baseline was 0.60 logMAR (range, -0.20 to 2.30 logMAR; Snellen equivalent, 20/80 [range, 20/12-hand movements]) in the right eye and 0.50 logMAR (range, -0.20 to 2.30 logMAR; Snellen equivalent, 20/63 [range, 20/12-hand movements]) in the left eye. Disease duration-matched BCVA was investigated in 12 of 17 families, and the median difference was 0.41 logMAR (range, 0.00-1.10 logMAR) for the right eye and 0.41 logMAR (range, 0.00-1.08 logMAR) for the left eye. We observed notable differences in time to severe visual impairment development in 7 families, ranging from 1 to 29 years. Median central retinal atrophy area was 11.38 mm2 in the right eye (range, 1.98-44.78 mm2) and 10.59 mm2 in the left eye (range, 1.61-40.59 mm2) and highly comparable between siblings. Similarly, qualitative FAF and SD OCT phenotypes were highly comparable between siblings. Phenotypic discordance between siblings with STGD1 carrying the same ABCA4 variants is a prevalent phenomenon. Although the FAF phenotypes are highly comparable between siblings, functional outcomes differ substantially. This complicates both sibling-based prognosis and genotype-phenotype correlations and has important implications for patient care and management.

Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease.

Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.

Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic ABCA4 Variant c.4539+2001G>A in Stargardt Disease.

Deep-sequencing of the ABCA4 locus has revealed that ~10% of autosomal recessive Stargardt disease (STGD1) cases are caused by deep-intronic mutations. One of the most recurrent deep-intronic variants in the Belgian and Dutch STGD1 population is the c.4539+2001G>A mutation. This variant introduces a 345-nt pseudoexon to the ABCA4 mRNA transcript in a retina-specific manner. Antisense oligonucleotides (AONs) are short sequences of RNA that can modulate splicing. In this work, we designed 26 different AONs to perform a thorough screening to identify the most effective AONs to correct splicing defects associated with c.4539+2001G>A. All AONs were tested in patient-derived induced pluripotent stem cells (iPSCs) that were differentiated to photoreceptor precursor cells (PPCs). AON efficacy was assessed through RNA analysis and was based on correction efficacy, and AONs were grouped and their properties assessed. We (a) identified nine AONs with significant correction efficacies (>50%), (b) confirmed that a single nucleotide mismatch was sufficient to significantly decrease AON efficacy, and (c) found potential correlations between efficacy and some of the parameters analyzed. Overall, our results show that AON-based splicing modulation holds great potential for treating Stargardt disease caused by splicing defects in ABCA4.

Detailed genetic characteristics of an international large cohort of patients with Stargardt disease: ProgStar study report 8

Background/aimsTo describe the genetic characteristics of the cohort enrolled in the international multicentre progression of Stargardt disease 1 (STGD1) studies (ProgStar) and to determine geographic differences based on the allele...

Identification of novel pathogenic ABCA4 variants in a Han Chinese family with Stargardt disease.

Stargardt disease (STGD1, OMIM 248200) is a common hereditary juvenile or early adult onset macular degeneration. It ultimately leads to progressive central vision loss. Here, we sought to identify gene mutations associated with STGD1 in a three-generation Han Chinese pedigree by whole exome sequencing and Sanger sequencing. Two novel potentially pathogenic variants in a compound heterozygous state, c.3607G>T (p.(Gly1203Trp)) and c.6722T>C (p.(Leu2241Pro)), in the ATP binding cassette subfamily A member 4 gene (ABCA4) were identified as contributing to the family’s STGD1 phenotype. These variants may impact the ABCA4 protein structure and reduce the retinal-activated ATPase activity, leading to abnormal all-trans retinal accumulation in photoreceptor outer segments and in retinal pigment epithelium cells. The present study broadens the mutational spectrum of the ABCA4 responsible for STGD1. A combination of whole exome sequencing and Sanger sequencing is likely to be a time-saving and cost-efficient approach to screen pathogenic variants in genetic disorders caused by sizable genes, as well as avoiding misdiagnosis. These results perhaps refine genetic counseling and ABCA4-targetted treatments for families affected by STGD1.

