Urine oligosaccharide screening by MALDI-TOF for the identification of NGLY1 deficiency.

N-glycanase deficiency (NGLY1 deficiency, NGLY1-CDDG), the first autosomal recessive congenital disorder of N-linked deglycosylation (CDDG), is caused by pathogenic variants in NGLY1. The majority of affected individuals have been identified using exome or genome sequencing. To date, no reliable, clinically available biomarkers have been identified. Urine oligosaccharide analysis was included as part of a routine evaluation for possible biomarkers in patients with confirmed NGLY1-CDDG. During the qualitative review of oligosaccharide profiles by an experienced laboratory director an abnormal analyte with a proposed structure of Neu5Ac1Hex1GlcNAc1-Asn was identified in NGLY1-CDDG patient urine samples. The same species has been observed in profiles from individuals affected with aspartylglucosaminuria, although the complete spectra are not identical. Additional studies using tandem mass spectrometry confirmed the analyte's structure. In addition to the known NGLY1-CDDG patients identified by this analysis, a single case was identified in a population referred for clinical testing who subsequently had a diagnosis of NGLY1-CDDG confirmed by molecular testing. Urine oligosaccharide screening by MALDI-TOF MS can identify individuals with NGLY1-CDDG. In addition, this potential biomarker might also be used to monitor the effectiveness of therapeutic options as they become available.

Enhanced stability of blood matrices using a dried sample spot assay to measure human butyrylcholinesterase activity and nerve agent adducts.

Dried matrix spots are safer to handle and easier to store than wet blood products, but factors such as intraspot variability and unknown sample volumes have limited their appeal as a sampling format for quantitative analyses. In this work, we introduce a dried spot activity assay for quantifying butyrylcholinesterase (BChE) specific activity which is BChE activity normalized to the total protein content in a sample spot. The method was demonstrated with blood, serum, and plasma spotted on specimen collection devices (cards) which were extracted to measure total protein and BChE activity using a modified Ellman assay. Activity recovered from dried spots was ∼80% of the initial spotted activity for blood and >90% for plasma and serum. Measuring total protein in the sample and calculating specific activity substantially improved quantification and reduced intraspot variability. Analyte stability of nerve agent adducts was also evaluated, and the results obtained via BChE-specific activity measurements were confirmed by quantification of BChE adducts using a previously established LC-MS/MS method. The spotted samples were up to 10 times more resistant to degradation compared to unspotted control samples when measuring BChE inhibition by the nerve agents sarin and VX. Using this method, both BChE activity and adducts can be accurately measured from a dried sample spot. This use of a dried sample spot with normalization to total protein is robust, demonstrates decreased intraspot variability without the need to control for initial sample volume, and enhances analyte stability.

Incidence, management, and outcome of high-grade transformation of nodular lymphocyte predominant Hodgkin lymphoma: long-term outcomes from a 30-year experience.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a rare form of Hodgkin lymphoma that typically presents as early stage, indolent disease in young adult males. The relationship between NLPHL and DLBCL is incompletely understood, and there remains a paucity of data with regard the incidence and management of high-grade transformation. We report the largest study to date describing the incidence, management and long-term outcome of 26 cases of high-grade transformation of NLPHL over a 30-year period. We report a transformation incidence of 17.0%. Bone marrow, splenic, and liver infiltration with DLBCL was frequent. Patients with an aa-IPI 2-3 have poorer OS and PFS (P = 0.034 and P = 0.009, respectively). Although the approach to treatment was somewhat variable, typically young, otherwise fit patients received anthracycline-based induction, platinum-based consolidation with stem cell harvesting, followed by autologous SCT with BEAM conditioning. Long-term (5 year) PFS was over 60% with this approach, and comparable to our de novo DLBCL historical age and time period-matched cohort largely treated with CHOP-like chemotherapy alone. The transformation rate of 17.0% highlights the importance of accurate initial diagnosis, long-term follow-up, and re-biopsy at relapse.

Simultaneous measurement of tabun, sarin, soman, cyclosarin, VR, VX, and VM adducts to tyrosine in blood products by isotope dilution UHPLC-MS/MS.

