Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XLRP)

The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene. In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification. In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls. A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries. Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region. So far, no point mutations in these putative exon sequences have been identified.      

Ten novel ORF15 mutations confirm mutational hot spot in the RPGR gene in European patients with X-linked retinitis pigmentosa

RGPR was the first gene found to be mutated in XLRP, the subtype of RP displaying the most severe form of retinal degeneration with partial or complete blindness in the third or fourth decade of life. Despite the RP3 locus on Xp21.1 accounting for 60-90% of XLRP, only 10-20% of identified RPGR mutations were reported in earlier analyses. This discrepancy appeared to be resolved when Vervoort et al. identified a mutational hot spot in a new purine-rich 3' exon (ORF15) that accounted for 60% of their XLRP patients [Vervoort et al., 2000]. In our mutation screening of 37 unrelated European XLRP patients we identified two recently described deletions and 10 novel mutations in exon ORF15 of RPGR, 4 of which were nonsense and 6 frameshift mutations. The latter included one duplication and 5 deletion mutations, all of which lead to a downstream premature termination. No mutations were detected in the additionally screened new exon ORF14. The data reported here, together with previous findings, document a significant clustering of mutations as well as polymorphisms in ORF15 of RPGR. In our unselected XLRP patient population, ORF15 mutations constitute 32% of cases, a finding that contradicts the results of Vervoort and coworkers [Vervoort et al., 2000] but is in agreement with a more recent study on North American XLRP patients [Breuer et al., 2002]. The observed prevalence is sufficient to justify an initial mutation screening of ORF15 in the genetically heterogeneous group of XLRP.     

Neanthes japonica (Iznka) fibrinolytic enzyme reduced cerebral infarction, cerebral edema and increased antioxidation in rat models of focal cerebral ischemia

X-linked retinitis pigmentosa (XLRP) is the most severe type of retinitis pigmentosa (RP), with patients consistently showing early onset and rapid deterioration. Obtaining a genetic diagnosis for a family with XLRP is important for counseling purposes. In this study, we aimed to identify disease-causing mutations in two unrelated XLRP families. Genetic analysis was performed on two unrelated XLRP families. Genomic DNA was extracted from peripheral blood or amniotic fluid samples. The coding regions and intron/exon boundaries of the Retinitis Pigmentosa GTPase Regulator (RPGR) and RP2 genes were amplified by PCR and then sequenced directly. A clinically unaffected pregnant female and the four month old fetus were found to have a hemizygous 2 base pair deletion (g.ORF15+484_485delAA) in the exon ORF15 of RPGR gene. In another XLRP family, a nonsense mutation (g.ORF15+810G>T) was identified. Neither mutation has been reported previously. Both are predicted to cause premature termination of the protein. In conclusion, we identified a micro-deletion through prenatal genetic diagnosis and another novel nonsense mutation in RPGR-ORF15. Identifying a disease-causing mutation facilitated early diagnosis and genetic counseling for the patients. Discovery of novel mutations also broadens knowledge of XLRP and the spectrum of its pathogenic genotypes.     

Genetic localisation of the RP2 type of X linked retinitis pigmentosa in a large kindred.

Genetic linkage and deletion studies have led to the proposal that there are at least two loci on the X chromosome which are responsible for X linked retinitis pigmentosa (XLRP). One locus (RP3) has been closely defined by genetic linkage and deletion analyses and localised to the region between the ornithine transcarbamylase (OTC) and chronic granulomatous disease (CYBB) loci in Xp21.1-p11.4. The other locus (RP2) has been assigned by linkage analysis alone to region Xp11.4-p11.2, but its localisation is less well defined. The results of a multipoint linkage analysis of a single large XLRP kindred using eight informative loci provide further evidence on the localisation of RP2 to this region. The maximum likelihood location of this locus shows a multipoint lod score of 7.17 close to DXS255 (in Xp11.22) and TIMP (in Xp11.3-p11.23), neither of which show recombination with RP2, in an area extending from 2 cM proximal to DXS7 to 1 cM distal to DXS14 (approximate 95% confidence limits).     

Novel frameshift mutations in the RP2 gene and polymorphic variants

Mutations in the RP2 gene located on Xp11.23 are associated with X-linked retinitis pigmentosa (XLRP), a severe form of progressive retinal degeneration which leads to complete loss of vision in affected males. To date, 14 different mutations in the RP2 gene have been reported to cause XLRP, the majority of which lead to a coding frameshift within the gene and predicted truncation of the protein product. We here report two novel frameshift mutations in RP2 identified in XLRP families by PCR-SSCP and direct sequencing, namely 723delT and 796-799del. Four single nucleotide polymorphisms (SNPs) within the coding region of RP2 are also described (105A>T, 597T>C, 844C>T, 1012G>T), the first polymorphisms to be reported within this gene of unknown function, two of which alter the amino acid sequence. The current study extends the XLRP mutation profile of RP2 and highlights non-pathogenic coding sequence variations which may facilitate both functional studies of the gene and analysis of intragenic allelic contribution to the phenotype.     

