High-Throughput Mass Spectrometry Assay for Quantifying ß-Amyloid 40 and 42 in Cerebrospinal Fluid.

BACKGROUND: The ratio of ß-amyloid 1-42 (Aß42) to Aß40 in cerebrospinal fluid (CSF) may be useful for evaluating Alzheimer disease (AD), but quantification is limited by factors including preanalytical analyte loss. We developed an LC-MS/MS assay that limits analyte loss. Here we describe the analytical characteristics of the assay and its performance in differentiating patients with AD from non-AD dementia and healthy controls. METHODS: To measure Aß42/Aß40, we used unique proteolytically derived C-terminal peptides as surrogate markers of Aß40 and Aß42, which were analyzed and quantified by LC-MS/MS. The assay was analytically validated and applied to specimens from individuals with clinically diagnosed AD (n = 102), mild cognitive impairment (n = 37), and non-AD dementias (n = 22), as well as from healthy controls (n = 130). Aß42/Aß40 values were compared with APOE genotype inferred from phenotype, also measured by LC-MS/MS. RESULTS: The assay had a reportable range of 100 to 25000 pg/mL, a limit of quantification of 100 pg/mL, recoveries between 93% and 111%, and intraassay and interassay CV <15% for both peptides. An Aß42/Aß40 ratio cutoff of <0.16 had a clinical sensitivity of 78% for distinguishing patients with AD from non-AD dementia (clinical specificity, 91%) and from healthy controls (clinical specificity, 81%). The Aß42/Aß40 ratio decreased significantly (P < 0.001) with increasing dose of APOE4 alleles. CONCLUSIONS: This assay can be used to determine Aß42/Aß40 ratios, which correlate with the presence of AD.

Author Correction: Applications and efficiencies of the first cat 63 K DNA array.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Author Correction: Applications and efficiencies of the first cat 63K DNA array.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Applications and efficiencies of the first cat 63K DNA array.

The development of high throughput SNP genotyping technologies has improved the genetic dissection of simple and complex traits in many species including cats. The properties of feline 62,897 SNPs Illumina Infinium iSelect DNA array are described using a dataset of over 2,000 feline samples, the most extensive to date, representing 41 cat breeds, a random bred population, and four wild felid species. Accuracy and efficiency of the array's genotypes and its utility in performing population-based analyses were evaluated. Average marker distance across the array was 37,741 Kb, and across the dataset, only 1% (625) of the markers exhibited poor genotyping and only 0.35% (221) showed Mendelian errors. Marker polymorphism varied across cat breeds and the average minor allele frequency (MAF) of all markers across domestic cats was 0.21. Population structure analysis confirmed a Western to Eastern structural continuum of cat breeds. Genome-wide linkage disequilibrium ranged from 50-1,500 Kb for domestic cats and 750 Kb for European wildcats (Felis silvestris silvestris). Array use in trait association mapping was investigated under different modes of inheritance, selection and population sizes. The efficient array design and cat genotype dataset continues to advance the understanding of cat breeds and will support monogenic health studies across feline breeds and populations.

Cost Effectiveness of Karyotyping, Chromosomal Microarray Analysis, and Targeted Next-Generation Sequencing of Patients with Unexplained Global Developmental Delay or Intellectual Disability.