This work describes a new specific, sensitive, and rapid stable isotope dilution method for the simultaneous detection of the organophosphorus nerve agents (OPNAs) tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VR, VX, and VM adducts to tyrosine (Tyr). Serum, plasma, and lysed whole blood samples (50 µL) were prepared by protein precipitation followed by digestion with Pronase. Specific Tyr adducts were isolated from the digest by a single solid phase extraction (SPE) step, and the analytes were separated by reversed-phase ultra high performance liquid chromatography (UHPLC) gradient elution in less than 2 min. Detection was performed on a triple quadrupole tandem mass spectrometer using time-triggered selected reaction monitoring (SRM) in positive electrospray ionization (ESI) mode. The calibration range was characterized from 0.100-50.0 ng/mL for GB- and VR-Tyr and 0.250-50.0 ng/mL for GA-, GD-, GF-, and VX/VM-Tyr (R(2) ≥ 0.995). Inter- and intra-assay precision had coefficients of variation of ≤17 and ≤10%, respectively, and the measured concentration accuracies of spiked samples were within 15% of the targeted value for multiple spiking levels. The limit of detection was calculated to be 0.097, 0.027, 0.018, 0.074, 0.023, and 0.083 ng/mL for GA-, GB-, GD-, GF-, VR-, and VX/VM-Tyr, respectively. A convenience set of 96 serum samples with no known nerve agent exposure was screened and revealed no baseline values or potential interferences. This method provides a simple and highly specific diagnostic tool that may extend the time postevent that a confirmation of nerve agent exposure can be made with confidence.

An enhanced butyrylcholinesterase method to measure organophosphorus nerve agent exposure in humans.

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be used to confirm exposure in humans. A highly accurate method to detect G- and V-series OPNA adducts to BChE in 75 µL of filtered blood, serum, or plasma has been developed using immunomagnetic separation (IMS) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). The reported IMS method captures > 88 % of the BChE in a specimen and corrects for matrix effects on peptide calibrators. The optimized method has been used to quantify baseline BChE levels (unadducted and OPNA-adducted) in a matched-set of serum, plasma, and whole blood (later processed in-house for plasma content) from 192 unexposed individuals to determine the interchangeability of the tested matrices. The results of these measurements demonstrate the ability to accurately measure BChE regardless of the format of the blood specimen received. Criteria for accepting or denying specimens were established through a series of sample stability and processing experiments. The results of these efforts are an optimized and rugged method that is transferrable to other laboratories and an increased understanding of the BChE biomarker in matrix.

Lambdoidal synostosis in dizygotic twins with a family history of an undiagnosed connective tissue disorder.

INTRODUCTION: Unilateral lambdoidal craniosynostosis is a rare disorder that occurs in approximately 3 % of all craniosynostosis phenotypes and only 0.03 % of one million live births. It is even more unusual for this type of synostosis to occur in siblings with only two other cases reported in the literature. CASE REPORT: We report a set of full-term dizygotic twins born with lambdoidal synostosis and a family history of connective tissue and cardiovascular anomalies. One of the twins also had concomitant bicoronal craniosynostosis. CONCLUSION: True familial lambdoidal synostosis is exceedingly rare. The present cases in dizygotic twins occurred in a family with a significant history of connective tissue disease suggesting a possible association.

Osmoregulation and branchial plasticity after acute freshwater transfer in red drum, Sciaenops ocellatus.

Red drum, Sciaenops ocellatus, is an estuarine-dependent fish species commonly found in the Gulf of Mexico and along the coast of the southeastern United States. This economically important species has demonstrated freshwater tolerance; however, the physiological mechanisms and costs related to freshwater exposure remain poorly understood. The current study therefore investigated the physiological response of red drum using an acute freshwater transfer protocol. Plasma osmolality, Cl⁻, Mg²âº and Ca²âº were all significantly reduced by 24h post-transfer; Cl⁻ and Mg²âº recovered to control levels by 7days post-transfer. No effect of transfer was observed on muscle water content; however, muscle Cl⁻ was significantly reduced. Interestingly, plasma and muscle Na⁺ content was unaffected by freshwater transfer. Intestinal fluid was absent by 24h post-transfer indicating cessation of drinking. Branchial gene expression analysis showed that both CFTR and NKCC1 exhibited significant down-regulation at 8 and 24h post-transfer, respectively, although transfer had no impact on NHE2, NHE3 or Na⁺, K⁺ ATPase (NKA) activity. These general findings are supported by immunohistochemical analysis, which revealed no apparent NKCC containing cells in the gills at 7days post transfer while NKA cells localization was unaffected. The results of the current study suggest that red drum can effectively regulate Na⁺ balance upon freshwater exposure using already present Na⁺ uptake pathways while also down-regulating ion excretion mechanisms.

