A multicentre randomised double masked clinical trial of a new formulation of topical cysteamine for the treatment of corneal cystine crystals in cystinosis.

AIM: To evaluate the safety and efficacy of a new topical cysteamine formulation, stable at room temperature, for the treatment of corneal cystine crystals in cystinosis. METHODS: 20 study subjects were enrolled in the safety study and 16 in the efficacy study. Both studies were randomised and double blind. The primary outcome for the safety study was the occurrence of predefined serious adverse reactions over 6 months and for the efficacy study the reduction of corneal cystine crystal score (CCCS) by 1.00 or more units on photographs graded by a reading centre using a standardised protocol. RESULTS: No study subject developed any serious adverse reactions. In the efficacy study, 47% of eyes receiving the standard formulation experienced a reduction in the CCCS of >/=1.00 after 1 year, while 7% of eyes on the new formulation experienced such a decrease (p=0.04). CONCLUSION: Although no serious adverse reactions were observed with either formulation, the new formulation was not as effective as the standard formulation.

Hermansky-Pudlak syndrome type 3 in Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and platelet storage-pool deficiency.

Hermansky-Pudlak syndrome (HPS), consisting of oculocutaneous albinism and a bleeding diathesis due to the absence of platelet dense granules, displays extensive locus heterogeneity. HPS1 mutations cause HPS-1 disease, and ADTB3A mutations cause HPS-2 disease, which is known to involve abnormal intracellular vesicle formation. A third HPS-causing gene, HPS3, was recently identified on the basis of homozygosity mapping of a genetic isolate of HPS in central Puerto Rico. We now describe the clinical and molecular characteristics of eight patients with HPS-3 who are of non-Puerto Rican heritage. Five are Ashkenazi Jews; three of these are homozygous for a 1303+1G-->A splice-site mutation that causes skipping of exon 5, deleting an RsaI restriction site and decreasing the amounts of mRNA found on northern blotting. The other two are heterozygous for the 1303+1G-->A mutation and for either an 1831+2T-->G or a 2621-2A-->G splicing mutation. Of 235 anonymous Ashkenazi Jewish DNA samples, one was heterozygous for the 1303+1G-->A mutation. One seven-year-old boy of German/Swiss extraction was compound heterozygous for a 2729+1G-->C mutation, causing skipping of exon 14, and resulting in a C1329T missense (R396W), with decreased mRNA production. A 15-year-old Irish/English boy was heterozygous for an 89-bp insertion between exons 16 and 17 resulting from abnormal splicing; his fibroblast HPS3 mRNA is normal in amount but is increased in size. A 12-year-old girl of Puerto Rican and Italian background has the 3,904-bp founder deletion from central Puerto Rico on one allele. All eight patients have mild symptoms of HPS; two Jewish patients had received the diagnosis of ocular, rather than oculocutaneous, albinism. These findings expand the molecular diagnosis of HPS, provide a screening method for a mutation common among Jews, and suggest that other patients with mild hypopigmentation and decreased vision should be examined for HPS.

Ophthalmic manifestations of Allgrove syndrome: report of a case.

A male patient with the ocular manifestations of Allgrove or triple-A syndrome is described. The need for early diagnosis based on alacrima, anisocoria and optic atrophy of this potentially fatal condition is stressed.

Immune response to retinal antigens in patients with gyrate atrophy and other hereditary retinal dystrophies.

PURPOSE: Gyrate atrophy (GA) is a rare hereditary disease that causes retinal destruction. Retinal damage in GA and other heredodegenerative diseases such as retinitis pigmentosa (RP) releases sequestered antigens and may trigger immune response to these molecules. Here, we studied the immune response to retinal antigens in patients with GA and RP and compared it with that of patients with inactive posterior uveitis and normal volunteers. PATIENTS AND METHODS: Peripheral blood was collected from 24 patients with RP, 10 patients with GA, 10 patients with inactive posterior uveitis, and 16 normal volunteers. Cell-mediated immune responses to human S-antigen (HS-Ag), bovine S-antigen (BS-Ag), and interphotoreceptor retinoid-binding protein (IRBP) were investigated by lymphocyte proliferation assay. In addition, serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were studied by ELISA. Immunologic data were correlated with clinical and electrophysiological findings. RESULTS: Patients with GA or RP responded to HS-Ag and BS-Ag more vigorously than patients with uveitis or healthy controls, as shown by higher mean stimulation indices and larger proportions of responders. Unlike S-Ag, IRBP stimulated low lymphocyte responses in only a small proportion of RP patients. The mean sVCAM-1 levels were significantly higher in the sera from patients with GA than in that from normal controls. CONCLUSION: An elevated cellular immune response to S-Ag is common in patients with GA and RP. This elevated cellular immune response to S-Ag may exacerbate retinal destruction in patients with GA and RP.

