Plants in Menstrual Diseases: A Systematic Study from Italian Folk Medicine on Current Approaches.

Background: Plant-based remedies have been used since antiquity to treat menstrual-related diseases (MD). From the late nineteenth to the early to mid-twentieth century, Italian folk remedies to treat "women's diseases" were documented in a vast corpus of literature sources. Aim: The purpose of this paper is to bring to light the plant-based treatments utilized by Italian folk medicine to heal clinical manifestations of premenstrual syndrome (PMS), dysmenorrhea, amenorrhea and menstrual disorders in an attempt to discuss these remedies from a modern pharmacological point of view. Moreover, we compare the medical applications described by Hippocrates with those utilized by Italian folk medicine to check if they result from a sort of continuity of use by over two thousand years. Results: Out of the 54 plants employed in Italian folk medicine, 25 (46.3%) were already documented in the pharmacopoeia of the Corpus Hippocraticum for treating MD. Subsequently, a detailed search of scientific data banks such as Medline and Scopus was undertaken to uncover recent results concerning bioactivities of the plant extracts to treat MD. About 26% of the plants used by Italian folk medicine, nowadays, have undergone human trials to assess their actual efficacy. At the same time, about 41% of these herbal remedies come back to in different countries. Conclusions: Active principles extracted from plants used by Italian folk healers could be a promising source of knowledge and represent strength candidates for future drug discovery for the management of MD.

LC-MALDI-TOF ISD MS analysis is an effective, simple and rapid method of investigation for histones characterization: Application to EBV lymphoblastoid cell lines.

This contribution is the result of our progressive engagement to develop and to apply a top-down liquid chromatography (LC) matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) (LC-MALDI-TOF) analysis for the histone post-translational modifications (PTMs) and variants characterization, mainly in order to provide comprehensive and fast results. The histone post-translational modifications and the differential expression of the histone variants play an essential role both in the DNA packaging mechanism in chromosomes and in the regulation of gene expression in different cellular processes, also in response to molecular agents of environmental origin. This epigenetic mechanism is widely studied in different field such as cellular differentiation, development and in the understanding of mechanisms underlying diseases. The characterization of histone PTMs has traditionally performed by antibodies-based assay, but immunological methods have significant limits, and today systems that use mass spectrometry are increasingly employed. We evaluated an in-source decay (ISD) analysis for the histone investigation on human lymphoblastoid cells, and by this approach, we were able to identify and quantify several PTMs such as the di-methylation in the lysine 20 and the acetylation in the lysine 16 in H4 and the mono-methylation, di-methylation and trimethylations at K9 of the histone H3.1. Moreover, we detected and quantified in the same H2B spectrum the prevalent H2B 1C/2E type but also the minor H2B 1D, 1M and 1B/1L/1N, 1O/2F, 1J/1K variants. In this work, we show that MALDI-ISD represents an excellent methodology to obtain global information on histone PTMs and variants from cells in culture, with rapidity and simplicity of execution. Finally, this is a useful approach to get label-free relative quantitative data of histone variants and PTMs.

NeuroArray: A Customized aCGH for the Analysis of Copy Number Variations in Neurological Disorders.

BACKGROUND: Neurological disorders are a highly heterogeneous group of pathological conditions that affect both the peripheral and the central nervous system. These pathologies are characterized by a complex and multifactorial etiology involving numerous environmental agents and genetic susceptibility factors. For this reason, the investigation of their pathogenetic basis by means of traditional methodological approaches is rather arduous. High-throughput genotyping technologies, including the microarray-based comparative genomic hybridization (aCGH), are currently replacing classical detection methods, providing powerful molecular tools to identify genomic unbalanced structural rearrangements and explore their role in the pathogenesis of many complex human diseases. METHODS: In this report, we comprehensively describe the design method, the procedures, validation, and implementation of an exon-centric customized aCGH (NeuroArray 1.0), tailored to detect both single and multi-exon deletions or duplications in a large set of multi- and monogenic neurological diseases. This focused platform enables a targeted measurement of structural imbalances across the human genome, targeting the clinically relevant genes at exon-level resolution. CONCLUSION: An increasing use of the NeuroArray platform may offer new insights in investigating potential overlapping gene signatures among neurological conditions and defining genotype-phenotype relationships.

5-HTTLPR, anxiety and gender interaction moderates right amygdala volume in healthy subjects.