Effect of Sleep Restriction on Adolescent Cognition by Adiposity: A Randomized Crossover Trial.

Importance: Pediatric obesity is associated with impaired cognitive function; however, the mechanisms underlying this association demand assessment. Sleep may be a relevant moderator, as poor sleep predicts both increased adiposity and impaired cognitive function. Objective: To determine the effects of adiposity and sleep on adolescent cognitive function. Design, Setting, and Participants: This single-blind randomized crossover trial was conducted from September 2020 to October 2022. Parents or caregivers provided demographic information for adolescent participants. Body mass index percentile and bioelectrical impedance analysis assessed adiposity. Adolescents completed 2 actigraphy-confirmed sleep conditions, adequate and restricted, followed by in-person cognitive assessment. No additional follow-up was provided. Data collection for this population-based study took place in a behavioral medicine clinic in Birmingham, Alabama. A total of 323 participants were assessed for eligibility (ages 14-19 years and healthy). Of the 244 eligible adolescents, 157 declined participation. Eighty-seven were randomized and 26 dropped out postenrollment. The final sample included 61 adolescents, 31 with healthy weight and 30 with overweight or obesity. Data were analyzed from April to October 2023. Interventions: Following a 2-day washout period of adequate sleep, adolescents completed 2 sleep conditions: adequate (mean [SD] duration, 8 hours, 54 minutes [58.0 minutes]) and restricted (mean [SD] duration, 4 hours, 12 minutes [50.7 minutes]). Main Outcomes and Measures: The National Institutes of Health Cognitive Toolbox assessed global and fluid cognition, cognitive flexibility, working and episodic memory, attention, and processing speed. The Stroop Task assessed inhibition. Results: The final sample included 61 adolescents (mean [SD] age, 16.3 [1.6] years; 35 [57.4%] female). Restricted sleep predicted poorer global cognition scores (restricted mean [SD], 98.0 [2.8]; adequate mean [SD], 103.2 [2.9]), fluid cognition scores (restricted mean [SD], 94.5 [3.2]; adequate mean [SD], 102.0 [3.6]), and cognitive flexibility scores (restricted mean [SD], 84.8 [3.0]; adequate mean [SD], 92.8 [3.0]) for adolescents with overweight or obesity. No differences emerged for adolescents with healthy weight. Adolescents with overweight or obesity also had poorer attention scores (mean [SD], 80.0 [2.3]) compared to adolescents with healthy weight (mean [SD], 88.4 [SD, 2.3]) following restricted sleep. No differences emerged following adequate sleep. Findings were similar for total body fat percentage (TBF%); however, for adolescents with TBF% above 42, restricted sleep also predicted poorer processing speed, and the association between sleep and attention did not vary based on TBF%. Conclusions and Relevance: Adolescents with overweight or obesity may be more vulnerable to negative cognitive effects following sleep restriction. Improved sleep hygiene and duration in this group may positively impact their cognitive health. Trial Registration: ClinicalTrials.gov Identifier: NCT04346433.

The effects of racial and socioeconomic disparities on time to diagnosis and treatment of pediatric functional seizures in the United States.