The metabolism of fatty acids in human Bietti crystalline dystrophy.

PURPOSE: To investigate the role of abnormal lipid metabolism in Bietti crystalline dystrophy. METHODS: Cultured human lymphocytes and fibroblasts from patients with Bietti crystalline dystrophy (BCD) were incubated in the presence of [(14)C]18:3n-3 or [(14)C]18:2n-6. Incorporation into the cellular lipid pools and further metabolism by desaturation or elongation were monitored by thin-layer chromatography and HPLC. Results were compared with those in normal control subjects and patients with Wolman disease (WD). RESULTS: Pulse-chase experiments with labeled fatty acids in all groups showed that, after 1 hour, radioactivity was largely confined to the triacylglyceride (TG) and choline phosphoglyceride (CPG) pools. However, after several hours, radioactivity was transferred from the TG and CPG pools, some going to the serine and ethanolamine phosphoglyceride (SPG and EPG) pools. Fibroblasts from all groups showed direct transfer of fatty acids (FAs) into CPG and EPG. Incorporation of labeled FAs into the EPG pool paralleled extensive desaturation and elongation of 18:2n-6 to 22:5n-6 and 18:3n-3 to 22:6n-3. Fibroblasts from patients with WD (a lysosomal acid lipase deficiency characterized by excessive lipid accumulation), showed higher incorporation of 18:2n-6 into TGs than did normal or BCD fibroblasts. Conversely, fibroblasts from patients with BCD showed lower conversion of 18:3n-3, but not of 18:2n-6, into polyunsaturated FAs (PUFAs) than those of normal subjects or patients with WD. This was true for total FAs, CPGs, and EPGs. Similar results were found in both fibroblasts and lymphocytes; however, unlike fibroblasts, lymphocytes from normal subjects showed similar levels of incorporation of FAs into EPGs and CPGs. In contrast, incorporation of 18:3n-3 into EPGs was decreased in lymphocytes from patients with BCD. CONCLUSIONS: BCD is characterized by a lower than normal conversion of FA precursors into n-3 PUFA, whereas there is a higher than normal level of n-6 and n-3 FAs incorporation into TGs in cells from patients with WD. These findings raise the possibility that abnormal lipid metabolism associated with BCD is the result of deficient lipid binding, elongation, or desaturation in contrast to the lysosomal acid lipase deficiency found in Wolman disease.

Assessment of visual function in patients with gyrate atrophy who are considered candidates for gene replacement.

OBJECTIVE: To assess the course of change of visual function outcome variables in 5 patients with gyrate atrophy before a gene replacement therapy clinical trial. METHODS: The outcome variables selected were visual field sensitivity and electroretinogram amplitude. The course of change of these outcome variables was determined by calculation of their half-lives. RESULTS: In the 4 to 6 years during which each patient was followed up for this study, median visual field half-lives were 17.0 years (static perimetry) and 11.4 years (kinetic perimetry). Median electroretinogram half-lives were 16.0 years (maximal response) and 10.7 years (flicker response). CONCLUSIONS: The course of the decline of visual function outcome variables is frequently slow. Thus, a long-term clinical trial would be required to assess the efficacy of the intervention in the preservation of visual function.

Genetic linkage of Bietti crystallin corneoretinal dystrophy to chromosome 4q35.