Genetic variants within the serotonin transporter gene (5-HTTLPR) impact the neurobiology and risk for anxiety-related behaviours. There are also gender differences in the prevalence of anxiety-related behaviours. Although numerous studies have investigated the influence of 5-HTTLPR genotype on the neural systems involved in emotional regulation, none have investigated how these effects are modulated by gender and anxiety. We investigated this issue using two complementary region of interest-based structural neuroimaging approaches (voxel-based morphometry and Freesurfer) in 138 healthy individuals categorized into 'no anxiety' and 'subclinical anxiety' groups based on the Hamilton Rating Scale for Anxiety (HAM-A). Preliminarily, using anxiety as a continuous variable, we found a significant interaction effect of genotype by gender on anxiety. Females homozygous for the Short allele showed the highest HAM-A scores and males the lowest. In addition, a three-way significant interaction among genotype, gender and anxiety category was found for the right amygdala volume. Post hoc tests revealed that homozygous females carrying the Short variant with a subclinical anxiety condition had larger volume. The reported interaction effects demonstrate that gender strongly modulates the relationship between 5-HTTLPR genotype and subclinical expression of anxiety acting on amygdala, one region of the emotional neural network specifically involved in the anxiety-like behaviours.

Amyotrophic lateral sclerosis: a new missense mutation in the SOD1 gene.

Copper-zinc superoxide dismutase-1 (SOD1) is the second most common mutated gene in amyotrophic lateral sclerosis (ALS). To date more than 150 missense mutations of SOD1 have been reported. The objective of this study was to describe a novel SOD1 mutation and its phenotypic expression. We describe a 74-year-old Caucasian man who began to complain of progressive weakness and atrophy of the right hand and over 10 months developed a severe tetraparesis, with atrophies of upper and lower limbs and neck muscles, dysphagia, and dyspnea that led to percutaneous endoscopic gastrostomy and tracheotomy. A diagnosis of ALS was made. Genetic analysis identified a heterozygous mutation in exon 4 of SOD1 that results in the amino acid substitution from arginine to cysteine at position 115 (p.R115C). We identified a novel pathogenic SOD1 mutation in a patient with a very rapid disease progression and aggressive phenotype providing additional information on the wide range of SOD1 mutations in apparently sporadic ALS and confirming the possibility of a strong genotype-phenotype correlation for distinct SOD1 mutations.

Ataxin-1 and ataxin-2 intermediate-length PolyQ expansions in amyotrophic lateral sclerosis.

OBJECTIVE: Recent evidence suggests that intermediate-length polyglutamine (PolyQ) expansions in the ataxin-2 (ATXN-2) gene are a risk factor for amyotrophic lateral sclerosis (ALS). This work was undertaken with the aim to investigate the frequency of ataxin-1 (ATXN-1) and ATXN-2 PolyQ expansions in a cohort of patients with sporadic ALS (sALS) and patients with familial ALS (fALS) from southern Italy. METHODS: We assessed the PolyQ lengths of ATXN-1 and ATXN-2 in 405 patients with sALS, 13 patients with fALS, and 296 unrelated controls without history of neurodegenerative disorders. RESULTS: We found significantly higher intermediate PolyQ expansions ≥ 32 for ATXN-1 alleles and ≥ 28 for ATXN-2 alleles in the sALS cohort (ATXN-1: ALS, 7.07% vs controls, 2.38%; p = 0.0001; ATXN-2: ALS, 2.72% vs controls, 0.5%; p = 0.001). ATXN-1 CAT and ATXN-2 CAA interruptions were detected in patients with ALS only. Age at onset, site of onset, and sex were not significantly related to the ATXN-1 or ATXN-2 PolyQ repeat length expansions. CONCLUSIONS: Both ATXN-1 and ATXN-2 PolyQ intermediate expansions are independently associated with an increased risk for ALS.

SMN1 gene copy number analyses for SMA healthy carriers in Italian population.

The routine molecular test for spinal muscular atrophy (SMA) diagnosis is based on the detection of a homozygous deletion of exons 7 and 8 of the telomeric copy of the survival motor neuron gene (SMN1). The presence of the centromeric copy of the SMN gene (SMN2) does not allow the detection of the hemizygous absence of the SMN1 gene, which characterizes the disease carriers. The demand for a quantitative SMN1 test is permanently growing because there is a high incidence of carriers. The disease is severe and to date there are no effective pharmacological treatments. Here, we present a non-radioactive assay based on real time quantitative polymerase chain reaction. We analyzed eight SMA patients, 14 SMA relatives and 50 health individuals from Southern Italy by real time quantitative method in order to identify haploid deletion occurring in SMA carriers. SMN1 copy number was determined by the comparative threshold cycle method (ΔΔCt). The results confirmed the deletion in all homozygous patients and permitted an evaluation of the number of alleles in the healthy carriers. This method is fast, reproducible, and enables us to discriminate carriers from healthy homozygous, which is impossible with normal techniques.

