Remapping of the RP15 locus for X-linked cone-rod degeneration to Xp11.4-p21.1, and identification of a de novo insertion in the RPGR exon ORF15

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe, because of their early onset, often leading to significant vision loss before the 4th decade. Previously, the RP15 locus was assigned to Xp22, by linkage analysis of a single pedigree with "X-linked dominant cone-rod degeneration." After clinical reevaluation of a female in this pedigree identified her as affected, we remapped the disease to a 19.5-cM interval (DXS1219-DXS993) at Xp11.4-p21.1. This new interval overlapped both RP3 (RPGR) and COD1. Sequencing of the previously published exons of RPGR revealed no mutations, but a de novo insertion was detected in the new RPGR exon, ORF15. The identification of an RPGR mutation in a family with a severe form of cone and rod degeneration suggests that RPGR mutations may encompass a broader phenotypic spectrum than has previously been recognized in "typical" retinitis pigmentosa.     

RP2 and RPGR mutations and clinical correlations in patients with X-linked retinitis pigmentosa

We determined the mutation spectrum of the RP2 and RPGR genes in patients with X-linked retinitis pigmentosa (XLRP) and searched for correlations between categories of mutation and severity of disease. We screened 187 unrelated male patients for mutations, including 135 with a prior clinical diagnosis of XLRP, 11 with probable XLRP, 30 isolate cases suspected of having XLRP, and 11 with cone-rod degeneration. Mutation screening was performed by single-strand conformation analysis and by sequencing of all RP2 exons and RPGR exons 1-14, ORF15, and 15a. The refractive error, visual acuity, final dark-adapted threshold, visual field area, and 30-Hz cone electroretinogram (ERG) amplitude were measured in each patient. Among the 187 patients, we found 10 mutations in RP2, 2 of which are novel, and 80 mutations in RPGR, 41 of which are novel; 66% of the RPGR mutations were within ORF15. Among the 135 with a prior clinical diagnosis of XLRP, mutations in the RP2 and RPGR genes were found in 9 of 135 (6.7%) and 98 of 135 (72.6%), respectively, for a total of 79% of patients. Patients with RP2 mutations had, on average, lower visual acuity but similar visual field area, final dark-adapted threshold, and 30-Hz ERG amplitude compared with those with RPGR mutations. Among patients with RPGR mutations, those with ORF15 mutations had, on average, a significantly larger visual field area and a borderline larger ERG amplitude than did patients with RPGR mutations in exons 1-14. Among patients with ORF15 mutations, regression analyses showed that the final dark-adapted threshold became lower (i.e., closer to normal) and that the 30-Hz ERG amplitude increased as the length of the wild-type ORF15 amino acid sequence increased. Furthermore, as the length of the abnormal amino acid sequence following ORF15 frameshift mutations increased, the severity of disease increased.     

Null RPGRIP1 alleles in patients with Leber congenital amaurosis

We isolated and characterized the entire coding sequence of a human gene encoding a protein that interacts with RPGR, a protein that is absent or mutant in many cases of X-linked retinitis pigmentosa. The newly identified gene, called "RPGRIP1" for RPGR-interacting protein (MIM 605446), is located within 14q11, and it encodes a protein predicted to contain 1,259 amino acids. Previously published work showed that both proteins, RPGR and RPGRIP1, are present in the ciliary structure that connects the inner and outer segments of rod and cone photoreceptors. We surveyed 57 unrelated patients who had Leber congenital amaurosis for mutations in RPGRIP1 and found recessive mutations involving both RPGRIP1 alleles in 3 (6%) patients. The mutations all create premature termination codons and are likely to be null alleles. Patients with RPGRIP1 mutations have a degeneration of both rod and cone photoreceptors, and, early in life, they experience a severe loss of central acuity, which leads to nystagmus.     

