Targeted next generation sequencing as a diagnostic tool in epileptic disorders

Purpose: Epilepsies have a highly heterogeneous background with a strong genetic contribution. The variety of unspecific and overlapping syndromic and nonsyndromic phenotypes often hampers a clear clinical diagnosis and prevents straightforward genetic testing. Knowing the genetic basis of a patient’s epilepsy can be valuable not only for diagnosis but also for guiding treatment and estimating recurrence risks. Methods: To overcome these diagnostic restrictions, we composed a panel of genes for Next Generation Sequencing containing the most relevant epilepsy genes and covering the most relevant epilepsy phenotypes known so far. With this method, 265 genes were analyzed per patient in a single step. We evaluated this panel on a pilot cohort of 33 index patients with concise epilepsy phenotypes or with a severe but unspecific seizure disorder covering both sporadic and familial cases. Key Findings: We identified presumed disease‐causing mutations in 16 of 33 patients comprising sequence alterations in frequently as well as in less commonly affected genes. The detected aberrations encompassed known and unknown point mutations (SCN1A p.R222X, p. E289V, p.379R, p.R393H; SCN2A p.V208E; STXBP1 p.R122X; KCNJ10 p.L68P, p.I129V; KCTD7 p.L108M; KCNQ3 p.P574S; ARHGEF9 p.R290H; SMS p.F58L; TPP1 p.Q278R, p.Q422H; MFSD8 p.T294K), a putative splice site mutation (SCN1A c.693A> p.T/P231P) and small deletions (SCN1A p.F1330Lfs3X [1 bp]; MFSD8 p.A138Dfs10X [7 bp]). All mutations have been confirmed by conventional Sanger sequencing and, where possible, validated by parental testing and segregation analysis. In three patients with either Dravet syndrome or myoclonic epilepsy, we detected SCN1A mutations (p.R222X, p.P231P, p.R393H), even though other laboratories had previously excluded aberrations of this gene by Sanger sequencing or high‐resolution melting analysis. Significance: We have developed a fast and cost‐efficient diagnostic screening method to analyze the genetic basis of epilepsies. We were able to detect mutations in patients with clear and with unspecific epilepsy phenotypes, to uncover the genetic basis of many so far unresolved cases with epilepsy including mutation detection in cases in which previous conventional methods yielded falsely negative results. Our approach thus proved to be a powerful diagnostic tool that may contribute to collecting information on both common and unknown epileptic disorders and in delineating associated phenotypes of less frequently mutated genes.     

Understanding the neural mechanisms involved in sensory control of voice production

Auditory feedback is important for the control of voice fundamental frequency (F0). In the present study we used neuroimaging to identify regions of the brain responsible for sensory control of the voice. We used a pitch-shift paradigm where subjects respond to an alteration, or shift, of voice pitch auditory feedback with a reflexive change in F0. To determine the neural substrates involved in these audio-vocal responses, subjects underwent fMRI scanning while vocalizing with or without pitch-shifted feedback. The comparison of shifted and unshifted vocalization revealed activation bilaterally in the superior temporal gyrus (STG) in response to the pitch shifted feedback. We hypothesize that the STG activity is related to error detection by auditory error cells located in the superior temporal cortex and efference copy mechanisms whereby this region is responsible for the coding of a mismatch between actual and predicted voice F0.     

MC1R variation and melanoma risk in relation to host/clinical and environmental factors in CDKN2A positive and negative melanoma patients

Host, environmental and genetic factors differently modulate cutaneous melanoma (CM) risk across populations. Currently, the main genetic risk determinants are germline mutations in the major known high-risk susceptibility genes, CDKN2A and CDK4, and variants of the low-risk gene MC1R, which is key in the pigmentation process. This case-control study aimed at investigating the influence of the main host and environmental risk factors and of MC1R variation on CM risk in 390 CDKN2A-negative and 49 CDKN2A-positive Italian individuals. Multivariate analysis showed that MC1R variation, number of nevi and childhood sunburns doubled CM risk in CDKN2A-negative individuals. In CDKN2A-positive individuals, family history of CM and presence of atypical nevi, rather than MC1R status, modified risk (20.75- and 2.83-fold, respectively). Occupational sun exposure increased CM risk (three to sixfold) in both CDKN2A-negative and CDKN2A-positive individuals, reflecting the occupational habits of the Ligurian population and the geographical position of Liguria.     

Inhaled colistin for lower respiratory tract infections

INTRODUCTION: Lower respiratory tract infections, due to Pseudomonas aeruginosa or Acinetobacter baumannii, are frequently encountered in patients with cystic fibrosis (CF) or in patients developing nosocomial pneumonias. Both of these conditions bear a high mortality risk and aggressive antibiotic therapy is necessary. Inhaled antibiotics might represent an effective therapeutic approach for these diseases as it has demonstrated good bactericidal efficacy and safety in both preclinical and clinical studies. This colistin formulation might be useful particularly in patients with respiratory tract infections due to multidrug-resistant Gram-negative bacteria. Its main advantages are a better safety profile with a minimal or absent risk of nephrotoxicity. AREAS COVERED: This paper discusses the available systemic formulations of colistin, with pharmacokinetic and safety profiles, followed by an overview of inhaled antibiotics in lower respiratory tract infections. EXPERT OPINION: Inhaled colistin should be used selectively as monotherapy in chronic infections with P. aeruginosa in CF patients, whereas in patients with hospital/ventilator-acquired pneumonia (HAP/VAP), it should be used in a combined regimen with systemic antibiotics.     

