Mutations in the small nuclear riboprotein 200 kDa gene (SNRNP200) cause 1.6% of autosomal dominant retinitis pigmentosa.

PURPOSE: The purpose of this project was to determine the spectrum and frequency of mutations in the small nuclear riboprotein 200 kDa gene (SNRNP200) that cause autosomal dominant retinitis pigmentosa (adRP). METHODS: A well-characterized adRP cohort of 251 families was tested for mutations in the exons and intron/exon junctions of SNRNP200 using fluorescent dideoxy sequencing. An additional 21 adRP families from the eyeGENE® Network were tested for possible mutations. Bioinformatic and segregation analysis was performed on novel variants. RESULTS: SNRNP200 mutations were identified in seven of the families tested. Two previously reported mutations, p.Arg681Cys and p.Ser1087Leu, were found in two families each. One family had the previously reported p.Arg681His mutation. Two novel SNRNP200 variants, p.Pro682Ser and p.Ala542Val, were also identified in one family each. Bioinformatic and segregation analyses suggested that these novel variants are likely to be pathogenic. Clinical examination of patients with SNRNP200 mutations showed a wide range of clinical symptoms and severity, including one instance of non-penetrance. CONCLUSIONS: Mutations in SNRNP200 caused 1.6% of disease in our adRP cohort. Pathogenic mutations were found primarily in exons 16 and 25, but the novel p.Ala542Val mutation in exon 13 suggests that variation in other genetic regions is also responsible for causing dominant disease. SNRNP200 mutations were associated with a wide range of clinical symptoms similar to those of individuals with other splice-factor gene mutations.

A Novel Dominant Mutation in SAG, the Arrestin-1 Gene, Is a Common Cause of Retinitis Pigmentosa in Hispanic Families in the Southwestern United States.

Purpose: To identify the causes of autosomal dominant retinitis pigmentosa (adRP) in a cohort of families without mutations in known adRP genes and consequently to characterize a novel dominant-acting missense mutation in SAG. Methods: Patients underwent ophthalmologic testing and were screened for mutations using targeted-capture and whole-exome next-generation sequencing. Confirmation and additional screening were done by Sanger sequencing. Haplotypes segregating with the mutation were determined using short tandem repeat and single nucleotide variant polymorphisms. Genealogies were established by interviews of family members. Results: Eight families in a cohort of 300 adRP families, and four additional families, were found to have a novel heterozygous mutation in the SAG gene, c.440G>T; p.Cys147Phe. Patients exhibited symptoms of retinitis pigmentosa and none showed symptoms characteristic of Oguchi disease. All families are of Hispanic descent and most were ascertained in Texas or California. A single haplotype including the SAG mutation was identified in all families. The mutation dramatically alters a conserved amino acid, is extremely rare in global databases, and was not found in 4000+ exomes from Hispanic controls. Molecular modeling based on the crystal structure of bovine arrestin-1 predicts protein misfolding/instability. Conclusions: This is the first dominant-acting mutation identified in SAG, a founder mutation possibly originating in Mexico several centuries ago. The phenotype is clearly adRP and is distinct from the previously reported phenotypes of recessive null mutations, that is, Oguchi disease and recessive RP. The mutation accounts for 3% of the 300 families in the adRP Cohort and 36% of Hispanic families in this cohort.

Visual maturation of term infants fed long-chain polyunsaturated fatty acid-supplemented or control formula for 12 mo.