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive retinal degeneration characterized by multiple glistening intraretinal dots scattered over the fundus, degeneration of the retina, and sclerosis of the choroidal vessels, ultimately resulting in progressive night blindness and constriction of the visual field. Although BCD has been associated with abnormalities in fatty-acid metabolism and absence of fatty-acid binding by two cytosolic proteins, the genetic basis of BCD is unknown. We report linkage of the BCD locus to D4S426 (maximum LOD score [Z(max)] 4.81; recombination fraction [straight theta] 0), D4S2688 (Zmax=3.97; straight theta=0), and D4S2299 (Zmax=5.31; straight theta=0), on chromosome 4q35-4qtel. Multipoint analysis confirmed linkage to the region telomeric of D4S1652 with a Z(max) of 5.3 located 4 cM telomeric of marker D4S2930.

Corneal crystals in nephropathic cystinosis: natural history and treatment with cysteamine eyedrops.

Although renal disease is the most prominent feature of the lysosomal storage disease cystinosis, corneal cystine crystal formation remains a major complication, leading to photophobia, corneal erosions, and keratopathies. Moreover, the extent of corneal crystal accumulation reflects the course and severity of the disease itself, and the cornea is accessible to direct examination. Therefore, we employed a scoring system, based on a library of slit-lamp photographs of corneas with increasing crystal densities (0.00-3.00), to assess the degree of crystal accumulation in 170 patients with nephropathic cystinosis examined at the National Institutes of Health between 1976 and 2000. None of the patients had received topical cystine-depleting therapy at the time of the evaluation. In this natural history study, infants in the first year of life had absent or minimal corneal crystals, i.e., a corneal cystine crystal score (CCCS) of 0 or 0.25. However, the CCCS increased linearly with age, such that every patient had visible crystals by 16 months of age, and plateaued at approximately 3.00 by early adolescence. Longitudinal studies in representative patients support the cross-sectional results. Individuals homozygous for the common 57-kb deletion involving the cystinosis gene (CTNS) displayed the same course of corneal crystal accumulation as did individuals not bearing the large deletion. Patients with ocular or nonnephropathic cystinosis had CCCSs that were, in general, half those expected for patients with nephropathic cystinosis of the same age. Administration of 0.55% cysteamine eyedrops, given 6 to 12 times per day, dissolved corneal cystine crystals in 10 representative patients with nephropathic cystinosis aged 1 to 32 years within 8 to 41 months.

A 5-base insertion in the gammaC-crystallin gene is associated with autosomal dominant variable zonular pulverulent cataract.

A seven-generation family with 30 members affected by highly variable autosomal dominant zonular pulverulent cataracts has been previously described. We have localized the cataracts to a 19-cM interval on chromosome 2q33-q35 including the gamma-crystallin gene cluster. Maximum lod scores are 4.56 (theta=0.02) with D2S157, 3.66 (theta=0.12) with D2S72, and 3.57 (theta=0.052) with CRYG. Sequencing and allele-specific oligonucleotide analysis of the pseudo gammaE-crystallin promoter region from individuals in the pedigree suggest that activation of the gammaE-crystallin pseudo gene is unlikely to cause the cataracts in the family. In addition, base changes in the TATA box but not the Sp1-binding site have been found in unaffected controls and can be excluded as a sole cause of cataracts. In order to investigate the underlying genetic mechanism of cataracts in this family further, exons of the highly expressed gammaC- and gammaD-crystallin genes have been sequenced. The gammaD-crystallin gene shows no abnormalities, but a 5-bp duplication within exon 2 of the gammaC-crystallin gene has been found in one allele of each affected family member and is absent from both unaffected family members and unaffected controls. This mutation disrupts the reading frame of the gammaC-crystallin coding sequence and is predicted to result in the synthesis of an unstable gammaC-crystallin with 38 amino acids of the first "Greek key" motif followed by 52 random amino acids. This finding suggests that the appropriate association of mutant betagamma-crystallins into oligomers is not necessary to cause cataracts and may give us new insights into the genetic mechanism of cataract formation.

Correlation of visual acuity and ocular pigmentation with the 16-bp duplication in the HPS-1 gene of Hermansky-Pudlak syndrome, a form of albinism.

