Dianna H. Wheaton

Impact of early dietary intake and blood lipid composition of long-chain polyunsaturated fatty acids on later visual development.

Background In contrast to human milk, current infant formulas in the United States do not contain ω3 and ω6 long-chain polyunsaturated fatty acids. This may lead to suboptimal blood lipid fatty acid profiles and to a measurable diminution of visual function in developing term infants. The need for docosahexaenoic acid and arachidonic acid supplementation in the infant diet was evaluated in a double-blind, randomized clinical trial. Methods Healthy term infants were randomized to diets of (1) commercial formula, (2) docosahexaenoic acid–enriched formula (0.35% of total fatty acids), or (3) docosahexaenoic acid– (0.36%) and arachidonic acid– (0.72%) enriched formula. Eighty-seven infants completed the 17-week nutritional trial, and 58 were observed until 52 weeks of life. A reference group was exclusively breast fed for at least 17 weeks (n = 29). Outcome measures included electroretinographic responses, visual evoked potentials, and blood fatty acid analysis in infants at birth and at 6, 17, and 52 weeks of age. Results Commercial formula-fed infants had 30% to 50% lower content of docosahexaenoic acid in total red blood cell lipids during the 17-week feeding trial compared with breast-fed infants. Significant differences persisted at the 1-year follow-up. Arachidonic acid content was consistently reduced in the commercial formula group by 15% to 20%. Infants fed long-chain polyunsaturated fatty acid–enriched formulas had docosahexaenoic acid and arachidonic acid blood lipid profiles resembling those of human milk-fed infants. Infants receiving this enriched formula had more mature electroretinographic responses than commercial formula-fed infants at 6 weeks of age. Human milk-fed and docosahexaenoic acid-enriched formula-fed infants had better visual acuity than commercial formula-fed infants at both 17 and 52 weeks of age. Early (17-week) fatty acid profiles in blood lipids were correlated with later (52-week) visual function development in study infants. Conclusions Results from this clinical trial demonstrate that long-chain polyunsaturated fatty acid supplementation of formula in term infants produces blood lipid fatty acid profiles that are similar to those observed in breast-fed infants. This supplementation leads to better visual function later in life (i.e., 1 year of age) than that shown by infants fed commercial formula.

Comprehensive molecular diagnosis of 179 Leber congenital amaurosis and juvenile retinitis pigmentosa patients by targeted next generation sequencing.

Background Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are inherited retinal diseases that cause early onset severe visual impairment. An accurate molecular diagnosis can refine the clinical diagnosis and allow gene specific treatments. Methods We developed a capture panel that enriches the exonic DNA of 163 known retinal disease genes. Using this panel, we performed targeted next generation sequencing (NGS) for a large cohort of 179 unrelated and prescreened patients with the clinical diagnosis of LCA or juvenile RP. Systematic NGS data analysis, Sanger sequencing validation, and segregation analysis were utilised to identify the pathogenic mutations. Patients were revisited to examine the potential phenotypic ambiguity at the time of initial diagnosis. Results Pathogenic mutations for 72 patients (40%) were identified, including 45 novel mutations. Of these 72 patients, 58 carried mutations in known LCA or juvenile RP genes and exhibited corresponding phenotypes, while 14 carried mutations in retinal disease genes that were not consistent with their initial clinical diagnosis. We revisited patients in the latter case and found that homozygous mutations in PRPH2 can cause LCA/juvenile RP. Guided by the molecular diagnosis, we reclassified the clinical diagnosis in two patients. Conclusions We have identified a novel gene and a large number of novel mutations that are associated with LCA/juvenile RP. Our results highlight the importance of molecular diagnosis as an integral part of clinical diagnosis.

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.

Mutations in RPGR and RP2 Account for 15% of Males with Simplex Retinal Degenerative Disease

PURPOSE To determine the proportion of male patients presenting simplex retinal degenerative disease (RD: retinitis pigmentosa [RP] or cone/cone-rod dystrophy [COD/CORD]) with mutations in the X-linked retinal degeneration genes RPGR and RP2. METHODS Simplex males were defined as patients with no known affected family members. Patients were excluded if they had a family history of parental consanguinity. Blood samples from a total of 214 simplex males with a diagnosis of retinal degeneration were collected for genetic analysis. The patients were screened for mutations in RPGR and RP2 by direct sequencing of PCR-amplified genomic DNA. RESULTS We identified pathogenic mutations in 32 of the 214 patients screened (15%). Of the 29 patients with a diagnosis of COD/CORD, four mutations were identified in the ORF15 mutational hotspot of the RPGR gene. Of the 185 RP patients, three patients had mutations in RP2 and 25 had RPGR mutations (including 12 in the ORF15 region). CONCLUSIONS This study represents mutation screening of RPGR and RP2 in the largest cohort, to date, of simplex males affected with RP or COD/CORD. Our results demonstrate a substantial contribution of RPGR mutations to retinal degenerations, and in particular, to simplex RP. Based on our findings, we suggest that RPGR should be considered as a first tier gene for screening isolated males with retinal degeneration.

Rates of decline in regions of the visual field defined by frequency-domain optical coherence tomography in patients with RPGR-mediated X-linked retinitis pigmentosa.

