Comprehensive Two-Dimensional Gas Chromatography-Mass Spectrometry as a Tool for the Untargeted Study of Hop and Their Metabolites.

Since hop secondary metabolites have a direct correlation with the quality of beer and other hop-based beverages, and the volatile fraction of hop has a complex composition, requiring effective separation, here we explore the application of headspace solid-phase microextraction as a sample preparation method, coupled with comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) analysis. The methodology involved the use of a DVB/PDMS fibre with 500 mg of hop cone powder, extracted for 40 min at 50 °C, for both GC-MS and GC×GC-MS. The varieties Azacca, Cascade, Enigma, Loral, and Zappa were studied comprehensively. The results demonstrate that GC×GC-MS increases the number of peaks by over 300% compared to classical GC-MS. Overall, 137 compounds were identified or tentatively identified and categorised into 10 classes, representing between 87.6% and 96.9% of the total peak area. The composition revealed the highest concentration of sesquiterpene hydrocarbons for Enigma, whilst Zappa showed a relatively significant concentration of monoterpene hydrocarbons. Principal component analysis for all compounds and classes, along with hierarchical cluster analysis, indicated similarities between Zappa and Cascade, and Azacca and Loral. In conclusion, this method presents an optimistic advancement in hop metabolite studies with a simple and established sample preparation procedure in combination with an effective separation technique.

Parents' Perspectives on the Utility of Genomic Sequencing in the Neonatal Intensive Care Unit.

BACKGROUND: It is critical to understand the wide-ranging clinical and non-clinical effects of genome sequencing (GS) for parents in the NICU context. We assessed parents' experiences with GS as a first-line diagnostic tool for infants with suspected genetic conditions in the NICU. METHODS: Parents of newborns (N = 62) suspected of having a genetic condition were recruited across five hospitals in the southeast United States as part of the SouthSeq study. Semi-structured interviews (N = 78) were conducted after parents received their child's sequencing result (positive, negative, or variants of unknown significance). Thematic analysis was performed on all interviews. RESULTS: Key themes included that (1) GS in infancy is important for reproductive decision making, preparing for the child's future care, ending the diagnostic odyssey, and sharing results with care providers; (2) the timing of disclosure was acceptable for most parents, although many reported the NICU environment was overwhelming; and (3) parents deny that receiving GS results during infancy exacerbated parent-infant bonding, and reported variable impact on their feelings of guilt. CONCLUSION: Parents reported that GS during the neonatal period was useful because it provided a "backbone" for their child's care. Parents did not consistently endorse negative impacts like interference with parent-infant bonding.

Contributions of rare and common variation to early-onset and atypical dementia risk.

We collected and analyzed genomic sequencing data from individuals with clinician-diagnosed early-onset or atypical dementia. Thirty-two patients were previously described, with 68 newly described in this report. Of those 68, 62 patients self-reported white, non-Hispanic ethnicity and 6 reported as African-American, non-Hispanic. Fifty-three percent of patients had a returnable variant. Five patients harbored a pathogenic variant as defined by the American College of Medical Genetics criteria for pathogenicity. A polygenic risk score (PRS) was calculated for Alzheimer's patients in the total cohort and compared to the scores of a late-onset Alzheimer's cohort and a control set. Patients with early-onset Alzheimer's had higher non-APOE PRSs than patients with late-onset Alzheimer's, supporting the conclusion that both rare and common genetic variation associate with early-onset neurodegenerative disease risk.

Lipase-catalysed changes in essential oils revealed by comprehensive two-dimensional gas chromatography.

Candida antarctica lipase A (CALA) was applied for the chemo-selective enzymatic transesterification of terpene and phenyl alcohols in 35 different essential oil samples. Comprehensive two-dimensional gas chromatography with mass spectrometry (GC×GC‒MS) analysis enabled the separation and tentative identification of a cohort of 125 compounds, allowing the instant visualisation of the reaction process changes, amid the complex chemical background of the samples. The results indicate that 42 out of 79 alcohols so-identified were fully or partially esterified within 48 h of reaction, with primary alcohols being the substrates of preference of the enzyme (90-100% conversion), followed by secondary alcohols (mostly ~ 80-100% conversion). No significant conversion of tertiary alcohols and phenols was observed using the tested conditions. Overall, the enzyme's performance was consistent for primary alcohol substrates identified in multiple samples of different compositions. The observed selectivity, efficiency, robustness, scalability (enzyme/substrate working concentration ratio > 1:160), potential reusability, mild reaction conditions, and other factors make this process a greener and more sustainable alternative for industry applications, particularly for the manufacture of novel flavours and fragrances.

