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DeBarber A.E.,Casey Molecular Diagnostic Laboratory | Luo J.,Casey Molecular Diagnostic Laboratory | Chiang J.,Oregon Health And Science University | Merkens L.S.,Chemistry and Consumables R and D | And 4 more authors.
Journal of Lipid Research | Year: 2014

Cerebrotendinous xanthomatosis (CTX) is a rare, diffi cult-to-diagnose genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be benefi cial because an effective oral therapy for CTX is available to prevent disease progression. There is no suitable test to screen newborn dried bloodspots (DBS) for CTX. Blood screening for CTX is currently performed by GC-MS measurement of elevated 5 - -cholestanol. We present here LC-ESI/MS/MS methodology utilizing keto derivatization with ( O -(3-trimethylammonium-propyl) hydroxylamine) reagent to enable sensitive detection of ketosterol BA precursors that accumulate in CTX. The availability of isotopically enriched derivatization reagent allowed ready tagging of ketosterols to generate internal standards for isotope dilution quantifi cation. Ketosterols were quantifi ed and their utility as markers for CTX was compared with 5 - -cholestanol. 7 - ,12 - - Dihydroxy-4-cholesten-3-one provided the best discrimination between CTX and unaffected samples. In two CTX, newborn DBS concentrations of this ketosterol (120-214 ng/ml) were - 10-fold higher than in unaffected newborn DBS (16.4 ± 6.0 ng/ml), such that quantifi cation of this ketosterol provides a test with potential to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefi t those affected with CTX by preventing morbidity and mortality. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.


Fakin A.,University College London | Fakin A.,Moorfields Eye Hospital | Robson A.G.,University College London | Robson A.G.,Moorfields Eye Hospital | And 13 more authors.
Investigative Ophthalmology and Visual Science | Year: 2016

PURPOSE. We describe the phenotypes associated with nullizigosity and nine splicing mutations in the ABCA4 gene. METHODS. The study included 19 patients with biallelic null mutations (Group A, nullizygous), 27 with splicing mutations in the homozygous state or in trans with a null mutation (Group B), and 20 with p.G1961E in trans with a null mutation (Group C, control). Ages at onset and visual acuities were determined from medical histories. Area of decreased autofluorescence within a 30° × 30° fundus autofluorescence (FAF) image was measured with the Region Finder (N = 58). Full-field electroretinography (ERG) was performed incorporating the International Society for Clinical Electrophysiology of Vision (ISCEV) standard (N = 40). RESULTS. For groups A to C, the median ages of onset were 6, 8, and 17, respectively. Kaplan Meier survival analysis estimated that 50% of patients reached visual acuity below 20/400 at the ages of 29, 48, and 66 years, respectively. The area of reduced FAF was estimated to increase by 1.5, 1.2, and 0.03 mm2per year, respectively, and cone–rod dystrophy was present in 10/12, 13/15, and 0/13 of cases, respectively. Splicing mutation c.5714+5G>A was associated with a significantly milder phenotype in comparison with nullizygous patients for all parameters. CONCLUSIONS. Nullizygosity for ABCA4 is associated with early onset cone–rod dysfunction with rapid progression shown by enlargement of central atrophy on FAF, decline of ERG amplitudes with age, and a high risk of reaching legal blindness by the fourth decade. Most studied splicing mutations were associated with a similarly severe phenotype. Estimated rates of progression may facilitate further genotype–phenotype correlations and inform the design of treatment trials. © 2016, Association for Research in Vision and Ophthalmology Inc. All rights reserved.


PubMed | University of California at San Francisco, Casey Molecular Diagnostic Laboratory, University College London and Keio University
Type: Journal Article | Journal: Investigative ophthalmology & visual science | Year: 2016

To determine the effect of 15 individual ABCA4 mutations on disease severity.Eighty-two patients harboring 15 distinct ABCA4 mutations in trans with null (hemizygous), 10 homozygous, and 20 nullizygous patients were recruited. Age of onset was determined from medical histories. Electroretinography (ERG) responses were classified into three groups (normal; cone dysfunction; cone and rod dysfunction). The dark-adapted bright-flash (DA 10.0) a-wave amplitudes and the light-adapted flicker ERG (LA 3.0 30 Hz) amplitudes were plotted against age and compared with the nullizygous patients. Fundus autofluorescence imaging (FAF) was assessed when available.Patients hemizygous for p.G1961E and p.R2030Q had normal ERGs. Patients harboring p.R24H, p.R212C, p.G863A/delG, p.R1108C, p.P1380L, p.L2027F, and c.5714+5G>A had abnormal ERGs (ERG group 2 or 3) at older ages, in most cases with significantly higher amplitudes than nullizygous patients. Mutations p.L541P+A1038V, p.E1022K, p.C1490Y, p.E1087K, p.T1526M, and p.C2150Y were associated with abnormal ERGs (group 2 or 3) and amplitudes comparable to those of nullizygous patients. The majority of patients, including those harboring p.G1961E, had foveal atrophy; while both patients harboring p.R2030Q had foveal sparing. Most patients harboring intermediate and null-like mutations displayed FAF abnormalities extending beyond the vascular arcades.In the hemizygous state, 2/15 ABCA4 alleles retain preserved peripheral retinal function; 7/15 are associated with either preserved or only mildly abnormal retinal function, worse in older patients; 6/15 behave like null mutations. These data help characterize the degree of dysfunction conferred by specific mutant ABCA4 proteins in the human retina.


