Detection of alterations in all three exons of the peripherin/RDS gene in Swedish patients with retinitis pigmentosa using an efficient DGGE system.

AIMS: To develop a sensitive mutation screening procedure suitable for routine analysis of the peripherin/RDS gene, and to estimate the nature and prevalence of peripherin/RDS gene mutations in Swedish patients with autosomal dominant retinitis pigmentosa. METHODS: To make the method as sensitive as possible, as many as eight segments, covering the three exons and the flanking intron sequences of the peripherin/RDS gene, were analysed by denaturing gradient gel electrophoresis. A group of 38 Swedish patients with a clinical diagnosis of autosomal dominant retinitis pigmentosa were screened for mutations in the peripherin/RDS gene. RESULTS: Three point mutations were found in four of the patients and five polymorphisms were defined. One mutation in exon 1, R172W, has been described previously in other ethnic groups as causing a macular degeneration. Another mutation, in exon 2 and causing the substitution F211L, was found in two unrelated patients. A third mutation, resulting in the likely non-pathogenic substitution S289L, as well as a polymorphism not reported previously, was found in exon 3. CONCLUSIONS: The screening procedure described allows detection of mutations in all of the exons, including the polymorphic 5' and 3' ends of the gene, and is therefore suitable for routine screening of peripherin/RDS gene defects in patients with autosomal dominant retinitis pigmentosa. The frequency of mutations found in the Swedish patient group indicates that defects in the peripherin/RDS gene might be a more common cause of autosomal dominant retinitis pigmentosa than was thought previously.     

Retinitis pigmentosa and related disorders: Phenotypes of rhodopsin and peripherin/RDS mutations

Retinitis pigmentosa comprises a group of clinically variable and genetically heterogeneous inherited disorders of the retina. It is estimated that approximately 1.5 million people throughout the world are affected by this disease. It is a slowly progressive disorder and causes loss of night vision and peripheral visual field in adolescence. It can be inherited through an autosomal dominant, recessive, or X-linked mode; the autosomal dominant form is considered to be the mildest form. Molecular genetic studies on the autosomal dominant disorder have shown that, in some families, genes encoding the rhodopsin and peripherin/RDS map very close to the disease loci identified previously by the systematic linkage analyses. These results, together with the observation that a recessive nonsense mutation in the Drosophila opsin gene causes photoreceptor degeneration, prompted an extensive search for the alterations in the human rhodopsin and peripherin/RDS genes in families with autosomal dominant retinitis pigmentosa. As a result, several distinct rhodopsin and peripherin/RDS mutations have been found in approximately 30% of all autosomal dominant cases. A wide variety of clinical expression of the disorder even within a family with the same mutation, its late onset, slow progression, and cone degeneration clearly suggest that some other factors or genes in addition to rhodopsin are responsible for the phenotypic expression of the disorder. In this article, an attempt is made to highlight some of these recent developments and to correlate the various mutations and the phenotypes. © 1994 Wiley-Liss, Inc.     

Polymorphic variation within "conserved" sequences at the 3' end of the human RDS gene which results in amino acid substitutions.

The human RDS gene, previously mapped to chromosome 6p, encodes a protein found in the outer disc membrane of the photoreceptor cells of the retina. The cDNA sequence of the human gene shows 85% identity with the bovine peripherin gene and the rds (retinal degeneration slow) genes from mouse and rat. Mutations in the RDS gene have recently been implicated in autosomal dominant retinitis pigmentosa (adRP) in some families. Here we present evidence that the third exon of this gene is subject to polymorphic variation in humans. The three sequence alterations described in this paper give rise to amino acid substitutions. However, as these missense mutations also occur in the normal population they are not implicated as causing adRP. Interestingly such sequence variation is not found within other species examined including mouse and bovine. These intragenic polymorphisms will be of future potential value in studies to locate further disease causing mutations in adRP patients in the RDS gene.     

Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies

Inherited retinopathies are a genetically and phenotypically heterogeneous group of diseases affecting approximately one in 2000 individuals worldwide. For the past 10 years, the Laboratory for Molecular Diagnosis of Inherited Eye Diseases (LMDIED) at the University of Texas-Houston Health Science Center has screened subjects ascertained in the United States and Canada for mutations in genes causing dominant and recessive autosomal retinopathies. A combination of single strand conformational analysis (SSCA) and direct sequencing of five genes (rhodopsin, peripherin/RDS, RP1, CRX, and AIPL1) identified the disease-causing mutation in approximately one-third of subjects with autosomal dominant retinitis pigmentosa (adRP) or with autosomal dominant cone-rod dystrophy (adCORD). In addition, the causative mutation was identified in 15% of subjects with Leber congenital amaurosis (LCA). Overall, we report identification of the causative mutation in 105 of 506 (21%) of unrelated subjects (probands) tested; we report five previously unreported mutations in rhodopsin, two in peripherin/RDS, and one previously unreported mutation in the cone-rod homeobox gene, CRX. Based on this large survey, the prevalence of disease-causing mutations in each of these genes within specific disease categories is estimated. These data are useful in estimating the frequency of specific mutations and in selecting individuals and families for mutation-specific studies.     