Contributions of rare and common variation to early-onset and atypical dementia risk.

We collected and analyzed genomic sequencing data from individuals with clinician- diagnosed early-onset or atypical dementia. Thirty-two patients were previously described, with sixty-eight newly described in this report. Of those sixty-eight, sixty-two patients reported Caucasian, non-Hispanic ethnicity and six reported as African American, non-Hispanic. Fifty-three percent of patients had a returnable variant. Five patients harbored a pathogenic variant as defined by the American College of Medical Genetics criteria for pathogenicity. A polygenic risk score was calculated for Alzheimer's patients in the total cohort and compared to the scores of a late-onset Alzheimer's cohort and a control set. Patients with early-onset Alzheimer's had higher non- APOE polygenic risk scores than patients with late onset Alzheimer's, supporting the conclusion that both rare and common genetic variation associate with early-onset neurodegenerative disease risk.

Quantitative assessment of enzymatic processes applied to flavour and fragrance standard compounds using gas chromatography with flame ionisation detection.

The performance of different enzymes towards the bioprocessing of aroma-related compounds was investigated and a strategy based on GC-FID analysis was developed to facilitate assessment of the stages of characterisation, screening and optimisation, including chiral ratio determination. Characterisation included activity assays (UV-Vis and GC-FID), protein quantification (NanoDrop spectrophotometry) and molar mass estimation (SDS-PAGE electrophoresis). Screening experiments assessed different enzymes, substrates, solvents, acyl donors or mediators. Aroma-related substrates comprised terpene and phenolic compounds. The enzymes tested included the lipases CALA (Sigma-Aldrich), NZ-435, LZ-TLIM, NC-ADL, LZ-CALBL and the laccases NZ-51003 and DL-IIS (all from Novozymes). Among those, NZ-435 and NZ-51003 had the highest activities in the characterisation stage and, along with CALA, achieved conversions above 70% for citronellol (lipases) or 50% for eugenol (laccases) at the screening stage. The lipases had preference for the primary alcohol and laccases for phenolic compounds, among the tested substrates. The transesterification reaction between the lipase CALA and the standards mixture (citronellol, menthol, linalool) was used to demonstrate the optimisation stage, where the best levels of temperature, enzyme and acyl donor concentrations were investigated. Optimum conditions were found to be 37-40 °C, 3-4 mg/mL of enzyme and 58-60% (v/v) vinyl acetate. Additional confirmation experiments using the same terpene standards mixture and citronella oil sample, gave a conversion of > 95% for citronellol after 1 h (for both, standards mixture and sample), and 20% or 74% for menthol after 1 h or 24 h, respectively. None of the tested enzymes demonstrated significant enantioselectivity under the tested conditions. The GC-FID approach demonstrated here was suitable to determine the reaction profiles and chiral ratio variations for biocatalysed reactions with aroma compounds in low complexity samples. Advanced separations will be applied to more complex samples in the future.

Heterozygous variants in CTR9, which encodes a major component of the PAF1 complex, are associated with a neurodevelopmental disorder.

