Scoliosis with cognitive impairment in a girl with 8q11.21q11.23 microdeletion and SNTG1 disruption.

Idiopathic scoliosis (IS) is an abnormality of the vertebral column with a spine curvature of at least 10 degrees. It is the most common spinal deformity in children with a prevalence of 2%-3%, and its aetiology is unknown. Genetic factors are known to play a role and a number of linkage analyses showed associations of various loci. Here we describe a new case of a de novo interstitial deletion 8q11.21q11.2 disrupting SNTG1 gene, identified by array-CGH in a girl with cognitive impairment and a scoliosis that 'appears' like to IS. SNTG1 encodes γ-1 Syntrophin protein that is part of the dystrophin associated protein complex and interacts directly with the C-terminal of dystrophin. Its expression is restricted to neurons and particularly in those areas of the brain that have been suggested to affect postural control. The involvement of SNTG1 gene in IS was already been reported in a family with a breakpoint between exons 10 and 11. Mutational analysis of SNTG1 exons in 152 sporadic IS patients had revealed changes in three patients. In conclusion, our data add a further line of evidence suggesting SNTG1 could represent an interesting candidate for its involvement in scoliosis.

17q23.3 de novo microdeletion involving only TANC2 gene: A new case.

Neurodevelopmental disorders (NDDs) show a wide range of overlapping clinical features. Intellectual disability (ID), developmental delay (DD), autism spectrum disorder (ASD), attention-deficit hyperactivity disorder (ADHD), language and communication disorders with or without motor abnormalities and/or epilepsy have been reported associated to single or multiple genes but in many cases the genetic basis remains unknown. The increasingly use of array-CGH has significantly improved the yield of diagnosing genomic disorders and led to the identification of several novel microdeletion and microduplication syndromes. TANC2 encodes a synaptic scaffold protein interacting with multiple neuropsychiatric disorder-related postsynaptic density (PSD) proteins in dendrites. Here, we describe a new case of TANC2 gene disruption in a 17q23.3 de novo microdeletion identified by array-CGH. The patient presented craniofacial dysmorphic features, hypotonia, and severe cognitive and motor impairment. In conclusion, our data add a further line of evidence supporting the role of TANC2 in NDDs and will help further researches to elucidate the regulatory mechanism of synaptic function and plasticity related to TANC2 haploinsufficiency.

Intervertebral disc and endplate cell characterisation highlights annulus fibrosus cells as the most promising for tissue-specific disc degeneration therapy.

Degenerative processes of the intervertebral disc (IVD) and cartilaginous endplate lead to chronic spine pathologies. Several studies speculated on the intrinsic regenerative capacity of degenerated IVD related to the presence of local mesenchymal progenitors. However, a complete characterisation of the resident IVD cell populations, particularly that isolated from the endplate, is lacking. The purpose of the present study was to characterise the gene expression profiles of human nucleus pulposus (NPCs), annulus fibrosus (AFCs) and endplate (EPCs) cells, setting the basis for future studies aimed at identifying the most promising cells for regenerative purposes. Cells isolated from NP, AF and EP were analysed after in vitro expansion for their stemness ability, immunophenotype and gene profiles by large-scale microarray analysis. The three cell populations shared a similar clonogenic, adipogenic and osteogenic potential, as well as an immunophenotype with a pattern resembling that of mesenchymal stem cells. NPCs maintained the greatest chondrogenic potential and shared with EPCs the loss of proliferation capability during expansion. The largest number of selectively highly expressed stemness, chondrogenic/tissue-specific and surface genes was found in AFCs, thus representing the most promising source of tissue-specific expanded cells for the treatment of IVD degeneration.

The impact of grape processing and carbonic maceration on the microbiota of early stages of winemaking.