Deletions in the 5'' region of dystrophin and resulting phenotypes.

Deletions in the dystrophin gene give rise to both Duchenne and Becker muscular dystrophies. Good correlation is generally found between the severity of the phenotype and the effect of the deletion on the reading frame: deletions that disrupt the reading frame result in a severe phenotype, while in frame deletions are associated with a milder disease course. Rare exceptions to this rule, mainly owing to frameshift mutations in the 5' region of the gene (in particular deletions involving exons 3 to 7) which are associated with a milder than expected phenotype, have been reported previously. In order to characterise better the relationship between genotype and phenotype as a result of mutations arising in the 5' region of the gene, we have studied a large cohort of patients with small in frame and out of frame deletions in the first 13 exons of the dystrophin gene. Fifty-five patients with a deletion in this area were identified; approximately one third of them had a phenotype different from that theoretically expected. Patients were divided into two groups: (1) patients with a severe clinical phenotype despite the presence of a small, in frame deletion and (2) patients with a mild phenotype and an out of frame deletion. Noticeable examples observed in the first group were Duchenne boys with a deletion of exon 5, of exon 3, and of exons 3-13. In the second group we observed several patients with an intermediate or Becker phenotype and out of frame deletions involving not only the usual exons 3-7 but also 5-7 and 3-6. These data indicate that a high proportion of patients with a deletion in the 5' end of the gene have a phenotype that is not predictable on the basis of the effect of the deletion on the reading frame. The N-terminus of dystrophin has at least one actin binding domain that might be affected by the small, in frame deletions in this area. The effect of the in frame deletions of exon 3, 5, and 3-13 on this domain might account for the severe phenotype observed in these patients. Other mechanisms, such as unexpected effect of the deletion on splicing behaviour, might, however, also be implicated in determining the phenotype outcome.     

Identification of novel RP2 mutations in a subset of X‐linked retinitis pigmentosa families and prediction of new domains

X‐linked Retinitis Pigmentosa (XLRP) shows a huge genetic heterogeneity with almost five distinct loci on the X chromosome. So far, only two XLRP genes have been identified, RPGR (or RP3) and RP2, being mutated in approximately 70% and 10% of the XLRP patients. Clinically there is no clearly significative difference between RP3 and RP2 phenotypes. In the attempt to assess the degree of involvement of the RP2 gene, we performed a complete mutation analysis in a cohort of patients and we identified five novel mutations in five different XLRP families. These mutations include three missense mutations, a splice site mutation, and a single base insertion, which, because of frameshift, anticipates a stop codon. Four mutations fall in RP2 exon 2 and one in exon 3. Evidence that such mutations are different from the 21 RP2 mutations described thus far suggests that a high mutation rate occurs at the RP2 locus, and that most mutations arise independently, without a founder effect. Our mutation analysis confirms the percentage of RP2 mutations detected so far in populations of different ethnic origin. In addition to novel mutations, we report here that a deeper sequence analysis of the RP2 product predicts, in addition to cofactor C homology domain, further putative functional domains, and that some novel mutations identify RP2 amino acid residues which are evolutionary conserved, hence possibly crucial to the RP2 function. Hum Mutat 18:109–119, 2001. © 2001 Wiley‐Liss, Inc.     

Five novel RPGR mutations in families with X-linked retinitis pigmentosa

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe because of their early onset, often leading to significant visual impairment before the fourth decade. RP3, genetically localized at Xp21.1, accounts for 70% of XLRP in different populations. The RPGR (Retinitis Pigmentosa GTPase Regulator) gene that was isolated from the RP3 region is mutated in 20% of North American families with XLRP. From mutation analysis of 27 independent XLRP families, we have identified five novel RPGR mutations in 5 of the families (160delA, 789 A>T, IVS8+1 G>C, 1147insT and 1366 G>A). One of these mutations was detected in a family from Chile. Hum Mutat 17:151, 2001.     

The human retinitis pigmentosa GTPase regulator gene variant database

X-linked retinitis pigmentosa (XLRP) is a genetically heterogeneous retinal degeneration. The major subtype of XLRP is RP3, which accounts for 6 to 20% of all RP cases. Mutations in the RP3 gene, called RP GTPase regulator (RPGR), cause a number of different retinopathies. An RPGR database has been created using the Leiden Open Source Variation Database (LOVD) software system and has comprehensive search and analysis tools. This database is a central resource of RPGR sequence variant data for investigators and will facilitate the interpretation of new mutations, variants, and polymorphisms when these are identified in patients. The database is available on the Internet (http://rpgr.hgu.mrc.ac.uk).     