PURPOSE: CTR9 is a subunit of the PAF1 complex (PAF1C) that plays a crucial role in transcription regulation by binding CTR9 to RNA polymerase II. It is involved in transcription-coupled histone modification through promoting H3K4 and H3K36 methylation. We describe the clinical and molecular studies in 13 probands, harboring likely pathogenic CTR9 missense variants, collected through GeneMatcher. METHODS: Exome sequencing was performed in all individuals. CTR9 variants were assessed through 3-dimensional modeling of the activated human transcription complex Pol II-DSIF-PAF-SPT6 and the PAF1/CTR9 complex. H3K4/H3K36 methylation analysis, mitophagy assessment based on tetramethylrhodamine ethyl ester perchlorate immunofluorescence, and RNA-sequencing in skin fibroblasts from 4 patients was performed. RESULTS: Common clinical findings were variable degrees of intellectual disability, hypotonia, joint hyperlaxity, speech delay, coordination problems, tremor, and autism spectrum disorder. Mild dysmorphism and cardiac anomalies were less frequent. For 11 CTR9 variants, de novo occurrence was shown. Three-dimensional modeling predicted a likely disruptive effect of the variants on local CTR9 structure and protein interaction. Additional studies in fibroblasts did not unveil the downstream functional consequences of the identified variants. CONCLUSION: We describe a neurodevelopmental disorder caused by (mainly) de novo variants in CTR9, likely affecting PAF1C function.

Genome sequencing as a first-line diagnostic test for hospitalized infants.

PURPOSE: SouthSeq is a translational research study that undertook genome sequencing (GS) for infants with symptoms suggestive of a genetic disorder. Recruitment targeted racial/ethnic minorities and rural, medically underserved areas in the Southeastern United States, which are historically underrepresented in genomic medicine research. METHODS: GS and analysis were performed for 367 infants to detect disease-causal variation concurrent with standard of care evaluation and testing. RESULTS: Definitive diagnostic (DD) or likely diagnostic (LD) genetic findings were identified in 30% of infants, and 14% of infants harbored an uncertain result. Only 43% of DD/LD findings were identified via concurrent clinical genetic testing, suggesting that GS testing is better for obtaining early genetic diagnosis. We also identified phenotypes that correlate with the likelihood of receiving a DD/LD finding, such as craniofacial, ophthalmologic, auditory, skin, and hair abnormalities. We did not observe any differences in diagnostic rates between racial/ethnic groups. CONCLUSION: We describe one of the largest-to-date GS cohorts of ill infants, enriched for African American and rural patients. Our results show the utility of GS because it provides early-in-life detection of clinically relevant genetic variations not detected by current clinical genetic testing, particularly for infants exhibiting certain phenotypic features.

Pharmacogenetic Predictors of Cannabidiol Response and Tolerability in Treatment-Resistant Epilepsy.

In patients with treatment-resistant epilepsy (TRE), cannabidiol (CBD) produces variable improvement in seizure control. Patients in the University of Alabama at Birmingham CBD Expanded Access Program (EAP) were enrolled in the genomic study and genotyped using the Affymetrix Drug Metabolizing Enzymes and Transporters plus array. Associations between variants and CBD response (≥50% seizure reduction) and tolerability (diarrhea, sedation, and abnormal liver function) was evaluated under dominant and recessive models. Expression quantitative trait loci (eQTL) influencing potential CBD targets was evaluated in the UK Brain Expression Consortium data set (Braineac), and genetic co-expression examined. Of 169 EAP patients, 112 (54.5% pediatric and 50.0% female) were included in the genetic analyses. Patients with AOX1 rs6729738 CC (aldehyde oxidase; odds ratio (OR) 6.69, 95% confidence interval (CI) 2.19-20.41, P = 0.001) or ABP1 rs12539 (diamine oxidase; OR 3.96, 95% CI 1.62-9.73, P = 0.002) were more likely to respond. Conversely, patients with SLC15A1 rs1339067 TT had lower odds of response (OR 0.06, 95% CI 0.01-0.56, P = 0.001). ABCC5 rs3749442 was associated with lower likelihood of response and abnormal liver function tests, and higher likelihood of sedation. The eQTL revealed that rs1339067 decreased GPR18 expression (endocannabinoid receptor) in white matter (P = 5.6 × 10-3 ), and rs3749442 decreased hippocampal HTR3E expression (serotonin 5-HT3E ; P = 8.5 × 10-5 ). Furthermore, 75% of genes associated with lower likelihood of response were co-expressed. Pharmacogenetic variation is associated with CBD response and influences expression of CBD targets in TRE. Implicated pathways, including cholesterol metabolism and glutathione conjugation, demonstrate potential interactions between CBD and common medications (e.g., statins and acetaminophen) that may require closer monitoring. These results highlight the role of pharmacogenes in fundamental biologic processes and potential genetic underpinnings of treatment-resistance.

