Gene mapping and mutation screeneng in candidate genes in a Chinese family of autosomal dominant retinitis pigmentosa

We wanted to find the gene defect in a Chinese pedigree with autosomal dominant form of retinitis pigmentosa (ADRP). A small Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite (STR) markers tightly linked to candidate genes for ADRP were selected for linkage analysis. We got a maximum LOD score of 0.87 between markers D19S210 and D19S418. Precursor mRNA-processing factor (PRPF) 31, 3, 8, rhodopsin (RHO), peripherin 2 (PRPH2 or RDS), rod outer segment protein 1 (ROM1), neural retina leucine zipper (NRL), cone-rod homeobox-containing (CRX), inosine-5-prime-monophosphate dehydrogenase, type I (IMPDH1) and retinitis pigmentosa 1 (RP1) were amplified by polymerase chain reaction (PCR) and screened by direct sequencing. One new sequence variation was found. It was the missence mutation c.148G > C (D50H) occurred in exon 1 of RDS gene which existed in all the effected individuals and one unaffected family member. The DNA sequence variation didn’t cosegregate with the RP disease. We considered this transition was one new polymorphism which we speculate involved in the pathogenesis of ADRP and increased the risk of ADRP. Further study should be conducted to confirm the causative gene of this family.     

Pro-347-Arg mutation of the rhodopsin gene in autosomal dominant retinitis pigmentosa.

It has been shown recently that autosomal dominant retinitis pigmentosa may be caused by point mutations of the rhodopsin gene in a portion of families. In this communication, a large six-generation family with autosomal dominant RP is described. Molecular analysis by PCR amplification followed by restriction digestion or heteroduplex analysis suggested a point mutation in codon 347, in which two different mutations (Pro-347-Ser and Pro-347-Leu) have already been reported. Direct sequencing of the patients' DNA revealed a previously undescribed CCG----CGG transversion in codon 347 predicting a Pro----Arg substitution. Ophthalmological data of the patients are summarized and compared to those of patients with other mutations in the rhodopsin gene.     

A novel Cys-214-Ser mutation in the peripherin/RDS gene in a Japanese family with autosomal dominant retinitis pigmentosa

We have screened for possible disease-causing mutations in the peripherin/retinal degeneration slow (RDS) gene in 13 Japanese families with autosomal dominant retinitis pigmentosa (ADRP). Using polymerase chain reaction-single strand conformation polymorphism analysis, a novel mutation at codon 214 was found in which the highly conserved cysteine was replaced with a serine in one family. The mutation at codon 214 was found in all three affected siblings of this family, but none of the 40 normal control individuals had this mutation. These results strongly suggest, that the mutation is pathogenic for RP in this family. The clinical phenotype for this family is a late-onset form of ADRP.     

A 3-bp deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa.

Autosomal dominant retinitis pigmentosa (ADRP) has recently been linked to locus D3S47 (probe C17), with no recombination, in a single large Irish family. Other ADRP pedigrees have shown linkage at zero recombination, linkage with recombination, and no linkage, demonstrating genetic heterogeneity. The gene encoding rhodopsin, the rod photoreceptor pigment, is closely linked to locus D3S47 on chromosome 3q. A point mutation changing a conserved proline to histidine in the 23d codon of the gene has been demonstrated in affected members of one ADRP family and in 17 of 148 unrelated ADRP patients. We have sequenced the rhodopsin gene in a C17-linked ADRP family and have identified in the 4th exon and in-frame 3-bp deletion which deletes one of the two isoleucine monomers at codons 255 and 256. This mutation was not found in 30 other unrelated ADRP families. The deletion has arisen in the sequence TCATCATCAT, deleting one of a run of three x 3-bp repeats. The mechanism by which this occurred may be similar to that which creates length variation in so-called mini- and microsatellites. Thus ADRP is an extremely heterogeneous disorder which can result from a range of defects in rhodopsin and which can have a locus or loci elsewhere in the genome.     