PubMed | University of California at San Francisco, Casey Molecular Diagnostic laboratory, University College London and Keio University
Type: Journal Article | Journal: Investigative ophthalmology & visual science | Year: 2016

We describe the phenotypes associated with nullizigosity and nine splicing mutations in the ABCA4 gene.The study included 19 patients with biallelic null mutations (Group A, nullizygous), 27 with splicing mutations in the homozygous state or in trans with a null mutation (Group B), and 20 with p.G1961E in trans with a null mutation (Group C, control). Ages at onset and visual acuities were determined from medical histories. Area of decreased autofluorescence within a 30 30 fundus autofluorescence (FAF) image was measured with the Region Finder (N = 58). Full-field electroretinography (ERG) was performed incorporating the International Society for Clinical Electrophysiology of Vision (ISCEV) standard (N = 40).For groups A to C, the median ages of onset were 6, 8, and 17, respectively. Kaplan Meier survival analysis estimated that 50% of patients reached visual acuity below 20/400 at the ages of 29, 48, and 66 years, respectively. The area of reduced FAF was estimated to increase by 1.5, 1.2, and 0.03 mm2 per year, respectively, and cone-rod dystrophy was present in 10/12, 13/15, and 0/13 of cases, respectively. Splicing mutation c.5714+5G>A was associated with a significantly milder phenotype in comparison with nullizygous patients for all parameters.Nullizygosity for ABCA4 is associated with early onset cone-rod dysfunction with rapid progression shown by enlargement of central atrophy on FAF, decline of ERG amplitudes with age, and a high risk of reaching legal blindness by the fourth decade. Most studied splicing mutations were associated with a similarly severe phenotype. Estimated rates of progression may facilitate further genotype-phenotype correlations and inform the design of treatment trials.


DeBarber A.E.,Oregon Health And Science University | Luo J.,Oregon Health And Science University | Giugliani R.,Hospital Of Clinicas Of Porto Alegre | Giugliani R.,Federal University of Rio Grande do Sul | And 7 more authors.
Clinical Biochemistry | Year: 2014

Objectives: Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Blood (normally serum or plasma) testing for CTX is performed by a small number of specialized laboratories, routinely by gas chromatography-mass spectrometry (GC-MS) measurement of elevated 5α-cholestanol. We report here on a more sensitive biochemical approach to test for CTX particularly useful for confirmation of CTX in the case of a challenging diagnostic sample with 5α-cholestanol that, although elevated, was below the cut-off used for diagnosis of CTX (10. μg/mL or 1.0. mg/dL). Design and methods: We have previously described liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) methodology utilizing keto derivatization to enable the sensitive quantification of plasma ketosterol BA precursors that accumulate in CTX. We have expanded this methodology to perform isotope dilution LC-ESI-MS/MS quantification of a panel of plasma ketosterol BA precursors, with internal standards readily generated using isotopically-enriched derivatization reagent. Results: Quantification of plasma ketosterol BA precursors (7α-hydroxy-4-cholesten-3-one, 7α,12α-dihydroxy-4-cholesten-3-one and 7α,12α-dihydroxy-5β-cholestan-3-one) in a single LC-ESI/MS/MS test provided better discrimination between a CTX-positive and negative samples analyzed (n = 20) than measurement of 5α-cholestanol alone. Conclusions: Quantification of plasma ketosterol BA precursors provides a more sensitive biochemical approach to discriminate between CTX negative and positive samples. A multiplexed LC-ESI-MS/MS test quantifying a panel of plasma ketosterols, with simple sample preparation, rapid analysis time and readily available internal standards, can be performed by most clinical laboratories. Wider availability of testing will benefit those affected with CTX. © 2014 The Canadian Society of Clinical Chemists.


PubMed | Oregon Health And Science University, Doernbecher Childrens Hospital, OHSU, Casey Molecular Diagnostic Laboratory and Federal University of Rio Grande do Sul
Type: Journal Article | Journal: Clinical biochemistry | Year: 2014

Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Blood (normally serum or plasma) testing for CTX is performed by a small number of specialized laboratories, routinely by gas chromatography-mass spectrometry (GC-MS) measurement of elevated 5-cholestanol. We report here on a more sensitive biochemical approach to test for CTX particularly useful for confirmation of CTX in the case of a challenging diagnostic sample with 5-cholestanol that, although elevated, was below the cut-off used for diagnosis of CTX (10 g/mL or 1.0 mg/dL).We have previously described liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) methodology utilizing keto derivatization to enable the sensitive quantification of plasma ketosterol BA precursors that accumulate in CTX. We have expanded this methodology to perform isotope dilution LC-ESI-MS/MS quantification of a panel of plasma ketosterol BA precursors, with internal standards readily generated using isotopically-enriched derivatization reagent.Quantification of plasma ketosterol BA precursors (7-hydroxy-4-cholesten-3-one, 7,12-dihydroxy-4-cholesten-3-one and 7,12-dihydroxy-5-cholestan-3-one) in a single LC-ESI/MS/MS test provided better discrimination between a CTX-positive and negative samples analyzed (n=20) than measurement of 5-cholestanol alone.Quantification of plasma ketosterol BA precursors provides a more sensitive biochemical approach to discriminate between CTX negative and positive samples. A multiplexed LC-ESI-MS/MS test quantifying a panel of plasma ketosterols, with simple sample preparation, rapid analysis time and readily available internal standards, can be performed by most clinical laboratories. Wider availability of testing will benefit those affected with CTX.

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