Three novel mutations of the RPGR gene exon ORF15 in three Japanese families with X-linked retinitis pigmentosa.

We describe three new mutations in a recently identified exon, ORF15, of the retinitis pigmentosa GTPase regulator gene (RPGR) in three unrelated Japanese families (Families 1-3) with X-linked retinitis pigmentosa (XLRP). The affected males had typical retinitis pigmentosa (RP), whereas the obligate carrier females showed a wide clinical spectrum, ranging from minor symptoms to severe visual disability. Some carrier females in Families 1 and 2 showed typical RP, most carriers manifested high myopia and astigmatism, and their corrected visual acuity was insufficient. They showed an impairment of cone function following the rod dysfunction and accompanied by refractive errors. Microsatellite analysis of Family 1 revealed that the RP in the family was linked to the RP3 locus. Although one patient in the family had no mutation in the previously published exons 1-19 including exon 15a, he had a single-nucleotide insertion in exon ORF15 (g.ORF15 + 753-754 insG). Likewise, patients in Families 2 and 3 had two-base insertion/deletion in the exon, i.e., g.ORF15 + 833-834delGG and g.ORF15 + 861-862insGG, respectively. These insertional/deletional mutations observed in the three families are all different and new, and are predicted to lead to a frameshift, resulting in a truncated protein. These findings may support the previous hypothesis that RPGR-ORF15 is a mutational hot spot.     

Mutations in the N-terminus of the X-linked retinitis pigmentosa protein RP2 interfere with the normal targeting of the protein to the plasma membrane

The X-linked retinitis pigmentosa (XLRP) gene, RP2, codes for a novel 350 amino acid protein of unknown function. We have identified putative sites for N-terminal acyl modification by myristoylation and palmitoylation in the RP2 protein. The RP2 protein is expressed ubiquitously in human tissues at relatively low levels (0.01% of total protein) and has a predominantly plasma membrane localization in cultured cells, as would be expected if the protein was subject to dual N-terminal acylation. Furthermore, mutagenesis of residues potentially required for N-terminal acylation prevents targeting of RP2 to the plasma membrane and the N-terminal 15 amino acids of the protein appear to be sufficient for this targeting. Our data suggest that the protein is dually acylated and that the palmitoyl moiety is responsible for targeting of the myristoylated protein from intracellular membranes to the plasma membrane. The effect of two mutations, which have been reported as causes of XLRP, R118H and DeltaS6, were investigated. The R118H mutation does not affect the normal plasma membrane localization of RP2; in contrast, the DeltaS6 mutation interferes with the targeting of the protein to the plasma membrane. Therefore, the DeltaS6 mutation may cause XLRP because it prevents normal amounts of RP2 reaching the correct cellular locale, whereas the R118H mutation is in a region of the protein that is vital for another aspect of RP2 function in the retina.     

Genetic mapping of loci for X-linked retinitis pigmentosa

Linkage analysis was performed in three Swedish families segregating for X-linked retinitis pigmentosa (XLRP), using five polymorphic DNA markers assigned to Xp. Individual recombination events were analyzed and two- and five-point linkage analysis was undertaken. In one family, a XLRP locus was mapped to the same position as OTC corresponding to RP3. In two families, a disease locus linked to OTC was excluded. In one family, recombination events indicate a locus for XLRP outside the interval (DXS84-OTC-DXS255-DXS14), most likely on the centromeric side of DXS14.     

Identification of a gene from Xp21 with similarity to the tctex-1 gene of the murine t complex

Long range physical mapping within the p21 region of the X chromosomeIdentified a CpG rich Island approximately 180 kb centromericto the chronic granulomatous disease (CGD) locus. The segmentsadjacent to the CpG Island hybridized to discrete bands In DNAsof several species and when used to screen retinal cDNA librariesled to the Identification of cDNAs that detected a mRNA of 2.1kb in many tissues. Molecular characterization of correspondinggenomic clones of this novel human gene confirmed the originof the cDNA clones and Indicated a genomic structure with fiveexons spanning a total of 9 kb. The complete cDNA sequence revealedthat this gene contained a putative open reading frame of 116amino acids with a 3' untranslated region of 1.74 kb. The aminoacid sequence shows a high degree of similarity to the predictedproduct of the tctex-1 gene of the mouse t complex. As linkagestudies and patients with deletions have Implicated the Xp21region as containing the retInltls plgmentosa defect (RP3),the gene was assessed as a candidate disease gene In RP3 families.A single base pair polymorphism was Identified within the codingregion but no disease associated changes were found by singlestrand conformational polymorphism and sequencing analysis ofamplified exons of 20 RP patients. Analysis of a dinucleotiderepeat polymorphism within this gene In families affected withRP3 suggested refinement of the RP3 region.     

