Rapid genome sequencing identifies a novel de novo SNAP25 variant for neonatal congenital myasthenic syndrome.

Congenital myasthenic syndrome (CMS) is a group of 32 disorders involving genetic dysfunction at the neuromuscular junction resulting in skeletal muscle weakness that worsens with physical activity. Precise diagnosis and molecular subtype identification are critical for treatment as medication for one subtype may exacerbate disease in another (Engel et al., Lancet Neurol 14: 420 [2015]; Finsterer, Orphanet J Rare Dis 14: 57 [2019]; Prior and Ghosh, J Child Neurol 36: 610 [2021]). The SNAP25-related CMS subtype (congenital myasthenic syndrome 18, CMS18; MIM #616330) is a rare disorder characterized by muscle fatigability, delayed psychomotor development, and ataxia. Herein, we performed rapid whole-genome sequencing (rWGS) on a critically ill newborn leading to the discovery of an unreported pathogenic de novo SNAP25 c.529C > T; p.Gln177Ter variant. In this report, we present a novel case of CMS18 with complex neonatal consequence. This discovery offers unique insight into the extent of phenotypic severity in CMS18, expands the reported SNAP25 variant phenotype, and paves a foundation for personalized management for CMS18.

Clinical Trial-Ready Patient Cohorts for Multiple System Atrophy: Coupling Biospecimen and iPSC Banking to Longitudinal Deep-Phenotyping.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease of unknown etiology characterized by widespread aggregation of the protein alpha-synuclein in neurons and glia. Its orphan status, biological relationship to Parkinson's disease (PD), and rapid progression have sparked interest in drug development. One significant obstacle to therapeutics is disease heterogeneity. Here, we share our process of developing a clinical trial-ready cohort of MSA patients (69 patients in 2 years) within an outpatient clinical setting, and recruiting 20 of these patients into a longitudinal "n-of-few" clinical trial paradigm. First, we deeply phenotype our patients with clinical scales (UMSARS, BARS, MoCA, NMSS, and UPSIT) and tests designed to establish early differential diagnosis (including volumetric MRI, FDG-PET, MIBG scan, polysomnography, genetic testing, autonomic function tests, skin biopsy) or disease activity (PBR06-TSPO). Second, we longitudinally collect biospecimens (blood, CSF, stool) and clinical, biometric, and imaging data to generate antecedent disease-progression scores. Third, in our Mass General Brigham SCiN study (stem cells in neurodegeneration), we generate induced pluripotent stem cell (iPSC) models from our patients, matched to biospecimens, including postmortem brain. We present 38 iPSC lines derived from MSA patients and relevant disease controls (spinocerebellar ataxia and PD, including alpha-synuclein triplication cases), 22 matched to whole-genome sequenced postmortem brain. iPSC models may facilitate matching patients to appropriate therapies, particularly in heterogeneous diseases for which patient-specific biology may elude animal models. We anticipate that deeply phenotyped and genotyped patient cohorts matched to cellular models will increase the likelihood of success in clinical trials for MSA.

Comprehensive variant calling from whole-genome sequencing identifies a complex inversion that disrupts ZFPM2 in familial congenital diaphragmatic hernia.

BACKGROUND: Genetic disorders contribute to significant morbidity and mortality in critically ill newborns. Despite advances in genome sequencing technologies, a majority of neonatal cases remain unsolved. Complex structural variants (SVs) often elude conventional genome sequencing variant calling pipelines and will explain a portion of these unsolved cases. METHODS: As part of the Utah NeoSeq project, we used a research-based, rapid whole-genome sequencing (WGS) protocol to investigate the genomic etiology for a newborn with a left-sided congenital diaphragmatic hernia (CDH) and cardiac malformations, whose mother also had a history of CDH and atrial septal defect. RESULTS: Using both a novel, alignment-free and traditional alignment-based variant callers, we identified a maternally inherited complex SV on chromosome 8, consisting of an inversion flanked by deletions. This complex inversion, further confirmed using orthogonal molecular techniques, disrupts the ZFPM2 gene, which is associated with both CDH and various congenital heart defects. CONCLUSIONS: Our results demonstrate that complex structural events, which often are unidentifiable or not reported by clinically validated testing procedures, can be discovered and accurately characterized with conventional, short-read sequencing and underscore the utility of WGS as a first-line diagnostic tool.

Evaluation of a serum ELISA for detection of bovine leukemia viral antibodies in milk samples.