OBJECTIVE: Patients with the Hermansky-Pudlak syndrome (HPS), a form of albinism, were studied. The first purpose of this investigation was to determine if visual acuity was related to the presence or absence of the 16-bp duplication in the HPS-1 gene. The second was to study the correlation between the degree of ocular pigmentation and visual acuity within the two genetic groups described above. DESIGN: Cross-sectional study of a series of consecutive patients. PARTICIPANTS: Forty-nine patients with HPS with or without the 16-bp duplication in HPS-1. METHODS: Best corrected visual acuity (VA) using Early Treatment Diabetic Retinopathy Study (ETDRS) charts, photographic gradings of iris transillumination and of visibility of choroidal vessels in the macula (macular transparency). MAIN OUTCOME MEASURES: Association between VA and the presence or absence of the 16-bp duplication in HPS-1 and correlation between VA and the degree of iris transillumination (iris score) and macular transparency (fundus score), as determined by masked reading of photographs, with respect to the presence or absence of the 16-bp duplication in HPS-1 were the main outcome measures. RESULTS: The VA of the better eye did not differ between the two genetic groups (P = 0.322, two-sided t test). Spearman's rank correlation between VA and iris scores in 39 eyes of 20 patients with the duplication was not statistically significant (P = 0.698) but was statistically significant in 36 eyes of 19 patients without the duplication (P < 0.001). Among all patients, the correlation was statistically significant (r = -0.36 in RE and r = -0.51 in LE). Spearman's rank correlation between VA and fundus scores in 36 eyes of 19 patients with and 34 eyes in 18 patients with and without the duplication was statistically significant (P = 0.035 and P = 0.008, respectively). Among all patients, it was also statistically significant (r = -0.39 in RE and r = -0.45 in LE). CONCLUSIONS: The mean VA of the better eye did not differ in patients with the 16-bp duplication compared with those without the duplication. There were statistically significant associations between VA and the iris score and the fundus score except for the VA and iris scores in patients with the 16-bp duplication. However, because of the variability of VA, these associations were not large enough for useful prediction of VA based on the degree of ocular pigmentation.

Ocular nonnephropathic cystinosis: clinical, biochemical, and molecular correlations.

Ocular nonnephropathic cystinosis, a variant of the classic nephropathic type of cystinosis, is an autosomal recessive lysosomal storage disorder characterized by photophobia due to corneal cystine crystals but absence of renal disease. We determined the molecular basis for ocular cystinosis in four individuals. All had mutations in the cystinosis gene CTNS, indicating that ocular cystinosis is allelic with classic nephropathic cystinosis. The ocular cystinosis patients each had one severe mutation and one mild mutation, the latter consisting of either a 928 G-->A (G197R) mutation or an IVS10-3 C-->G splicing mutation resulting in the insertion of 182 bp of IVS10 into the CTNS mRNA. The mild mutations appear to allow for residual CTNS mRNA production, significant amounts of lysosomal cystine transport, and lower levels of cellular cystine compared with those in nephropathic cystinosis. The lack of kidney involvement in ocular cystinosis may be explained by two different mechanisms. On the one hand (e.g. the G197R mutation), significant residual cystinosin activity may be present in every tissue. On the other hand (e.g. the IVS 10-3 C-->G mutation), substantial cystinosin activity may exist in the kidney because of that tissue's specific expression of factors that promote splicing of a normal CTNS transcript. Each of these mechanisms could result in minimally reduced lysosomal cystine transport in the kidneys.

Pilot trial of the safety, tolerability, and retinoid levels of N-(4-hydroxyphenyl) retinamide in combination with tamoxifen in patients at high risk for developing invasive breast cancer.