Molecular genetic study of autosomal dominant retinitis pigmentosa in Lithuanian patients

Lithuanian patients with visual problems were clinically examined for retinitis pigmentosa (RP). A total of 33 unrelated families with autosomal dominant RP (adRP) were identified. Screening for mutations in the rhodopsin (RHO) and peripherin/RDS (RDS) genes was performed using DNA heteroduplex analysis. Direct DNA sequencing in the cases of heteroduplex formation showed the presence of the following mutations and polymorphisms in 14 adRP patients: RHO gene - Lys248Arg (1 case), and Pro347Leu (2 cases); RDS gene - Glu304Gln (12 cases), Lys310Arg (5 cases), and Gly338Asp (12 cases). The presence of these mutations (except Lys248Arg in the RHO gene) was confirmed by relevant restriction enzyme digestion. The frequency of the RDS gene mutations Glu304Gln and Gly338Asp was estimated to be 36.4%, while mutation Lys310Arg was less frequent (15.2%). These 3 RDS gene mutations appear to be polypeptide polymorphisms not related to adRP.     

Novel rhodopsin mutation in an autosomal dominant retinitis pigmentosa family: phenotypic variation in both heterozygote and homozygote Val137Met mutant patients

A family affected with autosomal dominant retinitis pigmentosa (RP) is presented. Two clinically affected patients (mother and daughter) were heterozygous for the same novel missense mutation (Val137Met) of the rhodopsin gene (RHO). Both heterozygous and homozygous cases were observed among their few symptomatic relatives. Wide clinical variation was exhibited among the individuals with mutations in this family. None of the controls showed this change in RHO, nor has it been previously reported in other RP families. No other RHO mutation was observed. Additional genetic or environmental factors could play a role in modulating the penetrance and clinical expression of this RHO mutation. Received: 20 February 1995 / Revised: 1 September 1995, 27 November 1995, 3 February 1996     

Mutation screening of multiple genes in Spanish patients with autosomal recessive retinitis pigmentosa by targeted resequencing

Retinitis Pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. RP is the leading cause of visual loss in individuals younger than 60 years, with a prevalence of about 1 in 4000. The molecular genetic diagnosis of autosomal recessive RP (arRP) is challenging due to the large genetic and clinical heterogeneity. Traditional methods for sequencing arRP genes are often laborious and not easily available and a screening technique that enables the rapid detection of the genetic cause would be very helpful in the clinical practice. The goal of this study was to develop and apply microarray-based resequencing technology capable of detecting both known and novel mutations on a single high-throughput platform. Hence, the coding regions and exon/intron boundaries of 16 arRP genes were resequenced using microarrays in 102 Spanish patients with clinical diagnosis of arRP. All the detected variations were confirmed by direct sequencing and potential pathogenicity was assessed by functional predictions and frequency in controls. For validation purposes 4 positive controls for variants consisting of previously identified changes were hybridized on the array. As a result of the screening, we detected 44 variants, of which 15 are very likely pathogenic detected in 14 arRP families (14%). Finally, the design of this array can easily be transformed in an equivalent diagnostic system based on targeted enrichment followed by next generation sequencing.     

A molecular case report of autosomal dominant retinitis pigmentosa: RP1/RHO sequence variants in a Turkish family

Retinitis pigmentosa (RP) is an inherited progressive retinal disease with a complex inheritance pattern affecting about 1 in 3,500 people worldwide. To date, a large number of sequence changes in the causal contributor genes of wide-spectrum heterogeneous RP were reported, including deletions, insertions, or substitutions that lead missense mutations or truncations. Here we present an association between the clinical presentations of adRP and sequence variants involving novel M216L mutation in the RHO gene together with nonsynonimous sequence changes R872H, N985Y, A1670T, S1691P, C2033Y, and synonimous Q1725Q with novel, N1521N, and T1733T SNPs in the RP1 gene of uncertain pathogenicity in a Turkish family with autosomal dominant retinitis pigmentosa.     

