Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells.

High-throughput sequencing of immunoglobulin (Ig) repertoires (Ig-seq) is a powerful method for quantitatively interrogating B cell receptor sequence diversity. When applied to human repertoires, Ig-seq provides insight into fundamental immunological questions, and can be implemented in diagnostic and drug discovery projects. However, a major challenge in Ig-seq is ensuring accuracy, as library preparation protocols and sequencing platforms can introduce substantial errors and bias that compromise immunological interpretation. Here, we have established an approach for performing highly accurate human Ig-seq by combining synthetic standards with a comprehensive error and bias correction pipeline. First, we designed a set of 85 synthetic antibody heavy-chain standards (in vitro transcribed RNA) to assess correction workflow fidelity. Next, we adapted a library preparation protocol that incorporates unique molecular identifiers (UIDs) for error and bias correction which, when applied to the synthetic standards, resulted in highly accurate data. Finally, we performed Ig-seq on purified human circulating B cell subsets (naïve and memory), combined with a cellular replicate sampling strategy. This strategy enabled robust and reliable estimation of key repertoire features such as clonotype diversity, germline segment, and isotype subclass usage, and somatic hypermutation. We anticipate that our standards and error and bias correction pipeline will become a valuable tool for researchers to validate and improve accuracy in human Ig-seq studies, thus leading to potentially new insights and applications in human antibody repertoire profiling.

CGG Repeat-Induced FMR1 Silencing Depends on the Expansion Size in Human iPSCs and Neurons Carrying Unmethylated Full Mutations.

In fragile X syndrome (FXS), CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However, FXS siblings have been identified with more than 200 CGGs, termed unmethylated full mutation (UFM) carriers, without gene silencing and disease symptoms. Here, we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However, a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore, we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs, and expression of large unmethylated CGG repeats has phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population.

Rosuvastatin and thapsigargin modulate γ-secretase gene expression and APP processing in a human neuroglioma model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to slow neuronal loss in several brain regions. It is characterised by the presence of cerebral senile plaques comprised of aggregated amyloid-ß peptides. Transcriptional regulation of the γ-secretase complex, which cleaves the ß-amyloid precursor protein to produce Aß-peptides, could modulate the pathological phenotype of AD patients. This study investigates whether rosuvastatin, an HMG-CoA reductase inhibitor, modulates the expression of genes involved in the function of the γ-secretase complex, in a human cellular model for Aß peptide accumulation. In particular, we analysed the effect of the statin combined with apoptotic induction. Experimental apoptosis was induced by thapsigargin treatment, a drug that depletes intracellular calcium stores via inhibition of the calcium ATPase pump. Notably, systemic calcium dysregulation accompanies almost all of the brain pathology processes observed in AD. We found differential transcriptional regulation of some γ-secretase cofactors relative to rosuvastatin treatment, in cells expressing Swedish mutant APP. Interestingly, this statin down-regulated the transcription of some enzyme cofactors, similar to treatment with thapsigargin. However, rosuvastatin neither affected the basal Aß levels nor counteracted APP processing or Aß over-production triggered by the thapsigargin. Our results provide evidence that rosuvastatin alters gene expression of the γ-secretase complex without affecting enzyme activity.