Therapeutic implications of novel mutations of the RFX6 gene associated with early-onset diabetes.

Identification of the genetic defect underlying early-onset diabetes is important for determining the specific diabetes subtype, which would then permit appropriate treatment and accurate assessment of recurrence risk in offspring. Given the extensive genetic and clinical heterogeneity of the disease, high-throughput sequencing might provide additional diagnostic potential when Sanger sequencing is ineffective. Our aim was to develop a targeted next-generation assay able to detect mutations in several genes involved in glucose metabolism. All 13 known MODY genes, genes identified from a genome-wide linkage study or genome-wide association studies as increasing the risk of type 2 diabetes and genes causing diabetes in animal models, were included in the custom panel. We selected a total of 102 genes by performing a targeting re-sequencing in 30 patients negative for mutations in the GCK, HNF1α, HNF4α, HNF1ß and IPF1 genes at the Sanger sequencing analysis. Previously unidentified variants in the RFX6 gene were found in three patients and in two of them we also detected rare variants in WFS1 and ABCC8 genes. All patients showed a good therapeutic response to dipeptidyl peptidase-4 (DPP4) inhibitors. Our study reveals that next-generation sequencing provides a highly sensitive method for identification of variants in new causative genes of diabetes. This approach may help in understanding the molecular etiology of diabetes and in providing more personalized treatment for each genetic subtype.

Low bone density and bone metabolism alterations in Duchenne muscular dystrophy: response to calcium and vitamin D treatment.

UNLABELLED: Boys with Duchenne muscular dystrophy often have reduced bone mass and increased fracture risk. In this prospective study on 33 patients, calcifediol (25-OH vitamin D(3)) plus adjustment of dietary calcium to the recommended dose reduced bone resorption, corrected vitamin D deficiency, and increased bone mass in about two-thirds of cases. INTRODUCTION: Low BMC and BMD and bone metabolism alterations are frequent in boys with Duchenne muscular dystrophy (DMD), especially now that long-term glucocorticosteroid (GC) treatment is the standard of care. This prospective study was designed to evaluate the effects of a first-line treatment (25-OH vitamin D(3) [calcifediol] plus adjustment of dietary calcium to the recommended daily dose) on bone. METHODS: Thirty-three children with DMD on GC treatment were followed for 3 years: one of observation and two of treatment. MAIN OUTCOME: spine and total body BMC and BMD increase; secondary outcome: changes in bone turnover markers (C-terminal [CTx] and N-terminal [NTx] telopeptides of procollagen type I; osteocalcin [OC]). RESULTS: During the observation year, BMC and BMD decreased in all patients. At baseline and after 12 months, serum CTx and urinary NTx were higher than normal; OC and parathyroid hormone at the upper limit of normal; 25-OH vitamin D(3) significantly lower than normal. After 2 years of calcifediol and calcium-rich diet, BMC and BMD significantly increased in over 65% of patients, and bone metabolism parameters and turnover markers normalized in most patients (78.8%). During the observation year, there were four fractures in four patients, while during the 2 years of treatment there were two fractures in two patients. CONCLUSIONS: Calcifediol plus adequate dietary calcium intake seems to be an effective first-line approach that controls bone turnover, corrects vitamin D deficiency, and increases BMC and BMD in most patients with DMD. Lack of response seems related to persistently high bone turnover.

Type II CRISPR/Cas9 approach in the oncological therapy.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptable immune mechanism used by many bacteria and archaea to protect themselves from foreign nucleic acids. This complex system can recognize and cut non-self DNA in order to provide the prokaryotic organisms a strong defense against foreign viral or plasmid attacks and make the cell immune from further assaults. Today, it has been adapted to be used in vitro and in vivo in eukaryotic cells to perform a complete and highly selective gene knockout or a specific gene editing. The ease of use and the low cost are only two features that have made it very popular among the scientific community and the possibility to be used as a clinical treatment in several genetic derived pathologies has rapidly spread its fame worldwide. However, CRISPR is still not fully understood and many efforts need to be done in order to make it a real power tool for the human clinical treatment especially for oncological patients. Indeed, since cancer originates from non-lethal genetic disorders, CRISPR discovery fuels the hope to strike tumors on their roots. More than 4000 papers regarding CRISPR were published in the last ten years and only few of them take in count the possible applications in oncology. The purpose of this review is to clarify many problematics on the CRISPR usage and highlight its potential in oncological therapy.