BACKGROUND: Several studies found a benefit of long-chain polyunsaturated fatty acid (LCP) supplementation for visual or mental development, but others found no benefit. Likely contributors to differences among studies are the amount of LCP supplementation, functional outcomes, and sample size. OBJECTIVE: We evaluated LCP supplementation in amounts typical for human milk (based on local and worldwide surveys) in a large cohort of infants by using sweep visual evoked potential (VEP) acuity as the functional outcome. DESIGN: The study was a double-masked, randomized, controlled clinical trial in 103 term infants. By age 5 d, infants were randomly assigned to receive either formula with no docosahexaenoic acid (DHA) or arachidonic acid (ARA) or formula supplemented with DHA and ARA as 0.36% and 0.72%, respectively, of total fatty acids. Sweep VEP acuity was the primary outcome. Random dot stereoacuity, blood lipid profile, growth, and tolerance were secondary outcomes. RESULTS: VEP acuity in the LCP-supplemented group was significantly better than that in the control group at ages 6, 17, 26, and 52 wk. Stereoacuity in the LCP-supplemented group was significantly better than that in the control group at age 17 wk but not at ages 39 and 52 wk. By ages 17 and 39 wk, the red blood cell DHA concentration in the LCP-supplemented group was more than double and more than triple, respectively, that in the control group. Growth of infants fed LCP-supplemented and control formulas did not differ significantly, and both diets were well tolerated. CONCLUSION: LCP supplementation of term infant formula during the first year of life yields clear differences in visual function and in total red blood cell lipid composition.

Duration of long-chain polyunsaturated fatty acids availability in the diet and visual acuity.

BACKGROUND: Little is known about the critical period during which the dietary supply of long-chain polyunsaturated fatty acids (LCPUFAs) may influence the maturation of visual cortical function in term infants. AIM: To define the relationship between duration of dietary LCPUFA supply and visual acuity at 52 weeks of age. STUDY DESIGN: Data from 243 infants who participated in four randomized clinical trials of LCPUFA supplementation of infant formula at a single research center were combined. The primary outcome was visual acuity at 52 weeks of age as measured by swept visual evoked potentials (sweep VEP). RESULTS: Longer duration of LCPUFA supply was associated with better mean acuity at 52 weeks of age (r=-0.878; p<0.001). The relationship between duration of dietary LCPUFA supply and sweep VEP acuity at 52 weeks was similar whether the LCPUFAs were provided via formula containing 0.36% DHA and 0.72% ARA or human milk. Duration of breast-feeding was associated with individual infants' sweep VEP acuity outcomes at 52 weeks (r=-0.286; p<0.005). The duration of LCPUFA supply during infancy has a similar relationship to sweep VEP acuity at 52 weeks in breastfed infants regardless of birth order. CONCLUSION: A continued benefit from a supply of LCPUFAs is apparent in infants through 52 weeks of age, suggesting that the brain may not have sufficient stores of LCPUFAs from an early postnatal supply to support the optimal maturation of the visual cortex.

Biological safety assessment of docosahexaenoic acid supplementation in a randomized clinical trial for X-linked retinitis pigmentosa.

BACKGROUND: In a 4-year placebo-controlled trial to elevate blood docosahexaenoic acid levels in patients with X-linked retinitis pigmentosa (XLRP), the goal was to assess the potential benefit of docosahexaenoic acid supplementation in altering disease progression. However, docosahexaenoic acid (22:6omega3) is a highly unsaturated fatty acid and considered a target molecule for free-radical oxidative damage. Thus, nutritional provision of docosahexaenoic acid might lead to an increase in antioxidant stress. Additional concerns, such as decreased platelet aggregation, increased bleeding time, and alterations in lipoprotein cholesterol levels, have been reported in supplementation studies with long-chain polyunsaturates. OBJECTIVE: To assess the biological safety of long-term docosahexaenoic acid supplementation. DESIGN: Forty-four male patients (mean age, 16 years) enrolled in a randomized, double-masked, clinical trial and received docosahexaenoic acid, 400 mg/d, or placebo. Blood samples were collected every 6 months. Biological safety analysis included fatty acids, vitamin A and E concentrations, antioxidant capacity, platelet aggregation, alanine aminotransferase activity, and lipoprotein cholesterol and triglyceride profiles. RESULTS: Mean plasma docosahexaenoic acid levels were elevated 2.5-fold by supplementation compared with baseline. Patients receiving placebo capsules exhibited no change (P =.35) in plasma docosahexaenoic acid content. All adverse events reported were minor and equivalently distributed between groups. Plasma vitamin A concentrations remained unchanged during the trial. Mean plasma vitamin E concentrations were correlated with age (P =.005), such that as patients with XLRP matured, plasma vitamin E concentrations increased to approach normal values. There was a trend (P =.10) toward lower mean vitamin E concentrations in the docosahexaenoic acid-supplemented group after 4 years. Docosahexaenoic acid supplementation did not compromise plasma antioxidant capacity, platelet aggregation, liver function enzyme activity, or plasma lipoprotein lipid content in patients with XLRP. CONCLUSION: Long-term docosahexaenoic acid supplementation to patients with XLRP was associated with no identifiable safety risks in this 4-year clinical trial.