Spectrum of color gene deletions and phenotype in patients with blue cone monochromacy

Blue cone monochromacy (BCM) is an X-linked ocular disease characterized by poor visual acuity, nystagmus, and photodysphoria in males with severely reduced color discrimination. Deletions, rearrangements and point mutations in the red and green pigment genes have been implicated in causing BCM. We assessed the spectrum of genetic alterations in ten families with BCM by Southern blot, polymerase chain reaction, and sequencing analysis, and the phenotype was characterized by ophthalmoscopy, fluorescein angiography, and a battery of tests to assess color vision in addition to routine ophthalmological examination. All families showed clinical features associated with BCM. Acuities were reduced in all affected males, and photopic b-wave was reduced by more than 90% in seven families. In three families, however, the photopic b-wave response showed uncharacteristic relative preservation of 30-80% (of the clinical low-normal value). The color vision was unusually preserved in two affected males, but this was not correlated with photopic electroretinography retention. Progressive macular atrophy was observed in affected members of two BCM families while the rest of the families presented with normal fundus. In nine families deletions were identified in the gene encoding the red-sensitive photopigment and/or in the region up to 17.8 kb upstream of the red gene which contains the locus control region and other regulatory sequences. In the same nine families the red pigment gene showed a range of deletions from the loss of a single exon to loss of the complete red gene. In one family no mutation was found in the exons of the red gene or the locus control region but showed loss of the complete green gene. No association was observed between the phenotypes and genotypes in these families.     

Linkage analysis of X-linked cone-rod dystrophy: localization to Xp11.4 and definition of a locus distinct from RP2 and RP3.

Progressive X-linked cone-rod dystrophy (COD1) is a retinal disease affecting primarily the cone photoreceptors. The COD1 locus originally was localized, by the study of three independent families, to a region between Xp11.3 and Xp21.1, encompassing the retinitis pigmentosa (RP) 3 locus. We have refined the COD1 locus to a limited region of Xp11.4, using two families reported elsewhere and a new extended family. Genotype analysis was performed by use of eight microsatellite markers (tel-M6CA, DXS1068, DXS1058, DXS993, DXS228, DXS1201, DXS1003, and DXS1055-cent), spanning a distance of 20 cM. Nine-point linkage analysis, by use of the VITESSE program for X-linked disorders, established a maximum LOD score (17.5) between markers DXS1058 and DXS993, spanning 4.0 cM. Two additional markers, DXS977 and DXS556, which map between DXS1058 and DXS993, were used to further narrow the critical region. The RP3 gene, RPGR, was excluded on the basis of two obligate recombinants, observed in two independent families. In a third family, linkage analysis did not exclude the RPGR locus. The entire coding region of the RPGR gene from two affected males from family 2 was sequenced and was found to be normal. Haplotype analysis of two family branches, containing three obligate recombinants, two affected and one unaffected, defined the COD1 locus as distal to DXS993 and proximal to DXS556, a distance of approximately 1.0 Mb. This study excludes COD1 as an allelic variant of RP3 and establishes a novel locus that is sufficiently defined for positional cloning.     

Allelic heterogeneity and genetic modifier loci contribute to clinical variation in males with X-linked retinitis pigmentosa due to RPGR mutations

Mutations in RPGR account for over 70% of X-linked retinitis pigmentosa (XlRP), characterized by retinal degeneration and eventual blindness. The clinical consequences of RPGR mutations are highly varied, even among individuals with the same mutation: males demonstrate a wide range of clinical severity, and female carriers may or may not be affected. This study describes the phenotypic diversity in a cohort of 98 affected males from 56 families with RPGR mutations, and demonstrates the contribution of genetic factors (i.e., allelic heterogeneity and genetic modifiers) to this diversity. Patients were categorized as grade 1 (mild), 2 (moderate) or 3 (severe) according to specific clinical criteria. Patient DNAs were genotyped for coding SNPs in 4 candidate modifier genes with products known to interact with RPGR protein: RPGRIP1, RPGRIP1L, CEP290, and IQCB1. Family-based association testing was performed using PLINK. A wide range of clinical severity was observed both between and within families. Patients with mutations in exons 1–14 were more severely affected than those with ORF15 mutations, and patients with predicted null alleles were more severely affected than those predicted to make RPGR protein. Two SNPs showed association with severe disease: the minor allele (N) of I393N in IQCB1 (p = 0.044) and the common allele (R) of R744Q in RPGRIP1L (p = 0.049). These data demonstrate that allelic heterogeneity contributes to phenotypic diversity in XlRP and suggest that this may depend on the presence or absence of RPGR protein. In addition, common variants in 2 proteins known to interact with RPGR are associated with severe disease in this cohort.     