PURPOSE: The present study sought to assess the effects of racial and socioeconomic status in the United States on time to treatment and diagnosis of pediatric functional seizures (FS). METHODS: Eighty adolescents and their parent/guardian completed a demographics questionnaire and reported date of FS onset, diagnosis, and treatment. Paired samples t-tests compared time between FS onset and diagnosis, onset and treatment, and diagnosis and treatment based on race (White vs racial minority), annual household income (≤$79,999 vs ≥$80,000), maternal and paternal education (≤Associate's Degree vs Bachelor's Degree), and combined parental education (≤Post-graduate training vs Graduate degree). RESULTS: Adolescents with lower annual household income began treatment >6 months later than adolescents with greater annual household income (p = 0.049). Adolescents with lower maternal and paternal education (≤Associate's Degree vs Bachelor's Degree) began treatment >4 and ∼8.5 months later than adolescents with greater maternal and paternal education (p = 0.04; p = 0.03), respectively. Adolescents with lower maternal education also received a diagnosis >5 months later (p = 0.03). Adolescents without a mother or father with a graduate degree received a diagnosis and began treatment∼3 and >11 months later (p = 0.03; p = 0.01) than adolescents whose mother or father received a graduate degree, respectively. No racial differences were found. CONCLUSIONS: Adolescents with lower annual household income and/or parental education experienced increased duration between FS onset and treatment and diagnosis. Research is needed to clarify the mechanisms underlying this relationship, and action is needed to reduce these disparities given FS duration is associated with poorer prognosis and greater effects on the brain.

Cataract surgery using two 3D visualization systems: Complication rates, surgical duration & comparison with traditional microscopes.

PURPOSE: To compare the complication rates and surgical duration of cataract surgery using two 3D visualization systems and a traditional binocular microscope among experienced and inexperienced surgeons. METHODS: This retrospective case series included 571 eyes that received cataract surgery using either heads up cataract surgery, via a 3D head mounted system (N = 148-Group 1) or a 3D display screen (N = 338 eyes-Group 2), or traditional binocular microscope (N = 85 eyes-Group 3). The surgical records of consecutive patients who underwent cataract surgery by two groups of surgeons (experienced and inexperienced) were reviewed. Patients in all groups received either femtosecond laser assisted cataract surgery (FLACS) or traditional phacoemulsification. Complication rate, as well as duration of cataract surgery were evaluated between all three visualization approaches, between experienced and inexperienced surgeons. RESULTS: There was no statistically significant difference in duration of surgery between all 3 visualization approaches for both experienced and inexperienced surgeons (p < 0.05). Furthermore, the type of surgical technique (manual or FLACS) did not affect the surgical duration for both experienced and inexperienced surgeons (p < 0.05). No intraoperative complications were demonstrated in the current cohort. CONCLUSIONS: The implementation of heads up-3D visualization either through a screen or a head mounted platform for cataract surgery seems to offer similar safety and efficiency as the traditional binocular microscope, and both experienced and inexperienced surgeons demonstrate the same outcomes in terms of safety and efficiency.

Direct quantitation of methyl phosphonate adducts to human serum butyrylcholinesterase by immunomagnetic-UHPLC-MS/MS.

Hydrolysis of G- and V-series organophosphorus nerve agents (OPNAs) containing a phosphorus-methyl bond yields a methylphosphonic acid (MeP) product when adducted to human butyrylcholinesterase (BChE). The MeP adduct is considered a sign of "aging" and results in loss of the o-alkyl identifier specific to each nerve agent. After aging has occurred, common therapeutics such as oximes cannot reactivate the cholinesterase enzyme and relieve cholinergic inhibition. Until now, a direct, quantitative method for determination of the MeP adduct to BChE was unavailable. Aged adducts in serum samples were processed by immunomagnetic separation of BChE by antibody conjugated bead, isotope-dilution, pepsin digestion, followed by UHPLC separation and detection by conventional electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Ions were detected in selected reaction monitoring (SRM) mode, and transition m/z 874.3 → 778.3 was used for quantitation. The analytical response ratio was linearly proportional to the serum concentration of MeP-adducted peptide (MeP-P) over the nominal concentration range of 2.0-250 ng/mL, with a coefficient of determination of R(2) ≥ 0.997. Intrarun accuracy, expressed as %Relative Error (%RE), was ≤13.5%, 16.3%, and 3.20% at 2.0, 16, and 250 ng/mL, respectively; the corresponding precision expressed as %RSD was ≤11.9%, 6.15%, and 3.39%. Interday %RSD was ≤7.13%, 5.69%, and 1.91%. Recovery of MeP-P from serum was ≥68% across the validated concentration range, and contributions from matrix effects were minimal. The method provides a direct, quantitative measurement of MeP-P found in clinical samples suspected of nerve agent exposure and subjected to such post-sampling stresses as elevated temperature and extended shipping.