The heel-contact gait pattern of habitual toe walkers.

We used kinematic, kinetic and EMG analysis to compare the spontaneous heel-contact gait patterns of 13 children classified as habitual toe walkers (HTWs) and age-matched controls. In the HTWs, the incidence of spontaneous heel-contact strides during a single recording session ranged from 15% to 92%, with no correlation with age, passive ankle joint excursion, walking speed and trial order. Hallmarks of the heel-contact strides were premature heel-rise, reversal of the second rocker, relative shortening of the loading response and anticipation and enhancement of the electromyographic (EMG) activity normally observed in the triceps surae (TS) during the first half of the stance phase. This variant of the locomotor program is different from the walking patterns observed in normally developing toddlers and children with cerebral palsy (CP). It does not necessarily reflect a functional adaptation to changes in the rheological properties of the muscle-tendon complex.

Spectrum and distribution of MECP2 mutations in 64 Italian Rett syndrome girls: tentative genotype/phenotype correlation.

We report a direct DNA sequencing analysis of the MECP2 gene undertaken on a further 64 Italian patients with Rett syndrome by using a LICOR 4200 Automated Sequencer. All of the girls entering the study had a consistent clinical diagnosis for this disorder. All coding regions and the flanking intronic splice site sequences were amplified as three non-overlapping fragments by using both forward and reverse primers. The results were then compared to the MECP2 reference sequences published in GenBank. Mutations of the MECP2 gene were identified in 64 of 75 (85.33%) unrelated sporadic Rett syndrome girls. Genotype/phenotype correlation studies, in particular in groups of patients with the same mutation, did not offer definitive and interesting data.

A new mutation in EDA gene in X-linked hypohidrotic ectodermal dysplasia associated with keratoconus.

Hypohidrotic ectodermal dysplasia (HED) was first described in 1848 by Thurnam. HED belongs to ectodermal dysplasias (EDs), which are developmental impairments of ectodermal-derived tissues. X-linked hypohidrotic ectodermal dysplasia (XLHED) is the most common form of the EDs and consists in abnormal development of teeth, hair, and eccrine sweat glands. XLHED is determined by mutations in the ED1 gene, which is responsible for the coding of ectodysplasin-A(EDA-A), a protein that regulates ectodermal appendage formation. In the present study we found both in our proband and in the mother the same missense mutation in exon 9 (c.957 C>A), which resulted in an aminoacid change at position 319 (Ser319Arg). This latter anomaly might alter the charges in the TNF domain of EDA-A, affecting the stability of the protein and therefore the interaction with its receptor. The male propositus presented classical manifestations of HED except for keratoconus (KC) and, to the best of our knowledge, this association has not been previously described. The identification of this new mutation may contribute to evaluating the genotype/phenotype correlations. Finally, this report can give useful information about the genetic basis of KC and HED. Future studies will allow us to understand if a genetic bond exists between them.

The perception of involved professionals towards research feasibility and usefulness: lessons from the Multi-Site Trial on Efficacy of Constraint Induced Movement Therapy in Children with Hemiplegia.

BACKGROUND: In the last decades, the world of rehabilitation has been more and more calling for clear evidence to support intervention and numerous research programs have been developed. At stake, relatively little research on opinions and attitude of rehabilitation personnel involved in research conducted in real clinical settings has been carried out. AIM: To explore the opinion of professionals involved in a national clinical trial on research. DESIGN: Multicentre cross-sectional study. SETTING: 19 rehabilitation centres/services (4 research institutes, 15 local rehabilitation services). POPULATION: All professional participating to a multi-centre clinical trial on the effects of Constraint Induced Movement Therapy on children with hemiplegic cerebral palsy. METHODS: A 15-questions questionnaire inquiring feasibility, usefulness, products, costs, judgement and perceptions about clinical research in rehabilitation was administered. RESULTS: Among those working in one of the 19 rehabilitation centres part of the multicentric study, 76 professionals were asked to fill in the questionnaire. 68 professionals answered (89.4% of response rate). More than 75% of the sample thinks that its rehabilitation centre is suited to develop clinical research. Research results useful for the development of their daily activities (new tools for the assessment of children, to demonstrate the efficacy of a new treatment option and to learn a new way of working, and to strengthen the ties within the working team). Research is costly in terms of personal time and effort, but it can modify the rehabilitation praxis (assessment tools, the relationship with colleagues/patients). 98% of the interviewees declared the willingness to participate to other research projects. CONCLUSION AND CLINICAL REHABILITATION IMPACT: This survey highlights the importance of conducting research in local rehabilitation services, not only in terms of generation of new evidences, but also in terms of building networks, sharing experiences and knowledge, connecting with centers of excellence and providing a specific training for research conduction.