A missense mutation in DHDDS, encoding dehydrodolichyl diphosphate synthase, is associated with autosomal-recessive retinitis pigmentosa in Ashkenazi Jews

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations caused by mutations in at least 50 genes. Using homozygosity mapping in Ashkenazi Jewish (AJ) patients with autosomal-recessive RP (arRP), we identified a shared 1.7 Mb homozygous region on chromosome 1p36.11. Sequence analysis revealed a founder homozygous missense mutation, c.124A>G (p.Lys42Glu), in the dehydrodolichyl diphosphate synthase gene (DHDDS) in 20 AJ patients with RP of 15 unrelated families. The mutation was not identified in an additional set of 109 AJ patients with RP, in 20 AJ patients with other inherited retinal diseases, or in 70 patients with retinal degeneration of other ethnic origins. The mutation was found heterozygously in 1 out of 322 ethnically matched normal control individuals. RT-PCR analysis in 21 human tissues revealed ubiquitous expression of DHDDS. Immunohistochemical analysis of the human retina with anti-DHDDS antibodies revealed intense labeling of the cone and rod photoreceptor inner segments. Clinical manifestations of patients who are homozygous for the c.124A>G mutation were within the spectrum associated with arRP. Most patients had symptoms of night and peripheral vision loss, nondetectable electroretinographic responses, constriction of visual fields, and funduscopic hallmarks of retinal degeneration. DHDDS is a key enzyme in the pathway of dolichol, which plays an important role in N-glycosylation of many glycoproteins, including rhodopsin. Our results support a pivotal role of DHDDS in retinal function and may allow for new therapeutic interventions for RP.     

无脉络膜症的发病机制及诊疗进展

无脉络膜(CHM)是一种X染色体连锁的遗传性进行性视网膜色素上皮,光感受细胞和脉络膜血管逐渐退化,最终致盲的疾病。该病是由于位于Xq21上的REP-1缺失突变导致失活,导致CHM基因不能表达,从而出现脉络膜血管层发育障碍,进行性视网膜色素上皮和脉络膜营养不良,变性及进行性脉络膜萎缩消失。男性患者一般在十几岁至二十几岁时开始出现夜盲,周边视野逐渐丧失,形成管状视野,严重者仅剩5-10度的中央视野,最终失明。女性携带者大多无症状。     

Spectrum of Mutations in the RPGR Gene That Are Identified in 20% of Families with X-Linked Retinitis Pigmentosa

The RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%-15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis.     

Severe retinitis pigmentosa mapped to 4p15 and associated with a novel mutation in the PROM1 gene

Mutation in the PROM1 gene previously has been identified in one family with retinal degeneration for which neither ERG recordings nor detailed information about visual impairment is available. A large family with multiple individuals affected by retinal degeneration was ascertained in the Punjab province of Pakistan. The visual acuity of all affected patients in the family was severely compromised beginning in early childhood. The retinal disease in this family is a severe form of retinitis pigmentosa (RP) accompanied by macular degeneration. Fundus changes advanced with age. Choriocapillaris atrophy and posterior RPE atrophy were obvious allowing visualization of the large choroidal vessels in patients over 40 years of age. Rod and cone responses on ERG recordings were extinguished in patient’s teens. A genome-wide scan mapped the disease to a 34.7 cM region of chromosome 4p14–p16 between D4S1599 and D4S405. A maximum lod score of 3.96 with D4S403 and D4S391 is seen at θ = 0. Sequence analysis of PROM1 located in the linkage interval identified a c.1726C>T homozygous transition in exon 15: resulting in p.Gln576X in the translated protein. This mutation is found in a homozygous state in all six affected individuals and was heterozygous in five of the six unaffected family members examined. The mutation was not detected in 192 chromosomes of unrelated control individuals of the same ethnicity and from the same region. This delineates the phenotypic characteristics of retinopathy caused by mutations in PROM1. Qingjiong Zhang, Fareeha Zulfiqar, Xueshan Xiao, Sheikh Riazuddin and J. Fielding Hejtmancik contributed equally.     

X-linked dominant cone-rod degeneration: linkage mapping of a new locus for retinitis pigmentosa (RP 15) to Xp22.13-p22.11.