PURPOSE To determine whether annual decline in visual field sensitivity is greater in the transition zone at the edge of the frequency-domain optical coherence tomography (fdOCT) inner segment ellipsoid zone (EZ) than at other locations in the visual field. DESIGN Prospective, longitudinal, observational study. PARTICIPANTS Forty-four patients with X-linked retinitis pigmentosa (XLRP) resulting from a mutation in the RPGR gene. METHODS Static perimetric fields (Humphrey 30-2; Carl Zeiss Meditec, Dublin, CA) were obtained annually for 4 years. Beginning with year 2, fdOCT scans were obtained annually with a Heidelberg Spectralis HRA + OCT (Heidelberg Engineering, Heidelberg, Germany). MAIN OUTCOME MEASURES The rate of visual field decline at locations near the edge of the EZ compared with the rates for the macula and in the mid periphery. RESULTS Sensitivity just inside and outside the edge of the EZ declined at rates of 0.84 and 0.92 dB/year, respectively. By comparison, average sensitivity in the macula and mid periphery declined by 0.38 and 0.61 dB/year, respectively. CONCLUSIONS The edge of the EZ in each patient with XLRP indicates a transition zone between relatively healthy and relatively degenerate retina. The annual loss of sensitivity in the transition zone is more rapid than it is elsewhere in the retina.

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.

A novel GCAP1(N104K) mutation in EF-hand 3 (EF3) linked to autosomal dominant cone dystrophy

The GUCA1A gene encodes a guanylate cyclase activating protein (GCAP1) that is involved in regulation of phototransduction in the vertebrate retina. We discovered a novel C312A transversion in exon 2 of the human GUCA1A gene, replacing Asn-104 (N104) in GCAP1 with Lys (K), in two affected members of a family with dominant cone dystrophy. The mutation N104K is located in the third EF-hand motif (EF3) shown previously to be instrumental in converting Ca2+-free GCAP1 to a GC inhibitor in the Ca2+-bound form. In one patient, rod ERGs were fairly stable over a 12-year-period whereas 30 Hz flicker ERG and single-flash cone ERGs declined. In both patients, double-flash ERGs showed that rod recovery from an intense test flash was significantly delayed. The EC(50) for GC stimulation shifted from approximately 250 nM in wild-type GCAP1 to approximately 800 nM in the GCAP1(N104K) mutant suggesting inability of the mutant to assume an inactive form under physiological conditions. The replacement of N104 by K in GCAP1 is the first naturally occurring mutation identified in the EF3 loop. The rod recovery delays observed in double-flash ERG of affected patients suggest a novel dominant-negative effect that slows GC stimulation.

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%.

Docosahexaenoic acid in red blood cells of term infants receiving two levels of long-chain polyunsaturated fatty acids.

Objectives: A randomized, double-blind, prospective trial assessed effects of different formula levels of polyunsaturated fatty acids on blood phospholipid docosahexaenoic (DHA; 22:6&ohgr;3) and arachidonic acids (ARA; 20:4&ohgr;6) in term infants at 120 days of age. Methods: Healthy, formula-fed term infants (n = 78) were randomized to 1) routine milk-based formula with 8 mg DHA, 21 mg ARA, 110 mg &agr;-linolenic (ALA; 18:3&ohgr;3), and 1,000 mg linoleic acids (LA; 18:2&ohgr;6) per 100 kcal (Lower-long-chain polyunsaturated fatty acids [LCPUFA]; n = 39) or 2) routine milk-based formula with 17 mg DHA, 34 mg ARA, 85 mg ALA, and 860 mg LA per 100 kcal (Higher-LCPUFA; n = 39). Fatty acid methyl esters from red blood cell (RBC) and plasma phospholipid fractions were assessed using capillary column gas chromatography. Results: Compared with infants fed Lower-LCPUFA formula, the Higher-LCPUFA group had significantly greater percentages of fatty acids as DHA in RBC phosphatidylethanolamine (PE), RBC phosphatidylcholine (PC), total RBC, and plasma phospholipids (P < 0.001). Infants fed Lower-LCPUFA formula had higher percentages of precursor &ohgr;6 fatty acids in the desaturation/elongation pathway but lower percentages of ARA (RBC PE, RBC PC, and plasma phospholipid, P < 0.001; total RBC, P = 0.017) compared with the Higher-LCPUFA group. Conclusions: Greater amounts of dietary ALA do not produce as great an increase in DHA in blood lipids as preformed dietary DHA. Infants fed DHA at levels similar to human milk had significantly greater percentage of DHAat 120 days of age compared with the Lower-LCPUFA group despite higher precursor levels of ALA.

Diagnosis of a mild peroxisomal phenotype with next-generation sequencing

Peroxisomal biogenesis disorders (PBD) are caused by mutations in PEX genes, and are typically diagnosed with biochemical testing in plasma followed by confirmatory testing. Here we report the unusual diagnostic path of a child homozygous for PEX1 p.G843D. The patient presented with sensorineural hearing loss, pigmentary retinopathy, and normal intellect. After testing for Usher syndrome was negative, he was found to have PBD through a research sequencing panel. When evaluating a patient with hearing loss and pigmentary retinopathy, mild PBD should be on the differential regardless of cognitive function.