A novel homozygous mutation in the TRDN gene causes a severe form of pediatric malignant ventricular arrhythmia.

BACKGROUND: Triadin is a protein expressed in cardiac and skeletal muscle that has an essential role in the structure and functional regulation of calcium release units and excitation-contraction coupling. Mutations in the triadin gene (TRDN) have been described in different forms of human arrhythmia syndromes with early onset and severe arrhythmogenic phenotype, including triadin knockout syndrome. OBJECTIVE: The purpose of this study was to characterize the pathogenetic mechanism underlying a case of severe pediatric malignant arrhythmia associated with a defect in the TRDN gene. METHODS: We used a trio whole exome sequencing approach to identify the genetic defect in a 2-year-old boy who had been resuscitated from sudden cardiac arrest and had frequent episodes of ventricular fibrillation and a family history positive for sudden death. We then performed in vitro functional analysis to investigate possible pathogenic mechanisms underlying this severe phenotype. RESULTS: We identified a novel homozygous missense variant (p.L56P) in the TRDN gene in the proband that was inherited from the heterozygous unaffected parents. Expression of a green fluorescent protein (GFP)-tagged mutant human cardiac triadin isoform (TRISK32-L56P-GFP) in heterologous systems revealed that the mutation alters protein dynamics. Furthermore, when co-expressed with the type 2 ryanodine receptor, caffeine-induced calcium release from TRISK32-L56P-GFP was relatively lower compared to that observed with the wild-type construct. CONCLUSION: The results of this study allowed us to hypothesize a pathogenic mechanism underlying this rare arrhythmogenic recessive form, suggesting that the mutant protein potentially can trigger arrhythmias by altering calcium homeostasis.

Rare Pathogenic Variants Predispose to Hepatocellular Carcinoma in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a rising cause of hepatocellular carcinoma (HCC). We examined whether inherited pathogenic variants in candidate genes (n = 181) were enriched in patients with NAFLD-HCC. To this end, we resequenced peripheral blood DNA of 142 NAFLD-HCC, 59 NAFLD with advanced fibrosis, and 50 controls, and considered 404 healthy individuals from 1000 G. Pathogenic variants were defined according to ClinVar, likely pathogenic as rare variants predicted to alter protein activity. In NAFLD-HCC patients, we detected an enrichment in pathogenic (p = 0.024), and likely pathogenic variants (p = 1.9*10-6), particularly in APOB (p = 0.047). APOB variants were associated with lower circulating triglycerides and higher HDL cholesterol (p < 0.01). A genetic risk score predicted NAFLD-HCC (OR 4.96, 3.29-7.55; p = 5.1*10-16), outperforming the diagnostic accuracy of common genetic risk variants, and of clinical risk factors (p < 0.05). In conclusion, rare pathogenic variants in genes involved in liver disease and cancer predisposition are associated with NAFLD-HCC development.

A compendium of long non-coding RNAs transcriptional fingerprint in multiple myeloma.

Multiple myeloma (MM) is a clonal proliferation of bone marrow plasma cells characterized by highly heterogeneous genetic background and clinical course, whose pathogenesis remains largely unknown. Long ncRNAs (lncRNAs) are a large class of non-protein-coding RNA, involved in many physiological cellular and genomic processes as well as in carcinogenesis and tumor evolution. Although still in its infancy, the role of lncRNAs in MM is progressively expanding. Besides studies on selected candidates, lncRNAs expression at genome-wide transcriptome level is confined to microarray technologies, thus investigating a limited collection of transcripts. In the present study investigating a cohort of 30 MM patients, a deep RNA-sequencing analysis overwhelmed previous array studies and allowed the most accurate definition of lncRNA transcripts structure and expression, ultimately providing a comprehensive catalogue of lncRNAs specifically associated with the main MM molecular subgroups and genetic alterations. Despite the small number of analyzed samples, the high accuracy of RNA-sequencing approach for complex transcriptome processing led to the identification of 391 deregulated lncRNAs, 67% of which were also detectable and validated by whole-transcript microarrays. In addition, we identified a list of lncRNAs, with potential relevance in MM, co-expressed and in close proximity to genes that might undergo a cis-regulatory relationship.

Telomerase reverse transcriptase germline mutations and hepatocellular carcinoma in patients with nonalcoholic fatty liver disease.