Three novel mutations of the RPGR gene exon ORF15 in three Japanese families with X‐linked retinitis pigmentosa

We describe three new mutations in a recently identified exon, ORF15, of the retinitis pigmentosa GTPase regulator gene (RPGR) in three unrelated Japanese families (Families 1–3) with X‐linked retinitis pigmentosa (XLRP). The affected males had typical retinitis pigmentosa (RP), whereas the obligate carrier females showed a wide clinical spectrum, ranging from minor symptoms to severe visual disability. Some carrier females in Families 1 and 2 showed typical RP, most carriers manifested high myopia and astigmatism, and their corrected visual acuity was insufficient. They showed an impairment of cone function following the rod dysfunction and accompanied by refractive errors. Microsatellite analysis of Family 1 revealed that the RP in the family was linked to the RP3 locus. Although one patient in the family had no mutation in the previously published exons 1–19 including exon 15a, he had a single‐nucleotide insertion in exon ORF15 (g.ORF15 + 753–754 insG). Likewise, patients in Families 2 and 3 had two‐base insertion/deletion in the exon, i.e., g.ORF15 + 833–834delGG and g.ORF15 + 861–862insGG, respectively. These insertional/deletional mutations observed in the three families are all different and new, and are predicted to lead to a frameshift, resulting in a truncated protein. These findings may support the previous hypothesis that RPGR‐ORF15 is a mutational hot spot. © 2001 Wiley‐Liss, Inc.     

Genetic Analysis of FAM46A in Spanish Families with Autosomal Recessive Retinitis Pigmentosa: Characterisation of Novel VNTRs

Retinitis pigmentosa (RP) is a group of retinal dystrophies characterised primarily by rod photoreceptor cell degeneration. Exhibiting great clinical and genetic heterogeneity, RP be inherited as an autosomal dominant (ad) and recessive (ar), X-linked (xl) and digenic disorder. RP25 , a locus for arRP, was mapped to chromosome 6p12.1-q14.1 where several retinal dystrophy loci are located. A gene expressed in the retina, FAM46A , mapped within the RP25 locus, and computational data revealed its involvement in retinal signalling pathways. Therefore, we chose to perform molecular evaluation of this gene as a good candidate in arRP families linked to the RP25 interval. A comprehensive bioinformatic and retinal tissue expression characterisation of FAM46A was performed, together with mutation screening of seven RP25 families.
Herein we present 4 novel sequence variants, of which one is a novel deletion within a low complexity region close to the initiation codon of FAM46A . Furthermore, we have characterised for the first time a coding tandem variation in the Caucasian population.
This study reports on bioinformatic and moleculardata for the FAM46A gene that may give a wider insight into the putative function of this gene and its pathologic relevance to RP25 and other retinal diseases mapping within the 6q chromosomal interval.     

Mapping of X-linked progressive retinal atrophy (XLPRA), the canine homolog of retinitis pigmentosa 3 (RP3)

X-linked progressive retinal atrophy (XLPRA) in the Siberian husky dog is a naturally occurring X-linked retinopathy closely resembling X-linked retinitis pigmentosa (XLRP) in humans. In affected males, initial degeneration of rods is followed by cone degeneration and complete retinal atrophy; carrier females have random patches of rod degeneration consistent with random X chromosome inactivation. By typing the XLPRA pedigree with five intragenic markers [dystrophin, retinitis pigmentosa GTPase regulator ( RPGR ), tissue inhibitor of metalloproteinases 1, androgen receptor and factor IX], we established a linkage map of the canine X chromosome, and confirmed that the order of these five genes is identical to that on the human X. XLPRA was tightly linked to an intragenic RPGR polymorphism (LOD 11.7, zero recombination), thus confirming locus homology with RP3. We cloned the full-length canine RPGR cDNA and three additional splice variants. No disease-causing mutation was found in the RPGR-coding sequence of the four splice variants characterized, a finding similar to approximately 80% of human XLRP patients whose disease maps to the RP3 locus. In addition, there were no significant differences in the proportional expression of each splice variant in normal and pre-degenerate XLPRA-affected retina. Expression of all RPGR splice variants increased later in the disease, when retinas were undergoing active degeneration. The results provide further evidence of cross-species retention of a complex splicing pattern in the 3' portion of RPGR, the functional significance of which is unknown. In addition, the possibility of another disease locus in the RP3 region is supported.