The molecular basis of skeletal muscle weakness in a mouse model of inflammatory myopathy

Objective

It is generally believed that muscle weakness in patients with polymyositis and dermatomyositis is due to autoimmune and inflammatory processes. However, it has been observed that there is a poor correlation between the suppression of inflammation and a recovery of muscle function in these patients. This study was undertaken to examine whether nonimmune mechanisms also contribute to muscle weakness. In particular, it has been suggested that an acquired deficiency of AMP deaminase 1 (AMPD1) may be responsible for muscle weakness in myositis.

Methods

We performed comprehensive functional, behavioral, histologic, molecular, enzymatic, and metabolic assessments before and after the onset of inflammation in a class I major histocompatibility complex (MHC)–transgenic mouse model of autoimmune inflammatory myositis.

Results

Muscle weakness and metabolic disturbances were detectable in the mice prior to the appearance of infiltrating mononuclear cells. Force contraction analysis of muscle function revealed that weakness was correlated with AMPD1 expression and was myositis specific. Decreasing AMPD1 expression resulted in decreased muscle strength in healthy mice. Fiber typing suggested that fast‐twitch muscles were converted to slow‐twitch muscles as myositis progressed, and microarray results indicated that AMPD1 and other purine nucleotide pathway genes were suppressed, along with genes essential to glycolysis.

Conclusion

These data suggest that an AMPD1 deficiency is acquired prior to overt muscle inflammation and is responsible, at least in part, for the muscle weakness that occurs in the mouse model of myositis. AMPD1 is therefore a potential therapeutic target in myositis.

     

Anti-inflammatory changes of gene expression by Artemisia iwayomogi in the LPS-stimulated human gingival fibroblast: microarray analysis

The leaves and stems of Asteraceae Artemisia iwayomogi (Ai) for a long time have been known to inhibit inflammatory cytokine production and allergic reactions, and have been used to treat liver diseases. It needs to be elucidated in terms of global gene expression whether Ai has an influence as an anti-inflammatory agent on the cultured human gingival fibroblast stimulated with lipopolysaccharide (LPS). This study investigated the anti-inflammatory changes of the genes by Ai using the Affymetrix genechip human gene 1.0 ST array when the cultured human gingival fibroblast was treated with LPS. It was observed that the inflammation- and immune response-related genes were activated by LPS challenge in the cultured human gingival fibroblast. The array analysis showed that 65 of the 344 genes up-regulated by LPS stimulation, when compared to the control, were down-regulated by the Ai treatment. A number of inflammation- and immune response-related genes of the 65 genes were found. In addition, 78 of the 164 genes down-regulated by the LPS, when compared to the control, were up-regulated by the Ai treatment. The regulatory patterns of the representative genes were correlated with the real-time RT-PCR analysis. The Ai extract and its specific components, scopolin and scopoletin, significantly hindered the production of inflammatory mediators such as IL-6, TNF-α and nitrite in the LPS-challenged fibroblast. This study suggests that Ai can comprehensively inhibit the activation of the inflammation- and immune response-related genes and the inflammatory mediators in the human gingival fibroblast.     

Hepatic and pulmonary toxicogenomic profiles in mice intratracheally instilled with carbon black nanoparticles reveal pulmonary inflammation, acute phase response, and alterations in lipid homeostasis

Global pulmonary and hepatic messenger RNA profiles in adult female C57BL/6 mice intratracheally instilled with carbon black nanoparticles (NPs) (Printex 90) were analyzed to identify biological perturbations underlying systemic responses to NP exposure. Tissue gene expression changes were profiled 1, 3, and 28 days following exposure to 0.018, 0.054, and 0.162 mg Printex 90 alongside controls. Pulmonary response was marked by increased expression of inflammatory markers and acute phase response (APR) genes that persisted to day 28 at the highest exposure dose. Genes in the 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase pathway were increased, and those involved in cholesterol efflux were decreased at least at the highest dose on days 1 and 3. Hepatic responses mainly consisted of the HMG-CoA reductase pathway on days 1 (high dose) and 28 (all doses). Protein analysis in tissues and plasma of 0.162 mg Printex 90-exposed mice relative to control revealed an increase in plasma serum amyloid A on days 1 and 28 (p < 0.05), decreases in plasma high-density lipoprotein on days 3 and 28, an increase in plasma low-density lipoprotein on day 28 (p < 0.05), and marginal increases in total hepatic cholesterol on day 28 (p = 0.06). The observed changes are linked to APR. Although further research is needed to establish links between observations and the onset and progression of systemic disorders, the present study demonstrates the ability of NPs to induce systemic effects.