BACKGROUND: Genetic diagnosis of unexplained global developmental delay and intellectual disability (GDD/ID) often ends the diagnostic odyssey and can lead to changes in clinical management. OBJECTIVE: The objective of this study was to investigate the cost effectiveness of testing scenarios involving several methods used to diagnose GDD/ID: karyotyping, chromosomal microarray analysis (CMA), and targeted next-generation sequencing (NGS). METHODS: We used decision-tree models to estimate the number of genetic diagnoses, the cost from a payers' perspective in the USA, and the incremental cost per additional genetic diagnosis. Model parameters were taken from peer-reviewed literature and governmental fee schedules. RESULTS: CMA testing results in more genetic diagnoses at an incremental cost of US $2692 per additional diagnosis compared with karyotyping, which has an average cost per diagnosis of US $11,033. Performing both tests sequentially results in the same number of diagnoses, but the total cost is less when CMA testing is done first and karyotyping second. Furthermore, when CMA testing yields a variant of unknown significance, additional genetic diagnoses can be obtained at an incremental cost of US $4220 by CMA testing of both parents, and when parents are not available or the patient had a normal CMA result, targeted NGS of the patient can add diagnoses at a further incremental cost of US $12,295. CONCLUSION: These results provide a cost effectiveness rationale for the use of CMA as the first-tier test for the genetic diagnosis of unexplained GDD/ID and further indicate that testing of both parents may be cost effective when a variant of unknown significance is detected in the patient.

Glucocerebrosidase haploinsufficiency in A53T α-synuclein mice impacts disease onset and course.

Mutations in GBA1 encountered in Gaucher disease are a leading risk factor for Parkinson disease and associated Lewy body disorders. Many GBA1 mutation carriers, especially those with severe or null GBA1 alleles, have earlier and more progressive parkinsonism. To model the effect of partial glucocerebrosidase deficiency on neurological progression in vivo, mice with a human A53T α-synuclein (SNCAA53T) transgene were crossed with heterozygous null gba mice (gba+/-). Survival analysis of 84 mice showed that in gba+/-//SNCAA53T hemizygotes and homozygotes, the symptom onset was significantly earlier than in gba+/+//SNCAA53T mice (p-values 0.023-0.0030), with exacerbated disease progression (p-value <0.0001). Over-expression of SNCAA53T had no effect on glucocerebrosidase levels or activity. Immunoblotting demonstrated that gba haploinsufficiency did not lead to increased levels of either monomeric SNCA or insoluble high molecular weight SNCA in this model. Immunohistochemical analyses demonstrated that the abundance and distribution of SNCA pathology was also unaltered by gba haploinsufficiency. Thus, while the underlying mechanism is not clear, this model shows that gba deficiency impacts the age of onset and disease duration in aged SNCAA53T mice, providing a valuable resource to identify modifiers, pathways and possible moonlighting roles of glucocerebrosidase in Parkinson pathogenesis.

A new glucocerebrosidase-deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease.

Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1 Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1 To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba(-/-) mice and the control littermate (gba(+/+)) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba(-/-) neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba(+/+) neurons. This null allele gba(-/-) mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies.

DNA Methylation Signatures of Early Childhood Malnutrition Associated With Impairments in Attention and Cognition.

BACKGROUND: Early childhood malnutrition affects 113 million children worldwide, impacting health and increasing vulnerability for cognitive and behavioral disorders later in life. Molecular signatures after childhood malnutrition, including the potential for intergenerational transmission, remain unexplored. METHODS: We surveyed blood DNA methylomes (~483,000 individual CpG sites) in 168 subjects across two generations, including 50 generation 1 individuals hospitalized during the first year of life for moderate to severe protein-energy malnutrition, then followed up to 48 years in the Barbados Nutrition Study. Attention deficits and cognitive performance were evaluated with the Connors Adult Attention Rating Scale and Wechsler Abbreviated Scale of Intelligence. Expression of nutrition-sensitive genes was explored by quantitative reverse transcriptase polymerase chain reaction in rat prefrontal cortex. RESULTS: We identified 134 nutrition-sensitive, differentially methylated genomic regions, with most (87%) specific for generation 1. Multiple neuropsychiatric risk genes, including COMT, IFNG, MIR200B, SYNGAP1, and VIPR2 showed associations of specific methyl-CpGs with attention and IQ. IFNG expression was decreased in prefrontal cortex of rats showing attention deficits after developmental malnutrition. CONCLUSIONS: Early childhood malnutrition entails long-lasting epigenetic signatures associated with liability for attention and cognition, and limited potential for intergenerational transmission.

