The BRCA1 c.4096+1G>A Is a Founder Variant Which Originated in Ancient Times.

Approximately 30-50% of hereditary breast and ovarian cancer (HBOC) is due to the presence of germline pathogenic variants in the BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185) onco-suppressor genes, which are involved in DNA damage response. Women who carry pathogenic BRCA1 variants are particularly likely to develop breast cancer (BC) and ovarian cancer (OC), with a 45-79 percent and 39-48 percent chance, respectively. The BRCA1 c.4096+1G>A variant has been frequently ascertained in Tuscany, Italy, and it has also been detected in other Italian regions and other countries. Its pathogenetic status has been repeatedly changed from a variant of uncertain significance, to pathogenic, to likely pathogenic. In our study, 48 subjects (38 of whom are carriers) from 27 families were genotyped with the Illumina OncoArray Infinium platform (533,531 SNPs); a 20 Mb region (24.6 cM) around BRCA1, including 4130 SNPs (21 inside BRCA1) was selected for haplotype analysis. We used a phylogenetic method to estimate the time to the most recent common ancestor (MRCA) of BRCA1 c.4096+1G>A founder pathogenic variant. This analysis suggests that the MRCA lived about 155 generations ago-around 3000 years ago.

Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants.

Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in Saccharomyces cerevisiae increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants.

STAT3 induces breast cancer growth via ANGPTL4, MMP13 and STC1 secretion by cancer associated fibroblasts.

In the tumor microenvironment, Cancer Associated Fibroblasts (CAFs) become activated by cancer cells and increase their secretory activity to produce soluble factors that contribute to tumor cells proliferation, invasion and dissemination to distant organs. The pro-tumorigenic transcription factor STAT3 and its canonical inducer, the pro-inflammatory cytokine IL-6, act conjunctly in a positive feedback loop that maintains high levels of IL-6 secretion and STAT3 activation in both tumor and stromal cells. Here, we demonstrate that STAT3 is essential for the pro-tumorigenic functions of murine breast cancer CAFs both in vitro and in vivo, and identify a STAT3 signature significantly enriched for genes encoding for secreted proteins. Among these, ANGPTL4, MMP13 and STC-1 were functionally validated as STAT3-dependent mediators of CAF pro-tumorigenic functions by different approaches. Both in vitro and in vivo CAFs activities were moreover impaired by MMP13 inhibition, supporting the feasibility of a therapeutic approach based on inhibiting STAT3-induced CAF-secreted proteins. The clinical potential of such an approach is supported by the observation that an equivalent CAF-STAT3 signature in humans is expressed at high levels in breast cancer stromal cells and characterizes patients with a shorter disease specific survival, including those with basal-like disease.

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions.

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5'UTR and 3'UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband's group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3'UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.

Liver-Specific siRNA-Mediated Stat3 or C3 Knockdown Improves the Outcome of Experimental Autoimmune Myocarditis.

Myocarditis can lead to autoimmune disease, dilated cardiomyopathy, and heart failure, which is modeled in the mouse by cardiac myosin immunization (experimental autoimmune myocarditis [EAM]). Signal transducer and activator of transcription 3 (STAT3) systemic inhibition exerts both preventive and therapeutic effects in EAM, and STAT3 constitutive activation elicits immune-mediated myocarditis dependent on complement C3 and correlating with activation of the STAT3-interleukin 6 (IL-6) axis in the liver. Thus, liver-specific STAT3 inhibition may represent a therapeutic option, allowing to bypass the heart toxicity, predicted by systemic STAT3 inhibition. We therefore decided to explore the effectiveness of silencing liver Stat3 and C3 in preventing EAM onset and/or the recovery of cardiac functions. We first show that complement C3 and C5 genetic depletion significantly prevents the onset of spontaneous myocarditis, supporting the complement cascade as a viable target. In order to interfere with complement production and STAT3 activity specifically in the liver, we took advantage of liver-specific Stat3 or C3 small interfering (si)RNA nanoparticles, demonstrating that both siRNAs can significantly prevent myocarditis onset and improve the recovery of heart functions in EAM. Our data demonstrate that liver-specific Stat3/C3 siRNAs may represent a therapeutic option for autoimmune myocarditis and suggest that complement levels and activation might be predictive of progression to dilated cardiomyopathy.

A pilot study of D-chiro-inositol plus folic acid in overweight patients with type 1 diabetes.