The Therapeutic Odyssey: Positioning Genomic Sequencing in the Search for a Child's Best Possible Life.

Background: The desire of parents to obtain a genetic diagnosis for their child with intellectual disability and associated symptoms has long been framed as a diagnostic odyssey, an arduous and sometimes perilous journey focused on the goal of identifying a cause for the child's condition.Methods: Semi-structured interviews (N = 60) were conducted with parents of children (N = 59, aged 2-24 years) with intellectual disability and/or developmental delay (IDD) who underwent genome sequencing at a single pediatric multispecialty clinic. Interviews were conducted after parents received their child's sequencing result (positive findings, negative findings, or variants of unknown significance). Thematic analysis was performed on all interviews.Results: Parents reported that obtaining a genetic diagnosis was one important step in their overall goal of helping their child live their best life possible life. They intended to use the result as a tool to help their child by seeking the correct school placement and obtaining benefits and therapeutic services.Conclusions: For the parents of children with IDD, the search for a genetic diagnosis is best conceptualized as a part of parents' ongoing efforts to leverage various diagnoses to obtain educational and therapeutic services for their children. Cleaving parents' search for a genetic diagnosis from these broader efforts obscures the value that some parents place on a sequencing result in finding and tailoring therapies and services beyond the clinic. Interviews with parents reveal, therefore, that genomic sequencing is best understood as one important stage of an ongoing therapeutic odyssey that largely takes place outside the clinic. Findings suggest the need to expand translational research efforts to contextualize a genetic diagnosis within parents' broader efforts to obtain educational and therapeutic services outside clinical contexts.

Heterozygous variants that disturb the transcriptional repressor activity of FOXP4 cause a developmental disorder with speech/language delays and multiple congenital abnormalities.

PURPOSE: Heterozygous pathogenic variants in various FOXP genes cause specific developmental disorders. The phenotype associated with heterozygous variants in FOXP4 has not been previously described. METHODS: We assembled a cohort of eight individuals with heterozygous and mostly de novo variants in FOXP4: seven individuals with six different missense variants and one individual with a frameshift variant. We collected clinical data to delineate the phenotypic spectrum, and used in silico analyses and functional cell-based assays to assess pathogenicity of the variants. RESULTS: We collected clinical data for six individuals: five individuals with a missense variant in the forkhead box DNA-binding domain of FOXP4, and one individual with a truncating variant. Overlapping features included speech and language delays, growth abnormalities, congenital diaphragmatic hernia, cervical spine abnormalities, and ptosis. Luciferase assays showed loss-of-function effects for all these variants, and aberrant subcellular localization patterns were seen in a subset. The remaining two missense variants were located outside the functional domains of FOXP4, and showed transcriptional repressor capacities and localization patterns similar to the wild-type protein. CONCLUSION: Collectively, our findings show that heterozygous loss-of-function variants in FOXP4 are associated with an autosomal dominant neurodevelopmental disorder with speech/language delays, growth defects, and variable congenital abnormalities.

Non-coding and Loss-of-Function Coding Variants in TET2 are Associated with Multiple Neurodegenerative Diseases.

We conducted genome sequencing to search for rare variation contributing to early-onset Alzheimer's disease (EOAD) and frontotemporal dementia (FTD). Discovery analysis was conducted on 435 cases and 671 controls of European ancestry. Burden testing for rare variation associated with disease was conducted using filters based on variant rarity (less than one in 10,000 or private), computational prediction of deleteriousness (CADD) (10 or 15 thresholds), and molecular function (protein loss-of-function [LoF] only, coding alteration only, or coding plus non-coding variants in experimentally predicted regulatory regions). Replication analysis was conducted on 16,434 independent cases and 15,587 independent controls. Rare variants in TET2 were enriched in the discovery combined EOAD and FTD cohort (p = 4.6 × 10-8, genome-wide corrected p = 0.0026). Most of these variants were canonical LoF or non-coding in predicted regulatory regions. This enrichment replicated across several cohorts of Alzheimer's disease (AD) and FTD (replication only p = 0.0029). The combined analysis odds ratio was 2.3 (95% confidence interval [CI] 1.6-3.4) for AD and FTD. The odds ratio for qualifying non-coding variants considered independently from coding variants was 3.7 (95% CI 1.7-9.4). For LoF variants, the combined odds ratio (for AD, FTD, and amyotrophic lateral sclerosis, which shares clinicopathological overlap with FTD) was 3.1 (95% CI 1.9-5.2). TET2 catalyzes DNA demethylation. Given well-defined changes in DNA methylation that occur during aging, rare variation in TET2 may confer risk for neurodegeneration by altering the homeostasis of key aging-related processes. Additionally, our study emphasizes the relevance of non-coding variation in genetic studies of complex disease.

