Glutamine Supplementation as a Novel Metabolic Therapeutic Strategy for LIG3-Dependent Chronic Intestinal Pseudo-obstruction.

BACKGROUND & AIMS: We recently identified a recessive syndrome due to DNA ligase 3 (LIG3) mutations in patients with chronic intestinal pseudo-obstruction, leukoencephalopathy, and neurogenic bladder. LIG3 mutations affect mitochondrial DNA maintenance, leading to defective energy production. We aimed at identifying altered molecular pathways and developing possible targeted treatments to revert/ameliorate the cellular energy impairment. METHODS: Whole transcriptome analysis was performed on patient-derived fibroblasts total RNA and controls. Mitochondrial function, mitophagy, and l-glutamine supplementation effects were analyzed by live cell analysis, immunostaining, and Western blot. Patients were treated with Dipeptiven (Fresenius-Kabi) according to standard protocols. Patients' symptoms were analyzed by the Gastrointestinal Symptom Rating Scale questionnaire. RESULTS: We identified deregulated transcripts in mutant fibroblasts vs controls, including overexpression of genes involved in extracellular matrix development and remodeling and mitochondrial functions. Gut biopsy specimens of LIG3-mutant patients documented collagen and elastic fiber accumulation. Mutant fibroblasts exhibited impaired mitochondrial mitophagy indicative of dysfunctional turnover and altered Ca2+ homeostasis. Supplementation with l-glutamine (6 mmol/L), previously shown to increase mitochondrial DNA-defective cell survival, improved growth rate and adenosine 5'-triphosphate production in LIG3-mutant fibroblasts. These data led us to provide parenterally a dipeptide containing l-glutamine to 3 siblings carrying biallelic LIG3 mutations. Compared with baseline, gastrointestinal and extra-gastrointestinal symptoms significantly improved after 8 months of treatment. CONCLUSIONS: LIG3 deficiency leads to mitochondrial dysfunction. High levels l-glutamine supplementation were beneficial in LIG3-mutant cells and improved symptom severity without noticeable adverse effects. Our results provide a proof of concept to design ad hoc clinical trials with l-glutamine in LIG3-mutant patients.

Omics Technologies Improving Breast Cancer Research and Diagnostics.

Breast cancer (BC) has yielded approximately 2.26 million new cases and has caused nearly 685,000 deaths worldwide in the last two years, making it the most common diagnosed cancer type in the world. BC is an intricate ecosystem formed by both the tumor microenvironment and malignant cells, and its heterogeneity impacts the response to treatment. Biomedical research has entered the era of massive omics data thanks to the high-throughput sequencing revolution, quick progress and widespread adoption. These technologies-liquid biopsy, transcriptomics, epigenomics, proteomics, metabolomics, pharmaco-omics and artificial intelligence imaging-could help researchers and clinicians to better understand the formation and evolution of BC. This review focuses on the findings of recent multi-omics-based research that has been applied to BC research, with an introduction to every omics technique and their applications for the different BC phenotypes, biomarkers, target therapies, diagnosis, treatment and prognosis, to provide a comprehensive overview of the possibilities of BC research.

Mutant SPART causes defects in mitochondrial protein import and bioenergetics reversed by Coenzyme Q.

Pathogenic variants in SPART cause Troyer syndrome, characterized by lower extremity spasticity and weakness, short stature and cognitive impairment, and a severe mitochondrial impairment. Herein, we report the identification of a role of Spartin in nuclear-encoded mitochondrial proteins. SPART biallelic missense variants were detected in a 5-year-old boy with short stature, developmental delay and muscle weakness with impaired walking distance. Patient-derived fibroblasts showed an altered mitochondrial network, decreased mitochondrial respiration, increased mitochondrial reactive oxygen species and altered Ca2+ versus control cells. We investigated the mitochondrial import of nuclear-encoded proteins in these fibroblasts and in another cell model carrying a SPART loss-of-function mutation. In both cell models the mitochondrial import was impaired, leading to a significant decrease in different proteins, including two key enzymes involved in CoQ10 (CoQ) synthesis, COQ7 and COQ9, with a severe reduction in CoQ content, versus control cells. CoQ supplementation restored cellular ATP levels to the same extent shown by the re-expression of wild-type SPART, suggesting CoQ treatment as a promising therapeutic approach for patients carrying mutations in SPART.

Calcium and Reactive Oxygen Species Signaling Interplays in Cardiac Physiology and Pathologies.