AIMS: The work investigates the impact of grapes processing at the beginning of winemaking on the composition of microbiota during the oenological fermentations and on the composition of obtained wines. METHODS AND RESULTS: The experiments were conducted in a biodynamic winery to exclude interference due to microbial starters. Three winemaking protocols, with different pre-fermentative management of grapes, were followed by plate count and next generation sequencing on 16S for bacteria and internal transcribed spacer sequencing (ITS) for yeast. Chemical and sensory characterization of wine was performed. The grape processing influenced the evolution of microbiota (especially lactic and acetic acid bacteria) and the fermentation rate. The highest biodiversity was observed in the experiment carried out with whole grapes and carbonic maceration, with the presence of bacterial groups not usually found in winemaking (Bacteroidales, Clostridiales, Oscillospira). The different microbiotas influenced the organic acid profile of wines, the content of biogenic ammines and the perception of organoleptic descriptors linked to the vine cultivar (Syrah). CONCLUSIONS: Carbonic maceration impacts on the evolution of the microbiota and the wine features. The absence of addition of starters and sulphur dioxide would seem to be correlated with the high microbial biodiversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Carbonic maceration is a traditional winemaking practice, today there are difficulties in its managing because the anaerobiosis stimulates spoilage micro-organisms. The work elucidates the reasons of these difficulties and identified some microbial groups rarely associated with winemaking. The ratio of ethanol accumulation along with physical management of grapes and the supply of oxygen during the early stages of winemaking are powerful instruments of oenological variability, able to offer new possibilities to winemakers in order to defining the quality of red wines.

Detailed macro- and micromineral profile of milk: Effects of herd productivity, parity, and stage of lactation of cows of 6 dairy and dual-purpose breeds.

The aim of this study was to quantify the major sources of variation in the levels of 15 minerals in individual milk samples collected from cows raised in multibreed dairy herds. The herds (n = 27) were classified into 2 categories, according to milk productivity. Milk productivity was based on the net energy of lactating cows' average daily milk yield. Milk samples were collected from 240 cows of 6 different breeds: 3 specialized dairy (Holstein-Friesian, Brown Swiss, and Jersey) and 3 dual-purpose (Simmental, Rendena, and Alpine Grey). The samples were analyzed for macro-elements (Na, Mg, P, S, K, and Ca), essential micro-elements (Mn, Fe, Cu, Zn, and Se), and environmental micro-elements (B, Si, Sr, and Sn), using inductively coupled plasma-optical emission spectrometry. Data were analyzed using a linear mixed model that included fixed effects of days in milk (DIM), parity, breed, and herd productivity, and a random effect of herd-date within productivity level. Results showed that the effect of herd-date varied across minerals. It was especially large for environmental minerals (ranging from 47 to 91% of total variance) and ranged from 11 to 61% for macrominerals and essential microminerals. Milk samples collected from farms with a high level of herd productivity had a richer mineral profile than samples from low-productivity herds. Parity only influenced macrominerals, with the exception of S and Ca, while DIM influenced almost all minerals, with a few exceptions among the environmental elements. Differences in mineral profile were small between specialized and dual-purpose breeds, but they were large within the group of the specialized cows. These breed differences were reduced after adjusting for milk quality and yield, particularly in the case of milk Mg, S, Ca, Mn, and Zn levels. Milk samples from the Jersey and Brown Swiss cows had higher mineral levels (Sn excluded) than milk from the Holstein-Friesian cows; the other breeds of Alpine origin produced milk of intermediate quality. Our findings suggest that breed has a stronger effect on macrominerals and some of the essential microminerals than herd productivity, parity, and DIM. The modification of the mineral profile in milk seems possible for many minerals, but it likely depends on genetics (e.g., breed, selection) and on environmental and management factors in variable proportions according to the mineral considered.

Tailored-BODIPY/Amphiphilic Cyclodextrin Nanoassemblies with PDT Effectiveness.