Cross-sectional imaging of the torso reveals occult injuries in asymptomatic blunt trauma patients.

Background: High morbidity and mortality rates of trauma injuries make early detection and correct diagnosis crucial for increasing patient's survival and quality of life after an injury. Improvements in technology have facilitated the rapid detection of injuries, especially with the use of computed tomography (CT). However, the increased use of CT imaging is not universally advocated for. Some advocate for the use of selective CT imaging, especially in cases where the severity of the injury is low. The purpose of this study is to review the CT indications, findings, and complications in patients with low Injury Severity Scores (ISS) to determine the utility of torso CT in this patient cohort. Methods: A retrospective review of non-intubated, adult blunt trauma patients with an initial GCS of 14 or 15 evaluated in an ACS verified level 1 trauma center from July 2012 to June 2015 was performed. Data was obtained from the hospital's trauma registry and chart review, with the following data included: age, sex, injury type, ISS, physical exam findings, all injuries recorded, injuries detected by torso CT, missed injuries, and complications. The statistical tests conducted in the analysis of the collected data were chi-squared, Fischer exact test, and ANOVA analysis. Results: There were 2306 patients included in this study, with a mean ISS of 8. For patients with a normal chest exam that had a chest CT, 15% were found to have an occult chest injury. In patients with a negative chest exam and negative chest X-ray, 35% had occult injuries detected on chest CT. For patients with a negative abdominal exam and CT abdomen and pelvis, 16% were found to have an occult injury on CT. Lastly, 25% of patients with normal chest, abdomen, and pelvis exams with chest, abdomen, and pelvis CT scans demonstrated occult injuries. Asymptomatic patients with a negative CT had a length of stay 1 day less than patients without a corresponding CT. No incidents of contrast-induced complications were recorded. Conclusions: A negative physical exam combined with a normal chest X-ray does not rule out the presence of occult injuries and the need for torso imaging. In blunt trauma patients with normal sensorium, physical exam and chest X-ray, the practice of obtaining cross-sectional imaging appears beneficial by increasing the accuracy of total injury burden and decreasing the length of stay.

Genome sequencing for early-onset or atypical dementia: high diagnostic yield and frequent observation of multiple contributory alleles.

We assessed the results of genome sequencing for early-onset dementia. Participants were selected from a memory disorders clinic. Genome sequencing was performed along with C9orf72 repeat expansion testing. All returned sequencing results were Sanger-validated. Prior clinical diagnoses included Alzheimer's disease, frontotemporal dementia, and unspecified dementia. The mean age of onset was 54 (41-76). Fifty percent of patients had a strong family history, 37.5% had some, and 12.5% had no known family history. Nine of 32 patients (28%) had a variant defined as pathogenic or likely pathogenic (P/LP) by American College of Medical Genetics and Genomics standards, including variants in APP, C9orf72, CSF1R, and MAPT Nine patients (including three with P/LP variants) harbored established risk alleles with moderate penetrance (odds ratios of ∼2-5) in ABCA7, AKAP9, GBA, PLD3, SORL1, and TREM2 All six patients harboring these moderate penetrance variants but not P/LP variants also had one or two APOE ε4 alleles. One patient had two APOE ε4 alleles with no other established contributors. In total, 16 patients (50%) harbored one or more genetic variants likely to explain symptoms. We identified variants of uncertain significance (VUSs) in ABI3, ADAM10, ARSA, GRID2IP, MME, NOTCH3, PLCD1, PSEN1, TM2D3, TNK1, TTC3, and VPS13C, also often along with other variants. In summary, genome sequencing for early-onset dementia frequently identified multiple established or possible contributory alleles. These observations add support for an oligogenic model for early-onset dementia.