Mitochondria are key players in energy production, critical activity for the smooth functioning of energy-demanding organs such as the muscles, brain, and heart. Therefore, dysregulation or alterations in mitochondrial bioenergetics primarily perturb these organs. Within the cell, mitochondria are the major site of reactive oxygen species (ROS) production through the activity of different enzymes since it is one of the organelles with the major availability of oxygen. ROS can act as signaling molecules in a number of different pathways by modulating calcium (Ca2+) signaling. Interactions among ROS and calcium signaling can be considered bidirectional, with ROS regulating cellular Ca2+ signaling, whereas Ca2+ signaling is essential for ROS production. In particular, we will discuss how alterations in the crosstalk between ROS and Ca2+ can lead to mitochondrial bioenergetics dysfunctions and the consequent damage to tissues at high energy demand, such as the heart. Changes in Ca2+ can induce mitochondrial alterations associated with reduced ATP production and increased production of ROS. These changes in Ca2+ levels and ROS generation completely paralyze cardiac contractility. Thus, ROS can hinder the excitation-contraction coupling, inducing arrhythmias, hypertrophy, apoptosis, or necrosis of cardiac cells. These interplays in the cardiovascular system are the focus of this review.

Novel understanding on genetic mechanisms of enteric neuropathies leading to severe gut dysmotility.

The enteric nervous system (ENS) is the third division of the autonomic autonomic nervous system and the largest collection of neurons outside the central nervous system (CNS). The ENS has been referred to as "the brain in the gut" or "the second brain of the human body" because of its highly integrated neural circuits controlling a vast repertoire of gut functions, including absorption/secretion, splanchnic blood vessels, some immunological aspects, intestinal epithelial barrier, and gastrointestinal (GI) motility. The latter function is the result of the ENS fine-tuning over smooth musculature, along with the contribution of other key cells, such as enteric glia (astrocyte like cells supporting and contributing to neuronal activity), interstitial cells of Cajal (the pacemaker cells of the GI tract involved in neuromuscular transmission), and enteroendocrine cells (releasing bioactive substances, which affect gut physiology). Any noxa insult perturbing the ENS complexity may determine a neuropathy with variable degree of neuro-muscular dysfunction. In this review, we aim to cover the most recent update on genetic mechanisms leading to enteric neuropathies ranging from Hirschsprung's disease (characterized by lack of any enteric neurons in the gut wall) up to more generalized form of dysmotility such as chronic intestinal pseudo-obstruction (CIPO) with a significant reduction of enteric neurons. In this line, we will discuss the role of the RAD21 mutation, which we have demonstrated in a family whose affected members exhibited severe gut dysmotility. Other genes contributing to gut motility abnormalities will also be presented. In conclusion, the knowledge on the molecular mechanisms involved in enteric neuropathy may unveil strategies to better manage patients with neurogenic gut dysmotility and pave the way to targeted therapies.

Genetics of Familial Non-Medullary Thyroid Carcinoma (FNMTC).

Non-medullary thyroid carcinoma (NMTC) is the most frequent endocrine tumor and originates from the follicular epithelial cells of the thyroid. Familial NMTC (FNMTC) has been defined in pedigrees where two or more first-degree relatives of the patient present the disease in absence of other predisposing environmental factors. Compared to sporadic cases, FNMTCs are often multifocal, recurring more frequently and showing an early age at onset with a worse outcome. FNMTC cases show a high degree of genetic heterogeneity, thus impairing the identification of the underlying molecular causes. Over the last two decades, many efforts in identifying the susceptibility genes in large pedigrees were carried out using linkage-based approaches and genome-wide association studies, leading to the identification of susceptibility loci and variants associated with NMTC risk. The introduction of next-generation sequencing technologies has greatly contributed to the elucidation of FNMTC predisposition, leading to the identification of novel candidate variants, shortening the time and cost of gene tests. In this review we report the most significant genes identified for the FNMTC predisposition. Integrating these new molecular findings in the clinical data of patients is fundamental for an early detection and the development of tailored therapies, in order to optimize patient management.

Biallelic variants in LIG3 cause a novel mitochondrial neurogastrointestinal encephalomyopathy.

Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities.

Whole Genome Sequencing Prioritizes CHEK2, EWSR1, and TIAM1 as Possible Predisposition Genes for Familial Non-Medullary Thyroid Cancer.

