👤 Ryon Graf

Genome- and epigenome-wide association studies have associated variants and methylation status of CPT1a (carnitine palmitoyltransferase 1a) to reductions in VLDL (very low-density lipoprotein) cholesterol and triglyceride levels. The objective of this study was to determine the mechanisms by which CPT1a-dependent mitochondrial fatty acid oxidation influences hepatic and lipoprotein metabolism. Eight-week-old male and female We report significant associations between the presence of These studies provide mechanistic insight linking genetic variants and methylation status of Show less

The apolipoprotein E (APOE) gene is the best established genetic risk factor for Alzheimer's disease in later life, with the ε4 allele conferring higher risk. APOE disclosure is becoming increasingly common in the clinical care of people with Alzheimer's disease and in cognitively unimpaired adults. In this study, we aimed to describe changes in measures of genetic disease knowledge and psychiatric symptoms following APOE disclosure to cognitively unimpaired adults. Data were collected as part of the screening phase of the global, multicentre, Alzheimer's Prevention Initiative Generation Study 1 (NCT02565511). Eligible individuals were cognitively unimpaired (Mini-Mental State Exam total score ≥24), aged 60-75 years, and psychologically pre-screened for readiness (by measures of depressive symptoms and anxiety) to receive their APOE genotype from a health-care provider. Participants were assessed before disclosure, and 2-7 days, 6 weeks, 6 months, and 12 months after disclosure. Multivariable linear and ordinal logistic regressions were used to compare changes in genetic disease knowledge, anxiety, depression, and distress by APOE4 genotype status, adjusting for key covariates, with a focus on 2-7 days after disclosure. Multiple imputation by chained equations methods was used to account for missing outcome data. The trial took place between Nov 30, 2015, and Sept 23, 2019. In total, 9496 participants (including 790 APOE4 homozygotes, 4869 heterozygotes, and 3837 non-carriers) learned their APOE genotype from a health-care provider as part of Generation Study 1 screening. 4038 (42·5%) participants were in the 65-69-year age group, 5790 (61·0%) were female, 3706 (39·0%) were male, and 8862 (93·3%) self-identified as White. Increase in genetic disease knowledge 2-7 days after disclosure was greater in APOE4 homozygotes (mean 1·19 [SD 3·95]) than in heterozygotes (0·78 [3·95], p=0·042) and non-carriers (0·29 [3·96], p=0·0002). Disease-specific distress 2-7 days after disclosure increased more in homozygotes (2·25 [6·42]) than in heterozygotes (0·53 [5·08], p<0·0001) and non-carriers (0·79 [4·95], p<0·0001). Levels of anxiety 2-7 days after disclosure increased in homozygotes (0·17 [2·95]) but decreased in heterozygotes (-0·67 [2·68], p<0·0001) and non-carriers (-0·66 [2·67], p<0·0001). There were no significant changes in depressive symptoms following disclosure for any APOE4 group. Notably, for all APOE4 groups, increases in distress and anxiety were small and did not reach predefined levels of clinical concern. In cognitively unimpaired, psychologically pre-screened adults, APOE disclosure by a trained health-care provider was generally safe and well tolerated, consistent with results from previous studies. To our knowledge, this is the largest study experience of APOE disclosure to date, especially for homozygotes, and is notable for the older age of participants compared with previous research. These results are timely and important given anticipated increases in APOE disclosure to guide clinical decision making once an Alzheimer's disease prevention treatment is approved for cognitively unimpaired adults or if patients' family members are interested in genetic testing. Scalable approaches for returning Alzheimer's disease risk information are critical to meeting anticipated demand. Results from this study may be useful to bolster clinical translatability of disclosure programmes. The National Institute on Aging, Alzheimer's Association, Banner Alzheimer's Foundation, GHR Foundation, F-Prime Biomedical Research Initiative (FBRI), and Novartis Pharma. Show less