The Blossoming of Technology for the Analysis of Complex Aroma Bouquets-A Review on Flavour and Odorant Multidimensional and Comprehensive Gas Chromatography Applications.

Multidimensional approaches in gas chromatography have been established as potent tools to (almost) attain fully resolved analyses. Flavours and odours are important application fields for these techniques since they include complex matrices, and are of interest for both scientific study and to consumers. This article is a review of the main research studies in the above theme, discussing the achievements and challenges that demonstrate a maturing of analytical separation technology.

SYT1-associated neurodevelopmental disorder: a case series.

Synaptotagmin 1 (SYT1) is a critical mediator of fast, synchronous, calcium-dependent neurotransmitter release and also modulates synaptic vesicle endocytosis. This paper describes 11 patients with de novo heterozygous missense mutations in SYT1. All mutations alter highly conserved residues, and cluster in two regions of the SYT1 C2B domain at positions Met303 (M303K), Asp304 (D304G), Asp366 (D366E), Ile368 (I368T) and Asn371 (N371K). Phenotypic features include infantile hypotonia, congenital ophthalmic abnormalities, childhood-onset hyperkinetic movement disorders, motor stereotypies, and developmental delay varying in severity from moderate to profound. Behavioural characteristics include sleep disturbance and episodic agitation. Absence of epileptic seizures and normal orbitofrontal head circumference are important negative features. Structural MRI is unremarkable but EEG disturbance is universal, characterized by intermittent low frequency high amplitude oscillations. The functional impact of these five de novo SYT1 mutations has been assessed by expressing rat SYT1 protein containing the equivalent human variants in wild-type mouse primary hippocampal cultures. All mutant forms of SYT1 were expressed at levels approximately equal to endogenous wild-type protein, and correctly localized to nerve terminals at rest, except for SYT1M303K, which was expressed at a lower level and failed to localize at nerve terminals. Following stimulation, SYT1I368T and SYT1N371K relocalized to nerve terminals at least as efficiently as wild-type SYT1. However, SYT1D304G and SYT1D366E failed to relocalize to nerve terminals following stimulation, indicative of impairments in endocytic retrieval and trafficking of SYT1. In addition, the presence of SYT1 variants at nerve terminals induced a slowing of exocytic rate following sustained action potential stimulation. The extent of disturbance to synaptic vesicle kinetics is mirrored by the severity of the affected individuals' phenotypes, suggesting that the efficiency of SYT1-mediated neurotransmitter release is critical to cognitive development. In summary, de novo dominant SYT1 missense mutations are associated with a recognizable neurodevelopmental syndrome, and further cases can now be diagnosed based on clinical features, electrophysiological signature and mutation characteristics. Variation in phenotype severity may reflect mutation-specific impact on the diverse physiological functions of SYT1.

Genomic sequencing identifies secondary findings in a cohort of parent study participants.

PURPOSE: Clinically relevant secondary variants were identified in parents enrolled with a child with developmental delay and intellectual disability. METHODS: Exome/genome sequencing and analysis of 789 "unaffected" parents was performed. RESULTS: Pathogenic/likely pathogenic variants were identified in 21 genes within 25 individuals (3.2%), with 11 (1.4%) participants harboring variation in a gene defined as clinically actionable by the American College of Medical Genetics and Genomics. These 25 individuals self-reported either relevant clinical diagnoses (5); relevant family history or symptoms (13); or no relevant family history, symptoms, or clinical diagnoses (7). A limited carrier screen was performed yielding 15 variants in 48 (6.1%) parents. Parents were also analyzed as mate pairs (n = 365) to identify cases in which both parents were carriers for the same recessive disease, yielding three such cases (0.8%), two of which had children with the relevant recessive disease. Four participants had two findings (one carrier and one noncarrier variant). In total, 71 of the 789 enrolled parents (9.0%) received secondary findings. CONCLUSION: We provide an overview of the rates and types of clinically relevant secondary findings, which may be useful in the design and implementation of research and clinical sequencing efforts to identify such findings.