Retinitis pigmentosa is the name given to a heterogeneous group of hereditary retinal degenerations characterized by progressive visual field loss, pigmentary changes of the retina, abnormal electroretinograms, and, frequently, night blindness. In this study, we investigated a family with dominant cone-rod degeneration, a variant form of retinitis pigmentosa. We used microsatellite markers to test for linkage to the disease locus and excluded all mapped autosomal loci. However, a marker from the short arm of the X chromosome, DXS989, showed 0% recombination to the disease locus, with a maximum lod (log-odds) score of 3.3. On the basis of this marker, the odds favoring X-linked dominant versus autosomal dominant inheritance are > 10(5):1. Haplotype analysis using an additional nine microsatellite markers places the disease locus in the Xp22.13-p22.11 region and excludes other X-linked disease loci causing retinal degeneration. The clinical expression of the retinal degeneration is consistent with X-linked dominant inheritance with milder, variable effects of Lyonization affecting expression in females. On the basis of these data we propose that this family has a novel form of dominant, X-linked cone-rod degeneration with the gene symbol "RP15."     

Familial early onset macular degeneration in cynomolgus monkeys (Macaca fascicularis)

The mode of inheritance of macular degeneration was determined with 45 cynomolgus monkeys (18 females and 27 males) who were the offspring of one breeding male with typical macular degeneration. In the first generation, 27 offspring (10 females and 17 males) were born from mating between the macular degeneration-affected founder male and 5 normal female breeders. Among them, 18 monkeys (9 females and 9 males) were judged as having macular degeneration (affected). Next, the distribution of affected offspring was examined with 18 offspring who were born from 3 different mating pairs, normal vs normal, affected vs normal and affected vs affected, when they became 2 years old. All of the 9 monkeys (4 females and 5 males) obtained from the 2 pairs of normal vs normal were normal. On the other hand, 6 affected monkeys (3 females and 3 males) were detected in 8 offspring from the mating pair of affected vs normal, and the single offspring produced by the mating pair of affected vs affected was affected. These results showed that this degeneration must be early onset familial macular degeneration controlled by autosomal dominant gene(s).     

Disease mechanisms and neuroprotection by tauroursodeoxycholic acid in Rpgr knockout mice

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are the predominant cause of retinitis pigmentosa. RPGR plays a critical role as a scaffold protein in the regulation of protein trafficking from the basal body to the axoneme, where the cargoes are transported to the outer segments (OSs) of photoreceptors. This trafficking process is controlled directly by intraflagellar transport complexes and regulated by the RPGR protein complex, although the precise mechanisms have yet to be defined. We used an Rpgr conditional knockout (cko) mouse model to investigate the disease mechanisms during retinal degeneration and to evaluate the protective effects of tauroursodeoxycholic acid (TUDCA). Rhodopsin, cone opsins and transducin were mislocalized in Rpgr cko photoreceptors, while localization of NPHP4 to connecting cilia was absent, suggesting that RPGR is required for ciliary protein trafficking. Microglia were activated in advance of retinal degeneration in Rpgr cko mouse retinas. TUDCA treatment suppressed microglial activation and inflammation and prevented photoreceptor degeneration in Rpgr cko mice. Our data demonstrated that TUDCA has therapeutic potential for RPGR-associated RP patients.     

X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15

X-linked cone-rod dystrophy (COD1) is a retinal disease that primarily affects the cone photoreceptors; the disease was originally mapped to a limited region of Xp11.4. We evaluated the three families from our original study with new markers and clinically reassessed all key recombinants; we determined that the critical intervals in families 2 and 3 overlapped the RP3 locus and that a status change (from affected to probably unaffected) of a key recombinant individual in family 1 also reassigned the disease locus to include RP3 as well. Mutation analysis of the entire RPGR coding region identified two different 2-nucleotide (nt) deletions in ORF15, in family 2 (delAG) and in families 1 and 3 (delGG), both of which result in a frameshift leading to altered amino acid structure and early termination. In addition, an independent individual with X-linked cone-rod dystrophy demonstrated a 1-nt insertion (insA) in ORF15. The presence of three distinct mutations associated with the same disease phenotype provides strong evidence that mutations in RPGR exon ORF15 are responsible for COD1. Genetic heterogeneity was observed in three other families, including the identification of an in-frame 12-nt deletion polymorphism in ORF15 that did not segregate with the disease in one of these families.     