AIMS: To improve insulin sensitivity, insulin-sensitizing drugs such as metformin are commonly used in overweight and obese T1D patients. Similarly to metformin, D-chiro-inositol (DCI), as putative mediator of intracellular insulin action, can act as insulin sensitizer. The aim of this pilot study was to evaluate the hypothesis that DCI plus folic acid may improve glucose control reducing insulin resistance in overweight or obese T1D patients. METHODS: A 24-week randomized control trial was carried out in 26 overweight or obese T1D patients, undergoing intensive insulin therapy. Patients were randomized to 1 g DCI plus 400 mcg folic acid once daily (treated group) or to 400 mcg folic acid only once daily (control group). The primary end point was to evaluate the efficacy of DCI on metabolic control as assessed by HbA1c. As secondary endpoints, BMI and insulin requirement (IR) were evaluated. Paired t test (two tailed) and analysis of variance were used to evaluate differences in HbA1c, BMI and IR at different time points. RESULTS: A significant reduction in HbA1c levels in treated group versus control group (7.5% ± 0.9 vs. 7.9% ± 1.7, respectively, p < 0.05) was observed. However, no significant reduction in BMI and IR was observed [(BMI 25.7 ± 2.8 vs. 26.7 ± 1.0, respectively, p NS); (IR 0.52 ± 0.26 vs. 0.52 ± 0.19, respectively, p NS)]. CONCLUSIONS: This trial demonstrated for the first time that DCI plus folic acid oral supplementation can improve metabolic control in overweight T1D patients. CLINICALTRIAL. GOV ID: NCT02730949.

Efficacy and safety of otelixizumab use in new-onset type 1 diabetes mellitus.

INTRODUCTION: Type 1 diabetes (T1DM) is an immune-mediated disease induced by antigen-specific T cells infiltrating pancreatic beta cells leading to the progressive loss of endogenous insulin secretion. AREAS COVERED: The identification of specific components of the autoimmune response favoured the implementation of several immunomodulatory therapies including antiCD3 monoclonal antibody (mAb) called otelixizumab. Otelixizumab is a chimeric monoclonal antibody that targets the ε-chain of the CD3T-lymphocyte surface receptor that has been developed with the aim of short therapeutic courses capable of inducing a remission of T1DM. Clinical trials have been carried out with otelixizumab to evaluate its safety and efficacy, but despite positive results of Phase I and II studies, the results of Phase III studies have been contradictory. EXPERT OPINION: High doses of otelixizumab have shown beneficial effects on beta cell function whereas a lower dose, which was tested to avoid the adverse effects associated with higher doses, was not effective on beta cells preservation. We believe that otelixizumab is a drug of potential interest for treating new onset T1DM patients and its use in combination with other immunomodulatory agents should be considered as a solution to circumvent adverse effects while maintaining efficacy.

Therapy with proton pump inhibitors in patients with type 2 diabetes is independently associated with improved glycometabolic control.

AIMS: Experimental data demonstrated that gastrin has incretin-like stimulating actions on ß-cells, resulting in a promotion of glucose-induced insulin secretion. As proton pump inhibitors (PPIs) consistently increase plasma gastrin levels, a possible effect of this treatment on glucose-insulin homeostasis may be hypothesized. Therefore, the aim of this study was to evaluate the effect of chronic PPIs treatment on glycemic control in patients affected by type 2 diabetes. METHODS: This is an observational, retrospective study. A total of 548 consecutive patients with type 2 diabetes (mean age ± SD: 67.1 ± 10.9 years, M/F: 309/239, diabetes duration: 12.4 ± 9.8 years) referring to our diabetes outpatient clinics were enrolled; among them, 45 %were treated with PPIs longer than 2 years for preventive/therapeutic purposes. Fasting plasma glucose (FPG), glycated hemoglobin (HbA1c), serum lipids and transaminases were measured by standard laboratory methods. Major cardiovascular events and concomitant medications were recorded in all participants, and daily insulin requirement was calculated in insulin-treated subjects. RESULTS: PPIs-treated patients had significantly lower HbA1c (7.1 ± 1.07 %-54.1 ± 12 vs 7.4 ± 1.4 %-57.4 ± 8 mmol/mol, p = 0.011) and FPG (127 ± 36.9 vs 147.6 ± 49.4 mg/dl, p < 0.001) levels than those untreated. These differences increased in patients under insulin therapy and in those with concomitant PPIs + GLP-1-based therapy. The multivariate regression analysis demonstrated that the association between chronic PPIs treatment and HbA1c was independent from possible confounders (p = 0.01). CONCLUSIONS: PPIs treatment is associated with greater glycemic control in patients with type 2 diabetes, particularly in those on insulin- or GLP-1-based therapy. Our results suggest a role for PPIs in glucose-insulin homeostasis and may open a new scenario for diabetes therapy.