FUS mutations in sporadic amyotrophic lateral sclerosis: clinical and genetic analysis.

Fused in sarcoma (FUS) or translocation in liposarcoma (TLS), a DNA/RNA-binding protein, causes a dominant autosomal inherited form of amyotrophic lateral sclerosis (ALS), ALS 6. Its main role in neurodegeneration is highlighted by the presence of cytoplasmic accumulation of its mutant protein form in ALS patients. To further define the frequency and spectrum of FUS gene mutations, we have performed a molecular screening of a cohort of 327 Italian patients from Southern Italy with sporadic ALS (SALS). We identified 4 patients carrying 3 different missense mutations and several polymorphisms. Two different substitutions occurring in the same amino acidic position have been observed in 2 patients: R521G and R521C respectively; P525L mutation has been found in 2 additional cases. Most of the patients with FUS mutations showed early symptom onset and had short disease survival. We also detected 4 different polymorphic variants (3'-untranslated region [UTR] variant, c.*41G>A; c.523+3ins[GAGGTG]; c.335-15del[TTTT]; and rs13331793) in 9 patients from within our cohort. This study underlines the importance of population-based mutation screening of newly identified genes.

MAO A VNTR polymorphism and amygdala volume in healthy subjects.

The X-linked Monoamine Oxidase A (MAO A) gene presents a well known functional polymorphism consisting of a variable number of tandem repeats (VNTR) (long and short variants) previously associated with altered neural function of the amygdala. Using automatic subcortical segmentation (Freesurfer), we investigated whether amygdala volume could be influenced by this genotype. We studied 109 healthy subjects (age range 18-80 years; 59 male and 50 female), 74 carrying the MAO A High-activity allele and 35 the MAO A Low-activity allele. No significant effect of the MAO A polymorphism or interaction effect between polymorphism × gender was found on amygdalar volume. Thus, our findings suggest that the reported impact of the MAO A polymorphism on amygdala function is not coupled with consistent volumetric changes in healthy subjects. Future studies are needed to investigate whether the association between volume of the amygdala and the MAO A VNTR polymorphism is influenced by social/psychological variables, such as impulsivity, trauma history and cigarette smoking behaviour, not taken into account in this work.

A novel NF1 gene mutation in an Italian family with neurofibromatosis type 1.

PURPOSE: Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder with an estimated incidence of one in 3,500 births. Clinically, NF1 is characterized by café-au-lait (CAL) spots, neurofibromas, freckling of the axillary or inguinal region, Lisch nodules, optic nerve glioma, and bone dysplasias. NF1 is caused by inactivating mutations of the 17q11.2-located NF1 gene. We present a clinical and molecular study of an Italian family with NF1. METHODS: The proband, a 10-year-old boy, showed large CAL spots and freckling on the axillary region and plexiform neurofibromas on the right side only. His father (47 years old) showed, in addition to the similar signs, numerous neurofibromas of various sizes on his thorax, abdomen, back, and shoulder. Two additional family members (a brother and a sister of the proband) presented only small CAL spots. The coding exons of NF1 gene were analyzed for mutations by denaturing high-performance liquid chromatography and sequencing in all family members. RESULTS: The mutational analysis of the NF1 gene revealed a novel frameshift insertion mutation in exon 4c (c.654 ins A) in all affected family members. This novel mutation creates a shift on the reading frame starting at codon 218 and leads to the introduction of a premature stop at codon 227. CONCLUSIONS: The segregation of the mutation with the affected phenotype and its absence in the 200 normal chromosomes suggest that it is responsible for the NF1 phenotype.

Morphological correlates of MAO A VNTR polymorphism: new evidence from cortical thickness measurement.