Mutations P51U and G122E in retinal transcription factor NRL associated with autosomal dominant and sporadic retinitis pigmentosa

Retinitis pigmentosa (RP) is the most frequent form of inherited retinopathy. RP is genetically heterogeneous with autosomal dominant, autosomal recessive and X-linked forms. Autosomal dominant retinitis pigmentosa (adRP) accounts for about 20-25% of all RP cases. At least ten adRP loci have so far been mapped. However, mutations causing adRP have been identified only in four retina-specific genes: RHO (encoding rhodopsin) in approximately 20% of adRP families, peripherin/RDS (3-5% of adRP) and recently RP1 (Pierce et al., 1999, Sulivan et al., 1999) and NRL gene. Only one mutation in the NRL gene causing adRP has so far been reported (Bessant et al., 1999). Here we report a novel mutation Pro51Leu in an adRP Spanish family supporting that mutation in NRL is the cause of adRP. A second missense mutation Gly122Glu has been observed in a simplex RP patient that may represent a sporadic case of retinitis pigmentosa. Hum Mutat 17:520, 2001.     

Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa

BACKGROUND. Night blindness is an early symptom of retinitis pigmentosa. The rod photoreceptors are responsible for night vision and use rhodopsin as the photosensitive pigment. METHODS AND RESULTS. We found three mutations in the human rhodopsin gene; each occurred exclusively in the affected members of some families with autosomal dominant retinitis pigmentosa. Two mutations were C-to-T transitions involving separate nucleotides of codon 347; the third was a C-to-G transversion in codon 58. Each mutation corresponded to a change in one amino acid residue in the rhodopsin molecule. None of these mutations were found in 106 unrelated normal subjects who served as controls. When the incidence of these three mutations was added to that of a previously reported mutation involving codon 23, 27 of 150 unrelated patients with autosomal dominant retinitis pigmentosa (18 percent) were found to carry one of these four defects in the rhodopsin gene. All 27 patients had abnormal rod function on monitoring of their electroretinograms. It appears that patients with the mutation involving codon 23 probably descend from a single ancestor. CONCLUSIONS. In some patients with autosomal dominant retinitis pigmentosa, the disease is caused by one of a variety of mutations of the rhodopsin gene.     

Identification of 14 novel mutations in the long isoform of USH2A in Spanish patients with Usher syndrome type II

Mutations in USH2A gene have been shown to be responsible for Usher syndrome type II, an autosomal recessive disorder characterised by hearing loss and retinitis pigmentosa. USH2A was firstly described as consisting of 21 exons, but 52 novel exons at the 3' end of the gene were recently identified. In this report, a mutation analysis of the new 52 exons of USH2A gene was carried out in 32 unrelated patients in which both disease-causing mutations could not be found after the screening of the first 21 exons of the USH2A gene. On analysing the new 52 exons, fourteen novel mutations were identified in 14 out of the 32 cases studied, including 7 missense, 5 frameshift, 1 duplication and a putative splice-site mutation.     

Adult vitelliform macular dystrophy is frequently associated with mutations in the peripherin/RDS gene

Mutations in the peripherin/RDS gene, which encodes a photoreceptor-specific membrane glycoprotein, have been identified in a variety of retinal phenotypes. However, the mechanisms by which specific mutations in this gene can cause typical features of retinal dystrophies clinically as distinct as retinitis pigmentosa or macular degeneration are still unknown. Recently, a single case of adult vitelliform macular dystrophy (AVMD) has been associated with a Y258Stop mutation. To assess the frequency of peripherin/RDS mutations in the clinically heterogeneous group of AVMD, we analyzed the entire coding region of the gene in 28 unrelated patients. We identified five novel mutations including two presumed null allele mutations. Thus, our results demonstrate that a significant portion of AVMD patients (18%) carry point mutations in peripherin/RDS, suggesting that this gene is frequently involved in the pathogenesis of this macular disorder. In addition, this study shows that the variable phenotypes in AVMD are due, at least in part, to genetic heterogeneity and are likely to be caused by mutations in disease genes thus far unknown. Hum Mutat 10:301–309, 1997. © 1997 Wiley-Liss, Inc.     