Identification of two novel BRCA1-partner genes in the DNA double-strand break repair pathway.

M1775R and A1789T are two missense variants located within the BRCT domains of BRCA1 gene. The M1775R is a known deleterious variant, while the A1789T is an unclassified variant that has been analyzed and classified as probably deleterious for the first time by our group. In a previous study, we described the expression profile of HeLa G1 cells transfected with the two variants and we found that they altered molecular mechanisms critical for cell proliferation and genome integrity. Considering that the mutations in the BRCA1 C terminus (BRCT) domains are associated to a phenotype with an altered ability in the DNA double-strand break repair, we chose three of the genes previously identified, EEF1E1, MRE11A, and OBFC2B, to be tested for an homologous recombination (HR) in vitro assay. For our purpose, we performed a gene expression knockdown by siRNA transfection in HeLa cells, containing an integrated recombination substrate (hprtDRGFP), for each of the target genes included BRCA1. The knockdown of BRCA1, OBFC2B, MRE11A, and EEF1E1 reduces the HR rate, respectively, of 97.6, 28.6, 41.8, and 42.3 % compared to cells transfected with a scrambled negative control duplex and all these differences are statistically significant (P < 0.05; Kruskal-Wallis test). Finally, we analyzed the effect of target gene depletion both on HR that on cell survival after DNA-damage induction by ionizing radiation. The clonogenic assay showed that the down-regulation of the target genes reduced the cell survival, but the effect on the HR, is not evident. Only the BRCA1-siRNA confirmed the inhibition effect on HR. Overall these results confirmed the involvement of MRE11A in the HR pathway and identified two new genes, OBFC2B and EEF1E1, which according to these data and the knowledge obtained from literature, might be involved in BRCA1-pathway.

Effects on human transcriptome of mutated BRCA1 BRCT domain: a microarray study.

BACKGROUND: BRCA1 (breast cancer 1, early onset) missense mutations have been detected in familial breast and ovarian cancers, but the role of these variants in cancer predisposition is often difficult to ascertain. In this work, the molecular mechanisms affected in human cells by two BRCA1 missense variants, M1775R and A1789T, both located in the second BRCT (BRCA1 C Terminus) domain, have been investigated. Both these variants were isolated from familial breast cancer patients and the study of their effect on yeast cell transcriptome has previously provided interesting clues to their possible role in the pathogenesis of breast cancer. METHODS: We compared by Human Whole Genome Microarrays the expression profiles of HeLa cells transfected with one or the other variant and HeLa cells transfected with BRCA1 wild-type. Microarray data analysis was performed by three comparisons: M1775R versus wild-type (M1775RvsWT-contrast), A1789T versus wild-type (A1789TvsWT-contrast) and the mutated BRCT domain versus wild-type (MutvsWT-contrast), considering the two variants as a single mutation of BRCT domain. RESULTS: 201 differentially expressed genes were found in M1775RvsWT-contrast, 313 in A1789TvsWT-contrast and 173 in MutvsWT-contrast. Most of these genes mapped in pathways deregulated in cancer, such as cell cycle progression and DNA damage response and repair. CONCLUSIONS: Our results represent the first molecular evidence of the pathogenetic role of M1775R, already proposed by functional studies, and give support to a similar role for A1789T that we first hypothesized based on the yeast cell experiments. This is in line with the very recently suggested role of BRCT domain as the main effector of BRCA1 tumor suppressor activity.

Latent autoimmune diabetes in the adults (LADA) in Asia: from pathogenesis and epidemiology to therapy.

Diabetes mellitus is a metabolic disorder resulting from a defect in insulin secretion, insulin action or both. An effect of this process is chronic hyperglycaemia with disorder of carbohydrate, fat and protein metabolism and with long-term complications of diabetes including retinopathy, nephropathy and neuropathy. Latent autoimmune diabetes in adults (LADA) is a type of autoimmune diabetes that resembles Type 1 diabetes (T1D), however, it shows a later onset and slower progression towards insulin necessity. Epidemiological studies suggest that LADA may account for 2-12% of all cases of diabetes in adult population. The epidemiology and phenotypic characteristics of LADA may vary between Caucasian and Asian diabetic patients as lifestyle, food habits and body mass index differ between these two populations. Data on LADA from population-based studies in Asia are sparse and only few studies have looked at it. A number of attractive therapeutic interventions may be envisaged for prevention of beta-cell loss in LADA, including hypoglycaemic and immunomodulatory agents. Because the autoimmune process in LADA seems to be slower than in childhood T1D, there is a wider window of opportunities for intervention. In deciding the best therapeutic approach, features of LADA should guide therapy including presence of other comorbidities that may influence the therapeutic choice.

