In the current study, 21 ABCA4 mutant variants were found in 12 unrelated Han Chinese families with STGD1 by genetic testing. Among them, three novel variants were identified.
Except F5: II: 1, the onset age of all probands with STGD1 was relatively early. The mean onset age in 12 families was 12.25 years (range 2–34 years), which was younger than that of the previously reported patients with STGD1. The average age of onset in these reported cohorts is mostly around 20 years [18, 24, 25]. However, some studies have also shown that the onset age of STGD1 patients is earlier, with the average age of onset between 10 and 14 years [23, 26, 27].
In our previous research, c.101_106delCTTTAT p.Ser34_Leu35del, c.6563 T > C p.Phe2188Ser, and c.2894A > G p.Asn965Ser have been confirmed to be three prevalent ABCA4 variants in STGD1 patients mainly from eastern China . Interestingly, two frequent variants c.101_106delCTTTAT and c.2894A > G were also detected in 4 of 12 unrelated families in the present study. Based on the fact that all of the 12 families also come from eastern China, such results further illustrate the high frequency of the two mutant variants in this region. Our analyses have revealed that the allele frequencies of these three prevalent variants are about 20% , which are lower than those of some high frequency variants in STGD1 patients from Europe. For instance, the frequencies of p.Gly1961Glu and p.[Leu541Pro;Ala1038Val] may exceed 30% in European populations [25, 28]. Meanwhile, in another study of ABCA4 gene screening for STGD1 patients in Chinese population, the frequencies of the prevalent variants (namely, c.101_106delCTTTAT, c.2424C > G, c.2894A > G, and c.6563 T > C) are actually only 14% . According to the above findings, we speculated that the prevalence of STGD1 in China might be lower than that in European populations. So far, there is no exact statistical data on the STGD1 prevalence in Chinese populations. It is necessary to conduct the epidemiological investigation on a larger number of patients in the future, so as to better understand the disease characteristics of STGD1 in Chinese patients.
In addition, the common mutant variant c.5882G > A p.Gly1961Glu identified in eastern Africa and Europe [28, 29] was also detected in family 5, and c.1761-2A > G was detected in trans with c.5882G > A in proband F5:II:1. It has been reported that STGD1 cases harboring the p.Gly1961Glu allele tend to have a mild disease phenotype, and patients with variant p.Gly1961Glu in either the homozygous or heterozygous states show delayed onset of symptoms and later age of onset. Moreover, p.Gly1961Glu is closely related to the bull’s eye maculopathy phenotype of STGD1 [30,31,32]. Consistent with previous researches, the proband F5:II:1 carrying p.Gly1961Glu presented the highest age of onset (34 years) among all patients and the FAF imaging showed typical bull’s eye maculopathy.
Furthermore, variants c.3262C > A, c.4066C > T, and c.1892G > T that were first confirmed by our group  were also detected in three unrelated families in this study. In proband F10:II:2, c.1892G > T was found in compound heterozygosity with c.1229 T > C and both variants were located in the ECD1 domain. Variant c.3262C > A was detected in two families. In particular, c.3262C > A and c.4066C > T were detected in proband F3:II:1 who was identified as compound heterozygotes. These results further indicated the potential deleteriousness caused by the three ABCA4 mutants.
