Novel ABCA4 mutation leads to loss of a conserved C-terminal motif: implications for predicting pathogenicity based on genetic testing



Mutations in the ABCA4 gene result in a broad spectrum of severe retinal degeneration, including Stargardt macular dystrophy, fundus flavimaculatus, autosomal recessive retinitis pigmentosa, and cone-rod dystrophy. In addition to the detection of well-characterized mutations, genetic testing frequently yields novel variants of unknown significance. The purpose of this report is to describe an approach to aid in the assessment of genetic variants of unknown significance.

Case report

We report an 11-year-old girl with Stargardt disease harboring novel compound heterozygous deletions of ABCA4 (c.850_857delATTCAAGA and c.6184_6187delGTCT). The pathogenicity of these variants was otherwise unknown. Both deletions introduce premature stop codons and are localized within the open reading frame of ABCA4. The c.850_857delATTCAAGA occurs early in the gene and leads to a significantly truncated protein of only 317 amino acids. The c.6184_6187delGTCT, is localized to the 3’ terminus of the ORF and results in removal of the last 161 out of 2,273 amino acids of ABCA4, including the VFVNFA motif, which has been shown to be critical in ABCA4 protein function. Homology-based protein modeling of ABCA4 harboring this deletion suggests significant alterations in the protein structure and function.


Our analyses allowed us to classify novel variants in ABCA4 as being clearly loss-of-function mutations, and thus pathogenic variants. In cases of variants of unknown significance, appraising the protein structure-function consequences of genetic mutations using in silico tools may help to predict the clinical importance of variants of uncertain pathogenicity.

Post author correction

Article Type: CASE REPORT




Nutsuchar Wangtiraumnuay, Jenina Capasso, Mai Tsukikawa, Alex Levin, Esther Biswas-Fiss

Article History


Financial support: Supported in part by the Foerderer Fund (A.L.), the Robison D. Harley, MD Endowed Chair in Pediatric Ophthalmology and Ocular Genetics (A.L.), and a grant from the National Institutes of Health (NEI, EY 013113) (E.B.-F.).
Conflict of interest: None of the authors has conflict of interest with this submission.

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The ABCA4 gene when mutated results in a spectrum of retinal degeneration, including Stargardt macular dystrophy, fundus flavimaculatus, autosomal recessive retinitis pigmentosa, and cone-rod dystrophy (1). Over 800 disease-associated ABCA4 gene mutations have been reported have been reported. Diagnostic use of ABCA4 sequencing is increasingly used (2). Genotyping microarray chips for ABCA4 can identify >98% of the most common mutations. DNA sequencing may identify novel variants with uncertain significance or lack of consensus regarding pathogenicity. Mutations in other genes may result in Stargardt disease phenocopies (3). An ABCA4 sequence variant can therefore not be presumed to cause the patient’s phenotype. Understanding the functional implications of genetic variants at the protein level can allow prediction of protein loss of function or toxic gain of function.

In this report, we describe 2 novel ABCA4 variants in a patient with Stargardt disease. Bioinformatic and in silico analysis of the functional consequences of these variants provided compelling evidence for pathogenicity.

Case report

An 11-year-old otherwise healthy girl presented with gradual vision decrease in both eyes over the preceding 4 years. She was the product of an uncomplicated pregnancy born to a healthy Filipino mother and Italian/Irish father with no known family history of ocular disease. The mother and father were asymptomatic but not examined.

The proband underwent full consultative ophthalmic examination at the Ocular Genetics Clinic at Wills Eye Hospital including visual acuity, slit-lamp, and dilated fundus examination. Fundus autofluorescence and spectral-domain optical coherence tomography (Spectralis; Heidelberg Engineering), Goldmann visual field (Octopus 900 perimeter; Haag-Streit International), and intravenous fluorescein angiography were obtained. Full-field electroretinogram (Espion; Diagnosys LLC) and multifocal electroretinogram (Veris V.6.4.3; EDI Inc.) were performed in accordance with the International Society of Clinical Electrophysiology and Vision standards.

Best-corrected visual acuity was 20/125 right eye and 20/200 left eye. The patient demonstrated eccentric fixation. Pupillary responses were normal. Slit-lamp examination was normal. Fundus examination revealed healthy optic nerves and retinal blood vessels, bilateral macular geographic pigmentary stippling with subretinal flecks in and around this area, and a blunted internal limiting membrane reflex (Fig. 1). Peripheral retina was normal.