In an increasing proportion of cases, hepatocellular carcinoma (HCC) develops in patients with nonalcoholic fatty liver disease (NAFLD). Mutations in telomerase reverse transcriptase (hTERT) are associated with familial liver diseases. The aim of this study was to examine telomere length and germline hTERT mutations as associated with NAFLD-HCC. In 40 patients with NAFLD-HCC, 45 with NAFLD-cirrhosis and 64 healthy controls, peripheral blood telomere length was evaluated by qRT-PCR and hTERT coding regions and intron-exon boundaries sequenced. We further analyzed 78 patients affected by primary liver cancer (NAFLD-PLC, 76 with HCC). Enrichment of rare coding mutations (allelic frequency <0.001) was evaluated by Burden test. Functional consequences were estimated in silico and by over-expressing protein variants in HEK-293 cells. We found that telomere length was reduced in individuals with NAFLD-HCC versus those with cirrhosis (P = 0.048) and healthy controls (P = 0.0006), independently of age and sex. We detected an enrichment of hTERT mutations in NAFLD-HCC, that was confirmed when we further considered a larger cohort of NAFLD-PLC, and was more marked in female patients (P = 0.03). No mutations were found in cirrhosis and local controls, and only one in 503 healthy Europeans from the 1000 Genomes Project (allelic frequency = 0.025 vs. <0.001; P = 0.0005). Mutations with predicted functional impact, including the frameshift Glu113Argfs*79 and missense Glu668Asp, cosegregated with liver disease in two families. Three patients carried missense mutations (Ala67Val in homozygosity, Pro193Leu and His296Pro in heterozygosity) in the N-terminal template-binding domain (P = 0.037 for specific enrichment). Besides Glu668Asp, the Ala67Val variant resulted in reduced intracellular protein levels. In conclusion, we detected an association between shorter telomeres in peripheral blood and rare germline hTERT mutations and NAFLD-HCC.

Insulin resistance promotes Lysyl Oxidase Like 2 induction and fibrosis accumulation in non-alcoholic fatty liver disease.

In patients with non-alcoholic fatty liver disease (NAFLD), insulin resistance (IR) associates with fibrosis progression independently of the hepatic inflammation, but the mechanisms are still unclear. We modeled the independent contribution of inflammation (non-alcoholic steatohepatitis: NASH) by exploiting the methionine-choline deficient (MCD) diet, and that of IR by insulin receptor (InsR) haploinsufficiency (InsR+/-) in the pathogenesis of liver fibrosis in C57BL/6 mice. We confirmed the study findings in 96 patients with NAFLD. InsR+/- enhanced hepatic fat content and impaired hepatic insulin signaling leading to Forkhead box protein O1 (FoxO1) accumulation in MCD-fed mice. Remarkably, despite reduced inflammation and hampered transdifferentiation of hepatic stellate cells (HSCs), InsR+/- promoted hepatic fibrosis accumulation, which correlated with the induction of the Lysyl Oxidase Like 2 (Loxl2), involved in matrix stabilization. Loxl2 up-regulation was not a cell autonomous property of insulin resistant HSCs, but was dependent on microparticles (MPs) released specifically by insulin resistant hepatocytes (HEPs) exposed to fatty acids. The mechanism entailed FoxO1 up-regulation, as FoxO1 silencing normalized Loxl2 expression reversing fibrosis in InsR+/- MCD-fed mice. Loxl2 up-regulation was similarly detected during IR induced by obesity, but not by lipogenic stimuli (fructose feeding). Most importantly, LOXL2 up-regulation was observed in NAFLD patients with type 2 diabetes (T2D) and LOXL2 hepatic and circulating levels correlated with histological fibrosis progression. IR favors fibrosis deposition independently of the classic 'inflammation - HSC transdifferentiation' pathway. The mechanism entails a cross-talk between enhanced lipotoxicity in insulin resistant HEPs and Loxl2 production by HSCs, which was confirmed in patients with diabetes, thereby facilitating extracellular matrix (ECM) stabilization.

Glucose availability enhances lipopolysaccharide production and immunogenicity in the opportunistic pathogen Acinetobacter baumannii.

AIM: Acinetobacter baumannii can cause sepsis with high mortality rates. We investigated whether glucose sensing might play a role in A. baumannii pathogenesis. MATERIALS & METHODS: We carried out transcriptome analysis and extracellular polysaccharide determination in an A. baumannii clinical isolate grown on complex medium with or without glucose supplementation, and assessed its ability to induce production of inflammatory cytokines in human macrophages. RESULTS: Growth in glucose-supplemented medium strongly enhanced A. baumannii sugar anabolism, resulting in increasing lipopolysaccharide biosynthesis. In addition, glucose induced active shedding of lipopolysaccharide, in turn triggering a strong induction of inflammatory cytokines in human macrophages. Finally, hemolytic activity was strongly enhanced by growth in glucose-supplemented medium. CONCLUSION: We propose that sensing of exogenous glucose might trigger A. baumannii pathogenesis during sepsis.