PURPOSE: N-(4-hydroxyphenyl) retinamide (¿4-HPR, Fenretinide; R.W. Johnson Pharmaceutical Research Institute, Springhouse, PA) and tamoxifen (TAM) have synergistic antitumor and chemopreventive activity against mammary cancer in preclinical studies. We performed a pilot study of this combination in women at high risk for developing breast cancer. PATIENTS AND METHODS: Thirty-two women were treated with four cycles of 4-HPR, 200 mg orally (PO) for 25 days of each 28-day cycle, and TAM, 20 mg PO once daily for 23 months beginning after 1 month of 4-HPR alone. Tolerability, dark adaptometry, tissue biopsies, and retinoid plasma concentrations (Cp) were evaluated. RESULTS: Symptomatic reversible nyctalopia developed in two patients (6%) on 4-HPR, but 16 (73%) of 22 patients had reversible changes in dark adaptation, which correlated with relative decrease in Cp retinol (P

A randomized clinical trial of topical cysteamine disulfide (cystamine) versus free thiol (cysteamine) in the treatment of corneal cystine crystals in cystinosis.

In nephropathic cystinosis, corneal cystine crystals cause severe photophobia and corneal erosions. Topical cysteamine dissolves these crystals, but cannot be marketed because it rapidly oxidizes to the disulfide form, cystamine, at room temperature. Since cystamine itself could be used commercially, we compared the efficacy of cystamine and cysteamine with respect to cystine crystal dissolution in a randomized, double-masked clinical trial. One eye each of 14 patients with cystinosis was randomized to either cystamine or cysteamine, 0.5%, with 0.01% benzalkonium chloride; the companion eye was treated with the alternate preparation. Corneal crystals were photographed and a density score was assigned to each slide based on 13 standard slides. After 8-20 months, 6 patients showed significant reduction of the corneal crystal score in only one eye. In each case, the improved eye was the cysteamine-treated eye. Theoretically, cysteamine should dissolve both intracellular and extracellular crystals, whereas cystamine should dissolve only intracellular crystals because it must first be reduced to the free thiol by the cytoplasmic-reducing environment. Hence, the lack of efficacy of the disulfide cystamine suggests that some corneal cystine crystals in cystinosis patients are extracellular, and that another form of stable, topical cysteamine must be developed for cystinosis patients.

Identification, isolation, and characterization of a 32-kDa fatty acid-binding protein missing from lymphocytes in humans with Bietti crystalline dystrophy (BCD).

Bietti crystalline dystrophy (BCD) is an autosomal recessive retinal degeneration characterized by intraretinal lipid inclusions with degeneration of the retina and sclerosis of the choroidal vessels, resulting clinically in progressive night blindness and constriction of the visual fields. Characterization of fatty acid metabolism in Bietti crystalline dystrophy suggested that BCD might result from abnormalities in lipid-binding proteins or one or more enzymes active in fatty acid elongation and desaturation. To further investigate the first possibility, the docosahexaenoic acid-binding proteins (DHABPs) of human lymphocytes from patients with Bietti crystalline dystrophy were studied and compared with those of normal controls. For fatty acid-binding protein (FABP) identification, lymphocyte cytosol was first subjected to Lipidex 1000 chromatography. FABPs were then cross-linked with [14C]22:6n-3 and identified by HPLC and SDS-PAGE. Ten major peaks corresponding to calculated molecular weights of 13, 14, 32, 43, 45, 50, 64, 96, 105, and 186 kDa exhibit high-affinity binding of fatty acids. Significantly, peaks corresponding to two fatty acid-binding proteins of 32 and 45 kDa present in age-matched controls are absent from lymphocytes of patients with BCD. The 32-kDa fatty acid-binding protein present in normal individuals but absent from patients with BCD was isolated from cultured control human lymphocytes, its fatty acid-binding properties were characterized, and its amino acid composition was analyzed. It shows specific binding of 3n-3 fatty acids, consistent with the pattern of abnormalities of lipid metabolism demonstrated in patients with BCD. These results suggest that the 32- and 43-kDa FABPs are reasonable candidates for causing BCD.

Autosomal dominant zonular cataract with sutural opacities is associated with a splice mutation in the betaA3/A1-crystallin gene.

