Stefania Cheli

Remodeling of the chromatin structure of the facioscapulohumeral muscular dystrophy (FSHD) locus and upregulation of FSHD-related gene 1 (FRG1) expression during human myogenic differentiation

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (ANT1), FSHD-related gene 1 (FRG1), FRG2 and DUX4c, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (DUX4) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing FRG1 has been generated, displaying skeletal muscle defects.ResultsIn the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and FRG1 gene promoter, and FRG1 expression, in control and FSHD cells. The FRG1 gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of FRG1 expression. Using chromosome conformation capture (3C) technology, we revealed that the FRG1 promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes. The FRG1 promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation.ConclusionWe propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.

Expression Profiling of FSHD-1 and FSHD-2 Cells during Myogenic Differentiation Evidences Common and Distinctive Gene Dysregulation Patterns

BACKGROUND Determine global gene dysregulation affecting 4q-linked (FSHD-1) and non 4q-linked (FSHD-2) cells during early stages of myogenic differentiation. This approach has been never applied to FSHD pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS By in vitro differentiation of FSHD-1 and FSHD-2 myoblasts and gene chip analysis we derived that gene expression profile is altered only in FSHD-1 myoblasts and FSHD-2 myotubes. The changes seen in FSHD-1 regarded a general defect in cell cycle progression, probably due to the upregulation of myogenic markers PAX3 and MYOD1, and a deficit of factors (SUV39H1 and HMGB2) involved in D4Z4 chromatin conformation. On the other hand, FSHD-2 mytubes were characterized by a general defect in RNA metabolism, protein synthesis and degradation and, to a lesser extent, in cell cycle. Common dysregulations regarded genes involved in response to oxidative stress and in sterol biosynthetic process. Interestingly, our results also suggest that miRNAs might be implied in both FSHD-1 and FSHD-2 gene dysregulation. Finally, in both cell differentiation systems, we did not observe a gradient of altered gene expression throughout the 4q35 chromosome. CONCLUSIONS/SIGNIFICANCE FSHD-1 and FSHD-2 cells showed, in different steps of myogenic differentiation, a global deregulation of gene expression rather than an alteration of expression of 4q35 specific genes. In general, FSHD-1 and FSHD-2 global gene deregulation interested common and distinctive biological processes. In this regard, defects of cell cycle progression (FSHD-1 and to a lesser extent FSHD-2), protein synthesis and degradation (FSHD-2), response to oxidative stress (FSHD-1 and FSHD-2), and cholesterol homeostasis (FSHD-1 and FSHD-2) may in general impair a correct myogenesis. Taken together our results recapitulate previously reported defects of FSHD-1, and add new insights into the gene deregulation characterizing both FSHD-1 and FSHD-2, in which miRNAs may play a role.

DPD and UGT1A1 deficiency in colorectal cancer patients receiving triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan.

AIMS Triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan is a standard therapy for metastatic colorectal cancer (CRC). Single nucleotide polymorphisms (SNPs) in DPYD and UGT1A1 influence fluoropyrimdines and irinotecan adverse events (AEs). Low frequency DPYD variants (c.1905 + 1G > A, c.1679 T > G, c.2846A > T) are validated but more frequent ones (c.496A > G, c.1129-5923C > G and c.1896 T > C) are not. rs895819 T > C polymorphism in hsa-mir-27a is associated with reduced DPD activity. In this study, we evaluated the clinical usefulness of a pharmacogenetic panel for patients receiving triplet combinations. METHODS Germline DNA was available from 64 CRC patients enrolled between 2008 and 2013 in two phase II trials of capecitabine, oxaliplatin and irinotecan plus bevacizumab or cetuximab. SNPs were determined by Real-Time PCR. We evaluated the functional variants in DPYD (rare: c.1905 + 1G > A, c.1679 T > G, c.2846A > T; most common: c.496A > G, c.1129-5923C > G, c.1896 T > C), hsa-mir-27a (rs895819) and UGT1A1 (*28) genes to assess their association with grade 3-4 AEs. RESULTS None of the patients carried rare DPYD variants. We found DPYD c.496A > G, c.1129-5923C > G, c.1896 T > C in heterozygosity in 19%, 5% and 8%, respectively, homozygous rs895819 in hsa-mir-27a in 9% and homozygous UGT1A1*28 in 8%. Grade 3-4 AEs were observed in 36% patients and were associated with DPYD c.496A > G (odds ratio (OR) 4.93, 95% CI 1.29, 18.87; P = 0.021) and homozygous rs895819 in hsa-mir-27a (OR 11.11, 95% CI 1.21, 102.09; P = 0.020). Carriers of DPYD c.1896 T > C and homozygous UGT1A1*28 showed an OR of 8.42 (95% CI 0.88, 80.56; P = 0.052). Multivariate analysis confirmed an independent value for DPYD c.496A > G and c.1896 T > C. CONCLUSIONS Concomitant assessment of DPYD variants and the UGT1A1*28 allele is a promising strategy needing further validation for dose personalization.