A randomized, placebo-controlled clinical trial of docosahexaenoic acid supplementation for X-linked retinitis pigmentosa.

PURPOSE: Low docosahexaenoic acid (DHA) in X-linked retinitis pigmentosa (XLRP) may influence retinal function. The goals of this study were to elevate blood DHA levels and determine the effect on the rate of disease progression. DESIGN: In a 4-year prospective randomized clinical trial, male patients with XLRP (mean age = 16 years; range = 4-38 years) received DHA (400 mg/d; n = 23; +DHA group) or placebo (n = 21) capsules. METHODS: Red blood cell (RBC)-DHA concentrations were assessed every 6 months. Full-field cone electroretinograms (ERGs; the primary outcome measure), visual acuity, dark-adaptation, visual fields, rod ERGs, and fundus photos were recorded annually. RESULTS: In the +DHA group, RBC-DHA increased 2.5-fold over placebo levels (70 vs 28 mg DHA/l). Repeated measures analysis of variance for cone ERG showed a significant main effect of year (P <.0001) but not of group (P =.16). Preservation of cone ERG function correlated with RBC-DHA (P =.018), and there was less change in fundus appearance in the +DHA group (P =.04). Neither visual acuity nor visual fields were changed. In subset analysis, DHA supplementation was beneficial in reducing rod ERG functional loss in patients aged <12 years (P =.040) and preserving cone ERG function in patients > or =12 years (P =.038). CONCLUSIONS: Although DHA-supplemented patients had significantly elevated mean RBC-DHA levels, the rate of cone ERG functional loss was not significantly different between groups. Supplemental analyses provided evidence for a DHA benefit and a direction for subsequent investigations.

A dominant mutation in hexokinase 1 (HK1) causes retinitis pigmentosa.

PURPOSE: To identify the cause of retinitis pigmentosa (RP) in UTAD003, a large, six-generation Louisiana family with autosomal dominant retinitis pigmentosa (adRP). METHODS: A series of strategies, including candidate gene screening, linkage exclusion, genome-wide linkage mapping, and whole-exome next-generation sequencing, was used to identify a mutation in a novel disease gene on chromosome 10q22.1. Probands from an additional 404 retinal degeneration families were subsequently screened for mutations in this gene. RESULTS: Exome sequencing in UTAD003 led to identification of a single, novel coding variant (c.2539G>A, p.Glu847Lys) in hexokinase 1 (HK1) present in all affected individuals and absent from normal controls. One affected family member carries two copies of the mutation and has an unusually severe form of disease, consistent with homozygosity for this mutation. Screening of additional adRP probands identified four other families (American, Canadian, and Sicilian) with the same mutation and a similar range of phenotypes. The families share a rare 450-kilobase haplotype containing the mutation, suggesting a founder mutation among otherwise unrelated families. CONCLUSIONS: We identified an HK1 mutation in five adRP families. Hexokinase 1 catalyzes phosphorylation of glucose to glucose-6-phosphate. HK1 is expressed in retina, with two abundant isoforms expressed at similar levels. The Glu847Lys mutation is located at a highly conserved position in the protein, outside the catalytic domains. We hypothesize that the effect of this mutation is limited to the retina, as no systemic abnormalities in glycolysis were detected. Prevalence of the HK1 mutation in our cohort of RP families is 1%.

A randomized trial of DHA intake during infancy: school readiness and receptive vocabulary at 2-3.5 years of age.