Top row: right eye; bottom row: left eye. (A) Fundus photographs demonstrate lipofuscin deposits in the posterior macula. (B) Fundus autofluorescence shows hyperfluorescent flecks throughout the posterior pole and central geographic areas of hypofluorescence surrounded by a ring of hyperfluorescence. (C) Spectral-domain optical coherence tomography reveals foveal thinning, subretinal deposits, and loss of photoreceptors. (D) Multifocal electroretinogram demonstrates marked decrease in amplitudes with some peripheral recovery.

Fundus autofluorescence revealed hyperfluorescent flecks throughout the posterior pole extending to the midperiphery and a central geographic area of mixed hypofluorescence and hyperfluorescence surrounded by a hyperfluorescent ring (Fig. 1). Intravenous fluorescein angiography showed silent choroid. Multifocal electroretinogram showed severely decreased amplitude in both eyes centrally and paracentrally with mild improvement in the peripheral macula (Fig. 1). Full-field electroretinogram waveforms were normal in response to scotopic and combined flash stimuli in both eyes. The single flash photopic and 30-Hz flicker stimuli waveforms were mildly decreased in amplitude and delayed. Goldmann visual field suggested an area of relative scotoma centrally in each eye. Optical coherence tomography showed foveal thinning (119 µm right eye, 116 µm left eye), loss of the photoreceptors in the macular geographic areas, and subretinal flecks extending outward from this area (Fig. 1).

A macular dystrophy/degeneration/Stargardt disease sequencing panel (Emory Genetics Laboratory) including ABCA4, BEST1, CDH3, CNGB3, EFEMP1, ELOVL4, FSCN2, GUCA1B, PROM1, PRPH2, RBP4, RDH12, RPGR, RPGRIP1, and TIMP3 was used. Direct sequencing of the amplified captured regions for each gene was performed using next-generation short base pair read sequencing. The sequencing panel revealed 2 novel nonsense variants in ABCA4: a maternally inherited heterozygous c.850_857delATTCAAGA in exon 7 and a paternally inherited heterozygous c.6184_6187delGTCT in exon 45. Bioinformatic analysis of the structural and functional consequences of these variants was conducted using SWISS-MODEL and I-TASSER hierarchical protein structure prediction tools (4, 5).

Bioinformatic analysis demonstrated that both of these variants mapped to the ABCA4 open reading frame (ORF). Each introduces premature stop codons. The c.850_857delATTCAAGA occurs early in ABCA4 and leads to loss of a large portion of the polypeptide, 1,956 amino acids. The c.6184_6187delGTCT also leads to a premature stop codon, with loss of a total of 161 amino acid residues from the very C-terminal end, including the VFVNFA motif, which is highly conserved among ABCA transporters (Fig. 2). Additionally, as a consequence of the c.6184_6187delGTCT variation, in the region upstream of the premature truncation, 51 amino acid residues at the C-terminus were not homologous to the wild-type protein (Fig. 2). Superimposed homology models of the C-terminal nucleotide binding domain 2 (NBD2), developed using SWISS MODEL and I-TASSER tools, demonstrated loss of significant peptide sequences with β-sheet structure in the NBD2 domain of the truncated protein (Fig. 2).

Schematic representation of genetic variants and superimposed homology models of the C-terminal NBD2 domain of ABCA4. (A) Linear diagram of wild-type ABCA4 polypeptide alongside variants. (B) Homology models of mutant (red) and wild-type domains (deep blue and light blue) NBD2 in the c.6184_6187delGTCT variant. Regions where wild-type structure is homologous to the mutant is shown in light blue. The model demonstrates the loss of a significant β-sheet domain. (C) Protein sequence alignment displays the sequence from the amino acid residue 2051 to the end of each polypeptide chain, comparing c.6184_6187delGTCT (bottom) with wild-type sequence (top). Black shows normal sequence of ABCA4 protein while red shows the amino acid change and premature stop leading to loss of a C-terminal part of the protein.