A functional variant in the mono-amine oxidase A (MAO A) gene has been shown to impact neural function related to cognitive and affective processing and increase risk for conduct disorders. However, whether MAO A could be a candidate gene for structural variation in the human brain remains to be clarified. This study is the first to investigate the effect of this genotype on brain morphology by measuring cortical thickness. We genotyped 59 healthy male subjects (36 carrying the MAO A High-activity allele and 23 the MAO A Low-activity allele) who underwent structural MRI at 3T. Models of the grey-white and pial surfaces were generated for each individual's cortices, and the distance between these two surfaces was used to compute cortical thickness within a priori regions of interest of the orbitofrontal and cingulate cortices. Surface-based analysis of the cortical mantle showed that the MAO A genotype was associated with structural differences in the orbitofrontal cortex bilaterally, where the MAO A High-activity group showed the highest cortical thickness value and the MAO A Low-activity group the lowest. Otherwise, no significant difference was detected within the cingulate cortex. Thus, we confirm the hypothesis that the MAO A genotype has a specific impact on human brain morphology. In particular, thickness measurement of the orbitofrontal cortex provides new evidence about the biological impact of the MAO A genotype on neural systems relevant to the pathophysiology of behavioural disorders.

Mutation analysis of the SPG4 gene in Italian patients with pure and complicated forms of spastic paraplegia.

Mutations in the SPG4 gene are the most common causes of hereditary spastic paraplegia (HSP) accounting for up to 40% of autosomal dominant (AD) forms and 12-18% of sporadic cases. The phenotype associated with HSP due to mutations in the SPG4 gene tends to be pure. There is increasing evidence, however, of patients with complicated forms of spastic paraplegia in which SPG4 mutations were identified. A cohort of 38 unrelated Italian patients with spastic paraplegia, of which 24 had a clear dominant inheritance and 14 were apparently sporadic, were screened for mutations in the SPG4 gene. We identified 11 different mutations, six of which were novel (p.Glu143GlyfsX8, p.Tyr415X, p.Asp548Asn, c.1656_1664delinsTGACCT, c.1688-3C>G and c.*2G>T) and two exon deletions previously reported. The overall rate of SPG4 gene mutation in our patients was 36.8% (14/38); in AD-HSP we observed a mutation frequency of 45.8% (11/24), in sporadic cases the frequency was 21.4% (3/14). Furthermore, we found a mutational rate of 22.2% (2/9) and 41.4% (12/29) in the complicated and pure forms, respectively. The results underlie the importance of genetic testing in all affected individuals.

Further evidence that D90A-SOD1 mutation is recessively inherited in ALS patients in Italy.

Mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene have been reported to cause adult-onset autosomal dominant amyotrophic lateral sclerosis (FALS). In sporadic cases (SALS), de novo mutations in the SOD1 gene have occasionally been observed. All the SOD1 mutations are autosomal dominantly inherited with the exception of D90A. To date, in Italy, only two sporadic ALS cases carrying the D90A mutation have been reported in a homozygous state. We investigated for the presence of this mutation in 169 unrelated ALS patients from southern Italy. The genetic analysis revealed three ALS patients (1.8%) with mild phenotype carrying the homozygous D90A mutation.

Spastic paraplegia with thinning of the corpus callosum and white matter abnormalities: further mutations and relative frequency in ZFYVE26/SPG15 in the Italian population.

Spastic paraplegia with thinning of the corpus callosum (ARHSP-TCC) is a relatively frequent form of complicated hereditary spastic paraplegia in which mental retardation and muscle stiffness at onset are followed by slowly progressive paraparesis and cognitive deterioration. Although genetically heterogeneous, ARHSP-TCC is frequently associated with mutations in the SPG11 gene, on chromosome 15q. However, it is becoming evident that ARHSP-TCC can also be the clinical presentation of mutations in ZFYVE26 (SPG15), as shown by the recent identification of eight families with a variable phenotype. Here, we present an additional Italian ARHSP-TCC patient harboring two new, probably loss-of-function mutations in ZFYVE26. This finding, together with the report of a mutation in another Italian family, provides confirmation that ZFYVE26 is the second gene responsible for ARHSP-TCC in the Italian population.

MAO A VNTR polymorphism and variation in human morphology: a VBM study.

The X-linked monoamine oxidase A (MAO A) gene, coding for an enzyme especially involved in the serotonin catabolism, presents a well-characterized functional polymorphism (long and short variants) in the promoter region that alters the transcriptional activity of the gene and hence the function of the corresponding proteins. Using optimized voxel-based morphometry, we studied the effect of this functional polymorphism on brain morphology in normal individuals. Fifty-nine male healthy individuals (33 MAO A-high and 26 MAO A-low) were investigated. Voxel-based morphometry showed that the carriers of the long variant were significantly associated with loss of grey matter in orbitofrontal cortex, bilaterally. This study reveals pronounced genotype-related structural changes in a specific prefrontal region previously observed to mediate neurofunctional responses in behavioral tasks.

Ventro-lateral prefrontal activity during working memory is modulated by MAO A genetic variation.