Analysis of the ABCA4 c.[2588G>C;5603A>T] Allele in the Australian Population.

Inherited retinal diseases (IRDs) are genetically and phenotypically diverse, and they cause significant morbidity worldwide. Importantly, IRDs may be amenable to precision medicine strategies, and thus the molecular characterisation of causative variants is becoming increasingly important with the promise of personalised therapies on the horizon. ABCA4, involved in the translocation of visual cycle derivatives, is a well-established, frequent cause of IRDs worldwide, with pathogenic variants implicated in phenotypically diverse diseases. Identification of causative ABCA4 variants in some individuals, however, has been enigmatic, and resolution of this issue is currently a hotbed of research. Recent evidence has indicated that hypomorphic alleles, which cause disease under certain conditions, may account for some of the missing causal variants. It has been postulated that the ABCA4 c.5603A>T (p.Asn1868Ile) variant, previously considered benign, be reclassified as hypomorphic when in cis configuration with c.2588G>C (p.Gly863Ala/Gly863del), a variant previously considered to be pathogenic in its own right. We are exploring this relationship within an Australian cohort to test this theory.

Searching the second hit in patients with inherited retinal dystrophies and monoallelic variants in ABCA4, USH2A and CEP290 by whole-gene targeted sequencing

Inherited Retinal Dystrophies are clinically and genetically heterogeneous disorders affecting the photoreceptors. Although NGS has shown to be helpful for the molecular diagnosis of these conditions, some cases remain unsolved. Among these, several individuals harboured monoallelic variants in a recessive gene, suggesting that a comprehensive screening could improve the overall diagnosis. In order to assess the contribution of non-coding variations in a cohort of 29 patients, 25 of them with monoallelic mutations, we performed targeted NGS. The design comprised the entire genomic sequence of three genes (USH2A, ABCA4 and CEP290), the coding exons of 76 genes and two disease-associated intronic regions in OFD1 and PRPF31. As a result, likely causative mutations (8 novel) were identified in 17 probands (diagnostic rate: 58.62%), including two copy-number variations in USH2A (one deletion of exons 22–55 and one duplication of exons 46–47). Possibly damaging deep-intronic mutations were identified in one family, and another with a monoallelic variant harboured causal mutations in a different locus. In conclusion, due to the high prevalence of carriers of IRD mutations and the results obtained here, sequencing entire genes do not seem to be the approach of choice for detecting the second hit in IRD patients with monoallelic variants.

The Common ABCA4 Variant p.Asn1868Ile Shows Nonpenetrance and Variable Expression of Stargardt Disease When Present in trans With Severe Variants.

To assess the occurrence and the disease expression of the common p.Asn1868Ile variant in patients with Stargardt disease (STGD1) harboring known, monoallelic causal ABCA4 variants. The coding and noncoding regions of ABCA4 were sequenced in 67 and 63 STGD1 probands respectively, harboring monoallelic ABCA4 variants. In case p.Asn1868Ile was detected, segregation analysis was performed whenever possible. Probands and affected siblings harboring p.Asn1868Ile without additional variants in cis were clinically evaluated retrospectively. Two asymptomatic siblings carrying the same ABCA4 variants as their probands were clinically examined. The penetrance of p.Asn1868Ile was calculated using allele frequency data of ABCA4 variants in non-Finnish European individuals. The p.Asn1868Ile variant was found in cis with known variants in 14/67 probands. In 27/67 probands, we identified p.Asn1868Ile without additional variants in cis, in combination with known, mainly severe ABCA4 variants. In 23/27 probands, the trans configuration was established. Among 27 probands and 6/7 STGD1 siblings carrying p.Asn1868Ile, 42% manifested late-onset disease (>44 years). We additionally identified four asymptomatic relatives carrying a combination of a severe variant and p.Asn1868Ile; ophthalmologic examination in two persons did not reveal STGD1. Based on ABCA4 allele frequency data, we conservatively estimated the penetrance of p.Asn1868Ile, when present in trans with a severe variant, to be below 5%. A significant fraction of genetically unexplained STGD1 cases carries p.Asn1868Ile as a second variant. Our findings suggest exceptional differences in disease expression or even nonpenetrance of this ABCA4 variant, pointing toward an important role for genetic or environmental modifiers in STGD1.