The ABCA4 gene encodes an important protein involved in the retinoid cycle of rod and cone photoreceptors. The polypeptide consists of 2,273 amino acids including 2 transmembrane domains (TMD1, TMD2), 2 exocytoplasmic domains (ECD1, ECD2), and 2 nucleotide binding domains (NBD1, NBD2) (Fig. 3). ABCA4 functions in the transport of N-retinylidene-phosphatidylethanolamine (R-PE) from the luminal to the cytoplasmic side of disc membranes (6, 7). Loss of ABCA4 transport activity is believed to lead to the accumulation of toxic all-trans retinal derivatives (lipofuscin) in the rod and cone cells, leading to apoptosis of the supporting retinal pigment epithelium (RPE) and, eventually, the photoreceptors themselves.

Topologic organization of ABCA4. Pictorial representation shows soluble domains of ABCA4, nucleotide binding domain 1 (NBD1) aa 854-1375, nucleotide binding domain 2 (NBD2) aa 1897-2273, exocytoplasmic domain 1 (ECD1) aa 62-646, and exocytoplasmic domain 2 (ECD2) aa 1395-1680.

Genetic testing has become a valuable tool for diagnosis of many diseases. In silico prediction, segregation analysis information, and disease-associated databases are primary means to assess pathogenicity of sequence variants. Despite the availability of these tools, many variants are difficult to assess and classify. Molecular modeling comparisons of wild-type versus mutant proteins, although commonplace in biological research, are not routinely employed in clinical practice. The routine sequencing of DNA in ophthalmologic cases results in the detection of fully characterized mutations, with well-described phenotypes, as well as novel variants of unknown significance. Our study demonstrates that correlation of genotype with protein structure and function can help to comprehensively assess pathogenicity.

Our patient’s phenotype was consistent with Stargardt disease (8). Genetic testing revealed novel compound heterozygous ABCA4 deletions, c.850_857delATTCAAGA and c.6184_6187delGTCT. Segregation analyses showed that both parents are asymptomatic carriers. With no prior reports of these variants, the pathogenicity of these alterations is uncertain. Without this knowledge, diagnosis and genetic counseling were tentative.

Bioinformatic assessment of the c.850_857delATTCAAGA mutation showed that it resulted in a truncated 317 amino acid polypeptide, devoid of several essential domains of the ABCA4 transporter. The c.6184_6187delGTCT mutation led to a premature stop codon at the C-terminal end of the protein, resulting in a loss of a total of 161 amino acid residues. Although less than 7% of the protein was absent, the important VFVNFA motif, present within the last 30 amino acids of the NBD2 domain, was deleted (Fig. 2). This motif is known to be critical to ABCA4 protein function, is highly conserved among members of the ABCA transporter subfamily, and has also been linked to Tangier disease in the ABCA1 protein (9). Removal of this motif in ABCA4 leads to a loss of retinal stimulated ATPase in vitro and energy transduction of the transporter (9, 10). Protein modeling predicted a loss of an essential β-sheet, which significantly altered its structure. The NBD domains are sites of ATP hydrolysis that provide energy for transport of R-PE through rod outer segment membranes. Enzymatic studies suggest that the NBD2 domain in particular provides energy necessary for translocation of retinal derivatives generated in the visual cycle. The structural changes in NBD2 would affect ABCA4 transporter’s ability to transport retinoids, leading to accumulation of cytotoxic lipofuscin in RPE cells and ultimately photoreceptor cell death.

Bioinformatic analyses can enhance clinical genetic testing and improve assessment of novel variants. Our findings allowed for confident determination that the two ABCA4 sequence variants are pathogenic. The results of this study led to adequate explanation of the phenotype of each variant and family counseling.


Financial support: Supported in part by the Foerderer Fund (A.L.), the Robison D. Harley, MD Endowed Chair in Pediatric Ophthalmology and Ocular Genetics (A.L.), and a grant from the National Institutes of Health (NEI, EY 013113) (E.B.-F.).
Conflict of interest: None of the authors has conflict of interest with this submission.
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  • Pediatric Ophthalmology and Ocular Genetics, Wills Eye Hospital, Philadelphia, Pennsylvania - USA
  • Department of Ophthalmology, Queen Sirikit National Institute of Child Health, Bangkok - Thailand
  • Departments of Pediatrics and Ophthalmology, Sydney Kimmel Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania - USA
  • Department of Medical Laboratory Sciences, University of Delaware, Newark, Delaware - USA

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