ABCA4-associated Disease Research Articles

Quantitative Fundus Autofluorescence and Optical Coherence Tomography inPRPH2/RDS- andABCA4-Associated Disease Exhibiting Phenotypic Overlap

To assess whether quantitative fundus autofluorescence (qAF), a measure of RPE lipofuscin, and spectral-domain optical coherence tomography (SD-OCT) can aid in the differentiation of patients with fundus features that could either be related to ABCA4 mutations or be part of the phenotypic spectrum of pattern dystrophies. Autofluorescence images (30°, 488-nm excitation) from 39 patients (67 eyes) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference and were quantified as previously described. In addition, horizontal SD-OCT images through the fovea were obtained. Patients were screened for ABCA4 and PRPH2/RDS mutations. ABCA4 mutations were identified in 19 patients (mean age, 37 ± 12 years) and PRPH2/RDS mutations in 8 patients (mean age, 48 ± 13 years); no known ABCA4 or PRPH2/RDS mutations were found in 12 patients (mean age, 48 ± 9 years). Differentiation of the groups using phenotypic SD-OCT and AF features (e.g., peripapillary sparing, foveal sparing) was not reliable. However, patients with ABCA4 mutations could be discriminated reasonably well from other patients when qAF values were corrected for age and race. In general, ABCA4 patients had higher qAF values than PRPH2/RDS patients, while most patients without mutations in PRPH2/RDS or ABCA4 had qAF levels within the normal range. The high qAF levels of ABCA4-positive patients are a hallmark of ABCA4-related disease. The reason for high qAF among many PRPH2/RDS-positive patients is not known; higher RPE lipofuscin accumulation may be a primary or secondary effect of the PRPH2/RDS mutation.

Gene Therapy of ABCA4-Associated Diseases.

The ATP-binding cassette (ABC) transporter gene, ABCA4 (ABCR), was characterized in 1997 as the causal gene for autosomal recessive Stargardt disease (STGD1). Shortly thereafter several other phenotypes were associated with mutations in ABCA4, which now have collectively emerged as the most frequent cause of retinal degeneration phenotypes of Mendelian inheritance. ABCA4 functions as an important transporter (or "flippase") of vitamin A derivatives in the visual cycle. Several ways to alleviate the effects of the defective ABCA4 protein, which cause accumulation of 11-cis and all-trans-retinal in photoreceptors and lipofuscin in the retinal pigment epithelium, have been proposed. Although ABCA4 has proven to be a difficult research target, substantial progress through genetic, functional, and translational studies has allowed major advances in therapeutic applications for ABCA4-associated pathology, which should be available to patients in the (near) future. Here, we summarize the status of the gene therapy-based treatment options of ABCA4-associated diseases.

Genetic and clinical analysis of ABCA4-associated disease in African American patients.

Autosomal recessive Stargardt disease (STGD1) is caused by hundreds of mutations in the ABCA4 gene, which are often specific to racial and ethnic groups. Here, we investigated the ABCA4 variation and their phenotypic expression in a cohort of 44 patients of African American descent, a previously under-characterized racial group. Patients were screened for mutations in ABCA4 by next-generation sequencing and array-comparative genomic hybridization (aCGH), followed by analyses for pathogenicity by in silico programs. Thorough ophthalmic examination was performed on all patients. At least two (expected) disease-causing alleles in the ABCA4 gene were identified in 27 (61.4%) patients, one allele in 11 (25%) patients, and no ABCA4 mutations were found in six (13.6%) patients. Altogether, 39 different disease-causing ABCA4 variants, including seven new, were identified on 65 (74%) chromosomes, most of which were unique for this racial group. The most frequent ABCA4 mutation in this cohort was c.6320G>A (p.(R2107H)), representing 19.3% of all disease-associated alleles. No large copy number variants were identified in any patient. Most patients reported later onset of symptoms. In summary, the ABCA4 mutation spectrum in patients of West African descent differs significantly from that in patients of European descent, resulting in a later onset and “milder” disease.