Three novel RDS-peripherin mutations (689delT, 857del17, G208D) in Spanish familes affected with autosomal dominant retinal degenerations

Among 43 unrelated Spanish patients affected with autosomal dominant (AD) photoreceptor disorders a study of RDS-peripherin gene was performed. We found three different unreported mutations 689delT, 857del17, corresponding to two macular dystrophy families and G208D in a retinitis pigmentosa (RP) family giving us a proportion of about 20% of RDS mutations in autosomal dominant Spanish macular dystrophies and 3% in ADRP. Hum Mutat 12:70, 1998. © 1998 Wiley-Liss, Inc.     

Three novel RDS-peripherin mutations (689delT, 857del17, G208D) in Spanish families affected with autosomal dominant retinal degenerations. Mutations in brief no. 147. Online

Among 43 unrelated Spanish patients affected with autosomal dominant (AD) photoreceptor disorders a study of RDS-peripherin gene was performed. We found three different unreported mutations 689delT, 857del17, corresponding to two macular dystrophy families and G208D in a retinitis pigmentosa (RP) family giving us a proportion of about 20% of RDS mutations in autosomal dominant Spanish macular dystrophies and 3% in ADRP.     

常染色体显性遗传视网膜色素变性家系的基因筛查

目的：确定一个常染色体显性遗传视网膜色素变性(autosomal dominant retinitis pigmentosa,ADRP）家系的致病基因及其突变位点和类型。方法：应用聚合酶链反应－单链构象多态性结合DNA测序技术，对来自同一家系的4例RP患者及4名正常人外周血DNA进行分子遗传学分析，筛查3个候选基因共8个外显子。结果：来自同一家系的4例RP患者均发现有视紫红质基因（rhodopsin,RHO)第1外显子第52密码子存在TTC→TAC的点突变（Phe52Tyr），而4名正常人未发现这种突变。结论：在这个中国ADRP大家系中，发现RHO基因的致病突变，表明ADRP存在明显遗传异质性。     

Autosomal recessive retinitis pigmentosa in Spain: evaluation of four genes and two loci involved in the disease

Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous form of retinal degeneration. The genes for the β-subunit of rod phosphodiesterase (PDEB), rhodopsin (RHO), peripherin/RDS (RDS) and the rod outer segment membrane protein 1 (ROM1), as well as loci at 6p and 1q, have previously been reported as the cause of ARRP. In order to determine whether they are responsible for the disease in Spanish pedigrees, linkage and homozygosity studies using markers at these loci were carried out on 47 Spanish ARRP families. SSCP analysis was performed to search for mutations in the genes cosegregating with the disease in particular pedigrees. Three homozygous mutations in the PDEB gene were found, thus accounting for 6% of the cases. No other disease-causing mutation was observed in the other genes analysed, nor was significant evidence found for the involvement of the loci at 6p or 1q. On the basis of these data, it is unlikely that these genes and loci account for a considerable proportion of ARRP cases.     

NRL S50T mutation and the importance of 'founder effects' in inherited retinal dystrophies

The aim of this work was to identify NRL mutations in a panel of 200 autosomal dominant retinitis pigmentosa (adRP) families. All samples were subjected to heteroduplex analysis of the three exons of the NRL gene, and HphI restriction digest analysis of exon 2 (to identify the S50T mutation). Families found to have the S50T mutation, and six additional larger pedigrees (which had previously been excluded from the other nine adRP loci) underwent linkage analysis using polymorphic markers located in the region of 14q11. HphI restriction analysis followed by direct sequencing of the amplified NRL exon 2 product demonstrated the presence of the NRL S50T sequence change in three adRP families. Comparison of marker haplotypes in affected individuals from these families with those of affected members of the original 14q11 linked family revealed a common disease haplotype for markers within the adRP locus. Recombination events observed in these families define an adRP critical interval of 14.9 cM between D13S72 and D14S1041. Linkage analysis enabled all six of the larger adRP pedigrees to be excluded from the 14q11 locus. The NRL S50T mutation represents another example of a 'founder effect' in a dominantly inherited retinal dystrophy. Identification of such 'founder effects' may greatly simplify diagnostic genetic screening and lead to better prognostic counselling. The exclusion of several adRP families from all ten adRP loci indicates that at least one further adRP locus remains to be found.     

Detection and assignment of mutations and minihaplotypes in human DNA using peptide mass signature genotyping (PMSG): application to the human RDS/peripherin gene

Peptide mass-signature genotyping (PMSG) is a scanning genotyping method that identifies mutations and polymorphisms by translating the sequence of interest in more than one reading frame and measuring the masses of the resulting peptides by mass spectrometry. PMSG was applied to the RDS/peripherin gene of 16 individuals from a family exhibiting autosomal dominant macular degeneration. The method revealed an A-->T transversion in the 5' splice site of intron 2 that is the likely cause of the disease. It also revealed four different minihaplotypes in exon 3 that represent particular combinations of SNPs at four different locations. This study demonstrates the utility of PMSG for identifying and characterizing point mutations and local minihaplotypes that are not readily analyzed by other approaches.     