The first steps towards the era of personalised vaccinology: predicting adverse reactions

Until now, the occurrence of adverse reactions among individuals inoculated with identical vaccines has been ascribed to unpredictable stochastic processes. Recent advances in pharmacogenomics indicate that some features of host response to immunisation are influenced by genetic traits, henceforth predictable. The ability to predict the adverse reaction to vaccination would represent an important step towards the development of personalised vaccinology and could enhance public confidence in the safety of vaccines. Herein, we have reviewed all the available information on the association between genetic variants and the risk for healthy subjects to develop adverse reactions.

The predictive value of ABCB1, ABCG2, CYP3A4/5 and CYP2D6 polymorphisms for risperidone and aripiprazole plasma concentrations and the occurrence of adverse drug reactions

We investigated in ninety Caucasian pediatric patients the impact of the main polymorphisms occurring in CYP3A, CYP2D6, ABCB1 and ABCG2 genes on second-generation antipsychotics plasma concentrations, and their association with the occurrence of adverse drug reactions. Patients with the CA/AA ABCG2 genotype had a statistically significant lower risperidone plasma concentration/dose ratio (Ct/ds) (P-value: 0.007) and an higher estimated marginal probability of developing metabolism and nutrition disorders as compared to the ABCG2 c.421 non-CA/AA genotypes (P-value: 0.008). Multivariate analysis revealed that the ABCG2 c.421 CA/AA genotype was found associated to a higher hazard (P-value: 0.004) of developing adverse drug reactions classified as metabolism and nutrition disorders. The ABCB1 2677TT/3435TT genotype had a statistically significant lower aripiprazole Ct/ds if compared with patients with others ABCB1 genotypes (P-value: 0.026). Information obtained on ABCB1 and ABCG2 gene variants may result useful to tailor treatments with these drugs in Caucasian pediatric patients.

SLC29A1 polymorphism and prediction of anaemia severity in patients with chronic hepatitis C receiving triple therapy with telaprevir

OBJECTIVES The equilibrative nucleoside transporter 1 (ENT1) is the main protein involved in ribavirin cellular uptake. Polymorphisms at the SLC29A1 gene, encoding ENT1, may influence ribavirin-associated anaemia, which is observed at a higher incidence with telaprevir in combination with pegylated-IFNα and ribavirin than with pegylated-IFNα and ribavirin alone. In this study, we investigated the role of the rs760370 SLC29A1 variant in ribavirin-induced anaemia in chronic hepatitis C patients treated with telaprevir-based triple therapy. METHODS Forty patients infected with hepatitis C virus (HCV) genotype 1 and starting anti-HCV therapy with telaprevir in combination with pegylated-IFN/ribavirin were prospectively evaluated for SNPs at the SLC29A1 gene and inosine triphosphatase (ITPA) genes using a real-time PCR system. RESULTS 40% of patients developed severe anaemia with a haemoglobin (Hb) decline ≥ 5 g/dL from the pretreatment value. The SLC29A1 rs760370 GG genotype was associated with the severity of Hb decrease as expressed by the median (IQR) Hb nadir change from baseline [-5.4 (-5.6; -5.0) g/dL in GG versus -4.2 (-5.1; -3.4) in AA/AG genotype; P=0.05] and by the Hb decrease ≥ 5 g/dL by week 12 (77.8% of GG carriers versus 24% of AA/AG; P<0.01). In multivariate analysis, older age (P=0.03), lower baseline Hb concentration (P=0.02) and SLC29A1 rs760370 GG (P=0.02) were associated with the development of severe anaemia during treatment, whereas no association was found with ITPA SNPs in our population receiving telaprevir-based therapy. CONCLUSIONS In patients with chronic hepatitis C receiving telaprevir-based therapy, SNP rs760370A>G at the SLC29A1 gene influences the severity of ribavirin-induced anaemia, possibly mirroring the erythrocyte uptake of ribavirin.