Extremely hypomorphic and severe deep intronic variants in the ABCA4 locus result in varying Stargardt disease phenotypes.

Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of the ABCA4 locus in STGD1 patients identifies two expected disease-causing alleles in ∼75% of patients and only one mutation in ∼15% of patients. Recently, many possibly pathogenic variants in deep intronic sequences of ABCA4 have been identified in the latter group. We extended our analyses of deep intronic ABCA4 variants and determined that one of these, c.4253+43G>A (rs61754045), is present in 29/1155 (2.6%) of STGD1 patients. The variant is found at statistically significantly higher frequency in patients with only one pathogenic ABCA4 allele, 23/160 (14.38%), MAF = 0.072, compared to MAF = 0.013 in all STGD1 cases and MAF = 0.006 in the matching general population (P < 1 × 10−7). The variant, which is not predicted to have any effect on splicing, is the first reported intronic “extremely hypomorphic allele” in the ABCA4 locus; that is, it is pathogenic only when in trans with a loss-of-function ABCA4 allele. It results in a distinct clinical phenotype characterized by late onset of symptoms and foveal sparing. In ∼70% of cases the variant was allelic with the c.6006-609T>A (rs575968112) variant, which was deemed nonpathogenic. Another rare deep intronic variant, c.5196+1056A>G (rs886044749), found in 5/834 (0.6%) of STGD1 cases is, conversely, a severe allele. This study determines pathogenicity for three noncoding variants in STGD1 patients of European descent accounting for ∼3% of the disease. Defining disease-associated alleles in the noncoding sequences of the ABCA4 locus can be accomplished by integrated clinical and genetic analyses.

Identification and Rescue of Splice Defects Caused by Two Neighboring Deep-Intronic ABCA4 Mutations Underlying Stargardt Disease

Sequence analysis of the coding regions and splice site sequences in inherited retinal diseases is not able to uncover ∼40% of the causal variants. Whole-genome sequencing can identify most of the non-coding variants, but their interpretation is still very challenging, in particular when the relevant gene is expressed in a tissue-specific manner. Deep-intronic variants in ABCA4 have been associated with autosomal-recessive Stargardt disease (STGD1), but the exact pathogenic mechanism is unknown. By generating photoreceptor precursor cells (PPCs) from fibroblasts obtained from individuals with STGD1, we demonstrated that two neighboring deep-intronic ABCA4 variants (c.4539+2001G>A and c.4539+2028C>T) result in a retina-specific 345-nt pseudoexon insertion (predicted protein change: p.Arg1514Leufs∗36), likely due to the creation of exonic enhancers. Administration of antisense oligonucleotides (AONs) targeting the 345-nt pseudoexon can significantly rescue the splicing defect observed in PPCs of two individuals with these mutations. Intriguingly, an AON that is complementary to c.4539+2001G>A rescued the splicing defect only in PPCs derived from an individual with STGD1 with this but not the other mutation, demonstrating the high specificity of AONs. In addition, a single AON molecule rescued splicing defects associated with different neighboring mutations, thereby providing new strategies for the treatment of persons with STGD1. As many genes associated with human genetic conditions are expressed in specific tissues and pre-mRNA splicing may also rely on organ-specific factors, our approach to investigate and treat splicing variants using differentiated cells derived from individuals with STGD1 can be applied to any tissue of interest.