Whole-exome sequencing of primary plasma cell leukemia discloses heterogeneous mutational patterns.

Primary plasma cell leukemia (pPCL) is a rare and aggressive form of plasma cell dyscrasia and may represent a valid model for high-risk multiple myeloma (MM). To provide novel information concerning the mutational profile of this disease, we performed the whole-exome sequencing of a prospective series of 12 pPCL cases included in a Phase II multicenter clinical trial and previously characterized at clinical and molecular levels. We identified 1, 928 coding somatic non-silent variants on 1, 643 genes, with a mean of 166 variants per sample, and only few variants and genes recurrent in two or more samples. An excess of C > T transitions and the presence of two main mutational signatures (related to APOBEC over-activity and aging) occurring in different translocation groups were observed. We identified 14 candidate cancer driver genes, mainly involved in cell-matrix adhesion, cell cycle, genome stability, RNA metabolism and protein folding. Furthermore, integration of mutation data with copy number alteration profiles evidenced biallelically disrupted genes with potential tumor suppressor functions. Globally, cadherin/Wnt signaling, extracellular matrix and cell cycle checkpoint resulted the most affected functional pathways. Sequencing results were finally combined with gene expression data to better elucidate the biological relevance of mutated genes. This study represents the first whole-exome sequencing screen of pPCL and evidenced a remarkable genetic heterogeneity of mutational patterns. This may provide a contribution to the comprehension of the pathogenetic mechanisms associated with this aggressive form of PC dyscrasia and potentially with high-risk MM.

Comprehensive genomic characterization of cutaneous malignant melanoma cell lines derived from metastatic lesions by whole-exome sequencing and SNP array profiling.

Cutaneous malignant melanoma is the most fatal skin cancer and although improved comprehension of its pathogenic pathways allowed to realize some effective molecular targeted therapies, novel targets and drugs are still needed. Aiming to add genetic information potentially useful for novel targets discovery, we performed an extensive genomic characterization by whole-exome sequencing and SNP array profiling of six cutaneous melanoma cell lines derived from metastatic patients. We obtained a total of 3,325 novel coding single nucleotide variants, including 2,172 non-synonymous variants. We catalogued the coding mutations according to Sanger COSMIC database and to a manually curated list including genes involved in melanoma pathways identified by mining recent literature. Besides confirming the presence of known melanoma driver mutations (BRAF(V600E), NRAS(Q61R) ), we identified novel mutated genes involved in signalling pathways crucial for melanoma pathogenesis and already addressed by current targeted therapies (such as MAPK and glutamate pathways). We also identified mutations in four genes (MUC19, PAICS, RBMXL1, KIF23) never reported in melanoma, which might deserve further investigations. All data are available to the entire research community in our Melanoma Exome Database (at https://155.253.6.64/MExDB/). In summary, these cell lines are valuable biological tools to improve the genetic comprehension of this complex cancer disease and to study functional relevance of individual mutational events, and these findings could provide insights potentially useful for identification of novel therapeutic targets for cutaneous malignant melanoma.

Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms.

We studied mutations of MPL exon 10 in patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF), first investigating a cohort of 892 consecutive patients. MPL mutation scanning was performed on granulocyte genomic DNA by using a high-resolution melt assay, and the mutant allele burden was evaluated by using deep sequencing. Somatic mutations of MPL, all but one involving codon W515, were detected in 26/661 (4%) patients with ET, 10/187 (5%) with PMF, and 7/44 (16%) patients with post-ET myelofibrosis. Comparison of JAK2 (V617F)-mutated and MPL-mutated patients showed only minor phenotypic differences. In an extended group of 62 MPL-mutated patients, the granulocyte mutant allele burden ranged from 1% to 95% and was significantly higher in patients with PMF or post-ET myelofibrosis compared with those with ET. Patients with higher mutation burdens had evidence of acquired copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p in granulocytes, consistent with a transition from heterozygosity to homozygosity for the MPL mutation in clonal cells. A significant association was found between MPL-mutant allele burden greater than 50% and marrow fibrosis. These observations suggest that acquired CN-LOH of chromosome 1p involving the MPL location may represent a molecular mechanism of fibrotic transformation in MPL-mutated myeloproliferative neoplasms.

Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms.

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.