Next-generation sequencing of a large cohort of STGD1 patients, who had been screened previously with the ABCA4 array, discovered many new mutations in the coding region of ABCA4
. NGS revealed 57 new disease-associated variants in 59 patients, and three more possibly pathogenic intronic and synonymous variants in another 11 patients. Therefore, though the (updated) array still remains a cost- and time-efficient first-pass screening tool, sequencing on an NGS platform would be a much more comprehensive approach. Determining the pathogenicity of rare missense (and also splice-affecting and synonymous) variants remains a significant challenge, especially for the ABCA4
gene, for which close to 700 possibly disease-associated variants have now been identified. Because more than half of these have been detected only once, the unequivocal classification for disease association, which is usually accomplished by segregation and functional analyses, presents an almost impossible task. Given that ABCA4
-associated diseases are recessive, any given patient often represents the only affected member in a family with no siblings. Although determining the phase is often possible if parental samples are available, unequivocal segregation with the disease is often complicated or impossible. Functional analysis of the ABCA4
variants is also complicated because ABCA4
is expressed only in photoreceptors, which means no affected tissue can be obtained from patients, and because no direct functional test is available. Many frequent ABCA4
variants have been analyzed indirectly, such as by testing their effects on protein yield, folding, and ATP-binding and ATPase activity assays in mostly in vitro systems10,24
and Xenopus laevis
Performing these experiments for hundreds of rare variants is unrealistic at this time.
In the present study the new variants were analyzed in a multistep process. First, the frequent variants were removed from the analysis by filtering against public databases. Deleterious mutations (nonsense and ins/del) were considered pathogenic based on their truncating or frameshift effect, or both, on the ABCA4 protein. Missense mutations were analyzed with well-known in silico predictive programs, PolyPhen and SIFT, which have shown to be approximately 80% reliable in correctly predicting functional variants.26
If family members were available, variants were analyzed for segregation with the disease in pedigrees (). Some variants were also screened in a large control cohort (364 persons) from our AMD studies, which includes ethnically matched controls older than 60 years of age without any retinal pathology as documented by thorough eye examination. However, given that all but seven new variants were found only once in 159 STGD1 patients, the screening of 364 controls had limited value. Rare synonymous changes in the ABCA4
ORF were analyzed for their effect on splicing with in silico programs and for codon use. All rare intronic variants were analyzed with splicing prediction programs.
Screening of the ABCA4 gene with any method is still far from 100% efficient. Even after complete sequencing of ABCA4 coding region in patients with definitive clinical diagnosis of STGD1, approximately 25% to 30% of patients remain with one identified pathogenic mutation, and no mutations were found in approximately 15% to 20%. The three most likely reasons for not finding all mutations were that a small subset (~1%) of patients harbored CNVs undetected by PCR-based methods, a significant fraction of pathogenic mutations were outside the ABCA4 coding sequences, and some patients had diagnoses of ABCA4-associated diseases that were phenocopies (diseases caused by mutations in other known or yet to be discovered genes).
CNVs are predicted to be rare in the ABCA4 locus because several studies have found only a few cases (~1% of all patients) with large (entire exon or chromosomal segment) deletions that avoid PCR-based detection methods. However, for complete mutational scanning, CNV analysis with an array comparative genomic hybridization approach, or with multiplex ligation-dependent probe amplification, could be included.
The present study also determined that many pathogenic mutations are likely located outside the ABCA4 ORF because the second mutation, required for the genetic diagnosis of STGD1, was not found in approximately half of all patients with one mutation. Although one could argue that some of these patients could be carrying the ABCA4 variant by chance because of the high population frequency of ABCA4 variants (estimated 1:20), it is highly unlikely that a patient with a clinical diagnosis of STGD1 and carrying one mutation does not have the second pathogenic variant. Detection of disease-associated variants outside the ABCA4 coding sequences will be accomplished by sequencing of the entire 130-kb ABCA4 genomic locus in patients with one identified mutation, a study that is in progress.
Finally, in patients in whom no disease-associated variants are found, whole exome or genome approaches can be used to determine new gene mutations that cause STGD-mimicking phenotypes. This approach can be preceded by sequencing known genes, such as RDS/PRPH2
(gene for multifocal pattern dystrophy, 3 exons),27,28 ELOVL4
(dominant STGD-like disease gene, 6 exons),29,30 VMD2
(Best disease gene [recessive forms resemble STGD], 11 exons), RS1
(retinoschisis gene, 6 exons),31
(achromatopsia gene, 17 exons).32
In our studies, however, we have not found disease-associated mutations in RDS
genes in 30 to 40 STGD patients with no mutations in ABCA4
; therefore, the yield by this approach is expected to be limited.
The fraction of genocopies (i.e., clinical misdiagnoses) at a given clinic depends primarily on the depth of clinical analyses. At our centers patients have primarily undergone detailed clinical work-up with all the techniques shown in Materials and Methods; therefore, the fraction of genocopies is expected to be small. However, in an average clinic, diagnosis is based mainly on ophthalmoscopic examination (fundus photography) with only a few additional techniques (e.g., BCVA, OCT, microperimetry) yielding less stringent criteria for final diagnosis. Moreover, even exceptionally extensive clinical data are often not enough for pinpointing the possible genetic cause. As a reminder, depending on the severity of the ABCA4 mutation and the stage of the disease diagnosed, ABCA4-associated pathology presents in a wide range of phenotypes from mild fundus flavimaculatus to CRD and even RP-like phenotypes. The latter two phenotypes are caused by tens of distinct genes.
Given the substantially overlapping phenotypes and several treatment options currently in late stages of preclinical development or in clinical trials, the correct and comprehensive molecular diagnosis of ABCA4-associated diseases is crucial. The NGS platform is a time- and cost-efficient tool to analyze large and variable genes simultaneously in large cohorts and could be used for diagnostic applications.