C-peptide response and HLA genotypes in subjects with recent-onset type 1 diabetes after immunotherapy with DiaPep277: an exploratory study.

OBJECTIVE: To investigate whether lower risk HLA class II genotypes would influence the efficacy of DiaPep277 therapy in protecting ß-cell function evaluated by C-peptide secretion in recent-onset type 1 diabetic subjects. RESEARCH DESIGN AND METHODS: Data were collected from type 1 diabetic subjects enrolled in multicenter phase II studies with a randomized, double-blind, and placebo-controlled design in whom fasting and stimulated C-peptide levels were measured. HLA genotypes were classified in high, moderate, and low risk categories. RESULTS: A total of 146 subjects (aged 4.3 to 58.5 years) were enrolled, including 76 children (<18 years old) and 70 adults. At baseline, there was a significant increase in fasting, maximal, and area under the curve (AUC) C-peptide from high to moderate and low risk HLA genotypes in adults (P for trend <0.04) but not in children. Children showed a decrease of the three parameters over time regardless of therapy and HLA genotype. DiaPep277-treated adults with low risk genotype had significantly higher maximal and AUC C-peptide versus placebo at 12 months (0.04 ± 0.07 vs. -0.28 ± 0.09 nmol/L, P < 0.01, and 0.53 ± 1.3 vs. -4.59 ± 1.5 nmol/L, P < 0.05, respectively). In the moderate risk genotype group, Δmaximal and AUC C-peptide values were significantly higher in DiaPep277-treated versus placebo-treated patients (P < 0.01 and P < 0.05, respectively). CONCLUSIONS: This exploratory study demonstrates that type 1 diabetic adults with low and moderate risk HLA genotypes benefit the most from intervention with DiaPep277; the only subgroup with an increase of C-peptide at 12 months after diagnosis was the low risk DiaPep277-treated subgroup.

Clinical update on the use of immuno modulators (antiCD3, GAD, Diapep277, anti-IL1) in type 1 diabetes.

In type 1 diabetes, beta cells are attacked and destroyed by auto reactive T cells causing major impairment of blood glucose metabolism and, ultimately, the development of life-threatening complications. Currently, the treatment of this chronic disease is based on the use of endogenous insulin and no curative therapies are available. Treatment approaches in this respect need to be directed toward the primary causes of the disease tackling beta cells' auto reactive T cells. The goal of any curative intervention in type 1 diabetes is the preservation of insulin-secreting cells. This may be achieved by the abrogation of the pathogenic reactivity to beta cell auto antigens while preserving full capacity to generate a normal immune response against foreign antigens. In this review, some of the most promising drugs for immune intervention in type 1 diabetes are presented and discussed including phase 3 clinical trials that involve: DiaPep277, Anti-CD3 Otelixizumab, Glutamic Acid Decarboxylase (GAD) and anti-IL1 receptor antagonist. These approaches are currently being tested in international multicenter trials and all of them have a very similar outcome in terms of a beneficial effect on beta cells.

Double diabetes: a mixture of type 1 and type 2 diabetes in youth.

The increase in the incidence of type 1 diabetes (T1D), especially in children <5 years of age reported over the past decade can be attributed to changes in environmental factors, either quantitative or qualitative, rather than to an effect of genetic factors operating in such a short period of time. The notable increase in the incidence of type 2 diabetes (T2D) in children and adolescents is very likely the consequence of the increase in obesity and sedentary life style occurring in developed countries. The increase in the number of children and adolescents with a mixture of the two types of diabetes has recently come to light (i.e. subjects who are obese and/or with signs of insulin resistance as well as positive for markers of autoimmunity to beta cells). Under the current classification, it is difficult to define the type of diabetes affecting these young subjects, being classified as T2D because they are obese and insulin resistant, but also as T1D because of the presence of auto-antibodies to beta cells. There is no doubt that these subjects show an overlapping diabetes phenotype typical of both T1D and T2D suggesting that the current classification of diabetes should be revised taking into account this new form of diabetes which has called double diabetes or hybrid diabetes.