Generalized Choriocapillaris Dystrophy, a Distinct Phenotype in the Spectrum ofABCA4-Associated Retinopathies

We describe a particular form of autosomal recessive generalized choriocapillaris dystrophy phenotype associated with ABCA4 mutations. A cohort of 30 patients with identified ABCA4 mutations and a distinct phenotype was studied. A retrospective review of history, fundus photographs, electroretinography, visual field testing, dark adaptometry, and optical coherence tomography was performed. Genetic analyses were performed by ABCA4 microarray analysis, high resolution melting, and/or next generation sequencing of all protein-coding sequences of the ABCA4 gene. The earliest recorded manifestation of ABCA4-associated disease was a central bull's eye type of macular dystrophy that progressed to chorioretinal atrophy of the macula with coarse rounded hyperpigmentations and expanding involvement of the periphery. The mean age at first presentation was 10.3 years, the longest follow-up was 61 years. All patients had two ABCA4 mutations identified, confirming the molecular genetic diagnosis of an ABCA4-associated disease. Most patients harbored at least one mutation classified as "severe," the most common of which was the p.N965S variant that had been found previously at a high frequency among patients with ABCA4-associated retinal dystrophies in Denmark. Generalized choriocapillaris dystrophy is a progressive ABCA4-associated phenotype characterized by early-onset macular dystrophy that disperses and expands to widespread end-stage chorioretinal atrophy with profound visual loss. All cases in this study were confirmed as harboring two ABCA4 mutations. Most of the ABCA4 mutations were classified as "severe" explaining the early onset, panretinal degeneration, and fast progression of the disease.

ABCA4Gene Screening by Next-Generation Sequencing in a British Cohort

We applied a recently reported next-generation sequencing (NGS) strategy for screening the ABCA4 gene in a British cohort with ABCA4-associated disease and report novel mutations. We identified 79 patients with a clinical diagnosis of ABCA4-associated disease who had a single variant identified by the ABCA4 microarray. Comprehensive phenotypic data were obtained, and the NGS strategy was applied to identify the second allele by means of sequencing the entire coding region and adjacent intronic sequences of the ABCA4 gene. Identified variants were confirmed by Sanger sequencing and assessed for pathogenicity by in silico analysis. Of the 42 variants detected by prescreening with the microarray, in silico analysis suggested that 34, found in 66 subjects, were disease-causing and 8, found in 13 subjects, were benign variants. We detected 42 variants by NGS, of which 39 were classified as disease-causing. Of these 39 variants, 31 were novel, including 16 missense, 7 splice-site-altering, 4 nonsense, 1 in-frame deletion, and 3 frameshift variants. Two or more disease-causing variants were confirmed in 37 (47%) of 79 patients, one disease-causing variant in 36 (46%) subjects, and no disease-causing variant in 6 (7%) individuals. Application of the NGS platform for ABCA4 screening enabled detection of the second disease-associated allele in approximately half of the patients in a British cohort where one mutation had been detected with the arrayed primer extension (APEX) array. The time- and cost-efficient NGS strategy is useful in screening large cohorts, which will be increasingly valuable with the advent of ABCA4-directed therapies.

Outcome of ABCA4 Disease-Associated Alleles in Autosomal Recessive Retinal Dystrophies: Retrospective Analysis in 420 Spanish Families

To provide a comprehensive overview of all detected mutations in the ABCA4 gene in Spanish families with autosomal recessive retinal disorders, including Stargardt's disease (arSTGD), cone-rod dystrophy (arCRD), and retinitis pigmentosa (arRP), and to assess genotype-phenotype correlation and disease progression in 10 years by considering the type of variants and age at onset.Case series.A total of 420 unrelated Spanish families: 259 arSTGD, 86 arCRD, and 75 arRP.Spanish families were analyzed through a combination of ABCR400 genotyping microarray, denaturing high-performance liquid chromatography, and high-resolution melting scanning. Direct sequencing was used as a confirmation technique for the identified variants. Screening by multiple ligation probe analysis was used to detect possible large deletions or insertions in the ABCA4 gene. Selected families were analyzed further by next generation sequencing.DNA sequence variants, mutation detection rates, haplotypes, age at onset, central or peripheral vision loss, and night blindness.Overall, we detected 70.5% and 36.6% of all expected ABCA4 mutations in arSTGD and arCRD patient cohorts, respectively. In the fraction of the cohort where the ABCA4 gene was sequenced completely, the detection rates reached 73.6% for arSTGD and 66.7% for arCRD. However, the frequency of possibly pathogenic ABCA4 alleles in arRP families was only slightly higher than that in the general population. Moreover, in some families, mutations in other known arRP genes segregated with the disease phenotype.An increasing understanding of causal ABCA4 alleles in arSTGD and arCRD facilitates disease diagnosis and prognosis and also is paramount in selecting patients for emerging clinical trials of therapeutic interventions. Because ABCA4-associated diseases are evolving retinal dystrophies, assessment of age at onset, accurate clinical diagnosis, and genetic testing are crucial. We suggest that ABCA4 mutations may be associated with a retinitis pigmentosa-like phenotype often as a consequence of severe (null) mutations, in cases of long-term, advanced disease, or both. Patients with classical arRP phenotypes, especially from the onset of the disease, should be screened first for mutations in known arRP genes and not ABCA4.