BACKGROUND: Studies investigating the effects of docosahexaenoic acid (DHA) in infant formula on language development yield conflicting results. No study to date has investigated the effects of DHA in infant formula on school readiness. AIM: To determine the effects of different dietary concentrations of DHA provided during the first 12 months of life on language development and school readiness. DESIGN: This was a double-masked, randomized, controlled, prospective trial. A total of 182 infants were enrolled at 1-9 days of age and assigned randomly to receive infant formula with one of four levels of DHA: control (0% DHA), 0.32% DHA, 0.64% DHA, or 0.96% DHA. All formulas with DHA also contained 0.64% arachidonic acid. One hundred forty-one children completed the 12-month feeding trial and were eligible for this study. Consent was obtained from 131 participants. School readiness was assessed at 2.5 years using the Bracken Basic Concept Scale-Revised (BBCS-R) and receptive vocabulary was assessed at 2 and 3.5 years using the Peabody Picture Vocabulary Test-Third Edition (PPVT-III). RESULTS: There were no diet group differences on any of the BBCS-R subscales. On the PPVT-III, the control group had higher raw scores and standard scores than both the 0.32% and 0.96% groups at 2 years of age. These differences were not evident at 3.5 years. CONCLUSIONS: Dietary DHA during the first year of life did not enhance school readiness or language development. Children who consumed infant formula with 0.32% and 0.96% DHA showed lower receptive vocabulary scores than controls at 2 but not 3.5 years of age.

Cognitive function in 18-month-old term infants of the DIAMOND study: a randomized, controlled clinical trial with multiple dietary levels of docosahexaenoic acid.

BACKGROUND: Studies investigating cognitive outcomes following docosahexaenoic acid (DHA) supplementation of infant formula yield conflicting results, perhaps due to inadequate dietary concentrations. AIM: To determine the optimal DHA concentration in term formula to support cognitive maturation. DESIGN: This was a double-masked, randomized, controlled, prospective trial. A total of 181 infants were enrolled at 1-9 days of age and assigned randomly to receive one of four term infant formulas with one of four levels of docosahexaenoic acid: Control (0% DHA), 0.32% DHA, 0.64% DHA, or 0.96% DHA. All DHA-supplemented formulas contained 0.64% arachidonic acid (ARA). Infants were fed the assigned formulas until 12 months of age. One hundred forty-one children completed the 12-month feeding trial and were eligible for this study. Cognitive function was assessed in 131 children at 18 months of age using the Bayley Scales of Infant Development II (BSID II). RESULTS: There were no diet group differences on the Mental Development Index (MDI), the Psychomotor Development Index (PDI), or the Behavior Rating Scale (BRS) of the BSID II. However, when the scores of children who received any of the three DHA-supplemented formulas were combined and compared to control children, a significant difference emerged: the MDI scores of DHA-supplemented children were higher (104.1 v. 98.4; p=0.02). CONCLUSIONS: These results suggest that dietary supplementation of DHA during the first year of life leads to enhanced cognitive development at 18 months of age. DHA concentration of 0.32% is adequate to improve cognitive function; higher concentrations did not confer additional benefit.

Identification of disease-causing mutations in autosomal dominant retinitis pigmentosa (adRP) using next-generation DNA sequencing.

PURPOSE: To determine whether massively parallel next-generation DNA sequencing offers rapid and efficient detection of disease-causing mutations in patients with monogenic inherited diseases. Retinitis pigmentosa (RP) is a challenging application for this technology because it is a monogenic disease in individuals and families but is highly heterogeneous in patient populations. RP has multiple patterns of inheritance, with mutations in many genes for each inheritance pattern and numerous, distinct, disease-causing mutations at each locus; further, many RP genes have not been identified yet. METHODS: Next-generation sequencing was used to identify mutations in pairs of affected individuals from 21 families with autosomal dominant RP, selected from a cohort of families without mutations in "common" RP genes. One thousand amplicons targeting 249,267 unique bases of 46 candidate genes were sequenced with the 454GS FLX Titanium (Roche Diagnostics, Indianapolis, IN) and GAIIx (Illumina/Solexa, San Diego, CA) platforms. RESULTS: An average sequence depth of 70× and 125× was obtained for the 454GS FLX and GAIIx platforms, respectively. More than 9000 sequence variants were identified and analyzed, to assess the likelihood of pathogenicity. One hundred twelve of these were selected as likely candidates and tested for segregation with traditional di-deoxy capillary electrophoresis sequencing of additional family members and control subjects. Five disease-causing mutations (24%) were identified in the 21 families. CONCLUSION: This project demonstrates that next-generation sequencing is an effective approach for detecting novel, rare mutations causing heterogeneous monogenic disorders such as RP. With the addition of this technology, disease-causing mutations can now be identified in 65% of autosomal dominant RP cases.