Several lines of evidence have highlighted the role of the serotonergic system in working memory (WM) processes. The X-linked Mono-Amine Oxidase A (MAO A) gene, coding for an enzyme especially involved in the serotonin (5-HT) catabolism, presents a well-characterized functional polymorphism consisting in a variable number of tandem repeats (VNTR) in the promoter region with high activity and low activity variants. The high activity allele carriers have been associated with higher enzyme expression, lower amine concentration and altered prefrontal cortex (PFC) function during motor inhibition, but a direct effect of MAO A genotype on WM-related brain activity has not been demonstrated. We have studied the relationship of this polymorphism to brain activity elicited by a spatial working memory task (n-back) using blood oxygenation level-dependent functional magnetic resonance imaging in 30 healthy male individuals matched for a series of demographic and genetic variables (COMT Val108/158Met). We show that the high activity allele was significantly (p-level<0,001) associated with increased activity of the right ventro-lateral PFC (VLPFC, BA 47) during the high load condition of the n-back task. Our data reveal pronounced genotype-related functional changes in specific prefrontal region (VLPFC) subserving spatial working memory. Moreover, given the well-known role of this area in inhibitory control, our finding also provides new evidence for the involvement of 5-HT in PFC-mediated WM function.

Genetically dependent modulation of serotonergic inactivation in the human prefrontal cortex.

Previous research suggests that genetic variations regulating serotonergic neurotransmission mediate individual differences in the neural network underlying impulsive and aggressive behaviour. Although with conflicting findings, the monoamine oxidase-A (MAOA) and the serotonin transporter (5HTT) gene polymorphisms have been associated with an increased risk to develop impulsive and aggressive behaviour. Double knock-out mice studies have also demonstrated that MAOA and 5HTT genes strongly interact in the metabolic pathway leading to the serotonergic inactivation; however, their potential interactive effect in human brain remains uninvestigated. We used blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to assess the independent and interactive effects of both MAOA and 5HTT polymorphisms on the brain activity elicited by a response inhibition task in healthy volunteers. Multivariate analysis demonstrated an individual effect of both MAOA and 5HTT polymorphisms and a strong allele-allele interaction in the anterior cingulate cortex (ACC), a key region implicated in cognitive control and in the pathophysiology of impulsive and aggressive behaviour. These findings suggest that the MAOAx5HTT allelic interaction exerts a significant modulation on the BOLD response associated with response inhibition and contribute to validate haplotype models as useful tools for a better understanding of the neurobiology underlying complex cognitive functions.

A novel point mutation in PMP22 gene in an Italian family with hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant inherited disorder characterized by recurrent sensory or motor dysfunction. In 85% of HNPP cases the genetic defect is a 1.4 Mb deletion on chromosome 17p11.2, encompassing the PMP22 gene. Point mutations in the PMP22 gene responsible for HNPP phenotypes are rare. We investigated a 17-years-old girl who led to our detecting a novel mutation in PMP22 gene. The mutation was also detected in her father and corresponded to a deletion of one tymidine at position 11 in exon2 (c.11delT). This novel mutation creates a shift on the reading frame starting at codon 4 and leads to the introduction of a premature stop at codon 6.

Putative role of specific JAG1 gene exons in modulating clinical features in patients with leukoencephalopathy.

The aim of this study was to investigate the possible role of JAG1 gene mutations in modulating clinical features in patients with CADASIL-like phenotype which resulted negative for NOTCH3 gene mutations. Sixty-six CADASIL-like patients without NOTCH3 gene mutations were investigated for 5 out of 26 exons of the JAG1 gene, whose mutations were implicated in central nervous system vascular abnormalities. PCR was performed with primers specific for exons 3, 4, 13, 23 and 24 comprising the intron-exon boundaries. Amplicons were then analyzed by denaturing high performance liquid chromatography (DHPLC). The exons showing a variant DHPLC profile were directly sequenced. The sequence of exons 3, 4 and 23 revealed the presence of four already described polymorphisms in JAG1. 1001C/T (g.16015 C>T) in exon 4 was found in 9 patients, IVS23+18delT (g.33147 delT) in 29 patients, IVS3-15T/C (g.15852 T>C) in 17 patients, IVS2-43C/T (g.10532 C>T) in 1 patient; both the polymorphism 1001C/T and IVS3-15T/C were found in 3 patients. No mutations were found. These data demonstrate absence of correlation between mutations in specific JAG1 gene exons and clinical features in patients with CADASIL-like phenotype.