Undetected toxicity risk in pharmacogenetic testing for dihydropyrimidine dehydrogenase.

Fluoropyrimidines, the mainstay agents for the treatment of colorectal cancer, alone or as a part of combination therapies, cause severe adverse reactions in about 10%–30% of patients. Dihydropyrimidine dehydrogenase (DPD), a key enzyme in the catabolism of 5-fluorouracil, has been intensively investigated in relation to fluoropyrimidine toxicity, and several DPD gene (DPYD) polymorphisms are associated with decreased enzyme activity and increased risk of fluoropyrimidine-related toxicity. In patients carrying non-functional DPYD variants (c.1905+1G>A, c.1679T>G, c.2846A>T), fluoropyrimidines should be avoided or reduced according to the patients’ homozygous or heterozygous status, respectively. For other common DPYD variants (c.496A>G, c.1129-5923C>G, c.1896T>C), conflicting data are reported and their use in clinical practice still needs to be validated. The high frequency of DPYD polymorphism and the lack of large prospective trials may explain differences in studies’ results. The epigenetic regulation of DPD expression has been recently investigated to explain the variable activity of the enzyme. DPYD promoter methylation and its regulation by microRNAs may affect the toxicity risk of fluoropyrimidines. The studies we reviewed indicate that pharmacogenetic testing is promising to direct personalised dosing of fluoropyrimidines, although further investigations are needed to establish the role of DPD in severe toxicity in patients treated for colorectal cancer.

The role of Toll-like receptor 4 polymorphisms in vaccine immune response

Toll-like receptors (TLRs) are a class of pattern recognition receptors that are deputed to recognise a range of molecular structures in pathogens. One of the most studied members of this family is the TLR4, which is essential for the signalling of lipopolysaccharide. The gene encoding for TLR4 is highly polymorphic and this genetic variability may explain in part the interindividual variability observed in several clinical setting, including the response to vaccination. Herein, we review and systematise the available scientific evidence about the effect of TLR4 polymorphisms on vaccine response, including approved prophylactic, new therapeutic cancer vaccines and recently approved vaccine adjuvants. Data reviewed in this analysis indicate that TLR4 polymorphisms significantly affect vaccine response. If these results are confirmed by further analyses, the use of these genetic biomarkers may become a useful tool to tailor vaccination in specific subsets of patients.

Levofloxacin-induced seizures in a patient without predisposing risk factors: the impact of pharmacogenetics.

