To give a more detailed perspective, what follows is a look at the genes and mutations causing just 1 form of retinal disease, adRP. However, many of the conclusions from the study of adRP are broadly applicable to other inherited retinal diseases. Therefore, this section ends with observations that apply generally to all forms of RP.
In a recent survey, we tested a panel of affected individuals from 200 families with adRP for mutations in most of the known dominant RP genes ().23
To be included in the study, a family had to have a diagnosis of adRP by a knowledgeable clinical specialist and either 3 affected generations with affected females or 2 affected generations with male-to-male transmission. The latter requirement was to reduce the likelihood of including families with X-linked RP. This possibility arises because some mutations in the X-linked gene RPGR
affect female carriers; thus, the disease in these families can be misinterpreted as adRP.44-46
The cohort of patients with adRP was screened (largely by DNA sequencing) for mutations in the protein-coding regions and intronexon junctions of all adRP genes or gene regions causing at least 1% of cases. Open reading frame 15 (ORF15), the “hot spot” for dominant-acting mutations in RPGR
, was also tested in families without male-to-male transmission. Determining whether a novel, rare variant is pathogenic can be challenging.47
We used several computational and genetic tools for this purpose.23
Generally, once a definite disease-causing mutation was identified in a family, other genes were not tested further in these individuals.
We found definite or probable mutations in 53.5% of the families with adRP. In subsequent studies, we tested several of the remaining families for linkage to genetic markers within or close to the known adRP genes and to RPGR
The logic here was to uncover mutations that might have been missed by sequencing or to locate genes that have been mapped but not identified yet. In 1 large family, we found linkage to the PRPF31
gene, even though careful resequencing failed to disclose a DNA change. Further testing revealed that affected members of the family have a complex deletion and insertion in PRPF31
. This rearrangement was not detected earlier because only the nondeleted, homologous chromosome was sequenced; that is, the deletion is “invisible” to sequencing.
We then tested the remaining families for deletions in PRPF31
using multiplex ligation-dependent probe amplification (MLPA).48,49
Surprisingly, we found 4 large deletions, including 2 that encompass genes adjacent to PRPF31
This brings the fraction of detected mutations to 56% ().
These studies have a number of implications that go beyond just adRP. First, 14 different, common mutations account for up to 30% of the families with adRP in this survey; that is, each of these mutations accounts for at least 1% of the cases.23
Thus, screening for this handful of mutations alone will resolve at least 30% of the cases. Common mutations are found in other RP genes, and numerous inexpensive, high-throughput techniques exist for detecting these variants.50,51
Second, another 20% of mutations were novel and could only be detected by sequencing entire genes. Further, each novel mutation requires careful evaluation of pathogenicity. As a consequence, the main bottleneck in genetic testing of patients with RP is the need to screen and analyze many genes by expensive, time-consuming methods. Fortunately, promising high-throughput resequencing techniques, such as microarray gene chips, may relieve this bottleneck.52
Nonetheless, interpretation of novel, rare variants will still require professional evaluation.47
Third, some families thought to have adRP actually have digenic or X-linked mutations. Digenic RP is the result of 1 mutation in RDS
and a second in ROM1
Different individual mutations in RDS
can cause adRP, but each of the digenic mutations alone is not pathogenic. Digenic and polygenic inheritance is true of other forms of retinal disease, such as Bardet-Biedl syndrome, which can be “triallelic.”9
Another misleading mode of inheritance among families diagnosed with “adRP” is X-linked inheritance of RPGR
mutations with significant disease in carrier females.44-46
Both of these phenomena are important reminders that the molecular diagnosis can radically change genetic counseling.
Fourth, at least 2.5% of adRP mutations are genomic rearrangements or deletions in PRPF31
that are not detectable by conventional screening methods.24
Whether there are disease-causing deletions in other adRP genes or in recessive or X-linked genes is an active area of research. This is likely, though, because deletions are a common cause of other inherited and acquired diseases.54-56
For example, large deletions cause up to 17% of familial breast cancer.57
The existence of disease-causing deletions has significant implications for molecular testing of patients with RP. For one, routine testing methods may miss deletions (eg, sequencing does not detect the breast cancer deletions). For another, deletions may explain reported anomalies in the frequency and segregation of RP mutations. If so, here again, the molecular diagnosis will affect counseling. Finally, this finding suggests that there may be other subtle mutations in known RP genes that are missed by standard methods.
Fifth, there are definitely additional, unknown adRP genes. We failed to detect mutations in 40% of the families we tested. Some, but not all, of the remaining mutations may be deletions or subtle changes in known genes that have not been detected to date.58
Linkage mapping continues to locate new adRP genes—most recently RP31
Likewise, new recessive and X-linked genes are reported regularly.1
It is impossible to predict whether there are several or many more RP genes that have yet to be discovered. Completion of the Human Genome Project, new high-throughput screening methods, and development of powerful bioinformatic approaches have dramatically reduced the time it will take to find new genes. In spite of these technical advances, the need for thorough, knowledgeable, innovative clinical characterization of patients and families has never been greater.