Decreased cerebrospinal fluid (CSF) levels of synaptic proteins, possibly reflecting impaired synaptic function, have been observed in major depressive disorder (MDD). To investigate the diagnostic utility of the soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) complex protein, synaptosomal-associated protein of 25 kDa (SNAP-25), for MDD. Overall, 208 participants with one of MDD, schizophrenia (SCZ) or bipolar disorder (BD), and healthy controls (HCs) were retrospectively enrolled. CSF levels of SNAP-25 were assessed relative to MDD characteristics and the diagnostic potential was analysed. In subgroups of patients, CSF levels of presynaptic neurexin 3 (NRXN3), postsynaptic neurogranin (NRGN) and Alzheimer's disease biomarkers were measured for comparison. SNAP-25 levels, but not the levels of the other synaptic markers, were significantly decreased in MDD compared with HCs, allowing for discrimination with 68% sensitivity and 67% specificity. SNAP-25 was not associated with MDD severity or antidepressant medication. Compared with HCs, SCZ also displayed decreased SNAP-25 enabling discrimination with 64% sensitivity and 77% specificity. There were strong correlations between levels of synaptic proteins and established Alzheimer pathology markers, with subtle differences in the association pattern between disorders. Our data suggest that SNAP-25, NRXN3 and NRGN versus beta-amyloid and phosphorylated tau protein 181 (ptau) are regulated differentially across psychiatric disorders and that SNAP-25 has a moderate diagnostic potential for MDD and SCZ. We propose that CSF SNAP-25 level might represent an integrated readout of reduced synaptic function, rather than of synaptic degeneration, in MDD. Further studies are needed to analyse whether this potential can be increased by using multimarker measurements and whether it will be possible to subtype psychiatric disorders according to synaptic involvement in pathophysiology. SNAP-25 and other synaptic proteins in CSF might aid diagnosis and subtyping of MDD and SCZ. The current development of sensitive methods to also determine synaptic proteins in blood samples from patients will advance the validation of the biomarker potential and contribute to understanding of synaptic involvement in the pathophysiology of MDD and SCZ. Show less

Myasthenia gravis (MG) is a disorder of the neuromuscular junction, typically associated with autoantibodies (Abs) that impair neuromuscular transmission. However, approximately 10% of cases are seronegative. Emerging evidence suggests that seronegative MG (SNMG) may be mimicked by hereditary conditions, particularly congenital myasthenic syndromes (CMSs), which require different treatments. In this study, we aimed to determine the proportion of CMS among patients diagnosed with SNMG. We used whole-exome sequencing (WES) in adult patients (aged ≥18 years) diagnosed with SNMG who were enrolled at 3 Austrian tertiary neuromuscular centers between August 2022 and January 2024. Genetic testing was conducted in individuals who remained seronegative after comprehensive serologic testing to exclude Abs against (clustered) acetylcholine receptors, muscle-specific kinase, lipoprotein receptor-related protein 4, and voltage-gated calcium channels. Moreover, we aimed to analyze clinical and demographic factors associated with the likelihood of receiving a molecular diagnosis. A total of 50 patients with SNMG (35 [70%] female) were referred for exome-based genetic screening. The median age at disease onset was 35 years (interquartile range 24.0-46.0 years). Seven patients (14%) were genetically diagnosed with CMS through WES (4 with Our findings provide evidence that a considerable proportion of patients diagnosed with SNMG have an underlying hereditary etiology. Notably, a (subjective) response to immunotherapies does not exclude a molecular CMS diagnosis. In conclusion, offering genetic testing to seronegative patients with myasthenic syndromes may have profound therapeutic implications. Show less

Genome- and epigenome-wide association studies have associated variants and methylation status of carnitine palmitoyltransferase 1a (CPT1a) to reductions in very low-density lipoprotein (VLDL) cholesterol and triglyceride levels. We report significant associations between the presence of Loss-of-function SNPs in Show less

The Alzheimer's Prevention Initiative (API) Generation Studies evaluated the BACE inhibitor umibecestat for Alzheimer's disease (AD) prevention. The studies were terminated early, and the reversibility of umibecestat's side effects was assessed. Cognitively unimpaired 60- to 75-year-old apolipoprotein E (APOE) ε4 homozygotes and heterozygotes (the latter with elevated brain amyloid deposition) (n = 1556) received umibecestat (50 or 15 mg daily) or placebo for 7 months on average and were followed for a median (interquartile range) of 4 (3 to 6) months after washout. Compared to placebo, umibecestat-treated participants had small, non-progressive, but statistically significant decline in performance on certain cognitive batteries including Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and API Preclinical Composite Cognitive test, but not Clinical Dementia Rating-Sum of Boxes. RBANS differences were no longer significant at the end of follow-up. In people at genetic risk for AD, high-dose beta-site amyloid precursor protein cleaving enzyme (BACE) inhibition was associated with early mild cognitive worsening, which reversed shortly after washout, suggesting a symptomatic side effect not associated with neurodegeneration. Fully anonymized data, images, and samples are available upon request for further research on BACE inhibition. This is the first trial with blinded assessment of reversibility of BACE inhibitor side effects. Umibecestat was tested in cognitively unimpaired persons at genetic risk for AD. Umibecestat led to early mild cognitive decline that reversed shortly after washout. This suggests a potentially manageable effect not associated with neurodegeneration. Further research may determine the future of BACE inhibition in AD prevention. Show less