Sir, Fluoroquinolones (FQ) are a widely used class of antibacterials and can be considered to be safe and well-tolerated drugs. Central nervous system (CNS) disturbances are the second most commonly reported adverse event after gastrointestinal disorders, with an overall incidence of about 1–2 % [1, 2]. Symptoms like headache, dizziness, tremor, confusion, and drowsiness usually occur on the first day of therapy and resolve after discontinuation; in contrast, seizures are rarely reported [3]. Levofloxacin has one of the most favorable adverse reaction profiles of all the FQ [4]. Here we report a case of two episodes of generalized seizures following levofloxacin administration in a patient without known predisposing conditions and/or concomitant interacting therapies but with a documented genetic predisposition for enhanced penetration of the drug into the brain. A 45-year old Caucasian woman was directed to our Day Hospital with a history of relapsing infections of the abdominal subcutaneous tissue starting after the excision of an abdominal lipoma 8 months earlier. Written informed consent for patient management, genetic analysis, and data handling were obtained prior to her admission to the Division of Infectious Disease of the L. Sacco Hospital. Although all cultures of purulent material were negative for bacterial growth, she was prescribed several oral antibiotic regimens without complete remission of the infection. At admission the patient referred to abdominal pain, pus was leaking from the surgical wound, and there were several subcutaneous abdominal nodules. The microbiological culture of the pus showed growth of a coagulasenegative Staphylcoccus resistant to teicoplanin and rifampin. The patient was started on a new antibiotic regimen that included levofloxacin 500 mg and ertapenem 1 g, both given intravenously once daily. On the fourth day of therapy, the patient experienced an episode of loss of consciousness during levofloxacin administration (after the conclusion of ertapenem). The episode lasted few seconds and was followed by a sensation of psychomotor slowing. The infusion of levofloxacin was immediately stopped and the patient hospitalized. Laboratory test results were in the normal range. Neurological examination and brain computed tomography did not detect any abnormalities. No history of allergy to medications, alcohol intake, or neurological dysfunction was reported. The episode was initially attributed to ertapenem due to the welldocumented epileptogenic potential of carbapenemic agents [5], and levofloxacin was reintroduced into the therapeutic regimen the next day. However, a few minutes after starting drug infusion the patient experienced seizures consisting of approximately 30 s of tonic–clonic contractions of her upper and lower extremities and loss of consciousness. Levofloxacin was once again discontinued and diazepam was used for seizures control. As in the previous episode, all laboratory parameters, neurological examination, and also the electroencephalogram were normal. A skin biopsy was subsequently performed which showed histopathological characteristics compatible with subcutaneous panniculitis. The patient C. Gervasoni (*) : L. Milazzo :A. Riva III Division of Infectious Diseases, L. Sacco University Hospital, Via GB Grassi 74, 20157 Milan, Italy e-mail: cristina.gervasoni@unimi.it

Variant alleles in factor V, prothrombin, plasminogen activator inhibitor-1, methylenetetrahydrofolate reductase and risk of thromboembolism in metastatic colorectal cancer patients treated with first-line chemotherapy plus bevacizumab

Single-nucleotide polymorphisms (SNPs) related to hereditary thrombophilia were investigated as risk factors for thromboembolism in cancer patients. Their effect in metastatic colorectal cancer (mCRC) has never been explored so far. Our aim was to analyse the effect of coagulation factor V (FVL G1691A), prothrombin (PT G20210A), methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) and plasminogen activator inhibitor type 1 (PAI-1 5G/4G) allelic variants in this setting. Fifty-two patients treated with first-line chemotherapy plus bevacizumab who developed a thromboembolic event in their lifetime were initially genotyped. A contemporary cohort of 127 patients who did not experience any thromboembolic event was also analysed. DNA was extracted from peripheral blood and genotypes were determined by real-time PCR, using LightSNiP (TIB MOLBIOL) on LightCcler 480 (Roche). The association between thromboembolism and SNPs was investigated by univariable and multivariable analyses. All SNPs were in Hardy–Weinberg equilibrium (χ2 test P>0.20). FVL G1691A and PT G20210A were present only in heterozygosis in 4 (2.2%) and 7 (3.9%) patients, respectively; MTHFR C677T in homozygosis in 29 (16.2%), MTHFR A1298C in homozygosis in 13 (7.3%); PAI-1 5G/4G in 98 (54.7%) and 4G/4G in 41 (23%) patients. At univariable analysis, treatment duration was significantly associated with thromboembolism (P<0.001), whereas gender, age, obesity, platelets count and chemotherapy backbone were not. Similarly, FVL G1691A and PT G20210A as well as MTHFR C677T and PAI-1 4G allele were significantly associated, whereas MTHFR A1298C was not. At multivariable model including PT G20210A, MTHFR C677T and PAI-1 4G (age, obesity, treatment duration and chemotherapy backbone were included as adjustment factors), the three SNPs were significantlty associated with higher risk of thromboembolism (P=0.025, <0.0001 and P=0.033, respectively). Further validation studies are warranted in order to design a prospective trial of thromboprophylaxis in mCRC patients with high-risk genotypes.