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.     

A dominant mutation in RPE65 identified by whole-exome sequencing causes retinitis pigmentosa with choroidal involvement

Linkage testing using Affymetrix 6.0 SNP Arrays mapped the disease locus in TCD-G, an Irish family with autosomal dominant retinitis pigmentosa (adRP), to an 8.8 Mb region on 1p31. Of 50 known genes in the region, 11 candidates, including RPE65 and PDE4B, were sequenced using di-deoxy capillary electrophoresis. Simultaneously, a subset of family members was analyzed using Agilent SureSelect All Exome capture, followed by sequencing on an Illumina GAIIx platform. Candidate gene and exome sequencing resulted in the identification of an Asp477Gly mutation in exon 13 of the RPE65 gene tracking with the disease in TCD-G. All coding exons of genes not sequenced to sufficient depth by next generation sequencing were sequenced by di-deoxy sequencing. No other potential disease-causing variants were found to segregate with disease in TCD-G. The Asp477Gly mutation was not present in Irish controls, but was found in a second Irish family provisionally diagnosed with choroideremia, bringing the combined maximum two-point LOD score to 5.3. Mutations in RPE65 are a known cause of recessive Leber congenital amaurosis (LCA) and recessive RP, but no dominant mutations have been reported. Protein modeling suggests that the Asp477Gly mutation may destabilize protein folding, and mutant RPE65 protein migrates marginally faster on SDS-PAGE, compared with wild type. Gene therapy for LCA patients with RPE65 mutations has shown great promise, raising the possibility of related therapies for dominant-acting mutations in this gene.     

RP1 in Chinese: Eight novel variants and evidence that truncation of the extreme C-terminal does not cause retinitis pigmentosa

Heterozygous truncating mutations in the RP1 gene cause approximately 7% of autosomal dominant retinitis pigmentosa (RP) cases. To examine the role of RP1 mutations in RP, we screened 101 unrelated Chinese RP patients (unselected for mode of inheritance) and 190 elderly normal control subjects for sequence changes in the coding exons for the 2156 amino acid RP1 protein. One patient had a mutation, thus RP1 mutations cause about 0.0% to 5.4% (95% confidence interval) of all RP among Chinese. The mutation was R677X, the most common found in Americans. Five other known sequence changes were found. In addition, nine novel sequence alterations were identified: 746G>A (R249H), 1437G>T (M479I), 2116G>C (G706R), 3024G>A (Q1008Q), 3188G>A (Q1063R), 5797C>T (R1933X), 6423A>G (I2141M), and the variants 6542C>T and 6676T>A, both in the 3' untranslated region. One control subject and three members of a non-RP family were heterozygous for R1933X, which is therefore likely to be a non-disease-causing variant. The most C-terminal truncation previously reported was due to Tyr1053 (1-bp del) and occurred in RP patients. Thus the presence of a normal level of at least part of RP1 between amino acids 1052 and 1933 appears necessary to prevent RP. Hum Mutat 17:436, 2001.     

Mutations in the RP1 gene causing autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration that affects approximately 1 in 3500 people worldwide. Recently we identified the gene responsible for the RP1 form of autosomal dominant retinitis pigmentosa (adRP) at 8q11-12 and found two different nonsense mutations in three families previously mapped to 8q. The RP1 gene is an unusually large protein, 2156 amino acids in length, but is comprised of four exons only. To determine the frequency and range of mutations in RP1 we screened probands from 56 large adRP families for mutations in the entire gene. After preliminary results indicated that mutations seem to cluster in a 442 nucleotide segment of exon 4, an additional 194 probands with adRP and 409 probands with other degenerative retinal diseases were tested for mutations in this region alone. We identified eight different disease-causing mutations in 17 of the 250 adRP probands tested. All of these mutations are either nonsense or frameshift mutations and lead to a severely truncated protein. Two of the eight different mutations, Arg677X and a 5 bp deletion of nucleotides 2280-2284, were reported previously, while the remaining six mutations are novel. We also identified two rare missense changes in two other families, one new polymorphic amino acid substitution, one silent substitution and a rare variant in the 5'-untranslated region that is not associated with disease. Based on this study, mutations in RP1 appear to cause at least 7% (17/250) of adRP. The 5 bp deletion of nucleotides 2280-2284 and the Arg677X nonsense mutation account for 59% (10/17) of these mutations. Further studies will determine whether missense changes in the RP1 gene are associated with disease, whether mutations in other regions of RP1 can cause forms of retinal disease other than adRP and whether the background variation in either the mutated or wild-type RP1 allele plays a role in the disease phenotype.     

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.