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otal of 126 Bulgarian patients with WD were included in the study. Detailed history, physical and neurological examination, laboratory investigation of copper metabolism, slit-lamp examination, abdominal ultrasound, magnetic resonance imaging/computed tomography of the brain, molecular genetic testing, and statistical analysis were performed. RESULTS: Eighty-two patients demonstrated neurological signs. Tremor and dysarthria were most frequently observed. Rigidity, bradykinesia, and pyramidal signs were found in >25% of the patients. Dystonia, chorea, athetosis, ballismus, and epilepsy were rarely observed. We identified a total of 27 mutations of ATP7B. The most frequent mutation is p.H1069Q found on at least 1 allele in 78% of the patients. We did not find a significant correlation between p.H1069Q homozygosity and age of onset, ceruloplasmin level, and urinary copper excretion. The patients homozygous for p.H1069Q presented more frequently with hepatic signs. Mutations predicted to cause production of truncated protein are associated with earlier age at onset and lower ceruloplasmin level. In contrast to Bulgarian patients, Roma patients had an earlier disease onset and more frequent hepatic manifestation. CONCLUSIONS: WD presents with a variety of neurological signs. The mutation p.H1069Q is not uniformly associated with late onset and neurological presentation. Frameshift and nonsense mutations lead to severe phenotype. There are ethnic-specific differences in disease manifestation.

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ically regulates the activity of photoreceptor-specific genes. Mutations in the human NRL gene are associated with retinal degenerative diseases. Here we report functional analyses of 17 amino acid variations and/or mutations of NRL. We show that 13 of these lead to changes in NRL phosphorylation. Six mutations at residues p.S50 (c.148T>A, c.148T>C, and c.149C>T) and p.P51 (c.151C>A, c.151C>T, and c.152C>T), identified in patients with autosomal dominant retinitis pigmentosa, result in a major NRL isoform that exhibits reduced phosphorylation but enhanced activation of the rhodopsin promoter. The truncated NRL mutant proteins-p.L75fs (c.224_225insC) and p.L160fs (c.459_477dup)-do not localize to the nucleus because of the absence of bZIP domain. The p.L160P (c.479T>C), p.L160fs, and p.R218fs (c.654delC) mutant proteins do not bind to the NRL-response element, as revealed by electrophoretic mobility shift assays. These three and p.S225N (c.674G>A) mutant show reduced transcriptional activity and may contribute to recessive disease. The p.P67S (c.199C>T) and p.L235F (c.703C>T) variations in NRL do not appear to directly cause retinitis pigmentosa, while p.E63K (c.187G>A), p.A76V (c.227C>T), p.G122E (c.365G>A), and p.H125Q (c.375C>G) are of uncertain significance. Our results support the notion that gain-of-function mutations in the NRL gene cause autosomal dominant retinitis pigmentosa while loss-of-function NRL mutations lead to autosomal recessive retinitis pigmentosa. We propose that differential phosphorylation of NRL fine-tunes its transcriptional regulatory activity, leading to a more precise control of gene expression.

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. METHODS: Single-strand conformation analysis was used to survey 189 unrelated patients for mutations. The available relatives of index patients with mutations were also evaluated. In our clinical examination of patients, we measured visual acuity, final dark-adaptation threshold equivalent visual field diameter, and electroretinogram amplitudes among other parameters of visual function. We compared the clinical findings with those obtained earlier from similar evaluations of a group of 39 patients with the dominant rhodopsin mutation Pro23His and a group of 25 patients with the dominant rhodopsin mutation Pro347Leu. RESULTS: We identified 3 novel missense mutations in a total of 4 unrelated patients with dominant retinitis pigmentosa: Ser50Pro, Ser50Leu (2 patients), and Pro51Thr. Each mutation cosegregated with dominant retinitis pigmentosa. None of these mutations were found among 91 unrelated control individuals. The visual acuities among the 4 index patients and 3 relatives with NRL mutations who were clinically evaluated ranged from 20/20 (in a 9-year-old patient) to 20/200 (in a 73-year-old patient). All patients had bone-spicule pigment deposits in their fundi. Average rod-plus-cone and cone-isolated electroretinogram amplitudes were both decreased by 99% or more compared with normal amplitudes. The dark-adaptation thresholds, equivalent visual field diameters, and electroretinogram amplitudes (all corrected for age and refractive error) indicated that the disease caused by the NRL mutations was more severe than that caused by the dominant rhodopsin mutation Pro23His and was similar in severity to that produced by the rhodopsin mutation Pro347Leu. CONCLUSION: The 3 novel NRL mutations we discovered bring the total number of reported mutations in this gene to 6. Five of the 6 mutations affect residues 50 or 51, suggesting that these residues are important in a structural or functional domain of the encoded protein. CLINICAL RELEVANCE: Rod and cone function is affected to a similar degree in patients with these mutations. The disease caused by NRL mutations found in this study appears to be more severe than that caused by the rhodopsin mutation Pro23His and is similar in severity to that caused by the rhodopsin mutation Pro347Leu, even after correcting for age.

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, the phenotype associated with the loss of NRL function in humans has not been reported. We identified two siblings who carried two allelic mutations: a predicted null allele (L75fs) and a missense mutation (L160P) altering a highly conserved residue in the domain involved in DNA-binding-site recognition. In vitro luciferase reporter assays demonstrated that the NRL-L160P mutant had severely reduced transcriptional activity compared with the WT NRL protein, consistent with a severe loss of function. The affected patients had night blindness since early childhood, consistent with a severe reduction in rod function. Color vision was normal, suggesting the presence of all cone color types; nevertheless, a comparison of central visual fields evaluated with white-on-white and blue-on-yellow light stimuli was consistent with a relatively enhanced function of short-wavelength-sensitive cones in the macula. The fundi had signs of retinal degeneration (such as vascular attenuation) and clusters of large, clumped, pigment deposits in the peripheral fundus at the level of the retinal pigment epithelium (clumped pigmentary retinal degeneration). Our report presents an unusual clinical phenotype in humans with loss-of-function mutations in NRL.

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graphy, optical coherence tomography, and a best-corrected visual acuity test were performed to define the clinical features of the patients. We extracted the genomic DNA from peripheral blood samples. The proband's genomic DNA was submitted to the whole exome sequencing. RESULTS: Whole exome sequencing and the subsequent data analysis detected six candidate mutations in the proband of this pedigree. The novel c.146 C>T mutation in NRL was found to be the only mutation that co-segregated with the disease in this pedigree. This mutation resulted in a substitution of proline by a leucine at position 49 of NRL protein (p.P49L). Most importantly, the proline residue at position 49 of NRL is highly conserved from zebrafish to humans. The c.146 C>T mutation was not observed in 200 control individuals. What's more, we performed the luciferase activity assay to prove that this mutation we detected alters the NRL protein function. CONCLUSIONS: The c.146 C>T mutation in NRL gene causes autosomal dominant retinitis pigmentosa for this family. Our finding not only expands the mutation spectrum of NRL, but also demonstrates that whole-exome sequencing is a powerful strategy to detect causative genes and mutations in RP patients. This technique may provide a precise diagnosis for rare heterogeneous monogenic disorders such as RP.