William Watson Cheyne (1852-1932): a life in medicine and his innovative surgical treatment of congenital hydrocephalus.

William Watson Cheyne lived and trained during a period of great advances in medical knowledge and surgical techniques. Despite his various contributions to the fields of bacteriology and surgery, little is known about his career or his life apart from his affiliations with Joseph Lister. This article aims to identify Cheyne as a pioneer in the treatment of congenital hydrocephalus and sheds light on the man who existed in Lister's shadow for most of his life. Cheyne's technique for surgical intervention of hydrocephalus was a great turning point and contributes to the current treatment strategy utilized today for hydrocephalus.

Mutation rates and fitness consequences of mosaic chromosomal alterations in blood.

Mosaic chromosomal alterations (mCAs) are common in cancers and can arise decades before diagnosis. A quantitative understanding of the rate at which these events occur, and their functional consequences, could improve cancer risk prediction and our understanding of somatic evolution. Using mCA clone size estimates from the blood of approximately 500,000 UK Biobank participants, we estimate mutation rates and fitness consequences of acquired gain, loss and copy-neutral loss of heterozygosity events. Most mCAs have moderate to high fitness effects but occur at a low rate, being more than tenfold less common than equivalently fit single-nucleotide variants. Notable exceptions are mosaic loss of X and Y, which we estimate have roughly 1,000-fold higher mutation rates than autosomal mCAs. Although the way in which most mCAs increase in prevalence with age is consistent with constant growth rates, some mCAs exhibit different behavior, suggesting that their fitness may depend on inherited variants, extrinsic factors or distributions of fitness effects.

Sleep and Inflammation during COVID-19 Virtual Learning in Adolescents with Overweight or Obesity.

(1) Background: Adolescents present as a high-risk group for a range of adverse physical health outcomes during the pandemic, including sleep and C-reactive protein (CRP) levels. As adolescents with overweight or obesity (OWOB) present as an even higher risk group, the present study assessed relationships between sleep and CRP levels before and during COVID-19 in adolescents with OWOB. (2) Methods: Fourteen adolescents with OWOB participated in a pre-COVID1, pre-COVID2, and during-COVID-19 lab visit, measuring sleep and CRP levels. The sample size was limited by the number of participants who provided data before COVID-19 and who were enrolled in virtual school during the recruitment phase. However, our power analyses indicated needing a minimum of 10 participants to achieve adequate power. Pre-COVID1, pre-COVID2, and during-COVID-19 normative expected CRP levels were calculated based on age, sex, race, and body mass index percentile-matched data. Analyses compared pre-COVID1 and pre-COVID2 sleep with during-COVID-19 sleep, during-COVID-19 sleep and during-COVID-19 CRP levels, during-COVID-19 CRP levels with normative expected during-COVID-19 CRP levels, change in CRP levels from pre-COVID1 and pre-COVID2 to during-COVID-19 with normative expected CRP levels during those time periods, and change in CRP levels before COVID-19 with change in CRP levels during COVID-19. (3) Results. During COVID-19, participants experienced decreased sleep efficiency (p = 0.001), later wake time (p < 0.001), longer time in bed (p = 0.021), and onset latency (p = 0.004), compared to pre-COVID1, and decreased sleep efficiency (p = 0.002), longer onset latency (p = 0.006), and later wake time (p < 0.001) and bedtime (p = 0.016) compared with pre-COVID2. During-COVID-19 CRP levels were positively correlated with during-COVID-19 wake times (p = 0.01) and times in bed (p = 0.008). During-COVID-19 CRP levels were greater than normative expected CRP levels (p < 0.001). CRP levels increased more from pre-COVID1 and pre-COVID2 to during-COVID-19 than normative expected changes in CRP levels (p < 0.003). Changes in CRP levels before and during COVID-19 were not significantly different. (4) Conclusions. These findings highlight the consequential effects of COVID-19, including impairments in sleep, on adolescents with OWOB. CRP levels increased more (~5 mg/L) during COVID-19 than normative expected change.