An autosomal dominant retinitis pigmentosa family with close linkage to D7S480 on 7q

Retinitis pigmentosa is the most prevalent inherited disorder of the retina. It can be autosomal dominant (adRP), autosomal recessive (arRP) or X-linked (XLRP). A form of adRP mapping to chromosome 7q was reported in a large Spanish pedigree. We have typed DNA from the members of another Spanish family for polymorphic markers from the known candidate genes. Positive lod scores were obtained only for the markers located on 7q31-35, giving a maximum lod score of 2.98 (3.01 by multipoint analysis) at = 0.00 for D7S480. A brief clinical evaluation is given.     

Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa.

We inserted into the germline of mice either a mutant or wild-type allele from a patient with retinitis pigmentosa and a missense mutation (P23H) in the rhodopsin gene. All three lines of transgenic mice with the mutant allele developed photoreceptor degeneration; the one with the least severe retinal photoreceptor degeneration had the lowest transgene expression, which was one-sixth the level of endogenous murine rod opsin. Of two lines of mice with the wild-type allele, one expressed approximately equal amounts of transgenic and murine opsin and maintained normal retinal function and structure. The other expressed approximately 5 times more transgenic than murine opsin and developed a retinal degeneration similar to that found in mice carrying a mutant allele, presumably due to the overexpression of this protein. Our findings help to establish the pathogenicity of mutant human P23H rod opsin and suggest that overexpression of wild-type human rod opsin leads to a remarkably similar photoreceptor degeneration.     

Ubiquitin ligase activity of Cul3-KLHL7 protein is attenuated by autosomal dominant retinitis pigmentosa causative mutation

Substrate-specific protein degradation mediated by the ubiquitin proteasome system (UPS) is crucial for the proper function of the cell. Proteins are specifically recognized and ubiquitinated by the ubiquitin ligases (E3s) and are then degraded by the proteasome. BTB proteins act as the substrate recognition subunit that recruits their cognate substrates to the Cullin 3-based multisubunit E3s. Recently, it was reported that missense mutations in KLHL7, a BTB-Kelch protein, are related to autosomal dominant retinitis pigmentosa (adRP). However, the involvement of KLHL7 in the UPS and the outcome of the adRP causative mutations were unknown. In this study, we show that KLHL7 forms a dimer, assembles with Cul3 through its BTB and BACK domains, and exerts E3 activity. Lys-48-linked but not Lys-63-linked polyubiquitin chain co-localized with KLHL7, which increased upon proteasome inhibition suggesting that KLHL7 mediates protein degradation via UPS. An adRP-causative missense mutation in the BACK domain of KLHL7 attenuated only the Cul3 interaction but not dimerization. Nevertheless, the incorporation of the mutant as a heterodimer in the Cul3-KLHL7 complex diminished the E3 ligase activity. Together, our results suggest that KLHL7 constitutes a Cul3-based E3 and that the disease-causing mutation inhibits ligase activity in a dominant negative manner, which may lead to the inappropriate accumulation of the substrates targeted for proteasomal degradation.     

Evidence for a major gene (RP10) for autosomal dominant retinitis pigmentosa on chromosome 7q: linkage mapping in a second,unrelated family

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration, which has X-linked, autosomal recessive and autosomal dominant forms. The disease genes in families with autosomal dominant retinitis pigmentosa (adRP) have been linked to six loci, on 3q, 6p, 7p, 7q, 8q and 19q. In a large American family with late-onset adRP, microsatellite markers were used to test for linkage to the loci on 3q, 6p, 7p, 7q and 8q. Linkage was found to 7q using the marker D7S480. Additional microsatellite markers from 7q were then tested. In total, five markers, D7S480, D7S514, D7S633, D7S650 and D7S677, show statistically significant evidence for link-age in this family, with a maximum two-point lod score of 5.3 at 0% recombination from D7S514. These results confirm an earlier report of linkage to an adRP locus (RP10) in an unrelated family of Spanish origin and indicate that RP10 may be a significant gene for inherited retinal degeneration. In addition, we used recently reported microsatellite markers from 7q to refine the linkage map of the RP10 locus.     

