To our knowledge, this is the first report of ABCA4
mutations in Portuguese STGD patients. To date, over 490 variants have been reported in the largest gene of the ABC family: ABCA4
gene. Some of the variants that have been described are rare and may even be specific to certain specific geographic areas. Therefore different frequency distribution across populations have been reported. In this cohort of Portuguese patients, the most prevalent variant found in 19% of the families, Leu11Pro, is considered a rare mutation in other populations. Even in Spain, and in spite of its geographic proximity, Leu11Pro frequency is significantly lower (<1%) in macular degenerations associated with ABCA4
]. This likely moderate missense substitution, involving a conserved nonpolar amino acid residue, is located in the intracytoplasmic domain of the ABCA4 protein; it has been reported in FFM and in arCRD [12
]. The most frequent mutation in various European countries is p.Gly1961Glu. Although its frequencies range between 11%–21%, it seems to be less common in the Portuguese population (9.5%). Its prevalence is similar to the one found in other south European populations, namely 6.6% in Spanish STGD patients [7
]. Interestingly, the p.Arg1129Leu, which is the most frequent variant in Spain (14.5%) [20
], was not found in any of the 21 STGD Portuguese families studied. These findings are consistent with studies on genetic diversity. Those studies concluded that the Iberian population is not a genetic edge of European variation and might have a higher level of diversity than some neighboring populations, receiving significant North and sub-Saharian African influences at different times [24
]. Therefore, this study might provide further evidence of the importance of molecular analysis of this considerable large and polymorphic gene in different populations.
Also identified in our patient population were four putative pathogenic mutations, two of which are novel variants, that have not been reported in other populations: Family 9, c.2T>C (p.Met1Thr); Family 14, c.400C>T (p.Gln134X) and c.4036_4037delAC (p.Thr1346fs); and Family 12, c.4720G>T (p.Glu1574X). The p.Met1Thr substitution was detected in both chromosomes of a severely affected STGD patient, whose heterozygous mother presented subclinical impairment of retinal function even in absence of any fundus change, as described in our previous report [20
]. Interestingly, this variant is the second substitution residing in the Met1 residue. Previously, Briggs and colleagues [6
] described the p.Met1Val in a heterozygous STGD patient with no other sequence change identified. Therefore, this null mutation may lead to early disease onset, moderate central fundus changes (even as early as one year after disease onset), and residual visual acuity when in the homozygous state. Future functional studies should assess the relative severity of this variant to clarify whether this mutation can be pathologic even in heterozygous state as has been suggested for other ABCA4
mutations, namely p.Gly1961Glu [25
]. The novel nonsense mutation, a G>T transversion leading to p.Glu1574X, involves a highly conserved nucleotide in the ortholog bovine and mouse proteins. This sequence change causes a protein truncation before NBD-2, a functional domain that is believed to diminish ATP hydrolysis by NBD-1, without altering the basal ATPase activity [28
]. Segregation analysis was limited to available relatives: the mother, from whom the p.Val931Met al.lele was inherited, and the paternal aunt, who did not carry any of these disease alleles. The p.Val931Met mutation resides in the NBD-1, what according to the model proposed by Sun et al., has a severe impact in ABCA4 protein function, eliminating both basal and retinal-stimulated ATPase activity [28
]. Interestingly, clinical examination of this patient revealed that retinal damage was limited to the central 15 degrees, a relative late onset and moderate central fundus changes after the second year of disease onset. Therefore, the combination of the two alleles results in a relatively late disease onset with a moderate retinal dysfunction progression.
In Family 14, compound heterozygous of a nonsense mutation at exon 4 (p.Gln134X) and a frameshift in exon 27 (p.Thr1346fs), were associated to a very early disease onset. Even after only 4 years of disease onset, both patients were severely affected and shared dramatically reduced visual acuities. Their mfERG results revealed severely decreased response amplitudes (almost abolished) within the central 30 degrees of the retina and impaired color vision in all three main chromatic axes. This stop mutation affects a 100% conserved nucleotide, before any of the ATP binding domains, leading to a premature stop codon of 134 amino acid residue out of the 2,273 residues of ABCA4 protein that likely undergo nonsense-mediated decay. Additionally, in this family, we found a second null mutation that resides between the two homologous halves of ABCA4, a deletion of a dinucleotide AC at codon 1346. An insertion of a dinucleotide CA affecting the same codon has been detected and has been found to be associated with a severe phenotype (arCRD) [6
]. Therefore, both novel variants are compatible with the dramatically severe phenotype observed in these STGD patients.
Interestingly, in patients from families 9 and 14, two null mutant alleles were identified in each patient. It is worth noting that even considering the severe phenotype presented by those patients, they were diagnosed with STGD disease. However, according to the proposed model, the combination of two null alleles likely accounts to a more severe phenotype as retinitis pigmentosa or arCRD [29
]. Since in both families, STGD patients had short disease progression, we speculate whether their phenotype may evolve (in later stages) to a more severe retinal impairment such as arCRD.
In this study, genotype-phenotype correlations were addressed based on previous extensive phenotype characterization. However, the value of the ABCA4 model proposed for genotype-phenotype might be limited in larger families because of the intrafamiliar phenotypic variation and since the model is mainly based on large set of single patients, not on extensive families. Mutation analysis was performed with a combination of complementary techniques: ABCA4
gene chip, dHPLC, and direct sequencing. This was found to be a successful strategy resulting in high mutation rate detection (67%), compared to other surveys [6
]. It is worth to note the efficiency, specificity and high detection rate of the ABCR400 chip. However, in 3 out of the 21 studied families (14.3%; families 9, 12, and 14), the causal mutations were detected using a combination of dHPLC and direct sequencing technology.
Functional studies to evaluate the biochemical defects caused by the numerous variants identified will help understanding the relative impact of the complex (and single) heterozygous. Therefore, those studies will improve genetic counselling of families affected with ABCA4-related retinal diseases.