Neuroinflammation and α-synuclein accumulation in response to glucocerebrosidase deficiency are accompanied by synaptic dysfunction.

Clinical, epidemiological and experimental studies confirm a connection between the common degenerative movement disorder Parkinson's disease (PD) that affects over 1 million individuals, and Gaucher disease, the most prevalent lysosomal storage disorder. Recently, human imaging studies have implicated impaired striatal dopaminergic neurotransmission in early PD pathogenesis in the context of Gaucher disease mutations, but the underlying mechanisms have yet to be characterized. In this report we describe and characterize two novel long-lived transgenic mouse models of Gba deficiency, along with a subchronic conduritol-ß-epoxide (CBE) exposure paradigm. All three murine models revealed striking glial activation within nigrostriatal pathways, accompanied by abnormal α-synuclein accumulation. Importantly, the CBE-induced, pharmacological Gaucher mouse model replicated this change in dopamine neurotransmission, revealing a markedly reduced evoked striatal dopamine release (approximately 2-fold) that indicates synaptic dysfunction. Other changes in synaptic plasticity markers, including microRNA profile and a 24.9% reduction in post-synaptic density size, were concomitant with diminished evoked dopamine release following CBE exposure. These studies afford new insights into the mechanisms underlying the Parkinson's-Gaucher disease connection, and into the physiological impact of related abnormal α-synuclein accumulation and neuroinflammation on nigrostriatal dopaminergic neurotransmission.

Rare missense neuronal cadherin gene (CDH2) variants in specific obsessive-compulsive disorder and Tourette disorder phenotypes.

The recent finding that the neuronal cadherin gene CDH2 confers a highly significant risk for canine compulsive disorder led us to investigate whether missense variants within the human ortholog CDH2 are associated with altered susceptibility to obsessive-compulsive disorder (OCD), Tourette disorder (TD) and related disorders. Exon resequencing of CDH2 in 320 individuals identified four non-synonymous single-nucleotide variants, which were subsequently genotyped in OCD probands, Tourette disorder probands and relatives, and healthy controls (total N=1161). None of the four variants was significantly associated with either OCD or TD. One variant, N706S, was found only in the OCD/TD groups, but not in controls. By examining clinical data, we found there were significant TD-related phenotype differences between those OCD probands with and without the N845S variant with regard to the co-occurrence of TD (Fisher's exact test P=0.014, OR=6.03). Both N706S and N845S variants conferred reduced CDH2 protein expression in transfected cells. Although our data provide no overall support for association of CDH2 rare variants in these disorders considered as single entities, the clinical features and severity of probands carrying the uncommon non-synonymous variants suggest that CDH2, along with other cadherin and cell adhesion genes, is an interesting gene to pursue as a plausible contributor to OCD, TD and related disorders with repetitive behaviors, including autism spectrum disorders.

Skin ultrastructural findings in type 2 Gaucher disease: diagnostic implications.

BACKGROUND: Type 2 Gaucher disease is a rare and progressive subtype of this lysosomal storage disorder, marked by rapid, early-onset neurodegeneration. Distinguishing type 2 from types 1 and 3 Gaucher disease has remained challenging, due to the lack of a clear correlation between phenotype and enzymatic activity or genotype. ß-glucocerebrosidase, the enzyme deficient in Gaucher disease, also has an essential role in maintaining epidermal permeability function, by regulating the ratio of ceramides to glucosylceramides in the stratum corneum of the skin. OBJECTIVES: To further assess the diagnostic utility of epidermal evaluations in distinguishing patients with type 2 Gaucher disease in an expanded cohort. STUDY DESIGN: Epidermal samples were evaluated from twenty children with type 2, three patients with type 3 Gaucher disease and two adults with type 1 Gaucher disease with different clinical manifestations and genotypes. Electron microscopy on ruthenium tetroxide post-fixed tissue was performed. RESULTS: Compared to controls and subjects with type 1 and type 3 Gaucher disease, only patients with type 2 Gaucher disease displayed characteristic electron dense, non-lamellar clefts and immature-lamellar membranes. CONCLUSION: The appearance of characteristic alterations in epidermal ultrastructure provides an early and specific diagnostic tool to help in distinguishing type 2 from the other types of Gaucher disease.