Novel ACTG1 mutation causing autosomal dominant non-syndromic hearing impairment in a Chinese family

Theγ-actin(ACTG1)gene is a cytoplasmic nonmuscle actin gene,which encodes a major cytoskeletal protein in the sensory hair cells of the cochlea.Mutations in ACTG1 were found to cause autosomal dominant,progressive,sensorineural hearing loss linked to the DFNA 20/26 locus on chromosome 17q25.3 in European and American families,respectively.In this study,a novel missense mutation (c.364A>G;p.I122V)co-segregated with the affected individuals in the family and did not exist in the unaffected family members and 150 unrelated normal controls.The alteration of residue I1e122 was predicted to damage its interaction with actin-binding proteins,which may cause disruption of hair cell organization and function.These findings strongly suggested that the I122V mutation in ACTG1 caused autosomal dominant non-syndromic hearing impairment in a Chinese family and expanded the spectrum of ACTG1 mutations causing hearing loss.     

Mapping of locus for autosomal dominant retinitis pigmentosa on chromosome 6q23

Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal degenerative disorders resulting in severe visual loss and blindness that have remained incurable till date. We report the mapping of the disease locus in a 3-generation family of Indian origin with autosomal dominant RP (ADRP). Diagnosis of RP and recruitment was made after a complete clinical evaluation of all members. Manifestations of the disease included night blindness with blurred central vision in some cases, loss of peripheral vision, and diffuse degeneration of the retinal pigment epithelium. Linkage analysis using microsatellite markers was carried out on 34 members (14 affected). After testing for linkage to known retinal dystrophy loci as well as a subsequent genome-wide analysis, we detected linkage to markers on chromosome 6q23: D6S262 at 130 cM, D6S457 (130 cM) and D6S1656 (131 cM) gave significant 2-point LOD scores of 3.0–3.8. Multipoint LOD scores of ≥3.0 were obtained for markers between 121 and 130 cM. Haplotype analysis with several markers in the same region on chromosome 6 shows a disease-cosegregating region of about 25 Mb between 109 and 135 Mb. There are no known RP genes in this interval, which contains >100 genes. This study provides evidence for a novel ADRP locus on chromosome 6q23.     

Suppression of wild-type rhodopsin maturation by mutants linked to autosomal dominant retinitis pigmentosa

Autosomal dominant retinitis pigmentosa (ADRP) has been linked to mutations in the gene encoding rhodopsin. Most RP-linked rhodopsin mutants are unable to fold correctly in the endoplasmic reticulum, are degraded by the ubiquitin proteasome system, and are highly prone to forming detergent-insoluble high molecular weight aggregates. Here we have reported that coexpression of folding-deficient, but not folding-proficient, ADRP-linked rhodopsin mutants impairs delivery of the wild-type protein to the plasma membrane. Fluorescence resonance energy transfer and co-precipitation studies revealed that mutant and wild-type rhodopsins form a high molecular weight, detergent-insoluble complex in which the two proteins are in close (<70 A) proximity. Co-expression of ARDP-linked rhodopsin folding-deficient mutants resulted in enhanced proteasome-mediated degradation and steady-state ubiquitination of the wild-type protein. These data suggested a dominant negative effect on conformational maturation that may underlie the dominant inheritance of ARDP.     

Refined mapping of the gene for autosomal dominant retinitis pigmentosa (RP17) on chromosome 17q22

Linkage analysis was performed on a large Dutch family with autosomal dominant retinitis pigmentosa. Linkage was found to the RP17 locus on chromosome 17q22, which was previously described in two South African families by Bardien et al. (1995, 1997). Assuming that the disease phenotypes in these families are caused by the same gene, the RP17 critical region is refined to a 7.7-cM interval between markers D17S1607 and D17S948. Two positional candidate genes, the retina-specific amine oxidase (RAO) gene (AOC2) and the cone transducin γ gene (GNGT2), were excluded. Received: 7 September 1998 / Accepted: 23 November 1998     

A novel locus (RP33) for autosomal dominant retinitis pigmentosa mapping to chromosomal region 2cen-q12.1

