Stargardt macular degeneration [STGD/fundus flavimaculatus (FFM), OMIM #248200] is the most common type of hereditary macular dystrophy, which affects approximately 1 in 10,000 individuals (1–3). Characteristic features include the loss of central vision in the first or second decade of life (4), progressive atrophy of the macula and underlying retinal pigment epithelium and the frequent presence of yellow or white lipofuscin deposits in the posterior pole of the retina (5). To date, mutations in more than 3 genes have been identified as causing Stargardt’s disease. STGD1 is the most frequent type of juvenile macular dystrophy, which may be caused by the inheritance of ATP-binding cassette, subfamily A, member 4 (ABCA4) in an autosomal recessive manner (6). STGD3 may arise from the autosomal dominant inheritance of elongation of very long chain fatty acids 4 (ELOVL4) (7) and has a clinical profile that is very similar to STGD1. Mutations in the peripherin-2 (PRPH2) gene are an important cause of multifocal pattern dystrophy with Stargardt-like flecks (8).

The ABCA4 gene (OMOM: 601691) contains 50 exons and encodes a photoreceptor-specific ATP-binding cassette transporter (ABCR). To date, over 600 different ABCA4 mutations are known (9,10); however, the most frequent disease-associated ABCA4 alleles have each been described in only 10% of STGD patients (11). Complex ABCA4 alleles (2 pathogenic variants in ABCA4) are common. Furthermore, approximately 1 in 20 people across all populations carry a potentially disease-associated variant of this gene (12); therefore, the molecular scanning and analyses of ABCA4 are labor-intensive.

ELOVL4 (OMOM: 605512) contains 6 exons and encodes a putative protein of 314 amino acids which is involved in fatty acid chain elongation. Three disease-associated mutations in exon 6 of ELOVL4 have been reported (7,13,14). PRPH2 (OMOM:179605) contains 3 exons. Nine different disease mutations have been identified in PRPH2(8,15,16).

Mutation analyses have been performed as a cost-effective method of diagnosis (9,17). However, to date, mutation analyses of Chinese patients with STGD are rare (18). In order to evaluate the role that the ELOVL4, PRPH2 and ABCA4 genes play in STGD in Chinese patients, we analyzed the coding exons and the adjacent regions of the ELOVL4 and PRPH2 genes and 3 coding exons of the ABCA4 gene in 41 patients from unrelated Chinese families with STGD or suspected STGD.

After sequencing 3 exons of the ABCA4 gene from 41 STGD or suspected STGD patients, 3 novel heterozygous missense mutations in the ABCA4 gene were identified (Table II and Fig. 1A): c:2633C>A (p:Ser878X) mutation in exon 17, c:5646G>A (p:Met1882Ile) mutation in exon 40, and c:6389T>A (p:Met2130Lys) mutation in exon 47. All were absent from the 176 normal individuals and were predicted to be pathogenic. The C>A change at residue 2633 in exon 17 of the ABCA4 gene, where serine was replaced by a stop codon at codon 878, resulted in a premature termination. The 2 missense mutations (p:Met1882Ile and p:Met2130Lys) were predicted by PolyPhen2 to be damaging and were highly conserved for ABCA4 (Fig. 1B). In addition, a previously reported synonymous variation, c:5682G>C (p:Leu1894Leu), was observed in exon 40 of ABCA4(17).

There were 2 variations in ELOVL4 and 4 variations in the PRPH2 gene (Table II). For the novel variation c:699G>A in ELOVL4 observed in 2 patients, the substitution did not change the amino acid. Five single nucleotide polymorphisms in the ELOVL4 and PRPH2 genes (rs3812153, rs7764439, rs390659, rs434102 and c:929G>A) were detected. The homozygous variation (c:929G>A) has not been previously reported.

Patients with pathogenic mutations in ABCA4 had moderately reduced visual acuity in the second decade of life (Table III). All patients showed a blunted foveal reflex. The patient with a substitution of Ser878X had yellow or white lipofuscin deposits in the posterior pole of the retina, while the patient with Met2130Lys presented with a macula having a bronze metal appearance.

STGD presents in a wide range of phenotypes from mild FFM to multifocal pattern dystrophy. There is not an absolute STGD clinical phenotype; it has a wide degree of variability. All clinical features are also characteristic of age-related macular degeneration (21). The correct diagnosis and precise clinical data may be helpful for mutation-disease association studies. STGD1 may be distinguished from other macular degenerations by its autosomal recessive pattern of inheritance, and its hallmark feature is the presentation of a dark choroid during fluorescein angiography examination. There is not an absolute STGD3 clinical phenotype, except for its expression as an apparently dominant form. FFM is a late-onset, moderate form of STGD which is caused by mutations in PRPH2.

We screened the ELOVL4 and PRPH2 genes in 41 STGD patients or suspected STGD patients. Six variations were detected, but there were no pathogenic variations in ELOVL4 and PRPH2. ELOVL4 encodes an enlongase enzyme involved in the elongation of very long-chain fatty acids. All 3 known disease mutations are located in exon 6, resulting in a truncated protein missing the C-terminal segment (7,13,14). It is notable that all mutations were detected in large autosomal dominant families. In our study, most of the probands were sporadic, and there was no large autosomal dominant family in our STGD subjects. Although mutations in PRPH2 are a significant cause of multifocal pattern dystrophy with Stargardt-like flecks (7,8), there was no pathogenic variation in PRPH2 in our subjects. Similarly, Zernant et al did not find disease-associated mutations in the peripherin/RDS or ELOVL4 genes of 30 to 40 STGD patients with no mutations in ABCA4(22) and Lai et al did not find disease-associated mutations in the RDS or ELOVL4 genes in an autosomal dominant STGD3-like macular dystrophy pedigree (18). Extensive family data and typical clinical features may be helpful for pinpointing the possible genetic causes (23).

Mutations in ABCA4 are responsible for almost all cases of classic Stargardt’s disease (24). Frequent ethnic group-specific ABCA4 alleles have been identified (25). We screened the 3 exons (17, 40 and 47) of ABCA4 as disease-associated mutations in these exons have been reported more often in the Asian population (27–29). We found 3 novel mutations in the 3 exons: c:2633C>A (p:Ser878X), c:5646G>A (p:Met1882Ile) and c:6389T>A (p:Met2130Lys). The 3 missense variations were pathogenic, since the individuals with those mutations were STGD patients confirmed by precise clinical data. We found 3 novel variations in only 3 exons, which expanded the mutation spectrum of ABCA4. There are more than 600 variations and some complex alleles in ABCA4. Genetic analyses of ABCA4 are complicated. High-throughput and cost-effective screening tools are helpful, including the ABCA4 genotyping microarray (26) and next-generation sequencing strategy (22). We will screen all 50 exons of ABCA4 with high-flow analysis techniques in future studies.

In conclusion, our results not only reveal that no pathogenic variations in ELOVL4 and PRPH2 were detected in our Chinese STGD patients but also imply that ABCA4 is likely to be significant in Chinese STGD patients.