Early Patterns of Macular Degeneration in ABCA4-Associated Retinopathy

PurposeTo describe the earliest features of ABCA4-associated retinopathy.DesignCase series.ParticipantsChildren with a clinical and molecular diagnosis of ABCA4-associated retinopathy without evidence of macular atrophy.MethodsThe retinal phenotype was characterized by color fundus photography, OCT, fundus autofluorescence (FAF) imaging, electroretinography, and in 2 patients, adaptive optics scanning laser ophthalmoscopy (AOSLO). Sequencing of the ABCA4 gene was performed in all patients.Main Outcome MeasuresVisual acuity, OCT, FAF, electroretinography, and AOSLO results.ResultsEight children with ABCA4-associated retinopathy without macular atrophy were identified. Biallelic variants in ABCA4 were identified in all patients. Four children were asymptomatic, and 4 reported loss of VA. Patients were young (median age, 8.5 years; interquartile range, 6.8 years) with good visual acuity (median, 0.155 logarithm of the minimum angle of resolution [logMAR]; interquartile range, 0.29 logMAR). At presentation, the macula appeared normal (n = 3), had a subtly altered foveal reflex (n = 4), or demonstrated manifest fine yellow dots (n = 1). Fundus autofluorescence identified hyperautofluorescent dots in the central macula in 3 patients, 2 of whom showed a normal fundus appearance. Only 1 child had widespread hyperautofluorescent retinal flecks at presentation. OCT imaging identified hyperreflectivity at the base of the outer nuclear layer in all 8 patients. Where loss of outer nuclear volume was evident, this appeared to occur preferentially at a perifoveal locus. Longitudinal split-detector AOSLO imaging in 2 individuals confirmed that the greatest change in cone spacing occurred in the perifoveal, and not foveolar, photoreceptors. Electroretinography showed a reduced B-wave–to–A-wave ratio in 3 of 5 patients tested; in 2 children, recordings clearly showed electronegative results.ConclusionsIn childhood-onset ABCA4-associated retinopathy, the earliest stages of macular atrophy involve the parafovea and spare the foveola. In some cases, these changes are predated by tiny, foveal, yellow, hyperautofluorescent dots. Hyperreflectivity at the base of the outer nuclear layer, previously described as thickening of the external limiting membrane, is likely to represent a structural change at the level of the foveal cone nuclei. Electroretinography suggests that the initial site of retinal dysfunction may occur after phototransduction.

Localization and functional characterization of the p.Asn965Ser (N965S) ABCA4 variant in mice reveal pathogenic mechanisms underlying Stargardt macular degeneration.

ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) proteins that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across outer segment disc membranes thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptor cells. Mutations in the gene encoding ABCA4 are responsible for Stargardt disease (STGD1), an autosomal recessive retinal degenerative disease that causes severe vision loss. To define the molecular basis for STGD1 associated with the p.Asn965Ser (N965S) mutation in the Walker A motif of nucleotide binding domain 1 (NBD1), we generated a p.Asn965Ser knockin mouse and compared the subcellular localization and molecular properties of the disease variant with wild-type (WT) ABCA4. Here, we show that the p.Asn965Ser ABCA4 variant expresses at half the level of WT ABCA4, partially mislocalizes to the endoplasmic reticulum (ER) of photoreceptors, is devoid of N-Ret-PE activated ATPase activity, and causes an increase in autofluorescence and the bisretinoid A2E associated with lipofuscin deposits in retinal pigment epithelial cells as found in Stargardt patients and Abca4 knockout mice. We also show for the first time that a significant fraction of WT ABCA4 is retained in the inner segment of photoreceptors. On the basis of these studies we conclude that loss in substrate-dependent ATPase activity and protein misfolding are mechanisms underlying STGD1 associated with the p.Asn965Ser mutation in ABCA4. Functional and molecular modeling studies further suggest that similar pathogenic mechanisms are responsible for Tangiers disease associated with the p.Asn935Ser (N935S) mutation in the NBD1 Walker A motif of ABCA1.