Retinitis pigmentosa (RP) is a heterogeneous group of progressive degenerative disorders of the retina with a strong genetic component. Here, we report the clinical and genetic findings in a Chinese family in which autosomal dominant RP (adRP) was inherited by 13 affected members in four generations. Using a genome-wide linkage screening approach, a novel disease locus (RP33) was assigned to the long arm of chromosome 2. A maximum multi-point LOD score of 4.69 was reached at marker D2S2222 in 2q11.2. Meiotic recombination events in affected members placed RP33 in a 15.5 cM region between D2S329 and D2S2229. From meiotic recombinations in two unaffected members RP33 was further refined to a 4.8 cM (9.5 Mb) interval flanked by D2S2159 and D2S1343 in chromosomal region 2cen-q12.1. No disease-associated mutations were detected in the candidate genes SEMA4C, CNGA3 or HNK1ST from within the region. MERTK, a known disease gene for autosomal recessive RP located close to RP33 was similarly excluded. Clinically, the family presented relatively late onset of night blindness, gradually decreased visual acuity, progressive loss of peripheral visual field and typical RP fundus changes in the mid-periphery of the retina. In conclusion, a novel locus for adRP has been assigned to chromosomal region 2cen-q12.1, which in the present kindred was associated with a relatively late onset form of the disease.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Chen Zhao and Shasha Lu have contributed equally to this study     

Mutations in TOPORS cause autosomal dominant retinitis pigmentosa with perivascular retinal pigment epithelium atrophy

We report mutations in the gene for topoisomerase I-binding RS protein (TOPORS) in patients with autosomal dominant retinitis pigmentosa (adRP) linked to chromosome 9p21.1 (locus RP31). A positional-cloning approach, together with the use of bioinformatics, identified TOPORS (comprising three exons and encoding a protein of 1,045 aa) as the gene responsible for adRP. Mutations that include an insertion and a deletion have been identified in two adRP-affected families--one French Canadian and one German family, respectively. Interestingly, a distinct phenotype is noted at the earlier stages of the disease, with an unusual perivascular cuff of retinal pigment epithelium atrophy, which was found surrounding the superior and inferior arcades in the retina. TOPORS is a RING domain-containing E3 ubiquitin ligase and localizes in the nucleus in speckled loci that are associated with promyelocytic leukemia bodies. The ubiquitous nature of TOPORS expression and a lack of mutant protein in patients are highly suggestive of haploinsufficiency, rather than a dominant negative effect, as the molecular mechanism of the disease and make rescue of the clinical phenotype amenable to somatic gene therapy.     

Linkage mapping of autosomal dominant retinitis pigmentosa (RP1) to the pericentric region of human chromosome 8.

Linkage mapping in a large, seven-generation family with type 2 autosomal dominant retinitis pigmentosa (ADRP) demonstrates linkage between the disease locus (RP1) and DNA markers on the short arm of human chromosome 8. Five markers were most informative for mapping ADRP in this family using two-point linkage analysis. The markers, their maximum lod scores, and recombination distances were ANK1 (ankyrin)--2.0 at 16%; D8S5 (TL11)--5.3 at 17%; D8S87 [a(CA)n repeat]--7.2 at 14%; LPL (lipoprotein lipase)--1.5 at 26%; and PLAT (plasminigen activator, tissue)--10.6 at 7%. Multipoint linkage analysis, using a simplified pedigree structure for the family (which contains 192 individuals and two inbreeding loops), gave a maximum lod score of 12.2 for RP1 at a distance 8.1 cM proximal to PLAT in the pericentric region of the chromosome. Based on linkage data from the CEPH (Paris) reference families and physical mapping information from a somatic cell hybrid panel of chromosome 8 fragments, the most likely order for four of these five loci and the diseases locus is 8pter-LPL-D8S5-D8S87-PLAT-RP1. (The precise location of ANK1 relative to PLAT in this map is not established). The most likely location for RP1 is in the pericentric region of the chromosome. Recently, several families with ADRP with tight linkage to the rhodopsin locus at 3q21-q24 were reported and a number of specific rhodopsin mutations in families with ADRP have since been reported. In other ADRP families, including the one in this study, linkage to rhodopsin has been excluded. Thus mutations at two different loci, at least, have been shown to cause ADRP. There is no remarkable clinical disparity in the expression of disease caused by these different loci.