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Human Brain Lipidomics: Investigation of Formalin Fixed Brains.

ABSTRACT: Human brain lipidomics have elucidated structural lipids and lipid signal transduction pathways in neurologic diseases. Such studies have traditionally sourced tissue exclusively from brain bank biorepositories, however, limited inventories signal that these facilities may not be able to keep pace with this growing research domain. Formalin fixed, whole body donors willed to academic institutions offer a potential supplemental tissue source, the lipid profiles of which have yet to be described. To determine the potential of these subjects in lipid analysis, the lipid levels of fresh and fixed frontal cortical gray matter of human donors were compared using high resolution electrospray ionization mass spectrometry. Results revealed commensurate levels of specific triacylglycerols, diacylglycerols, hexosyl ceramides, and hydroxy hexosyl ceramides. Baseline levels of these lipid families in human fixed tissue were identified via a broader survey study covering six brain regions: cerebellar gray matter, superior cerebellar peduncle, gray and subcortical white matter of the precentral gyrus, periventricular white matter, and internal capsule. Whole body donors may therefore serve as supplemental tissue sources for lipid analysis in a variety of clinical contexts, including Parkinson's disease, Alzheimer's disease, Lewy body dementia, multiple sclerosis, and Gaucher's disease.

SUBMITTER: Beger AW

PROVIDER: S-EPMC9245516 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The advent of Next-Generation sequencing technologies, which significantly increases the throughput and reduces the cost of large-scale sequencing efforts, provides an unprecedented opportunity for discovery of novel gene mutations in human cancers. However, it remains a challenge to apply Next-Generation technologies to DNA extracted from formalin-fixed paraffin-embedded cancer specimens. We describe here the successful development of a custom DNA capture method using Next-Generation for detection of 140 driver genes in five formalin-fixed paraffin-embedded human colon cancer samples using an improved extraction process to produce high-quality DNA. Isolated DNA was enriched for targeted exons and sequenced using the Illumina Next-Generation platform. An analytical pipeline using 3 software platforms to define single-nucleotide variants was used to evaluate the data output. Approximately 250 × average coverage was obtained with >96% of target bases having at least 30 sequence reads. Results were then compared with previously performed high-throughput Sanger sequencing. Using an algorithm of needing a positive call from all three callers to give a positive result, 98% of the verified Sanger sequencing somatic driver gene mutations were identified by our method with a specificity of 90%. In all, 13 insertions and deletions identified by Next-Generation were confirmed by Sanger sequencing. We also applied this technology to two components of a biphasic colon cancer, which had strikingly differing histology. Remarkably, no new driver gene mutation accumulation was identified in the more undifferentiated component. Applying this method to profiling of formalin-fixed paraffin-embedded colon cancer tissue samples yields equivalent sensitivity and specificity for mutation detection as Sanger sequencing of matched cell lines derived from these cancers. This method directly enables high-throughput comprehensive mutational profiling of colon cancer samples, and is easily extendable to enable targeted sequencing from formalin-fixed paraffin-embedded material for other tumor types.

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Human Brain Lipidomics: Investigation of Formalin Fixed Brains.

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