Amphiphilic cyclodextrins (aCDs) are an intriguing class of carrier systems which, recently, have been proposed to deliver porphyrinoids and anticancer drugs or combined dose of both for dual therapeutic applications. The design of nanoassemblies based on aCD and photosensitizers (PSs) aims to preserve the photodynamic therapy (PDT) efficacy of PS, reducing the tendency of PS to self-aggregate, without affecting the quantum yield of singlet oxygen (1O2) production, and, not less importantly, minimizing dark toxicity and reducing photosensitization effects. With this idea in mind, in this paper, we focus on nanoassemblies between a non-ionic aCD (SC6OH) and halo-alkyl tailored iodinated boron-dipyrromethenes (BODIPY) dye, a class of molecules which recently have been successfully proposed as a stimulating alternative to porphyrinoids for their high photodynamic efficacy. Nanoassemblies of BODIPY/aCD (BL01I@SC6OH) were prepared in different aqueous media by evaporation of mixed organic film of aCD and BODIPY, hydration, and sonication. The nanostructures were characterized, measuring their hydrodynamic diameter and ξ-potential and also evaluating their time-stability in biological relevant media. Taking advantage of emissive properties of the not-iodinated BODIPY analogue (BL01), nanoassemblies based on aCD and BL01 were investigated as model system to get insight on entanglement of BODIPY in the amphiphile in aqueous dispersion, pointing out that BODIPY is well-entrapped in monomeric form (τ ≅ 6.5 ns) within the colloidal carriers. Also morphology and fluorescence emission properties were elucidated after casting the solution on glass. BL01@SC6OH is easily detectable in cytoplasm of HCT116 cell lines, evidencing the remarkable intracellular penetration of this nanoassembly similar to free BODIPY. On the same cell lines, the photodynamically active assembly BL01I/aCD shows toxicity upon irradiation. Despite the fact that free BL01I is more PDT active than its assembly, aCD can modulate the cell uptake of BODIPY, pointing out the potential of this system for in vivo PDT application.

Prematurity, ventricular septal defect and dysmorphisms are independent predictors of pathogenic copy number variants: a retrospective study on array-CGH results and phenotypical features of 293 children with neurodevelopmental disorders and/or multiple congenital anomalies.

BACKGROUND: Since 2010, array-CGH (aCGH) has been the first-tier test in the diagnostic approach of children with neurodevelopmental disorders (NDD) or multiple congenital anomalies (MCA) of unknown origin. Its broad application led to the detection of numerous variants of uncertain clinical significance (VOUS). How to appropriately interpret aCGH results represents a challenge for the clinician. METHOD: We present a retrospective study on 293 patients with age range 1 month - 29 years (median 7 years) with NDD and/or MCA and/or dysmorphisms, investigated through aCGH between 2005 and 2016. The aim of the study was to analyze clinical and molecular cytogenetic data in order to identify what elements could be useful to interpret unknown or poorly described aberrations. Comparison of phenotype and cytogenetic characteristics through univariate analysis and multivariate logistic regression was performed. RESULTS: Copy number variations (CNVs) with a frequency < 1% were detected in 225 patients of the total sample, while 68 patients presented only variants with higher frequency (heterozygous deletions or amplification) and were considered to have negative aCGH. Proved pathogenic CNVs were detected in 70 patients (20.6%). Delayed psychomotor development, intellectual disability, intrauterine growth retardation (IUGR), prematurity, congenital heart disease, cerebral malformations and dysmorphisms correlated to reported pathogenic CNVs. Prematurity, ventricular septal defect and dysmorphisms remained significant predictors of pathogenic CNVs in the multivariate logistic model whereas abnormal EEG and limb dysmorphisms were mainly detected in the group with likely pathogenic VOUS. A flow-chart regarding the care for patients with NDD and/or MCA and/or dysmorphisms and the interpretation of aCGH has been made on the basis of the data inferred from this study and literature. CONCLUSION: Our work contributes to make the investigative process of CNVs more informative and suggests possible directions in aCGH interpretation and phenotype correlation.

Targeted and untargeted characterisation of free and glycosylated simple phenols in cocoa beans using high resolution-tandem mass spectrometry (Q-Orbitrap).