Of the 21 disease-associated ABCA4 mutants detected in the current study, 18 known variants were detected in all probands, including 12 missense, two splicing, two frameshift, one small deletion, and one nonsense mutants. Among them, 16 known mutants were distributed in 12 exons of the ABCA4 gene. Analysis revealed three dissimilar mutants in exon 22; two dissimilar mutants each in exons 12 and 14; and one mutant each in exons 2, 4, 8, 9, 13, 19, 27, 38, and 42. In addition, two known splicing mutants were identified in two introns of ABCA4 (introns 12 and 39). As presented in Supplementary Table S2, the function prediction of coding variants was performed by four software prediction programs, including MutationTaster, SIFT, FATHMM, and LRT. Through the analyses with four online tools, the functional deleteriousness of 12 known missense mutants was verified by at least three of the four prediction programs. Three severe/null variants, namely, one nonsense mutant c.4066C > T and two frameshift mutants c.1561delG and c.2063_2064insA, could introduce a premature truncating codon in the protein during translation. The three severe/null variants and one inframe deletion variant c.101_106delCTTTAT were predicted to have deleterious effects through the MutationTaster analyses. Moreover, two known splicing variants, c.5584 + 5G > A and c.1761-2A > G, mainly affect the splice donor site and the splice acceptor site, respectively. The detected known variants of ABCA4 were distributed in all protein functional domains except for the exocytoplasmic domain 1 (ECD1). On basis of the online tools analyses and ABCA4 protein structure, p.Lys678Asn and p.Pro1776Leu located in the TMD1 and TMD2 domains might disrupt the transmembrane alpha-helices and give rise to the dysfunction of ABCA4 protein transport. Variants p.Pro143Leu, p.Tyr340Cys, p.Ile410Thr, p.Arg587Lys, and p.Gly631Val in the ECD1 domain might have an impact on the topologically associated domains outside the cell and lead to the loss of protein function. Variants p.Gly1961Glu, p.Arg1108Cys, p.Ser1096Leu, p.Pro1088Thr, and p.Asn965Ser in the NBD1 and NBD2 domains could affect the ATP hydrolysis function of protein through damaging the topologically associated domains in cells. The functional impacts of p.Asn965Ser have been demonstrated in the corresponding in vivo and in vitro studies [33, 34]. The pathogenicity of these variants was determined based on the ACMG guidelines, containing 5 likely pathogenic and 13 pathogenic mutants.
Three novel ABCA4 variants were found in this study, including one splicing and two missense variants. All three mutants showed the dramatically low allelic frequency in dbSNP, 1000 Genomes Project, ExAC, and ESP6500 databases and were not observed in 200 normal controls. Two novel missense mutants, c.3017G > A p.Gly1006Asp and c.5167 T > C p.Tyr1723His, co-segregated with the clinical phenotype of STGD1 in families 11 and 12. In silico analyses of missense variants revealed that c.3017G > A and c.5167 T > C could cause the functional damage in all of four software prediction programs (MutationTaster, LRT, SIFT, and FATHMM). Meanwhile, all of these two variants were detected in trans with the known pathogenic variants (c.3322C > T and c.101_106delCTTTAT), further indicating their possible pathogenicity. More significantly, c.3017G > A was located in a high incidence region of variants, namely, the NBD1 domain [18, 23]. Moreover, codons 1006 and 1723 of ABCA4 were strictly conserved amino acids among dissimilar species, suggesting that these amino acids were essential for the normal protein function. According to the ABCA4 protein structure and the online tools analyses, p.Gly1006Asp in the NBD1 domain might destroy the topologically associated domains in cells, thus interfering with the ATPase activity of the protein. And p.Tyr1723His in the TMD2 domain might bring about the ABCA4 transport dysfunction through breaking the extracellular topological domains. A novel null mutant, c.3051-1G > A in intron 21, was identified and co-segregated with the clinical phenotype of disease in family 7. As shown in Fig. 1, c.3051-1G > A was found in trans with c.2894A > G, which has been confirmed to be a high frequency variant in Chinese populations [18, 23]. According to HSF analyses, variant c.3051-1G > A affects the splice acceptor site. The above-mentioned analysis results confirmed the potential pathogenicity of these three novel mutants. In accordance with the ACMG guidelines, three novel mutants were categorized as likely pathogenic variants.
In the process of screening patients with the clinical phenotype of STGD1, we also detected the variants of other pathogenic genes instead of ABCA4 related to macular degeneration in another three Chinese families, namely, GUCY2D (c.2513G > A), PDE6C (c.967 T > C and c.1579C > T), and POC1B (c.1153G > A and c.458C > T). All of them are the causative genes for cone or cone-rod dystrophy [35,36,37]. The phenotypes of macular degeneration caused by these three genes are very similar to STGD1. The results also fully demonstrate the importance of genetic testing in clinical differential diagnosis.