Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin.

Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non-palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC6.

Differences in the molecular organisation of tumours along the colon are linked to interactions within the tumour ecosystem.

Tumours exhibit significant heterogeneity in their molecular profiles across patients, largely influenced by the tissue of origin, where certain driver gene mutations are predominantly associated with specific cancer types. Here, we unveil an additional layer of complexity: some cancer types display anatomic location-specific mutation profiles akin to tissue-specificity. To better understand this phenomenon, we concentrate on colon cancer. While prior studies have noted changes of the frequency of molecular alterations along the colon, the underlying reasons and whether those changes occur rather gradual or are distinct between the left and right colon, remain unclear. Developing and leveraging stringent statistical models on molecular data from 522 colorectal tumours from The Cancer Genome Atlas, we reveal disparities in molecular properties between the left and right colon affecting many genes. Interestingly, alterations in genes responsive to environmental cues and properties of the tumour ecosystem, including metabolites which we quantify in a cohort of 27 colorectal cancer patients, exhibit continuous trends along the colon. Employing network methodologies, we uncover close interactions between metabolites and genes, including drivers of colon cancer, showing continuous abundance or alteration profiles. This underscores how anatomic biases in the composition and interactions within the tumour ecosystem help explaining gradients of carcinogenesis along the colon.

Location and condition based reconstruction of colon cancer microbiome from human RNA sequencing data.

BACKGROUND: The association between microbes and cancer has been reported repeatedly; however, it is not clear if molecular tumour properties are connected to specific microbial colonisation patterns. This is due mainly to the current technical and analytical strategy limitations to characterise tumour-associated bacteria. METHODS: Here, we propose an approach to detect bacterial signals in human RNA sequencing data and associate them with the clinical and molecular properties of the tumours. The method was tested on public datasets from The Cancer Genome Atlas, and its accuracy was assessed on a new cohort of colorectal cancer patients. RESULTS: Our analysis shows that intratumoural microbiome composition is correlated with survival, anatomic location, microsatellite instability, consensus molecular subtype and immune cell infiltration in colon tumours. In particular, we find Faecalibacterium prausnitzii, Coprococcus comes, Bacteroides spp., Fusobacterium spp. and Clostridium spp. to be strongly associated with tumour properties. CONCLUSIONS: We implemented an approach to concurrently analyse clinical and molecular properties of the tumour as well as the composition of the associated microbiome. Our results may improve patient stratification and pave the path for mechanistic studies on microbiota-tumour crosstalk.

A fast, reliable and easy method to detect within-species DNA contamination.

BACKGROUND AND AIM: Next generation sequencing (ngs) is becoming the standard for clinical diagnosis. Different steps of NGS, such as DNA extraction, fragmentation, library preparation and amplification, require handling of samples, making the process susceptible to contamination. In diagnostic environments, sample contamination with DNA from the same species can lead to errors in diagnosis. Here we propose a simple method to detect within-sample contamination based on analysis of the heterozygous single nucleotide polymorphisms allele ratio (AR). METHODS: A dataset of 38000 heterozygous snps was used to estimate the ar distribution. The parameters of the reference distribution were then used to estimate the contamination probability of a sample. Validation was performed using 12 samples contaminated to different levels. RESULTS: Results show that the method easily detects contamination of 20% or more. The method has a limit of detection of about 10%, threshold below which the number of false positives increases significantly. CONCLUSIONS: The method can be applied to any type of ngs analysis and is useful for quality control. Being fast and easy to implement makes it ideal for inclusion in NGS pipelines to improve quality control of data and make results more robust.

appMAGI: A complete laboratory information management system for clinical diagnostics.

BACKGROUND: The increasing demand for genetic testing for clinical diagnosis and research challenges genetic laboratory capacity to track an increasing number of patient samples through all steps of analysis, from sample collection to report generation. This task is usually performed with the help of a laboratory information management system (LIMS), software that makes it possible to collect, store and retrieve laboratory and sample data. To date there are no open-source options that can manage the entire analytical flow of a genetic laboratory. appMAGI seeks to include all the management aspects of a clinical diagnostic laboratory, making it simpler to process many samples while maintaining the high security and quality standards required in clinical diagnostic practice. METHODS: appMAGI is written in python using Django. It is a web application that does not require local installation, making development, updates and maintenance a much easier task. appMAGI runs on the Ubuntu server and uses SQLite as engine database. RESULTS: In this work we describe an innovative LIMS called appMAGI designed to support all aspects of a clinical diagnostic laboratory. appMAGI can track samples throughout the diagnostic workflow and NGS analysis by virtue of a customizable bioinformatics pipeline. It can handle sample non-compliance, manage laboratory stocks, help generate reports and provide insights into sample data by means of special tools. CONCLUSIONS: appMAGI is a LIMS endowed with all the features required to manage thousands of samples. Allowing efficient management of patient samples from sample collection to diagnostic report generation.

CRB1-Related Cystic Maculopathy in Twins Conceived Through Heterologous Fertilization With Variant-Carrying Oocytes.

Cystic maculopathy has been associated with genetic disorders such as retinitis pigmentosa, X-linked retinoschisis, cone dystrophy, and foveal retinoschisis. Familial foveal retinoschisis was recently described as a rare disease caused by CRB1 variants. The authors report the phenotype-genotype pattern of a pair of dizygotic twins with early-onset cystic maculopathy due to CRB1 pathogenic variants. The twins were conceived by heterologous fertilization with variant-carrying oocytes. The probands were monitored for a period of 4 years. Next generation sequencing of a panel of genes responsible for retinal dystrophies was performed. Both children carried three pathogenic variants in CRB1: a novel heterozygous truncating variant p.(Val855*) inherited from the father and two known heterozygous missense variants, p.[(Phe144Val; Thr745Met)], inherited from the oocyte donor. The findings confirm that CRB1 variants can be responsible for foveal retinoschisis with variable clinical expressivity ranging from schitic macular alteration to early-onset forms of cystic maculopathy. The authors highlight the importance of exome analysis of gamete donors to assess the likelihood of recessively inherited disorders by means of a prediction algorithm able to combine parent and donor exome data. [J Pediatr Ophthalmol Strabismus. 2020;57:e19-e24.].

Monogenic hypertension.

Hypertension is a significant public health problem. Thirty percent of cases are caused by a single genetic mutation. Hypertension is the predominant and usually the only manifestation in monogenic hypertension Monogenic hypertension may involve mineralcorticoid-dependent or -independent increase in Na+ transport. Diagnosis is based on routine physical examination, blood pressure measurement and laboratory analysis of renin, aldosterone, cortisol and potassium. Genetic testing is useful for confirming diagnosis and for differential diagnosis. Monogenic hypertension has autosomal dominant or autosomal recessive inheritance.

Identification and dynamics of the human ZDHHC16-ZDHHC6 palmitoylation cascade.

S-Palmitoylation is the only reversible post-translational lipid modification. Knowledge about the DHHC palmitoyltransferase family is still limited. Here we show that human ZDHHC6, which modifies key proteins of the endoplasmic reticulum, is controlled by an upstream palmitoyltransferase, ZDHHC16, revealing the first palmitoylation cascade. The combination of site specific mutagenesis of the three ZDHHC6 palmitoylation sites, experimental determination of kinetic parameters and data-driven mathematical modelling allowed us to obtain detailed information on the eight differentially palmitoylated ZDHHC6 species. We found that species rapidly interconvert through the action of ZDHHC16 and the Acyl Protein Thioesterase APT2, that each species varies in terms of turnover rate and activity, altogether allowing the cell to robustly tune its ZDHHC6 activity.