Bovine leukemia virus (BLV) infection has worldwide distribution in both dairy and beef herds. Our study was initiated in order to encourage control of BLV infection by using milk samples, in lieu of serum samples, to readily test lactating animals prior to dry-off and calving. Two Holstein dairy herds (A and B), with known status of BLV infection as determined by serology, were sampled by the collection of serum and fresh milk samples. Selected samples were tested using a USDA-licensed BLV antibody ELISA kit (Bovine leukemia virus antibody test kit; VMRD, Pullman, WA) for serum. Forty-one lactating cows from each herd were sampled. Herd A was confirmed to have endemic BLV infection; herd B was confirmed to be free of BLV infection. The milk ELISA results demonstrated 100% identification of positive and negative animals compared with the serum results. The correlation of the ELISA values between serum and milk samples was 97%, which supports the use of this BLV ELISA on milk samples.

Identification of alkylpyrazines by gas chromatography mass spectrometry (GC-MS).

Pyrazines are an important group of natural products widely used as food additives and fragrants. Gas chromatography-mass spectrometry (GCMS) is the most widely applied analytical technique for characterization of alkylpyrazines. However, mass spectra of many positional isomers of alkylpyrazines are very similar. Consequently, an unambiguous identification of each positional isomer by spectral interpretation or database search protocols is practically unfeasible. In fact, there are many misidentifications in literature. To identify alkylpyrazines, chemists often resort to gas chromatographic retention indices (RIs). Although there are many compilations of retention indices of alkylpyrazines, these databases are often incomplete and the values reported are sometimes inconsistent. Herein, we present retention indices of fifty-six alkylpyrazines recorded on DB-1, ZB-5MS, DB-624, and ZB-WAXplus stationary phases, and compare them with those available in the NIST-2017 MS-RI database. Furthermore, we demonstrate how RI values can be used, together with mass spectral interpretations, to identify certain alkylpyrazines unambiguously.

Alkyl-Dimethylpyrazines in Mandibular Gland Secretions of Four Odontomachus Ant Species (Formicidae: Ponerinae).

Ponerine ants are known to contain mixtures of pyrazines in their mandibular glands. We analyzed the mandibular gland contents of four ponerine species (Odontomachus chelifer, O. erythrocephalus, O. ruginodis, and O. bauri) by gas chromatography coupled with mass spectrometry, and found that each species contains specific mixtures of trisubstituted alkylpyrazines among other volatiles. Attempts to identify alkylpyrazines solely by mass spectral interpretation is unrealistic because spectra of positional isomers are indistinguishable. To avoid misidentifications, we synthesized a large number of reference compounds and compared their mass spectral and gas chromatographic properties with those present in the Odontomachus species under investigation. Most of the compounds identified were 2-alkyl-3,5-dimethylpyrazines. Interestingly, when the third substituent was an isopentyl group, the two methyl groups were found to be located at the 2 and 5 ring positions. Using our data, we recognized several misidentifications in previous publications.

Cytochrome P450 diversification and hostplant utilization patterns in specialist and generalist moths: Birth, death and adaptation.

Across insect genomes, the size of the cytochrome P450 monooxygenase (CYP) gene superfamily varies widely. CYPome size variation has been attributed to reciprocal adaptive radiations in insect detoxification genes in response to plant biosynthetic gene radiations driven by co-evolution between herbivores and their chemically defended hostplants. Alternatively, variation in CYPome size may be due to random "birth-and-death" processes, whereby exponential increase via gene duplications is limited by random decay via gene death or transition via divergence. We examined CYPome diversification in the genomes of seven Lepidoptera species varying in host breadth from monophagous (Bombyx mori) to highly polyphagous (Amyelois transitella). CYPome size largely reflects the size of Clan 3, the clan associated with xenobiotic detoxification, and to some extent phylogenetic age. Consistently across genomes, families CYP6, CYP9 and CYP321 are most diverse and CYP6AB, CYP6AE, CYP6B, CYP9A and CYP9G are most diverse among subfamilies. Higher gene number in subfamilies is due to duplications occurring primarily after speciation and specialization ("P450 blooms"), and the genes are arranged in clusters, indicative of active duplicating loci. In the parsnip webworm, Depressaria pastinacella, gene expression levels in large subfamilies are high relative to smaller subfamilies. Functional and phylogenetic data suggest a correlation between highly dynamic loci (reflective of extensive gene duplication, functionalization and in some cases loss) and the ability of enzymes encoded by these genes to metabolize hostplant defences, consistent with an adaptive, nonrandom process driven by ecological interactions.