Progression of Stargardt Disease as Determined by Fundus Autofluorescence in the Retrospective Progression of Stargardt Disease Study (ProgStar Report No. 9)

ImportanceSensitive outcome measures for disease progression are needed for treatment trials of Stargardt disease.ObjectiveTo describe the yearly progression rate of atrophic lesions in the retrospective Progression of Stargardt Disease study.Design, Setting, and ParticipantsA multicenter retrospective cohort study was conducted at tertiary referral centers in the United States and Europe. A total of 251 patients aged 6 years or older at baseline, harboring disease-causing variants in ABCA4 (OMIM 601691), enrolled in the study from 9 centers between August 2, 2013, and December 12, 2014; of these patients, 215 had at least 2 gradable fundus autofluorescence images with atrophic lesion(s) present in at least 1 eye.ExposuresAreas of definitely decreased autofluorescence (DDAF) and questionably decreased autofluorescence were quantified by a reading center. Progression rates were estimated from linear mixed models with time as the independent variable.Main Outcomes and MeasuresYearly rate of progression using the growth of atrophic lesions measured by fundus autofluorescence.ResultsA total of 251 participants (458 study eyes) were enrolled. Images from 386 eyes of 215 participants (126 females and 89 males; mean [SD] age, 29.9 [14.7] years; mean [SD] age of onset of symptoms, 21.9 [13.3] years) showed atrophic lesions present on at least 2 visits and were graded for 2 (156 eyes), 3 (174 eyes), or 4 (57 eyes) visits. A subset of 224 eyes (123 female participants and 101 male participants; mean [SD] age, 33.0 [15.1] years) had areas of DDAF present on at least 2 visits; these eyes were included in the estimation of the progression of the area of DDAF. At the first visit, DDAF was present in 224 eyes (58.0%), with a mean (SD) lesion size of 2.2 (2.7) mm2. The total mean (SD) area of decreased autofluorescence (DDAF and questionably decreased autofluorescence) at first visit was 2.6 (2.8) mm2. Mean progression of DDAF was 0.51 mm2/y (95% CI, 0.42-0.61 mm2/y), and of total decreased fundus autofluorescence was 0.35 mm2/y (95% CI, 0.28-0.43 mm2/y). Rates of progression depended on the initial size of the lesion.Conclusions and RelevanceIn Stargardt disease with DDAF lesions, fundus autofluorescence may serve as a monitoring tool for interventional clinical trials that aim to slow disease progression. Rates of progression depended mainly on initial lesion size.

Highly sensitive measurements of disease progression in rare disorders: Developing and validating a multimodal model of retinal degeneration in Stargardt disease.

BackgroundEach inherited retinal disorder is rare, but together, they affect millions of people worldwide. No treatment is currently available for these blinding diseases, but promising new options—including gene therapy—are emerging. Arguably, the most prevalent retinal dystrophy is Stargardt disease. In each case, the specific combination of ABCA4 variants (> 900 identified to date) and modifying factors is virtually unique. It accounts for the vast phenotypic heterogeneity including variable rates of functional and structural progression, thereby potentially limiting the ability of phase I/II clinical trials to assess efficacy of novel therapies with few patients. To accommodate this problem, we developed and validated a sensitive and reliable composite clinical trial endpoint for disease progression based on structural measurements of retinal degeneration.Methods and findingsWe used longitudinal data from early-onset Stargardt patients from the Netherlands (development cohort, n = 14) and the United Kingdom (external validation cohort, n = 18). The composite endpoint was derived from best-corrected visual acuity, fundus autofluorescence, and spectral-domain optical coherence tomography. Weighting optimization techniques excluded visual acuity from the composite endpoint. After optimization, the endpoint outperformed each univariable outcome, and showed an average progression of 0.41° retinal eccentricity per year (95% confidence interval, 0.30–0.52). Comparing with actual longitudinal values, the model accurately predicted progression (R2, 0.904). These properties were largely preserved in the validation cohort (0.43°/year [0.33–0.53]; prediction: R2, 0.872). We subsequently ran a two-year trial simulation with the composite endpoint, which detected a 25% decrease in disease progression with 80% statistical power using only 14 patients.ConclusionsThese results suggest that a multimodal endpoint, reflecting structural macular changes, provides a sensitive measurement of disease progression in Stargardt disease. It can be very useful in the evaluation of novel therapeutic modalities in rare disorders.