Alzheimer's Prevention Initiative Generation Study 1 evaluated amyloid beta (Aβ) active immunotherapy (vaccine) CAD106 and BACE-1 inhibitor umibecestat in cognitively unimpaired 60- to 75-year-old participants at genetic risk for Alzheimer's disease (AD). The study was reduced in size and terminated early. Results from the CAD106 cohort are presented. Sixty-five apolipoprotein E ε4 homozygotes with/without amyloid deposition received intramuscular CAD106 450 μg (n = 42) or placebo (n = 23) at baseline; Weeks 1, 7, 13; and quarterly; 51 of them had follow-up Aβ positron emission tomography (PET) scans at 18 to 24 months. CAD106 induced measurable serum Aβ immunoglobulin G titers in 41/42 participants, slower rates of Aβ plaque accumulation (mean [standard deviation] annualized change from baseline in amyloid PET Centiloid: -0.91[5.65] for CAD106 versus 8.36 [6.68] for placebo; P < 0.001), and three amyloid-related imaging abnormality cases (one symptomatic). Despite early termination, these findings support the potential value of conducting larger prevention trials of Aβ active immunotherapies in individuals at risk for AD. This was the first amyloid-lowering prevention trial in persons at genetic risk of late-onset Alzheimer's disease (AD). Active immunotherapy targeting amyloid (CAD106) was tested in this prevention trial. CAD106 significantly slowed down amyloid plaque deposition in apolipoprotein E homozygotes. CAD106 was generally safe and well tolerated, with only three amyloid-related imaging abnormality cases (one symptomatic). Such an approach deserves further evaluation in larger AD prevention trials. Show less

Patients with metastatic colorectal cancer (mCRC) with This study utilized a de-identified mCRC clinicogenomic database from ∼280 US cancer clinics between March 2014 and April 2023. We examined real-world progression-free survival (rwPFS) and overall survival (rwOS) between patients with and those without pre-specified genomic alterations (PSGAs) by Cox models and an adjusted risk score. Genomic alterations were also compared between samples collected before and after EGFR mAb therapy. Nearly, one-third of microsatellite stable (MSS) Detection of genomic resistance alterations in MSS Show less

MYC is a commonly amplified, potentially targetable gene in prostate cancer (PCa). We sought to define the molecular, immunologic, and clinicodemographic landscape of MYC amplification (MYC Hybrid capture-based comprehensive genomic profiling (CGP) was performed on PCa tumor samples. MYC Of 12,528 hormone-sensitive and castrate-resistant (CRPC) samples, MYC MYC Show less

Genome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the role of liver-specific CPT1a on hepatic lipid metabolism. Male and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet (60% kcal fat) for 15 weeks. Mice were necropsied after a 16 h fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging, kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis (Plin2, Cidec, G0S2) and in polyunsaturated fatty acid metabolism (Elovl5, Fads1, Elovl2), while only female LKO mice increased genes involved in inflammation (Ly6d, Mmp12, Cxcl2). Kinase profiling showed decreased protein kinase A activity, which coincided with increased PLIN2, PLIN5, and G0S2 protein levels and decreased triglyceride hydrolysis in LKO mice. Liver-specific deletion of CPT1a promotes sexually dimorphic steatotic liver disease (SLD) in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury. Show less

Genome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the impact by which liver-specific CPT1a deletion impacts hepatic lipid metabolism. Six-to-eight-week old male and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet (HFD; 60% kcal fat) for 15 weeks. Mice were necropsied after a 16 hour fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase (ALT) levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in both whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis ( Liver-specific deletion of CPT1a promotes sexually dimorphic steatotic liver disease (SLD) in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury. Show less