Free simple phenols have positive effects on health and influence the organoleptic profile of cocoa products, contributing towards defining their aroma and nutritional properties. Glycosidically bound simple phenols can be hydrolysed during the production phase to the corresponding free forms, and thus potentially contribute to the final sensory profile. In this work, 60 samples of Forastero cocoa beans from all over the world were analysed, combining on-line solid phase extraction (SPE) clean-up with ultra-high performance liquid chromatography (UHPLC), coupled with high resolution mass spectrometry. Operating in negative ion mode and with a heated electrospray, 62 simple phenols were measured, of which four were glucosidic precursors, with quantification limits ranging from 0.04 to 40mgkg-1, calibration R2 of 0.99 for over 93% of compounds, and precision (R.S.D.%) always lower than 12%. On the basis of accurate mass, isotope pattern and MS/MS spectrum, 32 monoglycosylated simple phenols such as hexoside and pentoside precursors, and 14 diglycosylated simple phenols such as hexoside-hexoside, hexoside-pentoside and pentoside-hexoside precursors, were tentatively identified. The untargeted approach was validated using 3 glucosidic precursors synthesized by an external supplier. Honestly Significant Difference Tukey's test (p<0.05) and Discriminant Analysis showed it was possible to distinguish the geographical origin of cocoa beans. In particular, the absolute free phenol profile made it possible to characterise 4 out of 5 production macro-areas well, while an untargeted approach based on the ionisation profiles of glycosylated forms allowed complete characterisation of all the 5 macro-areas.

Clinical and molecular characterization of two patients with palmoplantar keratoderma-congenital alopecia syndrome type 2.

Palmoplantar keratoderma-congenital alopecia (PPKCA) syndrome is a rare genodermatosis, with two clinically recognizable forms: dominant (Type 1) and recessive (Type 2). Reports of only 18 patients have been published to date, and the molecular basis of the condition is unknown. We describe two cases with PPKCA Type 2 (PPKCA2), comprising a novel patient, originally reported as an example of autosomal ichthyosis follicularis-atrichia-photophobia syndrome, and the 6-year follow-up of a previously published case. Extensive molecular studies of both patients excluded mutations in all the known genes associated with PPK and partially overlapping syndromes. The striking similarities between these two patients confirm PPKCA2 as a discrete genodermatosis, of which the main features are congenital and universal alopecia, diffuse keratosis pilaris, facial erythema, and a specific PPK with predominant involvement of the fingertips and borders of the hands and feet, with evolution of sclerodactyly, contractures and constrictions. Clinical follow-up of these patients has demonstrated progressive worsening of the hand involvement and attenuation of facial erythema.

Free and glycosylated simple phenol profiling in Apulian Italian wines.

Free simple phenols have a significant role in defining the sensory and nutritional characteristics of wines, affecting the organoleptic profile and having positive effects on health, but glycosidically bound phenols can also be hydrolysed during the winemaking process, releasing the corresponding volatile compounds and making a possible contribution to the final sensory profile. In this work, application of on-line SPE liquid chromatography-high resolution mass spectrometry, operating in negative polarity with heated electrospray, allowed to detect over eighty free and glycosylated simple phenols in Primitivo di Manduria and Negroamaro wines. Sixty-one phenols, four of which phenolic glucosidic precursors, were quantified as having quantification limits ranging from 0.001 to 0.1µgmL(-1), calibration R(2) of 0.99 for over 92% of compounds, and precision (R.S.D.%) always lower than 12%. Twenty-four simple phenolic precursors were tentatively identified as hexoside, pentoside and hexoside-hexoside derivatives, on the basis of accurate mass, isotopic pattern and MS/MS fragmentation.

Evaluation of the oenological suitability of grapes grown using biodynamic agriculture: the case of a bad vintage.

AIMS: We compare the evolution of the microbiota of grapes grown following conventional or biodynamic protocols during the final stage of ripening and wine fermentation in a year characterized by adverse climatic conditions. METHODS AND RESULTS: The observations were made in a vineyard subdivided into two parts, cultivated using a biodynamic and traditional approach in a year which saw a combination of adverse events in terms of weather, creating the conditions for extensive proliferation of vine pests. The biodynamic approach was severely tested, as agrochemicals were not used and vine pests were counteracted with moderate use of copper, sulphur and plant extracts and with intensive use of agronomical practices aimed at improving the health of the vines. Agronomic, microbiological and chemical testing showed that the response of the vineyard cultivated using a biodynamic approach was comparable or better to that of vines cultivated using the conventional method. CONCLUSIONS: The work suggests that biodynamic cultivation of the grapevine may be sustainable even in difficult conditions, representing an interesting alternative to traditional vine-growing approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: This theme is topical and of interest to winemakers and consumers today, but is not easy to study due to the difficulties in finding vineyards with homogeneous characteristics, cultivated using different agronomical protocols. The particular climatic conditions observed in 2014 made this year a rare model, making it possible to verify the applicability of biodynamics to vine growing. The strict experimental plan gave results particularly useful for understanding the features of grape microbiota in a biodynamic context.