Visual function in breast-fed term infants weaned to formula with or without long-chain polyunsaturates at 4 to 6 months: a randomized clinical trial.

OBJECTIVE: Breast-fed infants receive docosahexaenoic acid (DHA) and arachidonic acid (ARA) in their diet. Upon weaning, infants lose this dietary source of long-chain polyunsaturates because many commercial formulas do not contain these important constituents for neural membrane biogenesis. We evaluated the benefits of postweaning dietary supplementation of DHA + ARA on visual maturation. STUDY DESIGN: Healthy term infants (n = 61) were breast-fed to 4 to 6 months, then were randomly assigned to commercial formula or formula supplemented with DHA (0.36%) + ARA (0.72%). Measurements of red blood cell (RBC) fatty acids, visually evoked potential (VEP) acuity, and stereoacuity were done before and after weaning. RESULTS: At 1 year of age, RBC-DHA in the commercial formula-fed group was reduced by 50% from the weaning level, whereas there was a 24% increase in the DHA + ARA-supplemented group. The primary outcome measure, VEP acuity, was significantly more mature in supplemented infants at 1 year of age. Elevated RBC-DHA levels were associated with more mature VEP acuity. There were no significant diet-related differences in stereoacuity. CONCLUSIONS: These data extend through the first year of life the critical period in which a dietary supply of DHA and ARA can contribute in optimizing visual development in term infants.

Maturation of visual acuity is accelerated in breast-fed term infants fed baby food containing DHA-enriched egg yolk.

Between 6 and 12 mo of age, blood levels of the (n-3) long-chain PUFA, docosahexaenoic acid (DHA), in breast-fed infants typically decrease due to diminished maternal DHA stores and the introduction of DHA-poor solid foods displacing human milk as the primary source of nutrition. Thus, we utilized a randomized, clinical trial format to evaluate the effect of supplemental DHA in solid foods on visual development of breast-fed infants with the primary outcome, sweep visual-evoked potential (VEP) acuity, as an index for maturation of the retina and visual cortex. At 6 mo of age, breast-fed infants were randomly assigned to receive 1 jar (113 g)/d of baby food containing egg yolk enriched with DHA (115 mg DHA/100 g food; n = 25) or control baby food (0 mg DHA; n = 26). Gravimetric measures were used to estimate the supplemental DHA intake which was 83 mg DHA/d in the supplemented group and 0 mg/d in controls. Although many infants in both groups continued to breast-feed for a mean of 9 mo, RBC DHA levels decreased significantly between 6 and 12 mo (from 3.8 to 3.0 g/100 g total fatty acids) in control infants, whereas RBC DHA levels increased by 34% from 4.1 to 5.5 g/100 g by 12 mo in supplemented infants. VEP acuity at 6 mo was 0.49 logMAR (minimum angle of resolution) and improved to 0.29 logMAR by 12 mo in controls. In DHA-supplemented infants, VEP acuity was 0.48 logMAR at 6 mo and matured to 0.14 logMAR at 12 mo (1.5 lines on the eye chart better than controls). At 12 mo, the difference corresponded to 1.5 lines on the eye chart. RBC DHA levels and VEP acuity at 12 mo were correlated (r = -0.50; P = 0.0002), supporting the need of an adequate dietary supply of DHA throughout 1 y of life for neural development.