Identification of cancer-predisposing germline variants in childhood cancer patients is important for therapeutic decisions, disease surveillance and risk assessment for patients, and potentially, also for family members. We investigated the spectrum and prevalence of pathogenic germline variants in selected childhood cancer patients with features suggestive of genetic predisposition to cancer. Germline DNA was subjected to exome sequencing to filter variants in 1048 genes of interest including 176 known cancer predisposition genes (CPGs). An enrichment burden analysis compared rare deleterious germline CPG variants in the patient cohort with those in a healthy aged control population. A subset of predicted deleterious variants in novel candidate CPGs was investigated further by examining matched tumor samples, and the functional impact of AXIN1 variants was analyzed in cultured cells. Twenty-two pathogenic/likely pathogenic (P/LP) germline variants detected in 13 CPGs were identified in 19 of 76 patients (25.0%). Unclear association with the diagnosed cancer types was observed in 11 of 19 patients carrying P/LP CPG variants. The burden of rare deleterious germline variants in autosomal dominant CPGs was significantly higher in study patients versus healthy aged controls. A novel AXIN1 frameshift variant (Ser321fs) may impact the regulation of β-catenin levels. Selection of childhood cancer patients for germline testing based on features suggestive of an underlying genetic predisposition could help to identify carriers of clinically relevant germline CPG variants, and streamline the integration of germline genomic testing in the pediatric oncology clinic. Show less

Early infantile epileptic encephalopathy (EIEE) is a severe neurologic and neurodevelopmental disease that manifests in the first year of life. It shows a high degree of genetic heterogeneity, but the genetic origin is only identified in half of the cases. We report the case of a female child initially diagnosed with Leber congenital amaurosis (LCA), an early-onset retinal dystrophy due to photoreceptor cell degeneration in the retina. The first examination at 9 months of age revealed no reaction to light or objects and showed wandering eye movements. Ophthalmological examination did not show any ocular abnormalities. The patient displayed mildly dysmorphic features and a global developmental delay. Brain MRI demonstrated pontine hypo-/dysplasia. The patient developed myoclonic epileptic seizures and epileptic spasms with focal and generalized epileptiform discharges on electroencephalogram (EEG) at the age of 16 months. Genetic screening for a potentially pathogenic DNA sequence variant by whole-exome sequencing (WES) revealed a novel, conserved, homozygous frameshift variant (c.5391delA, p.(Ala1798Leu Show less

Determine the impact of CETP (cholesteryl ester transfer protein) on the route of cholesterol elimination in mice. Approach and Results: We adapted our protocol for biliary cholesterol secretion with published methods for measuring transintestinal cholesterol elimination. Bile was diverted and biliary lipid secretion maintained by infusion of bile acid. The proximal small bowel was perfused with bile acid micelles. In high-fat, high-cholesterol-fed mice, the presence of a CETP transgene increased biliary cholesterol secretion at the expense of transintestinal cholesterol elimination. The increase in biliary cholesterol secretion was not associated with increases in hepatic SR-BI (scavenger receptor BI) or ABCG5 (ATP-binding cassette G5) ABCG8. The decline in intestinal cholesterol secretion was associated with an increase in intestinal Niemann-Pick disease, type C1, gene-like 1 mRNA. Finally, we followed the delivery of HDL (high-density lipoprotein) or LDL (low-density lipoprotein) cholesteryl esters (CE) from plasma to bile and intestinal perfusates. HDL-CE favored the biliary pathway. Following high-fat feeding, the presence of CETP directed HDL-CE away from the bile and towards the intestine. The presence of CETP increased LDL-CE delivery to bile, whereas the appearance of LDL-CE in intestinal perfusate was near the lower limit of detection. Biliary and intestinal cholesterol secretion can be simultaneously measured in mice and used as a model to examine factors that alter cholesterol elimination. Plasma factors, such as CETP, alter the route of cholesterol elimination from the body. Intestinal and biliary cholesterol secretion rates are independent of transhepatic or transintestinal delivery of HDL-CE, whereas LDL-CE was eliminated almost exclusively in the hepatobiliary pathway. Show less