The Effects of COVID-19 Virtual Learning on Body Fat and Insulin Resistance in Adolescents with Overweight or Obesity.

(1) Background: COVID-19 virtual learning reduced structural supports for adolescent physical activity and diet, threatening metabolic health, especially in teens with overweight or obesity (OWOB). (2) Methods: Adolescents (N = 14) with OWOB completed fasting blood draws (measuring insulin resistance, IR) and Dual Energy X-Ray Absorptiometry (DXA, measuring total body fat percent, TBF%) pre-COVID-19 and during COVID-19. Changes in TBF% and IR were calculated (1) pre-COVID-19 and (2) from pre-COVID-19 to during COVID-19. Age and body mass index (BMI) percentile-matched data assessed normative changes across similar, non-COVID-19 time periods. Paired t-tests compared TBF% change pre- to during COVID-19 with (1) TBF% change pre-COVID19 and (2) TBF% normative change. Two ANCOVAs compared IR change pre- to during COVID-19 with (1) IR change pre-COVID-19 controlling for BMI z-score and difference in time between assessments and (2) normative change in IR controlling for sex/race. (3) Results: The TBF% change pre-COVID-19 and the normative change were similar. The TBF% increased more (~six percentage points) during COVID-19 compared to normative change (p < 0.01). During COVID-19, IR increased more (~2.5 units) than change pre-COVID-19 (p = 0.03) and increased more (~3.5 units) than normative change (p = 0.01). (4) Conclusions: TBF% and IR increased exponentially during COVID-19 in teens with OWOB compared to pre-COVID-19 and normative changes.

The Impact of Longitudinal Patterns of Adolescent Sleep Duration on Adult C-Reactive Protein (CRP), Waist-To-Height Ratio, and Body Mass Index (BMI) Among Black and White Individuals.

PURPOSE: To assess the impact of longitudinal adolescent sleep duration on adult C-reactive protein (CRP), waist-to-height ratio (WtHR), and body mass index (BMI) by race. METHODS: Participants (N = 2,399; Mage = 15.7; 40.2% male; 79.2% White, 20.8% Black; Grades 7-12 at Wave I) from the Add Health database provided self-reported sleep duration in Waves I-IV. During Wave V, CRP, WtHR, and BMI were objectively measured. Trajectory analysis was performed using a group-based modeling approach. Chi-square test determined racial differences between groups. General linear models determined relationships between trajectory group, race, and group/race interaction with Wave V CRP, WtHR, and BMI. RESULTS: Three sleep trajectories emerged: Group 1 "shortest" (24.4%), Group 2 "stable recommended" (67.6%), and Group 3 "varied" (8%). Black individuals and older individuals were more likely to be in Group 1 compared with Group 2. Regardless of race, individuals with patterns of sleep duration increasing to above what is recommended across waves (Group 3) had elevated CRP. Individuals with stable patterns of adequate sleep (Group 2) had lower WtHR. Black individuals with consistently stable patterns of adequate sleep duration had lower BMI compared to those with low sleep duration. DISCUSSION: Black individuals were more likely to obtain chronically short sleep during the transition from adolescence to adulthood, highlighting a significant health disparity. Poor longitudinal sleep predicted elevated CRP and WtHR. Sleep only impacted BMI for Black individuals. This may relate to racial differences in BMI measurement.

Quantitative proteomics of cerebrospinal fluid from African Americans and Caucasians reveals shared and divergent changes in Alzheimer's disease.

