In order to determine the types and frequencies of RP1 mutations responsible for retinal disease we tested a total of 659 affected probands (). The first set of 56 unrelated probands were screened for mutations in the entire RP1 gene. All 56 probands were members of American families with autosomal dominant RP who tested negative for mutations in rhodopsin, peripherin/RDS and CRX. This initial screen identified two different RP1 mutations in three families. One of these is the nonsense mutation in codon 677 (Arg677X) that was initially identified in the UCLA-RP01 and Australian families and was found in one of the 56 probands tested. The other disease-associated mutation, a 5 bp deletion of bases 2280–2284, was found in two families and results in 16 incorrect amino acids after codon 761 with a termination codon present at 777. This mutation, also referred to as Leu762 (5 bp deletion), has already been observed in one additional adRP family (9
Clinical diagnosis of individuals screened for RP1 mutations
After preliminary results indicated that mutations seem to cluster in a small region of exon 4, we tested an additional 603 probands from American and European families for mutations in this small region. These probands have a range of retinal degenerations including autosomal dominant RP, autosomal recessive RP, isolated RP and autosomal dominant cone or cone–rod dystrophy. The segment tested spans 442 bp in exon 4 from nucleotide 1947 to 2388 (GenBank accession no. AF143222). Mutations in this small region of RP1 were found in 14 additional families with autosomal dominant RP.
We identified a total of eight different mutations in 17 of the 659 probands tested. Since all the mutations were found in probands with adRP, all frequency calculations are based on testing of the 250 probands with adRP. The remaining 409 probands are not included in these calculations since they have other forms of retinal degeneration. All disease-associated mutations and sequence variants identified in this study are listed in .
RP1 mutations and sequence variants detected
Mutation analysis revealed that five unrelated families had a nonsense mutation in codon 677 (Arg677X; 2029C→T) (). This mutation is predicted to result in a severely truncated protein and is the same as the one originally identified in UCLA-RP01 and Australian family D. The Arg677X mutation was seen in both American and European samples and is responsible for ~2% (5/250) of adRP.
Figure 1 Pedigrees of 17 families with retinitis pigmentosa grouped by RP1 mutation, with representative electropherogram for each different mutation. (A) Arg677X mutation; (B) 2280–22284del mutation; (C) 2168–2181del mutation; (D) 2303del mutation; (more ...)
Families with the Arg677X mutation exhibit a range of disease phenotypes. The two original families, UCLA-RP01 and Australian family D, have mild forms of disease. In general, affected members of these families have late onset of night blindness and slow loss of visual acuity and visual fields. All of the families with the Arg677X mutation identified in this study have more severe forms of disease. These families have earlier onset of night blindness and more severe loss of visual acuity and fields. Despite a more severe disease phenotype, two families show variable penetrance and expressivity. One family, Moorfields1512, like UCLA-RP01 and Australian family D, has an instance of non-penetrance, while family UTAD103 has an elderly male with mild symptoms despite the severe disease in his daughter and grandchild.
Mutation testing also identified a 5 bp deletion of bases 2280–2284 (2280–2284del) in five unrelated families with adRP (). This deletion results in 16 incorrect amino acids after codon 761 followed by a stop in codon 777. The 5 bp deletion was present in all affected family members that were tested and was not present in any of the unaffected members tested. This mutation is found in both American and European samples and appears to be responsible for ~2% (5/250) of adRP.
The majority of the families with the 2280–2284del mutation have mild disease with onset at 30–40 years of age, although a few family members have earlier onset and more severe disease. Of interest is the Moorfields1452 family. Even though the sister of the proband is affected also, this family was originally categorized as having an autosomal recessive pattern of inheritance since there was no other family history of disease. It is likely that one of the proband’s parents carries the 2280–2284del mutation, but due to non-penetrance or variable expressivity, has no discernable clinical symptoms.
Analysis of one affected member from two unrelated European families revealed a third type of RP1 mutation, a 14 bp deletion of bases 2168–2181 (2168–2181del) (). This deletion results in 10 incorrect amino acids after codon 722 followed by a premature stop in codon 733. One family with this deletion, Moorfields1614, has another example of apparent non-penetrance.
The remaining RP1 mutations that were identified in this study were each found in only one proband. A 1 bp deletion of nucleotide 2303, was identified in one affected member of RFS103 (). This deletion results in five incorrect amino acids after codon 768 with a premature termination codon at 774. This individual has mild disease with equal loss of rod and cone function. A second 1 bp deletion, 2029del, was found in seven affected members of Leeds356 (). This deletion leads to four incorrect amino acids after codon 676 followed by a stop at codon 681. This deletion is of the same nucleotide that is mutated in the Arg677X mutation.
One insertion mutation in RP1 was identified in this study. One proband was tested and found to have a 1 bp insertion (G) between nucleotides 2169 and 2170 (). This insertion leads to three incorrect amino acids after codon 724 followed by a premature stop codon. This individual has moderate disease with onset of visual field problems in childhood.
Two unique nonsense mutations were also identified. One affected member of Moorfields359 was tested and a nonsense mutation, Glu700X (2098G→T), was identified (). This individual has mild retinal disease that was diagnosed at 41 years of age. The second nonsense mutation, Cys744X (2232T→A), was identified in one proband (). This individual first noticed night blindness at 20 years of age.
Other possible disease-causing variants
Two missense variants were detected in affected probands. Leu1808Pro (5423T→C) was found in one affected member of RFS015 and a second variant, Lys663Asn (1989G→T), was detected in one affected member of Moorfields1474. In both instances no additional family members are currently available for testing. The Leu1808Pro was not seen in any of the other 56 probands tested through this region nor in a panel of 91 unaffected controls. The Lys663Asn variant was not seen in any of the other 658 samples tested.
Three previously unidentified variants were found in RP1. An Arg1595Gln (4784G→A) change in exon 4 was identified in one affected proband. Testing of unaffected controls revealed that this variant is present in ~1% of Caucasians, and therefore not a likely cause of disease. A silent substitution in exon 4, Ile2122Ile (6366T→C), was detected in one of the 56 probands tested through this region.
A third variant was identified in the 5′-untranslated region of exon 1. This variant, −315T→C was present in four affected probands, but not seen in any of the unaffected controls. Testing of additional family members in two of the four families revealed that the variant does not segregate with disease.
We designed PCR primers to flank four different microsatellite repeats that were located near the RP1 gene in BAC 18L28. These markers, D8S2607, D8S2608, D8S2609 and D8S2610, are located 3244, 25 314, 46 316 and 51 019 bp, respectively, from the 3′ end of the RP1 gene. Testing of these markers in CEPH parents showed that all four markers were polymorphic.
These four markers were analyzed in members of the families with the Arg677X, 2280–2284del and 2168–2181del RP1 mutations to determine whether families with the same mutation share a common ancestral haplotype or whether they represent independent mutation events. Due to the limited number of individuals from each family with the 2280–2284del and 2168–2181del RP1 mutations, the phase of marker alleles and the RP1 mutations could not be determined. The marker in phase with the Arg677X mutation could be determined in UCLA-RP01, Australian Family D, RFS137, UTAD103 and Moorfields1512. Three different D8S2607 and two different D8S2608 haplotypes were found among the five families tested (). These data suggest that the RP1 Arg677X mutations arose independently in some of the families or that this is an ancient mutation arising thousands of years ago. A mutation this old would allow for recombination between markers and the RP1 mutation, or for new marker alleles to arise, thus giving the appearance of a new mutation.
Genotype data from families with Arg677stop mutation in RP1