Cognitive and behavioural effects of migraine in childhood and adolescence.

Since cognitive and behavioural characteristics of paediatric migraine sufferers have yet to be adequately defined, in this study we assessed the effect of migraine on the interictal functioning of children and adolescents by comparing the performance of two patient groups, 17 migraine sufferers with aura (MA) and 31 without aura (MoA) and by correlating the duration of the disorder, the frequency of attacks and interictal period with neuropsychological and behavioural findings. Both patient groups had cognitive performance within normal range except for a significant delay in the reaction time (RT) task. Both MA and MoA revealed a behavioural phenotype characterized by internalizing problems on Child Behaviour Check List (CBCL) scales. Slower RT to simple visual stimuli may be an early sign of a subclinical neuropsychological dysfunction, significantly correlated with the frequency of headache attacks and interictal period. The lack of a control group and other methodological limitations, such as patient selection bias and unadjusted P-value for multiple testing, make it difficult to give this finding a clearcut meaning. Further studies are needed on larger samples compared with a control group.

Unilateral frontal lobe epilepsy affects executive functions in children.

Very few studies to date have investigated the neuropsychological changes detectable in children suffering from frontal lobe epilepsy (FLE). The aim of the present study was to assess the effects of FLE on cognitive and executive functions in childhood. The sample includes 17 children with a frontal epileptogenic focus (10 right and 7 left), with no evidence of anatomical brain damage. These subjects were assessed by means of a battery of tests to investigate executive functioning. The results emphasised the presence of selective impairments of frontal lobe functions without evidence of deficits in global intellectual functioning. No side-specific deficits were detected, while an earlier onset of epilepsy and the duration of the disorder, but not the seizures frequency, were found to correspond with more severe deficits in some specific frontal lobe functions.

Evolution of upper limb function in children with congenital hemiplegia.

Hand function deficits in hemiplegic children are a major cause of disability, but there is a lack of appropriate instruments for evaluating the evolution of this deficit over time and for verifying the efficacy of its treatment. We evaluated changes in upper limb function in relation to age and the course of individual rehabilitation treatment in 20 children (13 males and 7 females) who were first seen within the first four years of life and subsequently followed until a mean age of 13 years and four months (range, 11-17 years) in accordance with a diagnostic/rehabilitation program initiated in our division in 1989. All of the children were treated by us; those whose paretic upper limb functioned well were not treated in any specific or directed manner. The protocol involved a qualitative evaluation of the spontaneous use of the paretic hand and a quantitative evaluation of grip. Analysis of the results revealed an age-related global improvement over time, occurring within the first five years of life and more pronounced in terms of grip than spontaneous use. This finding makes our protocol more specific than those currently used because it more reliably establishes the real capacity to use the paretic hand in different situations of everyday life. The most important changes concerned the children with more impaired functional capacity, whereas the children who presented with good functional skill retained this capacity over time, thus confirming the initial decision not to treat them.

Infrared fluorescent automated detection of thirteen short tandem repeat polymorphisms and one gender-determining system of the CODIS core system.

We used an infrared (IR) automated fluorescence monolaser sequencer for the analysis of 13 autosomal short tandem repeat (STR) systems (TPOX, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, TH01, vWA, D13S317, D16S359, D18S51, D21S11) and the X-Y homologous gene amelogenin system. These two systems represent the core of the combined DNA index systems (CODIS). Four independent multiplex reactions, based on the polymerase chain reaction (PCR) technique and on the direct labeling of the forward primer of every primer pair, with a new molecule (IRDye800), were set up, permitting the exact characterization of the alleles by comparison with ladders of specific sequenced alleles. This is the first report of the whole analysis of the STRs of the CODIS core using an IR automated DNA sequencer. The protocol was used to solve paternity/maternity tests and for population studies. The electrophoretic system also proved useful for the correct typing of those loci differing in size by only 2 bp. A sensibility study demonstrated that the test can detect an average of 10 pg of undegraded human DNA. We also performed a preliminary study analyzing some forensic samples and mixed stains, which suggested the usefulness of using this analytical system for human identification as well as for forensic purposes.