Identification of plant-derived alkaloids with therapeutic potential for myotonic dystrophy type I

Myotonic dystrophy type I (DM1) is a disabling neuromuscular disease with no causal treatment available. This disease is caused by expanded CTG trinucleotide repeats in the 3 UTR of the dystrophia myotonica protein kinase gene. On the RNA level, expanded (CUG)n repeats form hairpin structures that sequester splicing factors such as muscleblind-like 1 (MBNL1). Lack of availableMBNL1leads to misregulated alternative splicing of many target pre-mRNAs, leading to the multisystemic symptoms in DM1. Many studies aiming to identify small molecules that target the (CUG)n-MBNL1 complex focused on synthetic molecules. In an effort to identify new small molecules that liberate sequesteredMBNL1from (CUG)n RNA, we focused specifically on small molecules of natural origin. Natural products remain an important source for drugs and play a significant role in providing novel leads and pharmacophores for medicinal chemistry. In a new DM1 mechanism-based biochemical assay, we screened a collection of isolated natural compounds and a library of over 2100 extracts from plants and fungal strains. HPLC-based activity profiling in combination with spectroscopic methods were used to identify the active principles in the extracts. The bioactivity of the identified compounds was investigated in a human cell model and in a mouse model of DM1.We identified several alkaloids, including the -carboline harmine and the isoquinoline berberine, that ameliorated certain aspects of theDM1pathology in these models. Alkaloids as a compound class may have potential for drug discovery in other RNA-mediated diseases

ACTN1 : identification of novel mutations in a cohort of Italian IMTP patients

Inherited macrothrombocytopenia (IMTP) is a highly heterogeneous group of inherited disorders characterized by a low platelet count and abnormally platelet size. Even though IMTP-causing mutations have been reported in several genes, only 50% of patients have to date a molecular diagnosis. In March 2013 Kunishima and colleagues identified alpha-actinin 1 (ACTN1) as a new gene responsible for IMTP which accounted for 5,5% of cases in Japanese population. To evaluate the frequency of ACTN1 mutations in the Italian population, we performed a screening in 160 probands in which all the known forms of IMTP were previously excluded. Ten, including 8 novel, different missense have been identified in 11 patients. All except one (p.D666V) segregated with the macrotrombocytopenia within the families. In vivo transfection experiments in Hela cells were performed to demonstrate the pathogenicity of the variants identified. Except for p.D666V, preliminary data indicate that the missense mutations are associated with disorganization of cytoskeleton, as determined different co-localization of mutant and wild type actinin-a with actin. Further studies will be needed to determine how these mutations may lead to the onset of disease

ACTN1 : identification of novel mutations in a cohort of Italian IMTP patients

Inherited macrothrombocytopenia (IMTP) is a highly heterogeneous group of inherited disorders characterized by a low platelet count and abnormally platelet size. Even though IMTP-causing mutations have been reported in several genes, only 50% of patients have to date a molecular diagnosis. In March 2013 Kunishima and colleagues identified alpha-actinin 1 (ACTN1) as a new gene responsible for IMTP which accounted for 5,5% of cases in Japanese population. To evaluate the frequency of ACTN1 mutations in the Italian population, we performed a screening in 160 probands in which all the known forms of IMTP were previously excluded. Ten, including 8 novel, different missense have been identified in 11 patients. All except one (p.D666V) segregated with the macrotrombocytopenia within the families. In vivo transfection experiments in Hela cells were performed to demonstrate the pathogenicity of the variants identified. Except for p.D666V, preliminary data indicate that the missense mutations are associated with disorganization of cytoskeleton, as determined different co-localization of mutant and wild type actinin-a with actin. Further studies will be needed to determine how these mutations may lead to the onset of disease

Celiac disease in patients with sporadic and inherited cardiomyopathies and in their relatives.

AIMS: To investigate celiac disease (CD) and related co-morbidity in patients with familial and sporadic cardiomyopathy and in their relatives. METHODS AND RESULTS: We screened anti-human-tissue-transglutaminase (IgA and IgG anti-h-tTG) and anti-endomysial antibodies (AEAs) in 238 consecutive adult patients with inherited or sporadic dilated cardiomyopathy (DCM), 418 relatives, and 2000 healthy blood donors. HLADQ2-DQ8 was tested in tTG-positive subjects. The IgA-tTG-positive patients with cardiomyopathy underwent duodenal biopsy. Twenty-six subjects were tTG-positive: five DCM patients (2.1\%), two of 28 (7.1\%) and three of 390 (0.7\%) relatives with and without echocardiographic abnormalities respectively, and 16 controls (0.8\%). Twenty-two of 26 subjects were AEA-positive, and 25 HLA-positive. Of the five patients with cardiomyopathy and biopsy-proven CD, four suffered iron-deficiency anaemia. Two CD-positive DCM patients and two tTG-positive relatives were from families with inherited disease in which CD did not co-segregate with DCM. CONCLUSIONS; The higher prevalence of CD in patients with sporadic or inherited DCM, and of tTG-positive serology in relatives with echocardiographic abnormalities, suggests that immune-mediated mechanisms are active in subsets of patients/families. However, gluten intolerance cannot be considered causative since CD seems to be associated but not co-segregated with DCM in familial cases

Celiac disease in patients with sporadic and inherited cardiomyopathies and in their relatives

AIMS: To investigate celiac disease (CD) and related co-morbidity in patients with familial and sporadic cardiomyopathy and in their relatives. METHODS AND RESULTS: We screened anti-human-tissue-transglutaminase (IgA and IgG anti-h-tTG) and anti-endomysial antibodies (AEAs) in 238 consecutive adult patients with inherited or sporadic dilated cardiomyopathy (DCM), 418 relatives, and 2000 healthy blood donors. HLADQ2-DQ8 was tested in tTG-positive subjects. The IgA-tTG-positive patients with cardiomyopathy underwent duodenal biopsy. Twenty-six subjects were tTG-positive: five DCM patients (2.1%), two of 28 (7.1%) and three of 390 (0.7%) relatives with and without echocardiographic abnormalities respectively, and 16 controls (0.8%). Twenty-two of 26 subjects were AEA-positive, and 25 HLA-positive. Of the five patients with cardiomyopathy and biopsy-proven CD, four suffered iron-deficiency anaemia. Two CD-positive DCM patients and two tTG-positive relatives were from families with inherited disease in which CD did not co-segregate with DCM. CONCLUSIONS; The higher prevalence of CD in patients with sporadic or inherited DCM, and of tTG-positive serology in relatives with echocardiographic abnormalities, suggests that immune-mediated mechanisms are active in subsets of patients/families. However, gluten intolerance cannot be considered causative since CD seems to be associated but not co-segregated with DCM in familial cases