Therapy for Gaucher disease: don't stop thinking about tomorrow.

While enzyme replacement therapy for Gaucher disease has been widely used and appears to be an efficacious and safe treatment, this success should not be a reason for complacency. Other treatment strategies currently under consideration for patients with Gaucher disease include gene therapy, substrate reduction therapy and chaperone therapy. Furthermore, improvements in enzyme therapy could also have a significant clinical impact. Individuals with Gaucher disease and other lysosomal disorders will greatly benefit from continual refinement and optimization of the current therapy, as well as from the development of new treatment modalities that offer improvements in efficacy, cost, safety and availability.

DNA targeting of rhinal cortex D2 receptor protein reversibly blocks learning of cues that predict reward.

When schedules of several operant trials must be successfully completed to obtain a reward, monkeys quickly learn to adjust their behavioral performance by using visual cues that signal how many trials have been completed and how many remain in the current schedule. Bilateral rhinal (perirhinal and entorhinal) cortex ablations irreversibly prevent this learning. Here, we apply a recombinant DNA technique to investigate the role of dopamine D2 receptor in rhinal cortex for this type of learning. Rhinal cortex was injected with a DNA construct that significantly decreased D2 receptor ligand binding and temporarily produced the same profound learning deficit seen after ablation. However, unlike after ablation, the D2 receptor-targeted, DNA-treated monkeys recovered cue-related learning after 11-19 weeks. Injecting a DNA construct that decreased N-methyl-d-aspartate but not D2 receptor ligand binding did not interfere with learning associations between the cues and the schedules. A second D2 receptor-targeted DNA treatment administered after either recovery from a first D2 receptor-targeted DNA treatment (one monkey), after N-methyl-d-aspartate receptor-targeted DNA treatment (two monkeys), or after a vector control treatment (one monkey) also induced a learning deficit of similar duration. These results suggest that the D2 receptor in primate rhinal cortex is essential for learning to relate the visual cues to the schedules. The specificity of the receptor manipulation reported here suggests that this approach could be generalized in this or other brain pathways to relate molecular mechanisms to cognitive functions.

Glucosylsphingosine accumulation in tissues from patients with Gaucher disease: correlation with phenotype and genotype.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of clinical manifestations including neuronopathic and non-neuronopathic forms. While the lipid glucosylceramide is stored in both patients with Gaucher disease and in a null allele mouse model of Gaucher disease, elevated levels of a second potentially toxic substrate, glucosylsphingosine, are also found. Using high performance liquid chromatography, glucosylsphingosine levels were measured in tissues from patients with type 1, 2, and 3 Gaucher disease. Glucosylsphingosine was measured in 16 spleen samples (8 type 1; 4 type 2; and 4, type 3) and levels ranged from 54 to 728 ng/mg protein in the patients with type 1 disease, 133 to 1200 ng/mg protein in the patients with type 2, and 109 to 1298 ng/mg protein in the type 3 samples. The levels of splenic glucosylsphingosine bore no relation to the type of Gaucher disease, the age of the patient, the genotype, nor the clinical course. In the same patients, hepatic glucosylsphingosine levels were lower than in spleen. Glucosylsphingosine was also measured in brains from 13 patients (1 type 1; 8 type 2; and 4 type 3). While the glucosylsphingosine level in the brain from the type 1 patient, 1.0 ng/mg protein, was in the normal range, the levels in the type 3 samples ranged from 14 to 32 ng/mg protein, and in the type 2 samples from 24 to 437 ng/mg protein, with the highest values detected in two fetuses with hydrops fetalis. The elevated levels found in brains from patients with neuronopathic Gaucher disease support the hypothesis that glucosylsphingosine may contribute to the nervous system involvement in these patients.