Recessive Mutations in KCNJ13, Encoding an Inwardly Rectifying Potassium Channel Subunit, Cause Leber Congenital Amaurosis

Inherited retinal degenerations, including retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), comprise a group of disorders showing high genetic and allelic heterogeneity. The determination of a full catalog of genes that can, when mutated, cause human retinal disease is a powerful means to understand the molecular physiology and pathology of the human retina. As more genes are found, remaining ones are likely to be rarer and/or unexpected candidates. Here, we identify a family in which all known RP/LCA-related genes are unlikely to be associated with their disorder. A combination of homozygosity mapping and exome sequencing identifies a homozygous nonsense mutation, c.496C>T (p.Arg166X), in a gene, KCNJ13, encoding a potassium channel subunit Kir7.1. A screen of a further 333 unrelated individuals with recessive retinal degeneration identified an additional proband, homozygous for a missense mutation, c.722T>C (p.Leu241Pro), in the same gene. The three affected members of the two families have been diagnosed with LCA. All have a distinct and unusual retinal appearance and a similar early onset of visual loss, suggesting both impaired retinal development and progressive retinal degeneration, involving both rod and cone pathways. Examination of heterozygotes revealed no ocular disease. This finding implicates Kir7.1 as having an important role in human retinal development and maintenance. This disorder adds to a small diverse group of diseases consequent upon loss or reduced function of inwardly rectifying potassium channels affecting various organs. The distinct retinal phenotype that results from biallelic mutations in KCNJ13 should facilitate the molecular diagnosis in further families.     

Gene therapy regenerates protein expression in cone photoreceptors in Rpe65(R91W/R91W) mice

Cone photoreceptors mediate visual acuity under daylight conditions, so loss of cone-mediated central vision of course dramatically affects the quality of life of patients suffering from retinal degeneration. Therefore, promoting cone survival has become the goal of many ocular therapies and defining the stage of degeneration that still allows cell rescue is of prime importance. Using the Rpe65(R91W/R91W) mouse, which carries a mutation in the Rpe65 gene leading to progressive photoreceptor degeneration in both patients and mice, we defined stages of retinal degeneration that still allow cone rescue. We evaluated the therapeutic window within which cones can be rescued, using a subretinal injection of a lentiviral vector driving expression of RPE65 in the Rpe65(R91W/R91W) mice. Surprisingly, when applied to adult mice (1 month) this treatment not only stalls or slows cone degeneration but, actually, induces cone-specific protein expression that was previously absent. Before the intervention only part of the cones (40% of the number found in wild-type animals) in the Rpe65(R91W/R91W) mice expressed cone transducin (GNAT2); this fraction increased to 64% after treatment. Correct S-opsin localization is also recovered in the transduced region. In consequence these results represent an extended therapeutic window compared to the Rpe65(-/-) mice, implying that patients suffering from missense mutations might also benefit from a prolonged therapeutic window. Moreover, cones are not only rescued during the course of the degeneration, but can actually recover their initial status, meaning that a proportion of altered cones in chromophore deficiency-related disease can be rehabilitated even though they are severely affected.     

Retinal cone and rod photoreceptor cells exhibit differential susceptibility to light-induced damage

All-trans-retinal and its condensation-products can cause retinal degeneration in a light-dependent manner and contribute to the pathogenesis of human macular diseases such as Stargardt's disease and age-related macular degeneration. Although these toxic retinoid by-products originate from rod and cone photoreceptor cells, the contribution of each cell type to light-induced retinal degeneration is unknown. In this study, the primary objective was to learn whether rods or cones are more susceptible to light-induced, all-trans-retinal-mediated damage. Previously, we reported that mice lacking enzymes that clear all-trans-retinal from the retina, ATP-binding cassette transporter 4 and retinol dehydrogenase 8, manifested light-induced retinal dystrophy. We first examined early-stage age-related macular degeneration patients and found retinal degenerative changes in rod-rich rather than cone-rich regions of the macula. We then evaluated transgenic mice with rod-only and cone-like-only retinas in addition to progenies of such mice inbred with Rdh8(-/-) Abca4(-/-) mice. Of all these strains, Rdh8(-/-) Abca4(-/-) mice with a mixed rod-cone population showed the most severe retinal degeneration under regular cyclic light conditions. Intense light exposure induced acute retinal damage in Rdh8(-/-) Abca4(-/-) and rod-only mice but not cone-like-only mice. These findings suggest that progression of retinal degeneration in Rdh8(-/-) Abca4(-/-) mice is affected by differential vulnerability of rods and cones to light.