The cationic, water-soluble quaternary trospium chloride (TC) is incompletely absorbed from the gut and undergoes wide distribution but does not pass the blood-brain barrier. It is secreted by the kidneys, liver, and intestine. To evaluate potential transport mechanisms for TC, we measured affinity of the drug to the human uptake and efflux transporters known to be of pharmacokinetic relevance. Affinity of TC to the uptake transporters OATP1A2, -1B1, -1B3, -2B1, OCT1, -2, -3, OCTN2, NTCP, and ASBT and the efflux carriers P-gp, MRP2 and MRP3 transfected in HEK293 and MDCK2 cells was measured. To identify relevant pharmacokinetic mechanisms in the bladder urothelium, mRNA expression of multidrug transporters, drug metabolizing enzymes, and nuclear receptors, and the uptake of TC into primary human bladder urothelium (HBU) cells were measured. TC was shown to be a substrate of OATP1A2 (Km = 6.9 ± 1.3 μmol/L; Vmax = 41.6 ± 1.8 pmol/mg·min), OCT1 (Km = 106 ± 16 μmol/L; Vmax = 269 ± 18 pmol/mg·min), and P-gp (Km = 34.9 ± 7.5 μmol/L; Vmax = 105 ± 9.1 pmol/mg·min, lipovesicle assay). The genetic OATP1A2 variants *2 and *3 were loss-of-function transporters for TC. The mRNA expression analysis identified the following transporter proteins in the human urothelium: ABCB1 (P-gp), ABCC1-5 (MRP1-5), ABCG2 (BCRP), SLCO2B1 (OATP2B1), SLCO4A1 (OATP4A1), SLC22A1 (OCT1), SLC22A3 (OCT3), SLC22A4 (OCTN1), SLC22A5 (OCTN2), and SLC47A1 (MATE1). Immuno-reactive P-gp and OATP1A2 were localized to the apical cell layers. Drug metabolizing enzymes CYP3A5, -2B6, -2B7 -2E1, SULT1A1-4, UGT1A1-10, and UGT2B15, and nuclear receptors NR1H3 and NR1H4 were also expressed on mRNA level. TC was taken up into HBU cells (Km = 18.5 ± 4.8 μmol/L; Vmax = 106 ± 11.3 pmol/mg·min) by mechanisms that could be synergistically inhibited by naringin (IC50 = 10.8 (8.4; 13.8) μmol/L) and verapamil (IC50 = 4.6 (2.8; 7.5) μmol/L), inhibitors of OATP1A2 and OCT1, respectively. Affinity of TC to OCT1 and P-glycoprotein may be the reason for incomplete oral absorption, wide distribution into liver and kidneys, and substantial intestinal and renal secretions. Absence of brain distribution may result from affinity to P-gp and a low affinity to OATP1A2. The human urothelium expresses many drug transporters and drug metabolizing enzymes that may interact with TC and other drugs eliminated into the urine. Show less

Current treatment protocols for Wilms tumor achieve 90% cure rates, but relapse risk and side effects from therapy remain challenging. Over the last decade, numerous markers have been proposed for classification and/or prediction of outcome. However, cohort sizes were quite variable and often small. We now provide a large-scale reassessment by real-time RT-PCR of 40 markers in 102 Wilms tumors followed by validation of potentially relevant markers in an independent set of 74 tumors. In the first data set, individual comparison with clinical data combined with adjustment for multiple testing and multivariate analysis revealed potentially relevant alteration of CA9, DKK1, EGR1, HEY2, MYC, MYCN, TERT, TOP2A, TRIM22, and VEGF expression in association with CTNNB1 mutation status, histological risk, response to chemotherapy, metastasis, relapse, or mortality. To further validate these data, potentially relevant genes for specific outcomes were reanalyzed in a second, independent tumor set. Here, univariate analysis confirmed the association of HEY2 with high-risk tumors and of TRIM22 with mortality. Even where significance levels could not be reached, the direction and extent of differential expression were generally reproducible. Multivariate analysis verified a weak correlation of TOP2A expression with metastasis and of TRIM22 with fatal outcome. Although we could corroborate only some of the previously reported associations of expression changes with clinical parameters, our results indicate that real-time RT-PCR analysis can facilitate further classification of Wilms tumor and prediction of outcome to adjust treatment accordingly. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. Show less