Familial inheritance in non-medullary thyroid cancer (NMTC) is an area that has yet to be adequately explored. Despite evidence suggesting strong familial clustering of non-syndromic NMTC, known variants still account for a very small percentage of the genetic burden. In a recent whole genome sequencing (WGS) study of five families with several NMTCs, we shortlisted promising variants with the help of our in-house developed Familial Cancer Variant Prioritization Pipeline (FCVPPv2). Here, we report potentially disease-causing variants in checkpoint kinase 2 (CHEK2), Ewing sarcoma breakpoint region 1 (EWSR1) and T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) in one family. Performing WGS on three cases, one probable case and one healthy individual in a family with familial NMTC left us with 112254 variants with a minor allele frequency of less than 0.1%, which was reduced by pedigree-based filtering to 6368. Application of the pipeline led to the prioritization of seven coding and nine non-coding variants from this family. The variant identified in CHEK2, a known tumor suppressor gene involved in DNA damage-induced DNA repair, cell cycle arrest, and apoptosis, has been previously identified as a germline variant in breast and prostate cancer and has been functionally validated by Roeb et al. in a yeast-based assay to have an intermediate effect on protein function. We thus hypothesized that this family may harbor additional disease-causing variants in other functionally related genes. We evaluated two further variants in EWSR1 and TIAM1 with promising in silico results and reported interaction in the DNA-damage repair pathway. Hence, we propose a polygenic mode of inheritance in this family. As familial NMTC is considered to be more aggressive than its sporadic counterpart, it is important to identify such susceptibility genes and their associated pathways. In this way, the advancement of personalized medicine in NMTC patients can be fostered. We also wish to reopen the discussion on monogenic vs polygenic inheritance in NMTC and instigate further development in this area of research.

BRAF Exon 15 Mutations in Papillary Carcinoma and Adjacent Thyroid Parenchyma: A Search for the Early Molecular Events Associated with Tumor Development.

BRAF exon 15 mutations are the most common molecular alterations found in papillary thyroid carcinoma (PTC). To date, there is no information regarding BRAF alterations in the thyroid parenchyma surrounding the tumor. To explore the early events associated with the development of PTC, we used massively parallel sequencing to investigate BRAF exon 15 in 30 PTCs and in 100 samples from the thyroid parenchyma surrounding the tumor. BRAF p.V600E was identified in 19/30 PTCs (63.3%). BRAF p.V600E mutations were identified in the tissue adjacent the PTC only in samples containing psammoma bodies. The other samples were either BRAF wild type (WT) or carried BRAF non p.V600E mutations. Specifically, BRAF p.G593D, -p.A598T, -p.V600M, -p.R603Q, -p.S607F, and -p.S607P were identified in 4 of 36 (11.1%) samples with follicular cell atypia, in 2 of 16 (12.5%) with follicular cell hyperplasia, and in 1 of 33 (3.0%) histologically normal samples-only in tissue surrounding BRAF p.V600E mutated PTCs. These mutations are predicted to affect protein function in silico but, in vitro, have kinase activity and BRAF phosphorylation levels similar to BRAF WT. No BRAF exon 15 mutations were identified in samples adjacent to PTCs that were BRAF WT. A mutagenic process affecting BRAF exon 15 occurs in a subset of thyroid glands that develop BRAF p.V600E mutated PTCs.

A novel mutation in SPART gene causes a severe neurodevelopmental delay due to mitochondrial dysfunction with complex I impairments and altered pyruvate metabolism.

Loss-of-function mutations in the SPART gene cause Troyer syndrome, a recessive form of spastic paraplegia resulting in muscle weakness, short stature, and cognitive defects. SPART encodes for Spartin, a protein linked to endosomal trafficking and mitochondrial membrane potential maintenance. Here, we identified with whole exome sequencing (WES) a novel frameshift mutation in the SPART gene in 2 brothers presenting an uncharacterized developmental delay and short stature. Functional characterization in an SH-SY5Y cell model shows that this mutation is associated with increased neurite outgrowth. These cells also show a marked decrease in mitochondrial complex I (NADH dehydrogenase) activity, coupled to decreased ATP synthesis and defective mitochondrial membrane potential. The cells also presented an increase in reactive oxygen species, extracellular pyruvate, and NADH levels, consistent with impaired complex I activity. In concordance with a severe mitochondrial failure, Spartin loss also led to an altered intracellular Ca2+ homeostasis that was restored after transient expression of wild-type Spartin. Our data provide for the first time a thorough assessment of Spartin loss effects, including impaired complex I activity coupled to increased extracellular pyruvate. In summary, through a WES study we assign a diagnosis of Troyer syndrome to otherwise undiagnosed patients, and by functional characterization we show that the novel mutation in SPART leads to a profound bioenergetic imbalance.-Diquigiovanni, C., Bergamini, C., Diaz, R., Liparulo, I., Bianco, F., Masin, L., Baldassarro, V. A., Rizzardi, N., Tranchina, A., Buscherini, F., Wischmeijer, A., Pippucci, T., Scarano, E., Cordelli, D. M., Fato, R., Seri, M., Paracchini, S., Bonora, E. A novel mutation in SPART gene causes a severe neurodevelopmental delay due to mitochondrial dysfunction with complex I impairments and altered pyruvate metabolism.