BACKGROUND: Despite being twice as likely to get Alzheimer's disease (AD), African Americans have been grossly underrepresented in AD research. While emerging evidence indicates that African Americans with AD have lower cerebrospinal fluid (CSF) levels of Tau compared to Caucasians, other differences in AD CSF biomarkers have not been fully elucidated. Here, we performed unbiased proteomic profiling of CSF from African Americans and Caucasians with and without AD to identify both common and divergent AD CSF biomarkers. METHODS: Multiplex tandem mass tag-based mass spectrometry (TMT-MS) quantified 1,840 proteins from 105 control and 98 AD patients of which 100 identified as Caucasian while 103 identified as African American. We used differential protein expression and co-expression approaches to assess how changes in the CSF proteome are related to race and AD. Co-expression network analysis organized the CSF proteome into 14 modules associated with brain cell-types and biological pathways. A targeted mass spectrometry method, selected reaction monitoring (SRM), with heavy labeled internal standards was used to measure a panel of CSF module proteins across a subset of African Americans and Caucasians with or without AD. A receiver operating characteristic (ROC) curve analysis assessed the performance of each protein biomarker in differentiating controls and AD by race. RESULTS: Consistent with previous findings, the increase of Tau levels in AD was greater in Caucasians than in African Americans by both immunoassay and TMT-MS measurements. CSF modules which included 14-3-3 proteins (YWHAZ and YWHAG) demonstrated equivalent disease-related elevations in both African Americans and Caucasians with AD, whereas other modules demonstrated more profound disease changes within race. Modules enriched with proteins involved with glycolysis and neuronal/cytoskeletal proteins, including Tau, were more increased in Caucasians than in African Americans with AD. In contrast, a module enriched with synaptic proteins including VGF, SCG2, and NPTX2 was significantly lower in African Americans than Caucasians with AD. Following SRM and ROC analysis, VGF, SCG2, and NPTX2 were significantly better at classifying African Americans than Caucasians with AD. CONCLUSIONS: Our findings provide insight into additional protein biomarkers and pathways reflecting underlying brain pathology that are shared or differ by race.

Quantitative Mass Spectrometry Analysis of Cerebrospinal Fluid Protein Biomarkers in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia, with cerebrospinal fluid (CSF) ß-amyloid (Aß), total Tau, and phosphorylated Tau (pTau) providing the most sensitive and specific biomarkers for diagnosis. However, these diagnostic biomarkers do not reflect the complex changes in AD brain beyond amyloid (A) and Tau (T) pathologies. Here, we report a selected reaction monitoring mass spectrometry (SRM-MS) method with isotopically labeled standards for relative protein quantification in CSF. Biomarker positive (AT+) and negative (AT-) CSF pools were used as quality controls (QCs) to assess assay precision. We detected 62 peptides (51 proteins) with an average coefficient of variation (CV) of ~13% across 30 QCs and 133 controls (cognitively normal, AT-), 127 asymptomatic (cognitively normal, AT+) and 130 symptomatic AD (cognitively impaired, AT+). Proteins that could distinguish AT+ from AT- individuals included SMOC1, GDA, 14-3-3 proteins, and those involved in glycolysis. Proteins that could distinguish cognitive impairment were mainly neuronal proteins (VGF, NPTX2, NPTXR, and SCG2). This demonstrates the utility of SRM-MS to quantify CSF protein biomarkers across stages of AD.

A protein panel in cerebrospinal fluid for diagnostic and predictive assessment of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease with heterogenous pathophysiological changes that develop years before the onset of clinical symptoms. These preclinical changes have generated considerable interest in identifying markers for the pathophysiological mechanisms linked to AD and AD-related disorders (ADRD). On the basis of our prior work integrating cerebrospinal fluid (CSF) and brain proteome networks, we developed a reliable and high-throughput mass spectrometry-selected reaction monitoring assay that targets 48 key proteins altered in CSF. To test the diagnostic utility of these proteins and compare them with existing AD biomarkers, CSF collected at baseline visits was assayed from 706 participants recruited from the Alzheimer's Disease Neuroimaging Initiative. We found that the targeted CSF panel of 48 proteins (CSF 48 panel) performed at least as well as existing AD CSF biomarkers (Aß42, tTau, and pTau181) for predicting clinical diagnosis, FDG PET, hippocampal volume, and measures of cognitive and dementia severity. In addition, for each of those outcomes, the CSF 48 panel plus the existing AD CSF biomarkers significantly improved diagnostic performance. Furthermore, the CSF 48 panel plus existing AD CSF biomarkers significantly improved predictions for changes in FDG PET, hippocampal volume, and measures of cognitive decline and dementia severity compared with either measure alone. A potential reason for these improvements is that the CSF 48 panel reflects a range of altered biology observed in AD/ADRD. In conclusion, we show that the CSF 48 panel complements existing AD CSF biomarkers to improve diagnosis and predict future cognitive decline and dementia severity.