DNA nanoparticle-mediated ABCA4 delivery rescues Stargardt dystrophy in mice

Mutations in the photoreceptor-specific flippase ABCA4 are associated with Stargardt disease and many other forms of retinal degeneration that currently lack curative therapies. Gene replacement is a logical strategy for ABCA4-associated disease, particularly given the current success of traditional viral-mediated gene delivery, such as with adeno-associated viral (AAV) vectors. However, the large size of the ABCA4 cDNA (6.8 kbp) has hampered progress in the development of genetic treatments. Nonviral DNA nanoparticles (NPs) can accommodate large genes, unlike traditional viral vectors, which have capacity limitations. We utilized an optimized DNA NP technology to subretinally deliver ABCA4 to Abca4-deficient mice. We detected persistent ABCA4 transgene expression for up to 8 months after injection and found marked correction of functional and structural Stargardt phenotypes, such as improved recovery of dark adaptation and reduced lipofuscin granules. These data suggest that DNA NPs may be an excellent, clinically relevant gene delivery approach for genes too large for traditional viral vectors.

Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy

Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4-/- mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4 -/- mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E. Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters. Abca4-/- mice were injected subretinally with 1 microl ( approximately 5.0 x 10(5) TU) of each EIAV vector in one eye at postnatal days 4 and 5. An injection of saline, an EIAV-null vector, or an uninjected contralateral eye served as a control. Mice were killed at various times after injection to determine photoreceptor (PR) transduction efficiency and A2E concentrations. EIAV-LacZ vectors transduced from 5 to 20% of the PRs in the injected area in mice. Most importantly, a single subretinal injection of EIAV-CMV-ABCA4 to Abca4-/- mouse eyes substantially reduced disease-associated A2E accumulation compared to untreated and mock-treated control eyes. Treated eyes of Abca4-/- mice accumulated 8-12 pmol per eye (s.d.=2.7) of A2E 1 year after treatment, amounts comparable to wt controls, whereas mock-treated or untreated eyes had 3-5 times more A2E (27-39 pmol per eye, s.d.=1.5; P=0.001-0.005). Although extrapolation to humans requires caution, the high transduction efficiency of both rod and cone photoreceptors and the statistically significant reduction of A2E accumulation in the mouse model of STGD1 suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases.

Quantifying fixation in patients with Stargardt disease

Fixational eye movements in 60 eyes of 30 patients with ABCA4-associated Stargardt disease were recorded by a Scanning Laser Ophthalmoscope (SLO). The results were quantified by two new fixation quality measures expressing the eccentricity of the preferred retinal locus (PRL) non-parametrically, and fixation stability by a dynamic index. 46 eyes (77%) fixated eccentrically; in 32 eyes (70% of the eccentrically fixating eyes) the PRL was located above the central retinal lesion. PRL eccentricity correlated positively with logMAR visual acuity ( r = .72; p < .0001) and negatively with fixation stability ( r = −.58; p < .0001). Multiple PRL were found only in three eyes.

ABCA4-Associated Retinal Degenerations Spare Structure and Function of the Human Parapapillary Retina

To study the parapapillary retinal region in patients with ABCA4-associated retinal degenerations. Patients with Stargardt disease or cone-rod dystrophy and disease-causing variants in the ABCA4 gene were included. Fixation location was determined under fundus visualization, and central cone-mediated vision was measured. Intensity and texture abnormalities of autofluorescence (AF) images were quantified. Parapapillary retina of an eye donor with ungenotyped Stargardt disease was examined microscopically. AF images ranged from normal, to spatially homogenous abnormal increase of intensity, to a spatially heterogenous speckled pattern, to variably sized patches of low intensity. A parapapillary ring of normal-appearing AF was visible at all disease stages. Quantitative analysis of the intensity and texture properties of AF images showed the preserved region to be an annulus, at least 0.6 mm wide, surrounding the optic nerve head. A similar region of relatively preserved photoreceptor nuclei was apparent in the donor retina. In patients with foveal fixation, there was better cone sensitivity at a parapapillary locus in the nasal retina than at the same eccentricity in the temporal retina. In patients with eccentric fixation, approximately 30% had a preferred retinal locus in the parapapillary retina. Human retinal degenerations caused by ABCA4 mutations spare the structure of retina and RPE in a circular parapapillary region that commonly serves as the preferred fixation locus when central vision is lost. The retina between fovea and optic nerve head could serve as a convenient, accessible, and informative region for structural and functional studies to determine natural history or outcome of therapy in ABCA4-associated disease.