Identification and quantification of 56 targeted phenols in wines, spirits, and vinegars by online solid-phase extraction - ultrahigh-performance liquid chromatography - quadrupole-orbitrap mass spectrometry.

Phenolic compounds seriously affect the sensory and nutritional qualities of food products, both through the positive contribution of wood transfer in barrel-aged products and as off-flavours. A new targeted analytical approach combining on-line solid-phase extraction (SPE) clean-up to reduce matrix interference and rapid chromatographic detection performed with ultrahigh-performance liquid chromatography coupled with quadrupole/high-resolution mass spectrometry (Q-Orbitrap), was developed for the quantification of 56 simple phenols. Considering the advantages of using on-line SPE and a resolving power of 140,000, the proposed method was applied to define phenolic content in red (N=8) and white (8) wines, spirits (8), common (8) and balsamic (8) vinegars. The final method was linear from the limits of quantification (0.0001-0.001µgmL(-1)) up to 10µgmL(-1) with R(2) of at least 0.99. Recovery, used to define method accuracy, ranged from 80 to 120% for 89% of compounds. The method was suitable for analytical requirements in the tested matrices being able to analyse 46 phenols in red wines, 41 phenols in white wines and in spirits, 42 phenols in common vinegars and 44 phenols in balsamic vinegars.

Autonomic nervous system dysregulation in irritable bowel syndrome.

BACKGROUND: Autonomic nervous system (ANS) regulation may be altered in functional diseases, including irritable bowel syndrome (IBS), but published data are not clear to date. The aim of the study was to analyze ANS function in IBS subjects classified by Rome III criteria and healthy controls using standardized technique. METHODS: ANS activity was evaluated by autoregressive spectral analysis of RR interval and systolic arterial pressure variabilities, to obtain indices of sympatho-vagal modulation of the heart and of spontaneous cardiac baroreflex (α index). A symptom list was used to score 18 somatic complaints (score 0-180) (4SQ). Fatigue and stress were assessed through the use of a global scoring index (0-10). KEY RESULTS: We enrolled 41 IBS subjects (29 F, age 40 ± 2 years) and 42 healthy matched controls. Heart rate was higher in IBS than control subjects (69 ± 2 vs 61 ± 1; p < 0.001). The total variance of RR interval variability, and α index, were significantly lower in IBS compared to controls (1983.12 ± 384.64 ms(2) vs 4184.55 ± 649.59 ms(2) ; 18.1 ± 2 ms/mmHg vs 29 ± 3 ms/mmHg; p < 0.01). The α index results showed an inverse correlation with stress scores and somatic symptoms. CONCLUSIONS & INFERENCES: IBS subjects display a significant reduction in α index, an established marker of cardiac baroreflex. ANS dysfunction appears to be involved in the pathophysiology of IBS and its assessment may open new perspectives for clinical management of patients suffering from IBS.

First-trimester euploid miscarriages analysed by array-CGH.