Westernization of the Filipino population resident in Rome: obesity, diabetes and hypertension.

BACKGROUND: Aims of the present study were to examine the anthropometrical and metabolic characteristics of the Filipino population migrant to the Southern European city of Rome, Italy. METHODS: A cross-sectional study was carried out in the city of Rome. Three hundred thirty-five Filipinos (95 M/240 F, mean age: 44.0+/-9.8 years, mean residence in Italy: 12.9+/-6.3 years) were studied. Data were collected by standardized questionnaires; anthropometrical parameters, arterial pressure, and fasting capillary blood glucose (FCG) were measured. RESULTS: Abdominal obesity was found in 52.5%, and BMI >or= 25 kg/m(2) in 44.5% of subjects. History of type 2 diabetes mellitus (T2DM) and hypertension were reported by 6.0 and 9.0% of subjects, respectively. Impaired fasting glucose was found in 13.1%, and FCG >or= 110 mg/dl in 18.5% of subjects. Altered systolic and/or diastolic blood pressure was found in 34.3% of subjects. About three-fourths of subjects were unaware of being diabetic and/or hypertensive. Years of Italy residence showed a direct significant correlation with the degree of changes in alimentary behaviours (rho=0.18, p=0.001), and with weight gain (rho=0.27, p<0.001). Multivariate analysis showed only age and waist circumference to be associated with both diabetes and hypertension. CONCLUSIONS: In the present study, the first to examine the metabolic disorders in a migrant Filipino population resident in Rome, a high prevalence of obesity, diabetes, and hypertension was found. The alarming results emerging from this study should be seriously considered by public health practitioners and decision makers, and made known to the Filipinos resident in Europe.

Menarche in type 1 diabetes is still delayed despite good metabolic control.

OBJECTIVE: To analyze the age at menarche of girls with type 1 diabetes (T1D) who were diagnosed with the disease before puberty and compare it with that of an age-matched group of normal girls. Previous studies on the appearance of menarche showed that the mean age of onset of menarche is delayed in girls affected by T1D compared with normal girls. DESIGN: Case-control study. SETTING: Patients and controls in an academic research environment. PATIENT(S): We studied, retrospectively, the charts of 162 consecutive girls with T1D born in a geographically defined region between 1984 and 1994 with a mean disease duration of 3-5 years, all of whom were on intensive insulin therapy since diagnosis of T1D. The control group consisted of 214 normal girls born between 1984 and 1994, who agreed to fill in an anonymous questionnaire regarding age at menarche and other clinical information. INTERVENTION(S): There was no intervention per se in the study. Age at menarche appears as a dependent variable of body mass index (BMI), HbA1c, and so on. MAIN OUTCOME MEASURE(S): BMI, HbA1c, and duration of T1D at menarche were considered among the potential factors affecting the age of menarche. RESULT(S): Age at menarche in girls with T1D was significantly delayed compared with control girls (12.6 +/- 1.5 years vs. 12.25 +/- 1.4 years, respectively). HbA1c levels and BMI did not influence the age at menarche. CONCLUSION(S): Despite intensive insulin therapy and good metabolic control since diagnosis of T1D, the age at menarche is still delayed in girls who develop T1D before puberty.

Double or hybrid diabetes associated with an increase in type 1 and type 2 diabetes in children and youths.

The increase in the incidence of type 1 diabetes (T1D), especially in children <5 yr of age, reported over the past decade can be attributed to changes in environmental factors (either quantitative or qualitative) rather than to an effect of genetic factors operating in such a short period of time. The notable increase in the incidence of type 2 diabetes (T2D) in children and adolescents is very likely the consequence of an increasing sedentary lifestyle and an increase in obesity, which has been occurring in developed countries. An increase in the number of children and adolescents with a mixture of the two types of diabetes has recently come to light (i.e., subjects who are obese and/or with signs of insulin resistance as well as positive for markers of autoimmunity to beta cells), although the epidemiological data supporting such a conclusion are sparse. Under the current classification, it is difficult to define the type of diabetes affecting these young subjects, who might be classified as T2D because they are obese and insulin resistant but also as T1D because of the presence of autoantibodies to beta cells. These subjects show an overlapping diabetes phenotype typical of both T1D and T2D, suggesting that the current classification of diabetes should be revised to take into account this new form of diabetes, which has been called 'double diabetes' or 'hybrid diabetes'. In this review, we report recent findings on the increasing rates of all forms of diabetes in the young population, including unpublished data collected in Russia.