PURPOSE: Congenital cataracts constitute a morphologically and genetically heterogeneous group of diseases that are a major cause of childhood blindness. Autosomal Dominant Zonular Cataracts with Sutural Opacities (CCZS) have been mapped to chromosome 17q11-q12 near the betaA3A1-crystallin gene (CRYBA1). The betaA3A1-crystallin gene was investigated as the causative gene for the cataracts. METHODS: The betaA3/A1-crystallin gene was sequenced in affected and control individuals. Base changes were confirmed and assayed in additional family members and controls using NlaIII restriction digestion of PCR amplified DNA sequences. Base changes were assessed for their effects on splicing by information analysis. RESULTS: The cataracts are associated with a sequence change in the 5' (donor) splice site of intron 3: GC(g->a)tgagt. The sequence change also creates a new NlaIII site. This base change cosegregates with the cataracts in this family, being present in every affected individual. Conversely, this base change was not seen in 140 chromosomes examined in 70 unaffected and unrelated individuals. Information theory mutational analysis shows that the base change lowers the information content of the splice site from 6.0 to -6.8 bits, so that splicing would not be expected to occur at the altered site. CONCLUSIONS: Taken together, these observations suggest that the observed mutation might be causally related to the cataracts in this family.

Three new mutations in a gene causing Hermansky-Pudlak syndrome: clinical correlations.

Hermansky-Pudlak syndrome (HPS) consists of oculocutaneous albinism, a platelet storage pool deficiency, and ceroid lipofuscinosis. HPS is common in northwest Puerto Rico, where affected individuals are homozygous for a 16-bp duplication in the gene HPS. Two other homozygous frameshift mutations in HPS were previously identified among non-Puerto Rican patients. Eighteen non-Puerto Rican HPS families were studied and HPS mutations in three of them identified. One mutation, T322insC, has been previously described. However, three additional mutations, E133X, T322delC, and S396delC, have not been reported. Two families exhibited compound heterozygosity for these mutations, although most previously reported HPS patients have been homozygous for a particular mutation. All the newly described mutations were associated with decreased or undetectable levels of HPS RNA by Northern blot analysis of fibroblasts, and all had significant pigment dilution. To date, all mutations in HPS result in a truncated protein, suggesting that the C-terminal portion of the HPS protein is functionally important.

Novel mutations in the XLRS1 gene may be caused by early Okazaki fragment sequence replacement.

PURPOSE: To determine whether two families diagnosed with X-linked retinoschisis contained mutations in the XLRS1 gene. METHODS: DNA from the patients was obtained from blood lymphocytes using commercially available kits. Single-strand conformation assay was performed in an electrophoresis apparatus using 10% acrylamide TBE gels at 10 degrees C. The gels were stained with SYB green II and were scanned in a phosphoimager. DNA was sequenced using an automated fluorescence sequencer. RESULTS: A deletion that eliminates exon 2 was found in one family. An abnormal sequence replacement in exon 4 was found in the other family. Both mutations have severe effects in the coding region by inserting premature stop codons. CONCLUSIONS: Both of the families have mutations in the XLRS1 gene. One of these mutations points to a novel mechanism. The mutation is caused by a replacement of 17 bp of a normal sequence with 20 bp of a sequence originating from two different places in the antisense strand. This suggests that early Okazaki fragments were incorporated into the sense strand of exon 4, replacing the normal sequence.

Effects of fenretinide (4-HPR) on dark adaptation.

OBJECTIVES: To assess the alterations in dark adaptation induced by low (200 mg/d) doses of fenretinide (4-HPR), to assess whether these effects were cumulative and whether they were reversible, and to attempt to elucidate the mechanism underlying the changes in night vision. DESIGN: Case series. SETTING: Outpatient eye clinic. PATIENTS: Twenty-two women enrolled in a breast cancer chemoprevention trial, and 18 normal control subjects. INTERVENTION: Measurements of absolute luminance thresholds during dark adaptation. MAIN OUTCOME MEASURES: Parameters of an exponential model of the dark-adaptation function before, during, and after administration of fenretinide. RESULTS: The most conspicuous effect of fenretinide on dark adaptation was a significant delay in the timing of the rod-cone break (P<.001). A minimal elevation of the final cone threshold was also observed. These effects were reversible after fenretinide therapy was discontinued and did not seem to be cumulative. An inverse relationship between delay of the rod-cone break and plasma retinol concentration was found. CONCLUSION: The dose of fenretinide used in this study produced clearly measurable, but not severe, changes in night vision, which were rarely symptomatic.