It is estimated that 10-15 % of all clinically recognised pregnancies results in a miscarriage, most of which occur during the first trimester. Large-scale chromosomal abnormalities have been found in up to 50 % of first-trimester spontaneous abortions and, for several decades, standard cytogenetic analysis has been used for their identification. Recent studies have proven that array comparative genomic hybridisation (array-CGH) is a useful tool for the detection of genome imbalances in miscarriages, showing a higher resolution, a significantly higher detection rate and overcoming problems of culture failures, maternal contamination and poor chromosome morphology. In this study, we investigated the possibility that submicroscopic chromosomal changes, not detectable by conventional cytogenetic analysis, exist in euploid miscarriages and could be causative for the spontaneous abortion. We analysed with array-CGH technology 40 foetal tissue samples derived by first-trimester miscarriages with a normal karyotype. A whole-genome microarray with a 100-Kb resolution was used for the analysis. Forty-five copy number variants (CNVs), ranging in size between 120 Kb and 4.3 Mb, were identified in 31 samples (24 gains and 21 losses). Ten samples (10/31, 32 %) have more than one CNV. Thirty-one CNVs (68 %) were defined as common CNVs and 14 were classified as unique. Six genes and five microRNAs contained within these CNVs will be discussed. This study shows that array-CGH is useful for detecting submicroscopic CNVs and identifying candidate genes which could account for euploid miscarriages.

Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011.

A precise guideline establishing chromosomal microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The controversial question is whether CMA technologies can or should soon replace standard karyotyping in prenatal diagnostic practice. A review of the recent literature and survey of the knowledge and experience of all members of the Italian Society of Human Genetics (SIGU) Committee were carried out in order to propose recommendations for the use of CMA in prenatal testing. The analysis of datasets reported in the medical literature showed a considerable 6.4% incidence of pathogenic copy number variations (CNVs) in the group of pregnancies with sonographically detected fetal abnormalities and normal karyotype. The reported CNVs are likely to have a relevant role in terms of nosology for the fetus and in the assessment of reproductive risk for the couple. Estimation of the frequency of copy number variations of uncertain significance (VOUS) varied depending on the different CMA platforms used, ranging from 0-4%, obtained using targeted arrays, to 9-12%, obtained using high-resolution whole genome single nucleotide polymorphism (SNP) arrays. CMA analysis can be considered a second-tier diagnostic test to be used after standard karyotyping in selected groups of pregnancies, namely those with single (apparently isolated) or multiple ultrasound fetal abnormalities, those with chromosomal rearrangements, even if apparently balanced, and those with supernumerary marker chromosomes.

Array-CGH and clinical characterization in a patient with subtelomeric 6p deletion without ocular dysgenesis.

Subtelomeric terminal 6p deletion has been recognized as a clinically identifiable syndrome including facial dysmorphism, malformation of the anterior eye chamber, hearing loss, heart defects, and developmental delay. Genotype-phenotype correlations of previously published patients have strongly suggested anterior eye segment anomalies as one of the major malformations of the syndrome if the critical 6p25 region contains the FOXC 1 gene. In addition, the presence in this region of one or more genes involved in hearing loss has been hypothesized. We report a patient with a 47,XYY karyotype and submicroscopic terminal 6p deletion. Further characterization of the deletion with array comparative genome hybridization also revealed a cryptic microduplication on chromosome 19. The patient showed dysmorphic features, neuromotor retardation, and profound language impairment, in absence of hearing loss and structural eye anomalies. As far as we know this is the first reported terminal 6p25.1 deletion case without eye dysgenesis precisely characterized by array-CGH. Our result suggests that the genes in this region may not be obvious candidates for hearing loss and demonstrate the need for further elucidation of the function of the genes involved in eye developmental processes.

Short communication: characterization of a monoclonal antibody for kappa-casein B of cow's milk.

A monoclonal antibody (antik-B) against an oligopeptide of 23 AA corresponding to the region 131-153 of bovine kappa-casein (kappa-CN) B was generated using the Human Combinatorial Antibody Library (HuCAL) technology. Both AA substitutions distinguishing kappa-CN A and B are located in that region (positions 136 and 148). In this study, the reactivity of antik-B to milk samples collected from cows previously genotyped as CSN3*AA, CSN3*AB, and CSN3*BB was tested. According to Western blot results, antik-B recognized kappa-CN B and it showed no cross-reactivity toward kappa-CN A and other milk proteins. Furthermore, a modified Western blot method, urea-PAGE Western blot, was set up to assess the reactivity of antik-B toward all isoforms of kappa-CN B. In conclusion, antik-B was specific to kappa-CN B in milk and it seemed to be reactive toward all its isoforms.